## FINAL REPORT

Study Title



## EVALUATION OF THE MU TAGENIC ACTIVITY OF FRET 09

- 0536

IN THE **SALMONELLA TYPHIMURI UM**  REVERSE MUTATION

ASSAY AND THE **ESCHERICHIA COLI** REVERSE MUTATION

ASSAY (WITH INDEPENDENT REPEAT)

Author

C.M. Verspeek

- Rip

Test Facilit y

WIL Research Europe B.V.

Hambakenwetering 7

5231 DD ‘s

- Hertogenbosch

The Netherlands

Laboratory Project Identification

Project 504369

Substance 205292 /A

- Page 1 of 38

-FRET 09

- 0536 Project 504369

- Page 2 - 1. CONTENTS

1. CONTENTS

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

2. STATEMENT OF GLP COM PLIANCE

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

3. QUALITY ASSURANCE ST ATEMENT

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

4. SUMMARY

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

5. INTRODUCTION

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

5.1. Preface

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

5.2. Aims of the study

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

5.3. Guidelines

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

5.4. Storage and retention of records and materials

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

6. MATERIALS AND METHOD S

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

6.1. Test substance

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

6.1 .1. Test substance information

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

6.1.2. Study specific test substance information

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

6.1.3. Test substance preparation

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

6.2. Reference substances

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

6.2.1. Negative control

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

6.2.2. Positive controls

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

6.3. Test system

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

6.4. Cell culture

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

6.5. Metabo lic activation system

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

6.5.1. Preparation of S9

- fraction

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

6.5.2. Preparation of S9

- mix

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

6.6. Study design

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

6.6.1. Dose range finding test

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

6.6.2. Mutation assay

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

6.6.3. Colony counting

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

6.7. Electronic data capture

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

6.8. Interpretation

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

6.8.1. Acceptability of the assay

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

6.8.2. Data evaluation and statistical procedures

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

6.9. List of deviations

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

6.9.1. List of protocol deviations

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

6.9.2. List of standard operating procedures deviations

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

7. RESULTS

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

7.1. Dose range finding test/first mutation experiment

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

7.2. Experiment 2

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

7.3. Experiment 3

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

8. DISCUSSION AND CONCL USION

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

9. REFERENCES

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

TABLES

T able 1 Toxicity of FRET 09

- 0536 in the dose range finding test/first mutation experiment

..... 15

Table 2 Toxicity of FRET 09

- 0536 in the second experiment

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

Table 3 Dose range finding test: Mutagenic response of FRET 09

- 0536 in the Sa lmonella

typhimurium reverse mutation assay and in the Escherichia coli reverse mutation

assay

............................................................................................................................. 18

Table 4 Experiment 1: Mutagenic response of FRET 09

- 0536 in the Salmonella

typhimurium reverse mutation assay

............................................................................ 19

Table 5 Experiment 2: Mutagenic response of FRET 09

- 0536 in the Salmonella

typhimurium reverse mutation assay and in the Escherichia coli reverse mutation

assay

............................................................................................................................. 20

Table 6 Experiment 3: Mutagenic response of FRET 09

- 0536 in the Escherichia coli

reverse mutation assay

................................................................................................ . 21FRET 09

- 0536 Project 504369

- Page 3 - APPENDICES

APPENDIX 1 SUMMARY TABLES

..................................................................................................... 18

APPENDIX 2 SUPPORTING MATERIALS AND METHOD

............................................................... 22

APPENDIX 3 DETAILED TABLES

...................................................................................................... 23

APPENDIX 4 HISTORICAL CONTROL DATA OF THE SOLVENT CONTROL

................................ 35

APPENDIX 5 HISTORICAL CONTROL DATA OF THE POSITIVE CONTROL SUBSTANCES

....... 36

APPENDIX 6 CERTIFICATE OF ANALYSIS

...................................................................................... 37

APPENDIX 7 SUBSTANCE IDENTITY INFORMATION DOCUMENT

.............................................. 38FRET 09

- 0536 Project 504369

- Page 6 - 4. SUMMARY

Evaluation of the mutagenic activit y of FRET 09

- 0536 in the Salmonella typhimurium reverse mutation

assay and the Escherichia coli reverse mutation assay (with independent repeat).

FRET 09

- 0536 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 Escherichia 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 a combination of phenobarbital and ß

- naphthoflavone or Aroclor ).

An additional experiment was performed with tester strain WP 2 uvrA in the absence of S9

- mix.

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

guidelines.

Batch RDIA5595 of FRET 09

- 0536 was a clear colourless liquid with a purity 89.6%

. The test

substance was dissolved in ethanol.

In the dose range finding test, FRET 09

- 0536 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. FRET 09

- 0536 precipitated on

the plates at dose levels of 1000 μg/plate and upwards. In tester strain TA100, toxicity was observed

at dose levels of 33 μg/plate and upwards in the absence of S9

- mix and 333 μg/plate and upwards in

the presence of S9

- mix. In tester strain WP 2 uvrA, toxicity was observed at dose levels o f

1000 μg/plate and upwards in the absence of S9

- mix and 3330 and 5000 μg/plate in the presence of

S9

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

Based on the results of the dose range finding test, FRET 09

- 0536 was tested in the first mutation

assay at a concentration range of 0.3 to 1 00 μg/plate in the absence of S9

- mix and at 3 to

1000 μg/plate in the presence of 5% (v/v) S9

- mix in tester strains TA1535, TA1537 and TA98. Toxicity

was observed in all three tester strains

In an independent repeat of the assay with additional parameters, FRET 09

- 0536 was tested at the

same concentration range as the first assay in the absence and presence of 10% (v/v) S9

- mix in tester

strains TA1535, TA1537, TA98 and TA100 . In tester strain WP 2 uvrA , FRET 09

- 0536 was tested at a

concentration range of 3 to 1000 μg/plate in the absence of S9

- mix and at 10 to 3330 μg/plate in the

presence of 10% (v/v) S9

- mix

. FRET 09

- 0536 precipitated on the plates at the dose level s of 1000 and

3 330 μg/plate in tester strains TA100 and WP 2 uvrA in the presence of S9

- mix

. Toxicity was observed

in all tester strains, except in WP 2 uvrA.

Since in tester strain WP 2 uvrA in the second experiment no dose level with toxicity or precipitate on

the plates was observed in the absence of S9

- mix, a third mutation experiment was performed with

this strain in the absence of S9

- mix. FRET 09

- 0536 was tested up to the dose level 5000 μg/plate.

FRET 09

- 0536 precipitated on the plates at the dose levels of 1000 and 3330 μ g/plate. Toxicity was

observed at dose levels of 3330 and 5000 μg/plate.

FRET 09

- 0536 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 tester strain WP 2 uvrA both in the absence and presence of S9

- metabolic

activation. These results were confirmed in independently repeated experiment s.

The negative control values were within the laboratory historical control data ranges

, except for TA1535 in

the absence of S9

- mix ( first experiment). However, since this value was just outside the limit of the range,

the validity of the test was considered to be not affected.FRET 09

- 0536 Project 504369

- Page 7 - The strain

- specific positive control v alues were at least three times the concurrent vehicle control

group mean indicating that the test conditions were adequate.

Based on the results of this study it is concluded that FRET 09

- 0536 is not mutagenic in the

Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.FRET 09

- 0536 Project 504369

- Page 8 - 5. INTRODUCTION

5.1. Preface

Sponsor International Flavors & Fragrances Inc.

800 Rose Lane

Union Beach, New Jersey 07735

U.S.A.

Study Monitor Vijyeta Kapur

Senior Regulatory Chemical Control Spec ialist

Test Facility WIL Research Europe B.V.

Hambakenwetering 7

5231 DD ‘s

- Hertogenbosch

The Netherlands

Study Director C.M. Verspeek

- Rip

ineke.verspeek@wilresearch.com

Sponsor International Flavors & Fragrances Inc.

800 Rose Lane

Union Beach, New Jersey 07735

U.S.A.

Study Plan Start : 06 January 2014

Completion : 10 February 2014

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 bacterial 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 Economi c 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.FRET 09

- 0536 Project 504369

- Page 9 - 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 Europe 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 made.

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 information

Identification FRET 09

- 0536

Structure

H

H O

+ H

H O

Molecular formula C 13 H 20 O

Molecular weight 192.1 5

CAS Number 1464989

- 16

- 9

Description Clear colourless liquid

Batch RDIA5595

Purity/Composition 89.6%

Test substance storage In refrigerator (2

- 8°C) in the dark under nitrogen

Stable under storage conditions until 01 November 2014 (expiry date)

Fo r Certificate of Analysis, see APPENDIX 6

. For the Substance Identity Information Document see

APPENDIX 7

.

6.1.2. Study specific test substance information

Purity/composition correction factor

required No

V olatile Yes, vapour pressure: 0.024 Pa at 298K

Reactivity Reactive to oxygen

Test substance handling

. Flush container with nitrogen after handling

Stability at higher temperatures Yes, maximum temperature: 60°C

Stability in vehicle Ethanol : Not indicat ed

Solubility in vehicle Ethanol : Not indicated

6.1.3. Test substance preparation

No correction was made for the purity/composition of the test compound

.

FRET 09

- 0536 was dissolved in ethanol (Merck, Darmstadt, Germany). Test substance concentrations

were used within 4 hours after preparation.FRET 09

- 0536 Project 504369

- Page 10

- 6.2. Reference substances

6.2.1. Negative control

The vehicle of the test substance

, which was ethanol

.

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) 15 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

TA15 35 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 sal ine ( 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, EC).

S ource 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)FRET 09

- 0536 Project 504369

- Page 11

- 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 a test.

Agar plates

Agar plates (ø 9 cm) contained 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

12. 5 μg/plate biotin ( Merck) and 15 μg/plate histidine ( Merck) and the agar plates for the test with the

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

Top agar

Milli

- Q water containing 0.6% (w/v) bacteriological agar ( Merck ) 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 35.9

– 39.2 °C) in the dark. Temporary deviations of maximally 2 hour s (in the range of

35.9

– 36.0 °C and 38.0

– 39.0°C ) occurred due to addition of plates (which were at room temperature)

to the incubator or due to opening and closing the incubator door. Based on laboratory historical data

these deviations are considered not to affect the study integrity. The temporary deviation from the

temperature (with a maximum of 39. 2 °C ) is explained in protocol deviation 1.

6.5. Metabolic activation system

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

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

intraperit oneally with Aroclor (500 mg/kg). Except in the Salmonella typhimurium tester strains in the

second experiment, where rat liver microsomal enzymes were routinely prepared from adult male

Wistar rats, which were obtained from Charles River, Sulzfeld, German y.

6.5.1. Preparation of S9

- fraction

The animals were housed at WIL Research Europe in a special room under standard laboratory

conditions, as described in the Standard Operating Procedures. The rats were orally dosed for three

consecutive days with a suspension of phenobarbital (Bufa B.V., IJsselstein, The Netherlands )

(80 mg/kg body weight) and ß

- naphthoflavone (Sigma Aldrich Chemie) (100 mg/kg body weight) in

corn oil (Fagron Farmaceutical, Capelle aan de IJssel, The Netherlands ) (they were denied access to

fo od for 3 to 4 hours preceding each dosing). One day after the final exposure (24 h), the rats were

sedated using oxygen/carbon dioxide and then killed by decapitation. The rats received a limitedFRET 09

- 0536 Project 504369

- Page 12

- quantity of food during the night before sacrifice. The livers of the rats were removed aseptically, and

washed in cold (0°C) sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM Na

2 - EDTA

(Merck). Subsequently the livers were minced in a blender and homogenized in 3 volumes of

phosphate buffer with a Potter homogenizer. The homogenate was centrifuged for 15 min at 9000 g.

The supernatant (S9) was transferred into sterile ampoules, which were stored in liquid nitrogen

(- 196°C) for a maximum of 1 year.

Before use, all S9 batches were characterised with the mutagens Benzo

- (a)

- pyrene (Sigma) and

2- aminoanthracene (Sigma), which require metabolic activation, in tester strain TA98 at concentrations

of 5 μg/plate and 1 μg/plate, respectively.

6.5.2. 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) 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 experi ment.

6.6. Study design

6.6.1. Dose range finding test

Se lection 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, 3, 10, 33, 100, 333,

1000, 3330 and 5000 μg/plate were tested in triplicate. The highest concentration of FRET 09

- 0536

used in the subsequent mutation assay was the level at which the test substance inhibited bacterial

growth unless the test substance exhibited limited solubility

.

6.6.2. Mutation assay

At least five different doses (increasin g with approximately half

- log steps) of the test substance were

tested in triplicate in each strain. In the first experiment FRET 09

- 0536 was tested both in the absence

and presence of 5% (v/v) S9

- mix in tester strains TA1535, TA1537 and TA98. In an indepe ndent

repeat of the assay with additional parameters, the test substance was tested both in the absence and

presence of 10% (v/v) S9

- mix in all tester strains. An additional experiment was performed with tester

strain WP 2 uvrA in the absence of S9

- mix.

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°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 ethanol 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

- ac tivation 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. Af ter this period revertant colonies (histidine independent (His + ) for Salmonella

typhimurium bacteria and tryptophan independent (Trp + ) for Escherichia coli were counted.

6.6.3. Colony counting

The revertant colonies wer e counted automatically with the Sorcerer C olony Counter. Plates with

sufficient test article precipitate to interfere with automated colony counting were counted manuallyFRET 09

- 0536 Project 504369

- Page 13

- and evidence of test article precipitate on the plates was recorded. The condition of the bacterial

background lawn was evaluat ed, both macroscopically and microscopically by using a dissecting

microscope.

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:

a) The negative control data (number of spontaneous revertants per plate) should be within the

laboratory historical range for each tester strain (see APPENDIX 4 )

. b) The positive control chemicals should produce responses in all tester strains, which are within the

laboratory historical range documented for each positive control substance. Furthermore, the mean

plate count should be at least three times the concurrent vehicle control group mean (see

APPENDIX 5 )

. c) 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.

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 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 independently repeated experiment.

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

a) The total number of revertants in 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 repeat 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 independently repeated

experiment.

The preceding criteria were not absolute and other modifying factors might enter into the final

evaluation decision.

6.9. List of deviations

6.9.1. List of protocol deviations

1. The temperature was recorded to be outside the range of 37.0 ± 1.0°C specified in the protocol

for approximately 1 hour in the second mutation experiment, tester strain TA1535, without S9

- mix

(with a maximum of 39.2°C).

Evaluation: This short term deviation was observed within three hours after initiation of the test

and was caused by adjustment of the temperature in the incubator after placing an amount of

selective agar plates in the incubator.FRET 09

- 0536 Project 504369

- Page 14

- 2. The mean plate count of the solvent control of TA 1535 (absence of S9

- mix) in the first

experiment, w as not within the laboratory historical range

. Evaluation: The value (28) was above the limit of the range (25). The mean plate count was just

above the limit of the range and clear negative results are observed in all experiments, therefore

this deviation in the mean plate count of the solvent control had no effect on the results of the

study.

The study integrity was not adversely affected by the deviations.

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.FRET 09

- 0536 Project 504369

- Page 15

- 7. RESULTS

7.1. Dose range finding test/first mutation experiment

FRET 09

- 0536 was tested in the tester strains TA100 and WP 2 uvrA with concentrations of 3, 10, 33,

100, 333, 1000, 3330 and 5000 μg/plate in the absence and presence of S9

- mix. Based on the results

of the dose range finding test, FRET 09

- 0536 was tested in the first mutation assay at a concentration

range of 0.3 to 100 μg/plate in the absence of S9

- mix and at a concentration range of 3 to

1000 μg/plate in the presence of 5% (v/v) S9

- mix in tester strains TA1535, TA1537 and TA98. The

results are shown in Table 3 and Table 4

. The individual data are presented in APPENDIX 3

.

Precipitate

Precipitation of FRET 09

- 0536 on the plates was observed at the start and at the end of the incubation

period at concentrations of 1000 μg/plate and upwards in tester strains TA100 and WP 2 uvrA (dose

range finding test)

. In tester strains TA1535, TA1537 and TA98 precipitation of FRET 09

- 0536 on the

plates was only observed at the start of the incubation period at the concentration of 1000 μg/plate

(first mutation experiment)

.

Toxicity

To determine the toxicity of FRET 09

- 0536 , 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

.

The reduction of the bacterial background lawn and the reduction in the number of revertants are

presented in Table 1

.

Table 1 Toxicity of FRET 09

- 0536 in the dose range finding test/first mutation experiment

(Reduction of the bacterial background lawn and in the number of revertant colonies)

Strain Without S9

- mix With S9

- mix

Dose Bacterial Revertant

(μg/plate) background lawn colonies Dose Bacterial Revertant

(μg/plate) background lawn colonies

TA100 33 slight slight

100 extreme microcolonies

333

- 5000 extreme comp lete 333 normal microcolonies

1 000 normal extreme

3330 normal complete

5000 normal extreme

WP 2 uvrA 1000

- 5000 extreme microcolonies 3330

- 5000 normal microcolonies

TA1535 100 extreme microcolonies 1 00 slight

- 1

333

- 1000 extreme microcolonies

TA1537 33 slight

- 1

100 extreme microcolonies 100 slight

- 1

3 33

- 1000 extreme microcolonies

TA98 100 moderate

- 1 333 slight 1

1000 extreme microcolonies

- 1 No reduction in the number of revertant colonies less than the minimal value of the historical

control data range

Mutagen icity

N o increase in the number of revertants was observed upon treatment with FRET 09

- 0536 under all

conditions tested.

In strain TA1535, fluctuations in the number of revertant colonies above the laboratory historical

control data range were observed i n the absence and presence of S9

- mix at several dose levels.

However, since the increases were not three

- fold (a maximum of 1.2

- fold was reached), these

increases were not considered to be relevant.FRET 09

- 0536 Project 504369

- Page 16

- 7.2. Experiment 2

To obtain more information about the possible mutagenicity of FRET 09

- 0536, a second mutation

experiment was performed in the absence of S9

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

- mix. The

following dose ranges w ere selected for the second mutation experiment:

TA1535, TA1537, TA98 and TA100

Witho ut S9

- mix: 0.3, 1, 3, 10, 33 and 100 μg/plate.

With S9

- mix: 3, 10, 33, 100, 333 and 1000 μg/plate.

WP 2 uvrA

Without S9

- mix: 3, 10, 33, 100, 333 and 1000 μg/plate.

With S9

- mix: 10, 33, 100, 333, 1000 and 3330 μg/plate.

The results are shown in Table 5

. The individual data are presented in APPENDIX 3

.

Precipitate

Precipitation of FRET 09

- 0536 on the plates was observed at the start of the incubation period at the

concentration of 1000 μg/plate and upwards. Precipitation of FRET 09

- 0536 on the plates at the end of

the incubation period was observed in tester strains TA100 and WP 2 uvrA at concentrations of

1000 μg/plate and upwards in the presence of S9

- mix

.

Toxicity

In tester strain WP 2 uvrA, no reduction of the bacterial background lawn and no biologically relevant

decrease in the number of revertants were observed.

The reduction of the bacterial background lawn and the reduction in the number of revertants in the

other tester strains are presented in Table 2 (See APPENDIX 2 for definitions).

Table 2 T oxicity o f FRET 09

- 0536 in the second experiment

(Reduction of the bacterial background lawn and in the number of revertant colonies)

Strain Without S9

- mix With S9

- mix

Dose Bacterial Revertant

(μg/plate) background lawn colonies Dose Bacterial Revertant

(μg/plate) background lawn colonies

TA1535 33 slight

- 1

100 extreme microcolonies 333 extreme microcolonies

1000 absent microcolonies

TA1537 33 slight

- 1

100 extreme microcolonies 100 slight

- 1

333 moderate

- 1

1000 absent complete

TA98 100 moderate

- 1 333 slight

- 1

1000 extreme microcolonies

TA100 1 00 extreme microcolonies 333 extreme microcolonies

1000 extreme microcolonies

- 1 No reduction in the number of revertant colonies less than the minimal value of the historical

c ontrol data range

Mutagenicity

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

with FRET 09

- 0536 under all conditions tested.

7.3. Experiment 3

Since in tester strain WP 2 uvrA in the second experiment no dose level with toxicity or precipitate on the

plates was observed in the absence of S9

- mix, a third mutation experiment was performed with this

strain in the absence of S9

- mix. FRET 09

- 0536 was tested up to the dose level 5000 μg/plate. The

results are shown i n Table 6

. The individual data are presented in APPENDIX 3

.FRET 09

- 0536 Project 504369

- Page 17

- Precipitate

Precipitation of FRET 09

- 0536 on the plates was observed at the start of the incubation period at

concentrations of 1000 μg/plate and upwards and at 3330 and 5000 μg/plate at t he end of the

incubation period.

Toxicity

An extreme reduction of the revertant colonies was observed at test substance concentrations of 3330

and 5000 μg/plate.

Mutagenicity

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

FRET 09

- 0536 under 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 independently repeated experiments.

The negative control values were within the laboratory historical control data ranges, except the

response for TA1535 in the absence of S9

- mix, second experiment (see protocol deviation 2).

The 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 FRET 09

- 0536 is not mutagenic in the

Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

9. REFERENCES

1 Leonardo, J.M., Dornfeld, S.S. and Peak, M.J., 1984, Evaluation of E. coli K12 343 \ 1 13 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 Trp + 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.FRET 09

- 0536 Project 504369

- Page 18

- APPENDIX 1 SUMMARY TABLES

Table 3 Dose range finding test: Mutagenic response of FRET 09

- 0536 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 Salmonella

typhimurium and one Escherichia coli strain

.

TA100 WP2uvrA

Without S9

- mix

Positive control 1108 ± 77 1438 ± 74

Solvent control 87 ± 10 n 28 ± 7 n

3 86 ± 2 48 ± 12

10 97 ± 19 n 41 ± 6

33 59 ± 12 s 36 ± 4

100 0 ± 0 e MC 29 ± 14

333 0 ± 0 e NP 24 ± 10 n NP

1000 0 ± 0 e SP 0 ± 0 n SP MC

3330 0 ± 0 e SP 0 ± 0 n SP MC

5000 0 ± 0 e SP 0 ± 0 n SP MC

With S9

- mix 1

Positive control 930 ± 160 187 ± 32

Sol vent control 98 ± 5 n 43 ± 2 n

3 97 ± 5 42 ± 7

10 91 ± 10 45 ± 9

33 101 ± 12 34 ± 7

100 69 ± 13 42 ± 6

333 0 ± 0 NP MC 45 ± 5 NP

1000 14 ± 9 SP 32 ± 15 SP

3330 1 ± 0 SP 0 ± 0 SP MC

5000 7 ± 8 n SP 0 ± 0 n S P MC

1 Plate incorporation assay (5% S9)

MC Microcolonies

NP No precipitate

SP Slight Precipitate

e Bacterial background lawn extremely reduced

n Normal bacterial background lawn

s Bacterial background lawn slightly reducedFRET 09

- 0536 Project 504369

- Page 19

- AP PENDIX 1 

## – continued 

## –

Table 4 E xperiment 1 : Mutagenic response of FRET 09

- 0536 in the **Salmonella**

**typhimurium** reverse mutation assay

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 747 ± 17 630 ± 43 1060 ± 140

Solvent control 28 ± 7 n NP 6 ± 3 n NP 25 ± 5 n NP

0.3 29 ± 3 10 ± 1 26 ± 3

1 28 ± 6 9 ± 2 32 ± 7

3.33 31 ± 6 9 ± 2 33 ± 3

10 23 ± 1 7 ± 3 n 29 ± 6

33 17 ± 6 n 6 ± 3 s 26 ± 6 n

100 0 ± 0 e NP MC 0 ± 0 e NP MC 21 ± 2 m NP

With S9

- mix 1

Positive control 282 ± 27 311 ± 34 747 ± 71

Solvent control 25 ± 5 n NP 8 ± 2 n NP 32 ± 8 n NP

3 25 ± 2 7 ± 3 32 ± 12

10 30 ± 4 8 ± 2 32 ± 8

33 24 ± 8 n 11 ± 3 n 37 ± 3

100 20 ± 2 s 12 ± 5 s 30 ± 4 n

333 0 ± 0 e NP MC 0 ± 0 e MC 23 ± 7 s

1000 0 ± 0 e NP MC 0 ± 0 e NP MC 0 ± 0 e NP MC

1 Plate incorporation assay (5% S9)

MC Microc olonies

NP No precipitate

e Bacterial background lawn extremely reduced

m Bacterial background lawn moderately reduced

n Normal bacterial background lawn

s Bacterial background lawn slightly reducedFRET 09

- 0536 Project 504369

- Page 20

- APPENDIX 1 

## – continued 

## –

Table 5 Experiment 2: Mutagenic response of FRET 09

- 0536 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

differe nt strains of Salmonella typhimurium and one Escherichia coli strain.

TA1535 TA1537 TA98 TA100 WP2uvrA

Without S9

- mix

Positive control 524 ± 42 900 ± 54 987 ± 104 1041 ± 28 1328 ± 113

Solvent control 19 ± 4 6 ± 2 17 ± 3 105 ± 5 26 ± 6

0.3 16 ± 5 7 ± 2 18 ± 3 114 ± 15

-

1 17 ± 5 8 ± 6 16 ± 1 108 ± 5

-

3 18 ± 3 4 ± 0 21 ± 11 120 ± 5 32 ± 7

10 23 ± 6 n 7 ± 7 n 19 ± 3 120 ± 22 27 ± 2

33 14 ± 2 s 4 ± 1 s 17 ± 2 n 95 ± 6 n 30 ± 7

100 0 ± 0 e NP MC 0 ± 0 e NP M C 13 ± 8 m NP 0 ± 0 e NP MC 31 ± 6

333

- - - - 29 ± 10

1000

- - - - 23 ± 6 n NP

With S9

- mix 1

Positive control 200 ± 58 449 ± 14 551 ± 69 1724 ± 72 195 ± 19

Solvent control 24 ± 5 8 ± 5 31 ± 6 113 ± 14 47 ± 9

3 25 ± 4 8 ± 3 32 ± 11 117 ± 24

-

10 23 ± 4 6 ± 4 31 ± 2 112 ± 20 38 ± 6

33 24 ± 6 6 ± 1 n 34 ± 4 96 ± 10 35 ± 7

100 13 ± 2 n 7 ± 4 s 24 ± 4 n 88 ± 21 n 30 ± 5

333 0 ± 0 e MC 5 ± 5 m 27 ± 6 s 0 ± 0 e NP MC 30 ± 7 NP

1000 0 ± 0 a NP MC 0 ± 0 a NP 0 ± 0 e NP MC 0 ± 0 e SP MC 27 ± 4 SP

3330

- - - - 19 ± 3 n SP

1 Plate incorporation assay (10% S9)

MC Microcolonies

NP No precipitate

SP Slight Precipitate

a Bacterial background lawn absent

e Bacterial background lawn extremely reduced

m Bacterial background lawn moderately reduced

n Normal bacterial background lawn

s Bacterial background lawn slightly reduced

- Not testedFRET 09

- 0536 Project 504369

- Page 21

- APPENDIX 1 

## – continued 

## –

Table 6 Experiment 3: Mutagenic response of FRET 09

- 0536 in the **Escherichia coli**

reverse mutation assay

Dose

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

the Escherichia coli strain WP2uvrA.

Without S9

- mix

Positive control 1476 ± 64

Solve nt control 19 ± 5

333 16 ± 6

1000 11 ± 1 NP

3330 3 ± 2 SP

5000 2 ± 2 n SP

NP No precipitate

SP Slight Precipitate

n Normal bacterial background lawnFRET 09

- 0536 Project 504369

- Page 22

- 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 ar e 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 Characteristics

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.FRET 09

- 0536 Project 504369

- Page 23

- APPENDIX 3 DETAILED TABLES

Individual plate counts; (following pages)

LIST OF ABBREVIATIONS

n Normal bacterial background lawn

s Bacterial background lawn slightly reduced

m Bacterial background lawn moderately reduc ed

a Bacterial background lawn absent

e Bacterial background lawn extremely reduced

MC Microcolonies

NP No precipitate

SP Slight precipitateFRET 09

- 0536 Project 504369

- Page 24

- APPENDIX 3 

## – continued 

## –

Dose range finding

Strain TA100

WITHOUT S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 1145 1160 1020 1108 ± 77

solvent control 91 n 94 n 76 n 87 ± 10

3 87 84 88 86 ± 2

10 113 n 103 n 76 n 97 ± 19

33 64 s 68 s 45 s 59 ± 12

100 0 e MC 0 e MC 0 e MC 0 ± 0

333 0 e NP 0 e N P 0 e NP 0 ± 0

1000 0 e SP 0 e SP 0 e SP 0 ± 0

3330 0 e SP 0 e SP 0 e SP 0 ± 0

5000 0 e SP 0 e SP 0 e SP 0 ± 0

WITH S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 1114 820 856 930 ± 1 60

solvent control 99 n 93 n 103 n 98 ± 5

3 94 103 94 97 ± 5

10 102 90 82 91 ± 10

33 87 106 109 101 ± 12

100 58 83 65 69 ± 13

333 0 NPMC 0 NPMC 0 NPMC 0 ± 0

1000 20 SP 19 SP 4 SP 14 ± 9

3330 1 SP 1 SP 1 SP 1 ± 0

5000 4 n SP 16 n SP 1 n SP 7 ± 8FRET 09

- 0536 Project 504369

- Page 25

- APPENDIX 3 

## – continued 

## –

Dose range finding

Strain WP2uvrA

WITHOUT S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 1520 1420 1375 1438 ± 74

solvent control 20 n 31 n 33 n 28 ± 7

3 38 46 61 48 ± 12

10 41 46 35 41 ± 6

33 31 39 38 36 ± 4

100 29 15 42 29 ± 14

333 14 n NP 24 n NP 33 n NP 24 ± 10

1000 0 n SPMC 0 n SPMC 0 n SPMC 0 ± 0

3330 0 n SPMC 0 n SPMC 0 n SPMC 0 ± 0

5000 0 n SPMC 0 n SPMC 0 n SPMC 0 ± 0

WITH S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 224 167 169 187 ± 32

solvent control 42 n 42 n 45 n 43 ± 2

3 39 50 38 42 ± 7

10 52 35 49 45 ± 9

33 27 34 41 34 ± 7

100 39 49 38 42 ± 6

333 50 NP 41 NP 44 NP 45 ± 5

1000 24 SP 49 SP 22 SP 32 ± 15

3330 0 SPMC 0 SPMC 0 SP 0 ± 0

5000 0 n SPMC 0 n SPMC 0 n SPMC 0 ± 0FRET 09

- 0536 Project 504369

- Page 26

- APPENDIX 3 

## – continued 

## –

Experiment 1

Strain TA1535

WITHOUT S9

- MIX

plate 1 2 3 MEAN S D

dose (μg/plate)

positive control 745 765 731 747 ± 17

solvent control 32 n NP 32 n NP 20 n NP 28 ± 7

0.3 26 30 31 29 ± 3

1 24 24 35 28 ± 6

3.33 31 26 37 31 ± 6

10 24 23 22 23 ± 1

33 11 n 23 n 16 n 17 ± 6

100 0 e NPMC 0 e NPMC 0 e NPMC 0 ± 0

WITH S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 298 297 250 282 ± 27

solvent control 29 n NP 26 n NP 19 n NP 25 ± 5

3 27 24 23 25 ± 2

10 30 34 27 30 ± 4

33 32 n 16 24 24 ± 8

100 19 s 22 s 19 s 20 ± 2

333 0 e NPMC 0 e NPMC 0 e NPMC 0 ± 0

1000 0 e NPMC 0 e NPMC 0 e NPMC 0 ± 0FRET 09

- 0536 Project 504369

- Page 27

- APPENDIX 3 

## – continued 

## –

Experiment 1

Strain TA1537

WITHOUT S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 679 599 611 630 ± 43

solvent control 8 n NP 8 n NP 3 n NP 6 ± 3

0.3 11 10 9 10 ± 1

1 10 7 9 9 ± 2

3.33 11 7 10 9 ± 2

10 7 n 10 n 5 n 7 ± 3

33 9 s 5 s 4 s 6 ± 3

100 0 e NPMC 0 e NPMC 0 e NPMC 0 ± 0

WITH S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 309 346 279 311 ± 34

solvent control 7 n NP 10 n NP 8 n NP 8 ± 2

3 5 7 10 7 ± 3

10 11 7 7 8 ± 2

33 12 n 13 n 7 n 11 ± 3

100 8 s 18 s 10 s 12 ± 5

333 0 e MC 0 e MC 0 e MC 0 ± 0

1000 0 e NPMC 0 e NPMC 0 e NPMC 0 ± 0FRET 09

- 0536 Project 504369

- Page 28

- APPENDIX 3 

## – continued 

## –

Experiment 1

Strain TA98

WITHOUT S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/pl ate)

positive control 1218 951 1011 1060 ± 140

solvent control 21 n NP 31 n NP 23 n NP 25 ± 5

0.3 23 27 28 26 ± 3

1 38 33 24 32 ± 7

3.33 33 31 36 33 ± 3

10 29 34 23 29 ± 6

33 33 n 23 n 22 n 26 ± 6

100 24 m NP 20 m NP 20 m NP 21 ± 2

WITH S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 744 677 819 747 ± 71

solvent control 37 n NP 23 n NP 35 n NP 32 ± 8

3 22 29 46 32 ± 12

10 35 23 39 32 ± 8

33 40 34 36 37 ± 3

100 28 n 27 n 35 n 30 ± 4

333 25 s 16 s 29 s 23 ± 7

1000 0 e NPMC 0 e NPMC 0 e NPMC 0 ± 0FRET 09

- 0536 Project 504369

- Page 29

- APPENDIX 3 

## – continued 

## –

Experiment 2

Strain TA1535

WITHOUT S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 571 491 511 524 ± 42

solvent control 18 16 23 19 ± 4

0.3 18 20 11 16 ± 5

1 20 20 11 17 ± 5

3 20 15 18 18 ± 3

10 22 n 29 n 18 n 23 ± 6

33 15 s 15 s 12 s 14 ± 2

100 0 e NPMC 0 e NPMC 0 e NPMC 0 ± 0

WITH S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 264 184 151 200 ± 58

solvent control 29 23 20 24 ± 5

3 23 22 29 25 ± 4

10 20 27 22 23 ± 4

33 29 18 24 24 ± 6

100 12 n 12 n 15 n 13 ± 2

333 0 e MC 0 e MC 0 e MC 0 ± 0

1000 0 a NPMC 0 a NPMC 0 a NPMC 0 ± 0FRET 09

- 0536 Project 504369

- Page 30

- APPENDIX 3 

## – continued 

## –

Experiment 2

Strain TA1537

WITHOUT S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positi ve control 911 948 842 900 ± 54

solvent control 4 8 5 6 ± 2

0.3 8 8 4 7 ± 2

1 3 7 14 8 ± 6

3 4 4 4 4 ± 0

10 15 n 4 n 1 n 7 ± 7

33 3 s 4 s 4 s 4 ± 1

100 0 e NPMC 0 e NPMC 0 e NPMC 0 ± 0

WITH S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 465 445 437 449 ± 14

solvent control 12 3 10 8 ± 5

3 10 5 8 8 ± 3

10 3 5 11 6 ± 4

33 5 n 5 n 7 n 6 ± 1

100 4 s 11 s 5 s 7 ± 4

333 1 m 4 m 11 m 5 ± 5

1000 0 a NP 0 a NP 0 a NP 0 ± 0FRET 09

- 0536 Project 504369

- Page 31

- APPENDIX 3 

## – continued 

## –

Experiment 2

Strain TA98

WITHOUT S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 1012 872 1076 987 ± 104

solvent control 20 16 14 17 ± 3

0.3 22 16 16 18 ± 3

1 16 16 15 16 ± 1

3 15 14 33 21 ± 11

10 18 16 22 19 ± 3

33 19 n 15 n 16 n 17 ± 2

100 10 m NP 22 m NP 7 m NP 13 ± 8

WITH S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

posit ive control 513 510 631 551 ± 69

solvent control 38 27 29 31 ± 6

3 39 19 38 32 ± 11

10 30 30 34 31 ± 2

33 33 31 38 34 ± 4

100 27 n 20 n 24 n 24 ± 4

333 30 s 20 s 30 s 27 ± 6

1000 0 e NPMC 0 e NPMC 0 e NPMC 0 ± 0FRET 09

- 0536 Project 504369

- Page 32

- APPENDIX 3 

## – continued 

## –

Experiment 2

Strain TA100

WITHOUT S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 1045 1011 1067 1041 ± 28

solvent control 109 107 99 105 ± 5

0.3 132 105 106 114 ± 15

1 102 112 109 108 ± 5

3 124 121 114 120 ± 5

10 146 109 106 120 ± 22

33 91 n 93 n 102 n 95 ± 6

100 0 e NPMC 0 e NPMC 0 e NPMC 0 ± 0

WITH S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive contro l 1790 1647 1734 1724 ± 72

solvent control 117 124 97 113 ± 14

3 97 144 110 117 ± 24

10 118 128 90 112 ± 20

33 97 86 106 96 ± 10

100 113 n 76 n 76 n 88 ± 21

333 0 e NPMC 0 e NPMC 0 e NPMC 0 ± 0

1000 0 e SPMC 0 e SPMC 0 e SPMC 0 ± 0FRET 09

- 0536 Project 504369

- Page 33

- APPENDIX 3 

## – continued 

## –

Experiment 2

Strain WP2uvrA

WITHOUT S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 1457 1247 1280 1328 ± 113

solvent control 22 33 24 26 ± 6

3 40 30 27 32 ± 7

10 27 29 26 27 ± 2

33 30 37 23 30 ± 7

100 37 31 26 31 ± 6

333 39 20 27 29 ± 10

1000 16 n NP 26 n NP 27 n NP 23 ± 6

WITH S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 207 173 205 195 ± 19

solvent control 54 49 37 47 ± 9

10 44 33 37 38 ± 6

33 33 42 29 35 ± 7

100 29 35 26 30 ± 5

333 37 NP 30 NP 23 NP 30 ± 7

1000 23 SP 30 SP 29 SP 27 ± 4

3330 22 n SP 16 n SP 19 n SP 19 ± 3FRET 09

- 0536 Project 504369

- Page 34

- APPENDIX 3 

## – continued 

## –

Experiment 3

Strain WP2uvrA

WITHOUT S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 1528 1495 1404 1476 ± 64

solvent control 15 19 24 19 ± 5

333 15 10 22 16 ± 6

1000 10 NP 11 NP 12 NP 11 ± 1

3330 2 SP 5 SP 3 SP 3 ± 2

5000 1 n SP 4 n SP 1 n SP 2 ± 2FRET 09

- 0536 Project 504369

- Page 35

- APPENDIX 4 HISTORICAL CONTROL DATA OF THE SOLVENT CONTROL

TA1535 TA1537 TA98 TA100 WP 2 uvrA

S9

- mix

- + - + - + - + - +

Range 2

- 25 2

- 25 2

- 22 2

- 32 7

- 49 7

- 56 63

- 195 55 - 205 7

- 51 8

- 62

Mean 10 8 5 5 20 24 110 98 24 26

SD 4 3 2 3 7 7 24 24 7 7

n 1296 1319 1108 1101 1302 1304 1373 1342 1169 1155

SD = Standard deviation

n = Number of observations

Historica l control data from experiments performed between July 2010 and July 2013.FRET 09

- 0536 Project 504369

- Page 36

- APPENDIX 5 HISTORICAL CONTROL DATA OF THE POSITIVE CONTROL SUBSTANCES

TA1535 TA1537 TA98

S9

- mix

- + - + - +

Range 250

- 1270 72

- 1016 113

- 1182 8 5

- 742 407

- 1518 153

- 1663

Mean 843 266 603 358 997 778

SD 130 120 243 106 147 280

n 1313 1317 1117 1118 1324 1307

TA100 WP 2 uvrA

S9

- mix

- + - +

Range 348

- 1417 229

- 1752 138

- 1479 122

- 1187

Mean 854 1113 1072 354

SD 120 238 200 126

n 133 8 1332 1166 1153

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between July 2010 and July 2013.FRET 09

- 0536 Project 504369

- Page 37

- APPENDIX 6 CERTIFICATE OF ANALYSISFRET 09

- 0536 Project 504369

- Page 38

- APPENDIX 7 SUBSTANCE IDENTITY INFORMATION DOCUMENT