## FINAL REPORT

Study Title



## EVALUATION OF THE MU TAGENIC ACTIVITY OF 1-

PROPANESULFONIC ACID , 2

- HYDROXY -3-(2- PROPEN -1-

YLOXY)

- , SODIUM SALT (1:1) IN THE **SALMONELLA**

**TYPHIMURIUM** REVERSE MUTATION ASS AY AND THE

**ESCHERICHIA COLI** REVERSE MUTATION ASS AY

(WITH INDEP ENDENT REPEAT)

Author

C.M. Verspeek

- Rip

Test Facility

WIL Research Europe B.V. Hambakenwetering 7

5231 DD ‘s

- Hertogenbosch

The Netherlands

Laboratory Project Identification

Project 500680

Substance 204240/A

- Page 1 of 29 -1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Pro ject 500680

- 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

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

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

- fraction

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

6.5.2. Prepar ation of S9

- mix

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

6.6. Study design

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

6.6.1. Dose range finding test

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

6.6.2. Mutation assay

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

6.6.3. Colony counting

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

6.7. Electronic data capture

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

6.8. Interpretation

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

6.8.1. Acceptability of the assay

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

6.8.2. Data evaluation and statistical procedures

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

6.9. List of deviations

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

6.9.1. List of protocol deviations

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

6.9.2. List of standard operating procedures deviations

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

7. RESULTS

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

7.1. Dose range finding test

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

7.2. Mutation assay

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

8. DISCUSSION AND CONCL USION

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

9. REFERENCES

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

TABLES

Table 1 Experiment 1: Mutagenic response of 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) in the Salmonella typhimurium reverse

mu tation assay and in the Escherichia coli reverse mutation assay

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

Table 2 Experiment 2: Mutagenic response of 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

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

APPENDICES

APPENDIX 1 SUMMARY TABLES

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

APPENDIX 2 SUPPORTING MATERIALS AND METHOD

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

APPENDIX 3 DETAILED TABLES

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

APPENDIX 4 HISTORICAL CONTROL DATA OF THE SOLVENT CONTROL

............................... 27

APPENDIX 5 HISTORICAL CONTROL DATA OF THE POSITIVE CONTROL SUBSTANCES

....... 28

APP ENDIX 6 CERTIFICATE OF ANALYSIS

................................................................................... 291- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Pro ject 500680

- Page 5 - 4. SUMMARY

Evaluation of the mutagenic activity of 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (with independent repeat).

1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) wa s 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 Escheric hia 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).

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

g uidelines.

Batch 12E1101 of 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) was a clear colourless liquid . 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1-yloxy)- , sodium salt (1:1) was

dissolved in Milli

- Q water

.

In the dose range finding test, 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt
(1:1) 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. 1- Propanesul fonic acid, 2

- hydroxy -3- (2

- propen -1-yloxy)- , sodium salt
(1:1) 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

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

Based on the results of the dose range finding test, 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)- , sodium salt ( 1:1) was tested in the first mutation assay at a concentration range of 100 to

5000 μg/plate in the absence and presence of 5% (v/v) S9

- mix in tester strains TA1535, TA1537 and
TA98. In an independent repeat of the assay with additional parameters, 1- Propa nesulfonic acid, 2

- hydroxy -3- (2

- propen -1-yloxy)- , sodium salt (1:1) 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,

TA100 and WP 2 uvrA. The bacterial backg round lawn was not reduced at any of the concentrations

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

1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) did not induce a significant

dose

- re lated 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 activatio n. These results were confirmed
in an independently repeated experiment.

In this study, 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 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen

1-yloxy)- , sodium salt (1:1) is not mutagenic in the Salmonella typhimurium reverse mutation assay

and in the Escherichia coli reverse mutation assay.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Pro ject 500680

- 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 Researc h Europe B.V. Hambakenwetering 7

5231 DD ‘s

- Hertogenbosch

The Netherlands

Study Director C.M. Verspeek

- Rip

Study Plan Start : 04 February, 2013 Completion : 14 February, 2013

5.2. Aims of the study

The objective of this study was to evaluate the tes t 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 ra nge 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. Th e 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 regu lation (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.

- Code of Federal Reg ulations, Title 40
– Protection of Environment. Section 799.9510 Toxic
Substances Control Act (TSCA) bacterial reverse mutation test.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Pro ject 500680

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

Records and materials pertaining to the study including protocol, raw da ta 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 R esearch Europe will retain information concerning decisions made.

WIL Research Europe will retain a test substance sample until the exp iry 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 1

- Propanesulfonic acid, 2

- hydroxy

- 3

- (2

- propen

- 1

- yloxy)

- , sodium salt (1:1) Synonyms COPS_1 or CS

- AHPS

Structure

O SO 3 -

OH Na +

Molecular formula C 6 H 1 1 NaO 5 S Molecular weight 218.22

CAS Number 52556

- 42

- 0 Description Clear colourless liquid

Batch 12E1101 Purity/Composition

Sodium -1- allyloxy -2- hydroxypropane sulphonate

3- allyl

- 1,2

- propanediol

Water CAS Number Percent

52556

- 42

- 0 38.64

123

- 34

- 2 2.26

7732

- 18

- 5 59.1 Test substance storage At room temperature in the dark Stability under storage conditions Stable Expiry date 13 August 2013

See APPENDIX 6 ; Certificate of analysis 1

.

6.1.2. Study speci fic test substance information

Purity/composition correction factor
required No

Stability at higher temperatures Yes, maximum temperature: 80 ° C,

maximum duration: 1 hour Stability in vehicle Water : 1 year Solubility in vehicle Water: 1

- 45%

1 The C of A provided for Lot No. 12E1101 is correct as originally provided. The value reported as Dry Residue is made up of two components which together add up to the 40.9 % reported as Dry Residue on the C of A. When doing the Dry Residue test only the water (59.1%) volatilizes, the sodium -1- alloxy -2- hydroxypropane sulphonate and the 3

- allyl

- 1,2

- propanediol together comprise the Dry Residue, respectively 94.5% and 5.5%. On the basis of the entire sample (water present) this pecentage will be 38.64 and 2.26 as reported previously.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Pro ject 500680

- Page 8 - 6.1.3. Test substance preparation

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

.

The test substance was dissolved in Milli

- Q water (Millipore Corp., Bedford, MA., USA). The stock
solution was filter (0.22 μm)

- sterilized. Test substance concentrations were used within 2 .5 hours after
preparation.

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) (Si gma) 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:

Strai n Concentration/plate Amount of S9

- mix Solvent

TA1535 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 2.5 μg 10% DMSO

WP 2 uvrA 10 μg 5 and 10% DMSO

Solvent s for reference substances Saline = physiological saline (B. Braun, Melsungen AG, Germany)
DMSO = dimethyl sulfoxide ( Merck )

6.3. Test system

Test system Salmonella typhimurium bacteria and Escherichia coli bacteria

Rationale Recommended test system in intern ational guidelines (e.g. OECD, 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

TA1 00 his G46/R

- factor* Base

- pair substitutions

*: R

- factor = plasmid pKM101 (increases error

- prone DNA repair)1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Pro ject 500680

- Page 9 - E ach tester strain contained the following additional mutations:

rfa : deep rough (defective lipopolysaccharide cellcoat)
gal : mutation in the gala ctose metabolism

chl : mutation in nitrate reductase

bio : defective biotin synthesis

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

- repair B gene)

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

- requirement, crystal violet sensitivity, 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 i s 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 -s ensitivity 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 nu trient broth (Oxoid LTD, Hampshire, England) 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 (Oxoid LTD) in Vogel

- Bonner Medium E, 20 g glucose (Fres e nius Kabi , Bad Homburg, Germany). The agar plates for the test with the Salmonel la 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 (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.

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

range 36.4 – 39.8 °C) in the dark. Temporary deviations of maximally 2 hour s (in the range of

38.0 – 39.0°C ) occurred due to additio n 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 39.8 °C ) is explained in protocol deviation 1.

6.5. Metabolic activation system

Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats, which were obtained from Charles River, Sulzfeld, Germany.

6.5.1. Preparation of S 9- 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 ( B ufa B.V., IJsselstein, The Netherlands )
(80 mg/kg body weight) and ß

- naphthoflavone ( Sigma Aldrich Chemie) (100 mg/kg body weight) in1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Pro ject 500680

- Page 10 - corn oil ( Roth, Karlsruhe, Germany) (they were denied access to food 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 limited quantity of food during the night before
sacrifice. The livers of the rats were removed aseptically, and washed in c old (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 str ain 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 ( R andox 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 t he 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

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, 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate were tested in triplicate. This dose range finding test was reported as a part of the first experiment of the mutation assay. The highest concentration of 1- Propanesulfonic acid,

2- hydroxy -3- (2

- propen -1-yloxy)- , sodium salt (1:1) used in the subsequent mutation assay was

5 000 μ g/plate

.

6.6.2. Mutation assay

At least four different doses (increasing with approximately half

- log steps , see protocol deviation 2 ) of
the test substance were tested in triplicate in each strain. In the first experiment 1- Propanesulfonic

acid, 2

- hydroxy -3- (2

- propen -1-yloxy)- , sodium salt (1:1) was tested both in the absence a nd presence of 5% (v/v) S9

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

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 to p 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

- 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.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Pro ject 500680

- Page 11 - 6.6.3. Colony counting

The revertant colonies (histidine independent or tryptophan independent) were counted manually if
less than 40 colonies per plate were present. If more than 40 colonies were present, these were counted automatically with a Biocount 4000 Pro -S- colony counter. Plates with abundant test

substance precipitate which interfered w ith automated colony counting were counted manually and the
evidence of test substance precipitate on the plates was recorded. The condition of the bacterial
background lawn was evaluated, both macroscopically and microscopically by using a dissecting

micr oscope.

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) : T emperature

.

6.8. Interpretati on

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 concentratio n 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 thre e (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.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Pro ject 500680

- Page 12 - 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 as specified in the protocol for approximately 2 hours in the first mutation assay ( with a maximum of 39.8 °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. The negative control data (numb er of spontaneous revertants per plate) were within the laboratory historical range for each tester strain, therefore this short deviation of the temperature had no effect on the results of the study.

2. In the second mutation assay in tester strain TA100 i n the absence of S9

- mix , four dose levels were tested instead of five dose levels as specified in the protocol. Evaluation: No bacteria were added to the lowest dose level of 100 μg/pl ate . Since the four highest dose levels were tested, and no toxicity and/or precipitate was observed, test ing of a lower dose level would have given no additional information. Therefore this deviation had no influence on the study result.

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.

7. RESULTS

7.1. Dose range finding t est

1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) 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.

This dose ra nge finding test is reported as a part of the first experiment of the mutation test ( Table 1 )

. The individual data are presented in APPENDIX 3

.

Precipitate Precipitation of 1- Propanesulfonic acid, 2

- hyd roxy-3- (2

- propen -1-yloxy)- , sodium salt (1:1) on the
plates was not observed at the start or at the end of the incubation period in both tester strains.

Toxicity To determine the toxicity of 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1)

, 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

.

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

revertants were observed.

Mutagenicity In the dose range finding test, no increase in the number of revertants was observed upon treatment

with 1- Propanesulfonic acid, 2

- hydr oxy-3- (2

- propen -1- yloxy)

- , sodium salt (1:1) under all conditions

tested.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Pro ject 500680

- Page 13 - 7.2. Mutation assay

Based on the results of the dose range finding test, 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)- , sodium salt (1:1) was tested up to concentrations of 5 000 μg/plate in the absence and
presence of S9

- mix in two mutation assays. The first mutation experiment was performed with the
strains TA1535, TA1537 and TA98 and the second mutation experiment was performed with the

strains TA1535, TA1537, TA98, TA100 an d WP 2 uvrA. The results are shown in Table 1 and Table 2

, the individual data are presented in APPENDIX 3

.

Precipitate
Precipitation of 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- pro pen -1-yloxy)- , sodium salt (1:1) on the plates was not observed at the start or at the end of the incubation period.

Toxicity In both mutation assays, 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 both mutation assays, no increase in the number of revertants was observed upon treatment with

1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) 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 two independently repeated 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

function ed properly.

Based on the results of this study it is concluded that 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen

1-yloxy)- , sodium salt (1:1) is not mutagenic in the Salmonella typhimurium reverse mutation assay

and in the Escherichia coli reverse mutat ion assay.

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 de tecting 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, Muta tion 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 propertie s. J. Biol. Chem., 218 , 97

- 106.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Pro ject 500680

- Page 14 - APPENDIX 1 SUMMARY TABLES

Table 1 Experiment 1: Mutagenic response of 1- Propanesulfonic acid, 2

- hydroxy -3- (2

-
propen -1- yloxy)

- , sodium salt (1:1) in the **Salmonella typhimu rium**  reverse mutation assay and in the **Escherichia coli** reverse mutation assay

Dose Mean number of revertant colonies/3 replicate plates (± S.D.) with (μg/plate) different strains of Salmonella typhimurium and one Escherichia coli strain

TA1535 TA1537 TA98 TA100 WP 2 uvrA

Without S9

- mix

**positive control 949 ±**  **46 538 ±**  **16 934 ±**  **49 702 ±**  **26 1185 ±**  **82**

**solvent control 13 ±**  **5 6 ±**  **1 17 ±**  **2 104 ±**  **6 28 ±**  **2**

**3 104 ±**  **14 36 ±**  **4**

**10 97 ±**  **12 33 ±**  **2**

**33 94**  **± 17 32**  **± 4** 

**100 13 ±**  **3 6 ±**  **1 16 ±**  **3 99 ±**  **13 34 ±**  **7**

**333 10 ±**  **6 6 ±**  **2 17 ±**  **4 105 ±**  **6 28**  **± 3** 

**1000 11 ±**  **1 5 ±**  **1 17 ±**  **2 104 ±**  **14 31 ±**  **4**

**3330 10 ±**  **3 8 ±**  **1 18 ±**  **2 115 ±**  **2 39 ±**  **6**

**5000 1 3**  **± 3 7**  **± 2 19**  **± 5 91**  **± 6 30**  **± 4** 

**------------------------------------------------------------------------------------------------------------**

With S9

- mix 1

**positive control 410 ±**  **6 478 ±**  **2 0 785**  **± 93 1048**  **± 52 458**  **± 25** 

**solvent control 13 ±**  **3 8 ±**  **1 24 ±**  **2 116 ±**  **8 35 ±**  **8**

**3 97 ±**  **21 41**  **± 1** 

**10 105 ±**  **16 39 ±**  **1**

**33 106 ±**  **5 31 ±**  **1**

**100 13 ±**  **1 7 ±**  **2 23 ±**  **5 94 ±**  **11 29 ±**  **4**

**333 8 ±**  **4 7 ±**  **1 25 ±**  **5 109 ±**  **14 30 ±**  **1**

**1000 9 ±**  **3 7 ±**  **3 27 ±**  **5 98**  **± 13 30**  **± 2** 

**3330 11 ±**  **4 5 ±**  **4 27 ±**  **4 100 ±**  **10 40 ±**  **5**

**5000 12 ±**  **5 6 ±**  **3 25 ±**  **2 106 ±**  **6 31 ±**  **3**

**------------------------------------------------------------------------------------------------------------**

Solvent control: 0.1 ml Milli

- Q water

1 The S9

- mix contained 5% (v/v) S9 fraction1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Pro ject 500680

- Page 15 - APPENDIX 1 

## – continued 

## –

Table 2 Experiment 2: Muta genic response of 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) in the **Salmonella typhimurium** reverse mutation assay and in the **Escherichia coli** reverse mutation assay

Dose Mean number of revertant colonies/3 replicate plates (± S.D.) with (μg/plate) different strains of Salmonella typhimurium and one Escherichia coli strain

TA1535 TA1537 TA98 TA100 WP 2 uvrA

Without S9

- mix

**positive control 822 ±**  **22 598 ±**  **4 1056 ±**  **105 743 ±**  **49 1024 ±**  **64**

**solvent control 11 ±**  **2 6 ±**  **2 15 ±**  **3 122 ±**  **18 26 ±**  **7**

**100 7 ±**  **4 5 ±**  **2 19 ±**  **4 2)**  **19 ±**  **3**

**333 10 ±**  **2 5 ±**  **3 19 ±**  **2 125 ±**  **6 18 ±**  **3**

**1000 10 ±**  **2 4 ±**  **1 21 ±**  **2 137 ±**  **33 17 ±**  **4**

**3330 10 ±**  **3 4 ±**  **1 21 ±**  **1 1 30**  **± 11 23**  **± 2** 

**5000 10 ±**  **4 3 ±**  **2 19 ±**  **4 119 ±**  **11 15 ±**  **3**

**------------------------------------------------------------------------------------------------------------**

With S9

- mix 1

**positive control 183 ±**  **21 474 ±**  **5 373 ±**  **39 339 ±**  **6 316 ±**  **33**

**solvent control 9 ±**  **1 4 ±**  **1 27 ±**  **7 109 ±**  **24 26 ±**  **7**

**100 7 ±**  **3 5**  **± 0 28**  **± 2 128**  **± 3 27**  **± 4** 

**333 7 ±**  **3 8 ±**  **3 21 ±**  **2 152 ±**  **19 27 ±**  **4**

**1000 7 ±**  **3 6 ±**  **3 28 ±**  **4 132 ±**  **15 33 ±**  **5**

**3330 6 ±**  **1 6 ±**  **3 27 ±**  **5 116 ±**  **13 32 ±**  **2**

**5000 10 ±**  **1 4 ±**  **2 28 ±**  **3 124 ±**  **11 17 ±**  **3**

**------------------------------------------------------------------------------------------------------------**

Solvent control: 0.1 ml Milli

- Q water

1 The S9

- mix contained 10% (v/v) S9 fraction

2 Not determined1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Pro ject 500680

- Page 16 - APPENDIX 2 SUPPORTING MATERIALS AND M ETHOD

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 Ch aracteristics

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 Distinguishe d 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 tes t 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 ar e 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.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Pro ject 500680

- Page 17 - APPENDIX 3 DETAI LED TABLES

Individual plate counts; (following pages)

**Experiment 1**

**Strain TA1535**

**-----------------------------------------------------------**

**WITHOUT S9**

**- MIX** 

**plate 1 2 3 MEAN SD**

**dose (μg /plate)** 

**positive control 934 912 1001 949 ±**  **46**

**solvent control 19 12 9 13 ±**  **5**

**100 10 14 16 13 ±**  **3**

**333 5 17 7 10 ±**  **6**

**1000 12 10 12 11 ±**  **1**

**3330 7 10 13 10 ±**  **3**

**5000 15 14 9 13 ±**  **3**

**-----------------------------------------------------------**

**WITH S9**

**- MIX** 

**plate 1 2 3 MEAN SD**

**dose (μg /plate)** 

**positive control 406 417 407 410 ±**  **6**

**solvent control 16 10 12 13 ±**  **3**

**100 14 13 13 13**  **± 1** 

**333 7 12 4 8 ±**  **4**

**1000 7 12 7 9 ±**  **3**

**3330 12 14 6 11 ±**  **4**

**5000 10 8 17 12**  **± 5** 

**-----------------------------------------------------------**1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Pro ject 500680

- Page 18 - APPENDIX 3 

## – continued 

## –

**Experiment 1**

**Strain TA1537**

**-----------------------------------------------------------**

**WITHOUT S9**

**- MIX** 

**plate 1 2 3 MEAN SD**

**dose (μg /plate)** 

**positive control 527 530 556 538 ±**  **16**

**solvent control 7 7 5 6 ±**  **1**

**100 6 5 7 6 ±**  **1**

**333 8 4 5 6 ±**  **2**

**1000 5 5 4 5 ±**  **1**

**3330 9 9 7 8 ±**  **1**

**5000 7 8 5 7 ±**  **2**

**-----------------------------------------------------------**

**WITH S9**

**- MIX** 

**plate 1 2 3 MEAN SD**

**dose (μg /plate)** 

**positive control 496 456 483 478 ±**  **20**

**solvent control 8 7 9 8**  **± 1** 

**100 6 7 9 7 ±**  **2**

**333 6 8 7 7 ±**  **1**

**1000 4 7 9 7 ±**  **3**

**3330 5 9 2 5**  **± 4** 

**5000 3 9 6 6 ±**  **3**

**-----------------------------------------------------------**1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Pro ject 500680

- Page 19 - APPENDIX 3 

## – continued 

## –

**Experiment 1**

**Strain TA98**

**-----------------------------------------------------------**

**WITHOUT S9**

**- MIX** 

**plate 1 2 3 MEAN SD**

**dose (μg /plate)** 

**positive control 912 990 901 934 ±**  **49**

**solvent control 19 16 17 17 ±**  **2**

**100 13 17 18 16 ±**  **3**

**333 14 15 22 17 ±**  **4**

**1000 17 15 18 17 ±**  **2**

**3330 19 19 16 18 ±**  **2**

**5000 20 24 14 19**  **± 5** 

**-----------------------------------------------------------**

**WITH S9**

**- MIX** 

**plate 1 2 3 MEAN SD**

**dose (μg /plate)** 

**positive control 823 679 854 785**  **± 93** 

**solvent control 25 21 25 24 ±**  **2**

**100 28 22 19 23 ±**  **5**

**333 19 27 28 25 ±**  **5**

**1000 33 23 25 2 7**  **± 5** 

**3330 27 24 31 27 ±**  **4**

**5000 24 27 25 25 ±**  **2**

**-----------------------------------------------------------**1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Pro ject 500680

- Page 20 - APPENDIX 3 

## – continued 

## –

**Experiment 1**

**Strain TA100**

**-----------------------------------------------------------**

**WITHOUT S9**

**- MIX** 

**plate 1 2 3 MEAN SD**

**dose (μg /plate)** 

**positive control 708 724 673 702 ±**  **26**

**solvent control 109 98 106 104 ±**  **6**

**3 96 120 95 104 ±**  **14**

**10 92 111 88 97 ±**  **12**

**33 110 95 76 94 ±**  **17**

**100 107 105 84 99**  **± 13** 

**333 109 98 109 105 ±**  **6**

**1000 95 120 98 104 ±**  **14**

**3330 115 113 117 115 ±**  **2**

**5000 86 97 90 91**  **± 6** 

**-----------------------------------------------------------**

**WITH S9**

**- MIX** 

**plate 1 2 3 MEAN SD**

**dose (μg /plate)** 

**positive control 996 1099 1050 1048 ±**  **52**

**solvent control 108 123 116 116 ±**  **8**

**3 76 118 96 97 ±**  **21**

**10 124 94 98 105 ±**  **16**

**33 101 110 106 106 ±**  **5**

**100 107 85 91 94 ±**  **11**

**333 118 116 93 109 ±**  **14**

**1000 103 107 83 98 ±**  **13**

**3330 109 90 101 100 ±**  **10**

**5000 99 111 107 106**  **± 6** 

**-----------------------------------------------------------**1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Pro ject 500680

- Page 21 - APPENDIX 3 

## – continued 

## –

**Experiment 1**

**Strain WP 2**  **uvrA**

**-----------------------------------------------------------**

**WITHOUT S9**

**- MIX** 

**plate 1 2 3 MEAN SD**

**dose (μg /plate)** 

**positive control 1098 1197 1261 1185 ±**  **82**

**solvent control 26 28 30 28 ±**  **2**

**3 32 37 39 36 ±**  **4**

**10 35 33 32 33 ±**  **2**

**33 35 28 32 32 ±**  **4**

**100 40 27 35 34 ±**  **7**

**333 28 25 31 28 ±**  **3**

**1000 34 33 27 31 ±**  **4**

**3330 34 39 45 39 ±**  **6**

**5000 26 29 34 30 ±**  **4**

**-----------------------------------------------------------**

**WITH S9**

**- MIX** 

**plate 1 2 3 MEAN SD**

**dose (μg /plate)** 

**positive control 485 437 451 458 ±**  **25**

**solvent control 28 44 33 35 ±**  **8**

**3 42 42 40 41 ±**  **1**

**10 40 39 38 39 ±**  **1**

**33 32 31 31 31 ±**  **1**

**100 30 25 32 29 ±**  **4**

**333 29 30 30 3 0**  **± 1** 

**1000 27 31 31 30 ±**  **2**

**3330 37 46 38 40 ±**  **5**

**5000 27 32 33 31 ±**  **3**

**-----------------------------------------------------------**1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Pro ject 500680

- Page 22 - APPENDI X 3 

## – continued 

## –

**Experiment 2**

**Strain TA1535**

**-----------------------------------------------------------**

**WITHOUT S9**

**- MIX** 

**plate 1 2 3 MEAN SD**

**dose (μg /plate)** 

**positive control 842 798 826 822 ±**  **22**

**solvent control 13 11 10 11 ±**  **2**

**100 8 3 11 7 ±**  **4**

**333 8 9 12 10 ±**  **2**

**1000 12 8 9 10**  **± 2** 

**3330 8 9 13 10 ±**  **3**

**5000 10 7 14 10 ±**  **4**

**-----------------------------------------------------------**

**WITH S9**

**- MIX** 

**plate 1 2 3 MEAN SD**

**dose (μg /plate)** 

**positive control 160 202 187 183 ±**  **21**

**solvent control 8 10 8 9 ±**  **1**

**100 4 6 10 7 ±**  **3**

**333 10 6 4 7 ±**  **3**

**1000 9 9 4 7 ±**  **3**

**3330 6 7 5 6 ±**  **1**

**5000 11 9 9 10 ±**  **1**

**-----------------------------------------------------------**1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Pro ject 500680

- Page 23 - APPENDIX 3 

## – continued 

## –

**Experiment 2**

**Strain TA1537**

**-----------------------------------------------------------**

**WITHOUT S9**

**- MIX** 

**plate 1 2 3 MEAN SD**

**dose (μg /plate)** 

**positive control 596 595 603 598 ±**  **4**

**solvent control 8 4 6 6 ±**  **2**

**100 6 3 5 5 ±**  **2**

**333 8 5 3 5**  **± 3** 

**1000 4 4 3 4 ±**  **1**

**3330 3 4 5 4 ±**  **1**

**5000 3 5 2 3 ±**  **2**

**-----------------------------------------------------------**

**WITH S9**

**- MIX** 

**plate 1 2 3 MEAN SD**

**dose (μg /plate)** 

**positive control 473 480 470 474 ±**  **5**

**solvent control 4 4 3 4 ±**  **1**

**100 5 5 5 5 ±**  **0**

**333 5 11 7 8 ±**  **3**

**1000 9 3 5 6 ±**  **3**

**3330 3 8 6 6 ±**  **3**

**5000 6 2 3 4**  **± 2** 

**-----------------------------------------------------------**1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Pro ject 500680

- Page 24 - APPENDIX 3 

## – continued 

## –

**Experiment 2**

**Strain TA98**

**-----------------------------------------------------------**

**WITHOUT S9 -M**  **IX**

**plate 1 2 3 MEAN SD**

**dose (μg /plate)** 

**positive control 1133 937 1098 1056 ±**  **105**

**solvent control 18 13 15 15 ±**  **3**

**100 18 16 23 19**  **± 4** 

**333 19 18 21 19 ±**  **2**

**1000 22 22 19 21 ±**  **2**

**3330 22 21 20 21 ±**  **1**

**5000 15 23 18 19 ±**  **4**

**-----------------------------------------------------------**

**WITH S9**

**- MIX** 

**plate 1 2 3 MEAN SD**

**dose (μg /plate)** 

**positive control 397 328 394 373 ±**  **39**

**solvent control 21 26 34 27**  **± 7** 

**100 27 27 31 28 ±**  **2**

**333 23 21 20 21 ±**  **2**

**1000 23 30 30 28 ±**  **4**

**3330 33 24 25 27**  **± 5** 

**5000 26 31 27 28 ±**  **3**

**-----------------------------------------------------------**1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Pro ject 500680

- Page 25 - APPENDIX 3 

## – continued 

## –

**Experiment 2**

**Strain TA100**

**-----------------------------------------------------------**

**WITHOUT S9**

**- MIX** 

**plate 1 2 3 MEAN SD**

**dose (μg /plate)** 

**positive control 800 709 721 743 ±**  **49**

**solvent control 138 126 103 122 ±**  **18**

**100 1)** 

**333 130 119 127 125 ±**  **6**

**1000 113 174 123 137 ±**  **33**

**3330 130 119 140 130 ±**  **11**

**5000 117 109 131 119 ±**  **11**

**-----------------------------------------------------------**

**WITH S9**

**- MIX** 

**plate 1 2 3 MEAN SD**

**dose (μg /plate)** 

**positive control 334 346 338 339**  **± 6** 

**solvent control 137 96 95 109 ±**  **24**

**100 126 126 131 128 ±**  **3**

**333 173 135 147 152 ±**  **19**

**1000 129 119 149 132 ±**  **15**

**3330 104 113 130 116 ±**  **13**

**5000 114 135 124 124 ±**  **11**

**----------------------------------------------------------- 1) Due to a technical error, plates could not be determimed** 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Pro ject 500680

- Page 26 - APPENDIX 3 

## – continued 

## –

**Experiment 2**

**Strain WP 2**  **uvrA**

**-----------------------------------------------------------**

**WITHOUT S9**

**- MIX** 

**plate 1 2 3 MEAN SD**

**dose (μg /plate)** 

**positive control 1023 1089 961 1024**  **± 64** 

**solvent control 20 34 24 26 ±**  **7**

**100 22 18 17 19 ±**  **3**

**333 19 20 15 18 ±**  **3**

**1000 12 19 20 17 ±**  **4**

**3330 23 25 21 23 ±**  **2**

**5000 16 18 12 15 ±**  **3**

**-----------------------------------------------------------**

**WITH S9**

**- MIX** 

**pla te 1 2 3 MEAN SD** 

**dose (μg /plate)** 

**positive control 346 281 322 316 ±**  **33**

**solvent control 24 21 34 26 ±**  **7**

**100 22 28 30 27 ±**  **4**

**333 23 31 28 27 ±**  **4**

**1000 34 27 37 33 ±**  **5**

**3330 34 31 32 32 ±**  **2**

**5000 15 17 20 17 ±**  **3**

**-----------------------------------------------------------**1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Pro ject 500680

- Page 27 - APPENDIX 4 HISTORICAL CONTROL DATA OF THE SOLVENT CONTROL

TA1535 TA1537 TA98 TA100 WP 2 uvrA

S9

- mix - + - + - + - + - +

Range 2 - 25 2 – 31 2 - 19 2 - 32 7 - 49 8 - 59 61 - 191 58 - 205 7 - 51 7 - 62

Mean 10 8 5 5 20 24 10 8 94 22 24

SD 4 3 2 2 6 7 24 24 7 7

n 1468 1 469 1253 1251 1453 1451 1 485 1 449 1309 1290

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between January 20 10 and January 20 13

.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Pro ject 500680

- Page 28 - APPENDIX 5 HISTORICAL CONTROL DATA OF THE POSITIVE CONTROL SUBSTANCES

TA1535 TA1537 TA98

S9

- mix - + - + - +

Range 245 - 1270 72 – 1016 113 - 1 182 85 - 677 40 7 - 1 518 202 - 166 3

Mean 8 41 2 59 539 3 53 1008 806

SD 122 11 6 265 103 134 290

n 1431 1437 1237 1242 1446 1428

TA100 WP 2 uvrA

S9

- mix - + - +

Range 348 - 1417 229 - 1752 138 - 1479 122 - 1233

Mean 872 111 4 10 58 3 58

SD 1 30 2 38 20 9 125

n 1452 152 1 280 1264

SD = Standard deviation

n = Number of obser vations

Historical control data from experiments performed between January 2010 and January 2013.1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Pro ject 500680

- Page 29 - APPENDIX 6 CERTIFICATE OF ANALYSIS