## FINAL REPORT

Study Title



## EVALUATION OF THE ABILITY OF 1- PROPANESULFONIC ACID

,

2- HYDROXY -3-(2-PROPEN-1-YLOXY)- , SODIUM SALT (1:1) TO

INDUCE CHROMOSOME AB ERRATIONS IN CULTURE D

PERIPHERAL HUMAN LYM PHOCYTES

(WITH REPEAT EXPERIM ENT)

Author

I.A.J. Verbaa n, PhD.

Test Facility

WIL Research Europe B.V.

Hambakenwetering 7

5231 DD ‘s

- Hertogenbosch

The Netherlands

Laboratory Project Identification

Project 500681

Substance 204240/A

- Page 1 of 30

-1- Propanesulfonic acid, 2

- hydro xy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500681

- Page 2 - 1. CONTENTS

1. CONTENTS

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

2. STATEMENT OF GLP COM PLIANCE

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

3. QUALITY ASSURANCE STATEMENT

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

4. SUMMARY

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

5. INTRODUCTION

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

5.1. Prefa ce

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

5.2. Aims of the study

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

5.3. Guidelines

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

5.4. Storage and retention of records and materials

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

6. MATERIALS AND METHOD S

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

6.1. Test substance

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

6.1.1. Test substance information

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

6.1.2. Study specific test substance information

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

6.1.3. Test substance preparation

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

6.2. Reference substances

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

6.2.1. Negative control

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

6.2.2. Positive controls

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

6.3. Test system

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

6.4. Cell culture

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

6.5. Metabolic activation system

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

6.5.1. Preparation of S9

- fraction

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

6.5.2. Preparation of S9

- mix

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

6.6. Study design

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

6.6.1. Dose range finding test

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

6.6.2. Cytogenetic as say

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

6.6.3. Chromosome preparation

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

6.6.4. Preparation of slides

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

6.6.5. Mitotic index/dose selection for scoring of the cytogenetic assay

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

6.6.6. Analysis of slides for chromosome aberrations

............................................................ 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

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

6.9.1. L ist of protocol deviations

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

6.9.2. List of standard operating procedures deviations

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

7. RESULTS

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

7.1. Dose range finding test

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

7.2. First cytogenetic assay

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

7.3. Second cytogenetic a ssay

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

7.4. Evaluation of the results

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

8. CONCLUSION

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

9. RE FERENCES

............................................................................................................................... 161- Propanesulfonic acid, 2

- hydro xy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500681

- Page 3 - TABLES

Table 1 Mitotic index of human lymphocyte cultures treated with 1

- Propanesulfonic acid,

2- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) in the dose range finding test

....... 17

Table 2 Mitotic index of human lymphocyte cultures treated with 1

- Propanesulfonic acid,

2- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) in the first cytogenetic assay

........ 18

Table 3 Chromosome aberrations in human lymphocyte cultures treated with 1

- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) in the

absence of S9

- mix in the fir st cytogenetic assay (3 h exposure time, 24 h fixation

time)

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

Table 4 Chromosome aberrations in human lymphocyte cultures treated with 1

- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) in the

presence of S9

- mix in the first cytogenetic assay (3 h exposure time, 24 h fixation

time)

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

Table 5 Mitotic index of human lymphocyte cultures treated with 1

- Propan esulfonic acid,

2- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) in the second cytogenetic

assay

............................................................................................................................. 21

Table 6 Chromosome aberrations in human lymphocyte cultures treated with 1

- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) in the

absence of S9

- mix in the second cytogenetic assay (24 h exposure time, 24 h

fixation time)

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

Table 7 Chromosome aberrations in human lymphocyte cultures treated with 1

- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) in the

absence of S9

- mix in the second cytogenetic assay (48 h exposure time, 48 h

fixation time)

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

Table 8 Chromosome aberrations in human lymphocyte cultures treated with 1

- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) in the

presence of S9

- mix in the second cytogenetic assay (3 h exposure time, 48 h

f ixation time)

.................................................................................................................. 24

APPENDICES

APPENDIX 1 TABLES

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

APPENDIX 2 DEFINITIONS OF CHROM OSOME ABERRATIONS SCORED IN METAPHASE

PORTRAITS

................................................................................................................. 25

APPENDIX 3 STATISTICAL EVALUATION OF THE TEST RESULTS

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

APPENDIX 4 HISTORICAL CONTROL DATA FOR LYMPHOCYTE CHROMOSOME

ABERRATION STUDIES

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

APPENDIX 5 HISTORICAL CONTROL DATA FOR NUMERICAL ABERRATIONS FOR

LYMPHOCYTE CHROMOSOME ABERRATION STUDIES

........................................ 29

APPENDIX 6 CERTIFICATE OF ANALYSIS

...................................................................................... 301- Propanesulfonic acid, 2

- hydro xy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500681

- Page 6 - 4. SUMMARY

Evaluation of the ability of 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1)

to induce chromosome aberrations in cultured peripheral human lymphocytes (with repeat

experiment).

This report describes the effect of 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium

salt (1:1) on the number of chromosome aberrations in cultured peripheral human lymphocytes in the

presence and absence of a metabolic activation system (phenobarbital and ß

- naphthoflavone induced

rat liver S9

- mix). The possible clastogenicity of 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1-

yloxy)

- , sodium salt (1:1) was tested in two independent experiments.

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

guidelines.

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

soluble in culture medium

.

In the first cytogenetic assay, 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt

(1:1) was tested up t o 2182 μ g/ml (= 0.01 M) for a 3 h exposure time with a 24 h fixation time in the

absence and presence of 1.8% (v/v) S9

- fraction. This is the highest concentration recommended for

testing in the guidelines.

In the second cytogenetic assay, 1- Propanesulfoni c acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt

(1:1) was tested up to 2182 μ g/ml for a 24 h and 48 h continuous exposure time with a 24 h and 48 h

fixation time in the absence of S9

- mix. In the presence of S9

- mix 1- Propanesulfonic acid, 2

- hydroxy -3-

(2- propen -1- yloxy)

- , sodium salt (1:1) was also tested up to 2182 μ g/ml for a 3 h exposure time with a

48 h fixation time.

The number of cells with chromosome aberrations found in the solvent control cultures was within the

laboratory historical control data range. Positive control chemicals, mitomycin C and

cyclophosphamide, both produced a statistically significant increase in the incidence of cells with

chromosome aberrations, indicating that the test conditions were adequate and that the metabolic

activ ation system (S9

- mix) functioned properly.

1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

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

significant or biologically relevant increase in the number of cells with chromosome aberrations in the

ab sence and presence of S9

- mix, in either of the two independently repeated experiments.

No effects of 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) on the

number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the

absence and presence of S9

- mix. Therefore it can be concluded that 1- Propanesulfonic acid,

2- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) does not disturb mitotic processes and cell cycle

progression and does not induce numerical chromosome aberrations under the experimental

conditions described in this report.

Finally, it is concluded that this test is valid and that 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1-

yloxy)

- , sodium salt (1:1) is not clastogenic in human lymphocytes under the experimental conditions

described in this report.1- Propanesulfonic acid, 2

- hydro xy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500681

- Page 7 - 5. INTRODUCTION

5.1. Preface

Sponsor Society of Chemical Manufacturers and Affiliates

1850 M Street, NW Suite 700

WASHINGTON, DC 20036

USA

Study Monitor Dr. C. T. Helmes

Test Facility WIL Research Europe B.V.

Hambakenwetering 7

5231 DD ‘s

- Hertogenbosch

The Netherlands

Study Director I.A.J. Verbaan, PhD.

Study Plan Start : 10 January 2013

Completion : 14 March 2013

5.2. Aims of the study

The objective of this study was to evaluate 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) for its ability to induce structural chromosome aberrations in cultured human

lymphocytes (1), either in the presence or absence of a metabolic activation system (S9

- mix).

Background of the test system

Whole blood samples obtained from healthy male subjects w ere treated with an anti

- coagulant

(heparin) and cultured in the presence of a mitogen (phytohaemagglutinin). These stimulated human

lymphocytes were used because they are sensitive indicators of clastogenic activity of a broad range

of chemicals (1

- 5).

The stimulated lymphocytes were exposed to 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) both in the absence and presence of a metabolic activation sy stem (S9

- mix). In

combination with this metabolic activation system indirect chemical mutagens, i.e. those requiring

metabolic transformation into reactive intermediates, can be tested for possible clastogenic effects in

vitro

.

At predetermined intervals after exposure of the stimulated human lymphocytes to 1- Propanesulfonic

acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1), cell division was arrested in the metaphase

stage of the cell cycle by addition of the metaphase

- arresting chemical colchicine. Cells were

harvested, stained and metaphase cells were analysed for the presence of structural chromosome

aberrations such as breaks, gaps, minutes, dicentrics and exchange figures. Results from cultures

treated with 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) were

compared with control (vehicle) treated cultures.

Chromosome aberrations were generally evaluated in the first post

- exposure mitosis (i.e. 24 hours

after exposure). However, since the appearance of the first po st

- exposure mitosis could be

considerably delayed due to toxic insult to the cells, cells were also harvested 48 hours after exposure

to cover the interval in which maximum aberration frequency was expected.

A test substance that induces a positive response in this assay is presumed to be a potential

mammalian cell clastogenic agent.1- Propanesulfonic acid, 2

- hydro xy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500681

- Page 8 - 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. 473: In Vitro Mammalian Chromosome Aberration 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.10: "Mutagenicity

- In Vitro

Mammalian Chromosome Aberration Test". Official Journal of the European Union No. L142, 3 1

May 2008.

- Code of Federal Regulations, Title 40

– Protection of Environment. Section 799.9537 Toxic

Substances Control Act (TSCA) in vitro mammalian chromosome aberration test.

5.4. Storage and retention of records and materials

Records and materials pertaining to the study, including protocol, raw data, specimens (except

specimens requiring refrigeration or freezing) and the final report, will be 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

Resea rch Europe will retain information concerning decisions made.

Those specimens requiring refrigeration or freezing will be retained by WIL Research Europe for as

long as the quality of the specimens permits evaluation but no longer than three months after

finalization of the report.

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 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 11 NaO 5 S

Molecular weight 218.22

CAS Nu mber 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- Propanesulfonic acid, 2

- hydro xy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500681

- Page 9 - 6.1.2. Study specific test substance information

Purity/co mposition correction factor

required No

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

maximum duration: 1 hour

Stability in vehicle Water: 1 year

Culture medium : Not indicated

Solubility in vehicle: Water: 1

- 45%

Culture medium : Not indicated

6.1.3. Test substance preparation

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

1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) was dissolved in culture

medium. 1- Propanesulfonic aci d, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) concentrations

were used within 2.5 hours after preparation and filter (0.22 μ m)

- sterilized.

The pH and the osmolarity of the culture medium containing the highest tested concentration were

recorded.

6.2. Reference substances

6.2.1. Negative control

The vehicle for the test substance was culture medium

.

6.2.2. Positive controls

Without metabolic activation (

- S9

- mix):

Mitomycin C (MMC

- C; CAS no. 50

- 07

- 7, Sigma, Zwijndrecht, The Netherlands) was used as a direct

acting mutagen at a final concentration of 0.5 and 0.75 μg/ml for a 3 h exposure period, 0.2 and

0.3 μg/ml for a 24 h exposure period and 0.1 and 0.15 μg/ml for a 48 h exposure period.

With metabolic activation (+S9

- mix):

Cyclophosphamide (CP; CAS no. 50

- 18

- 0. B axter B.V., Utrecht, The Netherlands) was used as an

indirect acting mutagen, requiring metabolic activation, at a final concentration of 10 μ g/ml for a 3 h

exposure period (24 h fixation time).

Solvent for positive controls

Hanks’ Balanced Salt Solution (HBSS) (Invitrogen Corporation, Breda, The Netherlands), without

calcium and magnesium.

All reference stock solutions were stored in aliquots at ≤

- 15°C in the dark. These solutions were

thawed immediately before use.

6.3. Test system

Cultured peripheral human lymphocytes were used as test system. Peripheral human lymphocytes are

recommended in international guidelines (e.g. OECD, E C).

Blood was collected from healthy adult, non

- smoking , male volunteers. The Average Generation Time

(AGT) of the cells and the age of the donor at the time the AGT was determined (December 20 12 ) are

presented below:

Dose range finding study: age 36 , AGT = 13.5 h1- Propanesulfonic acid, 2

- hydro xy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500681

- Page 10

- First cytogenetic assay: age 30 , AGT = 13.0 h

Second cytogenetic assay: age 33 , AGT = 12.9 h

6.4. Cell culture

Blood sample s

Blood samples were collected by ven i puncture using the Venoject multiple sample blood collecting

system with a suitable size sterile vessel containing sodium heparin (Vacuette, Greiner Bio

- One,

Alphen aan den Rijn, The Netherlands). Immediately after blood collection lymphocyte cultures were

started.

Culture medium

Culture medium consisted of RPMI 1640 medium (Invitrogen Corporation), supplemented with

20% (v/v) heat

- inactivated (56°C; 30 min) foetal calf serum (Invitrogen Corporation), L

- glutamine

(2 mM) (Invitrogen Corporation), penicillin/streptomycin (50 U/ml and 50 μ g/ml respectively)

(Invitrogen Corporation) and 30 U/ml heparin (Sigma, Zwijndrecht, The Netherlands).

Lymphocyte cultures

Whole blood (0.4 ml) treated with heparin was added to 5 ml or 4.8 ml culture medium (in the absence

and presence of S9

- mix, respectively). Per culture 0.1 ml (9 mg/ml) phytohaemagglutinin ( Remel,

Europe L td.

, U nited K ingdom ) was added.

Environmental conditions

All incubations were carried out in a controlled environment in the dark, in which optimal conditions

were a humid atmosphere of 80

- 100% (actual range 49

- 95 %), containing 5.0 ± 0.5% CO 2 in air, at a

temperature of 37.0 ± 1.0°C (actual range 35.9

- 37.4 °C). Temperature and humidity were

continuously monitored throughout the experiment. The CO 2 percentage was monitored once on each

working day. Temporary deviations from the temperature (in the range of 35.9

- 36.0°C ), humidity (with

a maximum of 3 0%) and CO 2 percentage (with a maximum of 1%) occurred that were caused by

opening and closing of the incubator door, but the time of these deviations did not exceed 2 hour s

. Based on laboratory historical data these deviations are considered not to affect the study integrity.

The temporary deviations from the humidi ty in the second cytogenetic assay with a minimum of 49 %

are e xplained in protocol deviation 1

.

6.5. Metabolic activation system

Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats (6), which were

obtained from Charles River (Sulzfeld, Germany).

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, and allowed to acclimatise for at least

5 days. The rats were orally dosed at three consecutive days with a suspension of phenobarbital

( 80 mg/kg body weight; Bufa B.V., IJsselstein, The Netherlands ) and ß

- naphthoflavone (100 mg/kg

body weight; Sigma) in corn oil (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 cold (0°C), sterile 0.1 M

sodium phosphate buffer (pH 7.4) containing 0.1 mM Na 2 - EDTA (Merck, Darmstadt, Germany). The

livers were minced in a blender and homogenised in 3 volumes of phosphate buffer with a Potter

homoge niser. The homogenate was centrifuged for 15 min at 9000 g . The supernatant (S9) was

transferred into sterile ampules, which were stored in liquid nitrogen (

- 196°C) for a maximum of 1 year.

The S9 batch was characterised in a bacterial reverse mutation as say in Salmonella typhimurium

tester strain TA98 with the mutagens Benzo

- (a)

- pyrene (Sigma) and 2

- aminoanthracene (Sigma) at

concentrations of 5 μg/plate and 1 μg/plate, respectively. These mutagens require metabolic activation1- Propanesulfonic acid, 2

- hydro xy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500681

- Page 11

- for exerting their mutagenic effects.

6.5.2. Preparation of S9

- mix

S9

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

- mix components contained per ml:

1.63 mg MgCl 2 .6H 2 O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose -6- phosphate (Roche, Mannheim,

Germany); 3.4 mg NADP (Randox Lab oratories Ltd., Crumlin, United Kingdom ); 4 μ mol HEPES

(Invitrogen Corporation).

The above solution was filter (0.22 μ m)

- sterilized. To 0.5 ml S9

- mix components 0.5 ml

S9

- fraction was added (50% (v/v) S9

- fraction) to complete the S9

- mix.

Metabolic activation was achieved by adding 0.2 ml S9

- mix to 5.3 ml of a lymphocyte culture

(containing 4.8 ml culture medium, 0.4 ml blood and 0.1 ml (9 mg/ml) phytohaemagglutinin). The

concentration of the S9

- fraction in the exposure medium was 1.8% (v/v).

6.6. Study design

6.6.1. Dose range finding test

In order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data

were obtained in a dose range finding test. 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) was tested in the absence and in the presence of 1.8% (v/v) S9

- fraction.

Lymphocytes (0.4 ml blood of a healthy male donor was added to 5 ml or 4.8 ml culture medium,

without and with metabolic activation respectively and 0.1 ml (9 mg/ml) Phytohaemagglutinin) were

c ultured for 48 h and thereafter exposed to selected doses of 1- Propanesulfonic acid, 2

- hydroxy -3- (2

-

propen -1- yloxy)

- , sodium salt (1:1) for 3 h, 24 h and 48 h in the absence of S9

- mix or for 3 h in the

presence of S9

- mix. A negative control was included at each exposure time.

The highest tested concentration was 2182 μ g/ml (= 0.01 M)

.

After 3 h exposure to 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) in the

absence or presence of S9

- mix, the cells were separated from the exposure medium by centrifugation

(5 min, 365 g ). The supernatant was removed and cells were rinsed with 5 ml HBSS. After a second

centrifugation step, HBSS was removed and cells were resuspended in 5 ml culture medium and

incubated for another 20

- 22 h (24 h fixation time). The cells that were exposed for 24 h and 48 h in

the absence of S9

- mix were not rinsed after exposure but were fixed immediately (24 h and 48 h

fixation time).

Cytotoxicity of 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) in the

lymphocyte cultures was determined using the mitotic index.

Based on the results of the dose range finding test an appropriate range of dose levels was chosen for

the cytogenetic assays considering the highest dose level was the recom mended 0.01 M.

6.6.2. Cytogenetic assay

The cytogenetic assay was carried out as described by Evans, 1984 (2) with minor modifications.

1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) was tested in the absence

and presence of 1.8% (v/ v) S9

- fraction in duplicate in two independent experiments.

First cytogenetic assay

Lymphocytes were cultured for 48 ± 2 h and thereafter exposed in duplicate to selected doses of

1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1 ) for 3 h in the absence and

presence of S9

- mix. After 3 h exposure, the cells were separated from the exposure medium by

centrifugation (5 min, 365 g ). The supernatant was removed and the cells were rinsed once with 5 ml

HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 ml1- Propanesulfonic acid, 2

- hydro xy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500681

- Page 12

- culture medium and incubated for another 20

- 22 h (24 h fixation time). Appropriate negative and

positive controls were included in the first cytogenetic assay.

Based on the mitotic index of the dose range finding test and the first cytogenetic assay appropriate

dose levels were selected for the second cytogenetic assay. The independent repeat was performed

with the following modifications of experimental conditions.

Second cytogenetic assay

Lym phocytes were cultured for 48 ± 2 h and thereafter exposed in duplicate to selected doses of

1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) for 24 h and 48 h in the

absence of S9

- mix and for 3 h in the presence of S9

- mix.

Afte r 3 h exposure, the cells exposed to 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) in the presence of S9

- mix were separated from the exposure medium by

centrifugation (5 min, 365 g ). The supernatant was removed and the cells were rinsed once with 5 ml

of HBSS and incubated in 5 ml culture medium for another 44

- 46 h (48 h fixation time).

The cells that were treated for 24 h and 48 h in the absence of S9

- mix were not rinsed after exposure

but were fixed immediately after 24 h an d 48 h (24 h and 48 h fixation time).

Appropriate negative and positive controls were included in the second cytogenetic assay.

6.6.3. Chromosome preparation

During the last 2.5

- 3 h of the culture period, cell division was arrested by the addition of the spind le

inhibitor colchicine (0.5 μ g/ml medium) ( Acros Organics, Geel, Belgium ). Thereafter the cell cultures

were centrifuged for 5 min at 365 g and the supernatant was removed. Cells in the remaining cell

pellet were swollen by a 5 min treatment with hypotonic 0.56% (w/v) potassium chloride (Merck)

solution at 37°C. After hypotonic treatment, cells were fixed with 3 changes of methanol (Merck):

acetic acid (Merck) fixative (3:1 v/v).

6.6.4. Preparation of slides

Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v)

ethanol (Merck)/ether (Merck) and cleaned with a tissue. The slides were marked with the WIL

Research Europe study identification number and group number. At least two slides were prepared

per culture. Slides were allowed to dry and thereafter stained for 10

- 30 min with 5% (v/v) Giemsa

(Merck) solution in tap water. Thereafter slides were rinsed in tap

- water and allowed to dry. The dry

slides were automatically embedded in a 1:10 mixture of xylene (Klinipath, Duiv en, The

Netherlands)/pertex (Klinipath) and mounted with a coverslip in an automated coverslipper ( Leica

Microsystems B.V., Rijswijk, The Netherlands ).

6.6.5. Mitotic index/dose selection for scoring of the cytogenetic assay

The mitotic index of each culture was determined by counting the number of metaphases from at least

1000 cells (with a maximum deviation of 5%). At least three analysable concentrations were used for

scoring of the cytogenetic assay. 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , s odium salt

(1:1) was not cytotoxic, the highest concentration analysed was the recommended 0.01 M.

6.6.6. Analysis of slides for chromosome aberrations

To prevent bias, all slides were randomly coded before examination of chromosome aberrations and

scored. An a dhesive label with WIL Research Europe study identification number and code was

placed over the marked slide. One hundr e d metaphase chromosome spreads per culture were

examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps

e xcluded, was ≥ 25 in 50 metaphases, no more metaphases were examined. Only metaphases

containing 46 ± 2 centromeres (chromosomes) were analysed. The number of cells with aberrations

and the number of aberrations were calculated.1- Propanesulfonic acid, 2

- hydro xy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500681

- Page 13

- 6.7. Electronic data capture

Obs ervations/measurements in the study were recorded electronically using the following

programme(s):

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

USA) : temperature and humidity

.

6.8. Interpretation

6.8.1. Acceptability of the assay

A chromosome aberration test is considered acceptable if it meets the following criteria:

a) The number of chromosome aberrations found in the solvent control cultures should

reasonably be within the laboratory historical control data range (see APPENDIX 4 )

. b) The positive control substances should produce a statistically significant (Chi

- square test, one

sided, p < 0.05) increase in the number of cells with chromosome aberrations.

c) A homogeneous response between the replicate cultures is observed.

d) A possible precipitate present on the slides should not interfere with the scoring of chromosome

aberrations.

6.8.2. Data evaluation and statistical procedures

A test substance was considered positive (clastogenic) in the chromosome aberration test if:

a) It induced a dose

- related statistically significant (Chi

- square test, one

- sided, p < 0.05) increase in

the number of cells with chromosome aberrations.

b) A statistically significant and biologically relevant increase in the frequencies of the number of cells

with chromosome aberrations was observed in the absence of a clear dose

- response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none

of the tested concentrations induced a stat istically significant (Chi

- square test, one

- sided, p < 0.05)

increase in the number of cells with chromosome aberrations.

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

decision.

The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or

excluded ) for each exposure group outside the laboratory historical control data range was compared

to that of the solvent control using Chi

- square statistics:

(N

- 1) ( ad

- bc) 2

X 2

= -------------------------------- (a+b) (c+d) (a+c) (b+d)

where b = the total number of aberrant cells in the control cultures.

d = the total number of non aberrant cells in the control cultures.

n 0 = the total number of cells scored in the control cultures.

a = the total number of aberrant cells in treated cultures to be compared with the control.

c = the total number of non aberrant cells in treated cultures to be compared with the

control.

n 1 = the total number of cells scored in the treated cultures.

N = sum of n 0 and n 1

(N

- 1) (ad

- bc) 2

If P X 2 >

------------------------------- (one

- tailed) is small (p< 0.05) the hypothesis that the

(a+b) (c+d) (a+c) (b+d)

incidence of cells with chromosome aberrations is the same for both the treated and the solvent

control group is rejected and the number of aberrant cells in the test group is considered to be1- Propanesulfonic acid, 2

- hydro xy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500681

- Page 14

- significantly different from the control group at the 95% confidence level.

6.9. List of deviations

6.9.1. List of protocol deviations

1. In the second cytogenetic assay t he humidity was several times below the range as mentioned

in the protocol (80

- 100%) with a minimum of 4 9% for approximately 3.5 hour s.

Evaluation: These deviations were only for a short time and were caused by opening and

closing of the incubator door during handling of the cell cultures

. The number of cells with

chromosome aberrations found in the negative control cultures was within the laboratory

historical negative control data range. Therefore these deviations of the humidity have no

effect on the results of the study.

The study integrity was not adve rsely affected by the deviation

.

6.9.2. List of standard operating procedures deviations

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

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

7. RESULTS

7.1. Dose range finding test

A concentration of 2182 μ g/ml (= 0.01 M) showed no precipitation in the culture medium and was used

as the highest concentration of 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt

(1:1)

.

The pH and osmolarity of a concentration of 2182 μg/ml were 7.95 and 285 mOs m/kg respectively

(compared to 7.93 and 284 mOsm/kg in the solvent control).

In the dose range finding test blood cultures were treated with 33, 100

, 333

, 1000 and 2182 μ g

1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1)/ml culture medium with and

without S9

- mix.

Table 1 ( APPENDIX 1 ) shows the mitotic index of cultures treated with various 1- Propanesulfonic acid,

2- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) concentrations or with the negative control

substance.

7.2. First cytogen etic assay

Based on the results of the dose range finding test the following dose levels were selected for the

cytogenetic assay:

Without and with S9

- mix: 10

, 33, 100, 1000 and 2182 μ g/ml culture medium

(3 h exposure time, 24 h fixation time).

Table 2 ( APPENDIX 1 ) shows the mitotic index of cultures treated with various 1- Propanesulfonic acid,

2- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) concentrations or with the positive or negative

control sub stances.

The following dose levels were selected for scoring of chromosome aberrations:1- Propanesulfonic acid, 2

- hydro xy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500681

- Page 15

- Without and with S9

- mix: 100

, 1000 and 2182 μ g/ml culture medium

(3 h exposure time, 24 h fixation time).

Both in the absence and presence of S9

- mix, 1- Propanes ulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) did not induce a statistically significant or biologically relevant increase in the number

of cells with chromosome aberrations ( APPENDIX 1 : Table 3

, Table 4 )

.

Both in the absence and presence of S9

- mix, 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) did not increase the number of polyploid cells and cells with endoreduplicated

chromosome s.

7.3. Second cytogenetic assay

To obtain more information about the possible clastogenicity of 1- Propanesulfonic acid, 2

- hydroxy -3-

(2

- propen -1- yloxy)

- , sodium salt (1:1), a second cytogenetic assay was performed in which human

lymphocytes were continuously e xposed to 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) in the absence of S9

- mix for 24 or 48 hours. In the presence of S9

- mix, cells were

fixed after 48 hours following a 3 hour exposure to 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1-

yloxy)

- , sodium salt (1:1). The following dose levels were selected for the second cytogenetic assay:

Without S9

- mix : 33

, 100, 1000, 1500 and 2182 μ g/ml culture medium

(24 h and 48 h exposure time, 24 h and 48 h fixation time).

With S9

- mix : 1

, 33, 100, 1000 and 2182 μ g/ml culture medium

(3 h exposure time, 48 h fixation time).

Table 5 ( APPENDIX 1 ) shows the mitotic index of cultures treated with various 1- Propanesulfonic acid,

2- hydrox y-3- (2

- propen -1- yloxy)

- , sodium salt (1:1) concentrations or with the positive or negative

control substances.

Based on these observations the following doses were selected for scoring of chromosome

aberrations:

Without S9

- mix : 33

, 1000 and 2182 μ g/ml culture medium

(24 h and 48 h exposure time, 24 h and 48 h fixation time).

With S9

- mix : 100, 1000 and 2182 μ g/ml culture medium

(3 h exposure time, 48 h fixation time).

Both in the absence and presence of S9

- mix, 1- Propanesulfonic acid, 2

- hydroxy -3-(2- propen -1- yloxy)

- , sodium salt (1:1) did not induce a statistically significant or biologically relevant increase in the number

of cells with chromosome aberrations ( APPENDIX 1 ; Tabl e 6

- 8).

Both in th e absence and presence of S9

- mix, 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) did not increase the number of polyploid cells and cells with endoreduplicated

chromosomes.

7.4. Evaluation of the results

The ability of 1- Propanesulf onic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) to induce

chromosome aberrations in human peripheral lymphocytes was investigated in two independent

experiments. The highest concentration analysed was 0.01 M.

The mitotic indices of cultures treated with various 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1-

yloxy)

- , sodium salt (1:1) concentrations or with the negative control substances are presented in

Table 1

, Table 2 and Table 5 ( APPENDIX 1 ) . The scores for the number of aberrant cells (gaps

included and excluded) and the number of the various types of chromosome aberrations at the various

concentrations of 1- Propanesulfonic acid, 2

- hyd roxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) are

presented in Table 3

, Table 4 and Table 6 – 8 ( APPENDIX 1 ) . Duplicate cultures are indicated by A1- Propanesulfonic acid, 2

- hydro xy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500681

- Page 16

- and B. The criteria according to which the aberrations were classified are outlined in APPENDIX 2

. APPENDIX 3 presents the statistical evaluations of the test results.

The number of cells with chromosome aberrations found in the solvent control cultures was within the

laboratory historical control data range (see APPENDIX 4 ) . The number of polyploid cells and cells

with endoreduplicated chromosomes in the solvent control cultures was within the laboratory historical

control data range (see APPENDIX 5 ) . The positive control chemicals (MMC

- C and CP) both

produced statistically significant increases in the frequency of aberrant cells. It was therefore

concluded that the test conditions were adequate and that the metabolic activation system (S9

- mix)

functioned properly.

Both in the absence and presence of S9

- mix 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) did not induce a statistically significant or biologically relevant increase in the number

of cells with chromosome aberrations in two independent experiments.

No effects of 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) on the

number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the

absence and presence of S9

- mix. Therefore it can be concluded that 1- Propanesulfonic acid,

2- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) does not disturb mitotic processes and cell cycle

progression and does not induce numerical chromosome aberrations under the experimental

conditions described in this report.

8. CONCLUSION

Finally, it is concluded that this test is valid and that 1- Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1-

yloxy)

- , sodium salt (1:1) is not clastogenic in human lymphocytes under the experimental conditions

described in this report.

9. REFERENCES

1. Evans H.J. (1976). Cytological methods for detecting chemical mutagens. In: Chemical

mutagens, principles and methods for their detection, Vol. 4, Ed. A. Hollaender (New York,

London, PlenumPress), 1

- 29.

2. Evans H.J. (1984). Human Peripheral Blood Lymphocytes for the Analysis of Chromosome

Aberrations in Mutagen Tests. In: Handbook of Mutagenicity Test Procedures, Eds . B.J. Kilbey,

M. Legator, W. Nichols and C. Ramel, 405

- 427, Elsevier Science Publishers B.V., Amsterdam.

3. Howard P.N., Bloom A.D., Krooth R.S. (1972). Chromosomal aberrations induced by N

- methyl

- N'

-

nitro -N- nitrosoguanidine in mammalian cells. In Vitro 7 : 359

- 365.

4. Preston R.J., Au W., Bender M.A., Brewen J.G., Carrano A.V., Heddle J.A., McFee A.F., Wolff S.,

Wassom J.S. (1981). Mammalian in vivo and in vitro cytogenetic assays: A report of the Gene

- tox

Program. Mutat Res 87 : 143

- 188.

5. Galloway S.M., Aardema M.J., Ishidate M.,Jr., Ivett, J.L., Kirkland D.J., Morita T., Mosesso P.,

Sofuni T. (1994). Report from Working Group on in Vitro Tests for Chromosomal Aberrations.

Mutat Res 312 : 241

- 261.

6. Ames B.N., McCann J. and Yamasaki, E. (1975). Methods for detecting carcinogens and

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

-

364.

7. Morita T., Nagaki T., Fukuda I. and Okumura K. (1992). Clastogenicity of low pH to various

cultured mammalian cells. Mutat R es 268 : 297

- 305.1- Propanesulfonic acid, 2

- hydro xy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500681

- Page 17

- APPENDIX 1 TABLES

Table 1 Mitotic index of human lymphocyte cultures treated with 1- Propanesulfonic

acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) in the dose range

finding test

1- Propanesulfonic acid, 2

- hydroxy -3- Number of metaphases

(2

- propen -1- yloxy)

- , sodium salt (1:1) Absolute Number Percentage

concentration (μg/ml) of cells scored of control

Without metabolic activation (

- S9

- mix)

3 h exposure time, 24 h fi xation time

Control a) 65 1000 100

33 43 1000 66

100 37 1000 57

333 28 1000 43

1000 30 1000 46

2182 30 1000 46

24 h exposure time, 24 h fixation time

Control a) 28 1000 100

33 27 1000 96

100 24 1000 86

333 23 1000 82

1000 16 1000 57

2182 11 1000 39

48 h exposure time, 48 h fixation time

Control a) 60 1000 100

33 55 1001 92

100 41 1000 68

333 44 1000 73

1000 36 1001 60

2182 36 1000 60

With metabolic activation (+S9

- mix)

3 h exposure time, 24 h fixation time

Control a) 52 1000 100

33 32 1000 62

100 38 1000 73

333 25 1004 48

1000 22 1000 42

2182 28 1000 54

a) Culture medium1- Propanesulfonic acid, 2

- hydro xy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500681

- Page 18

- Table 2 Mitotic index of human lymphocyte cultures treated with 1- Propanesulfonic

acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) in the first cytogenetic

assay

1- Propanesulfonic acid, 2

- hydroxy -3- Number of metaphases a)

(2

- propen -1- yloxy)

- , sodium salt (1:1) Absolute Number Percentage

concentration (μg/ml) of cells scored of control

Without metabolic activation (

- S9

- mix)

3 h exposure time, 24 h fixation time

Control b) 88

- 96 1000

- 1000 100

1 0 89

- 83 1003

- 1001 93

33 89

- 98 1001

- 1006 102

100 81

- 84 1000

- 1000 90

1000 98

- 90 1002

- 1002 102

2182 96

- 88 1000

- 1004 100

MMC

- C; 0.5 μg/ml 74

- 68 1000

- 1000 77

MMC

- C; 0. 7 5 μg/ml 52

- 63 1000

- 1001 63

With metabolic activation (+S9

- mix)

3 h exposure time, 24 h fixation time

Control b) 86

- 91 1000

- 1000 100

1 0 86

- 81 1002

- 1000 94

33 88

- 92 1000

- 1002 102

100 90

- 88 1000

- 1002 101

1000 89

- 90 1003

- 1000 101

2182 87

- 83 1003

- 1000 96

CP; 10 μg/ml 35

- 34 1000

- 1000 39

a) Duplicate cultures

b) Culture mediu m1- Propanesulfonic acid, 2

- hydro xy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500681

- Page 19

- Table 3 Chromosome aberrations in human lymphocyte cultures treated with 1-

Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) in the

absence of S9

- mix in the first cytogenetic assay (3 h exposure time, 24 h

fixation time)

Conc Culture medium 100

μ g/ml 1000

μ g/ml 2182

μ g/ml MMC -C

0.5 μ g/ml

Culture A B A+B A B A+B A B A+B A B A+B A B A+B

Mitotic

Index (%) 100 90 102 100 77

No. of

Cells

scored 100 100 200 100 100 200 100 100 200 100 100 200 1 00 100 200

No. of

Cells with

aberrations

(+ gaps) a) 1 0 1 0 1 1 1 0 1 0 0 0 23 18 *** )

41

No. of

Cells with

aberrations

(

- gaps) 1 0 1 0 0 0 1 0 1 0 0 0 22 16 *** )

38

g’ 1 2

g” 1

b’ 1 12 8

b” 1 6 3

m’

m”

exch. 8 5

dic

d’

misc.

total aberr

(+ gaps) 1 0 0 1 1 0 0 0 27 18

total aberr

(- gaps) 1 0 0 0 1 0 0 0 26 16

a) Abbreviations used for various types of aberrations are listed in APPENDIX 2

. misc. = (miscellaneous) aberrations not belonging to the ones mentioned above.

*) Significantly different from control group (Chi

- square test), * P < 0.05, ** P < 0.01 or *** P < 0.00 1.1- Propanesulfonic acid, 2

- hydro xy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500681

- Page 20

- Table 4 Chromosome aberrations in human lymphocyte cultures treated with 1-

Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) in the

presence of S9

- mix in the first cytogenetic assay (3 h exposure time, 24 h

fixation time)

Conc Culture medium 100

μ g/ml 1000

μ g/ml 2182

μ g/ml CP

10 μ g/ml

Culture A B A+B A B A+B A B A+B A B A+B A B A+B

Mitotic

Index (%) 100 101 101 96 39

No. of

Cells

scored 100 100 200 100 100 200 100 100 200 100 100 20 0 100 100 200

No. of

Cells with

aberrations

(+ gaps) a) 2 1 3 1 0 1 2 0 2 0 0 0 37 32 *** )

69

No. of

Cells with

aberrations

(

- gaps) 2 1 3 1 0 1 2 0 2 0 0 0 33 32 *** )

65

g’ 4 1

g” 4

b’ 2 22 19

b” 2 2 12 10

m’

m”

exch. 12 9

dic 2 1

d’

misc. intra

total aberr

(+ gaps) 2 2 2 0 2 0 0 0 54 41

total aberr

(- gaps) 2 2 2 0 2 0 0 0 46 40

a) Abbreviations used for various types of aberrations are listed in APPENDIX 2

. misc. = (miscellaneous) aberrations not belonging to the ones mentioned above.

*) Significantly different from control group (Chi

- square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.1- Propanesulfonic acid, 2

- hydro xy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500681

- Page 21

- Table 5 Mitotic index of human lymphocyte cultures treated with 1- Propanesulfonic

acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) in the second

cytogenetic assay

1- Propanesulfonic acid, 2

- hydroxy -3- Num ber of metaphases a)

(2

- propen -1- yloxy)

- , sodium salt (1:1) Absolute Number Percentage

concentration (μg/ml) of cells scored of control

Without metabolic activation (

- S9

- mix)

24 h exposure time, 24 h fixation time

Control b) 66

- 59 1000

- 1000 100

33 71

- 54 1000

- 1001 100

100 49

- 57 1000

- 1000 85

1000 38

- 53 1000

- 1000 73

1500 43

- 52 1000

- 1000 76

2182 35

- 37 1000

- 1000 58

MMC

- C; 0.2 μg/ml 26

- 24 1000

- 1000 40

MMC

- C; 0. 3 μg/ml 19

- 18 1005

- 1027 30

48 h exposure time, 48 h fixation time

Control b) 57

- 64 1000

- 1000 100

33 56

- 52 1001

- 1000 89

100 56

- 47 1000

- 1000 85

1000 d) 46

- 53 1000

- 1000 82

1500 51

- 52 1000

- 1000 85

2182 45

- 47 1000

- 1000 76

MMC

- C; 0.1 μg/ml 40

- 44 1009

- 1000 69

MMC

- C; 0. 1 5 μg/ml 23

- 30 1000

- 1000 44

With metabolic activation (+S9

- mix)

3 h exposure time, 48 h fixation time

Control b) 49

- 46 1000

- 1000 100

1 e) 44

- 52 1000

- 1000 101

33 54

- 45 1001

- 1000 104

100 54

- 49 1000

- 1002 108

1000 46

- 42 1000

- 1000 93

2182 41

- 46 1000

- 1000 92

CP ; 10 μg/ml 11

- 15 1000

- 1000

– c )

a) Duplicate cultures

b) Culture medium

c) CP was fixed after 24 hours. Therefore, the mitotic index could not be calculated as percen tage of

control.

d) Mean of two calculations (40 and 52 metaphases per 1000 cells).

e) Mean of two calculations (44 and 43 metaphases per 1000 cells)1- Propanesulfonic acid, 2

- hydro xy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500681

- Page 22

- Table 6 Chromosome aberrations in human lymphocyte cultures treated with 1-

Propanesulf onic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) in the

absence of S9

- mix in the second cytogenetic assay (24 h exposure time, 24 h

fixation time)

Conc Culture medium 33

μ g/ml 1000

μ g/ml 2182

μ g/ml MMC -C

0.2 μ g/ml

Culture A B A+B A B A+B A B A+B A B A+B A B A+B

Mitotic

Index (%) 100 100 73 58 40

No. of

Cells

scored 100 100 200 100 100 200 100 100 200 100 100 200 100 100 200

No. of

Cells with

aberrations

(+ gaps) a) 2 1 3 0 0 0 1 0 1 0 0 0 16 24 *** )

40

No. of

Cells with

a berrations

(

- gaps) 0 1 1 0 0 0 1 0 1 0 0 0 16 24 *** )

40

g’ 2

g” 1

b’ 1 1 10 19

b” 1 2

m’

m”

exch. 7 8

dic

d’

misc. po ly poly

total aberr

(+ gaps) 2 1 0 0 2 0 0 0 17 30

total aberr

(- gaps) 0 1 0 0 2 0 0 0 17 29

a) Abbreviations used for various types of aberrations are listed in APPENDIX 2

. misc. = (miscellaneous) aberrations not belonging to the ones mentioned above.

The numerical variation polyploidy (poly) w as not counted as an aberration.

*) Significantly different from control group (Chi

- square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.1- Propanesulfonic acid, 2

- hydro xy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500681

- Page 23

- Table 7 Chromosome aberrations in human lymphocyte cultures treated with 1-

Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) in the

absence of S9

- mix in the second cytogenetic assay (48 h exposure time, 48 h

fixation time)

Conc Cultu re medium 33

μ g/ml 1000

μ g/ml 2182

μ g/ml MMC -C

0.1 μ g/ml

Culture A B A+B A B A+B A B A+B A B A+B A B A+B

Mitotic

Index (%) 100 89 82 76 69

No. of

Cells

scored 100 100 200 100 100 200 100 100 200 100 100 200 100 100 200

No. of

Cells with

aberrations

(+ gaps) a) 3 0 3 0 1 1 0 0 0 1 0 1 14 11 *** )

25

No. of

Cells with

aberrations

(

- gaps) 0 0 0 0 1 1 0 0 0 1 0 1 14 11 *** )

25

g’ 2

g” 1

b’ 1 1 5 4

b” 3 1

m’

m”

exch. 4 1

dic

d’

misc. poly poly 4p 8p

poly

total aberr

(+ gaps) 3 0 0 1 0 0 1 0 16 14

total aberr

(

- gaps) 0 0 0 1 0 0 1 0 16 14

a) Abbreviations used for various types of aberrations are listed in APPENDIX 2

. misc. = (miscellaneous) aberrations not belonging to the ones mentioned above.

The numerical variation polyploidy (poly) w as not counted as an aberration.

*) Significantly different from control group (Chi

- square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.1- Propanesulfonic acid, 2

- hydro xy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500681

- Page 24

- Table 8 Chromosome aberrations in human lymphocyte cultures treated with 1-

Propanesulfonic acid, 2

- hydroxy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) in the

presence of S9

- mix in the second cytogenetic assay (3 h exposure time, 48 h

fixation time)

Conc Culture medium 100

μ g/ml 1000

μ g/ml 2182

μ g/ml CP

10 μ g/ml

Culture A B A+B A B A+B A B A+B A B A+B A B A+B

Mitotic

Index (%) 100 108 93 92

- b)

No. of

Cells

scored 100 100 200 100 100 200 100 100 200 100 100 200 100 100 200

No. of

Cells with

aberrations

(+ gaps) a) 0 0 0 1 1 2 0 0 0 0 0 0 23 22 *** )

45

No. of

Cells with

aberrations

(

- gaps) 0 0 0 1 1 2 0 0 0 0 0 0 23 22 *** )

45

g’

g” 1

b’ 1 1 22 25

b” 2 2

m’

m”

exch. 4 2

dic

d’

misc. intra

total aberr

(+ gaps) 0 0 1 1 0 0 0 0 28 31

total aberr

(- gap s) 0 0 1 1 0 0 0 0 28 30

a) Abbreviations used for various types of aberrations are listed in APPENDIX 2

. misc. = (miscellaneous) aberrations not belonging to the ones mentioned above.

b) CP was fixed after 24 hours. Therefore, the mitotic index could not be calculated as percentage of control.

*) Significantly different from control group (Chi

- square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.1- Propanesulfonic acid, 2

- hydro xy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500681

- Page 25

- APPENDIX 2 DEFINITIONS OF CHROM OSOME ABERRATIO NS SCORED IN METAPHA SE

PORTRAITS

Aberration Abbreviation Description

Chromatid gap g' An achromatic lesion which appears as an

unstained region in the chromatid arm, the size

of which is equal to or smaller than the width of

the chromatid and the appar ently "broken"

segments of the chromatid arm are in

alignment.

Chromosome gap g" An achromatic lesion which appears as an

unstained region in both chromatids at the

same position, the size of which is equal to or

smaller than the width of the chromatid and the

apparently "broken" segments of the

chromatids are in alignment.

Chromatid break b' An achromatic lesion in a chromatid arm, the

size of which is larger than the width of the

chromatid. The broken segments of the

chromatid arm are aligned or unalig ned.

Chromosome break b" An achromatic lesion in both chromatids at the

same position, the size of which is larger than

the width of the chromatid. The broken

segments of the chromatids are aligned or

unaligned.

Chromatid deletion d' Deleted material at the end of a chromatid arm.

Minute m' A single, usually circular, part of a chromatid

lacking a centromere.

Double minutes m" Two, usually circular, parts of a chromatid

lacking a centromere.

Dicentric chromosome dic A chromosome containing two centrome res.

Tricentric tric A chromosome containing three

chromosome centromeres.1- Propanesulfonic acid, 2

- hydro xy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500681

- Page 26

- APPENDIX 2

– continued

–

Aberration Abbreviation Description

Ring chromosome r A ring structure with a distinct lumen.

Exchange figure exch. An exchange(s) between two or mo re

chromosomes resulting in the formation of a tri

or more

- armed configuration.

Chromosome intra A chromosome intrachange is scored

intrachange after rejoining of a lesion within one

chromosome.

Pulverized p A fragmented or pulverized chromosome

chro mosomes

Multiple ma A metaphase spread containing ten or

aberrations more of the above mentioned aberrations

(chromatid and chromosome gaps not

included). ma is counted as 10 aberrations.

Polyploidy poly A chromosome number that is a multiple of the

nor mal diploid number.

Endoreduplication endo A form of polyploidy in which each centromere

connects two or four pairs of chromatids instead

of the normal one pair.1- Propanesulfonic acid, 2

- hydro xy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500681

- Page 27

- APPENDIX 3 STATISTICAL EVALUATION OF THE TEST RESULTS

Chi

- square Test

TOTAL NUMBER OF CELLS WITH ABERRATIONS; TREATMENT/CONTROL COMPARISON,

(INCLUSIVE/EXCLUSIVE GAPS) 1)

.

EXPOSURE S9

- MIX GAPS P- VALUE DECISION AT 95%

DOSE ( μ g/ml) one

- sided CONFIDENCE LEVEL

First cytogenetic assay

24 h fixation time

Positive cont rols

MMC

- C (0.5)

− + ≤0.0001 significant

− − ≤0.0001 significant

CP (10) + + ≤0.0001 significant

+

− ≤0.0001 significant

Second cytogenetic assay

24 h fixation time

Positive controls

MMC

- C (0.2)

− + ≤0.0001 significant

− − ≤0.0001 significant

CP (10) + + ≤0.0001 significant

+

− ≤0.0001 significant

48 h fixation time

Positive control

MMC

- C (0.1)

− + ≤0.0001 significant

− − ≤0.0001 significant

1) Only statistically significant results are presented.1- Propanesulfonic acid, 2

- hydro xy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500681

- Page 28

- APPENDIX 4 HISTORICAL CON TROL DATA FOR LYMPHOCYTE CHROMOSOME

ABERRATION STUDIES

Aberrant cells per 100 metaphases

Gaps included Gaps excluded

+ S9

- mix

- S9

- mix + S9

- mix

- S9

- mix

Range 0 - 5 0 - 6 0 - 5 0 - 5

Mean 0.8 0.8 0.7 0.7

SD 1.0 1.1 0.9 1.0

n 42 2 622 427 610

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between October 2009 and October 2012

.1- Propanesulfonic acid, 2

- hydro xy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500681

- Page 29

- APPENDIX 5 HISTORICAL CONTROL DATA FOR NUMERICAL ABERRATIONS FOR

LYMPHOCYTE CHROMOSOME A BERRATION STUDIES

Polyploid cells and endoreduplicated chromosomes per 100

metaphases

polyploidy endoreduplication

+ S9

- mix

- S9

- mix + S9

- mix

- S9

- mix

Range 0 - 2 0 - 2 0 - 2 0 - 1

Mean 0.07 0.09 0.02 0.01

SD 0.29 0.32 0.16 0.12

n 438 630 44 0 636

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between October 2009 and October 2012

.1- Propanesulfonic acid, 2

- hydro xy -3- (2

- propen -1- yloxy)

- , sodium salt (1:1) Project 500681

- Page 30

- APPENDIX 6 CERTIFICATE OF ANALYSIS