## FINAL REPORT

Study Title



## EVALUATION OF THE AB ILITY OF FRET 09

- 0536 TO INDUCE

CHROMOSOME ABERRATIO NS IN CULTURED PERIP HERAL

HUMAN LYMPHOCYTES (W ITH REPEAT EXPERIMEN T)

Author

I.A.J. Verbaan, PhD.

Test Facility

WIL Research Europe B.V.

Hambake nwetering 7

5231 DD ‘s

- Hertogenbosch

The Netherlands

Laboratory Project Identification

Project 504372

Substance 205292/A

- Page 1 of 33

-FRET 09-0536 Project 504372

- Page 2 - 1. CONTENTS

1. CONTENTS

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

2. STATEMENT OF GLP COMPLIANCE

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

3. QUALITY ASSURANCE STATEMENT

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

4. SUMMARY

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

5. INTRODUCTION

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

5.1. Preface

...................................................................................................................................... 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 METHODS

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

6.1. Test substance

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

6.1.1. Test substance information

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

6.1.2. Study specific test substance information

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

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

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

6.5. Metabolic activation system

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

6.5.1. Preparation of S9-fraction

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

6.5.2. Preparation of S9-mix

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

6.6. Study design

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

6.6.1. Dose range finding test

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

6.6.2. Cytogenetic assay

................................................................ ......................................... 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. List 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 assay

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

7.4. Evaluation of the results

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

8. CONCLUSION

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

9. REFERENCES

............................................................................................................................... 17FRET 09-0536 Project 504372

- Page 3 - TABLES

Table 1 Mitotic index of human lymphocyte cultures treated with FRET 09-0536 in the

dose range finding test

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

Table 2 Mitotic index of human lymphocyte cultures treated with FRET 09-0536 in the first

cytogenetic assay

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

Table 3 Chromosome aberrations in human lymphocyte cultures treated with FRET 09-

0536 in the absence of S9-mix in the first cytogenetic assay (3 h exposure time,

24 h fixation time)

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

Table 4 Chromosome aberrations in human lymphocyte cultures treated with FRET 09-

0536 in the presence of S9-mix in the first cytogenetic assay (3 h exposure time,

24 h fixation time)

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

Table 5 Mitotic index of human lymphocyte cultures treated with FRET 09-0536 in the

second cytogenetic assay

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

Table 6 Mitotic index of human lymphocyte cultures treated with FRET 09-0536 in

cytogenetic assay 2A

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

Table 7 Chromosome aberrations in human lymphocyte cultures treated with FRET 09-

0536 in the absence of S9-mix in the second cytogenetic assay (24 h exposure

time, 24 h fixation time)

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

Table 8 Chromosome aberrations in human lymphocyte cultures treated with FRET 09-

0536 in the absence of S9-mix in the second cytogenetic assay (48 h exposure

time, 48 h fixation time)

................................................................................................ . 26

Table 9 Chromosome aberrations in human lymphocyte cultures treated with FRET 09-

0536 in the presence of S9-mix in the second cytogenetic assay (3 h exposure

time, 48 h fixation time)

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

APPENDICES

APPENDIX 1 TABLES

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

APPENDIX 2 DEFINITIONS OF CHROMOSOME ABERRATIONS SCORED IN METAPHASE

PORTRAITS

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

APPENDIX 3 STATISTICAL EVALUATION OF THE TEST RESULTS

............................................. 30

APPENDIX 4 HISTORICAL CONTROL DATA FOR LYMPHOCYTE CHROMOSOME

ABERRATION STUDIES

.............................................................................................. 31

APPENDIX 5 HISTORICAL CONTROL DATA FOR NUMERICAL ABERRATIONS FOR

LYMPHOCYTE CHROMOSOME ABERRATION STUDIES

........................................ 32

APPENDIX 6 CERTIFICATE OF ANALYSIS

...................................................................................... 33FRET 09-0536 Project 504372

- Page 6 - 4. SUMMARY

Evaluation of the ability of FRET 09-0536 to induce chromosome aberrations in cultured peripheral

human lymphocytes (with repeat experiment).

This report describes the effect of FRET 09- 0536 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 FRET

09 -0536 was tested in two independent experiments.

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

guidelines.

Batch RDIA5595 of FRET 09-0536 was a clear colourless liquid with a purity of 89.6 %. FRET 09-0536

was dissolved in ethanol.

In the first cytogenetic assay, FRET 09-0536 was tested up to 70 and 100 μg/ml for a 3 h exposure

time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction, respectively

. Appropriate toxicity was reached at these dose levels.

In the second cytogenetic assay

, FRET 09-0536 was tested up to 45 μg/ml for a 24 h continuous

exposure time with a 24 h fixation time and up to 50 μg/ml for a 48 h continuous exposure time with a

48 h fixation time in the absence of S9-mix. In the presence of S9-mix FRET 09-0536 was tested up to

130 μg/ml for a 3 h exposure time with a 48 h fixation time. Appropriate toxicity was reached at these

dose levels.

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

activation system (S9-mix) functioned properly.

FRET 09-0536 did not induce a statistically significant or biologically relevant increase in the number

of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two

independently repeated experiments.

No effects of FRET 09- 0536 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 FRET 09-0536 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 FRET 09-0536 is not clastogenic in human

lymphocytes under the experimental conditions described in this report.FRET 09-0536 Project 504372

- Page 7 - 5. INTRODUCTION

5.1. Preface

Sponsor International Flavors & Fragrances Inc.

800 Rose Lane

Union Beach, New Jersey 07735

U.S.A.

Study Monitor Vijyeta Kapur

Senior Regulatory Chemica l Control Specialist

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 : 17 January, 2014

Completion : 06 April, 2014

5.2. Aims of the study

Th e objective of this study was to evaluate FRET 09-0536 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 were 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 FRET 09-0536 both in the absence and presence of a

metabolic activation system (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 FRET 09-0536, 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 FRET 09-0536 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 post-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 expect ed.

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

mammalian cell clastogenic agent.FRET 09-0536 Project 504372

- 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). Commission 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, 31

May 2008.

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

Research 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 METHODS

6.1. Test substance

6.1.1. Test substance information

Identification FRET 09

- 0536

Structure

Molecular for mula C 13 H 20 O

Molecular weight 192.15

CAS Number 1464989

- 16

- 9

Description Clear colourless liquid

Batch RDIA5595

Purity/Composition 89.6%

Test substance storage In refrigerator (2

- 8°C) in the dark under nitrogen

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

See APPENDIX 6; Certificate of analysis.

6.1.2. Study specific test substance information

Purity/composition correction factor

required No

Volatile Yes, vapour pressure: 0.024 Pa at 298K

Reactivity R eactive to oxygenFRET 09-0536 Project 504372

- Page 9 - Test substance handling

. Flush container with nitrogen after handling

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

Stability in vehicle Ethanol: Not indicated

Solubility in vehicle Ethanol: Not indicated

6.1.3. Test substance preparation

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

FRET 09-0536 was dissolved in ethanol absolute (Merck, Darmstadt, Germany). FRET 09-0536

concentrations were used within 2.5 hours after preparation

.

The final concentration of the solvent in the culture medium was 0.5% (v/v).

6.2. Reference substances

6.2.1. Negative control

The vehicle for the test substance was ethanol.

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. Baxter B.V., Utrecht, The Netherlands) was used as an

indirect acting mutagen, requiring metabolic activation, at a final concentration of 10 μg/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, EC).

Blood was collec te d 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 2013) are

presented below:

Dose range finding study: age 37 , AGT = 13.2 h

First cytogenetic assay: age 36 , AGT = 12.9 h

Second cytogenetic assay: age 31, AGT = 12.9 h

Cytogenetic assay 2A: age 35, AGT = 13.2 h

6.4. Cell culture

Blood samples

Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting

system with a suitable size sterile vessel containing sodium heparin (Vacuette, Greiner Bio-One,FRET 09-0536 Project 504372

- Page 10

- 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 μg/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 Ltd.

, United Kingdom) was added.

E nvironmental 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 42

- 91 %), containing 5.0 ± 0.5% CO 2 in air, at a

temperature of 37.0 ± 1.0°C (actual range 34.1 - 37.6 °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 3 4.1 - 36.0°C), humidity (with

a maximum of 30%) 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 humidity with a minimum of 42% are explained 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 Netherla nds ) and ß-naphthoflavone (100 mg/kg

body weight; Sigma) in corn oil (Fagron Farmaceuticals, Capelle a/d IJssel, the Netherlands) (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 homogeniser. 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 assay 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 activation

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 Laboratories Ltd., Crumlin, United Kingdom); 4 μmol HEPESFRET 09-0536 Project 504372

- Page 11

- (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. FRET 09-0536 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

cultured for 48 h and thereafter exposed to selected doses of FRET 09- 0536 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 determined by the solubility of FRET 09-0536 in the culture

medium at the 3 h exposure time. At the 24 and 48 h exposure time, FRET 09- 0536 was tested

beyond the limit of solubility to obtain adequate toxicity data.

After 3 h exposure to FRET 09-0536 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 FRET 09-0536 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 had an inhibition of the mitotic index of 50%

or greater whereas the mitotic index of the lowest dose level wa s approximately the same as the

mi totic index of the solvent control.

6.6.2. Cytogenetic assay

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

FRET 09-0536 was tested in the absence and presence of 1.8% (v/v) S9-fraction in duplicate in two

independent experiments. To be able to select appropriate dose levels for scoring of chromosome

aberrations a repeat assay had to be performed (cytogenetic assay 2A).

First cytogenetic assay

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

FRET 09-0536 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 ml culture medium and incubated for another 20 - 22 h

(24 h fixation time). Appropriate negative and positive controls were included in the first cytogenetic

assa y.

Based on the mitotic index of the dose range finding test and the first cytogenetic assay appropriateFRET 09-0536 Project 504372

- Page 12

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

with the following modifications of experimental conditions.

Second cytogenetic assay

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

FRET 09-0536 for 24 h and 48 h in the absence of S9-mix and for 3 h in the presence of S9-mix.

After 3 h exposure, the cells exposed to FRET 09-0536 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 and 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 spindle

inhibitor colchicine (0.5 μg/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 Sörensenbuffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry.

The dry slides were automatically embedded in a 1:10 mixture of xylene (Klinipath, Duiven, 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. Chromosomes of metaphase spreads were analysed from those

cultures with an inhibition of the mitotic index of about 50% or above whereas the mitotic index of the

lowest dose level was approximately the same as the mitotic index of the solvent control. Also cultures

treated with an intermediate dose were examined for chromosome aberrations.

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 adhesive label with WIL Research Europe study identification number and code was

placed over the marked slide. One hundred metaphase chromosome spreads per culture were

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

excluded, 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.FRET 09-0536 Project 504372

- Page 13

- 6.7. Electronic data capture

Observations/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, onesided, 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 statistically 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 beFRET 09-0536 Project 504372

- 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. The humidity was several times below the range as mentioned in the protocol (80-100%) with

a minimum of 42%, for a maximum of 4 hours.

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 dose range-finding test

was only used to determine the concentrations in the cytogenetic assays. In addition the

number of cells with chromosome aberrations found in the negative control cultures in the

cytogenetic assays was within the laboratory negative control data range. Therefore these

deviations of the humidity have no effects on the results of the study.

The study integrity was not adversely 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

At a concentration of 333 μg/ml FRET 09- 0536 precipitated in the culture medium. In the dose range

finding study, at the 3 h exposure time, blood cultures were treated with 3

, 10

, 33

, 100 and 333 μ g

FRET 09-0536/ml culture medium with and without S9-mix. At the 24 h and 48 h continuous exposure

time blood cultures were treated with 3

, 10

, 33

, 100

, 333

, 1000 and 1922 μ g FRE T 09- 0536 /ml culture

medium without S9-mix. FRET 09- 0536 was tested beyond the limit of solubility to obtain adequate

toxicity data.

Table 1 (APPENDIX 1 ) shows the mitotic index of cultures treated with various FRET 09- 0536

concentrations or with the negative control substance.

7.2. First cytogenetic assay

Further investigation showed that a concentration of 200 μg/ml already precipitated in the culture

medium. 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 : 30

, 50, 70, 100, 150, 200, 250 and 300 μg/ml culture medium

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

Table 2 (APPENDIX 1 ) shows the mitotic index of cultures treated with various FRET 09- 0536

concentrations or with the positive or negative control substances.

The following dose levels were selected for scoring of chromosome aberrations:

Without S9-mix : 30

, 50 and 70 μg/ml culture medium

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

With S9-mix : 30

, 70 and 100 μg/ml culture medium

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

Both in the absence and presence of S9-mix, FRET 09-0536 did not induce a statistically significant or

biologically relevant increase in the number of cells with chromosome aberrations (APPENDIX 1 :FRET 09-0536 Project 504372

- Page 15

- Table 3

, Table 4 )

. Both i n the absence and presence of S9-mix, FRET 09-0536 did not increase the number of polyploid

cells and cells with endoreduplicated chromosomes.

7.3. Second cytogenetic assay

To obtain more information about the possible clastogenicity of FRET 09-0536, a second cytogenetic

assay was performed in which human lymphocytes were continuously exposed to FRET 09-0536 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 FRET 09-0536. The following dose levels were selected for the second

cytogenetic assay:

Without S9-mix : 10

, 30, 50, 70 and 100 μg/ml culture medium

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

With S9-mix : 30, 50, 70, 100, 150, 200, 250 and 300 μg/ml culture medium

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

Table 5 (APPENDIX 1 ) shows the mitotic index of cultures treated with various FRET 09- 0536

concentrations or with the positive or negative control substances.

No appropriate dose levels could be selected for scoring of chromosome aberrations since in the

absence of S9-mix at the concentration of 30 μg/ml not enough cytotoxicity was observed (39 and

41%, respectively), whereas the next higher concentration of 50 μg/ml was too toxic for scoring (69

and 66%, respectively). In addition in the presence of S9-mix at the concentration of 100 μg/ml not

enough cytotoxicity was observed (12%), whereas the next higher concentration of 150 μg/ml was too

toxic for scoring (92%). The experiment was repeated in cytogenetic assay 2A.

The following dose levels were selected:

Without S9-mix : 1, 10, 30, 35

, 40, 45 and 50 μg/ml culture medium

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

With S9-mix : 50, 10 0, 110, 12 0, 130 and 14 0 μg/ml culture medium

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

Table 6 (APPENDIX 1) shows the mitotic index of cultures treated with various FRET 09-0536

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 : 1

, 30 and 45 μg/ml culture medium

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

1

, 30 and 50 μg/ml culture medium

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

With S9-mix : 50

, 110 and 130 μg/ml culture medium

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

Both in the absence and presence of S9-mix, FRET 09-0536 did not induce a statistically significant or

biologically relevant increase in the number of cells with chromosome aberrations (APPENDIX 1 ;

Table 7 - 8).

Both in the absence and presence of S9-mix, FRET 09-0536 did not increase the number of polyploid

cells and cells with endoreduplicated chromosomes.FRET 09-0536 Project 504372

- Page 16

- 7.4. Evaluation of the results

The ability of FRET 09-0536 to induce chromosome aberrations in human peripheral lymphocytes was

investigated in two independent experiments. The highest concentration analysed was selected based

on toxicity, inhibition of the mitotic index of about 50% or greater.

The mitotic indices of cultures treated with various FRET 09- 0536 concentrations or with the negative

control substances are presented in Table 1

, Table 2 and Tabl e 5 - 6 (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 FRET 09- 0536 are presented in Table 3

, Table 4 and Table 7 - 9 (APPENDIX 1) . Duplicate cultures are indicated by A 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 FRET 09- 0536 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 FRET 09- 0536 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 FRET 09-0536 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 FR ET 09-0536 is not clastogenic in human

lymphocytes under the experimental conditions described in this report.FRET 09-0536 Project 504372

- Page 17

- 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 Res 268: 297 - 305.FRET 09-0536 Project 504372

- Page 18

- APPENDIX 1 TABLES

Table 1 Mitotic index of human lymphocyte cultures treated with FRET 09- 0536 in the

dose range finding test

FRET 09-0536 Number of metaphases

concentration (μg/ml) Absolute Number Percentage

of cells scored of control

Without metabolic activation (- S9 -mix)

3 h exposure time, 24 h fixation time

Control a) 50 1005 100

3 53 1008 106

10 57 1048 114

33 49 1047 98

100 16 1036 32

333 b) c) c) c)

24 h exposure time, 24 h fixation time

Control a) 52 1028 100

3 40 1043 77

10 43 1006 83

33 41 1023 79

100 5 1026 10

333 b) c) c) c)

1000 b) c) c) c)

1922 b) c) c) c)

48 h exposure time, 48 h fixation time

Contro l a) 49 1040 100

3 44 1014 90

10 56 1004 114

33 38 1007 78

100 5 1026 10

333 b) c) c) c)

1000 b) c) c) c)

1922 b) c) c) c)

a) Ethanol

b) FRET 09-0536 precipitated in the culture medium.

c) Cell lysisFRET 09-0536 Project 504372

- Page 19

- Table 1

–

continued

–

FR ET 09-0536 Number of metaphases

concentration (μg/ml) Absolute Number Percentage

of cells scored of control

With metabolic activation (+S9-mix)

3 h exposure time, 24 h fixation time

Control a) 41 1005 100

3 46 1005 112

10 34 1008 83

33 48 1028 117

100 34 1004 83

333 b) c) c) c)

a) Ethanol

b) FRET 09-0536 precipitated in the culture medium.

c) Cell lysisFRET 09-0536 Project 504372

- Page 20

- Table 2 Mitotic index of human lymphocyte cultures treated with FRET 09- 0536 in the

first cytogenetic assay

FRET 09-0536 Number of metaphases a)

concentration (μg/ml) Absolute Number Percentage

of cells scored of control

Without metabolic activation (- S9 -mix)

3 h exposure time, 24 h fixation time

Control b) 71

- 69 1018

- 1021 100

30 70

- 63 1006

- 1025 95

50 59

- 50 1010

- 1018 78

70 33

- 22 1005

- 1015 39

100 10

- 13 1002

- 1006 16

150 0

- 0 1000

- 1000 0

200 c) 0

- 0 1001

- 1001 0

250 c) 0

- 0 1003

- 1001 0

300 c) 0

- 0 1011

- 1002 0

MMC

- C; 0.5 μg/ml 52

- 50 1017

- 1040 73

MMC

- C; 0. 7 5 μg/ml 29

- 30 1011

- 1015 42

With metabolic activation (+S9-mix)

3 h exposure time, 24 h fixation time

Control b) 71

- 66 1023

- 1015 100

30 60

- 71 1017

- 1012 96

50 52

- 54 1009

- 1044 77

70 54

- 50 1003

- 1007 76

100 27

- 28 1008

- 1006 40

150 14

- 11 1006

- 1022 18

200 c) 0

- 0 1002

- 1000 0

250 c) 0

- 0 1001

- 1001 0

300 c) 0

- 0 1001

- 1000 0

CP; 10 μg/ml 29

- 36 1010

- 1026 47

a) Duplicate cultures

b) Ethanol

c) FRET 09-0536 precipitated in the culture medium.FRET 09-0536 Project 504372

- Page 21

- Table 3 Chromosome aberrations in human lymphocyte cultures treated with FRET 09-

0536 in the absence of S9-mix in the first cytogenetic assay (3 h exposure time,

24 h fixation time)

Conc Ethanol

( 0. 5 % v/v) 30

μ g/ml 50

μ g/ml 70

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 95 78 39 73

No. of

Cells

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

No. of

Cells with

aberrat ions

(+ gaps) a) 1 0 1 0 1 1 0 0 0 2 1 3 29 30 *** )

59

No. of

Cells with

aberrations

(

- gaps) 1 0 1 0 0 0 0 0 0 2 1 3 29 30 *** )

59

g’ 1

g”

b’ 1 3 1 27 25

b” 2 1

m’

m”

exch. 8 8

dic

d’

misc. poly

total aberr

(+ gaps) 1 0 0 1 0 0 3 1 37 34

total aberr

(

- gaps) 1 0 0 0 0 0 3 1 37 34

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) was not counted as an aberration.

*) Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.FRET 09-0536 Project 504372

- Page 22

- Table 4 Chromosome aberrations in human lymphocyte cultures treated with FRET 09-

0536 in the presence of S9-mix in the first cytogenetic assay (3 h exposure time,

24 h fixation time)

C onc Conc

Ethanol

(0. 5 % v/v) 30

μ g/ml 70

μ g/ml 100

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 96 76 40 47

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 1 1 0 0 0 0 3 3 0 2 2 23 24 *** )

47

No. of

Cells with

aberrations

(

- gaps) 0 0 0 0 0 0 0 2 2 0 2 2 23 2 3 *** )

4 6

g’ 1 1 4

g”

b’ 2 1 21 25

b” 3 1

m’

m”

exch. 1 2 3

dic

d’

misc. poly

total aberr

(+ gaps) 0 1 0 0 0 3 0 2 26 33

total aberr

(

- gaps) 0 0 0 0 0 2 0 2 26 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.FRET 09-0536 Project 504372

- Page 23

- Table 5 Mitotic index of human lymphocyte cultures treated with FRET 09- 0536 in the

second cytogenetic assay

FRET 09-0536 Number of metaphases a)

concentration (μg/ml) Absolute Number Percentage

of cells scored of control

Without metabolic activation (- S9 -mix)

24 h exposure time, 24 h fixation time

Control b) 92

- 86 1003

- 1000 100

10 65

- 55 1000

- 1000 67

30 58

- 51 1001

- 1000 61

50 34

- 21 1000

- 1004 31

70 19

- 16 1000

- 1000 20

100 11

- 6 1002

- 1001 10

MMC

- C; 0.2 μg/ml 41

- 45 1003

- 1006 48

MMC

- C; 0. 3 μg/ml 25

- 24 1000

- 1000 28

48 h exposure time, 48 h fixation time

Control b) 78

- 76 1010

- 1000 100

10 77

- 79 1002

- 1001 101

30 48

- 43 1000

- 1000 59

50 29

- 24 1000

- 1001 34

70 4

- 5 1001

- 1000 6

100 0

- 0 1000

- 1000 0

MMC

- C; 0.1 μg/ml 32

- 40 1000

- 1003 47

MMC

- C; 0. 1 5 μg/ml 33

- 27 1002

- 1001 39

With metabolic activation (+S9-mix)

3 h exposure time, 48 h fixation time

Control b) 89

- 82 1000

- 1000 100

30 83

- 87 1000

- 1002 99

50 90

- 82 1002

- 1002 101

70 83

- 89 1000

- 1006 101

100 82

- 68 1003

- 1002 88

150 6

- 7 1001

- 1003 8

200 c) 0

- 0 1000

- 1000 0

250 c) e )

- e) e)

- e) e)

300 c) e )

- e) e)

- e) e)

CP ; 10 μg/ml 26

- 29 1000

- 1000

– d)

a) Duplicate cultures

b) Ethanol

c) FRET 09-0536 precipitated in the culture medium

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

control.

e) Cell lysisFRET 09-0536 Project 504372

- Page 24

- Table 6 Mitotic index of human lymphocyte cultures treated with FRET 09-0536 in

cytogenetic assay 2A

FRET 09-0536 Number of metaphases a)

concentration (μg/ml) Absolute Number Percentage

of cells scored of control

Without metabolic activation (- S9 -mix)

24 h exposure time, 24 h fixation time

Control b) 72

- 70 1010

- 1003 100

1 61

- 56 1014

- 1014 82

10 60

- 59 1005

- 1016 84

30 54

- 45 1003

- 1006 70

35 38

- 38 1007

- 1006 54

40 38

- 39 1007

- 1012 54

45 39

- 30 1030

- 1007 49

50 20

- 21 1020

- 1027 29

MMC

- C; 0.2 μg/ml 24

- 27 1010

- 1010 36

MMC

- C; 0.3 μg/ml 18

- 20 1019

- 1001 27

48 h exposure time, 48 h fixation time

Control b) 34

- 43 1040

- 1028 100

1 40

- 39 1006

- 1017 103

10 24

- 24 1008

- 1024 62

30 29

- 20 1013

- 1008 64

35 23

- 24 1003

- 1025 61

40 21

- 21 1015

- 1011 55

45 19

- 22 1009

- 1009 53

50 15

- 14 1009

- 1014 38

MMC

- C; 0.1 μg/ml 26

- 29 1003

- 1001 71

MMC

- C; 0.15 μg/ml 17

- 18 1013

- 1015 45

With metabolic activation (+S9-mix)

3 h exposure time, 48 h fixation time

Control b) 57

- 53 1037

- 1015 100

50 69

- 55 1034

- 1012 113

100 40

- 36 1017

- 1013 69

110 36

- 35 1002

- 1018 65

120 35

- 30 1036

- 1005 59

130 25

- 22 1016

- 1003 43

140 23

- 25 1018

- 1007 44

CP; 10 μg/ml 29

- 28 1020

- 1002

– c )

a) Duplicate cultures

b) Ethanol

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

control.FRET 09-0536 Project 504372

- Page 25

- Table 7 Chromosome aberrations in human lymphocyte cultures treated with FRET 09-

0536 in the absence of S9-mix in the second cytogenetic assay (24 h exposure

time, 24 h fixation time)

Conc Ethanol

(0. 5 % v/v) 1

μ g/ml 30

μ g/ml 45

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 82 70 49 36

No. of

Cells

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

No. of

C ells with

aberrations

(+ gaps) a) 0 1 1 0 0 0 1 1 2 0 0 0 20 28 *** )

48

No. of

Cells with

aberrations

(

- gaps) 0 1 1 0 0 0 0 0 0 0 0 0 20 27 *** )

47

g’ 1 1 2

g”

b’ 8 14

b” 1 2 9

m’

m”

exch. 10 11

dic

d’

misc.

total aberr

(+ gaps) 0 1 0 0 1 1 0 0 20 36

total aberr

(

- gaps) 0 1 0 0 0 0 0 0 20 34

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.FRET 09-0536 Project 504372

- Page 26

- Table 8 Chromosome aberrations in human lymphocyte cultures treated with FRET 09-

0536 in the absence of S9-mix in the second cytogenetic assay (48 h exposure

time, 48 h fixation time)

Conc Ethanol

(0. 5 % v/v) 1

μ g/ml 30

μ g/ml 50

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 (%) 100 103 64 38 71

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 1 2 0 1 1 25 31 *** )

56

No. of

Cells with

aberrations

(

- gaps) 1 1 2 0 0 0 0 1 1 0 1 1 24 31 *** )

55

g’ 1 1 2

g”

b’ 1 1 12 11

b” 1 1 17 13

m’

m”

exch. 6 15

dic

d’

misc.

total aberr

(+ gaps) 2 1 0 0 1 1 0 1 37 39

total aberr

(

- gaps) 1 1 0 0 0 1 0 1 35 39

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.FRET 09-0536 Project 504372

- Page 27

- Table 9 Chromosome aberrations in human lymphocyte cultures treated with FRET 09-

0536 in the presence of S9-mix in the second cytogenetic assay (3 h exposure

time, 48 h fixation time)

Conc Ethanol

(0. 5 % v/v) 50

μ g/ml 110

μ g/ml 130

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 (%) 100 113 65 43

- 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 0 1 1 1 1 2 1 0 1 36 22 *** )

58

No. of

Cells with

aberrations

(

- gaps) 0 0 0 0 1 1 0 1 1 1 0 1 34 22 *** )

56

g’ 1 3

g” 1

b’ 13 15

b” 1 1 1 12 4

m’

m”

exch. 13 8

dic

d’

misc.

total aberr

(+ gaps) 0 0 0 1 1 1 1 0 42 27

total aberr

(

- gaps) 0 0 0 1 0 1 1 0 38 27

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.FRET 09-0536 Project 504372

- Page 28

- APPENDIX 2 DEFINITIONS OF CHROMOSOME ABERRATIONS SCORED IN METAPHASE

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 apparently "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 unaligned.

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 o r

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 centromeres.

Tricentric tric A chromosome containing three

chromosome centromeres.FRET 09-0536 Project 504372

- Page 29

- 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 more

chromosomes resulting in the formation of a trior 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

chromosomes

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

normal 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.FRET 09-0536 Project 504372

- Page 30

- 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 (μg/ml) one -sided CONFIDENCE LEVEL

First cytogenetic assay

24 h fixation time

Positive controls

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.FRET 09-0536 Project 504372

- Page 31

- APPENDIX 4 HISTORICAL CONTROL 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

- 5 0

- 5 0

- 4

Mean 0.8 0.7 0.7 0. 6

SD 1.0 1. 0 0.9 1.0

n 378 558 382 541

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between September 20 10 and September 2013

.FRET 09-0536 Project 504372

- Page 32

- APPENDIX 5 HISTORICAL CONTROL DATA FOR NUMERICAL ABERRATIONS FOR

LYMPHOCYTE CHROMOSOME ABERRATION STUDIES

Polyploid cells and endoreduplicated chromosomes per 100

metaphases

polyploidy endoreduplication

+ S9

- mix

- S9

- mix + S9

- mix

- S9

- mix

Range 0

- 2 0

- 3 0

- 2 0

- 1

Me an 0.0 9 0. 10 0.02 0.03

SD 0. 31 0.3 6 0.1 5 0.1 6

n 329 470 335 475

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between September 2010 and September 2013.FRET 09-0536 Project 504372

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- APPENDIX 6 CERTIFICATE OF ANALYSIS