## FINAL REPORT

Study Title



## EVALUATION OF THE AB ILITY OF TO

INDUCE CHROMOSOME AB ERRATIONS IN CULTURE D

PERIPHERAL HUMAN LYM PHOCYTES

(WITH REPEAT EXPERIM ENT)

Author

Test Facility

Laboratory Project Identification

- Page 1 of 35

-- 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

...................................................................... 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 assay

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

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

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

6.6.6. Analysis of slides for chromosome aberrations

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

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

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

7. RESULTS

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

7.1. Dose range finding test

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

7.2. First cytogenetic assay

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

7.3. Second cytogenetic assay

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

7.4. Evaluation of the results

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

8. CONCLUSION

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

9. REFERENCES

............................................................................................................................... 18- Page 3 -

TABLES

Table 1 Mitotic index of human lymphocyte cultures treated with in

the dose range finding test

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

Table 2 Mitotic index of human lymphocyte cultures treated with in

the first cytogenetic assay

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

Table 3 Mitotic index of human lymphocyte cultures treated with in

cytogenetic assay 1A

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

Table 4 Chromosome aberrations in human lymphocyte cultures treated with

in the absence of S9-mix in cytogenetic assay 1A (3 h exposure

time, 24 h fixation tim e)

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

Table 5 Chromosome aberrations in human lymphocyte cultures treated with

in the presence of S9-mix in the first cytogenetic assay (3 h

exposure time, 24 h fixation time)

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

T able 6 Mitotic index of human lymphocyte cultures treated with in

the second cytogenetic assay

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

Ta ble 7 Mitotic index of human lymphocyte cultures treated with in

cytogenetic assay 2A

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

Table 8 Chromosome aberrations in human lymphocyte cultures treated with

in the absence of S9-mix in cytogenetic assay 2A (24 h exposure

time, 24 h fixation time)

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

Table 9 Chromosome aberrations in human lymphocyte cultures treated with

in the absence of S9-mix in cytogenetic assay 2A (48 h exposure

time, 48 h fixation time)

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

Table 10 Chromosome aberrations in human lymphocyte cultures treated with

in the presence of S9-mix in cytogenetic assay 2A (3 h exposure

time, 48 h fixation time)

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

APPENDICES

APPENDIX 1 TABLES

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

APPENDIX 2 DEFINITIONS OF CHROMOSOME ABERRATIONS SCORED IN

METAPHASE PORTRAITS

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

APPENDIX 3 STATISTICAL EVALUATION OF THE TEST RESULTS

............................................. 33

APPENDIX 4 HISTORICAL CONTROL DATA FOR LYMPHOCYTE CHROMOSOME

ABERRATION STUDIES

.............................................................................................. 34

APPENDIX 5 HISTORICAL CONTROL DATA FOR NUMERICAL ABERRATIONS FOR

LYMPHOCYTE CHROMOSOME ABERRATION STUDIES

........................................ 35- Page 6 -

4. SUMMARY

Evaluation of the ability of to induce chromosome aberrations in cultured

peripheral human lymphocytes (with repeat experiment).

This report describes the effect of 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 was tested in two independent experiments.

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

guidelines.

Batch of was a

.

was dissolved in dimethyl sulfoxide.

In the first cytogenetic assay, was tested up to 100 and 150 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, was tested up to 80 μg/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 was tested up to 200 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.

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 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 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 is not clastogenic in human

lymphocytes under the experimental conditions described in this report.- Page 7 -

5. INTRODUCTION

5.1. Preface

Sponsor

Study Monitor

(on behalf of the sponsor)

Test Facility

Study Director

Study Plan Start : 02 December 20 11

Completion : 28 March 2012

5.2. Aims of the study

The objective of this study was to evaluate 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 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

, 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 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 expected.

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

mammalian cell clastogenic agent.- 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 are retained in the

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. will retain

information concerning decisions made.

Those specimens requiring refrigeration or freezing will be retained by for as long as the

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

report.

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

Structure

Molecular formula

Molecular weight

CAS Number

Description

Batch

Purity

Test substance storage

Stability under storage conditions

Expiry date 17 October 2012- Page 9 -

6.1.2. Study specific test substance information

Test substance handling Flush container with nitrogen after ha ndling

Stability at higher temperatures Yes, maximum temperature: 130 ° C

maximum duration: 24 hours under nitrogen seal

Stability in vehicle Dimethyl sulfoxide: Unknown

Solubility in vehicle Dimethyl sulfoxide: Not indicated

6.1.3. Test substance preparation

was dissolved in dimethyl sulfoxide of spectroscopic quality (SeccoSolv, Merck,

Darmstadt, Germany). concentrations were used within 3 hours after

preparation.

The final concentration of the solvent in the culture medium was 1.0% (v/v).

6.2. Reference substances

6.2.1. Negative control

The vehicle for the test substance was dimethyl sulfoxide.

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 (see

protocol deviation 1), 0.2 μg/ml for a 24 h exposure period and 0.1 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 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

2010) are presented below:

Dose range finding study: age 34, AGT = 14.6 h

First cytogenetic assay: age 40, AGT = 14.4 h

Cytogenetic assay 1A: age 28, AGT = 14.5 h

Second cytogenetic assay: age 32, AGT = 14.7 h

Cytogenetic assay 2A: age 40, AGT = 15.3 h- Page 10

-

6.4. Cell culture

Blood samples

Blood samples were collected by venapuncture 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 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.

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 46 - 95%), containing 5.0 ± 0.5% CO 2 in air,

at a temperature of 37.0 ± 1.0°C (actual range 34.7 - 37.9°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 35.1 - 36.0°C),

humidity (with a maximum of 20%) 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 1 hour. Based on laboratory historical data these deviations are considered not to affect the

study integrity. The temporary deviations from the temperature with a minimum of 34.7°C and the

humidity with a minimum of 46% are explained in protocol deviation 2.

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).

In cytogenetic assay 2A the rat liver microsomal enzymes (S9 homogenate) was obtained from

Trinova Biochem GmbH, Giessen, Germany and is prepared from male Sprague Dawley rats that

have been injected intraperitoneal with Aroclor 1254 (500 mg/kg) (see protocol deviation 4).

6.5.1. Preparation of S9 -fraction

The animals were housed at 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 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.- Page 11

-

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

HEPES (Invitrogen Corporation).

The above solution was filter (0.22 μm)-sterilized. To 0.5 ml S9-mix components 0.5 ml

S9 -fraction (batches 11-1, 11-3 and 2740) 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. 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 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 in the

culture medium at the 3 h exposure time. At the 24 and 48 h exposure time,

was tested beyond the limit of solubility to obtain adequate toxicity data.

After 3 h exposure to 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 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 was approximately the same

as the mitotic index of the solvent control. If dose related cytotoxicity was observed at the 24 h and

48 h continuous exposure times, the highest dose level was based on toxicity irrespective of

solubility. However, the precipitate may not interfere with the scoring of chromosome aberrations.- Page 12

-

6.6.2. Cytogenetic assay

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

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 several repeat assays had to be performed.

First cytogenetic assay

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

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 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

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

for 24 h and 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix.

After 3 h exposure, the cells exposed to 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 1300 rpm (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

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, Duiven, The Netherlands)/pertex

(Klinipath) and mounted with a coverslip in an automated coverslipper (Leica Microsystems B.V.,

Rijswijk, The Netherlands).- Page 13

-

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 per

1000 cells. In the first and second cytogenetic assay, the mitotic index of each culture was

determined by counting the number of metaphases from 1000 cells (with a maximum deviation of

5%) (see protocol deviation 3). 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. In case

was not cytotoxic and/or difficult to dissolve in aqueous solutions, the highest

concentration analysed at the 3 h exposure time was determined by the solubility in the culture

medium. If dose related cytotoxicity was observed, the highest concentration analysed at the

24 and 48 h continuous exposure times was based on toxicity irrespective of the solubility of

in the culture medium. However, the extent of precipitation may not interfere

with the scoring of 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 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.

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

aber rations.

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.- Page 14

-

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 be

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 cytogenetic assay 1A the positive control cultures were exposed to two concentrations of

mitomycin C (0.5 and 0.75 μg/ml).

Evaluation: To be sure that a positive response was observed lymphocyte cultures were

exposed to two concentrations of mitomycin C. Since the concentration as mentioned in

the protocol gave a positive response the higher concentration was not used for scoring of

chromosome aberrations. Therefore the exposure of the lymphocytes to higher

concentrations of mitomycin C has no effect on the results of the study.

2. In cytogenetic assay 2A the temperature was below the range as mentioned in the protocol

(37.0 ± 1.0°C) with a minimum 34.7°C for approximately 1.5 hour. In addition the humidity

was several times below the range as mentioned in the protocol (80-100%) in the dose

range finding study for approximately 0.5 hour with a minimum of 46%.

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 of cytogenetic assay 2A

was within the laboratory historical negative control data range. In addition, the dose range

finding study was used to set the doses for both cytogenetic assays. Therefore these

deviations of the temperature and humidity have no effect on the results of the study.

3. In the first and second cytogenetic assay, the mitotic index of each culture was determined

by counting the number of metaphases from 1000 cells (with a maximum deviation of 5%).

Evaluation: Due to practical considerations, a deviation of +5% will be accepted.

This has no effect on the data integrity.- Page 15

- 4. The S9 -fraction used in cytogenetic assay 2A was obtained from Trinova Biochem GmbH,

Giessen, Germany and is prepared from male Sprague Dawley rats that have been

injected intraperitoneal with Aroclor 1254 (500 mg/kg).

Evaluation: Since rat liver S9-mix induced by Aroclor or a combination of phenobarbital and

ß-naphthoflavone are recommended by the guidelines and the response of the positive

control (CP) was within the laboratory historical positive control data range, this deviation of

the S9 homogenate had no effect on the results of the study.

The study integrity was not adversely affected by the deviations.

6.9.2. List of standard operating procedures deviations

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

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

7. RESULTS

7.1. Dose range finding test

At a concentration of 333 μg/ml 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 μg 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 1963 μg

ml culture medium without S9-mix. 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

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: 3, 30, 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

concentrations or with the positive or negative control substances.

In the absence of S9-mix no appropriate dose level could be selected for scoring of chromosome

aberrations since at the concentration of 100 μg/ml not enough cytotoxicity was observed (41%)

whereas the next higher concentration of 150 μg/ml was too toxic for scoring (89%). This part of the

experiment was repeated with the following dose levels:

Cytogenetic assay 1A:

Without S9-mix: 3, 50, 100, 120, 140 1) and 170 1) μg/ml culture medium

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

1) Precipitated in the culture medium.

Table 3 (APPENDIX 1) shows the mitotic index of cultures treated with various

concentrations or with the positive or negative control substances

.

The following dose levels were selected for scoring of chromosome aberrations:- Page 16

-

Without S9-mix : 3, 50 and 100 μg/ml culture medium

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

With S9-mix : 3, 100 and 150 μg/ml culture medium

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

Both in the absence and presence of S9-mix, did not induce a statistically

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

(APPENDIX 1 : Table 4

, T able 5 )

.

Both in the absence and presence of S9-mix, 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 a second

cytogenetic assay was performed in which human lymphocytes were continuously exposed to

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 The following dose

levels were selected for the second cytogenetic assay:

Without S9-mix : 1, 10, 20, 30, 40 and 50 μg/ml culture medium

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

With S9-mix : 3, 30, 100, 150, 200, 250 and 300 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

concentrations or with the positive or negative control substances.

In the absence of S9-mix no appropriate dose level could be selected for scoring of chromosome

since no concentration was available with an inhibition of the mitotic index of about 50%. In the

presence of S9-mix also no appropriate dose level could be selected for scoring of chromosome

aberrations since at the concentration of 150 μg/ml not enough cytotoxicity was observed (37%)

whereas the next higher concentration of 200 μg/ml was too toxic for scoring (67%). Therefore the

experiment was repeated with the following dose levels:

Cytogenetic assay 2A:

Without S9-mix : 10, 25, 50, 60, 70, 80, 90 and 100 μg/ml culture medium

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

With S9-mix : 100, 125, 150, 160, 170*, 180*, 190* and 200* μg/ml culture medium

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

* precipitated in the culture medium

Table 7 (APPENDIX 1) shows the mitotic index of cultures treated with various

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 : 50, 70 and 80 μg/ml culture medium

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

50, 60 and 80 μg/ml culture medium

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

With S9-mix : 150, 160 and 200 μg/ml culture medium

(3 h exposure time, 48 h fixation time).- Page 17

-

Both in the absence and presence of S9-mix, did not induce a statistically

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

(APPENDIX 1 ; Table 8

- 10).

Both in the absence and presence of S9-mix, did not increase the number of

polyploid cells and cells with endoreduplicated chromosomes.

7.4. Evaluation of the results

The ability of 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 concentrations or with the

negative control substances are presented in Table 1

, Table 2

, Table 3

, T able 6 and Table 7

(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

are presented in Table 4

, Table 5 and ; Table 8 - 10 (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 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 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 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 is not clastogenic in human

lymphocytes under the experimental conditions described in this report.- Page 18

-

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 Nmethyl- 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. Am es 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.- Page 19

-

APPENDIX 1 TABLES

Table 1 Mitotic index of human lymphocyte cultures treated with

in the dose range finding test

Period of treatment: From 14 December, 2011 to 16 December, 2011

Number of metaphases per 1000 cells

concentration (μg/ml) Absolute Percentage of control

Without metabolic activation (- S9 -mix)

3 h exposure time, 24 h fixation time

Control a) 39 100

3 36 92

10 33 85

33 25 64

100 19 49

333 b) 0 0

24 h exposure time, 24 h fixation time

Control a) 25 100

3 20 80

10 18 72

33 13 52

100 6 24

333 b) 0 0

1000 b) 0 0

1963 b) 0 0

48 h exposure time, 48 h fixation time

Control a) 40 100

3 34 85

10 27 68

33 21 53

100 3 8

333 b) 0 0

1000 b) 0 0

1963 b) 0 0

a) Dimethyl sulfoxide

b) precipitated in the culture medium.- Page 20

-

Table 1 - CONTINUED

Number of metaphases per 1000 cells

concentration (μg/ml) Absolute Percentage of control

With metabolic activation (+S9-mix)

3 h exposure time, 24 h fixation time

Control a) 42 100

3 35 83

10 33 79

33 32 76

100 31 74

333 b) 0 0

a) Dimethyl sulfoxide

b) precipitated in the culture medium.- Page 21

-

Table 2 Mitotic index of human lymphocyte cultures treated with

in the first cytogenetic assay

Period of treatment: From 11 January, 2012 to 12 January, 2012

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) 80

- 83 1011

- 100 9 100

3 79

- 81 1000

- 1003 98

30 78

- 70 1017

- 1011 91

100 51

- 45 1003

- 1012 59

150 8

- 10 1018

- 1009 11

200 c)

- d)

- - d)

- d)

- - d)

- d)

250 c)

- d)

- - d)

- d)

- - d)

- d)

300 c)

- d)

- - d)

- d)

- - d)

- d)

MMC

- C; 0.5 μg/ml 65

- 71 1001

- 1019 83

With metabolic activation (+S9-mix)

3 h exposure time, 24 h fixation time

Control b) 70

- 68 1019

- 1013 100

3 62

- 71 1017

- 1014 96

30 63

- 61 1002

- 1001 90

100 59

- 46 1000

- 1001 76

150 34

- 32 1001

- 1000 48

20 0 c) 10

- 9 1011

- 1001 14

250 c)

- d)

- - d)

- d)

- - d)

- d)

300 c)

- d)

- - d)

- d)

- - d)

- d)

CP; 10 μg/ml 34

- 30 1008

- 1001 46

a) Duplicate cultures

b) Dimethyl sulfoxide

c) precipitated in the culture medium.

d) Cell lysis- Page 22

-

Table 3 Mitotic index of human lymphocyte cultures treated with

in cytogenetic assay 1A

Period of treatment: From 7 March, 2012 to 8 March, 2012

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) 59

- 56 1000

- 1000 100

3 57

- 54 1000

- 1000 97

50 58

- 53 1000

- 1000 97

100 23

- 27 1000

- 1000 43

120 19

- 11 1000

- 1000 26

140 c) 3

- 2 1000

- 1000 4

170 c) 0

- 0 1000

- 1000 0

MMC

- C; 0.5 μg/ml 37

- 39 1000

- 1000 66

MMC

- C; 0.75 μg/ml 26

- 30 1000

- 1000 49

a) Duplicate cultures

b) Dimethyl sulfoxide

c) precipitated in the culture medium.- Page 23

-

Table 4 Chromosome aberrations in human lymphocyte cultures treated with

in the absence of S9-mix in cytogenetic assay 1A (3 h exposure

time, 24 h fixation time)

Conc DMSO

(1.0% v/v ) 3

μ g/ml 50

μ g/ml 100

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 97 97 43 66

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) 1 0 1 0 1 1 0 0 0 2 2 4 22 23 *** )

45

No. of

Cells with

aberrations

(

- gaps) 1 0 1 0 1 1 0 0 0 2 2 4 22 22 *** )

4 4

g’ 1

g” 1 1

b’ 1 1 2 8 10

b” 1 10 7

m’ 1 1 1

m”

exch. 6 6

dic

d’

misc. poly poly r

total aberr

(+ gaps) 1 0 0 2 0 0 2 2 27 25

total aberr

(

- gaps) 1 0 0 1 0 0 2 2 26 24

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.- Page 24

-

Table 5 Chromosome aberrations in human lymphocyte cultures treated with

in the presence of S9-mix in the first cytogenetic assay (3 h

exposure time, 24 h fixation time)

Conc Conc

DMSO

(1.0% v/v) 3

μ g/ml 100

μ g/ml 150

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 48 46

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 1 1 2 2 1 3 13 22 *** )

35

No. of

Cells with

aberrations

(

- gaps) 0 0 0 0 0 0 1 1 2 1 1 2 12 18 *** )

30

g’ 1 5

g” 1 1

b’ 1 5 6

b” 1 2 9

m’ 2 3

m” 1

exch. 2 6

dic

d’ 1 1

misc. poly poly

total aberr

(+ gaps) 0 1 0 0 1 1 2 1 13 29

total aberr

(

- gaps) 0 0 0 0 1 1 1 1 12 24

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.- Page 25

-

Table 6 Mitotic index of human lymphocyte cultures treated with

in the second cytogenetic assay

Period of treatment: From 25 January, 2012 to 27 January, 2012

Number of metaphases a)

concentration (μg/ml) Absolute Number Percentage

of cells scored of control

24 h exposure time, 24 h fixation time

Without metabolic activation (- S9 -mix)

Control b) 61

- 63 1000

- 1004 100

1 59

- 61 1010

- 1016 97

10 62

- 59 1012

- 1007 98

20 56

- 57 1004

- 1003 91

30 51

- 59 1001

- 1017 89

40 54

- 54 1017

- 1000 87

50 48

- 48 1007

- 1010 77

MMC

- C; 0.2 μg/ml 30

- 28 1011

- 1007 47

48 h exposure time, 48 h fixation time

Control b) 51

- 55 1003

- 1001 100

1 48

- 54 1016

- 1007 96

10 50

- 48 1009

- 1004 92

20 47

- 49 1018

- 1020 91

30 44

- 42 1016

- 1010 81

40 38

- 43 1018

- 1012 76

50 31

- 35 1000

- 1006 62

MMC

- C; 0.1 μg/ml 41

- 37 1009

- 1016 74

With metabolic activation (+S9-mix)

3 h exposure time, 48 h fixation time

Control b) 56

- 58 1011

- 1005 100

3 58

- 52 1009

- 1011 96

30 56

- 52 1023

- 1013 95

100 48

- 51 1022

- 1019 87

150 36

- 3 6 1017

- 1008 63

200 c) 20

- 18 1018

- 1000 33

250 c) 4

- 0 1010

- 1001 4

300 c)

- d)

- - d)

- d)

- - d)

- d)

CP; 10 μg/ml 34

- 30 1010

- 1011

– e)

a) Duplicate cultures

b) Dimethyl sulfoxide

c) precipitated in the culture medium

d) Cell lysis

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

of control.- Page 26

-

Table 7 Mitotic index of human lymphocyte cultures treated with

in cytogenetic assay 2A

Period of treatment: From 15 February, 2012 to 17 February, 2012

Number of metaphases a)

concentration (μg/ml) Absolute Number Percentage

of cells scored of control

24 h exposure time, 24 h fixation time

Without metabolic activation (- S9 -mix)

Control b) 48

- 47 1000

- 1000 100

10 49

- 48 1000

- 1000 102

25 42

- 43 1000

- 1000 89

50 43

- 44 1000

- 1000 92

60 42

- 38 1000

- 1000 84

70 30

- 35 1000

- 1000 68

80 16

- 16 1000

- 1000 34

90 15

- 15 1000

- 1000 32

100 8

- 10 1000

- 1000 19

MMC

- C; 0.2 μg/ml 35

- 30 1000

- 1000 68

48 h exposure time, 48 h fixation time

Control b) 24

- 27 1000

- 1000 100

10 17

- 25 1000

- 1000 82

25 28

- 25 1000

- 1000 104

50 25

- 24 1000

- 10 00 96

60 24

- 24 1000

- 1000 94

70 13

- 14 1000

- 1000 53

80 11

- 8 1000

- 1000 37

90 9

- 6 1000

- 1000 29

100 8

- 5 1000

- 1000 25

MMC

- C; 0.1 μg/ml 34

- 34 1000

- 1000 133

a) Duplicate cultures

b) Dimethyl sulfoxide- Page 27

-

Table 7 - CONTINUED

Number of metaphases a)

concentration (μg/ml) Absolute Number Percentage

of cells scored of control

With metabolic activation (+S9-mix)

3 h exposure time, 48 h fixation time

Control b) 45

- 39 1000

- 1000 100

1 00 46

- 37 1000

- 1000 99

125 40

- 40 1000

- 1000 95

150 40

- 41 1000

- 1000 96

160 32

- 39 1000

- 1000 85

170 c) 27

- 27 1000

- 1000 64

180 c) 26

- 25 1000

- 1000 61

190 c) 31

- 31 1000

- 1000 74

200 c) 16

- 20 1000

- 1000 43

C P; 10 μg/ml 50

- 50 1000

- 1000

– d)

a) Duplicate cultures

b) Dimethyl sulfoxide

c) precipitated in the culture medium

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

of control.- Page 28

-

Table 8 Chromosome aberrations in human lymphocyte cultures treated with

in the absence of S9-mix in cytogenetic assay 2A (24 h exposure

time, 24 h fixation time)

Conc DMSO

(1.0% v/v) 50

μ g/ml 70

μ g/ml 80

μ g/m l 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 92 68 34 68

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 1 1 0 0 0 19 13 *** )

32

No. of

Cells with

aberrations

(

- gaps) 0 1 1 0 0 0 0 0 0 0 0 0 19 13 *** )

32

g’ 1

g”

b’ 5 6

b” 1 7 4

m’

m”

exch. 9 3

dic

d’

misc.

total aberr

(+ gaps) 0 1 0 0 0 1 0 0 21 13

total aberr

(

- gaps) 0 1 0 0 0 0 0 0 21 13

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.- Page 29

-

Table 9 Chromosome aberrations in human lymphocyte cultures treated with

in the absence of S9-mix in cytogenetic assay 2A (48 h exposure

time, 48 h fixation time)

Conc DMSO

(1.0% v/v) 50

μ g/ml 60

μ g/ml 80

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 96 94 37 133

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) 1 0 1 0 0 0 0 0 0 0 0 0 21 26 *** )

47

No. of

Cells with

aberrations

(

- gaps) 1 0 1 0 0 0 0 0 0 0 0 0 20 26 *** )

46

g’ 1

g”

b’ 6 3

b” 1 9 17

m’

m”

exch. 8 7

dic

d’

misc.

total aberr

(+ gaps ) 1 0 0 0 0 0 0 0 24 27

total aberr

(

- gaps) 1 0 0 0 0 0 0 0 23 27

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.- Page 30

-

Table 10 Chromosome aberrations in human lymphocyte cultures treated with

in the presence of S9 -mix in cytogenetic assay 2A (3 h exposure

time, 48 h fixation time)

Conc DMSO

(1.0% v/v) 150

μ g/ml 160

μ g/ml 200

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 85 43

- b)

No. of

Cells

score d 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 0 0 1 0 1 0 2 2 14 16 *** )

30

No. of

Cells with

aberrations

(

- gaps) 0 0 0 0 0 0 1 0 1 0 0 0 13 16 *** )

29

g’ 2 1

g”

b’ 8 6

b” 1 4 9

m’

m”

exch. 2 4

dic

d’

misc.

total aberr

(+ gaps) 0 0 0 0 1 0 0 2 15 19

total aberr

(

- gaps) 0 0 0 0 1 0 0 0 14 19

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.- Page 31

-

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 br eak 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 centromeres.

Tricentric tr ic A chromosome containing three

chromosome centromeres.- Page 32

-

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

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

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.- Page 33

-

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.- Page 34

-

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

- 6 0

- 6 0

- 5 0

- 5

Mean 0.8 0.8 0.6 0.6

SD 1.1 1.0 0.9 0.9

n 402 587 405 597

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between January 2009 and December 2011.- Page 35

-

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

- 2 0

- 2 0

- 1

Mean 0.06 0.08 0.03 0.01

SD 0.25 0.31 0.20 0.10

n 414 606 420 61 2

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between January 2009 and December 2011.