FINAL REPORT

Study Title

EVALUATION OF THE ABILITY OF TO 

INDUCE CHROMOSOME ABERRATIONS IN CULTURED 

PERIPHERAL HUMAN LYMPHOCYTES 

(WITH REPEAT EXPERIMENT)

Author

Test Facility

 

Laboratory Project Identification

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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

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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 time)................................................................................................. 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

Table 6 Mitotic index of human lymphocyte cultures treated with in 

the second cytogenetic assay ......................................................................................25

Table 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

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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. 

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5. INTRODUCTION 

5.1. Preface 

Sponsor

Study Monitor

(on behalf of the sponsor)

Test Facility

Study Director

Study Plan Start : 02 December 2011

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. 

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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 

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6.1.2. Study specific test substance information 

Test substance handling Flush container with nitrogen after handling

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 

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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% CO2 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 CO2 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 CO2 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 Na2-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. 

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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 MgCl2.6H2O (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. 

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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). 

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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 

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. 

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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

 X2 = --------------------------------

 (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. 

 n0 = 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. 

 n1 = the total number of cells scored in the treated cultures. 

 N = sum of n0 and n1

 (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. 

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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, 1401) and 1701) 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: 

Final Report
- 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, Table 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). 

Final Report
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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, Table 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. 

Final 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. 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. 

Final Report
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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. 

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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. 

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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 metaphasesa)

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 - 1009 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

200

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 

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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 metaphasesa)

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. 

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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

μg/ml

50

μg/ml

100

μg/ml

MMC-C

0.5 μg/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

***)

44

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) was not counted as an aberration. 

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

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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

μg/ml

100

μg/ml

150

μg/ml

CP

10 μg/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. 

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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 metaphasesa)

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 - 36 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. 

Final Report
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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 metaphasesa)

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 - 1000 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 

Final Report
- Page 27 - 

Table 7 - CONTINUED 

 Number of metaphasesa)

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

100 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

CP; 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. 

Final Report
- 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

μg/ml

70

μg/ml

80

μg/ml

MMC-C

0.2 μg/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. 

Final Report
- 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

μg/ml

60

μg/ml

80

μg/ml

MMC-C

0.1 μg/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. 

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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

μg/ml

160

μg/ml

200

μg/ml

CP

10 μg/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 

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 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. 

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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 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 tric A chromosome containing three 

chromosome centromeres. 

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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. 

Final Report
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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. 

Final Report
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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. 

Final Report
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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 414 606 420 612

SD = Standard deviation 

n = Number of observations 

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

Final Report