FINAL REPORT

Study Title

EVALUATION OF THE ABILITY OF FRET 09-0536 TO INDUCE 

CHROMOSOME ABERRATIONS IN CULTURED PERIPHERAL 

HUMAN LYMPHOCYTES (WITH REPEAT EXPERIMENT)

Author

I.A.J. Verbaan, PhD.

Test Facility

WIL Research Europe B.V.

Hambakenwetering 7

5231 DD ‘s-Hertogenbosch

The Netherlands

 

Laboratory Project Identification

Project 504372

Substance 205292/A

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FRET 09-0536 Project 504372 

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

6.1.3. Test substance preparation ............................................................................................ 9

6.2. Reference substances .............................................................................................................. 9

6.2.1. Negative control .............................................................................................................. 9

6.2.2. Positive controls .............................................................................................................. 9

6.3. Test system ............................................................................................................................... 9

6.4. Cell culture ................................................................................................................................ 9

6.5. Metabolic activation system ....................................................................................................10

6.5.1. Preparation of S9-fraction .............................................................................................10

6.5.2. Preparation of S9-mix ...................................................................................................10

6.6. Study design ...........................................................................................................................11

6.6.1. Dose range finding test .................................................................................................11

6.6.2. Cytogenetic assay.........................................................................................................11

6.6.3. Chromosome preparation .............................................................................................12

6.6.4. Preparation of slides .....................................................................................................12

6.6.5. Mitotic index/dose selection for scoring of the cytogenetic assay ................................12

6.6.6. Analysis of slides for chromosome aberrations ............................................................12

6.7. Electronic data capture ...........................................................................................................13

6.8. Interpretation ...........................................................................................................................13

6.8.1. Acceptability of the assay .............................................................................................13

6.8.2. Data evaluation and statistical procedures ...................................................................13

6.9. List of deviations .....................................................................................................................14

6.9.1. List of protocol deviations .............................................................................................14

6.9.2. List of standard operating procedures deviations .........................................................14

7. RESULTS .......................................................................................................................................14

7.1. Dose range finding test ...........................................................................................................14

7.2. First cytogenetic assay ............................................................................................................14

7.3. Second cytogenetic assay ......................................................................................................15

7.4. Evaluation of the results ..........................................................................................................16

8. CONCLUSION ................................................................................................................................16

9. REFERENCES ...............................................................................................................................17

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FRET 09-0536 Project 504372 

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TABLES 

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

dose range finding test .................................................................................................18

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

cytogenetic assay .........................................................................................................20

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

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

24 h fixation time)..........................................................................................................21

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

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

24 h fixation time)..........................................................................................................22

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

second cytogenetic assay .............................................................................................23

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

cytogenetic assay 2A ....................................................................................................24

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

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

time, 24 h fixation time) .................................................................................................25

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

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

time, 48 h fixation time) .................................................................................................26

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

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

time, 48 h fixation time) .................................................................................................27

APPENDICES 

APPENDIX 1 TABLES ........................................................................................................................18

APPENDIX 2 DEFINITIONS OF CHROMOSOME ABERRATIONS SCORED IN METAPHASE 

PORTRAITS .................................................................................................................28

APPENDIX 3 STATISTICAL EVALUATION OF THE TEST RESULTS .............................................30

APPENDIX 4 HISTORICAL CONTROL DATA FOR LYMPHOCYTE CHROMOSOME 

ABERRATION STUDIES ..............................................................................................31

APPENDIX 5 HISTORICAL CONTROL DATA FOR NUMERICAL ABERRATIONS FOR 

LYMPHOCYTE CHROMOSOME ABERRATION STUDIES ........................................32

APPENDIX 6 CERTIFICATE OF ANALYSIS ......................................................................................33

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4. SUMMARY 

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

human lymphocytes (with repeat experiment). 

This report describes the effect of FRET 09-0536 on the number of chromosome aberrations in 

cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system 

(phenobarbital and ß-naphthoflavone induced rat liver S9-mix). The possible clastogenicity of FRET 

09-0536 was tested in two independent experiments. 

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

guidelines. 

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

was dissolved in ethanol. 

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

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

Appropriate toxicity was reached at these dose levels. 

In the second cytogenetic assay, FRET 09-0536 was tested up to 45 μg/ml for a 24 h continuous 

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

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

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

dose levels. 

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

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

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

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

activation system (S9-mix) functioned properly. 

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

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

independently repeated experiments. 

No effects of FRET 09-0536 on the number of polyploid cells and cells with endoreduplicated 

chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be 

concluded that FRET 09-0536 does not disturb mitotic processes and cell cycle progression and does 

not induce numerical chromosome aberrations under the experimental conditions described in this 

report. 

Finally, it is concluded that this test is valid and that FRET 09-0536 is not clastogenic in human 

lymphocytes under the experimental conditions described in this report. 

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5. INTRODUCTION 

5.1. Preface 

Sponsor International Flavors & Fragrances Inc.

800 Rose Lane

Union Beach, New Jersey 07735

U.S.A.

Study Monitor Vijyeta Kapur

Senior Regulatory Chemical Control Specialist

Test Facility WIL Research Europe B.V.

Hambakenwetering 7

5231 DD ‘s-Hertogenbosch

The Netherlands

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

Study Plan Start : 17 January, 2014

Completion : 06 April, 2014

5.2. Aims of the study 

The objective of this study was to evaluate FRET 09-0536 for its ability to induce structural 

chromosome aberrations in cultured human lymphocytes (1), either in the presence or absence of a 

metabolic activation system (S9-mix). 

Background of the test system 

Whole blood samples obtained from healthy male subjects were treated with an anti-coagulant 

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

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

of chemicals (1-5). 

The stimulated lymphocytes were exposed to FRET 09-0536 both in the absence and presence of a 

metabolic activation system (S9-mix). In combination with this metabolic activation system indirect 

chemical mutagens, i.e. those requiring metabolic transformation into reactive intermediates, can be 

tested for possible clastogenic effects in vitro. 

At predetermined intervals after exposure of the stimulated human lymphocytes to FRET 09-0536, cell 

division was arrested in the metaphase stage of the cell cycle by addition of the metaphase-arresting 

chemical colchicine. Cells were harvested, stained and metaphase cells were analysed for the 

presence of structural chromosome aberrations such as breaks, gaps, minutes, dicentrics and 

exchange figures. Results from cultures treated with FRET 09-0536 were compared with control 

(vehicle) treated cultures. 

Chromosome aberrations were generally evaluated in the first post-exposure mitosis (i.e. 24 hours 

after exposure). However, since the appearance of the first post-exposure mitosis could be 

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

to cover the interval in which maximum aberration frequency was 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, will be retained in the WIL Research 

Europe archives for a period of at least 2 years after finalization of the report. After this period, the 

sponsor will be contacted to determine how the records and materials should be handled. WIL 

Research Europe will retain information concerning decisions made. 

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

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

finalization of the report. 

WIL Research Europe will retain a test substance sample until the expiry date, but no longer than 

10 years after finalization of the report. After this period the sample will be destroyed. 

6. MATERIALS AND METHODS 

6.1. Test substance 

6.1.1. Test substance information 

Identification FRET 09-0536

Structure

H

H

O

+

H

H O

Molecular formula C13H20O

Molecular weight 192.15

CAS Number 1464989-16-9

Description Clear colourless liquid

Batch RDIA5595

Purity/Composition 89.6%

Test substance storage In refrigerator (2-8°C) in the dark under nitrogen

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

See APPENDIX 6; Certificate of analysis. 

6.1.2. Study specific test substance information 

Purity/composition correction factor 

required

No

Volatile Yes, vapour pressure: 0.024 Pa at 298K

Reactivity Reactive to oxygen

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Test substance handling. Flush container with nitrogen after handling

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

Stability in vehicle Ethanol: Not indicated

Solubility in vehicle Ethanol: Not indicated

6.1.3. Test substance preparation 

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

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

concentrations were used within 2.5 hours after preparation. 

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

6.2. Reference substances 

6.2.1. Negative control 

The vehicle for the test substance was ethanol. 

6.2.2. Positive controls 

Without metabolic activation (-S9-mix): 

Mitomycin C (MMC-C; CAS no. 50-07-7, Sigma, Zwijndrecht, The Netherlands) was used as a direct 

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

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

With metabolic activation (+S9-mix): 

Cyclophosphamide (CP; CAS no. 50-18-0. Baxter B.V., Utrecht, The Netherlands) was used as an 

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

exposure period (24 h fixation time). 

Solvent for positive controls 

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

calcium and magnesium. 

All reference stock solutions were stored in aliquots at ≤-15°C in the dark. These solutions were 

thawed immediately before use. 

6.3. Test system 

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

recommended in international guidelines (e.g. OECD, EC). 

Blood was 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 2013) are 

presented below: 

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

First cytogenetic assay: age 36, AGT = 12.9 h 

Second cytogenetic assay: age 31, AGT = 12.9 h 

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

6.4. Cell culture 

Blood samples 

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

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

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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 42 - 91%), containing 5.0 ± 0.5% CO2 in air, at a 

temperature of 37.0 ± 1.0°C (actual range 34.1 - 37.6°C). Temperature and humidity were 

continuously monitored throughout the experiment. The CO2 percentage was monitored once on each 

working day. Temporary deviations from the temperature (in the range of 34.1 - 36.0°C), humidity (with 

a maximum of 30%) 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 2 hours. 

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

The temporary deviations from the humidity with a minimum of 42% are explained in protocol 

deviation 1. 

6.5. Metabolic activation system 

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

obtained from Charles River (Sulzfeld, Germany). 

6.5.1. Preparation of S9-fraction 

The animals were housed at WIL Research Europe in a special room under standard laboratory 

conditions, as described in the Standard Operating Procedures, and allowed to acclimatise for at least 

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

(80 mg/kg body weight; Bufa B.V., IJsselstein, The Netherlands) and ß-naphthoflavone (100 mg/kg 

body weight; Sigma) in corn oil (Fagron Farmaceuticals, Capelle a/d IJssel, the Netherlands) (they 

were denied access to food for 3 to 4 hours preceding each dosing). One day after the final exposure 

(24 h), the rats were sedated using oxygen/carbon dioxide and then killed by decapitation. The rats 

received a limited quantity of food during the night before sacrifice. The livers of the rats were removed 

aseptically, and washed in cold (0°C), sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 

0.1 mM 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. 

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 

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(Invitrogen Corporation). 

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

S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix. 

Metabolic activation was achieved by adding 0.2 ml S9-mix to 5.3 ml of a lymphocyte culture 

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

concentration of the S9-fraction in the exposure medium was 1.8% (v/v). 

6.6. Study design 

6.6.1. Dose range finding test 

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

were obtained in a dose range finding test. FRET 09-0536 was tested in the absence and in the 

presence of 1.8% (v/v) S9-fraction. 

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

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

cultured for 48 h and thereafter exposed to selected doses of FRET 09-0536 for 3 h, 24 h and 48 h in 

the absence of S9-mix or for 3 h in the presence of S9-mix. A negative control was included at each 

exposure time. 

The highest tested concentration was determined by the solubility of FRET 09-0536 in the culture 

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

beyond the limit of solubility to obtain adequate toxicity data. 

After 3 h exposure to FRET 09-0536 in the absence or presence of S9-mix, the cells were separated 

from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells 

were rinsed with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were 

resuspended in 5 ml culture medium and incubated for another 20 - 22 h (24 h fixation time). The cells 

that were exposed for 24 h and 48 h in the absence of S9-mix were not rinsed after exposure but were 

fixed immediately (24 h and 48 h fixation time). 

Cytotoxicity of FRET 09-0536 in the lymphocyte cultures was determined using the mitotic index. 

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

the cytogenetic assays considering the highest dose level had an inhibition of the mitotic index of 50% 

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

mitotic index of the solvent control. 

6.6.2. Cytogenetic assay 

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

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

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

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

First cytogenetic assay 

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

FRET 09-0536 for 3 h in the absence and presence of S9-mix. After 3 h exposure, the cells were 

separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed 

and the cells were rinsed once with 5 ml HBSS. After a second centrifugation step, HBSS was 

removed and cells were resuspended in 5 ml culture medium and incubated for another 20 - 22 h 

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

assay.

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

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

with the following modifications of experimental conditions. 

Second cytogenetic assay 

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

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

After 3 h exposure, the cells exposed to FRET 09-0536 in the presence of S9-mix were separated 

from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and the 

cells were rinsed once with 5 ml of HBSS and incubated in 5 ml culture medium for another 44 - 46 h 

(48 h fixation time). 

The cells that were treated for 24 h and 48 h in the absence of S9-mix were not rinsed after exposure 

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

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

6.6.3. Chromosome preparation 

During the last 2.5 - 3 h of the culture period, cell division was arrested by the addition of the spindle 

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

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

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

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

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

6.6.4. Preparation of slides 

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

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

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

per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa 

(Merck) solution in Sörensenbuffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. 

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

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

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

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

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

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

scoring of the cytogenetic assay. Chromosomes of metaphase spreads were analysed from those 

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

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

treated with an intermediate dose were examined for chromosome aberrations. 

6.6.6. Analysis of slides for chromosome aberrations 

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

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

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

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

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

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

and the number of aberrations were calculated. 

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6.7. Electronic data capture 

Observations/measurements in the study were recorded electronically using the following 

programme(s): 

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

USA): temperature and humidity. 

6.8. Interpretation 

6.8.1. Acceptability of the assay 

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

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

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

b) The positive control substances should produce a statistically significant (Chi-square test, onesided, p < 0.05) increase in the number of cells with chromosome aberrations. 

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

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

aberrations. 

6.8.2. Data evaluation and statistical procedures 

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

a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in 

the number of cells with chromosome aberrations. 

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

with chromosome aberrations was observed in the absence of a clear dose-response relationship. 

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

of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) 

increase in the number of cells with chromosome aberrations. 

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

decision. 

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

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

to that of the solvent control using Chi-square statistics: 

 (N-1) (ad-bc)2

 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 X2 > ------------------------------- (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 

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

6.9. List of deviations 

6.9.1. List of protocol deviations 

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

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

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

closing of the incubator door during handling of the cell cultures.The dose range-finding test 

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

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

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

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

The study integrity was not adversely affected by the deviation. 

6.9.2. List of standard operating procedures deviations 

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

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

7. RESULTS 

7.1. Dose range finding test 

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

finding study, at the 3 h exposure time, blood cultures were treated with 3, 10, 33, 100 and 333 μg 

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

time blood cultures were treated with 3, 10, 33, 100, 333, 1000 and 1922 μg FRET 09-0536/ml culture 

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

toxicity data. 

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

concentrations or with the negative control substance. 

7.2. First cytogenetic assay 

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

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

for the cytogenetic assay: 

Without and with S9-mix : 30, 50, 70, 100, 150, 200, 250 and 300 μg/ml culture medium 

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

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

concentrations or with the positive or negative control substances. 

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

Without S9-mix : 30, 50 and 70 μg/ml culture medium 

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

With S9-mix : 30, 70 and 100 μg/ml culture medium 

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

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

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

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Table 3, Table 4).

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

cells and cells with endoreduplicated chromosomes. 

7.3. Second cytogenetic assay 

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

assay was performed in which human lymphocytes were continuously exposed to FRET 09-0536 in 

the absence of S9-mix for 24 or 48 hours. In the presence of S9-mix, cells were fixed after 48 hours 

following a 3 hour exposure to FRET 09-0536. The following dose levels were selected for the second 

cytogenetic assay: 

Without S9-mix : 10, 30, 50, 70 and 100 μg/ml culture medium 

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

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

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

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

concentrations or with the positive or negative control substances. 

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

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

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

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

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

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

The following dose levels were selected: 

Without S9-mix : 1, 10, 30, 35, 40, 45 and 50 μg/ml culture medium 

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

With S9-mix : 50, 100, 110, 120, 130 and 140 μg/ml culture medium 

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

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

concentrations or with the positive or negative control substances. 

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

aberrations: 

Without S9-mix : 1, 30 and 45 μg/ml culture medium 

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

 1, 30 and 50 μg/ml culture medium 

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

With S9-mix : 50, 110 and 130 μg/ml culture medium 

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

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

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

Table 7 - 8). 

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

cells and cells with endoreduplicated chromosomes. 

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7.4. Evaluation of the results 

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

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

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

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

control substances are presented in Table 1, Table 2 and Table 5 - 6 (APPENDIX 1). The scores for 

the number of aberrant cells (gaps included and excluded) and the number of the various types of 

chromosome aberrations at the various concentrations of FRET 09-0536 are presented in Table 3,

Table 4 and Table 7 - 9 (APPENDIX 1). Duplicate cultures are indicated by A and B. The criteria 

according to which the aberrations were classified are outlined in APPENDIX 2. APPENDIX 3 presents 

the statistical evaluations of the test results. 

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

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

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

control data range (see APPENDIX 5). The positive control chemicals (MMC-C and CP) both 

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

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

functioned properly. 

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

biologically relevant increase in the number of cells with chromosome aberrations in two independent 

experiments. 

No effects of FRET 09-0536 on the number of polyploid cells and cells with endoreduplicated 

chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be 

concluded that FRET 09-0536 does not disturb mitotic processes and cell cycle progression and does 

not induce numerical chromosome aberrations under the experimental conditions described in this 

report. 

8. CONCLUSION 

Finally, it is concluded that this test is valid and that FRET 09-0536 is not clastogenic in human 

lymphocytes under the experimental conditions described in this report. 

Final Report
FRET 09-0536 Project 504372 

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9. REFERENCES 

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

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

London, PlenumPress), 1 - 29.

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

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

M. Legator, W. Nichols and C. Ramel, 405 - 427, Elsevier Science Publishers B.V., Amsterdam. 

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

nitro-N-nitrosoguanidine in mammalian cells. In Vitro 7: 359 - 365. 

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

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

Program. Mutat Res 87: 143 - 188. 

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

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

Mutat Res 312: 241 - 261. 

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

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

364.

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

cultured mammalian cells. Mutat Res 268: 297 - 305. 

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APPENDIX 1 TABLES 

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

dose range finding test 

FRET 09-0536 Number of metaphases 

concentration (μg/ml) Absolute Number Percentage 

of cells scored of control 

Without metabolic activation (-S9-mix) 

3 h exposure time, 24 h fixation time 

Control a) 50 1005 100

3 53 1008 106

10 57 1048 114

33 49 1047 98

100 16 1036 32

333 b) c) c) c)

24 h exposure time, 24 h fixation time 

Control a) 52 1028 100

3 40 1043 77

10 43 1006 83

33 41 1023 79

100 5 1026 10

333 b) c) c) c)

1000 b) c) c) c)

1922 b) c) c) c)

48 h exposure time, 48 h fixation time 

Control a) 49 1040 100

3 44 1014 90

10 56 1004 114

33 38 1007 78

100 5 1026 10

333 b) c) c) c)

1000 b) c) c) c)

1922 b) c) c) c)

a) Ethanol 

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

c) Cell lysis 

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Table 1 – continued –

FRET 09-0536 Number of metaphases 

concentration (μg/ml) Absolute Number Percentage 

of cells scored of control 

With metabolic activation (+S9-mix) 

3 h exposure time, 24 h fixation time 

Control a) 41 1005 100

3 46 1005 112

10 34 1008 83

33 48 1028 117

100 34 1004 83

333 b) c) c) c)

a) Ethanol 

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

c) Cell lysis 

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

first cytogenetic assay 

FRET 09-0536 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) 71 - 69 1018 - 1021 100

30 70 - 63 1006 - 1025 95

50 59 - 50 1010 - 1018 78

70 33 - 22 1005 - 1015 39

100 10 - 13 1002 - 1006 16

150 0 - 0 1000 - 1000 0

200 c) 0 - 0 1001 - 1001 0

250 c) 0 - 0 1003 - 1001 0

300 c) 0 - 0 1011 - 1002 0

MMC-C; 0.5 μg/ml 52 - 50 1017 - 1040 73

MMC-C; 0.75 μg/ml 29 - 30 1011 - 1015 42

With metabolic activation (+S9-mix) 

3 h exposure time, 24 h fixation time 

Control b) 71 - 66 1023 - 1015 100

30 60 - 71 1017 - 1012 96

50 52 - 54 1009 - 1044 77

70 54 - 50 1003 - 1007 76

100 27 - 28 1008 - 1006 40

150 14 - 11 1006 - 1022 18

200 c) 0 - 0 1002 - 1000 0

250 c) 0 - 0 1001 - 1001 0

300 c) 0 - 0 1001 - 1000 0

CP; 10 μg/ml 29 - 36 1010 - 1026 47

a) Duplicate cultures 

b) Ethanol 

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

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

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

24 h fixation time) 

Conc Ethanol

(0.5% v/v)

30

μg/ml

50

μg/ml

70

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 95 78 39 73

No. of

Cells 

scored

100 100 200 100 100 200 100 100 200 100 100 200 100 100 200

No. of

Cells with

aberrations

(+ gaps) a)

1 0 1 0 1 1 0 0 0 2 1 3 29 30

***

)

59

No. of

Cells with

aberrations

(- gaps)

1 0 1 0 0 0 0 0 0 2 1 3 29 30

***

)

59

g’ 1

g”

b’ 1 3 1 27 25

b” 2 1

m’

m”

exch. 8 8

dic

d’

misc. poly

total aberr

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

total aberr

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

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

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

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

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

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

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

24 h fixation time) 

Conc Conc Ethanol

(0.5% v/v)

30

μg/ml

70

μg/ml

100

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 40 47

No. of

Cells 

scored

100 100 200 100 100 200 100 100 200 100 100 200 100 100 200

No. of

Cells with

aberrations

(+ gaps) a)

0 1 1 0 0 0 0 3 3 0 2 2 23 24

***

)

47

No. of

Cells with

aberrations

(- gaps)

0 0 0 0 0 0 0 2 2 0 2 2 23 23

***

)

46

g’ 1 1 4

g”

b’ 2 1 21 25

b” 3 1

m’

m”

exch. 1 2 3

dic

d’

misc. poly

total aberr

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

total aberr

(- gaps) 0 0 0 0 0 2 0 2 26 29

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

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

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

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

Final Report
FRET 09-0536 Project 504372 

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

second cytogenetic assay 

FRET 09-0536 Number of metaphasesa)

concentration (μg/ml) Absolute Number Percentage 

of cells scored of control 

Without metabolic activation (-S9-mix) 

24 h exposure time, 24 h fixation time 

Control b) 92 - 86 1003 - 1000 100

10 65 - 55 1000 - 1000 67

30 58 - 51 1001 - 1000 61

50 34 - 21 1000 - 1004 31

70 19 - 16 1000 - 1000 20

100 11 - 6 1002 - 1001 10

MMC-C; 0.2 μg/ml 41 - 45 1003 - 1006 48

MMC-C; 0.3 μg/ml 25 - 24 1000 - 1000 28

48 h exposure time, 48 h fixation time 

Control b) 78 - 76 1010 - 1000 100

10 77 - 79 1002 - 1001 101

30 48 - 43 1000 - 1000 59

50 29 - 24 1000 - 1001 34

70 4 - 5 1001 - 1000 6

100 0 - 0 1000 - 1000 0

MMC-C; 0.1 μg/ml 32 - 40 1000 - 1003 47

MMC-C; 0.15 μg/ml 33 - 27 1002 - 1001 39

With metabolic activation (+S9-mix) 

3 h exposure time, 48 h fixation time 

Control b) 89 - 82 1000 - 1000 100

30 83 - 87 1000 - 1002 99

50 90 - 82 1002 - 1002 101

70 83 - 89 1000 - 1006 101

100 82 - 68 1003 - 1002 88

150 6 - 7 1001 - 1003 8

200 c) 0 - 0 1000 - 1000 0

250 c) e)-

e) e)

-

e) e)

300 c) e)-

e) e)

-

e) e)

CP; 10 μg/ml 26 - 29 1000 - 1000 –

d)

a) Duplicate cultures

b) Ethanol 

c) FRET 09-0536 precipitated in the culture medium 

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

control. 

e) Cell lysis 

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

cytogenetic assay 2A 

FRET 09-0536 Number of metaphasesa)

concentration (μg/ml) Absolute Number Percentage 

of cells scored of control 

Without metabolic activation (-S9-mix) 

24 h exposure time, 24 h fixation time 

Control b) 72 - 70 1010 - 1003 100

1 61 - 56 1014 - 1014 82

10 60 - 59 1005 - 1016 84

30 54 - 45 1003 - 1006 70

35 38 - 38 1007 - 1006 54

40 38 - 39 1007 - 1012 54

45 39 - 30 1030 - 1007 49

50 20 - 21 1020 - 1027 29

MMC-C; 0.2 μg/ml 24 - 27 1010 - 1010 36

MMC-C; 0.3 μg/ml 18 - 20 1019 - 1001 27

48 h exposure time, 48 h fixation time 

Control b) 34 - 43 1040 - 1028 100

1 40 - 39 1006 - 1017 103

10 24 - 24 1008 - 1024 62

30 29 - 20 1013 - 1008 64

35 23 - 24 1003 - 1025 61

40 21 - 21 1015 - 1011 55

45 19 - 22 1009 - 1009 53

50 15 - 14 1009 - 1014 38

MMC-C; 0.1 μg/ml 26 - 29 1003 - 1001 71

MMC-C; 0.15 μg/ml 17 - 18 1013 - 1015 45

With metabolic activation (+S9-mix) 

3 h exposure time, 48 h fixation time 

Control b) 57 - 53 1037 - 1015 100

50 69 - 55 1034 - 1012 113

100 40 - 36 1017 - 1013 69

110 36 - 35 1002 - 1018 65

120 35 - 30 1036 - 1005 59

130 25 - 22 1016 - 1003 43

140 23 - 25 1018 - 1007 44

CP; 10 μg/ml 29 - 28 1020 - 1002 –

c)

a) Duplicate cultures

b) Ethanol 

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

control. 

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

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

time, 24 h fixation time) 

Conc Ethanol

(0.5% v/v)

1

μg/ml

30

μg/ml

45

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 82 70 49 36

No. of

Cells 

scored

100 100 200 100 100 200 100 100 200 100 100 200 100 100 200

No. of

Cells with

aberrations

(+ gaps) a)

0 1 1 0 0 0 1 1 2 0 0 0 20 28

***

)

48

No. of

Cells with

aberrations

(- gaps)

0 1 1 0 0 0 0 0 0 0 0 0 20 27

***

)

47

g’ 1 1 2

g”

b’ 8 14

b” 1 2 9

m’

m”

exch. 10 11

dic

d’

misc.

total aberr

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

total aberr

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

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

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

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

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

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

time, 48 h fixation time)

Conc Ethanol

(0.5% v/v)

1

μg/ml

30

μg/ml

50

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 (%) 100 103 64 38 71

No. of

Cells 

scored

100 100 200 100 100 200 100 100 200 100 100 200 100 100 200

No. of

Cells with

aberrations

(+ gaps) a)

2 1 3 0 0 0 1 1 2 0 1 1 25 31

***

)

56

No. of

Cells with

aberrations

(- gaps)

1 1 2 0 0 0 0 1 1 0 1 1 24 31

***

)

55

g’ 1 1 2

g”

b’ 1 1 12 11

b” 1 1 17 13

m’

m”

exch. 6 15

dic

d’

misc.

total aberr

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

total aberr

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

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

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

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

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

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

time, 48 h fixation time) 

Conc Ethanol

(0.5% v/v)

50

μg/ml

110

μg/ml

130

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 (%) 100 113 65 43 -

b)

No. of

Cells 

scored

100 100 200 100 100 200 100 100 200 100 100 200 100 100 200

No. of

Cells with

aberrations

(+ gaps) a)

0 0 0 0 1 1 1 1 2 1 0 1 36 22

***

)

58

No. of

Cells with

aberrations

(- gaps)

0 0 0 0 1 1 0 1 1 1 0 1 34 22

***

)

56

g’ 1 3

g” 1

b’ 13 15

b” 1 1 1 12 4

m’

m”

exch. 13 8

dic

d’

misc.

total aberr

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

total aberr

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

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

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

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

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

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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 trior more-armed configuration. 

Chromosome intra A chromosome intrachange is scored 

intrachange after rejoining of a lesion within one 

chromosome. 

Pulverized p A fragmented or pulverized chromosome 

chromosomes 

Multiple ma A metaphase spread containing ten or 

aberrations more of the above mentioned aberrations 

(chromatid and chromosome gaps not 

included). ma is counted as 10 aberrations. 

Polyploidy poly A chromosome number that is a multiple of the 

normal diploid number. 

Endoreduplication endo A form of polyploidy in which each centromere 

connects two or four pairs of chromatids instead 

of the normal one pair. 

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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. 

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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 - 5 0 - 5 0 - 5 0 - 4

Mean 0.8 0.7 0.7 0.6

SD 1.0 1.0 0.9 1.0

n 378 558 382 541

SD = Standard deviation 

n = Number of observations 

Historical control data from experiments performed between September 2010 and September 2013. 

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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 - 3 0 - 2 0 - 1

Mean 0.09 0.10 0.02 0.03

SD 0.31 0.36 0.15 0.16

n 329 470 335 475

SD = Standard deviation 

n = Number of observations 

Historical control data from experiments performed between September 2010 and September 2013. 

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APPENDIX 6 CERTIFICATE OF ANALYSIS 

Final Report