Endpoint study record: Genetic toxicity in 

vitro.chromosome aberration OECD 473 

Benzenesulfonyl chloride Lanxess 41201582 

Administrative Data 

Data source 

Reference 

Materials and methods 

Type of genotoxicity 

Type of study 

Test guideline 

UUID 

 

IUC5-db8f8fdc-1bf3-4529-b933-70a449e3c03a 

Dossier UUID 

 

0 

Author 

 

amorris / Harlan Laboratories Ltd. / Other / other:

Date 

 

2012-12-13 16:40:54 GMT 

Remarks 

 

 

Purpose flag robust study summary 

Study result type experimental 

result 

Study 

period 

The experimental phases of the study were performed

between 30 July 2012 and 01 November 2012. 

Reliability 1 (reliable without restriction) 

Rationale for 

reliability incl. 

deficiencies 

Study conducted to GLP and in compliance with agreed protocols, with no or minor 

deviations from standard test guidelines and/or minor methodological deficiencies, which 

do no effect the quality of the relevant results. 

Reference 

type 

Author Year Title Bibliographic 

source 

Testing 

laboratory 

Report 

no. 

Owner 

company 

Company 

study no. 

Report 

date 

study 

report 

Morris 

A 

2012 Benzenesulfonyl 

chloride: 

CHROMOSOME 

ABERRATION 

TEST IN 

HUMAN 

LYMPHOCYTES 

IN VITRO 

 

Harlan 

Laboratories 

Ltd 

Shardlow 

Business 

Park 

Shardlow 

Derbyshire 

DE72 2GD 

UK 

Telephone: 

+44 (0) 

1332 

792896 

Facsimile: 

+44 (0) 

1332 

799018 

41201582 Lanxess 

Corporation 

111 RIDC 

Park West 

Drive 

Pittsburgh 

PA 15275-

1112 

UNITED 

STATES 

OF 

AMERICA 

 

2012-

11-10 

chromosome aberration 

in vitro mammalian chromosome aberration test 

Qualifier Guideline Deviations 

according 

to 

OECD Guideline 473 (In vitro Mammalian Chromosome Aberration Test) no 

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GLP compliance 

Test materials 

Test material identity 

Test material form 

Details on test material 

Method 

Target gene 

Species/strain 

Test concentrations 

according 

to 

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test) no 

according 

to 

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test no 

according 

to 

other guideline: UK Department of Health Guidelines for Testing of Chemicals for 

Mutagenicity. 

no 

yes (incl. certificate) (The work described was performed in compliance with UK GLP standards (Schedule 1, 

Good Laboratory Practice Regulations 1999. Date of GLP Inspection 10/07/2012. Date of GLP Certificate 

30/11/2012.) 

Identifier Identity 

Common name Benzenesulfonyl chloride 

other: Clear colourless liquid 

Sponsor's identification : Benzenesulfonyl chloride 

Description : Clear colourless liquid 

Purity : 99.53% 

Batch number : KX120302.A 

Date received : 16 April 2012 

Expiry date : 12 April 2013 

Storage conditions : Room temperature in the dark 

Not applicable. 

Species/strain lymphocytes: human 

Details 

on 

mammalian 

cell 

lines 

(if 

applicable) 

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a 

volunteer who had been previously screened for 

suitability. The volunteer had not been exposed to high levels of radiation or hazardous 

chemicals and had not knowingly recently suffered from a 

viral infection. 

Additional 

strain 

characteristics 

not applicable 

Metabolic 

activation 

with and without 

Metabolic 

activation 

system 

phenobarbitone and beta-naphthoflavone induced rat liver, S9 

Preliminary toxicity test 

The dose range of test item used was 6.90, 13.80, 27.59, 55.19, 110.38, 220.75, 441.5, 883 and 1766 μg/ml. 

Experiment 1 

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Vehicle 

Controls 

Details on test system and conditions 

4-hour exposure to the test item without S9-mix followed by 20-hour culture in treatment-free media prior to cell 

harvest. The dose range of test item used 

was 13.75, 27.5, 55, 110, 220, 330 μg/ml. 

4-hour exposure to the test item with S9-mix followed by 20-hour culture in treatment-free media prior to cell 

harvest. The dose range of test item used was 13.75, 27.5, 55, 110, 220, 330 μg/ml. 

Experiment 2 

24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range of test item used 

was 3.44, 6.88, 13.75, 27.5, 55, 

110 μg/ml. 

4-hour exposure to the test item with S9-mix followed by 20-hour culture in treatment-free media prior to cell 

harvest. The dose range of test item used was 27.5, 55, 110, 165, 220, 330 μg/ml. 

- Vehicle(s)/solvent(s) used: Acetone 

- Justification for choice of solvent/vehicle: Acetone was selected as the solvent because the test material was 

readily soluble in it at the required 

concentrations. 

Negative 

controls 

no 

Solvent / 

vehicle 

controls 

yes (Acetone) 

True 

negative 

controls 

no 

Positive 

controls 

yes 

Positive 

control 

substance 

other: cyclophosphamide 

Remarks In the presence of S9 

Negative 

controls 

no 

Solvent / 

vehicle 

controls 

yes (Acetone) 

True 

negative 

controls 

no 

Positive 

controls 

yes 

Positive 

control 

substance 

mitomycin C (MMC) 

Remarks In the absence of S9 

METHOD OF APPLICATION: 

in medium 

DURATION 

- Preincubation period: 

48 hrs 

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Evaluation criteria 

Statistics 

Any other information on materials and methods incl. tables 

- Exposure duration: 

Experiment 1 - 4 hrs with and without S9. Experiment 2 - 24 hrs without S9, 4 hrs with S9. 

- Expression time (cells in growth medium): 

20 hrs for 4 hrs exposure. 

- Selection time (if incubation with a selection agent): 

Not applicable. 

- Fixation time (start of exposure up to fixation or harvest of cells): 

24 hrs. 

SELECTION AGENT (mutation assays): 

No selection agent. 

SPINDLE INHIBITOR (cytogenetic assays): 

Demecolcine 

STAIN (for cytogenetic assays): 

When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and coverslipped using 

mounting medium. 

NUMBER OF REPLICATIONS: 

Duplicate cultures 

NUMBER OF CELLS EVALUATED: 

100/culture 

DETERMINATION OF CYTOTOXICITY 

- Method: 

mitotic index - A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded 

and expressed as the mitotic 

index and as a percentage of the vehicle control value. 

-Scoring of Chromosome Damage: 

Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there 

was approximately 30% - 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 

44-48 chromosomes, any gaps, breaks or rearrangements were noted 

according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for 

mutagenicity testing. Cells with chromosome 

aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides. 

OTHER EXAMINATIONS: 

- Determination of polyploidy: 

Frequency of polyploid cells 

OTHER: 

None. 

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, 

markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest 

increases in aberration frequency a dose response relationship is generally 

required and appropriate statistical tests may be applied in order to record a positive response. 

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where 

necessary, with the concurrent vehicle control value using Fisher's Exact test. 

Cells

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been 

previously screened for suitability. The volunteer had not been exposed to high levels of radiation 

or hazardous chemicals and had not knowingly recently suffered from a viral infection. The 

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cell-cycle time for the lymphocytes from the donors used in this study was determined using 

BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third 

division metaphase cells and so calculate the average generation time (AGT). The average 

AGT for the regular donors used in this laboratory has been determined to be 

approximately 16 hours under typical experimental exposure conditions.

Cell Culture

Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with Lglutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS), at 37oC with 5% CO2

in 

humidified air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by 

the addition of phytohaemagglutinin (PHA).

Preparation of Test Item and Control Items

The test item was accurately weighed, dissolved in acetone and serial dilutions prepared. The molecular 

weight of the test item was supplied as 176.62; therefore the maximum dose level was 

1766 μg/ml, which was calculated to be equivalent to 10 mM, the maximum recommended 

dose level. The purity of the test item was 99.53% and therefore no correction was required 

when the test item formulations were prepared. There was no significant change in pH 

when the test item was dosed into media and the osmolality did not increase by more than 

50 mOsm (Scottet al1991). The pH and osmolality readings are presented in the following 

table.

- = not determined

The test item was formulated within two hours of it being applied to the test system. It is assumed that the 

formulation was stable for this duration.

No analysis was conducted to determine the homogeneity, concentration or stability of the test item 

formulation. This is an exception with regard to GLP and has been reflected in the GLP 

compliance statement.

Vehicle and positive controls were used in parallel with the test item. The positive control items were as 

follows:

In the absence of S9, mitomycin C (MMC) (Sigma, Batch No. 089K0731) was used at 0.4 and 0.2 μg/ml for 

cultures in Experiment 1 and 2 respectively. It was dissolved in Minimal Essential Medium.

In the presence of S9, cyclophosphamide (CP) (Acros, Batch No. A0302605) was used at 5 μg/ml in both 

experiments. It was dissolved in dimethyl sulphoxide.

Microsomal Enzyme Fraction

Lot Nos. PB/beta-NF S9 01/07/2012 and 16/09/2012 were prepared in-house from the livers of 

male Sprague-Dawley rats weighing approximately 250g. These had received three daily 

oral doses of a mixture of phenobarbitone (80 mg/kg) andbeta-naphthoflavone (100 mg/kg), 

prior to S9 preparation on the fourth day.

The S9 homogenate was produced by homogenising the liver in a 0.15 M KCl solution (1g liver to 3ml KCl) 

followed by centrifugation at 9000g. The protein content of the resultant supernatant was adjusted 

to 20 mg/ml using 0.15 M KCl and aliquots of the supernatant were frozen and stored in 

μg/ml 0 6.9 13.8 27.59 55.19 110.38 220.75 441.5 883 1766

pH 7.33 7.23 7.24 7.26 7.25 7.23 7.18 7.04 7.01 6.97

mOsm 411 416 421 421 422 424 402 420 411 409

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ampoules at approximately -196oC in a liquid nitrogen freezer.

Each batch of S9 has been routinely tested for its capability to activate known mutagens in the Ames test.

The above procedure was designed and conducted to cause the minimum suffering or distress to the animals 

consistent with the scientific objectives and in accordance with the Harlan Laboratories Ltd, Shardlow, UK policy 

on animal welfare and the requirements of the United Kingdom’s Animals (Scientific Procedures) Act 1986.

The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (10 to 20% (v/v)), 

MgCl2(8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and 

NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into 

culture media, was 2% in the Preliminary Toxicity Test and Experiment 1 and 1% in 

Experiment 2.

Culture Conditions

Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, 

giving, when dispensed into sterile plastic flasks for each culture:

8.05-9.15 ml MEM, 10% (FBS) 

0.1 ml Li-heparin 

0.1 ml phytohaemagglutinin 

0.65-0.75 ml heparinised whole blood

With Metabolic Activation (S9) Treatment

After approximately 48 hours incubation at 37oC, 5% CO2in humidified air, the cultures were transferred to tubes 

and centrifuged. Approximately 9 ml of the culture medium was removed, reserved, and 

replaced with the required volume of MEM (including serum) and 0.1 ml of the appropriate 

solution of vehicle control or test item was added to each culture. For the positive control, 

0.1 ml of the appropriate solution was added to the cultures. 1 ml of 20% S9 ̄mix (i.e. 2% 

final concentration of S9 in standard co-factors) was added to the cultures of the 

Preliminary Toxicity Test and of Experiment 1.

In Experiment 2, 1 ml of 10% S9-mix (i.e. 1% final concentration of S9 in standard co-factors), was added. All 

cultures were then returned to the incubator. The nominal final volume of each culture was 

10 ml.

After 4 hours at 37oC, 5% CO2in humidified air the cultures were centrifuged, the treatment medium removed by 

suction and replaced with an 8 ml wash of MEM culture medium. After a further centrifugation the wash 

medium was removed by suction and replaced with the original culture medium. The cells 

were then re-incubated for a further 20 hours at 37oC in 5% CO2in humidified air.

Without Metabolic Activation (S9) Treatment

In Experiment 1, after approximately 48 hours incubation at 37oC with 5% CO2in humidified air the cultures were 

decanted into tubes and centrifuged. Approximately 9 ml of the culture medium was removed and 

reserved. The cells were then resuspended in the required volume of fresh MEM (including 

serum) and dosed with 0.1 ml of the appropriate vehicle control, test item solution or 0.1 ml 

of positive control solution. The total volume for each culture was a nominal 10 ml.

After 4 hours at 37oC, 5% CO2in humidified air the cultures were centrifuged the treatment medium was removed 

by suction and replaced with an 8 ml wash of MEM culture medium. After a further centrifugation the 

wash medium was removed by suction and replaced with the reserved original culture 

medium. The cells were then returned to the incubator for a further 20 hours.

In Experiment 2, in the absence of metabolic activation, the exposure was continuous for 24 hours. Therefore, 

when the cultures were established the culture volume was a nominal 9.9 ml. After 

approximately 48 hours incubation the cultures were removed from the incubator and 

dosed with 0.1 ml of vehicle control, test item dose solution or 0.1 ml of positive control 

solution. The nominal final volume of each culture was 10 ml. The cultures were then 

incubated at 37oC, 5% CO2

in humidified air for 24 hours.

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The Preliminary Toxicity Test was performed using the methodology of Experiment 1 and Experiment 2 in the

absence of S9 except that positive controls were not included.

Preliminary Toxicity Test

A preliminary toxicity test was performed on cell cultures using a 4-hour exposure time with and without metabolic 

activation followed by a 20-hour recovery period, and a continuous exposure of 24 hours without metabolic 

activation. The dose range of test item used was 6.90, 13.80, 27.59, 55.19, 110.38, 220.75, 

441.5, 883 and 1766 μg/ml. Parallel flasks, containing culture medium without whole blood, 

were established for the three exposure conditions so that test item precipitate observations 

could be made. Precipitate observations were recorded at the beginning and end of the 

exposure periods.

Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, 

appropriate dose levels were selected for mitotic index evaluation. Mitotic index data was used to 

estimate test item toxicity and for selection of the dose levels for the main test.

Experiment 1

i) 4-hour exposure to the test item without S9-mix followed by 20-hour culture in 

treatment-free media prior to cell harvest. The dose range of test item used was 13.75, 

27.5, 55, 110, 220, 330 μg/ml.

ii) 4-hour exposure to the test item with S9-mix followed by 20-hour culture in treatmentfree media prior to cell harvest. The dose range of test item used was 13.75, 27.5, 55, 

110, 220, 330 μg/ml.

Experiment 2

i) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The 

dose range of test item used was 3.44, 6.88, 13.75, 27.5, 55, 110 μg/ml.

ii) 4-hour exposure to the test item with S9-mix followed by 20-hour culture in treatmentfree media prior to cell harvest. The dose range of test item used was 27.5, 55, 110, 165, 220, 

330 μg/ml.

Cell Harvest

Mitosis was arrested by addition of demecolcine (Colcemid 0.1 μg/ml) two hours before the required harvest 

time. After incubation with demecolcine, the cells were centrifuged, the culture medium was 

drawn off and discarded, and the cells resuspended in 0.075M hypotonic KCl. After 

approximately fourteen minutes (including centrifugation), most of the hypotonic solution 

was drawn off and discarded. The cells were resuspended and then fixed by dropping the 

KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was 

changed at least three times and the cells stored at approximately 4oC to ensure complete 

fixation.

Preparation of Metaphase Spreads

The lymphocytes were resuspended in several ml of fresh fixative before centrifugation and resuspension in a 

small amount of fixative. Several drops of this suspension were dropped onto clean, wet 

microscope slides and left to air dry. Each slide was permanently labelled with the 

appropriate identification data.

Staining

When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied 

using mounting medium.

Qualitative Slide Assessment

The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and 

extent of precipitation, if any, of the test item. These observations were used to select the dose 

levels for mitotic index evaluation.

Coding

The slides were coded using a computerised random number generator.

Mitotic Index

A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and 

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Results and discussions 

Test results 

Additional information on results 

expressed as the mitotic index and as a percentage of the vehicle control value.

Scoring of Chromosome Damage

Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there 

was approximately 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell 

had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the 

simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for 

mutagenicity testing (Appendix 1). Cells with chromosome aberrations were reviewed as 

necessary by a senior cytogeneticist prior to decoding the slides.

In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells 

(%) reported. Many experiments with human lymphocytes have established a range of 

aberration frequencies acceptable for control cultures in normal volunteer donors.

Species/strain lymphocytes: Human 

Metabolic 

activation 

with and without 

Test 

system 

strain/cell type: 

Genotoxicity negative 

Cytotoxicity yes (Refer to information on results and attached tables.) 

Vehicle 

controls 

valid 

yes 

Negative 

controls 

valid 

not applicable 

Positive 

controls 

valid 

yes 

TEST-SPECIFIC CONFOUNDING FACTORS 

- Effects of pH: There was no significant change in pH when the test material was dosed into media. 

- Effects of osmolality: The osmalality did not increase by more than 50 mOsm. 

- Evaporation from medium: Not applicable. 

- Water solubility: Not applicable, test material was dissolved in Acetone 

- Precipitation: Refer to results section 

RANGE-FINDING/SCREENING STUDIES: 

Preliminary Toxicity Test 

The dose range for the Preliminary Toxicity Test was 6.90, 13.80, 27.59, 55.19, 110.38, 220.75, 441.5, 883, 1766 

μg/ml. The maximum dose was the maximum recommended dose level, the 10 mM concentration. 

A greasy/oily precipitate of the test item was observed in the parallel blood-free cultures at the end of exposure, at 

and above 883 μg/ml, in the 4(20)-hour exposure group in the absence of S9 and in the 24-hour exposure group in 

the absence of S9, and at 1766 μg/ml in the 4(20)-hour exposure group in the presence of S9. 

Haemolysis was also noted in the blood cultures at the end of the exposure period from 110.38 μg/ml to 883 μg/ml 

in the 4(20)-hour exposure 

group in the absence of S9 and at and above 110.38 μg/ml in the 24-hour exposure group in the absence of S9. In 

the 4(20)-hour exposure group 

in the presence of S9 haemolysis was noted from 55.19 μg/ml to 883 μg/ml. Haemolysis is a common observation 

in chromosome aberration 

studies using whole blood, and is due to the action of the test items on the cell membranes of the red blood cells 

(erythrocytes) and not to the 

lymphocytes. It is considered that this effect is not fully related to the toxicity imparted on the lymphocytes and on 

occasions haemolysis has been 

observed at multiple dose levels where no toxicity to lymphocytes is observed and, therefore, has no effect on the 

end-point of this test. 

Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were 

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present up to 220.75 μg/ml in the 

4(20)-hour exposures in the presence and absence of metabolic activation (S9). The maximum dose with 

metaphases present in the 24-hour 

continuous exposure was 110.38 μg/ml. The mitotic index data are presented in the attached Table 1. The test 

item induced marked toxicity in all of the exposure groups. 

The selection of the maximum dose level was based on toxicity and was 330 μg/ml for the 4(20)-hour exposure 

groups and was 110 μg/ml for the 

continuous exposure group used in Experiment 2. 

 

Chromosome Aberration Test - Experiment 1 

The dose levels of the controls and the test item are given in the table below: 

Group Final concentration of Benzenesulfonyl chloride (μg/ml) 

4(20)-hour without S9 0*, 13.75, 27.5*, 55*, 110*, 220, 330, MMC 0.4* 

4(20)-hour with S9 (2%) 0*, 13.75, 27.5*, 55*, 110*, 220*, 330, CP 5* 

* Dose levels selected for metaphase analysis 

MMC = Mitomycin C 

CP = Cyclophosphamide 

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary 

Toxicity Test and that there were metaphases suitable for scoring present at 220 μg/ml in both exposure groups. 

No precipitate of the test item was observed at the end of exposure in either exposure group. Haemolysis was 

observed at and above 110 μg/ml in 

the 4(20)-hour exposure group in the presence of S9 at the end of the exposure period and at and above 110 

μg/ml in both exposure groups prior 

to harvesting the cultures. 

The mitotic index data are given in the attached Table 2. They confirm the qualitative observations in that a doserelated inhibition of mitotic index 

was observed, and that 42% and 99% mitotic inhibition was achieved at 110 and 220 μg/ml respectively in the 

absence of S9. In the presence of S9 

the toxicity curve was much steeper with 81% mitotic inhibition being achieved at 220 μg/ml. 

The maximum dose level selected for metaphase analysis was 110 μg/ml in the absence of S9 as the higher dose 

level of 220 μg/ml was considered 

too toxic for scoring. In the presence of S9 the maximum dose selected for scoring was 220 μg/ml as it was the 

only intermediate dose in a very 

steep toxicity curve. 

The chromosome aberration data are given in the attached Table 4 and Table 5. All of the vehicle control cultures 

had frequencies of cells with 

chromosome aberrations within the expected range. The positive control items induced statistically significant 

increases in the frequency of cells 

with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself 

was operating as expected. 

The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in 

the absence or presence of 

metabolic activation. 

The polyploid cell frequency data are given in the attached Tables 4 and 5. The test item did not induce a 

statistically significant increase in the 

numbers of polyploid cells at any dose level in either of the exposure groups. 

Chromosome Aberration Test - Experiment 2 

The dose levels of the controls and the test item are given in the table below: 

Group Final concentration of Benzenesulfonyl chloride (μg/ml) 

24-hour without S9 0*, 3.44, 6.88, 13.75*, 27.5*, 55*, 110, MMC 0.2* 

4(20)-hour with S9 (1%) 0*, 27.5*, 55*, 110*, 165, 220, 330, CP 5* 

* Dose levels selected for metaphase analysis 

MMC = Mitomycin C 

CP = Cyclophosphamide 

The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at 

the test item dose level of 220 μg/ml 

in the presence of S9. In the absence of S9 the maximum test item dose level with metaphases suitable for 

scoring was 55 μg/ml. No precipitate of 

the test item was observed at the end of exposure in either exposure group. Haemolysis was observed at the end 

of the exposure period at and 

above 110 μg/ml in the 4(20)-hour exposure group in the presence of S9 and at and above 55 μg/ml in the 

absence of S9. 

The mitotic index data are given in the attached the attached Table 3. They confirm the qualitative observations in 

that a dose-related inhibition of 

mitotic index was observed, and that 60% and 23% mitotic inhibition was achieved at 55 μg/ml and 27.5 μg/ml 

respectively in the 24-hour exposure 

group in the absence of S9. In the 4(20)-hour exposure group in the presence of S9 the toxicity was greater than 

that seen in Experiment 1 but this 

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Any other information on results incl. tables 

Overall remarks, attachments 

Attached background material 

Applicant's summary and conclusion 

Interpretation of results 

Conclusions 

Executive summary 

was considered to be due to the reduced S9 concentration and its protective effects. Mitotic inhibition of 66% and 

57% was demonstrated at 110 and 55 μg/ml respectively in the presence of S9. 

The maximum dose level selected for metaphase analysis was 110 μg/ml for the 4(20) hour exposure group in the 

presence of S9. In the absence of S9 the maximum dose selected for scoring was 55 μg/ml. 

The chromosome aberration data are given in the attached Table 6 and Table 7. All of the vehicle control cultures 

had frequencies of cells with 

chromosome aberrations within the expected range. The positive control items induced statistically significant 

increases in the frequency of cells 

with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself 

was operating as expected. 

The test item did not induce any statistically significant increases in the frequency of cells with chromosome 

aberrations in the presence of metabolic activation using a dose range which included dose levels which achieved 

greater than optimum toxicity. The 24 hour exposure group in the absence of metabolic activation demonstrated 

marked statistically significant increases in the frequency of cells with chromosome aberrations using a dose 

range which included dose levels which achieved slightly less than and greater than optimum toxicity. The highest 

frequency of aberrant cells (6.0%) was observed at 27.5 μg/ml where less than optimum toxicity was observed. 

With only 4.5% being observed at 55 μg/ml where the toxicity exceeded 

the optimum level of 50%. Both responses exceeded the upper limit of the historical control range for vehicle 

control culture. It is possible that an 

intermediate dose level between the two dose levels would have produced a bigger response and produced a true 

dose related effect. The aberrations seen in the 24-hour exposure group included complex exchange type

aberrations and these were distributed equally between the two replicate 

cultures. A clear statistically significant response was seen at 27.5 μg/ml where only 23% mitotic inhibition was 

achieved, well below the optimum 50% and it therefore cannot be discounted on the basis of toxicity. 

The polyploid cell frequency data are given in the attached Tables 6 and 7. The test item did not induce a 

statistically significant increase in the 

numbers of polyploid cells at any dose level in either of the exposure groups. 

For the attached tables of results mentioned above, please refer to the attached background material section for 

the following tables: 

Table 1 Mitotic Index - Preliminary Toxicity Test

Table 2 Mitotic Index - Experiment 1

Table 3 Mitotic Index - Experiment 2

Table 4 Results of Chromosome Aberration Test - Experiment 1 Without Metabolic Activation

Table 5 Results of Chromosome Aberration Test - Experiment 1 With Metabolic Activation

Table 6 Results of Chromosome Aberration Test - Experiment 2 Without Metabolic Activation

Table 7 Results of Chromosome Aberration Test - Experiment 2 With Metabolic Activation

Attached document Remarks 

41201582 Tables 1-7.pdf / 66.1 KB (application/pdf) 41201582 Tables 1-7 

positive without metabolic activation (In the 24-hour exposure group of Experiment 2 the test item induced 

statistically significant increases in the frequency of cells with aberrations at a dose level which achieved less than 

optimum toxicity.) 

The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in the 4

(20)-hour exposure groups in the absence or presence of S9 in Experiment 1 or in the 4(20)-hour exposure group 

in the presence of S9 in Experiment 2 using a dose range which exceeded optimum 

toxicity. In the 24-hour exposure group of Experiment 2 the test item induced statistically significant increases in 

the frequency of cells with aberrations at a 

dose level which achieved less than optimum toxicity. 

The test item was therefore considered to be clastogenic to human lymphocytes in vitro. 

Introduction. This report describes the results of anin vitrostudy for the detection of structural 

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chromosomal aberrations in cultured mammalian cells. It supplements microbial systems 

insofar as it identifies potential mutagens that produce chromosomal aberrations rather 

than gene mutations (Scottet al, 1990). The method used was designed to be compatible 

with that described in the OECD Guidelines for Testing of Chemicals (1997) No. 473 

"Genetic Toxicology: Chromosome Aberration Test", Method B10 of Commission 

Regulation (EC) No. 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS 

harmonised guideline 40 CFR 799.9537. The study design also meets the requirements of 

the UK Department of Health Guidelines for Testing of Chemicals for Mutagenicity.

Methods. Duplicate cultures of human lymphocytes, treated with the test item, were 

evaluated for chromosome aberrations at up to four dose levels, together with vehicle and 

positive controls. Four treatment conditions were used for the study; i.e. In Experiment 1, 

4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 

2% final concentration with cell harvest after a 20-hour expression period and a 4 hours 

exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In 

Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1% final S9 

concentration), whilst in the absence of metabolic activation the exposure time was 

increased to 24 hours.

The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were 

as follows:

Results. All vehicle (solvent) controls had frequencies of cells with aberrations within the 

range expected for normal human lymphocytes.

All the positive control items induced statistically significant increases in the frequency of cells with aberrations 

indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in the 4

(20)-hour exposure groups in the absence or presence of S9 in Experiment 1 or in the 4(20)-hour exposure group 

in the presence of S9 in Experiment 2 using a dose range which exceeded optimum toxicity. However in the 

24-hour exposure group of Experiment 2 the test item induced statistically significant 

increases in the frequency of cells with aberrations at a dose level which achieved less than 

optimum toxicity.

Conclusion. The test item was considered to be clastogenic to human lymphocytesin vitro.

Group Final concentration of test item (μg/ml)

4(20)-hour without S9 13.75, 27.5, 55, 110, 220, 330

4(20)-hour with S9 (2%) 13.75, 27.5, 55, 110, 220, 330

24-hour without S9 3.44, 6.88, 13.75, 27.5, 55, 110

4(20)-hour with S9 (1%) 27.5, 55, 110, 165, 220, 330

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