# 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 min or 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 Ab erration 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 Ch romosome Aberration Test) no

according to EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberr ation 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 per formed 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 dra wn 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 f rom 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, 2 7.59, 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 fol lowed by 20-hour culture in treatment-free media pr ior 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 followe d 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 withou t S9-mix prior to cell harvest. The dose range of t est 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 followe d 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: Aceto ne was selected as the solvent because the test mat erial 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 age nt): Not applicable.

- Fixation time (start of exposure up to fixation o r 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% Gi emsa 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 nucl ei were counted and the number of cells in metaphas e recorded and expressed as the mitotic index and as a percentage of the vehicle control va lue.

-Scoring of Chromosome Damage: Where possible the first 100 consecutive well-sprea d metaphases from each culture were counted, where there was approximately 30% - 50% of cells with aberratio ns, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangemen ts 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 tr eatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent contr ol, either with or without a clear dose-relationshi p. For modest increases in aberration frequency a dose response r elationship is generally required and appropriate statistical tests may be a pplied in order to record a positive response.

The frequency of cells with aberrations excluding g aps and the frequency of polyploid cells was compar ed, where necessary, with the concurrent vehicle control valu e using Fisher's Exact test.

Cells

For each experiment, sufficient whole blood was dra wn from the peripheral circulation of a volunteer w ho had been previously screened for suitability. 

## The volunteer had not been exposed to high levels o f radiation or hazardous chemicals and had not knowingly recent ly 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 th e number of first, second and third division metaphase cells and so calculate the avera ge 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 e xposure 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% C O 2 in humidified air. 

## The lymphocytes of fresh heparinised whole blood we re 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; the refore the maximum dose level was 1766 μg/ml, which was calculated to be equivalent t o 10 mM, the maximum recommended dose level. The purity of the test item was 99.53% and therefor e 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 osm olality did not increase by more than 50 mOsm (Scott et al 1991). 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 homogene ity, concentration or stability of the test item
formulation. 

## This is an exception with regard to GLP and has bee n 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, Bat ch 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-ho use from the livers of male Sprague-Dawley rats weighing approximately 250 g. These had received three daily oral doses of a mixture of phenobarbitone (80 mg/kg ) and beta 

## -naphthoflavone (100 mg/kg), prior to S9 preparation on the fourth day. The S9 homogenate was produced by homogenising the l iver in a 0.15 M KCl solution (1g liver to 3ml KCl) followed by centrifugation at 9000 g

. 

## The protein content of the resultant supernatant wa s adjusted to 20 mg/ml using 0.15 M KCl and aliquots of the su pernatant 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 nitrog en freezer. Each batch of S9 has been routinely tested for its ca pability to activate known mutagens in the Ames test

. The above procedure was designed and conducted to c ause the minimum suffering or distress to the anima ls consistent with the scientific objectives and in ac cordance with the Harlan Laboratories Ltd, Shardlow , UK policy on animal welfare and the requirements of the Unite d Kingdom’s Animals (Scientific Procedures) Act 1986

. The S9-mix was prepared prior to the dosing of the t est cultures and contained the S9 fraction (10 to 20 % (v/v)), MgCl 2 (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 T est and Experiment 1 and 1% in Experiment 2.
Culture Conditions Duplicate lymphocyte cultures (A and B) were establi shed for each dose level by mixing the following co mponents, 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% CO 2 in humidified air, the cultures were transferred to tubes and centrifuged. 

## Approximately 9 ml of the culture medium was remove d, 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) w as 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 co ncentration 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% CO 2 in humidified air the cultures were centrifuged, th e treatment medium removed by suction and replaced with an 8 ml wash of MEM cultur e 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 37 oC in 5% CO 2 

## in humidified air. Without Metabolic Activation (S9) Treatment In Experiment 1, after approximately 48 hours incuba tion at 37oC with 5% CO 2 in humidified air the cultures were decanted into tubes and centrifuged. 

## Approximately 9 ml of the culture medium was remove d and reserved. The cells were then resuspended in the required vol ume of fresh MEM (including serum) and dosed with 0.1 ml of the appropriate veh icle 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% CO 2 in humidified air the cultures were centrifuged the treatment medium was removed by suction and replaced with an 8 ml wash of MEM cul ture medium. 

## After a further centrifugation the wash medium was removed by suction and replaced wit h 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 activat ion, the exposure was continuous for 24 hours. 

## Therefore, when the cultures were established the culture volu me 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 dos e 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% CO 2 

## in humidified air for 24 hours. 

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13/12/2012 file://C:\Documents and Settings\amorris.EUROPE\Des ktop\index.htmlThe Preliminary Toxicity Test was performed using th e methodology of Experiment 1 and Experiment 2 in the absence of S9 except that positive controls were no t included. Preliminary Toxicity Test A preliminary toxicity test was performed on cell cu ltures using a 4-hour exposure time with and withou t metabolic activation followed by a 20-hour recovery period, a nd a continuous exposure of 24 hours without metabo lic
activation. 

## The dose range of test item used was 6.90, 13.80, 2 7.59, 55.19, 110.38, 220.75, 441.5, 883 and 1766 μg/ml. Parallel flasks, contain ing 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 begin ning and end of the exposure periods. Using a qualitative microscopic evaluation of the m icroscope slide preparations from each treatment cu lture, appropriate dose levels were selected for mitotic i ndex evaluation. 

## Mitotic index data was used to estimate test item toxicity and for selection of th e dose levels for the main test. Experiment 1 i) 

## 4-hour exposure to the test item without S9-mix fol lowed 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 follow ed by 20-hour culture in treatmentfree media prior to cell harvest. The dose range of test item used was 13.75, 27.5, 5 5, 110, 220, 330 μg/ml. Experiment 2 i) 

## 24-hour continuous exposure to the test item withou t 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 follow ed 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 (Co lcemid 0.1 μg/ml) two hours before the required har vest
time. 

## After incubation with demecolcine, the cells were c entrifuged, the culture medium was drawn off and discarded, and the cells resuspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifug ation), most of the hypotonic solution was drawn off and discarded. The cells were resuspended and then fixed by droppi ng the KCl cell suspension into fresh methanol/glacial ace tic acid (3:1 v/v). The fixative was changed at least three times and the cells stored a t approximately 4oC to ensure complete fixation. Preparation of Metaphase Spreads The lymphocytes were resuspended in several ml of f resh fixative before centrifugation and resuspensio n 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% Gi emsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.
Qualitative Slide Assessment The slides were checked microscopically to determin e the quality of the metaphases and also the toxici ty 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 n umber 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-sprea d metaphases from each culture were counted, where there was approximately 30 to 50% of cells with aberratio ns, slide evaluation was terminated at 50 cells. 

## If the cell had 44-48 chromosomes, any gaps, breaks or rearrang ements were noted according to the simplified system of Savage (1976) recommended in t he 1983 UKEMS guidelines for mutagenicity testing (Appendix 1). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decod ing the slides. In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of pol yploid cells (%) reported. 

## Many experiments with human lymphocytes have establ ished a range of aberration frequencies acceptable for control cultu res 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 t ables.)

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 incr ease by more than 50 mOsm. - Evaporation from medium: Not applicable. - Water solubility: Not applicable, test material w as dissolved in Acetone - Precipitation: Refer to results section

RANGE-FINDING/SCREENING STUDIES: Preliminary Toxicity Test The dose range for the Preliminary Toxicity Test wa s 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 obser ved in the parallel blood-free cultures at the end of exposure, at and above 883 μg/ml, in the 4(20)-hour exposure gro up 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)-h our 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 μ g/ml in the 24-hour exposure group in the absence o f S9. In the 4(20)-hour exposure group in the presence of S9 haemolysis was noted from 55.1 9 μg/ml to 883 μg/ml. Haemolysis is a common observ ation 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 n ot fully related to the toxicity imparted on the ly mphocytes and on occasions haemolysis has been observed at multiple dose levels where no toxicity to lymphocytes is observed and, therefore, has no e ffect on the end-point of this test. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells we re 

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13/12/2012 file://C:\Documents and Settings\amorris.EUROPE\Des ktop\index.htmlpresent 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 i ndex data are presented in the attached Table 1. Th e test item induced marked toxicity in all of the exposure groups. The selection of the maximum dose level was based o n toxicity and was 330 μg/ml for the 4(20)-hour exp osure 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 a re given in the table below: Group Final concentration of Benzenesulfonyl chlorid e (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 suitab le for scoring present at 220 μg/ml in both exposur e groups. No precipitate of the test item was observed at the end of exposure in either exposure group. Haemolys is 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 Ta ble 2. They confirm the qualitative observations in that a doserelated inhibition of mitotic index was observed, and that 42% and 99% mitotic inhibiti on was achieved at 110 and 220 μg/ml respectively i n the absence of S9. In the presence of S9 the toxicity curve was much steeper with 81% mitoti c inhibition being achieved at 220 μg/ml. The maximum dose level selected for metaphase analy sis was 110 μg/ml in the absence of S9 as the highe r dose level of 220 μg/ml was considered too toxic for scoring. In the presence of S9 the max imum 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 att ached Table 4 and Table 5. All of the vehicle contro l cultures had frequencies of cells with chromosome aberrations within the expected range. T he positive control items induced statistically sig nificant increases in the frequency of cells with aberrations. The metabolic activation system w as therefore shown to be functional and the test me thod itself was operating as expected. The test item did not induce any statistically sign ificant increases in the frequency of cells with ab errations 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 indu ce a statistically significant increase in the numbers of polyploid cells at any dose level in eit her of the exposure groups. Chromosome Aberration Test - Experiment 2 The dose levels of the controls and the test item a re given in the table below: Group Final concentration of Benzenesulfonyl chlorid e (μg/ml) 24-hour without S9 0*, 3.44, 6.88, 13.75*, 27.5*, 5 5*, 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 pr esent at the test item dose level of 220 μg/ml in the presence of S9. In the absence of S9 the maxi mum test item dose level with metaphases suitable f or scoring was 55 μg/ml. No precipitate of the test item was observed at the end of exposure i n 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 th e absence of S9. The mitotic index data are given in the attached th e attached Table 3. They confirm the qualitative ob servations in that a dose-related inhibition of mitotic index was observed, and that 60% and 23% mi totic 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 exposu re group in the presence of S9 the toxicity was gre ater 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 concentr ation and its protective effects. Mitotic inhibitio n of 66% and 57% was demonstrated at 110 and 55 μg/ml respective ly in the presence of S9. The maximum dose level selected for metaphase analy sis was 110 μg/ml for the 4(20) hour exposure group in the presence of S9. In the absence of S9 the maximum dos e selected for scoring was 55 μg/ml. The chromosome aberration data are given in the att ached Table 6 and Table 7. All of the vehicle contro l cultures had frequencies of cells with chromosome aberrations within the expected range. T he positive control items induced statistically sig nificant increases in the frequency of cells with aberrations. The metabolic activation system w as therefore shown to be functional and the test me thod itself was operating as expected. The test item did not induce any statistically sign ificant increases in the frequency of cells with ch romosome aberrations in the presence of metabolic activation using a dose range which included dose levels whic h achieved greater than optimum toxicity. The 24 hour exposure group in the absence of metabolic activation demon strated marked statistically significant increases in the f requency of cells with chromosome aberrations using a dose range which included dose levels which achieved sli ghtly 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 obs erved. With only 4.5% being observed at 55 μg/ml where the toxicity exceeded the optimum level of 50%. Both responses exceeded th e upper limit of the historical control range for v ehicle 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 betw een the two replicate cultures. A clear statistically significant response was seen at 27.5 μg/ml where only 23% mitotic inhi bition was achieved, well below the optimum 50% and it therefo re 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 indu ce a statistically significant increase in the numbers of polyploid cells at any dose level in eit her of the exposure groups.

For the attached tables of results mentioned above, please refer to the attached background material s ection 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) 4 1201582 Tables 1-7

positive without metabolic activation (In the 24-ho ur exposure group of Experiment 2 the test item ind uced statistically significant increases in the frequenc y of cells with aberrations at a dose level which a chieved less than optimum toxicity.)

The test item did not induce any statistically sign ificant increases in the frequency of cells with ab errations, in the 4 (20)-hour exposure groups in the absence or presenc e of S9 in Experiment 1 or in the 4(20)-hour exposur e group in the presence of S9 in Experiment 2 using a dose r ange which exceeded optimum toxicity. In the 24-hour exposure group of Experime nt 2 the test item induced statistically significan t increases in the frequency of cells with aberrations at a dose level which achieved less than optimum toxicit y. The test item was therefore considered to be clasto genic to human lymphocytes in vitro.

Introduction. 

## This report describes the results of an in vitro study 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 pr oduce chromosomal aberrations rather than gene mutations (Scott et al , 1990). The method used was designed to be compatible with that described in the OECD Guidelines for Test ing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test", M ethod 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 wi th 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 hom ogenate 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 s elected using data from the preliminary toxicity te st and were
as follows:

Results. 

## All vehicle (solvent) controls had frequencies of c ells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells wi th aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test item did not induce any statistically sign ificant increases in the frequency of cells with ab errations, in the 4 (20)-hour exposure groups in the absence or presenc e of S9 in Experiment 1 or in the 4(20)-hour exposur e group in the presence of S9 in Experiment 2 using a dose r ange which exceeded optimum toxicity. 

## However in the 24-hour exposure group of Experiment 2 the test ite m induced statistically significant increases in the frequency of cells with aberration s at a dose level which achieved less than optimum toxicity.

Conclusion. 

## The test item was considered to be clastogenic to h uman lymphocytes in 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 

# Page 11 of 11 Endpoint study record: Genetic toxicity in vitro.ch romosome aberration OECD 473

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