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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted with no reference guidelines but under a good methodology which do not affect the quality of the relevant results. The study report was conclusive, the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
n-Amyl bromide was tested in an in vitro cytogenetics assay using duplicate cultures of CHO cells. Treatments covering a broad range of doses, separated by narrow intervals, were performed. The test article was dissolved in dimethyl sulphoxide and the highest dose level used was 500 µg/ml.
GLP compliance:
yes
Type of assay:
other: in vitro CHO chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Description: clear colourless liquid
Batch number: 7-146-1
Conditions: storage at room temperature in the dark
Purity: 98.3 %
Acidity (HBr): < 5 mg/kg
Expiry date: August 1998

Method

Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CHO cells are maintained at Covence Laboratories in tissue culture flask containing McCoy's 5A medium including 10 % (v/v) foetal calf serum 5FCS) and 100 µg/ml gentamycin. They are subcultured regularly at low density, and before overgrowth occurs, to mainain low aberration frequencies. Stocks of cells preserved in liquid nitrogen are reconstitued for each experiment so as to maintain karyotypic stability. The cells are screened for mycoplasma contamination.
Cell sheets were removed from stock cultures using trysin/EDTA solution, and subcultured at 6.5 x 10E5 cells per flask into 75 sq cm tissue culture flasks (9.9 ml cell suspension per flask). After incubation for one day in an atmosphere of 5 % (v/v) CO2 in air, and at 37°C, cultures were of low confluence and considered suitable for treatment.
The test cultures, four for each solvent control treatment and two for all doses of test chemical or positive control chemical, were suitably labelled (using a colour-coded procedure) to clearly identify the study number, test chemical, positive and negative groups.
Metabolic activation:
without
Test concentrations with justification for top dose:
n-Amyl bromide was dissolved in DMSO to give 50 mg/ml. The stock solutin was membrane filter-sterilized and dilutions made using sterile DMSO. Test article solutions were used within approximately 2 hours of initial formulation.
Final concentrations from 7.206 µg/ml to 500 µg/ml.
Precipation was observed at 163.8 µg/ml and more.
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Details on test system and experimental conditions:
Treatment
One set of quadruplicate cultures for each of the treatment regimes was treated with the solvent and one set of duplicate cultures with the test article. Additional duplicate cultures were treated with 0.1 ml of the positive control chemicals. All treatments were performed in the absence of S-9.

Harvesting
Approximately 2 hours prior to harvest, colchicine was added to give final concentration of approximately 1 µg/ml to arrest dividing cells in metaphase. At the defined sampling time, the monolayers of these cultures were then removed using trypsin/EDTA.
Cell numbers were determined using a Coulter counter.
The suspension from each flask was transferred to a plastic centrifuge tube and the cells pelleted by centrifuging at 200 g for 5 minutes. The supernatant was carefully removed and the cells resuspended in 4 ml of 0.075 M KCl at 37°C for 5 minutes to allow swelling to occur.
Cells were fixed by dropping the KCl suspension into an equal volume of fresh, ice-cold methanol/glacial acetic acid (3:1 v/v). The fixative was changed by centrifugation(200 g for 2-3 minutes) and resuspension. This procedure was repeated several times until the cell pellets were clean.

Preparation of metaphase spreads
Cells wer kept in fixative in the refrigerator before slides were prepared but slides were not made on the day of harvest to ensure cells were adequately fixed. Cells were pelleted and resuspended in a minimal amount of fresh fixative so as to give a milky suspension. Several drops of 45 % v/v aqueous acetic acid were added to each suspension to enhance chromosome spreading, and several drops of suspension were transferred to clean microscope slides. Slides were flamed to enhance chromosome spreading.
After the slides had dried the cells were stained for 5 minutes in 4 % v/v filtered Giemsa strain in pH 6.8 buffer. The slides were rinsed, dried and mounted with coverslips.

Selection of doses for cytogenetic analysis
Cell number was detrmined using a Coulter counter. Slides were examined, uncoded, for mitotic index 5MI) that is, percentage of cells in mitosis. Slides from enough dose levels from each treatment group were scored to determine whether chemically induced mitotic inhibition has occured. This is defined as a clear decrease in mitotic index compared with negative control.

Rationale for dose selection
The highest dose for chromosome analysis was to be one at which at least 50 % reduction in cell number occured. Analysis of slides from highly cytotoxic concentrations is avoided, if possible. Slides from the highest selected dose and two lower doses, such that a range of mitotic inhibition from maximum to little or none was covered, were to be taken for microscope analysis.
For each treatment regime, two solvent control cultures were initially to be analysed for chromosome aberrations. Slides from the remaining solvent control cultures were only to be analysed if considered necessary, for exemple, to help resolve an equivocal result.

Scoring of aberrations
Slides from the selected treatments and controls were coded using randomly generated letters by a person not connected with the scoring of the slides. Labels bearing only the study reference number, experiment number, replicate number and the code were used to cover treatment details on the slides.
One hundred metaphases from each code were analysed for chromosome aberrations. Only cells with 19-23 chromosomes were considered acceptable for analysis of structural aberrations. Any cell with more than 23 chromosomes, that is polyploid, endoreduplicated and hyperdiploid cells, observed during this search was noted and recorded separately. Classification of structural aberrations was based on the scheme described by ISCN. Observations were recorded on raw data sheets with the microscope stage coodinates of any aberrant cell.
Slide analysis was performed off-site by analysis trained in accordance with Covance Standard Operating Procedures.

Analysis of results
a) Treatment of data
After completion of microscopic analysis, data were decoded. The aberrant cells in each culture were categorised as follows:
1) cells with structural aberrations including gaps
2) cells with structural aberrations excluding gaps
3) polyploid, endoreduplicated or hyperdiploid cells.
The totals for category 2 in negative control cultures were used to determine whether the assay was acceptable or not. The proportions of aberrant cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test.
The proportion of cells in category 2 for each test treatlent condition, were compared with the proportion in concurrent negative controls using Fisher's exact test. Probability values of p < 0.05 were accepted as significant. The proportions of cells in categories 1, 2 and 3 were examined in relation to the historical negative control range.
b) Acceptance criteria
The assay was to be considered valid if the following criteria were met:
1) the binomial dispersion test demonstrated acceptable heterogeneity between replicate cultures, particularly where no positive responses were seen, and
2) the proportion of cells with structural aberrations 'excluding gaps) in negative control cultures fell within the normal range, and
3) at least 160 cells out of an intended 200 were analysable at each dose level, and
4) the positive control chemicals induced statistically significant increases in the number of cells with structural aberrations.
Evaluation criteria:
The test article was to be considered as positive in this assay if:
1) a statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) occured et one or more concentrations, and
2) the proportion of cells with structural aberrations at such doses exceeded the normal range.
Increases in number of cells with gaps or increases in the proportions of cells with structural aberrations not exceeding the normal range or occuring only at very high or very toxic concentrations were likely to be concluded as "equivocal". Full assessmentof the biological importance of such increases is likely only to be possible with reference to data from other test systems. Cells with exchange aberrations or cells with greater than one aberration were to be considered of particular biological significance.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No evidence of mitotic accumulation was observed. A clear, dose-related decrease in cell number and increase in mitotic inhibition was apparent. At a dose of 163.8 µg/ml; approximately 74 %reduction in cell number was seen. This concentration and the next two lower doses, covering a range of 35 to 74 %reduction in cell number and 2 to 55 % mitotic inhibition, were analysed for chromosome aberrations.

Treatment of cultures with n-Amyl bromide resulted in frequencies of cells with aberrations which were similar to and not significantly different from those in concurrent negative controls. The number of aberrant cells fell within the historical negative control range in all treated cultures.
Normal frequencies (within the historical negative control range) were observed in all test article treated cultures.
Remarks on result:
other: strain/cell type: CHO cells
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative without metabolic activation

It is concluded that n-Amyl bromide did not induce chromosome aberrations in cultured Chinese Hamster Ovary cells when tested to its limit of toxicity.
Executive summary:

n-Amyl bromide was tested in an in vitro cytogenetics assay using duplicate cultures of CHO cells. Treatments covering a broad range of doses, separated by narrow intervals, were performed. The test article was dissolved in dimethyl sulphoxide and the highest dose level used was 500 µg/ml.

Treatment was performed in the absence of S-9 for 20 hours proir to harvest (20 +0). The test article dose levels for chromosome analysis were selected by evaluating the effect of n-Amyl bromide on cell number. Chromosome aberrations were analysed at three consecutive dose levels. The highest concentration chosen for analysis, 163.8 µg/ml, induced approximately 74 ù reduction in cell number and 58 % mitotic inhibition.

Appropriate negative (solvent) control cultures were included in the test system. The proportion of cells with structural aberrations in these cultures fell within the historical solvent control range. 4 -Nitroquinoline-1-oxide was employed as the positive control chemical and cells receiving this were sampled 20 hours after the start of the treatment. A clear and statistically significant increase in the proportion of cells with structural aberrations was observed.

Treatment of cultures with n-Amyl bromide resulted in frequencies of cells with aberrations which were similar to and significantly different from those in concurrent negative controls. The number of aberrant cells fell within the historical negative control range in all treated cultures.

It is concluded that n-Amyl bromide did not induce chromosome aberrations in cultured Chinese Hamster Ovary cells when tested to its limit of toxicity.