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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:
28 April to 10 June 1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1987
Report date:
1982

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
The assay was not repeated in an independent experiment and no continuous treatment without S9 mix was performed; only 50 metaphases per culture (i.e. total of 100 metaphases) were analysed.
Principles of method if other than guideline:
A confirmatory assay with S9 mix was performed because of positive results were obtained at the highest dose (90 µg/mL) in the first experiment.
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
Benzene, dibromoethyl Benzene, ethenyl-, ar-bromo derivs.
EC Number:
603-094-7
Cas Number:
125904-11-2
Molecular formula:
C8 H6 Br2
IUPAC Name:
Benzene, dibromoethyl Benzene, ethenyl-, ar-bromo derivs.
Test material form:
liquid

Method

Target gene:
Chromosome aberrations
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CHO-K1-BH4, Lot #A-12. Continous cell line with the modal number of 20 chromosomes with a population dubling time of 12-14 hours.
Metabolic activation:
with and without
Metabolic activation system:
Prepared immediately prior to treatment with microsomal preparation obtained from Aroclor 1254 induced rat liver.
Test concentrations with justification for top dose:
Following a preliminary cytotoxicity test utilising 5-bromo-2'deoxyuridine to detect any increase in the cell proliferation kinetics, dosages selected for analysis were 2.5, 10 and 35 µg/mL without S9 mix and 9, 50 and 90 µg/mL with S9 mix. As in the first experiment with S9 mix a statistically significant increase in the number of aberrations/cell was recorded at the highest concentration, a confirmatory assay was performed with S9 mix at concentrations of 80, 90 and 100 µg/mL.
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N-nitro-nitrosoguanidine (without S9 mix); N-nitrosodimethylamine (with S9 mix)
Details on test system and experimental conditions:
Duplicate cultures were treated for 5 hours, then washed three times with 5 mL of Saline-G and then supplied with fresh medium. The cultures were incubated for an additional 18 hours, with colcemid to arrest metaphases added 2-3 hours before harvest.
Evaluation criteria:
Assessment of a test article as positive is based upon its ability to produce a statistically significant increase in chromosome aberrations and proportion of aberrant metaphases, as compared to the solvent control or upon its ability to produce a dose response. If a weak positive response is detected at one dose point, a duplicate experiment bracketing (if possible) the dose level should be conducted to reproduce the positive results of the first assay.
The solvent control DMSO must have less than 10 spontaneous aberrations and the positive controls must show a significant (p < 0.05) increase in the aberration frequency as compared to the solvent controls.
Statistics:
To evaluate the proportion of cells with one or more aberrations, Chi-square analysis was done comparing each dose to its concurrent solvent control. Significant differences in aberrations/cell were determined by pooling the data from the two cultures per dose and conducting one-tailed t-tests comparing treated cultures with their concurrent solvent controls.

Results and discussion

Test resultsopen allclose all
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
only at the highest concentration of 90 µg/mL
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without S9 mix

The test substance was judged not-clastogenic under the experimental conditions.
Executive summary:

A preliminary cytotoxicity test using 5 -bromo-2'deoxyuridine to detect any increase in the cell proliferation kinetics in CHO cells was conducted at dosages ranging from 5 to 5000 µg/mL, with and without S9 mix. The results indicated that concentration of 50 to 5000 µg/mL without S9 mix were toxic to the cultures, resulting in no cell survival. In cultures treated with S9 mix dosages of 250 to 5000 µg/mL were toxic, resulting in no cell survival. On this basis 2.5, 10 and 35 µg/mL without S9 mix and 9, 50 and 90 µg/mL with S9 mix were evaluated in a first experiment. Negative results were obtained without S9 mix. Parwise t-test, but not Chi-square analysis, indicated a statistically significant increase in the proportion of aberrations/cell at the highest dose level with S9 mix and therefore a confirmatory assay was performed with S9 mix at dosages of 80, 90 and 100 µg/mL. Results of this second assay were negative.

The test substance was judged as not-clastogenic with and without S9 mix under the experimental conditions.