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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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study performed similarly to OECD Guideline 473 with deviations: no data on metabolic activation; no data on maintenance of cell cultures
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1995

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
no data on metabolic activation; no data on maintenance of cell cultures
Principles of method if other than guideline:
Not applicable
GLP compliance:
no
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Sodium arsenite
- Source: Sigma

Method

Target gene:
Not applicable
Species / strain
Species / strain / cell type:
lymphocytes: Human lymphocytes
Details on mammalian cell type (if applicable):
- Source: To investigate the aneugenic effect, peripheral blood was obtained from healthy donors (two males aged 28 years and two females aged 23 and 36 years). To investigate the induction of mitotic arrest, whole blood lymphocyte cultures were isolated from five healthy donors (two males and three females, four of them the same donors as before).
- Type and identity of media: 0.5 mL of blood in 6 mL of RPMI culture medium supplemented with 1% of L- glutamine and non essential amino acids, 32 µM of bromodeoxyuridine and 0.2 mL of phytohemagglutinin
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
not specified
Metabolic activation system:
not applicable
Test concentrations with justification for top dose:
1, 10-2, 10-4, 10-6, 10-8 and 10-10 µM
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: No data
Controls
Untreated negative controls:
yes
Remarks:
RPMI culture medium
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: colcemid, 10-2 µM
Remarks:
Aneugenic effect and mitotic arrest
Details on test system and experimental conditions:
METHOD OF APPLICATION: In RPMI medium

DURATION
- Exposure duration: 24 hours for aneugenic effect and 2 hours for mitotic arrest; 37 °C
- Fixation time (start of exposure up to fixation or harvest of cells): 72 hours; 37 °C

SPINDLE INHIBITOR (cytogenetic assays): Colcemid; incubated for 2 hours at 37 °C
STAIN (for cytogenetic assays): Aneugenic effect: Perry and Wolff (1974); Mitotic arrest: Gonsebatt et al. (1992)

NUMBER OF REPLICATIONS: Duplicates

NUMBER OF CELLS EVALUATED: 5000 mononuclear cells were analyzed for mitotic index, 200 metaphases at first and second division were analyzed for aneugenicity and 100 metaphases were analyzed for structural chromosomal aberrations

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index

OTHER: Breaks, rearrangements, multifragmentations and pulverizations were scored for chromosomal aberrations.
Evaluation criteria:
Aneugenicity: Metaphases were classified based on the number of chromosomes:
- Hypoploids: Fewer than 46 chromosomes
- Hyperploids: more than 46 chromosomes
-Tetraploids: Approximately 92 chromosomes

Mitotic arrest:
- Data obtained with colcemid treatment were taken as 100% pf accumulated metaphases compared with control/treatment groups.

Structural chromosomal aberrations:
- Metaphases with 46 centromeres were analyzed for chromosomal aberrations.
Statistics:
- Heterogeneity X2 test was used to compare the response of all the individuals on the three parameters such as heteroploid cells, cytostatic effect and structural chromosomal aberrations.
- Proportions of heteroploidy and cytostaticity were compared using student's t-test.
- Three way ANOVA was used to compare individuals considering all 3 parameters together.

Results and discussion

Test results
Species / strain:
lymphocytes: Human
Metabolic activation:
not specified
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1 µM in all experiments
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- Frequency of heteroploid cells in controls ranged 4.5 -5% in first division cells and 4.5 -7.5% in second division cells
- A dose-related effect was observed: highest concentration (10-4 µM) induces 28.33% and 22.44% hyperploid cells in first and second division respectively and 29% tetraploid cells.
- Sodium arsenite produced 40.24% and 12.93% of the colcemid effect (mitotic arrestant effect at 10-4 and 10-10µM respectively)
- Chromosomal aberrations scored at 10-2 µM of sodium arsenite treatment supported the data on the varying individual susceptibility between the donors of this study. The two donors had 66 and 30% aberrant cells and a total of 118 and 53 chromosome aberrations/ 100 cells, respectively. The most frequent types of damage found were chromatid breaks and cells with multiple chromosome and chromatid breaks.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive aneuploidogenic and mitotic arrestant

Under the test conditions, sodium arsenite was found to have an aneuploidogenic and a mitotic arrestant effect in cultured human lymphocytes.
Executive summary:

In an in vitro chromosomal aberration assay conducted similarly to OECD Guideline 473, whole blood cultures were incubated for 72 hours and treated with various concentrations (1-10-10 µM) of sodium arsenite for the last 24 hours. Also, mitotic arrest was evaluated in cultures treated for the last 2 hours. Cells were harvested and samples were processed for evaluation of aneuploidogenic and mitotic arrest. Number of chromosomes in 200 metaphases of first and second division cells was scored.

 

Positive controls (colcemid at 10-2 µM) induced the appropriate response. A dose-related effect was observed: highest concentration (10-2 µM sodium arsenite) induces 28.33 and 22.4% hyperploid cells in first and second division respectively and 29% tetraploid cells. Sodium arsenite produced 40.24 and 12.93% of the mitotic arrestant effect at 10-2 µM and 10-10 µM, respectively). A different individual susceptibility effect was observed in both parameters and confirmed with the chromosome aberrations levels induced by arsenic in the same donors.

 

Under the test conditions, sodium arsenite was found to have an aneuploidogenic and a mitotic arrestant effect in cultured human lymphocytes.

The authors of the report did not include a comment in their summary of the report, but a positive effect was also observed in terms of an increase in chromosome aberrations.