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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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Batch number: 15535

Method

Species / strain
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
CELLS USED
Human peripheral blood lymphocytes.
For this study (in each experiment) blood was collected only from a single donor to reduce inter-individual variability.

MEDIA USED
Complete Culture Medium:
RPMI 1640 medium supplemented with:
15 % fetal bovine serum (FBS)
100 U/100 µg/mL penicillin/streptomycin solution
0.24 g/mL PHA-L
Also used for the long-term treatment and the post incubation.

Treatment Medium (short-term exposure):
Complete culture medium without FBS.
All incubations were done at 37 °C in humidified atmosphere with 5% CO2.
Metabolic activation:
with and without
Metabolic activation system:
rat liver microsome preparations (S9 mix)
Test concentrations with justification for top dose:
On the basis of the data and the observations from the pre experiment and taking into account the recommendations of the guidelines, the following concentrations were evaluated for microscopic analysis.
Experiment I: without metabolic activation: 0.05, 0.1 and 0.25 mM; with metabolic activation: 0.5, 1 and 1.5 mM
Experiment II: without metabolic activation: 0.01, 0.1, 0.25 and 0.5 mM
Vehicle / solvent:
Vehicle: DMSO
The solvent was compatible with the survival of the cells and the S9 activity.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
Experimental Performance

Treatment
Experiment I: Short-term exposure 4 h (without and with S9 mix)
After 48 h the culture medium was replaced with serum-free medium containing the test item (without metabolic activation) and serum-free medium containing the test item with 50 µL/mL S9 mix (with metabolic activation). After 4 h the cells were spun down by gentle centrifugation for 10 min. The supernatant with the dissolved test item was discarded and the cells were resuspended in PBS. The washing procedure was repeated once as described. After washing, the cells were resuspended in complete cell culture medium. The cells were prepared 24 h after the beginning of the treatment.
Experiment II: Long-term exposure 24 h (without S9 mix):
After 48 h the culture medium was replaced with complete medium (with 15% FBS) containing the test item without S9 mix. The treated cells were prepared at the end of the treatment

Determination of Cell Cycle Disruption
For all experiments, the effect of the test item on cell cycle progression was investigated by addition of BrdU to the cultures. For each experiment the solvent and negative control and the highest dose group were evaluated to reassure the replication time of the cultured lymphocytes.
Evaluation criteria:
Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using microscopes with 100x oil immersion objectives. As structural chromosomal aberrations breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded.
If available, 300 well spread metaphases per concentration and validity controls were scored for cytogenetic damage.
To describe a cytotoxic effect the mitotic index (% cells in mitosis; by counting the number of mitotic cells in 1000 cells) was determined. Additionally the number of polyploid cells was scored.

Results and discussion

Test results
Species / strain:
lymphocytes: human peripheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In experiment II without metabolic activation, cytotoxic effects were observed at concentrations of 0.02 mM and higher
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The metaphases were prepared 24 h after start of treatment with the test item. The treatment interval was 4 h without and with metabolic activation (experiment I) and 24 h without metabolic activation (experiment II). Duplicate cultures were set up. Per culture 150 metaphases were scored for structural chromosomal aberrations.
The test item was diluted in DMSO and suspended in cell culture medium. Precipitate of the test item was noted at concentrations of 0.25 mM (withoutmetabolic activation) and 1.5 mM (with metabolic activation) and all higher tested concentrations after treatment. In experiment IIwithoutmetabolic activation, precipitation was observed starting at a concentration of 0.5 mM and higher concentrations.
In experiment I without and with metabolic activation, no biologically relevant decrease of the relative mitotic index was noted up to the highest evaluated concentrations of 0.25 mM and 1.5 mM, respectively. In experiment II without metabolic activation, cytotoxic effects were observed at concentrations of 0.02 mM and higher.
In both experiments, no biologically relevant increase of the aberration rates was noted after treatment with the test item without and with metabolic activation. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control.
In the experiments I and II without and with metabolic activation, no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the solvent controls.
The Fisher´s exact test was performed to verify the results in the experiment. No statistically significant increase (p < 0.05) of cells with chromosomal aberrations was noted in the dose groups of the test item evaluated in experiment I and II without and with metabolic activation.
The x² Test for trend was performed to test whether there is a concentration-related increase in chromosomal aberrations. No statistically significant increase was observed in all experimental conditions.
EMS (400 and 600 µg/mL) and CPA (5 µg/mL) were used as positive controls and induced distinct and biologically relevant increases of chromosomal aberrations, thus proving the efficiency of the test system to indicate potential clastogenic effects.

Any other information on results incl. tables

Experiments I and II:

Result tables are attched in box "Attached background material"

Applicant's summary and conclusion

Conclusions:
Molybdenum disilicide is considered to be non-clastogenic in this chromosome aberration test.
Executive summary:

In the OECD 473 in vitro chromosomal aberration test and under the experimental conditions reported, the test item Molybdenum disilicide did not induce structural chromosomal aberrations in human lymphocyte cells.