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Genetic toxicity in vitro

Description of key information

Three OECD guideline studies were performed to assess the genotoxicity potential of Molybdenum disilicide. The substance resulted not genotoxic in the bacterial reverse gene mutation assay (Ames test), non-mutagenic in an in vitro mammalian cell gene mutation test and not inducing structural chromosomal aberrations in an in vitro mammalian chromosome aberration test.

In conclusion, Molybdenum disilicide is not expected to exert genetic toxicity. 

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-04-28 to 2017-06-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name: Molybdenum disilicide
- CAS No: 12136-78-6
- Batch No: 86438
- Physical State: solid, powder
- Colour: grey
- Purity: >= 98.57 %
- Expiry Date: not provided by sponsor
- Storage conditions: room temperature, store sealed in original packaging in dry room

Target gene:
Histidine locusn
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells:
TA98, TA1535 and TA102: MOLTOX, INC., NC 28607, USA
TA100 and TA1537: Xenometrix AG, Switzerland

MEDIA USED:
Nutrient medium (per litre):
- 8 g Nutrient Borth
- 5 g NaCl
(solution of 125 µL ampicillin (10 mg/mL) (TA98, TA100, TA102) was added to retain phenotypic characteristics of strain)

Vogel-Bonner-salts (per litre)
- 10 g MgSO4 x 7 H2O
- 100 g citric acid
- 175 g NaNH4HPO4 x 4 H2O
- 500 g K2HPO4

Vogel-Bonner Medium E agar plates (per litre)
- 15 g Agar Agar
- 20 mL Vogel-Bonner salta
- 50 mL glucose-solution (40%)

Overlay agar (per litre)
- 7 g Agar Agar
- 6 g NaCl
- 10.5 mg L-histidine x HCl x H2O
- 12.2 mg biotin

Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
The test item concentrations in the main experiments were chosen according to the results of the pre-experiment:
Experiment 1 and 2: 31.6, 100, 316, 1000, 2000, 5000 µg/plate
Vehicle / solvent:
- Vehicle: A. dest.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Aqua dest.
True negative controls:
no
Positive controls:
no
Positive control substance:
sodium azide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Aqua dest.
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-NOPD
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Aqua dest.
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Aqua dest.
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION
- plate incorporation: Experiment 1; pre-incurbation: Experiment 2

EXPERIMENTAL PERFORMANCE
For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
- 100 µL Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control),
- 500 µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
- 100 µL Bacteria suspension (cf. Preparation of bacteria, pre-culture of the strain),
- 2000 µL Overlay agar.
For the pre-incubation method 100 μL of the test item preparation was pre-incubated with the tester strains (100 μL) and sterile buffer or the metabolic activation system (500 μL) for 60 min at 37 °C prior to adding the overlay agar (2000 μL) and pouring onto the surface of a minimal agar plate. After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: clearing or diminution of background lawn or reduction in number of revertants down to mutation factor of approximately < 0.5 in relation to solvent control.
Evaluation criteria:
The mutation factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and TA102 the number of reversions is at least twice as high
- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher than the reversion rate of solvent control.

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.
Statistics:
not applicable
Key result
Species / strain:
other: TA98, TA100, TA1535, TA1537, TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
No precipitation of test item was observed in any tester strain used in experiment 1 and 2 (with and without metabolic activation). No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in experiment 1 and 2.
No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Molybdenum dislicide at any concentration level, neither in the presence nor absence of metabolic activation in experiment 1 and 2.
All criteria of validity were met. The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiment. Only in experiment 2 in tester strain TA102 (with metabolic activation) a low mutation factor was found. Nevertheless, compared to the mutation factors found with the test item concentrations the increase can be considered as distinct. Thus, this effect was regarded as not biologically relevant.

Pre-experiment for toxicity and Main experiments I and II:

Result tables are attched in box "Attached background material"

Conclusions:
In conclusion, the test item is not genotoxic in the bacterial reverse gene mutation assay in the presence and absence of mammalian metabolic activation.
Executive summary:

In a reverse gene mutation assay in bacteria (OECD 471) strains of S. typhimurium (TA98, TA100, TA1535, TA1537, TA102) were exposed to Molybdenum disilicide (≥ 98.57 % purity) in Aqua dest. at concentrations of 31.6, 100, 316, 1000, 2000 and 5000 µg/plate (experiment 1: plate incorporation, experiment 2: pre-incubation) in the presence and absence of mammalian metabolic activation. Molybdenum disilicide was tested up to the limit dose (5.0 mg/plate). The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) 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
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
Specific details on test material used for the study:
Batch number: 15535
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.
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.
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.

Experiments I and II:

Result tables are attched in box "Attached background material"

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.

Endpoint:
in vitro gene mutation 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
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Specific details on test material used for the study:
Name: Molybdenum disilicide
CAS No.: 12136-78-6
Batch No.: 15535
Molecular Weight: 152.11 g/mol
Physical State: odourless powder
Colour: grey
Purity: >99.3%
Expiry Date: 29 November 2019
Storage Conditions: at room temperature; keep dry
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
rat liver microsome preparations (S9 mix)
Test concentrations with justification for top dose:
The test item was investigated at the following concentrations:

without metabolic activation:
0.04, 0.10, 0.75, 2 and 5 mM

and with metabolic activation:
0.025, 0.05, 0.10, 0.25 and 0.50 mM

The selection of the concentrations was based on data from the pre-experiments.
In the main experiment 5 mM (without metabolic activation) and 0.50 mM (with metabolic activation) were selected as the highest concentrations.
Untreated negative controls:
yes
Remarks:
treatment medium, duplicate cultures
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
The experiment with and without metabolic activation was performed as a 4 h short-term exposure assay
Evaluation criteria:
A test chemical is considered to be clearly negative if, in all experimental conditions examined,
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend-test, and
- all results are inside the distribution of the historical negative control data.

A test chemical is considered to be clearly positive if, in any of the experimental conditions examined
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- the increase is concentration-related when evaluated with an appropriate trend test, and
- any of the results are outside the distribution of the historical negative control data.
- if there is by chance a low spontaneous mutant frequency in the corresponding negative and solvent controls a concentration-related increase of the mutations within their range has to be discussed.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity

In the main experiment precipitation was observed at concentrations 5 mM (withoutmetabolic activations) and 0.50 mM (withmetabolic activation).

Toxicity:

No biologically relevant growth inhibition (reduction of relative survival below 70%) was observed after the treatment with the test item in experimentwithoutmetabolic activation (Table3).

In the experimentwithmetabolic activation a biologically relevant growth inhibition (reduction of relative survival below 70%) was observed after the treatment with the test item. The relative survival was 57% for the highest concentration (5 mM) evaluated (Table 3). Additionally, at concentration 0.75 mM the relative survival was 46%, but since the next lower and next higher concentration showed a relative survival above 70%, this effect was considered as an outlier and therefore not relevant.

 

Mutagenicity:

In the main experimentwithoutandwithmetabolic activation all validity criteria were met. The mutant values of the negative and solvent controls fall within the historical data range of the test facility and the cloning efficiencies of the negative and solvent controls are > 50%.

The positive controls, DMBA (1.0 µg/mL) and EMS (300 µg/mL) showed statistically significant increases in mutant frequency, thereby demonstrating both the sensitivity and validity of the test systems.

Experiment without metabolic activation

In the experimentwithoutmetabolic activationthemutant values of the negative, solvent controls and all mutant values of the test item concentrations found were within the historical control data of the test facilityEurofins Munich(about 8.5 - 40.2 mutants per 106cells).The positive control EMS induced a distinct increase in mutant frequency with 174.6 mutants/106cells (Table4).

The mutant frequencies induced by the test item did not show a biologically relevant increase (Table4). None of the observed mutant frequencies was statistically significantly increased over those of the solvent controls (Table7).

The highest mutant frequency was observed at a concentration of 0.10 mM (39.5 mutants per 106cells) with a relative survival of 71%.

 

           Experiment with metabolic activation

In the experimentwithmetabolic activationthe mutant values of the negative, solvent controls and all mutant values of the test item concentrations found were within the historical control data of the test facilityEurofins Munich(about 9.6 - 44.0 mutants per 106cells).The positive control DMBA induced a distinct increase in mutant frequency with 439.7 mutants/106cells (Table6).

The mutant frequencies induced by the test item did not show a biologically relevant increase (Table6). None of the observed mutant frequencies was statistically significantly increased over those of the negative controls (Table8).

The highest mutant frequency was observed at a concentration of 0.05 mM (39.5 mutants per 106cells) with a relative survival of 102%.

Conclusions:
The test item Molybdenum disilicide was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.
Under the experimental conditions reported, the test item Molybdenum disilicide is considered to be non-mutagenic at the HPRT locus using V79 cells of the Chinese Hamster.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the available data, Molybdenum disilicide does not warrant classification for mutagenicity.