Nickel sulphide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Meets generally accepted scientific standards, well documented and acceptable for publication.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

publication

Title:

Genotoxic activity of nickel subsulphide alpha-Ni3S2

Author:

Arrouijal FZ, Hildebrand HF, VophiI H, Marzin D

Year:

1990

Bibliographic source:

Mutagenesis. 5(6):583-589

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

V79 cells were exposed to 0-1000 ug Ni3S2 for 3 hours; mutants were counted 3 weeks later.

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Trinickel disulphide

EC Number:

234-829-6

EC Name:

Trinickel disulphide

Cas Number:

12035-72-2

Molecular formula:

Ni3S2

IUPAC Name:

Trinickel disulphide

Details on test material:

- Name of test material (as cited in study report): alpha-Ni3S2; nickel subsulphide
- Analytical purity: 99.5%
- Other: particle size < 2 um

Method

Target gene:

HPRT

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: H21 medium with 6% FCS, penicllin and streptomycin

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

without

Test concentrations with justification for top dose:

0, 30, 100, 300, 1000 ug Ni3S2/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: no data (medium assumed)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 3 hr
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 21 days


SELECTION AGENT (mutation assays): 6 thioguanine

NUMBER OF REPLICATIONS: 6 plates per dose x 2 assays


NUMBER OF CELLS EVALUATED: mutants per 10^6 cells

Evaluation criteria:

A compound was considered as positive if it induced a reproducible and dose-related increase of mutants more than twice the spontaneous rate of mutation.

Statistics:

Not reported.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1000 ug/mL

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Not applicable.

Remarks on result:

other: strain/cell type: Chinese hamster lung fibroblasts (V79)

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Mutagenic effects were examined at the HPRT locus in V79 cells after 3 hours of exposure to Ni3S2 concentrations ranging from 30 -1000 μg/mL. Survival was not reduced in cells treated with ≤ 300 μg/mL. In cells treated with the positive control ethylmethanesulphonate, mutants increased from 19.8 ± 9 in control cells to 236 ± 12 mutants per 10^6 cells. In contrast, exposure to 30 and 300 μg/mL resulted in 1.8 and 8.4 mutants per 10^6 cells, respectively (i.e. below background).

Ultrastructural analysis (via electron microscopy) of V79 cells treated with Ni3S2 for 6 hours revealed electron dense particles bound to the cell membrane, cytoplasmic vesicles and vacuoles, but were not observed on any other organelles, the cytoplasm, or the nucleus.

Applicant's summary and conclusion

Conclusions:

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

The HPRT test resulted in no significant difference between control and treated cells. Ultrastructural examination revealed that Ni3S2 bound to the cell membrane, and that very few particles were found in the cytoplasm and none in the nucleus.

Executive summary:

Arrouijal et al. (1990) examined the clastogenic and mutagenic effects of Ni3S2 using in vitro and in vivo systems in vivo results are described in the Genetic Toxicity In Vivo section). Mutagenic effects were assessed by Ames test in five strains (TA 1535, TA1537, TA1538, TA98, and TA100) at concentrations ranging from 5-1500 μg Ni3S2 /plate. High toxicity (growth inhibition) was observed at ≥ 500 μg/plate. The authors stated that no mutagenic activity was observed in any of the strains; however, they did note a statistically insignificant 2-fold increase in revertants/plate at 50 and 150 μg/plate, but not at higher (cytotoxic) doses. Mutagenic effects were also examined at the HPRT locus in V79 cells after 3 hours of exposure to Ni3S2 concentrations ranging from 30-1000 μg/mL. Survival was not reduced in cells treated with ≤ 300 μg/mL. In cells treated with the positive control ethylmethanesulphonate, mutants increased from 19.8 ± 9 in control cells to 236 ± 12 mutants per 10-6 cells. In contrast, exposure to 30 and 300 μg/mL resulted in 1.8 and 8.4 mutants per 10-6 cells, respectively. Ultrastructural analysis of V79 cells treated with Ni3S2 for 6 hours revealed electron dense particles bound to the cell membrane, cytoplasmic vesicles and vacuoles, but were not observed on any other organelles, the cytoplasm, or the nucleus. The authors noted that the lack of effects in V79 cells might be explained by insufficient time for Ni3S2 to reach the nucleus.

Clastogenic effects of Ni3S2 were assessed in freshly obtained human lymphocytes. For these experiments, Ni3S2 was first dissolved in medium, then cells were treated with total suspension or the soluble fraction obtained after centrifugation. The chromosomal aberrations at each dose level did not differ greatly between the two treatments, suggesting that the soluble fraction of Ni3S2 was responsible for the adverse effects. In cells treated with the positive control K2Cr2O7, the total number of aberrations (including gaps and breaks in chromatids and chromosomes) increased from 3 to 42 per 200 cells. Statistically significant increases in aberrations (generally ≥ 20 per 200 cells) were observed in all lymphocytes treated with ≥ 10 μg/mL Ni3S2 in media without FCS. The authors characterized the response as dose-dependent. These aberrations were also observed at higher frequencies in cells that were co-incubated with 20% FCS. Ultrastructural analysis of the lymphocytes treated with suspended Ni3S2 revealed the presence of electron dense particles in the cytoplasm and “especially in the nucleus.” These differences in Ni3S2 localization were posited to relate to differences in cell type and/or incubation time (i.e. 6 hr vs 24 hr). These clastogenic effects were supported by in vivo experiments that are described elsewhere (see above).

The authors concluded that their study demonstrated for the first time the in vitro clastogenic activity of α-Ni3S2 on human lymphocytes. This particular in vitro test system seems to suggest that only the soluble fraction is responsible for this activity. STUDY RATED BY AN INDEPENDENT REVIEWER