Trinickel disulphide

Endpoint summary

Additional information

A number of studies characterizing the genetic toxicity of nickel subsulphide were identified. Several multi-component studies that evaluated genotoxicity both in vivo and in vitro were included in the evaluation. Generally, all of the studies identified relied on test systems that were specific to evaluating genotoxicity.

A comprehensive study published by Arrouijal et al.(1990) demonstrated positive in vitro clastogenic activity of α-Ni₃S₂ in human lymphocytes. Statistically significant, dose-dependent increases in chromosomal aberrations (generally ≥ 20 per 200 cells) were observed in all lymphocytes treated with ≥ 10 μg/mL Ni₃S₂. In vitro gene mutation study in bacteria were also assessed by Ames test in five strains; high toxicity (growth inhibition) was observed at concentrations of ≥ 500 μg/plate, though no mutagenic activity was observed in any of the strains. In vitro gene mutation effects were also negative at the hprt locus in V79 mammalian cells under the conditions tested. The authors of this study concluded that the soluble fraction was responsible for genetic toxicities observed. The authors also included an in vivo gene mutation study that involved assessment of Ni₃S₂ using the micronucleus test in mice. Following a single i. p. injection, mice exhibited a significant increase of micronuclei frequency in polychromatic erythrocytes (PCEs) and a significant decrease in the number PCEs. Together with in vitro findings, the authors concluded that Ni₃S₂ when injected as a suspension can cause in vivo clastogenic effects. Intraperitoneal injection is a non physiological route of administration for a water insoluble nickel compound like Ni₃S₂.

In a multi-component study, Chakrabarti et al.(1999) reported dose-dependent formation of DNA-protein crosslinks (DPX) in isolated rat renal cortical slices (RCS). Additional evaluations of the potential mechanisms of toxicity suggested that Ni (from Ni₃S₂) binds to amino acids on proteins as well as other peptides such as glutathione. Ca²⁺ had no effect on the ability of Ni₃S₂ to induce DPXs. The authors also found that amino acids inhibited Ni uptake associated with Ni₃S₂ exposure, and that incubation of RCS with increasing doses of Ni₃S₂ resulted in a dose-dependent increase in reactive oxygen species. In a subsequent study, this group of researchers applied a similar approach in evaluating the genotoxicity in rat lymphocytes. Ni₃S₂ induced DPX in a dose- and time-dependent manner. The DPX were prevented by co-exposures to a number of amino acids, which was likely due to the inhibited uptake of Ni. Additionally, incubation of lymphocytes with increasing doses of Ni₃S₂ resulted in a dose-dependent and significant increase in ROS – this effect was also reduced by co-exposure to a number of amino acids. In a separate multi-part study, Kawanishi et al. (2002) demonstrated Ni₃S₂ exposure significantly increased the 8-hydroxy-2’-deoxyguanosine (8-OH-dG) content in HeLa cells, and further demonstrated that this response correlated with measures of 8-OH-dG in lung tissues of Wistar rats exposed to Ni₃S₂ via intratracheal instillation.

A number of studies evaluated genotoxicity in vitro, demonstrating that Ni₃S₂ is associated with cytotoxicity and genotoxicity (mostly clastogenicity) in a variety of laboratory cell culture models. For example, Ni₃S₂ induced point mutations that were likely due to DNA damage in a modified CHO cell line (Rossetto et al.1994). Data also demonstrate that Ni₃S₂ produced anchorage independence (AI; characteristic of transformation) in human foreskin cells (Biederman and Landolph 1987). Kargacin et al. (1993) found that the mechanisms of mutagenicity in vitro appeared to differ across transgenic cell lines as demonstrated by variable changes in cytotoxicity and mutation frequency in three hprt-deficient Chinese hamster V79 cells. It was later determined that the apparent mutagenicity observed in some transgenic cell lines was due to hypermethylation and not true mutations (Klein et al. 1994; Mayer et al. 1998). Lastly, a recent study that followed the OECD test guidelines for in vitro genotoxicity found equivocal evidence Ni₃S₂ genotoxicity (BSL, 2009). Using the mouse lymphoma thymidine kinase locus in the L5178Y cell line, the results demonstrated that Ni₃S₂ induced some elements of genotoxicity (elevated mutation frequency relative to controls with and without metabolic activation at high concentrations), but did not produce results that met all criteria associated with this genotoxicity model (concentrations at which elevated mutation frequency were observed often coincided with high cytotoxicity).

Mayer et al.(1998) also conducted a multi-component study to evaluate the mutagenicity and genotoxicity of Ni₃S₂ both in vitro and in vivo. Results indicated that Ni₃S₂ administration increased mutation frequency up to 4.5-fold during in vitro mutation studies using a BigBlueTM Rat 2 embryo fibroblast cell line (lac1 transgenic line). In the comet assay, Ni₃S₂ induced DNA fragmentation in a concentration dependent manner, though Ni₃S₂ did not affect the viability of the cells. The authors concluded that reactive oxygen species seem to be, at least in part, responsible for these in vitro effects. However, the authors further stated that while there was an indication of oxidative damage, nickel compounds have been negative in most bacterial mutagenicity assays, even in strains that specifically detect oxidative damage. Because of these conflicting findings assays were conducted in transgenic rodent mutation models to investigate the DNA damaging effect and mutagenic potential of nickel subsulphide in target cells of carcinogenesis. CD2F1 mice and lacZ transgenic CD2F1 mice (MutaMouse mice), as well as F344 rats and lac1 transgenic F344 rats (Big Blue rats), exposed to Ni₃S₂ via inhalation did not exhibit the same type of responses observed in vitro, and treatment-related genotoxic effects were generally related to DNA damage in mice. Thus, the authors suggested that the collective findings were supportive of a non-genotoxic model for nickel carcinogenesis. A repeated dose inhalation study with nickel subsulphide in rats can be read across to nickel sulphide (Benson et al., 2002). This study found that inhalation exposures to nickel sulphate and nickel subsulphide at toxic levels can cause genotoxicity in the respiratory tract in vivo. However, only Ni₃S₂ exposure (0.6 mg/m³, 0.44 mg Ni/m³) significantly increased epithelial and nonepithelial cell proliferation after 3 and 13 weeks.

Taken together, data were generally sufficient to characterize the genotoxicity of Ni₃S₂. A weight evidence evaluation indicates that genotoxic potential is dependent on the test system, though under the conditions reported, Ni₃S₂ is associated with a variety of genotoxicities including DNA damage. However, the mechanism and conditions inducing genotoxicity in vivo, and in non-laboratory species, are not clear based on available data.

The following information is taken into account for any hazard / risk assessment:

A weight of the evidence evaluation indicates that genotoxic potential is dependent on the test system, though under the conditions reported, Ni₃S₂ is associated with a variety of genotoxicities including DNA damage. However, the mechanism and conditions inducing genotoxicity in vivo, and in non-laboratory species, are not clear based on available data. Recently, nickel compounds have been recognized as genotoxic carcinogens with threshold mode of action in ECHA RAC opinion on nickel and nickel compounds OELs (see ECHA 2018 report discussion inAppendix C2).

Value used for CSA:Genetic toxicity: positive

Justification for classification or non-classification

Ni subsulphide is classified as Muta. 2; H341 according to the 1st ATP to the CLP Regulation. Background information can be found in the discussion section.