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A number of studies characterizing the genetic toxicity of nickel oxide were identified. Most of these were in vitro studies designed to assess (a) gene mutations at specific, traditionally evaluated loci, (b) chromosomal effects at the tk locus, (c) direct effects to DNA such as strand breaks or lesions, and (c) the influence of exposure on cytoxicity and morphological transformation. A single in vivo study evaluating direct effects to DNA associated with intratracheal instillation was also identified (note: a number of K3/K4 studies were also evaluated but were not addressed in this summary).

Three in vitro mammalian cell gene mutation studies evaluating three common loci were identified. Collective findings indicated that nickel oxide was not mutagenic under the conditions tested for the thymidine kinase locus (tk) or hypoxanthine-guanine phosphoribosyl transferase (hprt) locus. Though positive findings were noted in a cell line with a xanthineguanine phosphoribosyl transferase (gpt) locus sensitive to clastogens, these findings were later proven to be due to methylation and not gene mutation. The highest-rated study based on reliability was conducted by BSL Bioscience Laboratory (BSL 2008). This GLP, guideline-compliant in vitro mammalian cell gene mutation study utilized the mouse lymphoma cell line L5178Y to evaluate the potential for green nickel oxide to induce mutations at the thymidine kinase locus, both in the presence and absence of metabolic activation. No growth inhibition was observed (both with and without metabolic activation), no biologically relevant increases in mutation frequencies were observed, and all dose groups were considered as not clastogenic based on colony size. The study authors concluded that Nickel oxide green (substance ID N112) was non-mutagenic under the conditions tested.

Kargacin et al. (1993) examined the cytotoxicity and mutagenicity of black and green nickel oxides at the endogenous hprt gene in Chinese hamster V79 cells and at the gpt gene in two V79 transgenic variants (G12 and G10 cells). Both nickel oxide compounds caused dose-dependent increases in cytotoxicity and mutation frequency in G12 cells, but not in G10 or V79 cells. Similar findings were noted by Klein et al. (1994) in which nickel oxide was cytotoxic at doses tested, and that both black and green nickel oxides induced mutations in both G10 and G12 cells (but not in V79 cells) at approximately the same frequency (though the dose range for black nickel oxide was approximately 10-fold lower than that of green nickel oxide). Further analyses revealed that the phenotypic mutations in G12 cells were due to epigenetic changes in DNA methylation and not in DNA sequence.

Cytotoxicity and morphological transformation following exposure to nickel oxide has been evaluated in several cell types, including: baby hamster kidney cells, human foreskin cells, Syrian hamster embryo (SHE) cells, C3H/10T1/2 mouse embryo fibroblasts, rat tracheal epithelial (RTE) cells, and AS52 cells (modified CHO cells). A number of these studies also assessed the solubility of black and green nickel oxide relative to toxicity endpoints. Results generally indicated that although nickel oxide has genotoxic potential in vitro (based on cytoxicity and morphological transformation), the potency and mechanism of transformation by various forms of nickel is dependent on the physicochemical properties of each compound. Trends typically indicate that black nickel oxides are more potent than green nickel oxides. These studies also indicate that genotoxicity (e.g., transformation) occurs in the absence of cytotoxicity in vitro.

Sunderman et al. (1987) reported the most comprehensive evaluation on the genotoxicity of various nickel oxide compounds in their examination of the solubility, uptake, and cell transformation properties of six nickel oxides (as well as other nickel compounds) in vitro in relation to their physicochemical properties. Though there was no clear trend between phagocytosis and calcination temperature, trends were observed for cytotoxicity and transformation (as assessed in Syrian hamster embryo (SHE) cells). The two NiO compounds calcined at the lowest temperatures (identified as black and grey-black) demonstrated the greatest ability to transform SHE cells, though only the grey-black NiO exhibited a dose-related effect. Overall, green NiO was the least cytotoxic and induced the least amount of morphological transformation of all nickel oxide compounds evaluated, whereas black and grey-black were generally the most potent. The toxicity, uptake and mutagenicity of black and green nickel oxides were also evaluated in AS52 cells (modified CHO cells) by Fletcher et al. (1994). The LC50 (cytotoxicity) for black nickel oxide was much lower than that for green nickel oxide (23.3 µg/mL and 165.4 µg/mL, respectively) as measured 6-8 days after a 24-hour exposure of NiO. Mutation frequency was determined nine days after a 24-hour exposure; though both compounds exhibited mutagenic potential, a dose-related response was not observed.

Several studies reported morphological transformations whem testing a single form of nickel oxide (often as part of an evaluation of many nickel compounds). Miura et al. (1989) reported that C3H/10T1/2 mouse embryo fibroblasts treated with nickel oxide (5 μM to 1000 μM NiO) for 48 hours exhibited a reduced plating efficiency (indicative of cytotoxicity), did not take up NiO to a significant extent, and exhibited dose-dependent morphological transformations (including Type II foci), but did not induce base pair substitutions. Similar findings were reported by Patierno et al.(1993) in rat tracheal epithelial (RTE). In this study, NiO significantly increased transformation frequency. Together with an evaluation of phagocytic capacity, the authors concluded that the potency and mechanism of transformation by various forms of nickel may be different according to the physicochemical properties of each compound. Hansen and Stern (1984) reached a similar conclusion following their examination of the ability of several nickel compounds, including three doses of black NiO, to transform baby hamster kidney cells. Because equal transformation rates were observed at equal survival levels for nickel metal and all nickel compounds tested, the authors concluded that the nickel ion was the ultimate intracellular biologically active material independent of source. Relative to acetone-treated cells, a single dose of NiO induced a significant increase in frequency of anchorage independence, a characteristic of transformation thought to be an early marker of carcinogenic phenotypic alterations, in human foreskin cells (Biederman and Landolph 1987).

Multiple studies characterizing direct effects of NiO exposure to DNA were identified. First, using transmission electron microscopy, M’Bemba-Meka et al. (2005) explored the genotoxic potential (based on induction of DNA single strand breaks in chromosomal and nuclear chromatin) of several nickel compounds, including black NiO in human lymphocytes. Though exposure did not significantly reduce cell viability, NiO produced significantly higher induction of DNA single strand breaks (SSBs) than other nickel compounds tested in chromosomal chromatin. This group of researchers also reported an increase in sister chromatid exchange in human lymphocytes following a 2-hr exposure to 120 μM NiO (M’Bemba-Meka et al.2007). Second, the one study characterizing genotoxicity following in vivo exposure to nickel oxide also evaluated direct effects of exposure to DNA. Though the findings are difficult to interpret because only a single, relatively high dose was administered to rats via intratracheal instillation (a non-environmentally relevant route of exposure), findings indicate that both black and green NiO increased DNA damage as measured by 8-hydroxydeoxyguanosine (8-OH-dG) lesions in lung tissues 48 hours after exposure. This group reported conflicting findings in HeLa cells as NiO (black or green) did not induced 8-OH-dG damage in vitro.

Taken together, data indicate that nickel oxide has weak genotoxic potential (i.e., ability to induce DNA damage, oxidative damage, SCE) in a wide variety of cell types under the in vitro conditions studied; however, results were conflicting, which was partially due to the different nickel oxide compounds evaluated (and often not characterized by the authors). In vitro studies for gene mutations in mammalian cells were negative and did not suggest clastogenicity. Since nickel oxide is already classified as a category 1A carcinogen no furtherin vivotesting was conducted.

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

Taken together, data indicate that nickel oxide has weak genotoxic potential in a wide variety of cell types under thein vitroconditions studied; however, results were conflicting, which was partially due to the different nickel oxide compounds evaluated (and often not characterized by the authors). The highest-rated study based on reliability was conducted by BSL Bioscience Laboratory (BSL 2008). This GLP, guideline-basedin vitromammalian cell gene mutation study utilized the mouse lymphoma cell line L5178Y to evaluate the potential for green nickel oxide to induce mutations at the thymidine kinase locus, both in the presence and absence of metabolic activation. The study authors concluded that nickel oxide green (substance ID N112) was non-mutagenic under the conditions tested.

Value used for CSA:Genetic toxicity: negative

Justification for classification or non-classification

Ni oxide is not classified for mutagenicity according to the 1st ATP to the CLP regulation.