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Key value for chemical safety assessment

Additional information

Data for C.I. Pigment Green 50 (spinel pigment based on cobalt (II)/nickel (III)/zinc titanate) are not available. Thus read across was performed with C.I. Pigment Yellow 53 (nickel antimony titanium yellow). The two Nickel-containing pigments belong to a family of spinel and rutile pigments that have been tested for ion leaching (please refer to IUCLID section 7.9.3) and have been exempted from classification based on non-availability of ion toxicophores. The heavy metal oxides (used for Pigment manufacturing) are absorbed by the spinel resp. rutile lattice and thus lose their chemical, physical, and physiological properties. Both pigments show a very low water solubility (< 0.05 mg/L) being practically physiologically inert. Thus, it can be concluded, that the chemical behaviour towards the different toxicological endpoints is similar for both pigments. Therefore all toxicological endpoints were addressed with C.I. Pigment Yellow 53.

Genetic toxicity in bacteria

Key study

In a reverse mutation assay in bacteria according to OECD guideline 471, strains of S. typhimurium (TA 98, TA 100, TA 1535, TA 1537, TA 1538) and E. coli(WP2 uvrA) were exposed to C.I. Pigment Yellow 53 at concentrations of 100, 250, 500, 1000, 2500 and 5000 µg/plate in the presence and absence of metabolic activation (plate incorporation) (Corning Hazleton, 1995). The results indicate that under the conditions of this study, the test article, Nickel Antimony Titanate, did not cause a positive increase in the number of revertants per plate of any of the tester strains either in the presence or absence of microsomal enzymes prepared from Aroclor-induced rat liver (S9).

Supporting study

In a reverse mutation assay in bacteria according to OECD guideline 471, strains of S. typhimurium (TA 100, TA 1535, TA 98, TA 1537) and E. coli (WPs uvrA) were exposed to C.I. Pigment Yellow 53 at concentrations of 156, 313, 625, 1250, 2500 and 5000 µg/plate in the presence and absence of metabolic activation (pre-incubation) (MHLW, 2002). The test substance was tested up to the limit concentration. There was no evidence or a concentration related positive response of induced mutant colonies over background.

Gene mutagenicity in mammalian cells

Key study

In an in vitro mammalian cell gene mutation assay according to OECD guideline 476, L5178Y cells cultured in vitro were exposed to C.I. Pigment Yellow 53 at concentrations of 3.13, 6.25, 12.5, 25, 50 and 100 µg/mL (suspension with DMSO) in the presence and absence of mammalian metabolic activation (Aroclor-induce rat liver (S9)). Higher concentrations were not tested because of the insoluble nature of the test article. Doses were included where all visible precipitate could be removed with washing and the dosing period could therefore be controlled. Two trials of the non-activation and the S9 metabolic activation mutation assays were performed but the first trial was unacceptable because of problems in the cell cultures. In Trial 2, six treatments from 3.13 µg/mL to 100 µg/mL were initiated and all doses were cloned for mutant analysis. No cytotoxicity was observed under either activation condition. None of the six analyzed treatments with or without metabolic activation induced a mutant frequency that exceeded the minimum criterion for a positive response. Nickel Antimony Titanate was therefore evaluated as negative with and without metabolic activation at the TK locus in L5178Y mouse lymphoma cells under the conditions used in this study.

Cytogenic toxicity in mammalian cells

Key study

In a mammalian chromosomal aberration test according to OECD guideline 473 (MHLW, 2002), Chinese hamster lung cells (CHL/IU) were exposed to C.I. Pigment Yellow 53 at concentrations of:

-         9.79, 19.5, 39.1 µg/mL without metabolic activation (short term treatment)

-          19.5, 39.1, 78.1 µg/mL with metabolic activation (short term treatment)

-         4.88, 9.75, 19.5 µg/mL without metabolic activation (continuous treatment, 24 hours)

The S9 mix was composed of phenobarbital- and 5,6-benzoflavone-induced rat liver. No increase in chromosomal aberrations was observed in the test with either the short term treatment (-S9 mix and +S9 mix) or the continuous treatment.

Supporting publications

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) direct effects to DNA such as strand breaks or adducts, and (c) the influence of exposure on cytotoxicity and morphological transformation. A single in vivo study evaluating direct effects to DNA associated with intratracheal instillation was also identified.

Three gene mutation tests 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-based 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 in hprt-deficient Chinese hamster V79 cells and 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 cytotoxicity 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 calcinated 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 significant dose-related trend was not observed.

Several studies reported morphological transformations in studies that evaluated 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 for cytotoxicity), did not take up NiO, 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 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) adducts 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 genotoxic potential in a wide variety of cell types under the in vitro conditions studied; however, results were conflicting, which was likely due to the different nickel oxide compounds evaluated (and often not characterized by the authors). Data clearly indicate that the potency and mechanism of toxicity induced by various forms of nickel is dependent on the physicochemical properties (e.g., speciation and surface area) of each compound. Under a guideline-based test to screen for possible mammalian mutagens and carcinogens, green nickel oxide was not mutagenic.

 

Conclusions

Study data with the read across substance Pigment Yellow 53 did not reveal any genotoxic effects in vitro with or without metabolic activation further proving the inert character of pigments. Additionally, a guideline compliant mammalian cell gene mutation test with Nickel oxide did not reveal any mutagenicity. The above publications showed mostly conflicting results. It is therefore concluded that Pigment Green 50 is most likely regarded as not mutagenic based on the above results.


Short description of key information:
Data for C.I. Pigment Green 50 are not available. Read across was performed to studies conducted with C.I. Pigment Yellow 53. The read across substance did not reveal any genotoxic effects in vitro with or without metabolic activation further proving the inert character of pigments. However, because the substance contains the impurity nickel titanate, additional data are necessary to characterize the endpoint. No data were available for nickel titanate, instead, bridging to nickel oxide was performed. The pigment was not mutagenic in bacteria or mammalian cells in vitro and showed no cytogenetic potential. Taken together, data indicate that nickel oxide has genotoxic potential in a wide variety of cell types under the in vitro conditions studied; however, results were conflicting, which was likely 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-based 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. The study authors concluded that Nickel oxide green (substance ID N112) was non-mutagenic under the conditions tested.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for genotoxicity under Directive 67/548/EEC.

 

 

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008.