Zinc oxide

Endpoint summary

Additional information

Several in vitro studies and one in vivo study are available on the genotoxicity of zinc oxide. Data on other zinc compounds have also to be taken into account, as the basic assumption is made that after intake all zinc compounds (including metallic zinc) are changed (at least in part) to the ionic species and that it is this zinc cation that is the determining factor for the biological activities of the zinc compounds.

The genotoxicity of soluble and slightly soluble zinc compounds have been extensively investigated in a wide range of in vitro and in vivo studies. The in vitro investigations included non-mammalian and mammalian test systems covering the endpoints of gene mutation, chromosomal aberrations, sister chromatide exchange, unscheduled DNA synthesis (UDS), as well as cell transformation. Availablein vivogenotoxicity assays included the micronucleus test, sister chromatide exchange (SCE) and chromosomal aberration test and the dominant lethal mutation assay in mouse or rat as well as investigations for sex-linked recessive lethal mutation in drosophila melanogaster.

The investigated zinc compounds did not increase the mutation frequencies in the majority of bacterial or mammalian cell culture systems. For example, zinc chloride, zinc sulphate, zinc bis(dihydrogen phosphate), zinc oxide or zinc monoglycerolate were consistently negative in the Ames test. While zinc chloride was also negative for gene mutations in the mouse lymphoma assays, there was some evidence that zinc oxide, zinc acetate or zinc monoglycerolate induced in the absence of metabolic activation the formation of mutation colonies. Several reviewers noted, however, that these mutations were observed at cytotoxic concentrations and that the analysis did not distinguish between big and small colonies which could be caused by gene mutation or chromosomal aberrations (Thompson et al.,1989, WHO, 2001; EU RAR, 2004; MAK, 2009).

Conflicting information was further found when zinc compounds were examined for their potential to induce chromosomal aberrations or sister chromatide exchange in mammalian cell systems or when evaluated in the cell transformation assay. Positive as well as negative results were obtained in these cell systems with either soluble or slightly soluble zinc compounds. In those studies where chromosomal aberrations or sister chromatide exchange has been observed, these were generally considered to be weak and occurred only at high, often cytotoxic concentrations. Moreover, these positive in vitro findings have also to be seen in context of the impact that changes in zinc levels can have on cell system processes that are controlled by a strict metal homeostasis. A change of this metal homeostasis due to increased zinc levels, may lead to a binding of zinc to amino acids like cystein and therefore to an inhibition of certain enzymes. This can lead to interactions with the energy metabolism, signal transmission and apoptotic processes which can lead to the observed clastogenic or aneugenic effects in in vitro systems (EU RAR, 2004; MAK, 2009).

In addition to above mentioned in vitro investigations, various soluble and slightly soluble zinc compounds have also been studied in a range of in vivo studies including the micronucleus test, SCE and chromosomal aberration test or dominant lethal mutation assay in mice or rats as well as in the Drosophila Melanogaster SLRL test. The zinc compounds were consistently negative in the micronucleus and in the assay with Drosophila Melanogaster. Zinc sulphate was further negative in a dominant lethal assay in rats.

Equivocal and sometimes contradicting results were found for the induction of chromosomal aberrations which have been studied in bone marrow cells harvest from animals exposed to zinc compounds zinc chloride, and zinc oxide. Negative findings for chromosome aberrations have been produced after intraperitoneal injection of zinc chloride into mice (Vilkina et al., 1978) or when rats were given zinc sulphate by gavage once or daily for 5 consecutive days (Litton Bionetics, 1974). In contrast, increased aberrations have been reported in rats after inhalation exposure to zinc oxide (Voroshilin et al., 1978), in rats after oral exposure to zinc chloride and in mice after multiple intraperitoneal injections of zinc chloride (Gupta et al., 1991). Moreover, increased chromosomal aberrations were found in calcium-deficient mice when fed zinc (in form of zinc chloride) via the diet (Deknudt, 1982).These equivocal finding likely a reflection of inter-study differences in routes, levels, and duration of zinc exposure, the nature of lesions scored (gaps compared to more accepted structural alterations) and great variability in the technical rigour of individual studies (WHO, 2001). The German MAK committee reviewed the existing in vivo evidence and concluded that particularly those studies indicating clastogenic effects involved a lot of methodological uncertainties which do not allow overruling those in vivo studies which did not provide any evidence for chromosomal aberrations in vivo. Moreover, the Dutch rapporteur of EU risk assessment of zinc compounds under the EU existing substance legislation considered the positive in vitro findings for chromosomal aberration and SCE assays to be overruled by the overall weight of evidence of negativein vivotests for this endpoint (EU RAR, 2004).

 

Human information:

The only identified publicly available genotoxicity study in humans related to the identification of chromosomal aberrations in lymphocytes of 24 workers in a zinc smelting plant (Bauchinger et al.,1976). This study was, however, not suitable to draw any conclusions to the association of these effects with zinc exposure, as the workers displayed also increased blood levels of lead and cadmium, and the clastogenic effects were predominantly attributed to cadmium exposure.
There were no further reports in the accessible literature on genotoxic effects of zinc compounds in human populations.

 

Zinc oxide nanomaterial:

In vitro:

Nano-ZnO did not increase the mutation frequencies in bacterial cell culture systems: it was consistently negative in the Ames test. There was some evidence that nano-ZnO induced the formation of mutation colonies. However,significantly increased mutation frequencies were always linked to acute cytotoxicity. For this reason and the limited significance of the test system for particles, the test results should more likely be judged as equivocal/questionable in L5178Y/TK cells under the conditions and restrictions of this assay, implying a possible false positive result.

Nano-ZnO data have shown that DNA damage was detected in the comet assay in the absence of a metabolic activation system after exposure to uncoated ZnO nanoparticles; there was evidence that these effects were mediated by generation of oxygen species and oxidative stress. Other studies have also showninduction of DNA damage but these occurred only at high, often cytotoxic concentrations.
Conflicting information was found when nano-ZnO was examined for its potential to induce chromosomal aberrations or micronucleus induction.A chromosome aberration test according to OECD Guideline 473 was performed with V79 cells using Z-COTE® HP1, Z-COTE®, and micronised zinc oxide. No increase in structural chromosome aberrations and no aneugenic activity were detected. In contrast, other data revealed clastogenic activity also below the threshold for cytotoxicity. 

In vivo:

Systemic clastogenic and aneugenic effects were investigated in a sub-acute nose-only inhalation toxicity study in male and female Wistar rats using the bone marrow micronucleus assay (according to OECD Guideline 474). No systemic chromosome mutagenic activity was detected after inhalation exposure toZ-COTE^® HP1 or uncoated ZnO nanoparticles or micronised ZnO at dose levels inducing local effects (see section repeated dose toxicity(Bellmann, 2011)) in the lung.

Z-COTE HP1 tested in thein vivomicronucleus test in bone marrow cells of mouse after single intraperitoneal injection (according to OECD Guideline 474) does not induce any chromosome damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

In mice administered via intragastric gavage both ZnO nanoparticles and ZnO microparticles, it was shown that both ZnO nanoparticles and ZnO microparticles were not considered to be clastogenic in the in vivo micronucleus assay.

 

DNA strand breaks and oxidative DNA damage were analysed ex vivo in BAL cells of Wistar rats using the hOGG1-modified Comet assay. DNA damage was not observed 24h after last exposure, but occurred after 14 days of recovery. The available data on genotoxicity in vitro partly supported evidence for local genotoxic effects. However, it is questionable, whether BAL cells are a suitable surrogate for epithelial cells in the lung, which are the relevant cells for tumour development. No oxidative DNA damage was detected in epithelial cells of the lung parenchyma using immunohistochemical methods indicating lack of local genotoxicity in relevant cells mediated by Reactive oxygen species (ROS). Although an increase in pro-inflammatory cytokines (coated ZnO) was observed, there is no evidence of substance specific genotoxic potential.

 

In conclusion, for nano-ZnO no nano-specific mutagenic/genotoxic effects could be identified. Zn²⁺ion determines the toxicity of ZnO and read across between various forms of ZnO (micro-scale, nano, coated or not) is fully supported. 

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The overall weight of the evidence from the existing in vitro and in vivo genotoxicity assays suggests that zinc compounds do not have biologically relevant genotoxic activity. This conclusion is in line with those achieved by other regulatory reviews of the genotoxicity of zinc compounds (WHO, 2001; SCF, 2003; EU RAR, 2004, MAK, 2009). Hence, no classification and labelling for mutagenicity are required.

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