Concentrates of lead and zinc compounds with sulfur resulting from hydrometallurgy (hot acid leaching, super-hot acid leaching and flotation)

Endpoint summary

Additional information

The health hazard assessment was conducted based on the most critical constituents of the substance. This substance is an UVCB substance and can be described as a moist solid powder which is insoluble to water. According to the chemical composition analysis, the main phases of the substance are lead sulphate and zinc sulphide. The product consists primarily of sulphur (ca. 35 %), lead (ca. 25 %) and zinc (ca. 17 %) together with minor trace elements such as silver, silicon, aluminium, calcium and iron.

The transformation/dissolution study (OECD guidance 29) was conducted for the substance and the results of this study were used for the chemical safety assessment. The results of that study indicated that the release at pH 6 was higher for all studied elements compared to release at pH 8. Therefore the following 7 and 28 day studies were conducted at pH 6. Based on the screening test results (loading rate 100 mg/L), the most critical components for the assessment were lead and zinc, with releases of 8282 µg/L and 75.4 µg/L, respectively. The other minor leachable metals were silver (34.7 µg/L), cadmium (0.48 µg/L) and copper (17.2 µg/L).

Results from the 7 day T/D test (loading rate 100 mg/L, pH 6) showed similar trend in release rates: 12333 µg/L (Pb), 91.4 µg/L (Zn), 15.6 µg/L (Cu), 31.4 µg/L (Ag) and 0.056 µg/L (Cd). In the 28 day test with lower loading rate (1 mg/L, pH 6), only concentrations of Pb (362.4 µg/L) and Zn (3.2 µg/L) were over the detection limits or blank sample values.

According to T/D study results, the most soluble and critical components of this substance are lead and zinc. Therefore, the studies for this endpoint have been selected as a read-across data for the critical constituents. The read-across justification is presented in CSR annex I. All read-across data for toxicology are based on test data using either soluble Pb or Zn salts or measured (dissolved) Pb or Zn concentrations. The weight of evidence approach was used to make conclusions on the key value for CSA. Conclusion for this endpoint is based on read-across data from zinc and lead compounds.

Lead compounds

The genotoxic profile of lead is mixed. Bacterial mutagenesis assays produce negative results while conflicting (positive and negative) observations have been made in mammalian cell mutagenesis systems. In the absence of confirmation that lead was in fact taken up by bacteria, negative results in bacterial systems will not be assigned significance in a weight of evidence evaluation.

With few exceptions, in vitro studies of lead’s effects upon eukaryotic cells in vitro have employed high concentrations of soluble lead compounds producing significant levels of cytotoxicity and only weak genotoxic responses. A central issue that requires resolution is whether mechanisms for in vitro genotoxicity possess physiological relevance, by virtue of the mechanisms involved or the concentrations required to produce effects. For example, induction of genotoxic effects in cultured cells at lead concentrations in the µM or mM range would have limited relevance to in vivo exposures wherein the concentration of lead available for transfer to the soft tissues is in the nM range or lower. Extrapolation of the effects of soluble lead compounds to the compounds that are the subject of this risk assessment is further complicated by the sparingly soluble nature of the metal and its’ compounds. The compounds, while largely untested for mutagenicity in vitro, will not undergo dissolution in neutral aqueous media to an extent that will yield lead ion concentrations adequate to induce the weak effects reported for soluble compounds.In vitro mutagenicity assay results would thus be expected to be negative if tests were conducted using sparingly soluble compounds but the lead cation itself appears to have weak genotoxic potential. This activity does not appear to entail direct interaction with DNA – instead indirect mechanisms have been proposed to mediate genotoxicity.

Multiple indirect mechanisms have been proposed for lead genotoxicity in vitro but not all are concordant with the genotoxicity response profiles observed. For example, although some studies have suggested that noncytotoxic lead concentrations can interfere with the mitotic spindle and induce aneuploidy that manifests as micronucleus induction, the concentrations required to disrupt spindle formation are higher than those that induce micronuclei. Conversely, interference with spindle formation would not be expected to produce the DNA damage or point mutations that have been reported to accompany micronucleus induction in other studies. There is thus inconsistency between the dose responses for genotoxic effects observed and some of the underlying mechanisms that have been proposed to produce them. Although lead may be capable of inducing genotoxicity by multiple mechanisms, it is not yet possible to ascertain which mechanism, or group of mechanisms, is of greatest importance and/or of physiological relevance in producing the spectrum of changes suggested by in vitro studies.

In vivo studies using experimental animals are similarly characterised by conflicting results for endpoints such as DNA damage, chromosome aberrations, micronuclei and sister chromatid exchange induction. In most studies responses have occurred after lead compounds were administered via exposure routes (e. g. i. v., i. p. or s. c. injection) that have limited relevance to normal exposure routes and/or that are difficult to compare on a dosimetric basis to lead administered via ingestion. Furthermore, in many instances, only single doses have been studied and dose response relationships that help to validate the significance of a positive finding cannot be evaluated. When multiple doses have been evaluated, especially in injection studies, the dose response for genotoxic effects has either been weak, non-existent or inverse. Poor dose dependency under such circumstances is likely an indication of systemic or tissue toxicity that limits response. Injection routes of administration bypass the normal toxicokinetic processes responsible for the uptake and distribution of lead – 99% of the lead taken up into the blood following oral or inhalation exposure is bound within the red blood cell and only a small fraction (~1%) of lead in the blood is available for transfer to the soft tissues. Studies have not documented the free or biologically available lead in blood concentrations that result from i. p. or i. v. administration routes but the concentrations are likely far higher, perhaps by three orders of magnitude, than those that can be achieved via physiological routes of administration prior to the onset of lethality or other severe manifestations of systemic toxicity. For this reason, the dosimetry for genotoxic effects from injection studies is difficult to compare to other effects of concern such as carcinogenicity. Studies evaluating the comparative toxicokinetics of lead after oral and i. v. administration are ongoing and should assist in resolving this issue.

Given the preceding concerns regarding dosimetry for effects and the induction of indirect mechanisms with physiological relevance, studies conducted using physiologically relevant routes of exposure are especially important. Oral or inhalation exposure to high levels of soluble lead compounds produce negative or equivocal responses in all but one study. Assays for chromosome damage are either negative or report effects (e. g. weak induction of chromosome gaps) that are not now believed to be true indicators of a mutagenic response. Induction of weak positive responses can also require non-standard test conditions (e. g. extreme calcium deficiency) that make results difficult to interpret. A single study reported aberrations in bone marrow cells and spermatocytes, but the levels of Pb administration were high and cytotoxicity was not monitored. Lack of information regarding systemic lead levels precludes comparison of the study results from studies with similar dosing levels but negative findings. In this instance, the weight of evidence derived from four negative studies of high and comparable study quality would indicate that chromosomal aberrations are not induced by oral lead administration.

Several findings of micronucleus induction were reported but are difficult to interpret. One observed a low level response in polychromatic ethrythrocytes after prolonged exposure to high levels of lead – the response observed may have been an artifactual positive produced by anaemia. Other studies observed micronuclei following the administration of high levels of lead but did not adequately control for cytotoxicity. Only one germ cell mutagenesis assay was found reported in the literature, but although the administration of lead in drinking water did not produce a response in the dominant lethal assay, the dose administered only produced a modest elevation of blood lead.

When studies are ranked by overall study quality, negative response is generally seen in the higher quality studies and suggestions of effects generally are relegated to low quality studies. Responses in so-called indicator assays (SCE induction or the Comet assay) have been reported as positive with greater frequency but are difficult to interpret in light of the mostly negative findings from true mutagenicity assays and a failure if indicator assay studies to adequately monitor apoptosis and, in most instances, cytotoxicity.

This inconsistent response profile extends to observational studies in humans where endpoints such as chromosome aberrations, micronuclei and sister chromatid exchange induction have been evaluated. Both positive and negative studies exist, but even positive studies are characterised by weak or non-existent dose responses and small effect sizes. Furthermore, almost without exception, studies in humans have failed to monitor potential impacts of lead upon apoptosis or cellular toxicity and measurements are generally lacking of co-exposures to other substances in the workplace that may have genotoxic potential. The lack of a cohesive response profile, combined with technical inadequacies in most studies, does not support the presence of significant in vivo genotoxic activity in humans.

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 chromatid exchange, unscheduled DNA synthesis (UDS), as well as cell transformation. Available in vivo genotoxicity assays included the micronucleus test, sister chromatid 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, 2008; MAK, 2009).

Conflicting information was further found when zinc compounds were examined for their potential to induce chromosomal aberrations or sister chromatid 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, 2008; 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.

As discussed in section 5.7.1.2, equivocal and sometimes contradictory results were obtained in the in vivo chromosomal aberration assays. 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 negative in vivo tests for this endpoint (EU RAR, 2008).

Conclusions

The assessment of the potential mutagenicity of this UVCB substance is based on the overall weight of evidence on the composition of the substance and the bioavailability and the genotoxicity of the most critical components (zinc and lead). According to the in vitro and in vivo results from these components the substance is not expected to cause genotoxicity. Thus, no classification for this endpoint is warranted.

Justification for selection of genetic toxicity endpoint
No study conducted for the target UVCB substance. The hazard assessment conclusion is based on the overall weight of evidence of the results of several in vitro and in vivo tests conducted for read-across substances (ie. bioavailable lead and zinc compounds).

Short description of key information:
According to transformation/dissolution study (OECD guidance 29) conducted for the substance, the most critical constituents leachable to water from this UVCB substance are lead and zinc compounds. Therefore, the chemical safety assessment focuses on the properties of these constituents and the key values for CSA are selected based on the read-across data on the most bioavailable compounds of Pb and Zn.

The overall weight of the evidence from the existing in vitro and in vivo genotoxicity assays conducted for read-across substances suggest that this UVCB substance does not have biologically relevant genotoxic activity.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based upon the weight of evidence of in vivo and in vitro tests from read-across lead and zinc compounds permits the conclusion to be drawn that no classification for mutagenicity is required.