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Genetic toxicity in vitro:

Only two studies are available with zirconium basic carbonate. Therefore, read across was performed using studies performed with zirconium dioxide, a similar non-soluble zirconium compound, and zirconium acetate, a soluble zirconium compound. The read across justification is added in Section 13 of IUCLID.

Bacterial reverse mutation assay: Two studies are included to cover this endpoint using a 'weight-of-evidence approach': one study performed with zirconium basic carbonate and a second one with zirconium dioxide.

BIBRA (1987) performed an Ames test (OECD 471) with Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 with and without metabolic activation. The test item was zirconium carbonate.

Following concentrations were tested:

Method 1: 0, 1, 10, 100, 1000, 10000 µg/plate (preliminary toxicity study)

Method 2: 0, 0.2, 1.0, 5.0, 20.0, 100 µg/plate (plate incorporation test)

Method 3: 0, 0.2, 1.0, 5.0, 20.0, 100 µg/plate (plate incorporation test)

Method 4: 0, 0.2, 1.0, 5.0, 20.0, 100 µg/plate (liquid incubation test)

Number of replicates tested: Method 1: duplicate; Method 2: triplicate; Method 3: triplicate; Method 4: triplicate.

No increase in the number of revertants was seen in either of the strains, with or without S-9, with exposure to the test substance.

Negative controls and positive controls were run in parallel and were considered to be valid.

According to the results of the study, the test substance is not mutagenic in the Ames test with and without metabolic activation.

The above-mentioned study was performed according to the OECD 471 guideline adopted in 1983 where only 4 strains were required and not Salmonella typhimurium strains TA102 or E. coli strain. Although the test is reliable and relevant to cover this enpoint, the strains used may not detect certain oxidising mutagens, cross-linking agents and hydrazines. Therefore, read-across to zirconium dioxide, another 'insoluble' zirconium compound, is proposed. Indeed, comparison of the water solubility can be used as surrogate to assess the bioavailability of metals, metal compounds and other inorganics compounds. So, information on other ‘insoluble’ zirconium salts as zirconium dioxide can be used as source of a common metal ion Zr+4.

Laus (2008) performed a bacterial reverse mutation study according to OECD guideline 471 and EU method B13/14 Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 were exposed to the read-across substance zirconium dioxide at concentrations between 50 to 4998 µg/plate with and without metabolic activation in 2 independent experiments. Vehicle and positive controls were valid. Zirconium dioxide did not induce mutation with and without metabolic activation and no cytotoxicity was observed. This study was scored as reliable with restrictions (K2) to reflect the read-across proposal. The read across justification is included in Section 13 of IUCLID.

Based on the data abovementioned and applying a 'weigh-of-evidence' approach between these two 'insoluble' zirconium compounds as similar source of zirconium ion, it can be concluded that zirconium basic carbonate is not mutagenic to bacteria with and without metabolic activation.

Chromosome Aberration:

Ciliutti (2013) performed an in vitro Chromosome Aberration test in Chinese hamster ovary cells (OECD 473 and EU Method B.10). Two experiments were performed using different concentrations (10 different concentrations up to and including 5.35 mM; corresponding to 3038 µg/mL zirconium basic carbonate hydrate and 1650 µg/mL zirconium basic carbonate anhydrous)

with and without S9 activation (3h exposure and harvested at 20h in experiment I; continuous exposure until harvest at 20h for experiment II). Both negative and positive controls were considered to be valid. On the basis of the results obtained, it was concluded that zirconium basic carbonate did not induce structural chromosome aberrations in this in vitro test, with and without metabolic activation, and under the reported conditions.

 

In vitro mammalian cell gene mutation:

Two studies are included to cover this endpoint using a 'weight-of-evidence approach': one study performed with zirconium acetate and a second one with zirconium dioxide.

Bisini (2013) performed an in vitro mammalian gene mutation assay in L5178Y TK+/- lymphoma cells using the fluctuation method according to OECD Guideline 476 and EU Method B.17. The test substance was zirconium acetate, a water soluble zirconium compound. Two experiments were performed using different concentrations with and without metabolic S9 activation (experiment I) and without metabolic S9 activation (experiment II). Plates were tested in duplicate. Plates were exposed for 3hr (experiment I) and 24 hr (experiment II). The positive and negative (vehicle) controls were considered to be valid. It was concluded that the test substance did not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation under the experimental conditions.

Taking into account the concept that the more water soluble is the substance the higher is its potential for systemic bioavailability, it can be assumed that zirconium basic carbonate (an insoluble zirconium compound) will be of an even lower concern than zirconium acetate (a 'water-soluble' zirconium compound) for mutageniticy to mamalian cell genes.

This lack of mutagenicity is confirmed by a reliable mouse lymphoma test performed with the read-across substance zirconium dioxide (another insoluble zirconium compound), according to OECD guideline 476 (NOTOX B.V., 2010. In this study mouse lymphoma L5178Y cells were exposed to 0.03, 0.1, 1, 3, 10, 33 and 100 µg/mL zirconium dioxide with and without metabolic activation. In a first experiment, cell cultures were exposed for 3 hours to zirconium dioxide in exposure medium in the absence and presence of S9-mix. In a second experiment, cell cultures were exposed to zirconium dioxide in exposure medium for 24 hours in the absence of S9-mix and for 3 hours in the presence of S9-mix. Zirconium dioxide tested negative in both experiments with and without metabolic activation. No cytotoxicity was observed and positive and vehicle controls were considered valid.

Based on the data abovementioned and applying a 'weight-of-evidence' approach between these two zirconium compounds as source of zirconium ion, it can be concluded that zirconium basic carboante is not mutagenic, when tested in vitro in mammalian cells, with and without metabolic activation.

Both studies were scored as reliable with restrictions (K2) to reflect the read-across proposal. The read across justification is included in Section 13 of IUCLID.

 

Genetic toxicity in vivo:

According to REACH Annex IX section 8.4, column 2, no further in vivo testing is required as no positive results were obtained in any of the three in vitro studies performed according to REACH Annexes VII and VIII section 8.4.


Justification for selection of genetic toxicity endpoint
There are 5 in vitro key studies available: one key study and one weight of evidence with the substance and three read-across studies also used in a 'weight-of-evidence' approach. The read-across substances are zirconium acetate and zirconium dioxide.

Short description of key information:
Genetic toxicity in vitro:
Bacterial reverse mutation assay: two studies are used in a 'weight-of-evidence' approach.
One study was performed with zirconium basic carbonate and according to the OECD Guideline 471 (adopted in 1983), in S. typhimurium TA98, TA100, TA1535, TA1537 and TA1538 (BIBRA, 1987). The test item was not mutagenic with and without metabolic activation. A second study (LAUS, 2008) was performed with the read-across substance zirconium dioxide, a similar unsoluble zirconium compound. This bacterial reverse mutation study was performed according to OECD guideline 471 and EU method B13/14 with the read-across substance zirconium dioxide. The substance was negative with and without metabolic activation in S. typhimurium strains TA97a, TA98, TA100, TA102 and TA1535.
In vitro mammalian chromosome aberration test: performed with zirconium basic carbonate and according to OECD Guideline 473 in Chinese hamster ovary cells (Ciliutti, 2013). The test item was not clastogenic with and without metabolic activation.
In vitro mammalian cell gene mutation test: Two studies are used ('weight-of-evidence'). One study was performed with the read across substance zirconium acetate, a water soluble zirconium compound. The test item was tested for mutation using L5178Y mouse lymphoma cells (fluctuation method) according to OECD Guideline 476 (Bisini, 2013). The test item was not mutagenic under the experimental conditions, with and without metabolic activation. In a second study (NOTOX, 2010) the read-across substance zirconium dioxide (a similar non-soluble zirconium compound) was tested according to OECD 476. The substance was negative in mouse lymphoma L5178Y cells with and without metabolic activation.
Genetic toxicity in vivo: According to REACH Annex IX section 8.4, column 2, no further in vivo testing is required as no positive results were obtained in any of the three in vitro studies performed according to REACH Annexes VII and VIII section 8.4.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available data and according to the criteria of the DSD and CLP Regulation, zirconium basic carbonate should not be classified for mutagenicity.