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Genetic toxicity in vitro:

Bacterial reverse mutation assay:

Scarcella (2013) performed an Ames test (OECD 471) with S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E. coli strain WP2 uvrA with and without metabolic activation.

Following test concentrations were applied in triplicate: 313, 625, 1250 and 2500 µg zirconium acetate/plate for experiment I; 313, 625, 1250, 2500, 5000 µg of zirconium acetate/plate and an additional dose level of 156 µg/plate with TA100 tester strain both in the absence and presence of S9 metabolism for experiment II. For experiment III:

- TA1535, TA1537, TA98 without S9 at 156, 78.1, 39.1, 19.5, 9.77, 4.88, 2.44 µg of zirconium acetate/plate
- TA100 without S9 at 78.1, 39.1, 19.5, 9.77, 4.88, 2.44, 1.22 µg of zirconium acetate/plate
- WP2 uvrA without S9 at 156, 78.1, 39.1, 19.5, 9.77, 4.88 µg of zirconium acetate/plate
- TA98 with S9 at 156, 78.1, 39.1, 19.5, 9.77, 4.88 µg of zirconium acetate/plate
- TA1535, TA1537, WP2 uvrA with S9 at 313, 156, 78.1, 39.1, 19.5, 9.77 µg of zirconium acetate/plate
- TA100 with S9 at 78.1, 39.1, 19.5, 9.77, 4.88, 2.44 µg of zirconium acetate/plate

Negative controls and positive controls were run in triplicate and were considered to be valid. Main assay: the first assay was performed using a plate-incorporation method. The second and third assays were performed using a pre-incubation method.

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

 

Chromosome Aberration:

Ciliutti (2013) performed an in vitro Chromosome Aberration test in Chinese hamster ovary cells (OECD 473).

Two experiments were performed using different test concentrations with and without S9 activation (3 h exposure and harvested at 20 h in experiment I; continuous exposure until harvest at 20 h for experiment II):

For the first main experiment, the maximum dose level of 10.0 mM (corresponding to 2230 µg/mL of zirconium acetate and 912 µg/mL of zirconium) was selected in agreement with the study protocol and on the basis of the solubility of the test item in the treatment medium. Lower dose levels of 5.00, 2.50, 1.25, 0.625, 0.313, 0.156, 0.0781, 0.0391 and 0.0195 mM (corresponding to 1120, 558, 279, 139, 69.7, 34.8, 17.4, 8.71 and 4.36 µg/mL) were used in the absence and presence of S9 metabolism. The dose range used in the second experiment was modified taking into account the severe toxicity observed in first experiment. The dose levels of 5.00, 2.50, 1.25, 0.625, 0.313, 0.156, 0.0781, 0.0391, 0.0195 and 0.00977 mM were used (corresponding to 1120, 560, 280, 140, 70.0, 35.0, 17.5, 8.75, 4.38 and 2.19 µg/mL).

Both negative and positive controls were considered to be valid.

On the basis of the results obtained, it was concluded that zirconium acetate did not induce structural chromosome aberrations after in vitro treatment and under the reported conditions, in the presence and in the absence of metabolic activation.

In vitro mammalian cell gene mutation:

Bisini (2013b) performed an in vitro mammalian gene mutation assay in L5178Y TK+/- lymphoma cells using the fluctuation method according to OECD Guideline 476. 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 3 h (experiment I) and 24 h (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.

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
One study cannot be selected as there are 3 in vitro key studies available.

Short description of key information:
Genetic toxicity in vitro:
- Bacterial reverse mutation assay: performed according to OECD Guideline 471 in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A (Scarcella, 2013). The test item was not mutagenic with and without metabolic activation.
- In vitro mammalian chromosome aberration test: performed according to OECD Guideline 473 in Chinese hamster ovary cells (Ciliutti, 2013). The test item was neither mutagenic nor clastogenic with and without metabolic activation.
- In vitro mammalian cell gene mutation test: the test item was tested for mutation using L5178Y TK+/- mouse lymphoma cells (fluctuation method). The test was performed according to OECD Guideline 476 (Bisini, 2013b). The test item was not mutagenic under the experimental conditions, with and without metabolic activation.

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 acetate should not be classified for mutagenicity.