Hydroxido potassium zirconium carbonate, where there are 0, 1 or 2 hydroxide groups and 2, 3 or 4 potassium atoms and 2, 3 or 4 carbonate groups.

Endpoint summary

Additional information

In vitro Data

Bacterial Reverse Mutation in Bacteria

The test material was tested for mutagenic activity in Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 at concentrations ranging from 33 to 10000 µg in the presence and absence of metabolic activation. Concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the S9 mix. No mutagenic activity was observed in any of the 5 bacterial strains used, in any condition. There was no toxicity to the bacteria and no precipitation of the test material was observed. It was concluded that the test material was not mutagenic in Salmonella typhimurium.

Chromosome Aberration

Cell cultures of Chinese hamster ovaries were treated with the test material in the presence and absence of a post-mitochondrial supernatant fluid preparation (S9 mix). Three main aberration tests were carried out. In each test, duplicate cells cultures were treated for 6 h in the presence and 22 h in the absence of S9 mix. Cultures were harvested at 24 h (tests 1, 2 and 3) and 48 h (test 2 only). Test 3 was included because of a poor response to positive control cultures in test 1. Under the conditions of the test there was no evidence that the test material induced aberrations in either the presence or absence of S9 mix. In the absence of S9 mix, the test material was shown to cause polyploidy in cells exposed to concentrations between 75 -200 µg/ml. This is a common finding in this type of study and does not directly indicate that the test material is cytogenic. There is sufficient evidence to suggest that the test material is not mutagenic, as no structural chromosome breakage was observed in this study. Additionally a negative in vivo mouse micronucleus study has been provided which has been performed on the read-across substance, ammonium zirconium carbonate.

Mouse Lymphoma

 

The test material was tested for mutagenic activity in the mouse lymphoma L5178Y assay in both the presence and absence of a rat liver preparation and co-factors required for mixed function oxidase activity (S9 mix).

A preliminary toxicity test was conducted with an alternative test material, potassium zirconium carbonate (KZC). This showed that, while KZC was soluble in water, it precipitated at all concentrations above 50 µg/ml when added to the treatment medium. It also raised the pH of the exposure medium. A 50 % solution of KZC was substituted for the main study. In the main study, test material was toxic in the range 1000 to 2000 µg/ml, it precipitated only in the middle of this concentration range (900 to 1500 µg/ml) and in addition raised the pH of the culture medium.

In 4 independent mutation assays (2 in the absence and 2 in the presence of S9 mix), results were obtained where the final concentrations of the test material in the treatment medium ranged between 200 and 2400 µg/ml in the absence of the S9 mix, and between 200 and 1800 µg/ml in the presence of S9 mix. Positive controls demonstrated the sensitivity of the assay and the effectiveness of the S9 mix.

Both assays in the absence of S9 mix resulted in increased mutant fractions only in cultures treated with 1800 µg/ml. In the first of these, survival was relatively high (mean = 26.5 % of controls), while in the second, survival was low (mean = 3.5 % of controls). The biological significance of the increase in the second assay is questionable therefore, and while the first experiment was classified positive, the second was classified inconclusive.

The first assay in the presence of S9 mix gave no evidence of mutagenic activity, although results were not obtained at critically toxic concentrations. The second assay in the presence of S9 mix gave a marginal response at 1800 µg/ml, at a level of survival that was also marginal in terms of biological significance (mean = 11 % of controls). This experiment was classified inconclusive.

As there were not two experiments that met the criteria for a positive result, it could not be concluded that the test material is mutagenic in the test system. The evidence suggested that there was a weak potential to induce mutations in mouse lymphoma L5178Y cells in the absence of S9 mix, and less so in the presence of S9 mix. The level of survival at which responses were obtained tended to be sufficiently low as to cast doubt on the biological significance of the effect (one response was recorded at a higher level of survival).

The significance of the result was further compounded by 2 physical properties of the test material:

1. The test material reacted with the culture medium causing precipitates to be formed in the middle of the concentration range.

2. The test material raised the pH of the exposure medium

The significance of the latter effect was not known, but was readily investigated. Further experiments were conducted using potassium carbonate.

A toxicity test showed that potassium carbonate was toxic to a portion of the exposed cell populations at 625 µg/ml, and caused total cell death at 1250 µg/ml. The pH of the treatment medium was sufficiently elevated at these concentrations to be able to attribute the toxicity to the increase in pH.

Potassium carbonate was assessed for mutagenic activity at concentrations ranging between 675 and 1050 µg/ml. The results gave dose-related mutagenic responses in both the absence and presence of S9 mix. There is therefore strong evidence to suggest that the mutagenic activity recorded with the test material was due to an artefact of the test system. The cause would seem to be elevated pH during exposure and/or excessive potassium levels during exposure.

In Vivo Data

 

Mouse Micronucleus 

 

This study has been provided on a surrogate substance, used on the basis of read-across. The test material was administered by intraperitoneal injections at dosages of 1000, 500 and 250 mg/kg in male ICR mice. Clinical signs of toxicity and reductions in the PCE/NCE ratio confirmed exposure of the target organ. The test material was found not to produce a statistically significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice in comparison to the control. Therefore under the conditions of the test, the test material is considered to be non-genotoxic.

This study demonstrates sufficient evidence of the absence of genotoxic activity of the test material.

Justification for selection of genetic toxicity endpoint
No study selected since all in vitro and in vivo studies are necessary to address the genotoxic risk of exposure to the test material (see below discussion).

The key in vivo study (Marr, 2010) was performed on a substance similar to the one being registered, potassium zirconium carbonate, and has been provided on the basis of read-across given that the substances have common ionic components, ZrO2. The study was performed using ammonium zirconium carbonate in a GLP compliant study conducted according to the standardised guidelines OECD 474, EU Method B.16 and EPA OPPTS 870.5395. The study has been assigned a reliability score of 2 according to the principles for assessing data quality set out by Klimisch (1997).

Three key in vitro studies have been provided, a bacterial reverse mutation assay (Dillion, 1994), a chromosome aberration assay (Leddy, 1994) and a mouse lymphoma assay (Riach & Willington, 1994). The studies were GLP compliant and conducted according to OECD guidelines 471, 473 and 467, respectively. All three studies have been assigned a reliability score of 1 according to the principles for assessing data quality set out by Klimisch (1997).

Short description of key information:
IN VITRO GENETIC MUTATION STUDY IN BACTERIA
Dillon (1994); Bacterial reverse mutation in bacteria: Negative (Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 with and without S9 mix).

IN VITRO GENE MUTATION STUDY IN MAMMALIAN CELLS
Leddy (1994); In vitro chromosome aberration: Negative (Chinese hamster ovaries with and without S9 mix).

IN VITRO MAMMALIAN CELL GENE MUTATION
Riach & Willington (1994); Gene mutation in mammalian cells: Negative (mouse lymphoma L5178Y with and without S9 mix), signs of polyploidy.

IN VIVO MOUSE MICRONUCLEUS GENE MUTATION
Flanders (2010); Chromosome aberration micronucleus assay: Negative.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In accordance with criteria for classification as defined in Annex I, Regulation 1272/2008, the test material does not require classification for genetic toxicity based on the overall negative response noted in the available in vitro and in vivo genetic toxicity studies.