Calcium dihydroxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation study in bacteria:
Calcium dihydroxide - OECD TG 471 - negative with and without metabolic activation
Calcium carbonate - OECD TG 471 - negative with and without metabolic activation
 In vitro chromosome aberration study in mammalian cells:
Calcium dihydroxide - OECD TG 473 - negative with and without metabolic activation
Calcium dihydroxide - non-mutagenic is a study performed using D824 cells
Calcium carbonate - OECD 473 - negative with and without metabolic activation
 In vitro gene mutation study in mammalian cells:
Calcium dihydroxide - OECD TG 476 - negative
Calcium carbonate - OECD TG 476 - negative.

Additional information

In vitro gene mutation study in bacteria:

In a reliable GLP OECD TG 471 study [Sokolowski, 2007] calcium dihydroxide (purity = 98.2 %) was tested in a bacterial reverse mutation assay in Salmonella typhimurium (strains TA 98, TA 100, TA 1535 and TA 1537) and Escherichia coli WP2 uvrA with and without metabolic activation (S9). The concentrations tested were 117.2, 234.4, 468.75, 937.5, 1875 and 3750 µg/plate. No mutations occurred.

Similarly, in a reliable GLP OECD TG 471 study [Thompson, 2010], calcium carbonate (nano) was tested in a bacterial reverse mutation assay in Salmonella typhimurium (strains TA 98, TA 100, TA 1535 and TA 1537) and Escherichia coli WP2 uvrA with and without metabolic activation (S9). The concentrations tested were 50, 150, 500, 1,500 and 5,000 μg/plate. No mutations occurred.

 In vitro chromosome aberration study in mammalian cells:

An in vitro study for the detection of structural chromosomal aberrations was performed under GLP in accordance with OECD TG 473 using calcium dihydroxide (purity = 98 %) [Morris, 2015].

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated using a 4-hour exposure in the presence and absence of a standard metabolizing system (S9 at a 2% final concentration) and a 24-hour exposure in the absence of metabolic activation.

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited predominantly on precipitate. The dose level range used for the main experiment were 4.38 - 140 μg/mL.

All vehicle (MEM) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control items induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item was marginally toxic and did not induce a statistically significant increase in the frequency of cells with aberrations, using a dose range that was limited by the lowest precipitating dose level. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro.

The ability of calcium dihydroxide to induce chromosome aberrations in human dental pulp cells (D824) has been investigated [Nishimura, 2008]. The D824 cells were exposed to calcium dihydroxide at test concentrations of 30, 100 and 300 µM in glycerol for 3 or 30 hours. Calcium dihydroxide did not induce chromosome aberrations after treatment for either time period and was considered to be non-mutagenic under the conditions of the test.

In a reliable GLP OECD TG 473 study [Lacey & Durwood, 2010], calcium carbonate (nano) was tested for its ability to induce structural chromosomal aberrations in cultured mammalian cells (human lymphocytes) in the presence and absence of metabolic activation. Calcium carbonate (nano) did not induce any statistically significant increases in the frequency of cells with aberrations or in the numbers of polyploid cells, in either the absence or presence of metabolic activation and was therefore considered to be non-mutagenic under the conditions of the test.

 In vitro gene mutation study in mammalian cells:

A GLP study was conducted according to OECD TG 476, Method B17 of Commission Regulation (EC) No. 440/2008 and US EPA OPPTS 870.5300, and in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances, in order to assess the potential mutagenicity of calcium dihydroxide (purity = 98 %) on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line [Flanders, 2015].

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle (R0 medium), and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at up to ten dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24-hour exposure group in the absence of metabolic activation.

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose level ranges plated out for viability and expression of mutant colonies were as follows: Experiment 1 - 34.73 - 277.88 μg/mL (4 -h, -S9); 17.37 - 555.75 μg/mL (4 -h, +S9); Experiment 2 - 17.25 - 230 μg/mL (24-h, -S9); 17.37 - 555.75 (4 -h, +S9).

The maximum dose levels used in the mutagenicity test were limited by a combination of pH increase and test item-induced toxicity. Precipitate of the test item was observed at and above 34.5 μg/mL in the Mutagenicity Test. The vehicle controls had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control treatment induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system.

The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels, either with or without metabolic activation, in either the first or the second experiment and was therefore considered to be non-mutagenic under the conditions of the test.

In a reliable GLP OECD TG 476 study [Flanders, 2010], calcium carbonate (nano) was tested for its ability to induce mutations in mouse lymphoma L5178Y cells in the presence and absence of metabolic activation. Calcium carbonate (nano) did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and was therefore considered to be non-mutagenic under the conditions of the test.

Other data:

Several comet assays have been performed using calcium dihydroxide-containing endodontic materials [Ribeiro, 2004; Ribeiro, 2005; da Silva, 2007]. The results of these assays were all negative with respect to DNA breakage, demonstrating lack of genotoxic potential.

Finally, calcium is an essential mineral nutrient omnipresent in the human body, with daily dietary requirements for adults of approximately 1000 mg, varying by developmental status and age.

On the basis of the available data, it may be concluded that all grades of calcium dihydroxide including those containing up to 35% calcium carbonate are not genotoxic.

Justification for classification or non-classification

The results of in vitro gene mutation studies in bacteria, in vitro chromosome aberration studies in mammalian cells and in vitro gene mutation studies in mammalian cells performed using calcium dihydroxide or calcium carbonate were all negative. It is concluded that both calcium dihydroxide and calcium carbonate are not genotoxic and neither chemical nor mixtures of these chemicals warrants classification for mutagenicity under CLP.