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EC number: 218-235-4
CAS number: 2090-05-3
The in vitro genetic toxicity of Calcium
dibenzoate has been evaluated in a bacterial reverse gene mutation assay
(acc. to OECD TG 471) and in a mammalian cell gene mutation assay (acc.
to OECD TG 476). These studies were performed according to the current
guidelines and in accordance with GLP and were evaluated to be reliable
without restrictions. The cytogenetic potential in mammalian cells is
addressed with data on the individual moieties, calcium and benzoic
acid. The cytogenetic potential in mammalian cells for the moiety
benzoic acid is addressed with a micronucleus test and a chromosome
aberration assay, both of which were performed in the pre-guideline and
pre-GLP era but are considered to be reliable with restrictions. For the
moiety calcium, the data is considered to be not scientifically
Mouse lymphoma L5178Y cells were used in this study. Three types of
treatment were carried out parallel: two treatments without metabolic
activation with or without a recovery period after 24 h continuous
treatment and one treatment with metabolic activation by S9 mix. The
concentrations of the test substance were 1000, 500, 250 μg/mL in the
three type of treatment. Mitomycin C was used as a positive control in
the absence of S9 mix and cyclophosphamide was used as a positive
control in the presence of S9 mix.
The test substance gave unequivocal negative responses both with or
without metabolic activation or cytotoxicity.
Chromosomal aberration test was carried out on the test substance, using
a Chinese hamster fibroblast cell line, CHL.
The test substance was ambiguous in the chromosomal aberration test.
No in vivo genetic toxicity study with Calcium dibenzoate is available, thus the genetic toxicity will be addressed with existing data on the individual moieties calcium and benzoate.
In vitro gene mutation in bacteria:
Chang (2021) investigated the mutagenic potential of Calcium dibenzoate in a bacterial reverse mutation assay according to OECD TG 471 (2020) under GLP. The test was performed in S. typhimurium TA 98, TA 100, TA 1535, and TA 1537 as well as E. coli WP2 uvrA (pKM101) in two independent experiments using triplicate cultures. The cell cultures were exposed to the test material, both in absence and presence of metabolic activation, up to the recommended maximum concentration of 5 mg/plate. The two independent experiments were performed according to the plate incorporation and pre-incubation procedure. Treatment with Calcium dibenzoate did not result in a mutagenic response at all concentrations tested both in absence and presence of S9 metabolic activation. Appropriate positive controls demonstrated the activity of the metabolic activation system and the sensitivity of the test system.
In vitro gene mutation in mammalian cells:
Sokolowski (2021) examined the gene mutation potential of Calcium dibenzoate in an in vitro mammalian cell gene mutation test at the Hprt locus according to OECD TG 476 (2016) and under GLP. Chinese hamster lung fibroblasts (V79) were exposed for 4 hours to Calcium dibenzoate up to a precipitating concentration of 2 mg/mL (recommended maximum concentration) without metabolic activation and 1 mg/mL with metabolic activation (S9 fraction from phenobarbital/β-naphthoflavone induced rat livers). The outcome of this study was judged as clearly negative both in the absence and presence of S9 metabolic activation. Appropriate positive controls demonstrated the activity of the metabolic activation system and the sensitivity of the test system.
In vitro cytogenicity:
Considering the overall weight of evidence, such as in vitro data and the crucial role of calcium in cellular processes such as DNA-polymerase and cell cycle functioning as well as its role in approved in vitro genotoxicity experiments show that calcium does not exert any genotoxic or mutagenic effects to mammalian cells. Based on the above existing information on the genotoxicity of calcium substances and the weight of evidence information, calcium is considered as not genotoxic.
In vitro studies
For cytogenetic effects, a chromosome aberration test was ambiguous without metabolic activation (Ishidate, 1983). Benzoic acid was clearly negative in an in vitro micronucleus test (with and without metabolic activation) (Nesslany, 1999).
Additional studies were identified, but were not available for review. The available results confirm that benzoic acid is not likely to induce effects on the chromosomal level.
In vivo studies
A battery of in vivo tests (Fabrizio, 1974) was conducted with the structurally related Sodium benzoate and these all clearly indicated no cytogenetic effects. These studies are considered to fully address and over-ride the ambiguous results found in the in vitro data set. The toxicokinetic evaluation on Sodium benzoate concluded that the substance will not be taken up as the salt, but rather as its moieties benzoate and sodium (NOTOX 2010). Therefore it can be concluded that the results for benzoic acid will not differ from those found in in vivo tests with Sodium benzoate.
Based on the clear negative in vivo results, it is concluded that benzoic acid does not induce genetic toxicity.
Calcium dibenzoate is not expected to be genotoxic, since Calcium dibenzoate has neither shown any gene mutation potential in bacteria or mammalian cells. A cytogenetic potential is not anticipated, since the two moieties, calcium and benzoate are either considered not cytogenic due to their crucial role in cell function, as in the case of calcium, or, as in the case of benzoate, are clearly negative in existing in vivo studies. Further testing is not required. Thus, Calcium dibenzoate is not to be classified according to regulation (EC) 1272/2008 and its subsequent amendments as genetic toxicant. For further information on the toxicity of the individual moieties, please refer to the relevant sections in the IUCLID and CSR.
Calcium dibenzoate showed no genotoxic effect neither in a bacterial reverse mutation test nor in a mammalian cell gene mutation test (Hprt assay). Moreover, data from both moieties, calcium and benzoate, indicated no cytogenetic potential.
The classification criteria acc. to regulation (EC) 1272/2008 and its subsequent amendments as germ cell mutagen are not met, thus no classification is required.
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