Calcium diformate

Endpoint summary

Additional information

Only one bacterial mutagenicity test with calcium diformate is known to exist, but results on other formate salts or formic acid may be extrapolated as briefly outlined below and justified in detail in section 7.1.1.

Experimental data

Genetic toxicity in vitro; bacteria

Calcium diformate (10-12500 µg/plate) was negative in a valid Ames test (2 independet experiments; 4 replicates each; conducted according to OECD guideline 471 and under GLP conditions) using Salmonella typhimurium strains TA1535, TA100, TA1537, and TA98, both in the presence and absence of metabolic activation (S9 liver fraction from Aroclor induced male Sprague Dawley rats). Cytotoxicity was seen at 4000 µg/plate and above. Positive and solvent controls performed as expected (Bayer, 1989).

 

This study is considered to be valid and acceptable for assessment.

Data from supporting chemicals

Genetic toxicity in vitro

Mutagenicity in bacteria:

Formic acid was tested in an in-vitro genotoxicity test using bacteria (TA97, TA98, TA100, and TA1535) with and without metabolic activation (supernatant from induced male rat and Syrian hamster liver) at concentrations of 0, 10, 33, 100, 333, 1000, and 3333 µg/plate in accordance with OECD Guideline No. 471. Solvent and positive controls were included and performed as expected. Tests were conducted in triplicate, and two independent experiments were conducted.The number of revertants was not increased in any strain with or without metabolic activation up to and including the top dose of formic acid.Bacteriotoxicitywas seen at 1000 µg/plate and above (Zeiger, 1992).

 

Conclusions:

1) Formic acid lacked genotoxicity in a valid bacterial cell in-vitro test performed according to OECD Guideline No. 471.

2) Calcium diformate is not mutagenic in bacteria. Reason: data on formic acid may be used to assess formate salts, because formic acid is almost exclusively present as formate anion in aqueous solution at neutral pH.

Mutagenicity in mammalian cells:

In a mammalian cell gene mutation assay (HPRT locus), Chinese Hamster ovary cells cultured in vitro were exposed to formic acid (85.3%) at concentrations of 0, 31.25, 62.5, 125, 250, and 500μg/mL in the presence, and of0, 25, 50, 100, 200, and 400μg/mL in theabsence of mammalian metabolic activation. 

 

Formic acidwas tested up to cytotoxic concentrations (i.e., 200 to 400 µg/mL in the absence, and 400 to 500 µg/mL in the presence of metabolic activation) without increasing mutation frequency at any concentration.  The positive controls did induce the appropriate response as did the vehicle control. There was no evidence of induced mutant colonies over background.

 

This study is classified as acceptable because it meets the requirements of GLP and current test guidelines.  This study satisfies the requirement for Test Guideline OECD 476 and EEC Directive 2000/32, B.17 for in vitro mutagenicity (mammalian forward gene mutation) data.

 

Conclusions:

1) Formic acid did not induce forward mutations in vitro in the CHO/HPRT assay, with or without metabolic activation.

2) Calcium diformate is not mutagenic in mammalian cells. Reason: data on formic acid may be used to assess formate salts, because formic acid is almost exclusively present as formate anion in aqueous solution at neutral pH.

Chromosome aberration

In a mammalian cell cytogenetics assay (Chromosome aberration, conducted similar to OECD Test Guideline No. 473) CHO cell cultures were exposed to formic acid dissolved in F12 cell culture medium at concentrations of 6 to 14 mM, i.e. 0, 276, 368, 460, 552, and 644 µg/mL with and without metabolic activation. In a series of subsequent experiments the influence of confounding factors, i.e. pH and osmolality) on the incidence of aberrant cells (%) was examined at concentrations of 20, 25, 27.5, and 30 mM, i.e. at 920, 1150, 1266, and 1380 µg/mL.

 

Formic acid was tested up to cytotoxic concentrations. Overt cytotoxicity and increased numbers of aberrant cells were seen when the initial pH of the incubation medium was approximately 6 or less. The number of aberrant cells was not increased by formic acid up to 14 mM, i.e. 644 mg/mL, if the initial pH was adequate (pH 7.2). Moreover, no positive response was seen with concentrations up to 20 mM (920 µg/mL) with two different buffer systems as long as the buffer capacity was not exhausted. At 25 to 30 mM formic acid an increasing positive response and cytotoxicity were both seen. It was concluded that this results from the combined inadequately low pH and high osmolarity of the incubation medium.

  

Positive controls were not included. Acetic and lactic acid were included and showed similar results. There was no evidence of Chromosome aberration induced over background by formic, acetic or lactic acid themselves. Pseudo-positive reactions attributable to non-physiological pH could be eliminated by either neutralisation of the treatment medium or enhancing the buffer capacity (Morita, 1990).

 

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OECD 473 in  Chinese Hamster ovary cells for in vitro cytogenetic mutagenicity data. 

Conclusions:

1) Formic acid itself is not clastogenic. A pseudo-positive response was attributable to non-physiologically low pH.

2) The same applies to calcium diformate. Data on formic acid may be used to assess formate salts, because formic acid is almost exclusively present as formate anion in aqueous solution at neutral pH.

Genetic toxicity in vivo

Formic acid and sodium formate were both tested for genetic toxicity in a multigenerational test in Drosophila melanogaster similar to the OECD Guideline No. 477 (Genetic Toxicology: Sex-linked Recessive Lethal Test in Drosophila melanogaster). Following exposure to 0.1% formic acid vapour, the number of mutants was significantly increased compared to historical controls (p<0.001).

An increase was also seen with 0.1% formic acid in a subsequent feeding experiment, but without gaining statistical significance.  Sodium formate(produced by neutralization of formic acid) at the same molar concentration in the feed was negative in the Drosophila SLRL test.The authors concluded that the mutations observed with formic acid were related to the acidic pH, rather than to the acid or the formate molecule itself (Stumm-Tegethoff, 1969). As above, this result may be extrapolated to calcium diformate.

Conclusions:

Formic acid and sodium formate did not induce mutations in the Drosophila SLRL test in vivo.

Overall, formate salts including calcium diformate lack genotoxic properties. This is also supported by the finding that another formate salt, potassium diformate, lacks carcinogenicity in rats and mice.

 

Short description of key information:
Calcium diformate is not genotoxic. It was negative in a valid Ames test using S. typhimurium strains, with and without metabolic activation. Sodium formate and formic acid lack genotoxic properties in in vitro and in vivo assays. This can be extrapolated to other formate salts including calcium diformate. This is supported by the finding that another formate salt, potassium diformate, lacked oncogenicity in rats and mice (cf. respective section).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

No classification required. Criteria of regulations 67/548/EC and 1272 /2008/EC not met.