Formic acid

Endpoint summary

Additional information

Genetic toxicity tests in-vitro

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. Bacteriotoxicity was seen at 1000 µg/plate and above. Formic acid lacked genotoxicity in a valid bacterial cell in-vitro test performed according to OECD Guideline No. 471

 

In a mammalian cell gene mutation assay (HPRT locus) performed according to the OECD Guideline No. 476 and under GLP conditions, 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 500 μg/mL in the presence, and of 0, 25, 50, 100, 200, and 400 μg/mL in the absence of mammalian metabolic activation. Formic acid was 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 in vitro in the CHO/HPRT assay, with or without metabolic activation.

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 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. It is therefore concluded that formic acid itself is not clastogenic. A pseudo-positive response was attributable to non-physiologically low pH.

In a mammalian cell cytogenetics assay (SCE),Chinese hamster V79 cell cultures were exposed to formic acid at concentrations of 0, 18.4, 27.6, 46.0, 92.0 µg/mL (i. e. 0, 0.4, 0.6, 1.0, 2.0 mM) with and without metabolic activation, S9-mix containing Arochlor 1254-induced male rat liver supernatant. Formic acid was tested in a concentration range that was not cytotoxic. Negative controls performed as expected. Positive controls (10 mM dimethylnitrosamine) induced the appropriate response. There was no evidence of SCE induced by formic acid over background. This study satisfies the requirement for Test Guideline OECD 479 for in vitro cytogenetic mutagenicity data. It is concluded that formic acid did not induced Sister Chromatid Exchange in V79 cells, with or without metabolic activation.

 

In another  mammalian cell cytogenetics assay (SCE) human lymphocyte cultures were exposed to formic acid at concentrations of 0, 29, 58, 115, 230, and 460  µg/mL (i.e. 0.63, 1.25, 2.5, 5, and 10 mM) without metabolic activation. Formic acid was tested up to cytotoxic concentrations, as evidenced by a significantly decreased replication index at 10 mM.  There was no increase of SCEs at 0.63, 1.25, 2.5 and 5 mM.  A slight, yet statistically significant increase of SCEs was seen at 10 mM (p<0.01), but this was attributable to a confounding pH effect which caused marked cytotoxicity. Positive controls were not reported to be included, negative controls performed as expected. There was no evidence of SCE induced over background by formic acid itself. This study satisfies the requirement for Test Guideline In vitro sister chromatid exchange assay in mammalian cells, OECD 47, for in vitro cytogenetic mutagenicity data. It is therefore concluded that formic acid itself lacked genotoxicity, and that pseudo-positive results are attributable to the confounding effect of cytotoxicity related to low pH values.

 

Genetic toxicity tests in-vivo

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

 

 

 

 

 

Short description of key information:
Formic acid was negative in all in-vitro and in-vivo genetic toxicity tests. Pseudo-positive results obtained in some of the in-vitro tests were demonstrated to have resulted from the confounding effect of low pH values at high acid concentrations exceeding the buffering capacity of the test system. Formic acid itself is therefore not genotoxic. Likewise, formate ion from solute formate salts was not genotoxic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Formic acid was not genotoxic in valid in-vitro assays for gene mutation using bacteria (TA97, TA98, TA100, and TA1535) and mammalian cells (HPRT), with and without metabolic activation, and it was negative in cytogenetic assays in -vitro as it did not induce Chromosome Aberration or Sister Chromatid Exchange in mammalian cells. Formic acid was negative in the Sex-linked Recessive Lethal Test in Drosophila melanogaster in-vivo.

If there were any pseudo-positive results, these were attributable to the confounding effects of low pH values. In aqueous solution at near neutral pH formic acid is mainly present as the formate anion, which was negative in all genetic toxicity studies known to exist.

The formate salts are therefore also not considered to be genotoxic because they dissociate into the formate anion and the respective counter ion in aqueous solution.

In conclusion, the criteria of regulations 67/548/EC and 1272/2008/EC for classification are not met.