Formic acid, manufacture of, by-products from

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Data are available for the product components

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Data are available for formate

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The reaction mass of 2,2-bis(formyloxymethyl)propane-1,3-diyl diformate and formic acid consists of formic acid, propylidynetrimethanol-esters and pentaerythritol-esters. The toxicity of the propylidynetrimethanol-esters and pentaerythritol-esters is predicted to be comparable to propylidynetrimethanol and pentaerythritol respectively. Genotoxicity data on formic acid, propylidynetrimethanol and pentaerythritol are considered appropriate to meet the REACH Annex VII-X data requirements for the reaction mass of 2,2-bis(formyloxymethyl)propane-1,3-diyl diformate and formic acid.

Formic acid

The potential mutagenicity of formic acid in bacterial cells has been assessed in a bacterial reverse mutation assay (Ames test) conducted according to OECD Test Guideline 471. No evidence of mutagenicity was observed inS. typhimuriumusing test strains TA97, TA98, TA100 and TA1535 with or without metabolic activation(SRI International; Zeigeret al, 1992).

The cytogenic potential of formic acid in mammalian cellsin vitrohas been investigated in three studies: a chromosome aberration test and two sister chromatid exchange studies.

The clastogenic potential of formic acid in mammalian cellsin vitrohas been assessed in a chromosome aberration test conducted according to OECD Test Guideline 473 (Morita et al, 1990). In the study Chinese Hamster Ovary 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/mLwith and without metabolic activation. In a series of subsequent experiments the influence of confounding factors, (i.e. pH and osmolarity) 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 by formic, acetic or lactic acid. Pseudo-positive reactions attributable to non-physiological pH could be eliminated by either neutralisation of the treatment medium or enhancing the buffer capacity.It was therefore concluded that formic acid is not clastogenic. A pseudo-positive response was attributable to non-physiologically low pH.

 

In a sister chromatid exchange assay conducted according to OECD Test Guideline 476, 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 (Basler, 1985). 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. In the study, formic acid was not found to induce sister chromatid exchange in V79 cells, with or without metabolic activation.

 

In another sister chromatid exchange (SHE) assay conducted according to OECD Test Guideline 476 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 (Sipi, 1992).   Formic acid was tested up to cytotoxic concentrations, as evidenced by a significantly decreased replication index at 10 mM. No increase in SCE was observed at 0.63, 1.25, 2.5 and 5 mM.  A slight, yet statistically significant increase in SCEs was seen at 10 mM (p<0.01), but this was attributable to a confounding pH effect which caused marked cytotoxicity at elevated concentrations exceeding the buffer capacity. Positive controls were not reported to be included, negative controls performed as expected.  In the study, formic acid was not found to induce sister chromatid exchange in mammalian cells in vitro; pseudo-positive results were attributable to the confounding effect of cytogenicity related to low pH values.

 

The potential for formic acid to cause forward gene mutations in mammalian cells in vitro has been investigated in the hypoxanthine-guanine phosphoribosyl transferase locus (HPRT) in Chinese Hamster Ovary (CHO) cells in a study conducted according to OECD Test Guideline 476 (BASF, 2002). In the study CHO 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 400μg/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 this study, formic acid did not induce forward mutations in vitro in the CHO/HPRT assay, with or without metabolic activation.

 

The genotoxicity of formic acidin vivohas been investigated in a multi-generational study using Drosophila melanogaster comparable to the OECD Guideline No. 477 (Genetic Toxicology: Sex-linked Recessive Lethal Test in Drosophila melanogaster; Stumm-Tegethof, 1969). 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. Based on these findings, formic acid and sodium formate were not considered to induce mutations in the Drosophila SLRL test in vivo.

Propylidynetrimethanol

The mutagenic potential of propylidynetrimethanol in bacterial cell systems (S. typhimuriumTA 98, TA 100, TA 1535, TA 1537 and E coli WP2 uvrA) was investigated in an Ames test conducted according to OECD Test Guideline 471 and GLP (MHLW 1994). No mutagenic activity was observed with or without exogenous metabolic activation up to 5000 µg/plate, but cytotoxicity was not reached.

Negative results were also obtained in anotherin vitro genotoxicity test in bacterial cells conducted according to Ames et al. Mut Res 31, 347-364 (1975) and GLP (Bayer, 1989). In the study doses of propylidynetrimethanol at up to 5000 µg per plate were tested on Salmonella typhimurium strains TA98, TA100, TA1535 and TA 1537. There was no evidence of mutagenic activity of propylidynetrimethanol with and without mutagenic activation, but cytotoxicity was not reached.

The cytogenic potential of propylidynetrimethanol was investigated in a chromosomal aberration test conducted according to OECD TG 473 and GLP (MHLW, 1994). In the study, Chinese Hamster Lung (CHL) cells were treated with propylidynetrimethanol at concentrations up to 1.34 mg/ml in the presence and in the absence of a metabolic activation system. There was no evidence for clastogenic activity neither with nor without S9 -mix (MHLW 1994).

The potential for propylidynetrimethanol to induce gene mutations at the HPRT locus in Chinese hamsters V79 cells was investigated in a study conducted according to OECD TG 476 and GLP (Harlan CCR, 2010). In the study, V79 cells were treated with propylidynetrimethanol at concentrations up to 1400 µg/ml in the presence and in the absence of S9 -mix. Cytotoxicity was determined in preliminary experiments. Negative results were obtained: propylidynetrimethanol did not induce gene mutations at the HPRT locus in V79 cells.

Pentaerythritol

The mutagenic potential of pentaerythritol in bacterial test systems has been investigated in a reverse mutation assay (Ames test) conducted according to OECD Test Guideline 471 (Shibuya, 1994). No evidence of mutagenicity was found using the substance in S. typhimuriumstrains TA98, TA100, TA1535, TA1537 and TA1538 with and without metabolic activation at test concentrations upto 5000 μg/plate

Negative results were also obtained in another OECD TG 471 Ames test using the substance in S. typhimurium strains TA98, TA100, TA1535 and TA1537 and in E. coli WP2 (Shimizu, 1985).

In anin vitrocytogenicity study, conducted according to OECD Test Guideline 473, the claustogenic potential of pentaerythritol was investigated in cultured Chinese hamster lung cells using the chromosome aberration test (Tanka, 1994). The test substance was tested up to a concentration of 1.4 mg/ml, both in the presence and absence of metabolic activation. There were no increases in the number or type of chromosome aberrations in treated cultures, compared to solvent controls. No evidence of clastogenicity was seen under the conditions of this assay.

The potential for pentaerythritol to induce gene mutation in mammalian cells in vitro was investigated in a mouse lymphoma assay conducted according to OECD TG 476 (Riach, 2010). In the study, no evidence of mutagenic potential was observed in mouse lymphoma L5178Y cells both in the presence and in the absence of metabolic activation.

The reaction mass of 2,2-bis(formyloxymethyl)propane-1,3-diyl diformate and formic acid.

No evidence of mutagenicity was seen for formic acid in a bacterial reverse mutation assay (Ames) test or inin vitrocytogenicity or gene mutation studies using mammalian cells. Formic acid was not genotoxicin vivoin Drosophia melanogaster. Neither propylidynetrimethanol nor pentaerythritol are mutagenic in bacterial or in mammalian tests systems in vitro. Based on read-across propylidynetrimethanol-esters and pentaerythritol-esters are similarly predicted to lack genotoxic potential. Due to the lack of genotoxic potential demonstrated for its components, no genotoxic potential is predicted for the reaction mass of 2,2-bis(formyloxymethyl)propane-1,3-diyl diformate and formic acid.

Justification for selection of genetic toxicity endpoint
No study was selected since negative results were obtained in the available in vitro and in vivo tests for the formic acid, propylidynetrimethanol and pentaerythritol

Short description of key information:
No evidence of mutagenicity was seen for formic acid in a bacterial reverse mutation assay (Ames) test or in in vitro cytogenicity or gene mutation studies using mammalian cells. Formic acid was not genotoxic in vivo in Drosophia melanogaster. Neither propylidynetrimethanol nor pentaerythritol are mutagenic in bacterial or in mammalian tests systems in vitro: negative results were obtained in respective Ames tests, in chromosome aberration assays and gene mutation assays. Due to the lack of genotoxic potential demonstrated for its components, no genotoxic potential is predicted for the reaction mass of 2,2-bis(formyloxymethyl)propane-1,3-diyl diformate and formic acid.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Due to the lack of genotoxic potential demonstrated for its components, the reaction mass of 2,2-bis(formyloxymethyl)propane-1,3-diyl diformate and formic acid does not therefore meet the criteria for classification for genotoxicity according to Regulation. 1272/2008/EC.