Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

Justification for grouping of substances and read-across

There are no data available on the potential in vitro cytogenicity and induction of gene mutation in mammalian cells of Ethylene diformate. In order to fulfil the standard information requirements set out in Annex VIII, 8.4, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006, read-across from structurally related substances is conducted.

In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across).

Having regard to the general rules for grouping of substances and read-across approach laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006, whereby physicochemical, toxicological and ecotoxicological properties may be predicted from data for reference substance(s) by interpolation to other substances on the basis of structural similarity, ethane-1,2-diol (ethylene glycol) (CAS 107-21-1) and formic acid (CAS 64-18-6) are selected as reference substances for assessment of in vitro cytogenicity and gene mutation in mammalian cells.

The read-across is based on the metabolism of Ethylene diformate, in particular on the fact that the substance undergoes enzymatic ester hydrolysis resulting in the formation of ethylene glycol and formic acid. A detailed analogue approach justification is provided in the technical dossier (see IUCLID Section 13).

Overview of in vitro genetic toxicity

CAS#

Chemical name

Molecular weight

In vitro gene mutation in bacteria

In vitro cytogenicity

In vitro gene mutation in mammalian cells

629-15-2 (a)

Ethylene diformate

118.09

Experimental result:

negative

WoE:

RA: CAS 107-21-1

RA: CAS 64-18-6

WoE:

RA: CAS 107-21-1

RA: CAS 64-18-6

107-21-1 (b)

Ethane-1,2-diol

(Ethylene glycol)

62.07

--

Experimental result:

negative

Experimental result:

negative

64-18-6

Formic acid

46.03

--

Experimental result:

negative

Experimental result:

negative

(a) The substance subject to registration is indicated in bold font.

(b) Reference (read-across) substances are indicated in normal font. Lack of data for a given endpoint is indicated by “--“.

 

Gene mutation in bacteria

The potential induction of gene mutation in bacteria by Ethylene diformate was investigated in a GLP-compliant study conducted according to OECD guideline 471 (Thompson, 2012). Following a preliminary toxicity assay, the tester strains Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 were treated with the test substance at five concentrations from 50 to 5000 µg/plate in two independent experiments in the presence and absence of metabolic activation (S9 mix). Concurrent untreated, solvent (DMSO) and positive controls were included in the test. No increase in the number of revertant colonies was observed in any of the bacterial strains, both with and without metabolic activation. The included negative and positive controls showed the expected results.

Under the conditions of the study, the test substance was not mutagenic in bacteria.

In vitro cytogenicity

CAS 107-21-1

For ethylene glycol, an in vitro mammalian chromosome aberration test was performed in Chinese hamster ovary (CHO) cells similarly to OECD Guideline 473 (National Toxicology Program, 1993). The occurrence of chromosome aberrations was investigated in the presence and absence of metabolic activation (S9-mix from rats treated with Aroclor 1245). Test substance concentrations of 160, 500, 1600 and 5000 µg/mL were used. Cells were treated for 10 h without metabolic activation and for 2 h with metabolic activation. Positive controls significantly increased the rate of chromosome aberrations indicating the sensitivity of the assay. In the absence and presence of metabolic activation, no increase in chromosomal aberrations was observed at any test substance concentration.

Thus, under the conditions of the study, ethylene glycol did not show clastogenic activity in this chromosomal aberration test with and without metabolic activation performed in CHO cells in vitro.

CAS 64-18-6

One study with formic acid is available, in which cytogenicity in mammalian cells was investigated. The in-vitro mammalian chromosome aberration test was conducted similarly to OECD guideline 473 (Morita, 1989). The induction of structural chromosome aberrations was evaluated in Chinese hamster ovary (CHO) cells incubated for 24 h with and without a metabolic activation system (S9-mix from rats treated with Phenobarbital/5,6-benzoflavone). Concentrations of 8-14 mM (without S9 mix) and 6-12 mM (with S9 mix) of formic acid in water were applied in a first series in F12 medium. Afterwards, the effects of the use of an enhanced buffering system on the induction of chromosome aberrations were investigated. The concentrations tested were 20, 25, 27.5 and 30 mM formic acid in F12 medium containing 34 mM NaHCO3 (without S9 mix) and 10, 20, 25 and 30 mM formic acid in F12 medium containing 30 mM HEPES (without S9 mix). In the first experiment (without buffering), the frequency of aberrant cells with and without metabolic activation was increased up to 15.9 and 20.5% without and with metabolic activation. Concomitantly, an increase in cytotoxicity and a lowered pH value was observed at these concentrations. The lowest observed pH value in this experiment without enhanced buffer abilities was 5.8. In the following experimental series using enhanced buffering systems, no increase in chromosomal aberrations was observed. These results indicated that the chromosomal aberrations in the first experiment were attributed to acidification of the medium and thus formic acid itself is non-clastogenic. Hence, the confounding factors do not allow an interpretation of the observed chromosomal damages as test-substance specific.

Therefore, under the conditions of the study, formic acid itself did not show clastogenic activity in this chromosomal aberration test with and without metabolic activation performed in CHO cells in vitro.

In vitro gene mutation in mammalian cells

CAS 107-21-1

Ethylene glycol was tested for induction of gene mutations in mammalian cells in a study conducted similarly to OECD guideline 476 (National Toxicology Program, 1993). Gene mutations in the thymidine kinase locus were investigated in L5178Y mouse lymphoma cells in the presence and absence of a metabolic activation system (S9-mix from rats treated with Aroclor 1254).

Cells were exposed for 4 h to ethylene glycol at up to 5000 µg/mL without metabolic activation in three independent experiments. In a fourth and fifth experiment, cells were exposed to 1000-5000 µg/mL for 4 h with metabolic activation. No cytotoxicity was observed up to the highest tested concentration of 5000 µg/mL. The vehicle and positive controls in the study showed the expected results. In the first and second experiment, a significant increase in the number of forward mutations was observed at 4000 µg/mL and at 4000 and 5000 µg/mL, respectively, without clear concentration dependency. The repeat experiment (experiment three), did not shown an increase in the number of forward mutations and thus did not confirm the results of experiments I and II. The experiments with metabolic activation did not show an increase in the number of forward mutations at any concentration. Thus, in the absence of a metabolic activation system, inconsistent responses were seen among the three independent experiments. A positive response in experiment II was observed in the two highest concentrations. However, this was not confirmed in two other independent experiments (experiment I and III) without metabolic activation. In addition, two experiments with a metabolic activation system were negative as well.

Thus, the test substance was considered to not induce gene mutation in this test performed in L5178Y mouse lymphoma cells in vitro.

CAS 64-18-6

An in vitro mammalian cell gene mutation study was carried out with formic acid similarly to OECD guideline 476 (Engelhardt, 2002). CHO cells were exposed for 4 h to concentrations from 31.25 to 500 µg/mL without metabolic activation and from 25 to 400 µg/mL with metabolic activation. Cytotoxicity was observed from approximately 400 µg/mL with and from 200 µg/mL without metabolic activation. The pH-value was reduced to the minimal value of 5.5. The vehicle and positive controls in the study showed the expected results. In the experiments, no significant increase in the number of forward mutations was observed at the tested concentrations with and without a metabolic activation system.

Thus, formic acid was not considered to show gene mutation activity in this test performed in CHO cells in vitro.

Conclusions for in vitro genetic toxicity

Ethylene diformate has been tested negative in a bacterial gene mutation assay. There are no data available on the potential induction of cytogenicity or gene mutation in mammalian cells.

Ethylene diformate is anticipated to undergo enzymatic ester hydrolysis within the body resulting in the formation of ethylene glycol and formic acid, as indicated by an in vitro hydrolysis test (Butler, 2013). Thus, the available data on the hydrolysis products was used for assessment by means of read-across applying an analogue approach.

Ethylene glycol and formic acid have been tested for the induction of cytogenicity and gene mutations in mammalian cells. All available studies were negative. Thus, the substances are considered to be not clastogenic and not mutagenic in mammalian cells.

Overall, based on substance specific data and read-across from the hydrolysis products, Ethylene diformate is considered to be not mutagenic and not clastogenic in vitro.


Justification for selection of genetic toxicity endpoint
Hazard assessment is conducted using substance specific data and by means of read-across from structural analogues/surrogates. No study was selected, since all available in vitro genetic toxicity studies were negative. All available studies are adequate and reliable based on the identified similarities in structure and intrinsic properties between source and target substances and overall quality assessment (refer to the endpoint discussion for further details).

Short description of key information:
Based on substance specific data and read-across from ethylene glycol (CAS No. 107-21-1) and formic acid (CAS No. 64-18-6):
In vitro gene mutation in bacteria: negative with and without metabolic activation
In vitro cytogenicity in mammalian cells: negative with and without metabolic activation
In vitro gene mutation in mammalian cells: negative with and without metabolic activation

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on substance specific data and read-across following an analogue approach, the available data on the genetic toxicity of Ethylene diformate do not meet the classification criteria according to Regulation (EC) 1272/2008 or according to Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.