Ethyl acrylate

Endpoint summary

Additional information

 In-vitro studies: Bacterial systems 

 

Ethyl acrylate, similar to acrylic acid and all the acrylate esters within the category members are non-mutagenic in bacterial cells (i.e. Ames test).

 

Ethyl acrylate was tested in the Salmonella reverse mutation (SRM) assay withSalmonella typhimuriumTA98, TA100, Ta 1535, TA1537 and TA1538 and in Saccharomyces cerevisiae strain D4 at concentrations between 0.001 and 5.0 μL/plate with and without metabolic activation. Cytotoxicity was not observed at any dose. No mutagenic effects were found (Rohm & Haas 1977, 1981). 

 

In addition, there are several other Ames assays in strains TA 97, TA 98, TA 100, TA 1535, TA 1537, and TA 1538 as well as Sacharomyces cerevisiae strain D4 which were all negative except for one weakly positive result in TA100 (NTP, 1981). In the latter study numbers of revertants were increased by factor 1.6 at most and there was no clear dose-dependency. In later studies by NTP (1984, 1986) this weakly positive result was not verified. Thus, the overall result of assays by NTP was considered negative. 

 

Testing in Salmonella typhimurium strain TA 102 or Escherichia coli strain WP2 uvrA was not included in any of the available studies. Nonetheless, since all tests were negative with and without metabolic activation, there is no indication for a mutagenic potential of the test substance in bacterial cells. 

 

In-vitro studies: Mammalian cell gene mutation test 

 

Similar to Acrylic acid and other acrylate esters, EA was positive in some of the in vitro clastogenicity and mammalian gene mutation tests. However, positive results were due to excessive cytotoxicity. Available studies include a negative HPRT assay, positive mouse lymphoma TK mutation assay with small mutant colonies. Again in a 2019 mouse lymphoma TK mutation assay, EA was demonstrated to be negative at the same test concentrations by avoiding the previously observed cytotoxicity with supplemental GSH. There are several negative in vivo mouse micronucleus assays and chromosome aberration tests. In addition ethyl acrylate was tested in the in vivo gene mutation assay in gpt Delta Mice according to OECD TG 488. The mutant frequencies (6-thioguanine and Spi- selection) in the liver and stomach of all groups treated by oral gavage with ethyl acrylate were not increased; therefore ethyl acrylate was negative for genotoxicity in this test system.

 

Five mouse lymphoma assays (Dearfield et al. 1981, Rohm & Haas 1984, Moore et al. 1988, 1989, NTP, no date given) and two HGPRT assays in CHO cells (Moore et al. 1989, 1991) were performed. All the mouse lymphoma tests were positive with and without metabolic activation. The HGPRT tests were negative without metabolic activation. 

 

In the earlier mouse lymphoma TK studies, positive results occurred mostly at concentrations that led to a clearly reduced cell survival rate, and the increase in the number of mutants while dose-dependent, did not attain a doubling in mutant frequency in cultures showing at least 50 % relative total growth. Colony size was not evaluated in most assays and therefore it cannot be determined whether the mutants were derived from point mutations or chromosomal aberrations. 

 

Moore et al. (1989) evaluated four acrylates including EA in the mouse lymphoma assay and in a suspension adapted CHO assay for their mutagenicity. In addition to the specific locus mutagenesis analysis, mouse lymphoma and CHO cells were evaluated for the frequency of gross chromosomal aberrations. EA induced almost exclusively small-colony TK mutants and very few if any HGPRT mutants. Aberration analysis revealed that both the mouse lymphoma and CHO cells responded to the clastogenicity of the compound and that neither cell line was clearly more sensitive to the test compound. It is significant that all four acrylates including ethyl and methyl acrylate gave no evidence of genotoxicity when evaluated using selection for HGPRT-deficient mutants, but were clastogenic to the same cells in the same experiment. Percent survival in these experiments was between 60 and 15 %.  The ability of EA to induce gene mutations at the thymidine kinase (TK) locus or structural chromosome aberrations at chromosome 11 in L5178Y TK+/- mouse lymphoma cells in vitro was recently assessed in the presence of 1 mM Glutathione was recently assessed (BAMM 2019). One experiment carried out without the addition of liver S9 mix from phenobarbital and β-naphthoflavone induced rats (exogenous metabolic activation), but with or without the addition of 1mM Glutathione. In this study, the vehicle control with the addition of Glutathione gave mutant frequencies within the range expected for the L5178Y TK+/- mouse lymphoma cell line. The positive control methyl methanesulfonate (MMS) led to the expected increase in the frequencies of forward mutations. A dose-dependent cytotoxicity indicated by reduced relative total growth (RTG) of below 20% of control was observed in the absence of 1mM Glutathione at the highest applied concentration. In the presence of Glutathione, no relevant cytotoxicity was observed up to the highest tested concentration. Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies without S9 mix after adding 1 mM Glutathione. Thus, under the experimental conditions described, Ethylacrylate did not induce forward mutations or structural chromosome aberrations in vitro in the mouse lymphoma assay with L5178Y TK+/- cells in the presence of 1mM Glutathione without the addition of a metabolic activation.

 

Taking into account the negative results from the HGPRT and glutathione-repleated L5178Y TK+/- mouse lymphoma cell assays, and the finding that EA induced almost exclusively small colonies in the mouse lymphoma TK assay, it can be concluded that EA does not show mutagenic potential in mammalian cell cultures but indications of in vitro clastogenicity at doses which cause significant cytotoxicity associated with glutathione depletion. 

 

 In-vitro studies: Genotoxicity tests 

 

There is a considerable number of chromosome aberration tests in vitro available for EA (Ishidate et al. 1981, Moore et al. 1988, 1989, Tennant et al. 1987, NTP 1985, 1986). Assays were performed with CHL cells, L5178Y mouse lymphoma cells and CHO cells with and without metabolic activation. All assays gave positive or equivocal results at doses which reduced cell survival to 60 % or lower. There were no doses tested for chromosome aberrations which resulted in 70 % cell survival or more. Thus, there is no experimental evidence that EA might cause chromosome aberrations at non-cytotoxic doses. 

 

More recent studies have indicated that there is an association between chromosomal aberrations and cytotoxicity at exposure concentrations which reduce cell growth to less than 50% of the control value (Galloway, 2000 and references cited therein). These data suggest that the increase in mutagenicity reported in the cytogenicity assays with ethyl acrylate may be an artifact of the experimental method. The lack of mutagenicity when cell growth was within the acceptable range (50-100 % of the control value) indicates that this compound does not induce chromosomal aberrations under exposure conditions that prevent cytotoxicity. 

 

 Conclusion of in-vitro studies 

 

Ethyl acrylate has shown no evidence of mutagenic potential in Salmonella typhimurium in a number of tests. In mammalian cells in culture, indications of genotoxicity (clastogenicity) were restricted to experimental conditions that allowed only a low level of cell survival associated with depletion of glutathione. HGPRT assays conducted in CHO cells without metabolic activation gave no indication for a mutagenic potential in mammalian cells. Additionally, glutathione-repleated L5178Y TK+/- mouse lymphoma cells did not induce forward mutations or structural chromosome aberrations in vitro in cells without metabolic activation. 

 

 In vivo studies 

 

Ethyl acrylate has been tested in the mouse micronucleus assay for chromosomal abberations and in the mouse transgenic rodent assay for mutations.   

 

Ethyl acrylate was tested in the mouse micronucleus assay. Five animals/sex/group of C57B16J mice were administered with a single dose of ethyl acrylate via intraperitoneal injection at 0, 461 or 738 mg/kg bw in corn oil, corresponding to 50 or 80 % of the previously determined mean lethal dose and bone marrow cells were collected at 24, 48 and 72 hours post dosing. A separate group was dosed with 738 mg/kg bw of ethyl acrylate in water two times, 24 hours apart and cells were collected 6 hours after the last dose. No biologically significant increases in the incidence of micronucleated polychromatic erythrocytes (MPE) among polychromatic erythrocytes (PE) occurred in any test group. In high dose males (all time points) and females (48 and 72 hour time points) a statistically significant depression in erythropoiesis occurred (Ashby et al., 1989). 

 

In addition, there are several in vivo mouse micronucleus assays and chromosome aberration tests which were all negative (Hara et al. 1994, Tice et al. 1997, NTP 1988, 1990). 

 

The only study with a “positive” result for these in vivo studies was that reported first by Przybojewska et al. (1984). There were significant methodological deficiencies in this study, and the positive response was not confirmed in several reliable studies by Ashby et al. (1989). Therefore, this study is considered invalid, and ethyl acrylate is considered to be negative in in vivo chromosomal abberation studies. 

 

A gene mutation assay with transgenic mice (gpt delta mouse) was conducted to assess the potential of ethyl acrylate to induce gene point mutations and deletion mutations using the gpt gene and the red/gam genes (Spi⁻selection), respectively, as mutation reporter genes in the liver and stomach. 

 

In dose range-finding study in C57BL/6JJmsSlc mice treated for 28 days with 0, 25.0, 50.0, 100, or 200 mg/kg/day of ethyl acrylate in corn oil, gross observation revealed white nodules in the forestomach in all mice receiving 200 mg/kg/day and in one of the 3 mice receiving 100 mg/kg/day. Histopathological examination of animals in the 100 and 200 mg/kg/day groups revealed that there was an inflammatory response in the forestomach (consisting of squamous cell hyperplasia, fibrosis of mucosa, and infiltration of inflammatory cells). In addition, erosion was observed in 1 animal in the 200 mg/kg/day group. A local inflammatory response may interfere with the outcome of the mutagenicity data and should thus be avoided. Metabolism of ethyl acrylate occurs through carboxylesterases and through conjugation with glutathione. The latter metabolic reaction becomes saturated between 20 and 100 mg/kg, while dosages ≥ 100 mg/kg exhibit complete saturation. Therefore, a dosage of 50.0 mg/kg/day was selected as the high dose as this dose was without a potentially confounding inflammatory response and is anticipated to be at the threshold for saturation of GSH metabolism, based on previous rodent studies. Two additional dosage levels including 20.0 and 8.00 mg/kg/day were selected as lower doses in the present study. 

 

The test substance was administered to male transgenic mice orally for 28 consecutive days by gavage and after 3 days of the manifestation period, the liver, stomach and testis were removed and mutant frequencies in the liver and stomach were determined. 

 

The results showed that the mutant frequencies (6-thioguanine and Spi-selection) in the liver and stomach of all groups treated with ethyl acrylate were negative for genotoxicity under the conditions of this study. Furthermore, the mutant frequencies in the liver and stomach of all groups treated with ethyl acrylate did not show any statistically significant increases as compared with the negative control group. 

 

The mutant frequencies in the liver and stomach of the positive control group, which was treated with benzo[a]pyrene (B[a]P, dosage level of 125 mg/kg/day), increased in both thegptand Spi-assays and these increases were statistically significant compared with that of the negative control group. 

 

 Conclusion 

 

Based on the presented data, ethyl acrylate is considered to not be damaging to chromosomes and to be non-mutagenic in vivo. 

Short description of key information:

Ethyl acrylate was negative in bacterial mutation tests. EA seems to have some potential for genotoxicity in mammalian cells, presumably by a clastogenic mechanism. This effect is limited to doses with moderate to strong cytotoxicity and associated with glutathione depletion. Furthermore, this potential has not been expressed in vivo with Ethyl acrylate being negative in several in vivo mouse micronucleus assays, chromosome aberration tests, and a mouse transgenic rodent assay. Thus, taking the negative test results in vivo for EA into consideration, it can be assumed that EA will not cause any DNA damage, i.e. genotoxicity in vivo.  

Endpoint Conclusion:

Detailed information on the Mode of Action is available in Annex III.

Justification for classification or non-classification

EU classification according to Annex VI of Directive 67/548/EEC: no classification required

GHS classification (GHS UN rev.3, 2009): no classification required