Mecrilate

Endpoint summary

Additional information

General:

For the induction of mutagenicity, two principle mechanistic actions can be distinguished: the potential to induce either mainly gene- or chromosome mutations. For the conclusion on the mutagenic potential of a substance, both modes of action have to be investigated with suitable test systems. With regard to methyl 2-cyanoacrylate (MCA) and its structural homologues (ethyl 2-cyanoacrylate (ECA) and buyl 2-cyanoacrylate (BUCA)), both types of studies are available, allowing a comprehensive assessment of genetic toxicity. 

Data obtained with structural homologues ECA and BUCA are taken into account for the following reasons:

·        The homologues contain the same functional units and differ only in the length of the side chain (+CH₂ in the case of ECA, +C₃H₆ in the case of BUCA)

·        MCA, ECA and BUCA polymerize within seconds in the presence of small amounts of water (even air humidity is sufficient to trigger this process)

·        For the homologues, the same breakdown products are expected after hydrolytic degradation, i.e. the corresponding cyanoacetate and formaldehyde. However, a faster degradation was observed for MCA compared to longer-chained homologues.

·        The exposure to MCA, ECA and BUCA is comparable, as they are used as instant adhesives (superglue).

In order to avoid polymerization, cyanoacrylates usually contain small amounts of stabilizers. With regard to MCA and ECA, hydroquinone is common, whereas BUCA is often stabilized with butylhydroxytoluene. Furthermore, MCA differs from the two homologues by a stronger irritation potential that seems to decrease with elongation of the carbon chain length.

Genetic toxicity in vitro:

Regarding bacterial mutagenicity, several assays have been conducted with either MCA or MCA-based adhesives. The protocols followed the plate incorporation or the preincubation method, or were conducted as spot test for volatile compounds. In addition, further bacterial mutagenicity studies with structural homologues (ECA and BUCA) are published in literature.

In the studies with MCA, the tested Salmonella typhimurium strains comprised TA98, TA100, TA1535, TA1537 and 1538. In a part of the experiments, MCA increased the rate of reverse mutations. This effect was observed only for TA100, in the presence and absence of metabolic activation (rat liver S9 or hamster liver S9). The tests with the four other strains were negative. In contrast to MCA, the two homologues ECA and BUCA were not mutagenic in all tested Salmonella typhimurium strains, including TA100.

In the table below, the results with TA100 are summarized:

Reference	Test item	Concentration (plate incorporation), mg/plate	Spot test for volatile compounds		Plate incorporation or preincubation protocol		Remarks
			- S9	+ S9	- S9	+ S9
RT 930160 (Key)	MCA, purity not specified	0-1.62	(pos)	n.a.	neg	n.a.	Toxicity >= 0.54 mg/plate (ECA, ECA-based adhesive, BUCA: not mutagenic)
RT 930159 (Supp)	MCA, purity not specified, with 1-25 fold content of stabilizer	0-1.62	pos	n.a.	neg	n.a.	Toxicity >= 0.54 mg/plate No influence of stabilization on toxicity or mutagenicity
Zeiger et al. (Supp)	MCA, purity not specified	0-16.7	n.a.	n.a.	pos	pos	Both hamster and liver S9 mix were used
Andersen et al. (Supp)	Two commercial MCA adhesives (composition not specified)	0-5	pos	pos	pos	n.a.	Toxicity >= 0.5 mg/plate Visible precipitation of the polymer >= 1 mg/plate (ECA/BUCA adhesives: not mutagenic, toxicity >2 mg/plate) Tetrachloromethane extract (presumably containing additives of the adhesives, but not the MCA polymer) was negative Polymerized adhesive was negative
Rietveld et al. (Supp)	MCA (>98% purity) and two MCA adhesives (composition not specified)	0-0.5	pos	pos	pos	Pos	Toxicity >= 0.5 mg/plate (ECA: toxicity > 2 mg/plate; negative in TA 100)

 

The interpretation of the results for bacterial mutagenicity is hampered by several aspects:

1)    Toxicity of MCA
While toxicity started at appr. 0.5 mg/plate in most of the studies (RT 930160, RT 930159, Andersen et al., Rietveld et al.), Zeiger et al. did not explicitly describe whether toxicity was observed and analyzed the induction of mutations in the dose range of 1-16.7 mg/plate.

2)    Purity of the test item/composition of the tested product
The purity of the applied cyanoacrylate and the composition of the tested adhesive are not specified in any of the studies. Thus, it remains unclear whether the observed mutagenic effects were caused by the MCA monomer or by side components, e.g. hydroquinone.

3)    Precipitation
Andersen et al. reported about precipitations at 1 mg/plate and higher. Though concentrations up to 16.7 mg/plate were used in the assay of Zeiger et al., the authors did not mention the occurrence of precipitations. As automatic colony counters were used in this study, the question remains whether precipitations caused the high number of revertant colonies, especially as a reduction of overall colonies should be expected in light of the bacteriotoxic effects at this high concentration range used.

4)    Decomposition
During the relatively long incubation period in the bacterial mutagenicity test (48-72 h) and pushed by the temperature of 37°C, cyanoacrylates could theoretically undergo a hydrolytic degradation. The resulting cyanoacetate would not be expected to contribute to a mutagenic potential, but the released formaldehyde is also known to induce reverse mutations in Salmonella typhimurium. As MCA is more prone to decompose than longer-chained homologues, this might explain the discrepancy between MCA results (positive and negative) and ECA/BUCA results (negative).

Altogether, no final conclusion can be drawn for the induction of bacterial mutagenicity. Due to the rapid polymerization of cyanoacrylates in presence of small amounts of water, a repetition of the test would not deliver clearer results than already obtained in the available studies.

Regarding mammalian cells, an in vitro chromosome aberration test (according to OECD TG 473) and a mouse lymphoma test (according to OECD TG 476) are available, both with ECA as test item. Two unpublished HGPRT assays conducted with MCA are cited by the MAK commission.

In the chromosome aberration test, peripheral human lymphocytes were incubated with solutions of ECA in acetone. No effects on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9 -mix. Based on this result, it was concluded that ethyl 2-cyanoacrylate does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations.

In the mutagenicity test with L5178Y mouse lymphoma cells, the effect of ECA on the induction of forward mutations at the thymidine-kinase locus (TK-locus) was investigated. The test was performed in two independent experiments in the absence and presence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone). In parallel, hydroquinone was tested as additional control at a concentration of 1.28 µg/ml, representing the concentration of hydroquinone in the test system at the highest tested initial concentration (load rate) of the test substance. The content of hydroquinone in the tested sample of ECA according to the specification is in the range of just below 1000 ppm (equal to approx. 0.1%). Based on the results of this study, it is concluded that the tested sample of Ethyl 2-cyanoacrylate, (containing nearly 0.1% hydroquinone) has shown a mutagenic response in the mouse lymphoma L5178Y test system under the experimental conditions described in this report. The mutagenicity was confined only to incubations without metabolic activation at the prolonged treatment period. However, hydroquinone alone induced the same increase in the mutation frequency of 2.6-fold, when tested as additional control at the same concentration. Hydroquinone is known to induce positive responses in gene mutation tests in vitro at comparable concentrations. Due to the pattern of the identical response of hydroquinone under the test conditions performed, it has to be assumed that hydroquinone is the agent responsible for the detected mutagenic activity. The observed effect should therefore not be attributed to ethyl-2-cyanoacrylate as substance.

The MAK reported about two independent HGPRT tests with Chinese hamster V79 cells. The concentrations of Eurecryl 2400®, a MCA-based adhesive, were 5, 15, 25, 35 µg/ml without and 20, 50, 70, 90 µg/ml with S9 mix. No induction of a mutagenic potential was observed, neither with nor without metabolic activation.

 

Genetic toxicity in vivo:

In addition to the in vitro experiments, two in vivo tests are available for the assessment of the genetic toxicity potential of MCA. The drosophila sex-linked recessive lethal assay was conducted with MCA, whereas the structural homologue BUCA served as test item in the mammalian erythrocyte micronucleus test. Furthermore, the WHO cites an unpublished in vivo micronucleus study with MCA.

In an in vivo micronucleus study following OECD TG 474, an extract of polymerized BUCA was tested for its ability to induce a statistically significant increase in the number of micronucleated cells in rodent bone marrow. The undiluted extract obtained by incubation with 0.9% USP sodium chloride for 24 hours at 70±2°C was administered by injection to Swiss albina mice. No statistical significant increase in micronucleated cells was observed as compared to the negative control at 24 and 48 hours after dosing. The positive control, Mitomycin C, caused a statistically significant increase in micronucleated cells as compared to the negative control. Eight animals out of the test article group showed signs of lethargy and clonic convulsions immediately after injections. These signs of clinical toxicity, which indicate that the test item was systemically available, were resolved by the 24-hour observation and had no impact on the formation of micronuclei. No other clinical signs of toxicity were observed for any of the test or control animals. Based on the results of this test, a mutagenic potential is not expected for butyl 2-cyanoacrylate, which is a structural homologue to MCA.

According to the OECD TG 477, the drosophila sex-linked recessive lethal (SLRL) assay is suited to detect the “occurrence of mutations, both point mutations and small deletions”. Undiluted Super Bonder (containing 94.2% MCA) or dilutions in acetone were tested for their ability to induce lethal germ cell mutations in insects. The test substance solutions were given either as feed (test compound series I) or via inhalation (test compound series IIa, IIb, III) to male drosophila. In the first part of the study, the toxic concentration was determined using various concentrations. All males in test compound series I survived, no apparent differences were observed compared to the control group. In contrast, lethality up to 100% was observed if the inhalation protocol was followed. For the main study, four concentrations of the test item were applied by inhalation with varying exposure periods (test compound series V and VI). Following exposure, males were assigned a number and mated three times with three sets of virgin females for each 2-3 days, resulting in brood 1-3. After an approximate two-week incubation period, the F1 progeny were subjected to a round of brother-sister mating. The progeny (F2) of F1 females were scored after incubation. No statistically significant differences in percent lethals were noted between the test groups and the negative air control group. In contrast, mutagenic activity was detected with the positive control, dimethylnitrosamine, proving the sensitivity and responsiveness of the tester system. Under the conditions of this test, the MCA-based adhesive provided no evidence of a mutagenic effect.

As reported by the WHO, a MCA-based adhesive containing appr. 99% MCA was subjected to an in vivo micronucleus study. Based on a range-finder test, in which mortality at 750 mg/kg body weight or more was observed, a dose of 600 mg/kg b.w. was chosen for the main study. The MCA-based adhesive was suspensed in mineral oil and given by single intraperitoneal injection to groups of five male and five female mice. The animals  were sacrificed at 30, 48, and 72 h, and bone marrow samples were obtained for analysis of micronuclei. All animals showed clinical signs of systemic toxicity, such as decreased activity and decreased body tone. There were no statistically significant increases in the number of micronucleated polychromatic erythrocytes in any of the MCA-treated groups at any sacrifice time. A significant decrease in the ratio of polychromatic to normochromatic erythrocytes (P/N ratio) was observed at 48 h among mice receiving the test material. The WHO concluded that in a well-conducted micronucleus assay producing signs of bone marrow toxicity and other clinical signs of toxicity, MCA produced no evidence of mutagenicity.

Based on the in vivo tests with MCA and the close homologue BUCA, no evidence for a mutagenic effect of MCA was detected.

 

Overall conclusion:

On a weight-of-evidence basis, MCA can be regarded as not genotoxic. Though bacterial mutagenicity tests with MCA were not concordant, higher-tier in vitro tests with mammalian cells as well as animal studies gave no indication for a mutagenic potential.

Short description of key information:
see discussion

Endpoint Conclusion: No adverse effect observed (negative)