N,N-dimethylacrylamide

Endpoint summary

Additional information

In vitro gene mutation test in bacteria

The mutagenic activity of N,N-dimethylacrylamide was examined in the reverse mutation test by using bacterial strains Salmonella typhimurium TA1535, TA1537, TA98 and TA100 and Escherichia coli WP2uvrA (USEPA, 1995). The GLP-compliant study was performed comparable to OECD guideline 471. The reverse mutation test was composed of a dose range finding and a main test. The plate incorporation method was used for all bacterial strains in both the presence and the absence of metabolic activation using ten concentrations ranging from 6.67 to 5000 µg/plate (dose range finding) and 100, 333, 1000, 3330 and 5000 µg/plate (main test). No increase in revertant colonies was observed in the tested strains. Furthermore, no cytotoxicity was observed with any of the tester strains in either the presence or absence of metabolic activation as evidenced by a normal background lawn and no decrease in the number of revertants per plate. It is concluded that under the conditions of this study N,N-dimethylacrylamide is not mutagenic.

 

N,N-dimethylacrylamide was also negative in a reverse mutation test using bacterial strain Salmonella typhimurium TA1535, TA1537, TA1538, TA98 and TA100 (Hashimoto, 1985). In this test, at least 5 concentrations within the range 5 -5000 µg/plate were tested both in the presence and absence of metabolic activation.

 

In vitro chromosome aberration test with mammalian cells

The ability of N,N-dimethylacrylamide to induce chromosomal aberrations was investigated by using Chinese ovary cells in a study comparable to OECD guideline 473 (USEPA, 1995). Based on the results of a range finding test, the concentrations of the test substance in the chromosomal aberration test were set at 458, 610, 915, 1220, 1520, 2270, 3020 and 4020 µg/ml without metabolic activation, and 2020, 2770, 3520, 4270 and 5020 µg/ml with metabolic activation. Significant increases in cells with chromosomal aberrations were observed in the cultures treated with 1520 and 2270 µg/plate without metabolic activation and a weakly positive response was observed at a single concentration, 5020 µg/plate, with metabolic activation. No cytotoxicity was observed. The test article, N,N-dimethylacrylamide, was considered positive for inducing chromosomal aberrations in Chinese hamster ovary cells under the nonactivation conditions and weakly positive under the activation conditions of this assay.

 

In vitro mammalian cell gene mutation test

The ability of N,N-dimethylacrylamide to induce forward mutations at the thymidine kinase (TK) locus in the mouse lymphoma L5178Y cell line was investigated in a study comparable to OECD guideline 476 (USEPA, 1995). Based on the results of a range finding test, the concentrations of the test substance were set at 9 concentrations ranging from 500 to 5000 µg/ml with and without metabolic activation. Under nonactivation conditions, six treatments from 2000 µg/ml to 5000 µg/ml were chosen for mutant induction. The mutation assay was more cytotoxic than the rangefinding assay and several of the dose levels were excessively cytotoxic. However, all treatments, including a moderately cytotoxic dose with 41.3% relative growth, induced mutant frequencies that exceeded the minimum criterion for a positive response. Increases that exceeded the minimum criterion for a positive response were also induced in the presence of metabolic activation. The test article was also more cytotoxcic with activation and only four treatments from 500 µg/ml to 2000 µg/ml were available for analysis. The four treatments induced weak to very high cytotoxicities and the two highly cytotoxic treatments (1500 µg/ml and 2000 µg/ml) induced increases in the mutant frequency that exceeded the minimum criterion for a positive response.

The test material, N,N-dimethylacrylamide, was therefore evaluated as positive (clastogenic) for inducing forward mutations at the TK locus in L5178Y mouse lymphoma cells under the nonactivation and activation conditions of this assay.

Analysis of colony size showed that the increases in mutant frequency with and without metabolic activation were due to the presence of both small and large mutant colonies, with a slightly higher number of small colonies at the highest concentrations analysed. The induction of small colony mutants is known to be associated with chemicals that induce gross chromosome aberrations. Less seriously affected mutant cells grow at rates similar to the parental cells and form large colonies. As the number of small colonies increased with dose, the positive result in this test represents clastogenicity.

 

In vitro mammalian cell gene mutation test

The potential of N,N-Dimethylacrylamide to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro was assessed in a GLP compliant study according to OECD guideline 476 (BASF SE, 2013). Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation).

Based on a pretest for toxicity in which 1,000 µg/mL (about 10 mM) was used as top concentration, the following doses were tested in the first experiment: 0; 125.0; 250.0; 500.0; 1 000.0 μg/mL (4-hour exposure, with and without S9 mix). The doses tested in the second experiment were: 0; 62.5; 125.0; 250.0; 500.0; 1 000.0 μg/mL (24-hour exposure, without S9 mix) and 0; 175.0; 350.0; 700.0; 1 000.0 μg/mL (4-hour exposure, with S9 mix).

Following attachment of the cells for 20-24 hours, cells were treated with the test substance for 4 and 24 hours in the absence of metabolic activation and for 4 hours in the presence of metabolic activation. Subsequently, cells were cultured for 6-8 days and then selected in 6-thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted.

The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, EMS and DMBA, led to the expected increase in the frequencies of forward mutations.

In this study in the absence and the presence of metabolic activation no cytotoxicity was observed up to the highest required concentration tested for gene mutations.

Based on the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other.

Thus, under the experimental conditions of this study, the test substance N,N-dimethylacrylamide is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

 

In vivo mammalian erythrocyte micronucleus test

A GLP compliant micronucleus assay in bone marrow cells according to OECD guideline 474 was performed with mice (Harlan CCR, 2013). Male and female NMRI mice received a single dose of the test item in sterile water by oral gavage at dose levels of 100, 200 and 400 mg/kg body weight (males) and 50, 100 and 200 mg/kg body weight (females). The animals treated with the test item showed clinical signs such as reduced spontaneous activity, eyelid closure and/or ruffled fur.

After treatment with the test item the number of PCEs per 2000 erythrocytes was not substantially decreased as compared to the mean value of PCEs per 2000 erythrocytes of the vehicle control thus indicating that the substance did not exert a cytotoxic effect in the bone marrow.

In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used.

The negative and positive controls fulfilled the requirements for determination of a valid test.

Under the conditions of this assay N,N-Dimethylacrylamide did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Therefore, N,N-Dimethylacrylamide is considered to be non-mutagenic in this micronucleus assay.

Short description of key information:
The substance was negative in an Ames test and positive in both an in vitro chromosomal aberration test and a mouse lymphoma assay. This result was indicative of in vitro clastogenicity. The HPRT assay was negative, as was the in vivo micronucleus test.
Based on these available studies it can be concluded that N,N-Dimethylacrylamide is not mutagenic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the results of the in vitro and in vivo tests, N,N-Dimethylacrylamide does not need to be classified according to Directive 67/548/EEC and according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.