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Bacterial Reverse Mutation assay

The test substance was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2UvrA. Two independent tests using duplicate cultures were carried out for both assays. The study was conducted according to OECD guidelines 471 and 472. The test substance was tested up to concentrations of 5000 µg/plate in the absence and presence of S9 mix. No induction of the number of revertant colonies was observed in all strains tested with or without S9 mix.

Therefore it was concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and not mutagenic in the Escherichia coli reverse mutation assay. (Hatano, 1999)

 

In vitro Chromosome Aberration Assay

The test substance was tested in a Chromosome Aberration Assay in CHL/IU cells according to OECD Guideline 473. The test item was dissolved in Dimethylsulphoxide (DMSO) and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (with and without metabolic activation using S9 mix). At least 200 well-spread metaphase cells were analysed at concentrations and incubation/expression intervals given below, ranging from little to maximum (40 - 50 % survival) toxicity:

-S9 mix (continuous exposure 24 and 48 hours); 0, 0.019, 0.038, 0.075, 0.15, 0.30 mg/mL

-S9 mix (short-term exposure 6 hours): 0, 0.019, 0.038, 0.075, 0.15, 0.30 mg/mL

+S9 mix (short-term exposure 6 hours): 0, 0.038, 0.075, 0. 15, 0.30, 0.60 mg/mL

 

During continuous treatment of 24 and 48 hours, there were no biologically significant increases in the number of cells showing structural chromosome aberrations in the absence of metabolic activation, up to and including cytotoxic concentrations.

Cytogenetic effects were seen as follows: Structural chromosome aberrations (included gap) were induced at 0.3 and 0.6 mg/mL during short term exposure (mid and high concentrations, 6 % and 24.5 %, respectively) in the presence of metabolic activation. Polyploidy was induced under the following conditions: 24 h continuous treatment (0.15 an 0.3 mg/mL: mid and high concentrations, 1.75 and 7.25 %, respectively); 48 h continuous treatment (0.3 mg/mL, 5.18 %); short term treatment without metabolic activation (0.3 mg/mL, 7.13 %).

The validity of the test was shown using Mitomycin C (0.05μg/mL) and Cyclophosphamide (5.0μg/mL) as concurrent positive controls. It was concluded from the above results that the test substance induced structural aberrations of chromosomes and polyploid cells in the CHL/IU cells of Chinese hamsters. (Hatano, 1999)

 

In vitro Mammalian Cell Gene Mutation Test

A mammalian cell gene mutation test was not available for the test substance. Therefore read across was performed with a structural very similar substance, 2 -(dimethylamino)ethyl methacrylate (CAS 2867-47-2).

The in vitro potential mutagenic activity of the test substance was investigated in V79 Chinese hamster cells according to OECD guideline 476. After a preliminary cytotoxicity assay to define the concentrations to be used in the mutagenicity study, the test substance was tested both in the absence and in the presence of a metabolic activation system. Two independent tests using duplicate cultures were carried out on 2 separate occasions. The concentrations were:

+S9 mix: 62.5, 125, 250, 500 and 1000 µg/mL for the 1st test,

+S9 mix: 250, 500, 1000, 1500 and 2000 µg/mL for the 2nd test,

-S9 mix: 31.25, 62.5, 125, 250 and 500 µg/mL for both tests.

 

Without S9 mix, the test substance showed some signs of cytotoxicity for the V79 cells at 250 and 500 µg/mL: some cells were round and refringent after 3 hours of treatment; at 500 µg/mL, the cloning efficiency was decreased by 85 % and 100 % in the first and the second assay respectively.

With S9 mix, round and refringent cells were observed at concentrations greater than or equal to 250 µg/mL and the cloning efficiency was decreased by 90 % at 2000 µg/mL. No mutagenic effect was observed in this test system V79/HPRT either with and without metabolic activation at the concentrations used. The mutation frequency of cells treated with the positive controls (N-methyl-N-nitro-N-nitrosoguanidine without S9 mix or benzo(a)- pyrene with S9 mix) was higher than the mutation frequency of nontreated cells, indicating the sensitivity of this test system as well as the efficacy of the metabolic activation system in this study.

In conclusion, under the experimental conditions, the test substance did not show mutagenic activity in this HPRT gene mutation assay in V79 Chinese hamster cells. (Atochem, 1992)

 

In vivo Micronucleus Assay

The clastogenic activity of the test substance was assessed in a micronucleus test by oral gavage admistration in ICR mice according to OECD guideline 474. A preliminary test was conducted to derive the maximum tolerable doses. Male and female mice were subjected to double oral gavage administrations of 250, 500, 1000, and 2000 mg/kg/day doses of methacrylic acid, 2-(diethylamino)ethyl ester at 24-hour intervals. At 2000 mg/kg bw/d two male animals died and clinical signs of toxicity were seen in the other individuals. Female animals did not show any signs of toxicity up to 2000 mg/kg bw/d.

Based on the results of the preliminary toxicity test, in the main micronucleus test, male mice were subjected to double oral gavage administrations of 250, 500, and 1000 mg/kg/day doses of methacrylic acid, 2-(diethylamino)ethyl ester at 24-hour intervals. Bone marrow smears were prepared after 24 hours of the final administration. No statistically significant increase in micronuclei was observed in any of the dose groups. As for the percentage of polychromatic erythrocytes in erythrocytes, no statistically significant differences were recognized between the negative control group and the dose groups, and there were no antiproliferative activities of bone marrow cells resulting from administration of the test substance. The negative and positive controls fell well within the variation range of the historical control data.

In conclusion, under the experimental conditions, the test substance did not show clastogenic effects in bone narrow cells of mice after oral administration.(Hatano, 2006)


Short description of key information:
No mutagenic effects were seen in the bacterial reverse mutation assay and HPRT (read across). The test substance induced chromosome aberrations in chinese hamster lung cells. However, this positive result was not confirmed in an in vivo micronucleus assay; the test substance did not show clastogenic effects in bone narrow cells of mice after oral administration. Thus, the test substance was not considered to be genotoxic / clastogenic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for genetic toxicity under Directive 67/548/EEC, as amended for the 31st time in Directive 2009/2/EG.

 

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No. 1272/2008, as amended for the sixth time in Regulation EC 605/2014.