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Key value for chemical safety assessment

Additional information

Four genotoxicity studies are available for assessment: an Ames test, an in vitro chromosome aberration test, an in vitro TK assay and an in vivo micronucleus test.

In vitro gene mutation test in bacteria

The mutagenic activity of the substance was examined in the reverse mutation test by using bacterial strains Salmonella typhimurium TA100, TA1535, TA98 and 1537 and Escherichia coli WP2uvrA (Genetic Laboratory, 2005). The GLP-compliant study was performed according to OECD guideline 471. The reverse mutation test was composed of the preliminary, the main and the confirmatory test, and the reappearance of these test results was confirmed. The pre-incubation method was used for all bacterial strains in both the presence and the absence of metabolic activation using the following concentrations: 156, 313, 625, 1250, 2500, 5000 µg/plate.

The test substance did not show statistically significant dose-related increase in the number of the revertant colonies compared with the negative control in any bacterial strains regardless of the presence or the absence of metabolic activation. In addition, the reappearance of the test results between the main and the confirmatory test was confirmed.

The values of the negative controls and the positive controls were appropriate in comparison with the historical data of our laboratory. Furthermore, all of the positive controls, such as 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, sodium azide, 9-aminoacridine, benzo[a]pyrene and 2-aminoanthracene, increased the number of the revertant colonies two-fold or more compared with the negative control in all bacterial strains, respectively. These results indicate that the test has been properly carried out.

From the foregoing results, it is concluded that the mutagenic activity of EEA-NH4 is considered negative under the test conditions employed.

In vitro chromosome aberration test with mammalian cells

The ability of EEA-NH4 to induce chromosomal aberrations was investigated by using Chinese hamster lung fibroblasts (CHLIIU cells) (Hita Laboratory, 2006). Based on the results of the cell growth inhibition test, the doses in the chromosomal aberration test were set at 723, 868, 1040, 1250, 1500 and 1800 µg/mL in short-term treatment (treatment for 6 hours followed by 18 hours culture) without S9 mix and at 603, 723, 868, 1040, 1250, 1500 and 1800 µg/mL in short-term treatment (treatment for 6 hours followed by 18 hours culture) with S9 mix.

In observation of specimens in the chromosomal aberration test, the highest dose for observation was selected at the minimum dose that the cell growth rate showed below 50%, and a total of 3 doses were selected for the observation doses of the test substance. Accordingly, the doses for observation were selected at 1250, 1500 and 1800 µg/mL for short-term treatment without S9 mix and at 868, 1040 and 1250 µg/mL in short-term treatment with S9 mix. In the observation, the frequencies of cells with structural aberrations and of numerical aberration cells were scored.

As a result of observation of specimens, the maximum frequencies of cells with structural aberration was over 10% in the short-term treatments without and with S9 mix, and the frequencies were recognized as a dose-related increase. Therefore, structural aberration was judged to be positive. The frequencies of numerical aberration cells showed below 5% at all doses of the test substance in the short-term treatments without and with S9 mix, therefore, numerical aberration were judged to be negative.

On the other hand, the frequencies of cells with structural aberrations and of numerical aberration cells in the negative control treated for distilled water showed below 5%, and the frequencies of cells with structural aberrations in the positive controls (mitomycin C or cyclophosphamide) showed above 20%, indicating the proper performance of the present study.

It is concluded that EEA-NH4 does not induce numerical aberrations but it induces structural aberrations under the present test conditions.

In vitro gene mutation test with mammalian cells

The test substance was examined for its potential to induce gene mutations at the TK-locus of cultured mouse lymphoma L5178Y cells, in both the absence and the presence of a metabolic activation system (S9-mix) in a GLP compliant study according to OECD guideline 476 (TNO Quality of Life, 2010). Two assays were conducted; in the first assay single cultures were treated for 24 hours and 4 hours in the absence and presence of S9-mix, respectively, and in the second assay cultures were treated for 4 hours in both the absence and presence of S9-mix. The test substance was dissolved in culture medium without serum prior to testing.

The highest concentrations of the substance evaluated for mutagenicity in the absence of S9-mix after 4 and 24 hours treatment were 3.1 mmol/L and 1.8 mmol/L, respectively. In the presence of S9-mix the highest concentrations evaluated for mutagenicity were 2.4 mmol/L and 2.9 mmol/L in the first and second assay, respectively. The maximum concentrations were limited by cytotoxicity.

the substance was cytotoxic in both the absence and presence of S9-mix. In the absence of S9-mix cytotoxicity, resulting in a reduction in initial cell yield and suspension growth, was observed at and above 0.39 mmol/L and 1.6 mmol/L. The relative total growth (RTG) values at the highest concentrations evaluated for mutagenicity (3.1 and 1.8 mmol/L) were 7% and 11% after 4 and 24 hours treatment. In the presence of S9-mix cytotoxicity was observed at and above 1.7 mmol/L; the RTG at the highest concentration (2.4 mmol/L and 2.9 mmol/L) were 37% and 10% in the presence of S9-mix in the first and second assay, respectively.

In both the absence and presence of S9-mix no increase in mutant frequency was observed at any test substance concentration evaluated. All data were within the range of the negative control and the historical background.

Methyl methanesulphonate (MMS) and 3-methylcholanthrene (MCA) were used as positive control substances in the absence and presence of the S9-mix, respectively; culture medium without serum served as negative control. The negative controls were within historical background ranges and treatment with the positive control yielded the expected significant increase in mutant frequency compared to the negative controls.

It is concluded that under the conditions used in this study, the test substance EEA-NH4 is not mutagenic at the TK-locus of mouse lymphoma L5178Y cells.

In vivo micronucleus study with rats

A GLP compliant micronucleus study performed according to OECD guideline 474 was performed with rats (TNO Quality of Life, 2010). The study consisted of a preceding Dose Range Finding (DRF) acute toxicity test, in which, by a step-wise treatment approach, the Maximum Tolerated Dose (MTD) was determined for the main micronucleus test. The preceding Dose Range Finding (DRF) acute toxicity test was followed by the main micronucleus test. The rats were dosed once daily on two consecutive days (interval ca. 24 hours).

Regarding the DRF, as a result of treatment with the test substance, clinical signs were observed in both sexes at all dose levels used (500, 750 and 1000 mg/kg bw/day) and all rats showed loss of weight. At the lowest dose level (500 mg/kg bw/day), clinical signs were observed in both sexes but judged as not severe. At the two higher dose levels (750 and 1000 mg/kg bw/day), severe clinical signs were observed in both sexes. At these two dose levels rats were found dead or were sacrificed for ethical reasons. Based on the clinical signs obtained in the preceding dose range finding acute toxicity test it was decided to perform the main micronucleus test with male and female rats and dose levels of 500 mg/kg bw/day (MTD), 250 mg/kg bw/day (50% of the MTD) and 125 mg/kg bw/day (25% of the MTD) were adopted.

In the main micronucleus test, animals were treated twice orally, on two successive days, with an interval of 24 hours, with three graded dose levels of the test substance (500, 250 and 125 mg/kg bw/day). In the vehicle control group each animal was dosed in a similar way with the vehicle control (sterile water for injection). Each group consisted of 5 animals per sex. A positive control group consisted of 5 males, and each animal was given a single intraperitoneal dose of mitomycin C at 1.5 mg/kg bw.

Due to severe clinical signs, as a result of treatment with the highest dose level of 500 mg/kg bw, two females were sacrificed for ethical reasons and one female was found dead before scheduled sacrifice. One female was replaced by a reserve female. Therefore, three instead of five females were available for analysis in the high dose group. At 24 hours after the final treatment, all remaining rats were euthanised; bone marrow cells were collected from one femur and processed into smears for microscopic examination.

The number of polychromatic erythrocytes (PE) per 200 erythrocytes (E) and the number of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE) were counted for each rat.

The incidence of MPE per 2000 PE, found in the positive control group, were statistically significantly higher compared to those of the negative control group (**p<0.01). The mean numbers of MPE per 2000 PE in the negative control group were within the historical control data. Thus, the test system was considered valid.

The incidence of PE per 200 E in male and female rats, treated with three dose levels of the test substance and the positive control mitomycin C, showed no statistically significant difference from those found in the vehicle (water for injection) controls. This indicates that treatment with the substance, up to 500 mg/kg bw/day, did not result in cytotoxicity to the bone marrow of male and female rats. Nevertheless, systemic availability of the substance can be assumed based on the systemic effects observed in the 28-day oral repeated dose toxicity study and in the reproduction/developmental toxicity screening study.

The incidence of MPE per 2000 PE in male and female rats, treated with three dose levels of the test substance (125, 250 and 500 mg/kg bw/day) showed no statistically significant difference from those found in the vehicle (water for injection) controls. This indicates that treatment with the substance, up to 500 mg/kg bw/day, did not result in damage to the chromosomes and/or to the spindle apparatus of the bone marrow cells of male and female rats.

In conclusion, EEA-NH4 is not considered to be a genotoxic substance. Negative results were obtained regarding gene mutations. Concerning chromosomal aberrations, while a positive result was obtained in vitro, the available in vivo micronucleus study was negative indicating that EEA-NH4 does not cause chromosomal aberrations in vivo.


Short description of key information:
The substance was negative in an Ames test, positive in an in vitro chromosomal aberration, negative in a TK assay and negative in an in vivo micronucleus test.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available data, classification for mutagenicity is not needed according to EU Directive 67/584/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.