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EC number: 700-323-3
CAS number: 908020-52-0
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
mutagenic activity of the substance was examined in the reverse mutation
test by using bacterial strains Salmonella
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.
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
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.
In vitro chromosome aberration test with
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
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
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).
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.
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
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.
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.
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
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
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.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
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