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Key value for chemical safety assessment

Additional information

1) in vitro

 

Ames-test:

In a reverse gene mutation assay the bacteria strains TA 1535, TA 1537, TA 98 and TA 100 of S. typhimurium  were exposed to AEEA (99.0 % a.i.) at concentrations of 0, 20, 100, 500, 2000, 2500, 4000, 5000, 6000 or 8000 µg/plate (3 plates/dose) in the presence and absence of mammalian metabolic activation applying both the standard plate and the pre-incubation method (BASF AG, Department of Toxicology, 1991). AEEA was completely soluble in water and tested up to 8000 µg/plate. Cytotoxicity was only observed in the pre-incubation test depending on the strain at 2500 and 5000 µg/plate. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable and satisfies the requirement of OECD Test Guideline 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

AEEA (98.9 %) was tested in the Ames test using Salmonella typhimurium TA100, TA1535, TA98, and TA1537 with and without metabolic activation at 0, 33, 100, 33, 1000, 1800, 2800, 3333, 3500, and 4000 μg/plate (Zeiger, 1987). Concentrations higher than 2800 µg/ml were slightly toxic. AEEA was negative in TA98, TA1537, and TA100 with or without metabolic activation. In TA1535 with metabolic activation at 3333, 3500, and 4000 μg/plate the number of revertants was ca. 2.5 -fold increased compared to the solvent control. The authors concluded that AEEA was "weakly mutagenic" in TA1535. However, as the increase was less than 3 -fold, which is commonly used as an evaluation criteria for a positive response in this strain, and no historical control data have been presented in the publication, the conclusion of authors is questionable. Furthermore, the purity of the test material used in this test was less than in the test performed by BASF (1991), where AEEA was clearly negative in TA 1535, so that it cannot be excluded that impurities might be responsible for the slight increased revertant frequency. Therefore, the test by Zeiger et al. is less reliable than the test performed by BASF (1991).

Taking these facts into account, the overall conclusion is that AEEA is not mutagenic in the bacterial reverse mutation assay.

HGPRT-test:

In a mammalian cell gene mutation assay for the HGPRT locus CHO cells cultured in vitro were exposed to AEEA (97.1 - 99.4 % a.i.) at concentrations of 0, 0.125, 0.25, 0.5, 1, 2, 4, or 8 µl/mL corresponding to 0.13, 0.26, 5.16, 1.03, 2.06, 4.12, or 8.24 mg/mL in the presence and absence of mammalian metabolic activation (S9 -mix). AEEA was tested up to cytotoxic concentrations (Leung, 1994). It was cytotoxic at 4 and 8μg/mL(< 20 % growth relative to water control).The positive controls did induce the appropriate response. There was no evidence of induced mutant colonies over background. This study is classified as acceptable and satisfies the requirement of OECD Test Guideline 476 for in vitro mutagenicity (mammalian forward gene mutation) data. 

Sister Chromatid Exchange:

In a mammalian cell cytogenetics assay (SCE) CHO cell cultures were exposed to AEEA (97.1 - 99.4 % a.i.) at concentrations of 0 , 0.13 , 0.26 , 0.52 , 1.03 , 2.06 or 4.12 mg/ml with and without metabolic activation employing S9 rat liver homogenate (Leung, 1994). Positive controls induced the appropriate response. A single, sporadic, statistically significant increase in the frequency of SCE was observed at dose level 0.26 µg/ml with metabolic activation. However, there was no evidence for a concentration related positive effect of SCE induced over background. Thus, it is concluded that AEEA is negative in the sister chromatid exchange assay.

This study is classified as acceptable and satisfies the requirement of OECD Test Guidelinine 479 for in vitro cytogenetic mutagenicity (in vitro sister chromatid exchange assay in mammalian CHO cells) data.

Unscheduled DNA synthesis

In an unscheduled DNA synthesis assay (Leung, 1994), primary rat hepatocyte cultures were exposed to AEEA, (97.1 – 99.4 % a.i.) at concentrations 0, 0.001024, 0.01024, 0.0307, 0.1024, 0.307, or 1024 µg/mL for 1 hour. The positive controls induced 4-NQO and DMN appropriate response. There was no evidence that unscheduled DNA synthesis, as determined by radioactive tracer was induced.

Chromsome aberration

In a mammalian cell cytogenetics chromosome aberration assay CHL cell cultures were exposed to AEEA (99.9 % a.i.) at concentrations of 0, 0.25, 0.5, 1.0 mg/mL with and without metabolic activation and examined for structural changes after 6 and 18 hours (Tanaka, 1996). Additionally, cells were incubated in the absence of metabolic activation for 24 and 48 hours. The applied positive controls gave the expected results.There was no increase of structural changes noted in any of the experiments. Polyploidy was statistically significantly increased at 0.5 and 1 mg/mL (0.88% and 4.0%, respectively) without S9 after 48 hours, but not after 24 hours. However, no repeat experiment was conducted to verify the finding after 48h exposure. Also, this effect was not observed in the other genotoxcitiy experiments with or without metabolic activation.

2) in vivo

 

SLRL-test:

In a Sex-linked Ressessive Lethal Test male Drosophila melanogaster were exposed to AEEA (99.8 % a.i.) in a feeding test at a concentration of 52000 ppm for 3 days (Foureman, 1994). Since the authors regarded the lethality of 0.14 % as equivocal (concurrend control: 0.06 %) an injection test at a concentration of 3400 ppm was conducted following the feeding test. The lethality in the injection test was 0.04 % lethals compared to 0.09 % lethals in the control group.

However, the lethality following a very high exposure to AEEA in the feeding experiment was well in the range of lethality that was seen in control groups of the same laboratory (0.0 % - 0.21 %). Therefore, AEEA is regarded to be negative in the feeding test and in the injection test.

This study is classified as acceptable and satisfies the requirement of OECD Test Guideline 477 for in vivo mutagenicity data.

 

Micronucleus Assay:

In a Crj:BDF1 mouse bone marrow micronucleus assay, 5 animals/sex/dose were treated by gavage with AEEA (99.9 % a.i.) at doses of 0, 500, 1000, or 2000 mg/kg bw (Shibuya, 1996). The vehicle was water. Bone marrow cells were harvested 24 hours post-treatment and analysed for the occurrence of miceonucleated PCEs. The number of 2000 PCEs was counted per animal. The proportion of PCEs in 500 NCE was recorded. There were no signs of toxicity during the study. The positive control (CPA) induced the appropriate response. There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow up to the dose of 2000 mg/kg bw after treatment time.

This study is classified as acceptable and satisfies the requirement for OECD Test Guideline 474 for in vivo cytogenetic mutagenicity data.


Justification for selection of genetic toxicity endpoint
No study was selected since all studies were negative.

Short description of key information:
No potential for genetic toxicity of AEEA was indicated in the Ames Test including doses that were bacteriotoxic. In the mammalian cell forward mutation assay using Chinese hamster ovary cells (HGPRT) AEEA was not genotoxic even at cytotoxic concentrations both with and without metabolic activation. This also holds for Sister Chromatid Exchanges in the same cell line and UDS tested in rat hepatocytes. No increase of structural chromosomal changes was noted in Chinese Hamster lung cells. In this assay a slight but statistically significant increased polyploidy was noted in one experiment.
AEEA was tested in male Drosophila melanogaster using the Sex-Linked Recessive Lethal assay (SLRL, OECD TG 477) and regarded as being negative.
There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in a micronucleus assay.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the negative in vitro results of the Ames test, the HGPRT test and SCE assay supported by negative in vivo testing (SLRL test and bone marrow micronucleus assay), AEEA has not to be classified and labelled mutagenic according to EU Regulation No. 1272/2008 and EU Directive 67/548/EEC.