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Key value for chemical safety assessment

Genetic toxicity in vivo

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: F. Winkelmann, Borchen, Germany
- Strain: Bor: NMRI (SPF Han)
- Age at study initiation: approximately 8-12 weeks
- Weight at study initiation: 28-41 g
- Housing: females in groups of a maximum of 3 mice, males singly
- Diet and water: ad libitum
- Acclimation period: at least one week
Route of administration:
intraperitoneal
Vehicle:
The test substance was used purely without vehicle.
Details on exposure:
The test substance was injected intraperitoneally.
Duration of treatment / exposure:
Animals were treated once.
Frequency of treatment:
Single intraperitoneal administration of the test substance or the positive or negative control substance.
Post exposure period:
The animals were sacrificed for bone marrow preparation 16, 24 and 48 hours after the test substance administration. (For positive and negative control: 24 hours.)
Remarks:
Doses / Concentrations:
5 ml/kg bw
Basis:
actual ingested
i.p. administration
No. of animals per sex per dose:
5 per sex, dose and per each bone marrow preparation time
Control animals:
other: yes, for negative control physiological saline solution... (see attached file)
Positive control(s):
Cyclophosphamide, dissolved in deionized water, administered i. p. with 20 mg/kg bw. Animals were sacrificed 24 hours after administration of substance.
Tissues and cell types examined:
Bone marrow smears examined.
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION: Schmid's method (1975) was used to produce the smears. At least one intact femur was prepared from each sacrificed animal.

METHOD OF ANALYSIS: 1000 polychromatic erythrocytes were counted per animal. The ratio of polychromatic to normochromatic erythrocytes was determined. The number of normochromatic erythrocytes showing micronuclei was established.
Evaluation criteria:
A test was considered positive if, at any of te intervals, there was a relevant and significant increase in the number of polychromatic erythrocytes showing micronuclei in comparison to the negative control.
A test was considered negative if there was no relevant or significant increase in the rate of micronucleated polychromatic erythrocytes at any time. A test was also considered negative if there was a significant increase in that rate which, according to the laboratory’s experience was within the range of negative controls.
In addition, a test was considered equivocal if there was an increase of micronucleated polychromatic erythrocytes above the range of historical negative controls, provided the increase was not significant and the result of the negative control was not closely related to the data of the respective treatment group. In this case, a second test had to be performed at the most sensitive interval.
Statistics:
Wilcoxon's non-parametric rank sum test; A variation was considered statistically significant if its error probability was below 5% and the treatment group figure was higher than that of the negative control.
One-sided Chi²-test used for determination of the rate of normochromatic erythrocytes containing micronuclei, in case of the micronuclear rate for polychromatic erythrocytes was already increased.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
, at doses of 5.0 mg/kg bw
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
Groups of 5 animals (including both males and females) were intraperitoneally administered 5, 10 and 20 ml/kg. Additionally 2500 mg/kg were formulated in corn oil and injected intraperitoneally. All animals died in the 10 and 20 ml/kg groups, the animals of the other groups exhibited symptoms (apathy, reduced motility, roughened fur, staggering gait, spasm, twitching, difficulty in breathing) and also mortality (5/5 animals died within 6 days in one of the 5 ml/kg group; no animal died in the other 5 ml/kg group). 5 ml/kg bw pure substance was choosen for the main study.

RESULTS OF DEFINITIVE STUDY
- Observations: clinical symptoms: apathy, roughened fur, distended abdomen, staggering gait, spasms, twitching, difficulty in breathing, eyelids stuck together, reduced discharge of faeces. Their feeding behavior was normal. There were no substance-induced mortalities.
- Induction of micronuclei (for Micronucleus assay): No biologically important or statistically significant variations with respect to the incidence of micronucleated polychromatic erythrocytes (1.6/1000 in the negative control; 1.8/1000 in the 16 hours-group; 2.0/1000 in the 24 hours-group; 1.0/1000 in the 48 hours-group).
- Ratio of PCE/NCE: the ratio was altered by the treatment with the test substance, being 1000/631 in the negative control; 1000/2109 in the 16 hours-group; 1000/1147 in the 24 hours group; 1000/1797 in the 48 hours group.
- The number of micronucleated normochromatic erythrocytes did not increase relevantly in any of the groups.

Study was performed with Aspartic acid, N,N'-[methylenebis(2-methyl-4,1-cyclohexanediyl)]bis-, 1,1',4,4'-tetraethyl ester which is a structural analogue to Aspartic acid, N,N'-(methylenedi-4,1-cyclohexanediyl)bis-, 1,1',4,4'-tetraethyl ester. Both substances are diethyl esters of aspartic acid linked to a dicyclohexylmethyldiamine moiety. The difference between these two substances is merely the presence of two methyl groups connected to the cyclohexane rings. This structural analogy was confirmed by the Member State responsible for the notification of both substances under the NONS regulation. The Member State decided that test results obtained for one substance can be transferred to the other substance and that testing of both substances is usually not required. This decision is in accordance with the grouping of substances and read-across approach in Annex XI, 1.5 of the REACH Regulation.

Executive summary:

In a mouse bone marrow micronucleus test (OECD TG 474) on 5 male and 5 female mice receiving each a single intraperitoneal injection of 5 ml/kg bw of aspartic acid, N,N'-[methylenebis(2-methyl-4,1-cyclohexanediyl)]bis-,1,1',4,4'-tetraethyl ester no indications of a clastogenic effect were found after evaluation of femoral marrow smears, obtained at 16, 24 and 48 hours after test substance administration. Relevant systemic exposure was demonstrated by symptoms of toxicity and an altered PCE/NCE ratio.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

The structural analogue aspartic acid, N,N'-[methylenebis(2-methyl-4,1-cyclohexanediyl)]bis-,1,1',4,4'-tetraethyl ester did not show genetic toxicity in two in vitro testings (OECD TG 471, Herbold, 1991; OECD TG 473, Herbold, 2002; both with and without metabolic activation) and in an in vivo mammalian erythrocyte micronucleus test (OECD TG 474, Herbold, 2004).

All studies investigating mutagenicity were performed with aspartic acid, N,N’-[methylenebis(2-methyl-4,1-cyclohexanediyl)]bis-, 1,1',4,4'-tetraethyl ester which is a structural analogue to aspartic acid, N,N’-(methylenedi-4,1-cyclohexanediyl)bis-, 1,1',4,4'-tetraethyl ester. Both substances are diethyl esters of aspartic acid linked to a dicyclohexylmethyldiamine moiety. The difference between these two substances is merely the presence of two methyl groups connected to the cyclohexane rings. This structural analogy was confirmed by the Member State responsible for the notification of both substances under the NONS regulation. The Member State decided that test results obtained for one substance can be transferred to the other substance and that testing of both substances is usually not required. This decision is in accordance with the grouping of substances and read-across approach in Annex XI, 1.5 of the REACH Regulation.


Justification for selection of genetic toxicity endpoint
Highest tier genotoxicity study available.

Justification for classification or non-classification

No classification required according to EU-Directive 67/548/EEC, Annex I.

No classification required according to Regulation (EC) No 1272/2008, Annex VI.

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