Registration Dossier

Administrative data

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:

Test animals

Details on test animals or test system and environmental conditions:
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Strain: Hsd/Win: NMRI
- Age at study initiation: approx. 6-12 weeks
- Weight at study initiation: 37-44 g
- Housing: singly
- Diet and water: ad libitum
- Acclimation period:at least 5 days

Administration / exposure

Route of administration:
corn oil
Details on exposure:
The test substance was injected intraperitoneally.
The selection of doses was based on a pilot test.
The application volume for the treated and negative control groups was 20 ml/kg bw.
Duration of treatment / exposure:
Each animals received two intraperitoneal administrations of the test substance, separated by 24 hours
Frequency of treatment:
The test substance and the negative control substance was administered twice. (The positive control substance was administered only once.)
Post exposure period:
The femoral marrow of all groups was prepared 24 hours after the last administration.
Doses / concentrations
Doses / Concentrations:
625, 1250, 2500 mg/kg bw
actual ingested
i.p. administration of 2x 625, 2x 1250, 2x 2500 mg/kg bw; injections separated by 24 hours.
No. of animals per sex per dose:
5 males per dose
Control animals:
other: yes, for negative control corn oil (20 ml/kg bw) was given... (see attached file)
Positive control(s):
Cyclophosphamide, dissolved in deionized water, administered i. p. with 10 ml/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: Schmidt's method was used to produce the smears. At least one intact femur was prepared from each sacrificed animal.

METHOD OF ANALYSIS: Normally, 2000 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. 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 historical 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.
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.

Results and discussion

Test results
>/= 625 mg/kg bw
Vehicle controls validity:
Negative controls validity:
not applicable
Positive controls validity:
Additional information on results:
Groups consisting each of three males and three females received two i.p. injections of 2500 mg/kg separated by 24 h using an application volume of 10 ml/kg . In addition, two males were treated with an application volume of 20 ml/kg.Symptoms were recorded for up to 48 h (for males: apathy, roughened fur, emaciation, loss of weight, sunken flanks, spasm, difficulty in breathing, eyelids stuck together, slitted eyes, closed eyes, lachrymation, white tears and diarhoea; for females: apathy, roughened fur, emaciation, loss of weight, sunken flanks, spasm, shivering, slitted eyes, closed eyes) None of the animals died. Due to the results it was concluded, that there were no substantial differences between sexes. Therefore, no females were used in the definite study.

- Compound-related symptoms observed after i.p. administation until sacrifice, starting at doses of 625 mg/kg bw:
apathy, roughened fur, emaciation, loss of weight, spasm, shivering, induration of abdomen, difficulty in breathing, eyelids stuck together, slitted eyes, closed eyes and lachrymation; no substance-induced mortalities.
- PCE/NCE: the ratio was altered by the treatment with the test substance, being 2000/2026 in the negative control; 2000/2309 in the 625 mg/kg group; 2000/4169 in the 1250 mg/kg group; 2000/3784 in the 2500 mg/kg group.
- No biologically important or statistically significant variations between the negative control and the substance-treated groups, with respect to the incidence of micronucleated polychromatic erythrocytes (3.4/2000 in the negative control; 2.2/2000; 3.0/2000; 3.2/2000 in the substance-treated groups).
- No biologically significant variation between the negative control and the substance-treated groups in the number of micronucleated normochromatic erythrocytes.

Applicant's summary and conclusion

Executive summary:
A mouse bone marrow micronucleus test according to OECD TG 474 was conducted with 5 male mice per dose group, receiving each two intraperitoneal injections of 0, 625, 1250 or 2500 mg/kg bw, separated by 24 hours. No indications of a clastogenic effect were found after evaluation of femoral marrow smears, which were obtained 24 hours after the last test substance administration. Relevant systemic exposure was demonstrated by symptoms of toxicity and an altered PCE/NCE ratio.