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in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start: 02.02.2021, Experimental completion: 18 March 2021
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
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)
Version / remarks:
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
Cas Number:
Molecular formula:
Test material form:

Test animals

Details on species / strain selection:
The mouse is an animal that has been used for many years as a suitable experimental animal in cytogenetic investigations. There are many data available from such investigations, which may be helpful in the interpretation of results from the micronucleus test.
Details on test animals or test system and environmental conditions:
Source: Charles River Laboratories, Research Models and Services Germany GmbH, Sulzfeld, Germany
Age: 6 to 10 weeks
Acclimation: 5 days
Body weight: 34.8 (31.2 to 37.2) g at start, 35.6 (32.2 to 38.8) g at end of experiment
Housing: individually in Macrolon Type II/III cage with wire mesh top, soft wood bedding
Feed: pellet standard diet ad libitum
Water: tap water ad libitum
Temperature: 22 ± 2°C
Humidity: 45 to 65%
Ventilation: at least 8 air changes per hour
Light: 12 hours light to 12 hours darkness cycles

Administration / exposure

Route of administration:
oral: gavage
Corn oil
Details on exposure:
Animals received the oral dose by gavage, using a stainless steel feeding needle with rounded tip (1.2 Gauge) and disposable syringe at a dose volume of 10 mL/kg bw.
Duration of treatment / exposure:
Sampling of bone marrow after 24 and 48 hours
Frequency of treatment:
Single oral dose by gavage
Post exposure period:
Animals were sacrificed 24 and 48 hours after oral administration of the test substance or control substances.
Doses / concentrationsopen allclose all
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Preliminary study: two male and two female mice
Main study: 6 males per dose, 5 males for negative and positive controls
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CPA), administered at 40 mg/kg bw in distilled water, administered in a volume of 10 mL/kg bw


Tissues and cell types examined:
Samples were prepared from bone marrow cells obtained from the femora of test animals. Per animal 6000 polychromatic erythrocytes (PCE) were analysed for micronuclei.
Details of tissue and slide preparation:
The animals were sacrificed using CO2 followed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum using a disposable syringe. The cell suspension was centrifuged at 1500 rpm (3900 x g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald / Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.
Evaluation criteria:
A test substance is classified as positive in the assay if
a) at least one of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control,
b) this increase is dose-related at least at one sampling time when evaluated with an appropriate trend test, and
c) any of these results are outside the distribution of the historical negative control data (e.g., Poisson-based 95% control limits).
There is no requirement for verification of a clearly positive or negative response. In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgment and/or further investigations.
A test item that fails to produce a biologically relevant increase in the number of micronucleated polychromatic erythrocytes, applying the above mentioned criteria, is considered negative in this system, given that there is evidence for bone marrow exposure.
Statistical methods (nonparametric Mann-Whitney test, linear regression analysis) were used as an aid in evaluating the results.

Results and discussion

Test results
Key result
Vehicle controls validity:
Negative controls validity:
not applicable
Positive controls validity:
Additional information on results:
One animal dosed at 125 mg/kg bw showed piloerection, partially closed eyes and decreased activity in the first hour following oral administration. All animals dosed at 500 mg/kg bw exhibited signs of acute toxicity. All treated animals showed piloerection for up to 4 hours after dosing. A smaller number of animals treated with 500 mg/kg bw showed partially closed eyes, hunched posture, decreased activity and prostation during the first hour following administration.

Any other information on results incl. tables

Table: summary of micronucleus test results

Test groupDose in mg/kg bwSampling time (h)Mean MN/4000 PCESD MN/4000 PCEMean % MNMinMaxRatio PCE/total erythroc.% ratio vehicle
Vehicle control0243.21.20.08250.582100.00
Test item125243.11.40.08250.57999.48
Test item250245.01.60.13370.601103.26
Test item500245.22.00.13280.613105.33
Positive control4024114.141.42.85691670.643110.48
Vehicle control0483.20.70.08240.609100.00
Test item500486.02.50.15390.57594.42

Applicant's summary and conclusion

The substance did not induce micronuclei in polychromatic erythrocytes of mice in this in vivo bone marrow micronucleus assay. It is considered to be non-genotoxic (non-clastogenic and non-aneugenic).
Executive summary:

The genotoxicity potential of the substance was studied under GLP in an in vivo bone marrow microncleus assay in the mouse to OECD TG 474 (2016). The test substance was dissolved in corn oil and administered to male NMRI mice (aged 6 to 10 weeks) by oral gavage. A single dose of 125, 250 and 500 mg/kg bw was administered orally to six animals per group at a volume of 10 mL/kg bw. The highest dose was estimated to be a suitable maximum tolerated dose based on two pre-experiments with two male and two female mice. Clinical signs observed were indicative of systemic exposure to the test substance. The bone marrow of treated animals was collected 24 hours (all dose groups) and 48 hours (500 mg/kg bw) after oral administration. Groups of five animals received the vehicle without test substance (negative vehicle control) and were sacrificed after 24 and 48 hours. Another group of five animals received cyclophosphamide at a single oral dose of 40 mg/kg in distilled water (volume of 10 mL/kg bw) as the positive control substance. Bone marrow was obtained from the femora of test animals and smears on slides were air-dried and then stained with May-Grünwald / Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample. The slides were evaluated for the occurrence of micronuclei. Per animal at least 6000 polychromatic erythrocytes (PCEs) were scored for micronuclei. After treatment with the test substance the number of PCEs per total erythrocytes was not substantially decreased as compared to the mean value of PCEs per total erythrocytes of the vehicle control, shwowing that the substance did not exert any significant cytotoxic effects in the bone marrow. In comparison to the corresponding vehicle controls, there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test substance at any dose level used. For all treatment groups the mean values of micronuclei observed after treatment with the substance were below or near the corresponding vehicle control value and well within the the 95% control limit of the historical vehicle control data. Treatment with the positive control substance produced a substantial increase of induced micronucleus frequency.

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