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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
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study following official guidelines, GLP compliant, performed on a mixture containing the substance and other analogue substances

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
In deviation to the protocol for a short period the relative humidity was higher than 70 % (highest value 88 %). Three of the males exceeded the upper limit of the body weight range slightly 40.1 g, 40.4 g and 42.0 g instead of 40 g)
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Test material form:
solid: particulate/powder
migrated information: powder
Details on test material:
batch: 9300001solubility: > 2g/l in waterstorage: room temperatureexpiration date: February 1998

Test animals

Details on test animals or test system and environmental conditions:
Source:BRL, CH-4414 FüllinsdorfNumber of Animals:60 (30 males/30 females)Initial Age at Start of Acclimatization: 8-12 weeksAcclimatization: minimum 5 daysInitial Body Weight at Start of Treatment: males mean value 36.5 g (SD ± 2.4 g), females mean value 28.8 g (SD ± 2.2 g)According to the suppliers assurance the animals were in healthy condition. The animals were under quarantine in the animal house of RCC - CCR for a minimum of five days after their arrival. During this period the animals did not show any signs of illness or altered behaviour. The animals were distributed into the test groups at random and identified by cage number.HusbandryThe animals were kept conventionally. The experiment was conducted under standard laboratory conditions.Housing:singleCage Type:Makrolon Type I, with wire mesh top, (EHRET GmbH, D-79302 Emmendingen)Bedding:granulated soft wood bedding (ALTROMIN, D-32791 Lage/Lippe)Feed: pelleted standard diet, ad libitum (ALTROMIN 1324, D-32791 Lage/Lippe)Water:tap water, ad libitum, (Gemeindewerke, D-64380 Roßdorf)Environment: temperature 21 ±3°C relative humidity 30-88 % artificial light 6.00 a.m. - 6.00 p.m.

Administration / exposure

On the day of the experiment, the test article was formulated in deionised water. The vehicle was chosen to its non-toxicity for the animals. All animals received a single standard volume of 10 ml/kg body weight orally.
Details on exposure:
gavageAt the start of the experiment the animals were weighed and the administed volume was adjusted to the weight of the animal
Frequency of treatment:
Doses / concentrations
Doses / Concentrations:0, 200, 67 and 2000 mg/kg bwBasis:nominal in water
No. of animals per sex per dose:
5 male and 5 females per dose
Positive control(s):
cyclo phosphamide administed at 40 mg/kg bw


Tissues and cell types examined:
bone marrow polychromatic erytrocytes
Details of tissue and slide preparation:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (MERCK, D-64293 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg). At least one slide was made from each bone marrow sample.Analysis of Cells: Evaluation of the slides was performed using NIKON microscopes with 1 OOx oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 2000 the PCEs. The analysis was performed with coded slides. Five animals per sex and group were evaluated as described.
Evaluation criteria:
A test article is classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant positive response for at least one of the test points.A test article producing neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant positive response at any of the test points is considered non-mutagenic in this system.This can be confirmed by means of the nonparametric Mann-Whitney test (8)

Results and discussion

Test results
Vehicle controls validity:
Positive controls validity:

Any other information on results incl. tables

 test group dose (mg/kg bw)   sampling time (h)  PCEs with micronuclei (%)   PCE/NCE 
 vehicle  0  24  0.09    2000/1508
 test article 200  24  0.06 2000/1533
 test article  670  24  0.120   2000/1488 
 test article  2000  24  0.110    2000/1571
 cyclophosphamide  40  24  1.29    2000/2108
 test article  2000  48  0.045    2000/2252

Applicant's summary and conclusion

The test item was tested for in vivo chromosome aberration test following OECD 474. Under the epxerimental conditions the item did not show any mutagenic potential.
Executive summary:

The test article was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test article was formulated in deionised water. Deionised water was used as vehicle control. The volume administered orally was 10 ml/kg b.w.. 24 h and 48 h after a single administration of the test article the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 2000 PCE. The following dose levels of the test article were investigated: 24 h preparation interval: 200, 670, and 2000 mg/kg b.w.. 48 h preparation interval: 2000 mg/kg b.w.. The highest guideline-recommended dose (2000 mg/kg) was estimated by a pre-experiment to be suitable. The animals expressed slight toxic reactions. The mean number of normochromatic erythrocytes was increased after treatment with the test article as compared to the mean value of NCEs of the vehicle controls at preparation interval 48 hours, indicating that the test item had cytotoxic properties in the bone marrow.

In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test article. The mean values

of micronuclei observed after treatment with test item were near to the range of the vehicle control group. 40 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.