Registration Dossier

Administrative data

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
read across substance (disodsium and calcium salt)

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
according to guideline
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
according to guideline
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
GLP compliance:
yes (incl. QA statement)
testing lab.
Type of assay:
mammalian bone marrow chromosome aberration test

Test material

Constituent 1
Reference substance name:
Reference substance 001

Test animals

Details on test animals or test system and environmental conditions:
Animals with a mean weight of about 28 g (with an age range of about 5 - 8 weeks according to the information from the breeder) were used for the study.
For the duration of at least 5 days the animals were housed in Makrolon cages, separately according to sex. During this time the animals were accommodated in fully air-conditioned rooms in which central air conditioning guaranteed a temperature range of 20 - 24°C and a relative humidity range of 30 - 70%. Before the start of the treatment the animals were transferred to Makrolon cages, type Ml, and housed individually under the same conditions until the end of the test.
The day/night rhythm was 12 hours (12 hours light from 6.00 - 18.00 hours and 12 hours darkness from 18.00 - 6.00 hours).
Standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland) and drinking water from bottles were available ad libitum.

Administration / exposure

Route of administration:
oral: gavage
- Vehicle(s)/solvent(s) used: In comparison to other commonly used vehicles (e.g. DMSO, olive oil, etc.) water is the most suitable. Therefore, an aqueous CMC formulation was selected as the vehicle.
- Concentration of test material in vehicle: 2.5 g/100 ml (500 mg/kg bw); 5.0 g/100 ml (1000 mg/kg bw); 10 g/100 ml (2000 mg mg/kg bw).
- Application volume: The usual application volume of 10 ml/kg body weight led to an non-applicable mass and therefore the volume was increased to 20 ml/kg body weight.
Details on exposure:
For the determination of the test substance concentrations in the vehicle, 3 samples of each dose were taken from the test substance preparations, kept at room temperature until the treatment of the last animal (approximately 1 hour) and then deep-frozen until they were determined analytically. The determination of the concentrations in the vehicle was carried out by means of Spectrophotometer Lambda 900 (Perkin Elmer).
Duration of treatment / exposure:
two administrations
Frequency of treatment:
twice orally, with a 24-hour interval between administrations
Post exposure period:
24 hours
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
Dose / conc.:
2 000 mg/kg bw/day
No. of animals per sex per dose:
Control animals:
Positive control(s):
The following positive controls, both dissolved in purified water were administered to male animals once orally (CPP) or intraperitoneally (VCR) each in a volume of 10 ml/kg body weight:
- Cyclophosphamnide (CPP); 20 mg CPP (Baxter Oncology GmbH, Reg. No. E 432-1)/kg body weight for clastogenic effects
- Vincristine Sulphate (VCR); 0.15 mg VCR (SIGMA - V 8879)/kg body weight for aneugenic effects.
The stability of CPP and VCR is weII-defined under the selected conditions, since both positive control articles are well- established reference clastogens and aneugens respectively.


Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
The bone marrow was prepared according to the method described by SCHMID, W. 1976, 1977 and SALAMONE, M. et al. 1980 (Mut.Res.,74,347).
The two femora of the animals sacrificed by cervical dislocation were prepared by dissection and removing all soft tissues. After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum which was at 37°C (about 2 mI/femur). The suspension was mixed thoroughly with a pipette, centrifuged at 300 x g for 5 minutes, the supernatant was removed and the precipitate was resuspended in about 50 µl fresh FCS. 1 drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.
The animals were sacrificed and the bone marrow of the two femora was prepared 24 hours after the second administration. After staining of the preparations, 2000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2000 polychromatic erythrocytes were also recorded.
Evaluation criteria:
The mouse micronucleus test is considered valid if the following criteria are met: The quality of the slides must allow the identification and evaluation of a sufficient number of analyzable cells, i.e. > 2000 PCEs and a clear differentiation between PCEs and NCEs. The ratio of PCEs/NCEs in the untreated animals (negative control) has to be within the normal range for the animal strain selected. The number of cells containing micronuclei in negative control animals has to be within the range of the historical control data both for PCEs and for NCEs. The two positive control substances have to induce a significant increase in the number of PCEs containing small and large micronuclei within the range of the historical control data or above.
A finding is considered positive if the following criteria are met: Significant and dose-related increase in the number of PCEs containing micronuclei. The number of PCEs containing micronuclei has to exceed both the concurrent negative control and the highest value of the historical control range.
A test substance is considered negative if the following criteria are met: The number of cells containing micronuclei in the dose groups is not significantly above the negative control and is within the historical control data.
The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there were significant differences between the control group and dose groups with regard to the micronucleus rate in polychromatic erythrocytes. The relative frequencies of cells containing micronuclei of each animal was used as a criterion for the rank determination for the U test. Significances were identified as follows: * p < 0.05, ** p < 0.01.

Results and discussion

Test results
no effects
Vehicle controls validity:
Negative controls validity:
Positive controls validity:
Additional information on results:
Both of the positive controls chemicals i.e cyclophosphamide for clastogenicity and vincristine for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei.
According to the results of the present study, the two oral administrations of Paliotol Gelb K 1700 did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always within the same range as that of the concurrent negative control in all dose groups and within the range of the historical control data.

Applicant's summary and conclusion

Under the experimental conditions chosen here, the test substance does not have any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.