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Toxicological information

Genetic toxicity: in vivo

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Administrative data

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:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Please refer to the section "Confidential details on test material" below.

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River GmbH, Wiga, Sulzfeld, Germany
- Weight at study initiation: mean 27 g
- Assigned to test groups randomly: yes, under following basis: computerized
- Fasting period before study: none
- Housing: Makrolon cages, individually
- Diet: standardized pellet feed, Kliba, Klingentalmuehle, Kaiseraugst, Switzerland; ad libitum
- Water: ad libitum
- Acclimation period: about 1 week

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 30-70 %
- Photoperiod: 12 hours dark / 12 hours light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle/solvent used: water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was dissolved at dose levels of 375, 750 and 1500 mg/kg bw immediately before administration.
Duration of treatment / exposure:
24 and 48 hours
Frequency of treatment:
single application
Post exposure period:
none
Doses / concentrations
Remarks:
Doses / Concentrations:
375, 750, 1500 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5 males, 5 females used for each dose level of the test substance;
5 animals in total were used for each positive control substance
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide or Vincristine were used as positive control.
- Route of administration: orally or ip
- Doses/concentrations: 20 mg/kg bw (Cyclophosphamide) or 0.15 mg/kg bw (Vincristine)

Examinations

Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
Pretests for dose selection
In a pretest for the determination of the acute oral toxicity, deaths were observed down to a dose of 1750 mg/kg bw whereas 1500 mg/kg bw were survived by all animals. Therefore, a dose of 1500 mg/kg bw was selected as the highest dose in the present cytogenetic study. Dose levels of 750 and 375 mg/kg bw were administered as further doses.

The test substance to be administered was dissolved in purified water.

- Test group 3 was given 375 mg/kg bw or 10 mL/kg bw of a solution with a concentration of 3.75 g/100 mL.
- Test group 4 was given 750 mg/kg bw or 10 mL/kg bw of a solution with a concentration of 7.5 g/100 mL.
- Test groups 5 and 6 were given 1500 mg/kg bw or 10 mL/kg bw of a solution with a concentration of 15.0 g/100 mL.

Preparation of the bone marrow
The bone marrow was prepared according to the method described by Schmid (1976, 1977).
- The two femora were prepared from the animals, and all soft tissues were removed.
- 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 mL/femur).
- The suspension was mixed thoroughly with a pipette, centrifuged at 1500 rpm for 5 min, the supernatant was removed except for a few drops, and the precipitate was resuspended.
- 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.

Staining of the slides
The slides were stained in Eosin and Methylene blue solution for 5 min, rinsed in purified water and then placed in fresh purified water for 2 or 3 min. They were finally stained in Giemsa solution for 12 min. After being rinsed twice in purified water and clarified in Xylene, the preparations were embedded in Corbit-Balsam.

References
- Schmid W (1976). The micronucleus test for cytogenetic analysis. In: Hollaender A (editor). Chemical mutagens. Principles and methods of their detection. Vol 4, Plenum Press, New York.
- Schmid W (1977). The micronucleus test. In: Kilby et al. (editors). Handbook of mutagenicity test procedures. Elsevier Scientific Publishing Company, Amsterdam, New York, Oxford.
Evaluation criteria:
Microscopic evaluation
In general, 1000 polychromatic erythrocytes (PCE) from each of the male and female animals of every test group were evaluated and investigated for micronuclei (MN). The normochromatic erythrocytes (NCE), which occur, were also scored. The following parameters were recorded:
- Number of PCE
- Number of PCE containing micronuclei
The increase in the number of micronuclei in PCE of treated animals as compared with the solvent control group provided an index of a chromosome-breaking (clastogenic) effect or of a spindle activity of the substance tested.
- Number of NCE
- Number of NCE containing micronuclei

The number of micronuclei in NCEs at the early sacrifice intervals represents the situation before test substance administration and may serve as a control value. A substance-induced increase in the number of micronuclei in NCEs may be found with an increase in the duration of the sacrifice intervals.
- PCE/NCE. This ratio indicates an influence of the test substance specifically on the bone marrow.
- Number of small micronuclei (d < D/4) and of large micronuclei (d > D/4) (d = diameter of micronucleus, D = cell diameter)
The size of micronuclei may give an indication on the possible mode of action of the test substance, i.e. a clastogenic or a spindle poison effect. Slides were coded before microscopic analysis.
Statistics:
The statistical evaluation of the data was carried out using the program system MUKERN (BASF AG). The number of micronuclei in PCEs was analyzed. A comparison of the dose group with the vehicle control was carried out using the Wilcoxon test for the hypothesis of equal medians. Here, the relative frequencies of cells with micronuclei of each animal were used. If the results of this test were significant, labels (+ for p ≤ 0.05, ++ for p ≤ 0.01) were printed with the group means in the tables. This test was performed one-sided. This analysis was done separately for each sex and combined for both sexes.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
2 females of the 48-hour 1500 mg/kg bw group died.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF PRETEST FOR DOSE SELECTION
In a pretest for the determination of the acute oral toxicity, deaths were observed down to a dose of 1750 mg/kg bw whereas 1500 mg/kg bw were survived by all animals.

RESULTS OF DEFINITIVE STUDY
The single oral administration of purified water in a volume of 10 mL/kg bw led to 1.7 ‰ PCEs containing micronuclei after the 24-hour sacrifice interval or to 1.4 ‰ after the 48-hour sacrifice interval.

After the single administration of the highest dose of 1500 mg/kg bw, 1.2 ‰ PCEs containing micronuclei were found after 24 hours and 1.3 ‰ after 48 hours. In the two lower dose groups rates of micronuclei of about 2.1 ‰ (750 mg/kg bw) and 1.0 ‰ (375 mg/kg bw) were detected after a sacrifice interval of 24 hours in each case.

With 11.4 ‰ the positive control substance for clastogenicity (Cyclophosphamide) led to the expected increase in the number of PCEs containing mainly small micronuclei at a dose level of 20 mg/kg bw. With 112 ‰ the positive control substance for spindle poison effects (Vincristine) also led to a clearly enhanced number of micronuclei containing PCEs with the expected amount of large micronuclei, i.e . 27.2 ‰.

The number of NCEs containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups at any of the sacrifice intervals. Thus, the test substance did not lead to any increase in the rate of micronuclei. The number of NCEs or PCEs containing small micronuclei (d < D/4) or large micronuclei (d > D/4) did not deviate from the solvent control value at any of the sacrifice intervals. A slight inhibition of erythropoiesis induced by the treatment of mice with the test substance was detected at the high dose of 1500 mg/kg bw both after a sacrifice interval of 24 and 48 hours.

CLINICAL EXAMINATIONS
The single oral administration of the vehicle in a volume of 10 mL/kg bw was tolerated by all animals without any signs or symptoms. In the treatment groups the following signs of toxicity were observed:
375 mg/kg bw: piloerection after about 30 min; no symptoms any longer after 3 hours
750 mg/kg bw: piloerection, squatting posture and irregular respiration after about 30 min to 2 hours; piloerection was observed even the day after treatment.
1500 mg/kg bw: piloerection, squatting posture and irregular respiration after about 30 min to 5 hours; the general state of the animals was poor. Piloerection was still observed the day after treatment and 4 animals were found dead.

Neither the single administration of the positive control substance Cyclophosphamide in a dose of 20 mg/kg bw nor that of Vincristine at 0.15 mg/kg bw caused any evident signs of toxicity.

Any other information on results incl. tables

According to the results of the present study, there were no biologically relevant, significant differences in the frequency of erythrocytes containing micronuclei either between the solvent control and the 3 dose groups (375, 750, 1500 mg/kg bw) or between the two sacrifice intervals (24 and 48 hours). Thus, under the experimental conditions chosen, the test substance showed no chromosome-damaging (clastogenic) effect nor did it lead to any impairment of chromosome distribution in the course of mitosis.

Applicant's summary and conclusion