Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January to 04 June 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
see "Principles of method if other than guideline" for further information
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
not specified
Principles of method if other than guideline:
The study is compliant with TG474 with just one exception: only 1000 and not 4000 PCE have been assessed for MN. The recent TG474 update increased the number of cells to be scored from 2000 PCE to 4000 PCE due to the low background MN frequency in the assay. This increase should assure that at least 1 MN per animal is identified in the vehicle controls and therefore increase the statistical power of the test. In this study the incidence of vehicle control animals with 0MN PCE is low so the study can be considered sufficiently sensitive to detect a biologically relevant increase in micronuclei.
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Appearance: clear, straw-colored liquid, containing a very small number of small, black, free-floating particles
- Storeage: ambient temperature
- Readily miscible with corn oil at a maximum concentration of 500 mg/mL.
Specific details on test material used for the study:
- Dose preparations were freshly formulated in corn oil on the day of dosing, each concentration being individually formulated, and mixed prior to use.
- No allowance was made for purity or activity below 100 %. No determinations of homogeneity or concentration were performed on the dose preparations.

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 4-5 weeks old
- Weight at study initiation: 17.9 - 25.7 g
- Housing: Animals were in single-sex groups of 2 or 5 animals per cage in high density polypropylene cages with stainless steel tops. Cages housing animals of different treatment groups were distributed so that the effects of any spatially-variable components were equalised between groups as much as possible.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 4 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23 °C
- Humidity: 40-70 %
- Air changes: 15 air changes per hour
- Photoperiod: 12-hour light/dark cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Corn oil was used as the vehicle.

- Justification for choice of solvent/vehicle: Test material was formulated in corn oil and therefore degradation due to hydrolysis prior to dose administration would not have occurred.

Clinical signs, especially those suggesting CNS effects, confirm systemic (and therefore bone marrow) exposure. In addition, weight loss was also seen in the main study and bone marrow toxicity in several animals in the MN DRF support that adequate systemic exposure was observed in the main study.
Details on exposure:
- A preliminary toxicity test was conducted. Test material dosages of 250, 500, 1000, and 2000 mg/kg were administered to groups of 4 test animals (2 males and 2 females). Volume of dosage was 10 mL/kg for both the preliminary test as well as the main micronucleus test.
- All animals dosed at 1000 and 2000 mg/kg, and 3 of the 4 dosed at 500 mg/kg, showed severe reactions to treatment and only three survived to the end of the 72-hour exposure period. All animals dosed at 250 mg/kg survived to termination. Slides were prepared and stained from all animals. Some bone marrow toxicity was apparent in individual animals dosed at 2000, 1000, and 500 mg/kg, despite the short time period between dosing and death or sacrifice. No real toxicity was apparent in the bone marrow of animals treated at 250 mg/kg. Therefore test material doses of 10, 50, and 250 mg/kg were chosen for subsequent testing. Concentrations of the test material were administered once orally (by gavage, in corn oil) to test animals (5 male/5 female in 10 and 50 mg/kg bw dose groups; 15 male/15 female in 250 mg/kg bw dose group). Control animals (15 male/15 female) were given corn oil at 10 ml/kg bw. Chlorambucil (30 mg/kg in aqueous 10 % ethanol; administered orally) served as the positive control (5 male/5 female). 5 males and 5 females per group were sacrificed at 24 hours following treatment. An additional 5 males and 5 females per test group in the vehicle control and the 250 mg/kg dose group, were sacrificed at 48 and 72 hours. Bone marrow smears were prepared from each animal, then stained and examined.
Duration of treatment / exposure:
single exposure
Frequency of treatment:
single oral dose
Post exposure period:
Preliminary toxicity test: 72 hours after treatment
Main micronucleus test: 24, 48, and 72 hours
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw (total dose)
Dose / conc.:
50 mg/kg bw (total dose)
Dose / conc.:
250 mg/kg bw (total dose)
No. of animals per sex per dose:
Preliminary toxicity test: 2 mice per sex per dose

Main micronucleus test: 5 mice per sex per dose per exposure duration
Control animals:
yes, concurrent vehicle
Positive control(s):
Main micronucleus test: Chlorambucil 30 mg/kg administered to 5 males and 5 females for 24 hours.

Examinations

Tissues and cell types examined:
Bone marrow erythrocytes were examined.
Details of tissue and slide preparation:
After the animals were sacrificed, femurs from each animal were dissected out and their marrows extracted. Single drops of the cell suspension were transferred to clean, dry slides, two or three smears (for the preliminary toxicity test and the main micronucleus test, respectively) were prepared, and the slides air-dried, fixed with methanol, and then stained using the Schmid (May-Grunwald and Giemsa) staining technique.
Evaluation criteria:
Slides were examined under the light microscope. A minimum of 2000 erythrocytes per animal were examined for the presence of micronuclei. The frequencies of micronucleated cells per 1000 erythrocytes were then calculated. The ratio of polychromatic to mature cells was also calculated for each animal as an indicator of gross toxicity - a decrease in this ratio may indicate inhibition of cell division following treatment. The incidence of micronuclei in the mature cell population 24 hours after treatment was also calculated and reflected the pretreatment situation, since most of these cells were produced before treatment.
Statistics:
Calculated values of micronuclei per 1000 polychromatic erythrocytes were analysed statistically using the Mann-Whitney U test. Data from males and females within each group were compared using a two-tailed test. Where there was no significant difference within the group, the sexes were pooled for further analysis. For each sampling time (24, 48, or 72 hours), each treated group was compared with concurrent vehicle controls using a one-tailed test.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
No animals dosed at 10 or 50 mg/kg showed adverse reactions to treatment; however, some instances of weight loss were apparent in both groups. Only one animal treated at 50 mg/kg showed a marked weight loss. At 250 mg/kg, 6 of 15 males and one female showed reactions to treatment including rales, hunched posture, and/or piloerection on the day of dosing only. A single male showed these signs for the 24 hours until termination. At the 24 hour termination time, 6 of 10 animals had lost weight, and one failed to gain weight; at the 48 hour termination time, 5 of 10 animals lost weight; at the 72 hour termination, no animal showed weight loss. Of the 10 mice (5 males/5 females) given Chlorambucil (i.e., the positive control group), 7 lost weight during the 24-hour period before termination.
Among mice sacrificed at 24 hours following treatment, the mean incidence of micronucleated polychromatic erythrocytes (MPE) (per 1000 polychromatic cells scored) was 1.6 for the vehicle control, and 0.7, 1.1, and 1.0 for the 10, 50, and 250 mg/kg dose groups, respectively. The mean incidence of MPE for the positive control was 49.7. The ratio of polychromatic cells to mature cells was 1.0 for the vehicle control; 1.0,  1.1, and 0.9 for the 10, 50, and 250 mg/kg test material groups; and 0.8 for the positive control. 
Among mice sacrificed at 48 and 72 hours following treatment, the mean incidence of MPE was 0.6 and 1.4 for the vehicle control, and 1.3 and 1.8 for 250 mg/kg test material. The ratio of polychromatic cells to mature cells at 48 and 72 hours was 0.8 and 0.7 for the vehicle control, and 0.9 and 0.8 for 250 mg/kg test material.
A statistically significant increase in the incidence of micronucleated polychromatic cells was seen in mice in the 250 mg/kg dose group and sacrificed 48 hours later. However, the increase was not considered biologically relevant. The individual incidences of micronucleated  polychromatic cells in animals in this group were small and within the range observed in the vehicle control animals throughout the study. No biologically or statistically significant increase was recorded for animals treated with the test material and killed 24 or 72 hours later.
Under these experimental conditions, there is no evidence of induced chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes of treated mice at 24, 48, or 72 hours after oral administration of the test material.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, there was no evidence of induced chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes of treated mice at 24, 48, or 72 hours after oral administration of the test material.
Executive summary:

The effect of the test material on chromosome structure in bone marrow erythrocytes following acute oral administration in mice was investigated using the micronucleus assay. The test was performed in accordance with the standardised guidelines OECD 474 and EU Method B.12, under GLP conditions.

A preliminary toxicity test, using doses between 250 and 2000 mg/kg, guided the choice of doses for the main micronucleus test, which were 10, 50 and 250 mg/kg body weight, along with the vehicle (corn oil) control. Chlorambucil was administered at a dose of 30 mg/kg and served as the positive control. Animals were sacrificed 24 hours after treatment. In addition, some animals in the control and 250 mg/kg-dose groups were sacrificed at 48 and 72 hours.

Clinical signs observed at 250 mg/kg confirmed systemic exposure to MBTC following oral administration. Furthermore, evidence of bone marrow toxicity at doses >250 mg/kg was identified in the preliminary toxicity test. Exposure of the bone marrow is considered to have occurred in this study. Frequencies of micronucleated polychromatic erythrocytes in the treated groups were similar to those in control groups with all treatment durations. A statistically significant increase was observed in the 250 mg/kg dose group sacrificed at 48 hours. However, this increase was not considered to be biologically relevant. Statistically significant increases over controls were seen in positive controls given Chlorambucil.

Under the conditions of this study, there was no evidence of induced chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes of treated mice at 24, 48, or 72 hours after oral administration of the test material.