Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome and genome mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented publication/ study report which meets basic scientific principles

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
Procedure according to Heddle, 1973; Hayashi et al., 1994; Mavournin et al. 1990.
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
ICR
Details on species / strain selection:
ICR mice from Harlan Sprague Dawley, Inc.
Sex:
male/female
Details on test animals and environmental conditions:
Age 6 to 8 weeks old
Body weight range : Male: 25.0-29.6 g, Female: 25.2-29.7 g

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Details on exposure:
Volume injected: 20 ml/kg bw
Duration of treatment / exposure:
Bone marrow was collected 24 and 48 hours after dose administration.
Frequency of treatment:
single application
Post exposure period:
24, 48 h
Doses / concentrations
Remarks:
Doses / Concentrations:
462.5, 925, or 1850 mg/kg body weight
Basis:
nominal conc.
No. of animals per sex per dose:
5 animals per sex per dose
Control animals:
not specified
Positive control(s):
- Cyclophosphamide
- Route of administration: IP injection
- Doses / concentrations: 2.5 mg/ml bw

Examinations

Tissues and cell types examined:
erythrocytes for the bone marrow of the femurs
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
24 and 48 h

DETAILS OF SLIDE PREPARATION:
Immediately following sacrifice, the femurs were exposed, cut just above the knee and the bone marrow was aspirated into a syringe containing fetal bovine serum. The cells were centrifuged; the supernatant was drawn off and then resuspended. The bone marrow suspension was spread onto a clean glass slide (two to four slides were prepared for each mouse). The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.

METHOD OF ANALYSIS:
2000 polychromatic erythrocytes were scored for the presence of micronuclei. The number of micronucleated normochromatic erythrocytes in the field of 2000 polychromatic erythrocytes was enumerated. The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified

Any other information on results incl. tables

No effects, slight to moderate reduction (up to 35%) in the ratio of  
polychromatic erythrocytes to total erythrocytes were observed in some of  the 
test article-treated groups relative to the respective vehicle controls. 
A statistically significant increase in micronucleated  polychromatic 
erythrocytes (8 MNPCE/10000 PCE) was observed in male mice 24 hrs after 
treatment with 925 mg/kg. 
However, this response is not considered biologically relevant (each of the 
five animals had no more  than 3 MPCE, which are within the range of historical 
solvent control:  0-7 MN/2000 PCE/animal). 
No significant increase and no dose responsiveness increase was observed in any 
other test article treated group regardless of dose level, sex, or bone marrow 
collection time.

Applicant's summary and conclusion

Conclusions:
The results of the assay indicate that under the conditions described in this report, the test articl, MEthyl ionone did not induce a significant increase in micronucleated polychromatic erythrocytes in either male or female mice. Methyl ionone was concluded to be negative in the mouse micronucleus assay.
Executive summary:

A mouse micronucleus assay (Hayashi et al., Heddle, 1973; Mavournin et al., 1990) was conducted in male and female ICR mice (5/sex/dose). Methyl ionone in corn oil, the vehicle alone or the positive control (2.5 mg/ml cyclophosphamide in sterile distilled water) were administered by intraperitoneal injection at a constant volume of 20 ml/kg body weight. The test animals were dosed with 462.5, 925, or 1850 mg/kg body weight and bone marrow was collected 24 and 48 hours after dose administration. Mortality was observed in only 1/15 male mice receiving 1850 mg/kg. This animal was replaced at the time of bone marrow collection with a replacement animal that also

received 1850 mg/kg. Immediately following sacrifice, the femurs were exposed, cut just above the knee and the bone marrow was aspirated into a syringe containing fetal bovine serum. The cells were centrifuged; the supernatant was drawn off and then resuspended. The bone marrow suspension was spread onto a clean glass slide (two to four slides were prepared for each mouse). The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted. Using oil immersion, 2000 polychromatic erythrocytes were scored for the presence of micronuclei. The number of micronucleated normochromatic erythrocytes in the field of 2000 polychromatic erythrocytes was enumerated. The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes.

Slight to moderate reduction (up to 35%) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test article-treated groups relative to the respective vehicle controls. A statistically significant increase in micronucleated polychromatic erythrocytes (8 MNPCE/10000 PCE) in test article-treated group relative to the respective vehicle control group was observed in male mice 24 hrs after treatment with 925 mg/kg. However, this response is not considered biologically relevant, since each of the five animals had no more than 3 MPCE. These numbers of MNPCE are within the range of historical solvent control (0-7 MN/2000 PCE/animal). No significant increase and no dose responsiveness increase was observed in any other test article treated group regardless of dose level, sex, or bone marrow collection time. It was concluded that methyl ionone did not induce a significant increase in micronucleated polychromatic erythrocytes in either male or female mice.