Registration Dossier

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:
key study
Study period:
1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study without detailed documentation, for read-across substance

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1984
Report Date:
1984

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
- only one dose tested instead of 3.
GLP compliance:
no
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Methyl ethyl ketone
- Physical state: Clear liquid
- Analytical purity: 99.91%
- Storage condition of test material: Room temperature under nitrogen

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratory
- Age at study initiation: 6-8 weeks old
- Weight at study initiation: 28-37 g (males) and 22-26 g (females)
- Housing: Housed in an AAALAC-accredited facility up to 6/cage
- Diet (e.g. ad libitum): Certified laboratory rodent chow ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: Quarantined for 10-14 days after receipt


ENVIRONMENTAL CONDITIONS
- Temperature: 74 ± 6 ºF
- Humidity (%): 50 ± 20%
- Photoperiod (hrs dark / hrs light): 12 hours:12 hours

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Details on exposure:
The test article-vehicle mixture and the vehicle alone were administered by I.P. injection at a rate of 10 ml/kg body weight. The positive control, TEM, was injected IP at a dose level of 0.25 mg/kg. All mice in the experimental and control groups were weighed immediately prior to dose administration and the dose volume based on individual body weights. Animals were observed after dose administration for clinical signs of chemical effect.
At the scheduled sacrifice time, five mice per sex were sacrificed by cervical dislocation.
Duration of treatment / exposure:
Single exposure.
Frequency of treatment:
Single exposure.
Doses / concentrations
Remarks:
Doses / Concentrations:
1.96 mL/kg
Basis:
nominal conc.
No. of animals per sex per dose:
5/sex/dose/timepoint
Control animals:
yes, concurrent vehicle
Positive control(s):
triethylenemelamine
- Route of administration: IP injection
- Doses / concentrations: 0.25 mg/kg

Examinations

Tissues and cell types examined:
Bone marrow cells from the femurs.
Details of tissue and slide preparation:
Immediately following sacrifice, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing FBS. The bone marrow cells were transferred to a capped centrifuge tube containing approximately 1 ml cold FBS. The bone marrow cells were pelleted by centrifugation at approximately 1000 rpm for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended by aspiration with a capillary pipet and a small drop of bone marrow suspension was spread onto a clean glass slide. Two to four slides were prepared from each animal. The slides were aged overnight and stained with May-Gruenwald-Giemsa and permanently mounted.

Slides were coded using a random number table. Using medium magnification, an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion, 1000 polychromatic erythrocytes were scored for the presence of micronuclei. The number of micronucleated normocytes in the field of 1000 polychromatic erythrocytes was also enumerated. The ratio of normochromatic to polychromatic erythrocytes was recorded as the proportion of polychromatic erythrocytes per total erythrocytes.
Evaluation criteria:
The incidence of micronucleated erythrocytes per 1000 polychromatic erythrocytes must not exceed 0.5% in the negative (vehicle) control. The incidence of micronucleated polychromatic erythrocytes in the positive control group must be at least twice that of the negative control and significantly increased (p<=0.05) relative to the negative control using t-test statistics.
Statistics:
The ratio of normochromatic to polychromatic erythrocytes is presented for each animal and treatment group as the proportion of polychromatic erythrocytes per total erythrocytes. The incidence of micronuleated polychromatic erythrocytes per 1000 polychromatic erythrocytes is presented for each animal and treatment group. An analysis of variance was used to compare the incidence of micronucleated polychromatic erythrocytes of the treatment group and solvent control groups. The test article is considered to induce a positive response if a treatment-related increase (p<0.05, one-way ANOVA, Duncan’s multiple range test) in micronucleated polychromatic erythrocytes is observed relative to the vehicle control. All data, including reproducibility, was evaluated and a final judgment made on sound scientific basis.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Mortalities started at 2 mL/kg in the dose-range finding study. Test article-treated animals in the micronucleus assay appeared heavily sedated immediately following dose administration (1.96 mL/kg)
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative