4,4-dimethyloxazolidine

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 March 1989 - 22 May 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

May 26, 1983

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

September 19, 1984

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

4,4-dimethyloxazolidine

EC Number:

257-048-2

EC Name:

4,4-dimethyloxazolidine

Cas Number:

51200-87-4

Molecular formula:

C5H11NO

IUPAC Name:

4,4-dimethyl-1,3-oxazolidine

Test material form:

liquid

Specific details on test material used for the study:

Batch Number E-03099
Purity: not indicated
Storage Conditions: Room temperature in the dark

Test animals

Species:

mouse

Strain:

Swiss

Remarks:

CD-1 (SPF-quality)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Date of arrival of animals: March 21, 1989
Number of animals per test: 5 male and 5 female mice per treatment group.
Age at start of treatment: Approximately 8 weeks
Body weight at start of treatment: 20-27 g females, 27-34 g males
Identification: By unique cage number and a mark on the tail (for a unique animal number).
Acclimatisation: At least 6 days under laboratory conditions.
Allocation: Allocated to treatment groups as they came to hand from delivery boxes.

On arrival and prior to final assignment to study, all animals have undergone a detailed clinical examination to ensure selected animals were in a good state of health.

Conditions: Standard Laboratory Conditions. Air-conditioned room, 7.5 air changes per hour, temperature 21±3 °C, relative humidity around 40-70%, 12 hours artificial fluorescent light/12 hours dark.

Accommodation: In groups of 5 per sex in polycarbonate cages.
Bedding: Purified sawdust (Woody Clean, from the Broekmann Institute, Someren, The Netherlands).
Diet: Standard laboratory animal diet RMH-B, pellet diameter 10 mm, Hope Farms, Woerden, The Netherlands. Feed was withheld overnight prior to dosing approximately 3-4 hours after administration the test substance. The feed was analysed for contaminants by the manufacturer, and the resultswere archived.
Water: Tap water, ad libitum. Results of chemical and contaminant analyses were archived.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Milli-RO water (Millipore Corp., Bedford,Mass., USA).

Frequency of treatment:

Single dosing

Doses / concentrations

Dose / conc.:

500 mg/kg bw (total dose)

Remarks:

Dosing volume: 10ml/kg bw

No. of animals per sex per dose:

5 Female, 5 male

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (CAS 50-18-0); Endoxan, Asta-Werke, 50mg/kg body weight dissolved in 0.9% NaCl (Merck) in Milli-RO water.

Examinations

Tissues and cell types examined:

Bone marrow smears

Details of tissue and slide preparation:

The animals were sacrificed by cervical dislocation at 24, 48 and 72 hours after dosing of the test substance and the vehicle and at 48 hours after dosing of the positive control. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened untill a small opening to the marrow canal became visible. The bone was flushed with approximately 2 ml of foetal calf serum. The cell suspension was collected and centrifuged at 1000 rpm (approximately 100 g) for 5 min.
The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed by aspiration with the serum. A drop of the cell suspension was placed on the end of a slide which was previously cleaned (24 hours immersed in a 1:1 mixture of 96% ethanol/ether and cleaned with a tissue) and marked (with the RCC NOTOX study identification number and the animal number). The drop was spread by moving a clean slide with round-whetted sides at an angle of 45°C over the slide with the drop of bone marrow suspension. The preparations were then air-dried and thereafter fixed for 5 min in 100% methanol and air-dried overnight. Two slides were prepared per animal.

Staining of the bone marrow smears:
The slides were stained for 3 min in undiluted May-Grünwald solution Followed by 2 min in May-Grünwald solution diluted 1:1 with Sörensen buffer pH 6.8. Thereafter slides were rinsed in this buffer and stained for 25 min in 5% (v/v) Giemsa solution in Sörensen buffer pH 6.8. The preparations were rinsed for 1 min in running tap-water and blotted dry between filter paper. The dry slides were cleared by dipping them in xylol before they were embedded in DePeX and mounted with a coverslip.

Analysis of the bone marrow smears for micronuclei:
All slides were randomly coded before examination. An adhesive label with RCC NOTOX study identification number and code was stuck over the marked slide. At first the slides were screened at a magnification of 100 x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000 x. The number of micronuclei was counted in 1000 polychromatic erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes.

Evaluation criteria:

The micronucleus test is considered acceptable if it meets the following criteria:
a) The positive control substance induced a statistically significant (Wilcoxon Rank Sum Test, two-sided test at P < 0.05) increase in the frequency of micronuclei.
b) The incidence of micronuclei in the control animals should reasonably fall within the laboratory historical control data range.

A test substance is considered positive in the micronucleus test if:
a) It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test; two-sided test at p < 0.05) increase in the frequency of micronuclei (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups separately.

A test substance is considered negative in the micronucleus test if:
a) None of the tested concentrations or sampling times showed a statistically significant (P < 0.05) increase in the incidence of micronuclei neither in the combined data for both sexes nor in the data for male or female groups alone.

Statistics:

Averages and standard deviations were calculated.
Wilcoxon rank-sum test; two-sided test at p > 0.05.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No increase in the frequency of micronuclei was observed.
The incidence of micronuclei in the control animals was found to be in the range of historical data (0.66 ± 0.93; mean ± standard deviation, N = 910).
The groups that were treated with Cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes, which reflects a toxic effect of this compound on the erythropoiesis . The positive control substance induced in both sexes a statistically significant increase in the number of micronuclei.

Applicant's summary and conclusion

Conclusions:

It is concluded that this test is valid and that OXABAN A (4,4-dimethyl-1,3-oxazolidine, CAS 51200-87-4) can be considered as not mutagenic in the Micronucleus Test under the experimental conditions described in this report

Executive summary:

OXABAN A (4,4-dimethyl-1,3-oxazolidine, CAS 51200-87-4) was tested in the Micronucleus Test in mice. Three groups (D to F), each comprising 5 males and 5 females, received a single oral dose of 500 mg/kg body weight. Bone marrow was sampled at 24, 48 and 72 hours after dosing. Corresponding vehicle treated groups (A to C) served as negative controls. Bone marrow from a positive control group (G), treated with a single oral dose of cyclophosphamide (CP) at 50 mg/kg body weight, was harvested at 48 hours after dosing only. The test substance was found to respond negatively in the Micronucleus Test, whereas the positive control substance (CP) produced a statistically significant increase in the incidence of micronuclei in polychromatic erythrocytes.

It is concluded that OXABAN A (4,4-dimethyl-1,3-oxazolidine, CAS 51200-87-4) can be considered as not mutagenic in the Mouse Micronucleus Test under the experimental conditions described in this report.