Methoxycarbonyloxycyclooct-4-ene

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:

October 01, 1985 - November 14, 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

(1982)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Methoxycarbonyloxycyclooct-4-ene

EC Number:

401-620-8

EC Name:

Methoxycarbonyloxycyclooct-4-ene

Cas Number:

87731-18-8

Molecular formula:

C10H16O3

IUPAC Name:

(1R,3aS,6aS)-octahydropentalen-1-yl methyl carbonate; (1S,3aS,6aS)-octahydropentalen-1-yl methyl carbonate; (3Z)-cyclooct-3-en-1-yl methyl carbonate; (4Z)-cyclooct-4-en-1-yl methyl carbonate; bicyclo[3.2.1]octan-8-yl methyl carbonate

Test material form:

liquid

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River U.K. Limited, Margate, Kent, UK.
- Age at study initiation: no data available
- Weight at study initiation: between 22 and 24 grams on arrival
- Assigned to test groups randomly: yes
- Fasting period before study: no data available
- Housing: 2 or 5 animals in plastic disposable cages
- Diet (e.g. ad libitum): free access to Labsure LAD 1 rodent diet
- Water (e.g. ad libitum): free access to tap water
- Acclimation period: approximately 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): no data available
- Air changes (per hr): 30
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: corn oil (obtained from Macarthy's Ltd.)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Solutions of cyclooctenyl methyl carbonate were prepared just prior to use.
- Concentrations of the dosing formulations prepared were as follows:
For the preliminary toxicity test: 2, 4, 8, 16 and 32 mg/mL/400, 800, 1600, 3200 and 6400 mg/kg (phase I), and 16, 20, 25 and 31.25 mg/mL/3200, 4000, 5000 and 6250 mg/kg (phase II)
For the micronucleus test: 142.5 mg/mL
- All animals in all groups were dosed with the standard volume of 20 mL/kg bodyweight.

Duration of treatment / exposure:

24, 48 and 72 hours (solvent control and test substance)
24 hours (postive control)

Frequency of treatment:

Once

Doses / concentrations

Dose / conc.:

2 850 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5/time point

Control animals:

yes, concurrent vehicle

Positive control(s):

- Mitomycin C
- Route of administration: oral gavage
- Doses / concentrations: 8 mg/kg bodyweight / 0.4 mg/mL

Examinations

Tissues and cell types examined:

- Incidence of micronucleated cells per 1000 polychromatic erythrocytes.
- The ratio of polychromatic to normochromatic erythrocytes by examination of at least 1000 erythrocytes.
- The number of micronucleated normochromatic erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
From the results obtained in the preliminary toxicity study, a dosage of 2850 mg per kg bodyweight was chosen for the micronucleus test.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Five males and five females from the negative control and test compound groups were sacrificed 24, 48 and 72 hours after dosing.
The positive control group was sacrificed 24 hours after dosing.

DETAILS OF SLIDE PREPARATION:
The animals were killed by cervical dislocation and both femurs dissected out from each animal. The femurs were cleared of tissue and one epiphysis removed from each bone. A direct bone marrow smear was made onto a slide containing a drop of calf serum. One smear was made from each femur. The prepared smears were air-dried and fixed in methanol. After fixation, the smears were air-dried and stained using Giemsa's technique. After rinsing in buffered distilled water (pH 6.8), the slides were air-dried and mounted with coverslips using DPX.

METHOD OF ANALYSIS:
The stained smears were examined (under code) by light microscopy.

Evaluation criteria:

Any statistically significant increases in the number of micronucleated polychromatic erythrocytes at any sampling time.

Statistics:

Wilcoxon's sum of ranks test.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

at the 48 hour sampling time

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY:
The mortality data were subjected to probit analysis which indicated that a dose of 2850 mg/kg would be expected to kill approximately 10% of the animals over the 72 hour exposure period (LD 50 was estimated as 4485 mg/kg). A dosage of 2850 mg per kg bodyweight was chosen for use in the main test.

RESULTS OF DEFINITIVE STUDY:
- Mortalities: No animals died after treatment with cyclooctenyl methyl carbonate in the main study.
- Clinical signs included: pilo-erection, hunched posture, lethargy, decreased respiratory rate, ptosis, pallor of extremities and ataxia.
- Micronucleated polychromatic erythrocyte counts: the substance did not cause any statistically significant increases in the number of micronucleated polychromatic erythrocytes at any of the three sampling times - P>0.05 using Wilcoxon's sum of ranks test. Mitomycin C caused large, highly significant increases (P<0.001) in the frequency of micronucleated polychromatic erythrocytes.
- Micronucleated normochromatic erythrocytes: the substance did not cause any substantial increases in the incidence of micronucleated normochromatic erythrocytes at any of the three sampling times.
- Ratio of polychromatic to normochromatic erythrocytes: At the 48 hour sampling time, slight but statistically significant decreases in the ratio were observed after treatment with the substance. Significant decreases were not obtained at the 24 and 72 hour sampling times. The results suggest that the substance may have a slight cytotoxic activity towards bone marrow. Mytomycin C caused statistically significant decreases in the ratio.

Applicant's summary and conclusion

Conclusions:

The substance was not clastogenic in the in vivo micronucleus test (OECD TG 474).

Executive summary:

The substance was tested in the in vivo micronucleus test (OECD TG 474). After a preliminary test to determine test concentrations, male and female mice were dosed with 2850 mg/kg. Three sampling times (24, 48 and 72 hours) were used. No animals died after treatment. The substance did not cause any substantial increases in the incidence of micronucleated normochromatic or polychromatic erythrocytes. The substance only caused slight but statistically significant decreases in the ratio of polychromatic to normochromatic erythrocytes at the 48 h sampling times suggesting that the substance has a cytotoxic activity towards bone marrow. Based on the results, the substance is not clastogenic in this test.