cis-4-[3-(p-tert-butylphenyl)-2-methylpropyl]-2,6-dimethylmorpholine

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:

2. Nov. 1993 - 28. Oct. 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

No positive response in positive control of Exp.1 with Cyclophosphamide. No analytical verification of substance bloodlevel

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian bone marrow chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

cis-4-[3-(p-tert-butylphenyl)-2-methylpropyl]-2,6-dimethylmorpholine

EC Number:

266-719-9

EC Name:

cis-4-[3-(p-tert-butylphenyl)-2-methylpropyl]-2,6-dimethylmorpholine

Cas Number:

67564-91-4

Molecular formula:

C20H33NO

IUPAC Name:

(2R,6S)-4-[3-(4-tert-butylphenyl)-2-methylpropyl]-2,6-dimethylmorpholine

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River GmbH, WIGA, Sulzfeld,
- Age at study initiation: Not specified
- Weight at study initiation: Animals witha mean weight of about 28g were used for the study.
- Assigned to test groups randomly: yes, under following basis: according to a randomization plan prepared with an appropriate computer program.
- Fasting period before study: Not specified
- Housing: During the test animals were housed individually in Makrolon cages type MI
- Diet: Standardized pelleted feed (Kliba Haltungs Diät, Klingenthalmühle AG, Kaiseraugst, Switzerland)
- Water: ad libitum
- Acclimation period: 2 weeks in groups of 5

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): Not specified
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: Not specified

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

Aqueous solution of 0.5% carboxy methyl cellulose

Details on exposure:

Route = intraperitoneal

Duration of treatment / exposure:

once

Post exposure period:

24h and 48h

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 000 mg/kg bw (total dose)

Remarks:

Experiment 2
24h and 48h sacrifice interval

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw (total dose)

Remarks:

Experiment 1
24h and 48h sacrifice interval

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw (total dose)

Remarks:

Experiment 1
24h sacrifice interval

Doses / concentrations 4

Dose / conc.:

250 mg/kg bw (total dose)

Remarks:

Experiment 1
24h sacrifice interval

No. of animals per sex per dose:

5 males and 5 females

Control animals:

yes, concurrent vehicle

Positive control(s):

20 mg Cyclophosphamide
0.15 mg Vincristine

Examinations

Tissues and cell types examined:

Bone marrow smears

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In a pretest for the determination of the acute intraperitoneal toxicity deaths were observed down to a dose of 1250 mg/kg bodyweight. 1000 mg/kg were survived by all animals but led to clinical signs such as apathy and abdominal position; the general state of the animals was poor. Therefore, a dose of 1000 mg/kg bodyweight was selected as the highest dose in the present cytogenetic study. 500 mg/kg and 250 mg/kg bodyweight were administered as further doses.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Sampling was performed 24 h and/ or 48 h after treatment

DETAILS OF SLIDE PREPARATION:
The bone marrow was prepared according to the method described by SCHMID, W. The two femora were prepared from the animals, and all soft tissues were removed. After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum which was at 37°C (about 2 ml/femur). The suspension was mixed thoroughly with a pipette, centrifuged at 1500 rpm for 5 minutes, the supernatant was removed except for a few drops, and the precipitate was resuspended.
1 drop of this suspension was dropped onto clean microscopic slides, using a Pasteurpipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained. The slides were stained in eosin and methylene blue solution for 5 minutes, rinsed in aqua dest. and then placed in fresh aqua dest. for 2 or 3 minutes. They were finally stained in Giemsa solution for 12 minutes. After being rinsed twice in aqua dest. and clarified in xylene, the preparations were embedded in Corbit-Balsam.


METHOD OF ANALYSIS:
In general, 1000 polychromatic erythrocytes from each of the male and female animals of every test group are evaluated and investigated for micronuclei. The normochromatic erythrocytes (= normocytes), which occur, are also scored. The following parameters are recorded:
-Number of polychromatic erythrocytes
-Number of polychromatic erythrocytes containing micronuclei The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the solvent control group provides an index of a chromosome breaking (clastogenic) effect or of a spindle activity of the substance tested.
-Number of normochromatic erythrocytes
-Number of normochromatic erythrocytes containing micronuclei The number of micronuclei in normochromatic erythrocytes at the early sacrifice intervals represents the situation before test substance administration and may serve as a control value. A substance induced increase in the number of micro nuclei in normocytes may be found with an increase in the duration of the sacrifice intervals.
-Ratio of polychromatic to normochromatic erythrocytes: this ratio indicates an influence of the test substance specifically on the bone marrow. Number of small micronuclei (d < D/4) and of large micronuclei (d > D/4) (d= diameter of micronucleus, D = cell diameter) The size of micronuclei may give an indication on the possible mode of action of the test substance, i.e. aclastogenic or a spindle poison effect. Slides were coded before microscopic analysis.

OTHER:

Statistics:

The statistical evaluation of the data was carried out using the program system MUKERN (BASF AG). The number of micronuclei in polychromatic erythrocytes were analysed. A comparison of the dose group with the vehicle control was carried out using the Wilcoxon test for the hypothesis of equal medians. Here, the relative frequencies of cells with micronuclei of each animal were used. If the results of this test were significant, labels (+ for p < 0.05, ++for p < 0.01) were printed with the group means in the tables. This test was performed one sided. This analysis was done separately for each sex and combined for both sexes.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

other: No positive response with Cyclophosphamid in Exp.1