Morpholine

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Transplacental mutagenesis of products formed in the stomach of golden hamsters given sodium nitrite and morpholine.

Author:

Inui N, Nishi Y, Taketomi M, Mori M, Yamamoto M, Yamada T & Tanimura A

Year:

1979

Bibliographic source:

Int. J. Cancer 24: 365-372.

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Hamster embryos were exposed in utero to the action of sodium nitrite and Morpholine or Morpholine alone administered by stomach tube to the mothers on the 11th or 12th day of pregnancy. Embryo cells were examined for chromosomal aberrations, micronucleus formation, morphological or malignant transformation and drug resistance mutations. For detection of induced mutations, the embryo cells were cultured in normal medium for 72 h and then transferred to medium containing 10 or 20 µg/mL of 8-azaguanine or 1 mM ouabain.

GLP compliance:

not specified

Type of assay:

other: chromosomal aberration and micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Morpholine

EC Number:

203-815-1

EC Name:

Morpholine

Cas Number:

110-91-8

Molecular formula:

C4H9NO

IUPAC Name:

morpholine

Test material form:

liquid

Test animals

Species:

hamster

Strain:

other: Syrian golden hamster

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 150 ± 30 g
- Age at study initiation: young adult (based on body weight)
- Housing: closed colony in a clean barrier system room
- Diet: sterilized standard laboratory chow (Japan Clea Ltd., Tokyo, Japan), ad libitum
- Water: ad libitum

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Morpholine was dissolved at a concentration of 500 mg/mL in distilled water immediately before use.

Duration of treatment / exposure:

single treatment on the 11th or 12th day of pregnancy

Frequency of treatment:

once

Post exposure period:

Twenty-four hours after treatment, hamster embryos were excised.

Doses / concentrations

Dose / conc.:

500 mg/kg bw (total dose)

No. of animals per sex per dose:

no numbers given; all foetuses from each litter were used

Control animals:

yes, concurrent vehicle

Positive control(s):

N-nitrosomorpholine (Dr. Okada, Tokyo Biochemical Institute, Tokyo, Japan)
- Justification for choice of positive control: active compound
- Route of administration: oral gavage
- Doses / concentrations: 100 or 200 mg/kg bw; 500 mg/mL

Examinations

Tissues and cell types examined:

Primary cultures of trypsinized cells

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:

Preparation of samples for examination of chromosomes and micronucleus formation:
For preparation of chromosomes, some cells were treated with 0.3 µg/mL of colcemid for 3 h, within 24 h of initiation of the primary culture, so that mitotic cells could then be studied in the first cell cycle in culture. Chromosome preparations were made by the usual air-dry method with a slight modification (Rothfels & Simonovitch, 1958; Yoshida et al., 1970) and stained with Giemsa. The numbers and types of chromosomal aberrations were assessed by 200 well-spread metaphase plates. For examination of micronucleus formation, samples were made by a modification of the method of Schmid (1975); 36 h after initiation of cell cultures, the cells were collected with 0.1 % trypsin, smeared on glass slides and fixed with methanol for 30 min. The slides were dried and stained with Giemsa. Per experiment, over 5000 resting nuclei were examined for micronucleus formation.

Selection of resistant mutant cells:
For selection of drug-resistant mutant cells, standard-incubated primary cultures were inoculated into medium containing 10 or 20 µg/mL of 8-azaguanine (8AG) or 1 mM ouabain (Oua) in 60 mm plastic dishes. At least five independent experiments were made. For selection of 8AG-resistant mutations, the medium containing 8AG was changed every day for the first 3 days and then, when cells that could not produce colonies had died, the medium was changed once every 3 days. For selection of Oua-resistant mutants, the medium containing 1 mM Oua was changed after 1 week. After total cultivation periods of 15 to 20 days (8AG) and 30 days (Oua), dishes were fixed and stained, and the number of resistant colonies was investigated. Embryonic cells were obtained from mothers treated with Morpholine; embryonic cells from untreated mothers were used as controls. Control cells were cultured and selected in the same way as those in experimental group.

Evaluation criteria:

Not indicated

Statistics:

Statistically significant differences between treatment and control groups were determined at p<0.05 and p<0.01. The statistical test method chosen was not specified.

Results and discussion

Test results

Key result

Sex:

female

Genotoxicity:

negative

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table 1: Results Chromosome Aberration Test

	Dose (mg/kg bw)	No. of metaphases observed	Cells with aberrations	Normal diploid	cells with over 44 chromosomes (%)
control	0	200	4 (2%)	89.0%	4.0
morpholine	500	200	3 (1.5%)	89.0%	3.0

Table 1: Results Micronucleus Test

	Dose (mg/kg bw)	No. of cells observed	No. of cells with micronucleus	% of cells with micronucleus	Induced ratio*
control	0	5000	21	0.42	-
morpholine	500	5000	27	0.52	x 1.29

* The induced ratio means the ratio of the induced number to the number in control cells

Treatment with Morpholine at 500 mg/kg bw induced no increases in chromosomal aberrations or frequency of micronuclei in primary embryonic cell cultures.