Registration Dossier

Administrative data

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Remarks:
Type of genotoxicity: 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:
other: Acceptable publication which meets basic scientific principles; limited information on test animals.

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

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: choromosome aberration assay and micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: Morpholine
- Supplier: Wako Chemical Industries Ltd., Tokyo, Japan

Test animals

Species:
hamster
Strain:
other: Syrian golden hamster
Sex:
female
Details on test animals 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
Remarks:
Doses / Concentrations:
500 mg/kg bw
Basis:
actual ingested
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
Sex:
female
Genotoxicity:
negative
Toxicity:
not examined

Any other information on results incl. tables

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

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of this study, transplacental exposure to Morpholine did not induce genetic toxicity on the basis of chromosomal aberrations or micronuclei formation.
Executive summary:

In this transplacental mutagenesis study (Inui et al., 1979), sodium nitrite with Morpholine or Morpholine (500 mg/kg bw) alone were administered by single oral gavage to pregnant Syrian golden hamsters on day 11 or 12 of pregnancy. Twenty-four hours after treatment, the hamster embryos were excised and examined for chromosomal aberrations, micronucleus formation, morphological or malignant transformation and drug resistance mutation. Cells exposed in utero to Morpholine showed no increases in the numbers of chromosomal aberrations, micronuclei, 8 -azaguanine- or ouabain-resistant mutants, or transformation rates. The number of resistant colonies was markedly increased after administration of sodium nitrite together with Morpholine only, showing that Morpholine alone has no mutagenic effect in vivo under the conditions of this study. In this valid in vivo study there was no evidence for mutagenic effects of Morpholine over background.

This study is classified as acceptable.