4-[(morpholinothio)thioxomethyl]morpholine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1979

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

Protocol references indicate a standard procedure described in the Litton Bionetics Inc. Screening Programe for the Identification of Potential Mutagens and Carcinogens.
The purpose of the study was to evaluate the ability of test article to induce chromosome aberrations in Chinese Hamster Ovary (CHO) cells, with and without metabolic activation using S9 mix. Cell line used American Type Culture Collection Repository, No. CCL61. Chromosome aberrations were scored against known DNA patterns by the type, frequency and correlation to dose trends.

GLP compliance:

not specified

Remarks:

signed QA statement provided

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

4-[(morpholinothio)thioxomethyl]morpholine

EC Number:

237-335-9

EC Name:

4-[(morpholinothio)thioxomethyl]morpholine

Cas Number:

13752-51-7

Molecular formula:

C9H16N2O2S2

IUPAC Name:

morpholin-4-yl morpholine-4-carbodithioate

Constituent 2

Reference substance name:

Cure-Rite® 18 (purified)

IUPAC Name:

Cure-Rite® 18 (purified)

Details on test material:

- Name of test material (as cited in study report): Cure-rite 18 (purified)
- Physical state: solid
- Analytical purity: 97.5%
- Lot/batch No.: not given
- Expiration date of the lot/batch:
- Storage condition of test material: not specified

Method

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

S9 rat liver activation system

Test concentrations with justification for top dose:

2.5, 5.0, 10.0, 15.0, and 20.0 µg/mL

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

media

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Controls 2

Positive controls:

yes

Positive control substance:

triethylenemelamine

Details on test system and experimental conditions:

CHO cells were obtained from the American Culture Collection, Repository No. CCL61, Rockville, MD. CHO cells were grown in McCoy's 5a medium
supplemented with 10% fetal calf serum, L-glutamine, and penicillinstreptomycin. Cultures were set up approximately 24 hours prior to
treatment by seeding 8x105 cells per 75-cm2 plastic flask in 10 ml of fresh medium.
The test chemical were dissolved in DMSO. Untreated and solvent (1% final concentration) control cultures were used and the positive controls
were triethylenemelamine (0.5, 0.75, or 1.0 μg.ml) without activation or cyclophosphamide (62.25, 130.5, or 261 μg/ml) with S-9 activation. The
dose range was selected on the basis of survival of L5178Y cells 24 hours after treatment in the mouse lymphoma forward mutation assay, considering that the exposure period in the CHO assay (2 hours) is shorter than the mouse lymphoma assay (4 hours). The highest dose for cytogenetics assay was selected to produce little or no toxicity. In the nonactivation assay, approximately 2-3 x 106 cells were treated with the test chemical for two hours at 37° C in growth medium. The exposure period was terminated by washing the cells twice with saline containg 10% FBS and then adding fresh medium. Incubation continued for 17 hours. Colecemid was added for the last tow hours of incubation (2 x 10-7 M final concentration), and metaphase cells were collected by mitotic shake-off. The cells were treated with 0.075 M KCl hypotonic solution, washed three times in fixative (methanol:acetic acid, 3:1< v/v), dropped into slides, and air-dried. The slides were stained with 10% Giemsa at pH 6.8. Fifty or 100 cells were scored at each dose level.
The activation assay differed from the nonactivation assay in that the S-9 reaction mixture (2.4 mg NADP, 4.5 mg isocitric acid and 15 μl S-9 fraction
per ml) was added to the growth medium, together with the test chemical, for two hours. After exposure the cultures were treated as above.

Statistics:

statistical evaluations were conducted using the Student t-test.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: strain/cell type: CCL61 CHO cells

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive

The test material is considered to be mutagenic. Cure-rite 18 (commercial grade) induces chromosome aberrations in CHO cells at 2.5 and
5.0 µg/mL. Metabolic activation is not necessary for the positive response.

Executive summary:

Cure-rite 18 (Purified) was clastogenic when evaluated in CHO cells. The effect was increased slightly when an activation system was included. The evaluation suggests some metabolism to an active breakdown product.