4-methylmorpholine 4-oxide, monohydrate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 14, 1981 to May 22, 1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1981

Report date:

1981

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

Positive controls are wrongly chosen. No colony sizing performed on negative and positive controls.

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-methylmorpholine 4-oxide, monohydrate

EC Number:

231-391-8

EC Name:

4-methylmorpholine 4-oxide, monohydrate

Cas Number:

7529-22-8

Molecular formula:

C5H11NO2

IUPAC Name:

4-methyl-4λ⁵-morpholin-4-one

Specific details on test material used for the study:

- Name of test material (as cited in study report): 4236-40-5 Order No. J-64
- Substance type: clear, nearly-colorless liquid
- Physical state: liquid

Method

Target gene:

thymidine kinase

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: The cells are maintained in Fischer's mouse leukemia medium supplemented with L-glutamine, sodium pyruvate, and horse serum (10% by volume).
- Properly maintained: yes
- Stocks are maintained in liquid nitrogen and laboratory cultures are periodically checked for the absence of mycoplasma contamination by culturing methods.
- To reduce the negative control frequency (spontaneous frequency) of TK -/- mutants to as low level as possible, cell cultures are exposed to conditions which select against the TK -/- phenotype (exposure to methotrexate) and are then returned to normal growth medium for three or more days before use.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced Fischer 344 or SD adult male rat liver S9 homogenate

Test concentrations with justification for top dose:

Dose range finding:
nonactivation and activation assay: 0.625, 1.250, 2.500, 5.000 and 10.000 uL/mL
Final test:
nonactivation and activation assay: 6.0, 8.0 and 10.0 µL/mL of culture medium

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The substance was miscible with deionized water up to 10µl/ml. Therefore, just prior to use, stock solutions were prepared by performing serial dilutions of the test material in water. The stock solutions were then diluted 1:10 into tubes of culture medium containing the cells to initiate the treatments.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

0.5 µL/mL (nonactivation assay)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Remarks:

0.3 µL/mL (activation assay)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 2-3 days (mostly 2 days)
- Selection time (if incubation with a selection agent): 10 days of incubation

SELECTION AGENT (mutation assays): Selection medium is cloning medium containing 100 µg/mL of BrdU or 3 µg/mL of TFT. Cloning medium consists of growth medium with the addition of agar to a final concentration of 0.35% to achieve a semisolid state.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

NUMBER OF CELLS EVALUATED
3 x 10^6 cells are seeded in selection medium and after 10 days mutant colonies are counted.

Evaluation criteria:

See field 'Any other information on materials and methods incl. tables.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

treatments up to a highly concentrated 10 µL/mL were moderately toxic with and without S9 microsomal activation

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

First trial (without S9): 0.00061 - 10 µl/ml tested. Test substance was non-toxic to moderately toxic, but not dose-related. Test substance was negative in this trial.
First trial (with S9): 0.0061 - 10 µl/ml tested. Test substance was highly toxic. No mutant analysis possible. Instead of Fisher 344 rat derived S9, which was used in the first trial, an S9 mix derived from SD rats was used in the next trials.
Second trial (without S9): 3 - 10 µl/ml tested. Still only moderate toxicity observed. Test substance was also negative in this trial.
Second trial (with S9): 3 - 10 µl/ml tested. Less toxicity observed than when tested without S9. Test substance was negative in this trial (only one slightly positive response).
Third trial (with S9): 3 - 10 µl/ml tested. Still nontoxic to moderate toxicity. Test substance was negative in this trial.

Percent relative growth (relative to controls) ranged from 62.7 to 106.6% in the activated trial, and from 26.8 to 77.6% in the non-activated trial. When compared to the untreated cultures, NMMO failed to induce a statistically significant number of mutant colonies in both the activated and non-activated trials. Cell survival and observed mutational frequencies, for both positive and negative controls in activated and non activated trials, fell within acceptable ranges for this assay based on historical data for this cell line and laboratory. NMMO is considered to be negative, or non-mutagenic, in this assay.

Applicant's summary and conclusion

Conclusions:

The substance did not induce repeatable increases in the mutant frequency at the TK locus in L5178Y mouse lymphoma cells. Treatments up to the highly concentrated 10 µL/mL were only moderately toxic with and without S9 microsomal activation and did not induce significant increases in the mutant frequency. The substance was therefore considered to be inactive under conditions of moderate toxicity in the mouse lymphoma forward mutation assay.

Executive summary:

In a mammalian cell gene mutation assay conducted similar to OECD guideline 490, L5178Y mouse lymphoma cells cultured in vitro were exposed to 4-methylmorpholine 4-oxide (NMMO), dissolved in distilled water at concentrations of 6.0, 8.0 and 10.0 µl/mL in the absence and presence of mammalian metabolic activation.

NMMO was tested up to limit concentrations.

When compared to the untreated cultures, NMMO failed to induce a statistically significant number of mutant colonies in both the activated and non-activated trials. Cell survival and observed mutational frequencies, for both positive and negative controls in activated and non activated trials, fell within acceptable ranges for this assay based on historical data for this cell line and laboratory and therefore did induce the appropriate response.

There was no evidence or a concentration related positive response of induced mutant colonies over background.

NMMO is considered to be negative, or non-mutagenic, in this assay.

This study is classified as acceptable and satisfies the requirement for Test Guideline OECD 490 for in vitro mutagenicity (mammalian forward gene mutation) data.