4-ethylmorpholine

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:

1980-09-08 to 1980-09-26

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

Well documented non-GLP study, according to a method equivalent to OECD guideline 490 with following deviations: positive control substances different from the ones indicated in OECD Guideline 490, triplicate cultures instead of single or duplicate cultures no information reported on historical control data for negative and positive controls, no statistical method reported.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1980

Report date:

1980

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 490 (In vitro mammalian cell gene mutation test using the thymidine kinase gene) adopted on 28 July 2015

Deviations:

yes

Remarks:

see rationale for reliability

Principles of method if other than guideline:

The methodology described in the study report was based on following publication:
Clive, D. and Spector J.F.S.: Laboratory procedure for assessing specific locus mutations at the TK locus in cultured L5178Y mouse lymphoma cells. Mutation Res., 31: 17-29. 1975.

GLP compliance:

no

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-ethylmorpholine

EC Number:

202-885-0

EC Name:

4-ethylmorpholine

Cas Number:

100-74-3

Molecular formula:

C6H13NO

IUPAC Name:

4-ethylmorpholine

Test material form:

liquid

Details on test material:

- Alternative names of reference substance: Jeffcat NEM; N-ethylmorpholine; 4-ethylmorpholin; Morpholine, 4-ethyl-
- Molecular formula: C6H13NO
- Molecular weight: 115.17
- SMILES notation: CCN1CCOCC1
- InChl: InChI=1/C6H13NO/c1-2-7-3-5-8-6-4-7/h2-6H2,1H3

Method

Target gene:

TK locus in L5178Y mouse lymphoma cells

Rationale: Thymidine kinase (TK) is a cellular enzyme that allows cells to salvage thymidine from the surrounding medium for use in DNA synthesis. If a thyimidine analog such as BrdU is included in the growth medium, the analog will be phosphorylated via the TK pathway and be incorporated into DNA, eventually resulting in cellular death. Cells which are heterozygous at the TK locus (TK+/-) may undergo a single step forward mutation to the TK -/- genotype in which little or no TK activity remains. Such mutants are as viable as the heterozygotes in normal medium because DNA synthesis proceeds by de novo synthetic pathways that do not involve thymidine as an intermediate. The basis for selection of the TK-/- mutants is the lack of any ability to utilize toxic analogs of thymidine, which enables only the TK-/- mutants to grow in the presence of BrdU. Cells which grow to form colonies in the presence of BrdU are therefore assumed to have mutated, either spontaneously or by the action of a test substance, to the TK-/- genotype.

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). Cloning medium consists of the preceding growth medium with the addition of agar to a final concentration of 0.35% to achieve a semisolid state. Selection medium is cloning medium containing 100 µg/mL of BrdU or 3 µg/mL of TFT.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes, laboratory cultures are periodically checked for the absence of mycoplasma contamination by culturing methods
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: yes, 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 growh medium for three or more days before use.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

The test item dissolved easily in deionized water at a concentration of 100 µL/mL. This stock was serially diluted with water, and the treatments in a preliminary cytotoxicity test were initiated by diluting the solutions 1:10 into tubes of culture medium containing the cells.
Without metabolic activation: 78, 2500, 3000, 4000 and 5000 nL/mL
With metabolic activation: 78, 156, 1250, 2000 and 2500 nL/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: no data

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation, 0.5 µL/mL

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Remarks:

With metabolic activation, 0.3 µL/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h
- Expression time (cells in growth medium): 48 to 72h
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 5-bromo-2'-deoxyuridine (BrdU) or 5-trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: Triplicate cultures

NUMBER OF CELLS EVALUATED: 100 cells/plate (except with metabolic activation at 2000 and 2500 ngL/mL: culture not split back to 3 x 10E6 cells: the entire culture was assayed and 370 and 340 cells were seeded for viable clone count respectively).

DETERMINATION OF CYTOTOXICITY
- Method: Relative cytotoxicity is expressed as the reduction in growth compared to the growth of untreated cells

Evaluation criteria:

A test item will be evaluated as non-mutagenic in a single assay only if the minimum increase in mutant frequency (MF) is not observed for a range of applied concentrations that extends to toxicity causing 5% to 10% relative suspension growth. If the test material is relatively nontoxic, the maximum applied concentrations will normally be 10 mg/mL (or 10 µL/mL) for water-soluble materials or 1 mg/mL (or 1 µL/mL) for materials in organic solvents. If a repeat assay does not confirm an earlier, minimal response, as discussed above, the test material will be evaluated as nonmutagenic in this assay system.
The following test results must be obtained to evaluate a test material as a mutagen: see 'Any other information on materials and methods incl. tables'

Statistics:

no statistic method specified

Results and discussion

Test results

Test results 1

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

toxicity with concentrations exceeding 1250 nL/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

test material highly toxic at 5000 nL/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The test material was dissolved easily in deionized water at a concentration of 100 µL/mL. This stock was serially diluted with water, and the treatments in a preliminary cytotoxicity test were initiated by diluting the solutions 1:10 into tubes of culture medium containing the cells. The test material remained soluble in the culture medium at all tested concentrations. However, a pinkish coloration (due to phenol red component of the medium) indicated a slightly alkaline pH. At 10 µL/mL the medium turned purple, indicating a pH greater than 8.
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: The test material was dissolved easily in deionized water at a concentration of 100 µL/mL. This stock was serially diluted with water, and the treatments in a preliminary cytotoxicity test were initiated by diluting the solutions 1:10 into tubes of culture medium containing the cells. The test material remained soluble in the culture medium at all tested concentrations.
- Precipitation: no data

RANGE-FINDING/SCREENING STUDIES:
The test material was not toxic to 24-hour cell growth for treatment with concentrations up to 1250 nL/mL. A small reduction in growth was observed at 2500 nL/mL, and treatment with 5000 nL/mL was completely lethal. These results were used to select a concentration range of 5000 nL/mL to 78 nL/mL for the mutation assay, using small concentration steps down to 1250 nL/mL and two-fold dilutions thereafter in an effort to cover a wide range of toxic action.

COMPARISON WITH HISTORICAL CONTROL DATA: no comparison with historical control data available

ADDITIONAL INFORMATION ON CYTOTOXICITY: Under nonactivation conditions, the test material was highly toxic at 5000 nL/mL, reducing the relative growth to only 7.4%. In spite of the high toxicity, the mutant frequency in the treated culture was not significantly altered from the negative control values (solvent and untreated). A frequency of 37.9 x 1E-06 was considered to be the minimum criterion for indicating mutagenesis by a given treatment. This value was just exceeded in the culture exposed to 4000 nL/mL, but the increased frequency was isolated and too small to be considered as evidence for mutagenesis. No increases in mutant frequency were observed at 3000 nL/mL and 78 nL/mL. A frequency could not be calculated for the 2500 nL/mL treatment due to the loss of two of the three mutant selection dishes to mold contamination. However, the number of mutant colonies in the remaining dish was equal to the numbers observed for the contiguous treatments, so no increase in the frequency would have been expected. Since no dose-related or toxicity-related increase in mutant frequency was obtained, the test material was considered to be nonmutagenic under nonactivation conditions.
In the presence of the S9 metabolic activation mix, greater toxicity was observed for treatments with concentrations exceeding 1250 nL/mL. This result suggested an interaction with the activation system and the possible consequent formation of mutagenic products. The mutant frequencies in the assayed cultures remained similar to the negative controls, however, and did not approach the value of 78.7 x 1E-06 used to indicate mutagenesis in this portion of the assay. The toxicity of treatment spanned the range from no apparent toxicity at 78 nL/mL to very high toxicity at 2000 and 2500 nL/mL. Thus, any metabolic products formed in the presence of the S9 mix were not detectably mutagenic over a wide range of toxic action.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Results on cloning efficiency of negative and positive controls:

The average cloning efficiencies for the negative controls (solvent and untreated) varied from 75% without activation to 76% with activation, which demonstrated good culturing conditions for the assay. The negative control mutant frequencies were all in the normal range, and the positive control compounds induced normal mutant frequencies that were greatly in excess of the negative controls.

Applicant's summary and conclusion

Conclusions:

The test material, 4236-30-5, did not induce significant changes in the mutant frequency at the TK locus in L5178Y mouse lymphoma cells. Concentrations up to 5000 nL/mL without activation and 2500 nL/mL with rat liver S9 activation became highly toxic without causing dose-related increases in the mutant frequency. Therefore, the test material was considered to be inactive in the Mouse Lymphoma Forward Mutation Assay.