Morpholinium toluene-4-sulphonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study is scientifically valid and followed the principles of OECD Test Guideline 476.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1979

Report date:

1979

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

no

Type of assay:

mammalian cell gene mutation 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

Details on test material:

- Name of test material: Morpholine (7H-4892/LOS-0575)
- Physical state: clear colorless liquid

Method

Target gene:

thymidine kinase locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: The cells were maintained in Fischer's mouse leukemia medium supplemented with L-glutamine, sodium pyruvate, and horse serum (10 % by volume). Cloning medium consisted of the preceding growth medium with the addition of agar to a final concentration of 0.35 % to achieve a semisolid state. Section medium was cloning medium containing 100 µg/mL of BrdU or 3 µg/mL of TFT.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

other: heterozygous at the thymidine kinase locus

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 (S9 mix)

Test concentrations with justification for top dose:

Without metabolic activation:
Experiment 1: 0.078, 0.500, 0.625, 1.000, 1.250 µL/mL
Experiment 2: 0.500, 0.625, 0.750, 1.000, 1.250 µL/mL
Experiment 3: 0.750, 1.000, 1.200 µL/mL

Metabolic activation:
Experiment 1: 0.156, 0.500, 0.625, 1.000, 1.250 µL/mL
Experiment 2: 0.500, 0.625, 0.750, 1.000, 1.250 µL/mL
Experiment 3: 0.750, 1.000 µL/mL

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent/vehicle: due to a technical error in the initial solubility testing, the test substance was described as insoluble at 100 uL/mL in deionized water. The substance was, in fact, soluble and water would have been the preferred solvent.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

see positive controls below

Remarks:

with and without metabolic activation

Controls 2

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

not specified

Remarks:

no data was provided on the solvent which the positive control was dissolved in.

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Migrated to IUCLID6: 0.5 µL/mL

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

Remarks:

No data was provided on the solvent which the positive control was dissolved in.

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: dimethylnitrosamine (0.3 µL/mL)

Remarks:

with metabolic activation

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
- Selection time (if incubation with a selection agent): 10 days
- Fixation time (start of exposure up to fixation or harvest of cells): cells were harvested at the end of the selection time.

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

NUMBER OF REPLICATIONS: 1-2

NUMBER OF CELLS EVALUATED: Mutation frequency was based on 10 EXP 6 cells.

DETERMINATION OF CYTOTOXICITY
- Method: % relative total growth

Evaluation criteria:

The test substance was considered mutagenic in the assay if:
- A dose response relationship was observed over 3 of the 5 dose levels employed.
- The minimum increase at the low level of the dose response curve was at least 2.5 times greater than the solvent and/or negative control values.
- The solvent and negative control data were within the normal range of the spontaneous background for the TK locus.
All evaluations of mutagenic activity were based on consideration of the concurrent solvent and negative control values run with the experiment in question. Positive control values were not used as reference points, but were included to ensure that the current cell population responded to direct and promutagens under the appropriate treatment conditions.
Occasionally, a single point within a concentration range was expected to show an increase 2.5 times greater than the spontaneous background. If the increase was at the high dose, was reproducible, and if an additional higher dose level was not feasible because of toxicity, the substance was possibly considered mutatgenic. If the increase was internal within the dose range and was not reproducible, the increase was considered aberrant. If the internal increase was reproducible, several doses clustered around the positive concentration were examined to either confirm or reject the reliability of the effect.

Statistics:

Not indicated.

Results and discussion

Test results

Test results 1

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: The test substance was tested for cytotoxicity at an applied dose range of 0.01-5 uL/mL. After an exposure time of four hours, the cells were washed and a viable cell count was obtained the next day. Relative cytotoxicities expressed as the reduction in growth compared to the growth of untreated cells were used to select seven to ten doses that covered the range from 0 to 50-90 % reduction in 24 hour growth. These selected doses were subsequently applied to cell cultures prepared for mutagenicity testing, but only four or five of the doses would be carried through the mutant selection process. The test substance remained soluble in the growth medium, but an alkaline pH was obtained at concentrations above about 0.3 µL/mL. Twenty four hours after treatment, the cell count was greatly reduced at 1.25 µL/mL and doses of 2.5 µL/mL and higher were completely lethal.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Four trials of the mutation assay were initiated, but the first trial became contaminated before any mutagenesis data could be collected. Under the nonactivation conditions, the test substance was lethal at 2.0 µL/mL and five treatments from 1.25 -0.078 µL/mL were assayed for mutant induction. The 1.25 µL/mL treatment was highly toxic (9.4 % relative growth) and did not induce a significant increase in mutant frequency. A 2.5 fold increase over the background was considered the minimum criterion for demonstrating mutagenesis at any given dose level. The other four treatments were moderately toxic and also yielded mutant frequencies similar to the background, except for the 1 µL/mL treatment. In this case, a 2.8 fold increase was observed. An increase of this small magnitude that is not followed by a similar or larger increase for more toxic treatments is normally regarded as spurious. Nevertheless, a repeat assay was performed to test the repeatability of the response. In the second nonactivation assay, the toxicity of the test substance was more variable as a function of the applied concentration. Three of the assayed treatments were highly toxic and all of these induced mutant frequency increases near the 2.5 fold criterion used for evaluation as mutagenic. Thus, 2.2 fold, 2.4 fold and 2.7 fold increases were obtained for 0.625, 1 and 1.25 µL/mL, respectively. These results suggested that the test substance may be weakly mutagenic at the limit of detectability for this assay. A third nonactivation assay was performed which yielded similar behavior. One treatment with 0.75 µL/mL and another at 1.2 µL/mL induced 2.5 and 2.7 fold increases in the mutant frequency. However, duplicate treatments at these two doses, both of which were highly toxic, caused no significant change from the background frequency. The results from all three trials therefore indicated that the test substance can be weakly mutagenic for highly toxic treatments in the 0.625 to 1.25 µL/mL dose range. The lack of mutagenic activity for some toxic treatments in this dose range suggested competition between toxic and mutagenic modes of action. In the presence of metabolic activation, the test substance was not as toxic, indicating a reaction with the activating system. Only moderately toxic treatments were achieved in the first trial for applied concentrations up to 1.25 µL/mL (35.5 % relative growth). The mutant frequencies in the treated cultures remained similar to the background. The next highest applied concentration of 2 µL/mL was completely lethal to the cells. A repeat assay gave similar results and confirmed the lack of detectable mutagenesis for moderately toxic treatments obtained with concentrations up to 1.25 µL/mL. In this assay, the next highest dose was 1.5 µL/mL, which was completely lethal. A third activation assay was performed in which most of the applied concentrations (from 1.2 to 2 µL/mL) were lethal and only two dose levels (in duplicate) were available for analysis. One of the 0.75 µL/mL treatments was moderately toxic (37.3 % relative growth), whereas the other treatments had no apparent effect on the cells. The mutant frequencies remained similar to the one solvent negative control that was not contaminated. Thus, the test substance had little to moderate toxicity in the presence of activation until a threshold concentration was reached where lethality suddenly became excessive. This threshold occurred between 1.25 and 1.5 µL/mL in trial 2 and between 1.0 and 1.2 µL/mL in trial 3. Since the test substance was alkaline in the culture medium at these concentrations, the sharp increase in lethality may be pH related rather than test substance induced. Any mutagenic activity that might be associated with highly toxic treatments would therefore occur over a very narrow concentration range just preceding complete lethality. Such information was probably not biologically meaningful, so further attempts to achieve this toxicity range did not seem warranted. The average cloning efficiencies for the solvent and untreated negative control varied from 105 to 108% without activation and from 106 to 124 % with activation, which demonstrated excellent culturing conditions for the assays. The negative control mutant frequencies were all in the normal range, and the positive control substance yielded normal mutant frequencies that were greatly in excess of the background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD .

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
positive without metabolic activation

Morpholine was evaluated for mutagenic potential in the mouse lymphoma forward mutation assay using L5178Y TK +/- cells in the presence and absence of metabolic activation (S9 mix). Under the conditions of this study, the test substance was considered to be weakly mutagenic in the assay without metabolic activation.