4-morpholinecarbaldehyde

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:

2011-09-08 to 2011-12-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Guideline study according GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-morpholinecarbaldehyde

EC Number:

224-518-3

EC Name:

4-morpholinecarbaldehyde

Cas Number:

4394-85-8

Molecular formula:

C5H9NO2

IUPAC Name:

morpholine-4-carbaldehyde

Test material form:

other: liquid

Details on test material:

- Name of test substance: N-Formylmorpholin
- Batch identification: O 2891
- CAS No.: 4394-85-8
- Purity: 99.96 area-% (corrected)
- Homogeneity: The homogeneity of the test substance was guaranteed on account of the high purity and was ensured by mixing
before preparation of the test substance solutions.
- Storage stability: The stability of the test substance under storage conditions over the test period was guaranteed until 16 May 2013

ADDITIONAL TEST SUBSTANCE INFORMATION
- Date of production: 16 May 2011
- Molecular weight: 115.13 g/mol
- Physical state, appearance: Liquid, colorless, clear
- Storage conditions: Room temperature

Method

Target gene:

- hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Proliferation rate: doubling time of about 12 - 16 hours
- Plating efficiency: about 90 %
- Karyotype with a modal number of 20 chromosomes

- Stocks of the cell line (1-mL portions) were maintained at -196°C in liquid nitrogen using 7 % (v/v) DMSO in culture medium as a cryoprotectant. Each batch used for mutagenicity testing was checked for mycoplasma contamination.
- Culture medium: Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10%
(v/v) fetal calf serum (FCS) supplemented with: 1 % (v/v) penicillin/streptomycin (stock solution: 10 000 IU / 10 000 μg/mL) 1 % (v/v) amphotericine B (stock solution: 250 μg/mL)

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from induced rats

Test concentrations with justification for top dose:

1st Experiment:
without S9 mix (4-hour exposure period)
0; 150; 300; 600; 1200 µg/mL

with S9 mix (4-hour exposure period)
0; 150; 300; 600; 1200 µg/mL


2nd Experiment:
without S9 mix (24-hour exposure period)
0; 150; 300; 600; 1200 µg/mL

with S9 mix (4-hour exposure period)
0; 200; 400; 800; 1200 µg/mL

Vehicle / solvent:

- Due to the good solubility of the test substance in water, culture medium (Ham's F12) was used as most suitable vehicle.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9 mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

with S-9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION

- Exposure duration: 4 hours both with and without metabolic activation (Exp 1) and 24 hours without metabolic activation and 4 hours with metabolic activation (Exp 2)
- Expression time (cells in growth medium): 6-8 days
- Selection time: 1 week


SELECTION AGENT (mutation assays): 6-TG (10 µg/mL)

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Duplicate cultures were used for all experimental groups.

NUMBER OF CELLS EVALUATED: 10E6

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency


OTHER EXAMINATIONS:
- pH:
Changes in the pH were recorded by a change in the indicator color in the culture medium (phenol red: no color change from pH 6.7 - 8.3). The pH was measured, at least for the two top doses and for the negative controls with and without S9 mix.
- Osmolarity:
Osmolarity was measured, at least for the top dose and for the negative controls with and without S9 mix.
- Solubility:
Test substance precipitation was checked immediately after treatment of the test cultures and at the end of treatment.
- Cell morphology:
The test cultures of all test groups were examined microscopically at the end of exposure period with regard to cell morphology, which allows conclusions to be drawn about the attachment of the cells.

Evaluation criteria:

The HPRT assay is considered valid if the following criteria are met:
- The absolute cloning efficiencies of the negative/vehicle controls should not be less than 50 % (with and without S9 mix).
- The background mutant frequency in the negative/vehicle controls should fall within the laboratory's historical negative control data range of 0 – 15.95 mutants per 10E6 clonable cells
- The positive controls both with and without S9 mix must induce distinctly increased mutant frequencies
- At least 4 dose levels ranging up to a toxic concentration or up to or beyond the limit of solubility under culture conditions should be tested. Freely soluble and apparently non-toxic substances are not tested at concentrations higher than 5 mg/mL or 10 mM.


A finding is assessed as positive if the following criteria are met:
- Increase of the corrected mutation frequencies both above the concurrent negative control values and the historical negative control data range
- Evidence of reproducibility of any increase in mutant frequencies.
- A statistically significant increase in mutant frequencies and the evidence of a doseresponse relationship.

Isolated increases of mutant frequencies above historical negative control range (i.e. 15 mutants per 10E6 clonable cells) or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

The test substance is considered non-mutagenic according to the following criteria:
- The corrected mutation frequency in the dose groups is not statistically significantly increased above the concurrent negative control and is within the historical negative control data range.

Statistics:

Due to the clearly negative findings, a statistical evaluation was not carried out.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no, not influenced by test substance treatment
- Effects of osmolality: no, not influenced by test substance treatment
- Precipitation: no precipitation in culture medium was observed up to the highest applied test substance concentration
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES:
In the pretest for toxicity based on the purity and the molecular weight of the test substance 1200 μg/mL (approx. 10 mM) N-Formylmorpholin was used as top concentration both with and without S9 mix at 4-hour exposure time and without S9 mix at 24-hour exposure time.
The pretest was performed following the method described for the main experiment. The cloning efficiency (survival) was determined as toxicity indicator for dose selection and various parameters were checked for all or at least for some selected doses.
In the pretest the parameters pH value and osmolarity were not relevant influenced by the addition of the test substance preparation to the culture medium at the concentrations measured. In culture medium no test substance precipitation occurred up to the highest applied concentration at the end of treatment in the absence and presence of S9 mix.


COMPARISON WITH HISTORICAL CONTROL DATA:
In both experiments after 4 and 24 hours treatment with the test substance the values for the corrected mutation frequencies were within the respective vehicle control values and clearly within the range of the historical negative control data.


ADDITIONAL INFORMATION ON CYTOTOXICITY:
No cytotoxic effects indicated by clearly reduced cloning efficiencies of below 20 % of control were observed under all experimental conditions of this study when tested up to the highest required concentration.

Remarks on result:

other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:

The test substance did not cause any relevant increase in the mutant frequencies both without S9 mix and after adding a metabolizing system in two experiments performed independently of each other.

Executive summary:

Under the experimental conditions of this study, the test substance N-formylmorpholin is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.