4-morpholinecarbaldehyde

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames (OECD 471): negative (BASF, 1985)

CA (OECD 473): negative (CCR, 1997)

HPRT (OECD 476): negative (BASF, 2012)

Link to relevant study records

Reference

Reference 1

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

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

Target gene:

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

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.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9 mix

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.

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1984-08-020to 1984-10-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study, comparable to Guideline Study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Remarks:

There was the following deviation from the requirements of GLP principles:The stability of the test substance throughout the study period has not been determined analytically.

Type of assay:

bacterial reverse mutation assay

Target gene:

not applicable

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix from Aroclor 1254 induced Sprague-Dawley rats

Test concentrations with justification for top dose:

20 - 5000 µg/plate

Vehicle / solvent:

- Vehicle / solvent used: aqua dest.
- Justification for choice of solvent/vehicle: the test substance was completely soluble in water

Untreated negative controls:

yes

Remarks:

solvent

Negative solvent / vehicle controls:

yes

Remarks:

sterility

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene in DMSO (10 µg for Salmonella strains, 60 µg for E.coli)

Remarks:

with S-9 mix for strains TA100, TA98, TA1537, TA1535 and E.coli WP2 uvrA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitroso-guanidine (5 µg in DMSO)

Remarks:

without S-9 mix for strain TA100 and TA1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine (10 µg in DMSO)

Remarks:

without S-9 mix for strain TA98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S-9 mix for strain TA1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S-9 mix for strain E.coli WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours at 37 °C


NUMBER OF REPLICATIONS: 3 plates per dose or control, each in 2 independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

In general, a substance characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results

Statistics:

no data

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

bacteriotoxic only in strain TA1535 without S-9 mix at 5000 µg/plate, artefact

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: complete solubility of the test substance in aqua dest .
- Precipitation: no
- Other confounding effects: none


ADDITIONAL INFORMATION ON CYTOTOXICITY:

A strong bacteriotoxicity, which was detected with the strain TA 1537 in the 2nd experiment without S-9 mix could not be observed in the first study and could also not be confirmed in a 3rd study. Therefore, the results of the 2nd experiment with TA 1537 were considered as an artefact and not as an indication for bacteriotoxicity

Remarks on result:

other: all strains/cell types tested

Conclusions:

An increase in the number of his+ or trp+ revertants could not be observed either without S-9 mix or after addition of a metabolizing system .

Executive summary:

Under the experimental conditions of this study, the test substance N-formylmorpholine is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Reference 3

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

Study period:

1997-07-28 to 1997-11-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline Study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable, no target gene

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Minimal Essential Medium (MEM) supplemented with 10% fetal calf serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Mammalian microsomal fraction (S-9 mix)

Test concentrations with justification for top dose:

0.0, 37.5, 75.0, 150.0, 300.0, 600.0 or 1150 µg/mL (18 h experiments)
0.0, 150.0, 600.0 or 1150 µg/mL (28 h experiments)

(only concentrations of 150.0, 600.0 and 1150 µg/ml were evaluated)

Vehicle / solvent:

- Vehicle / solvent used: deionised water
- Justification for choice of solvent / vehicle: The test substance was completely soluble in the vehicle chosen

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

solvent (deionised water)

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S-9 mix Migrated to IUCLID6: 600 μg/mL = 4.8 mM

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S-9 mix Migrated to IUCLID6: 0.71 μg/mL = 2.5 μM

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours or 18 hours or 28 hours
- Expression time (cells in growth medium): 18 or 28 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 18 hours or 28 hours

SPINDLE INHIBITOR (cytogenetic assays): colcemid ( 0.2 µg/mL), 2.5 h before harvesting
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: dublicate cultures, 2 inependent experiments
NUMBER OF CELLS EVALUATED: 100 metaphases per culture

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

The chromosome aberration assay is considered acceptable if it meets the following criteria:

a) The number of structural aberrations found in the negative and/or solvent controls falls within the range of the historical laboratory control data:
0.00 % - 4.00 %.
b) The positive control substances should produce significant increases of the number of cells with structural chromosome aberrations.

A test article is classified mutagenic if it induces reproducibly either a significant concentration-related increase in the number of structural
chromosome aberrations or a significant and reproducible positive response for at least one of the test points.
A test article producing reproducibly neither a significant concentration-related increase in the number of structural chromosome aberrations nor a
significant and reproducibly positive response at any one of the test points is considered non-mutagenic in this system.

Statistics:

Statistical significance was confirmed by means of the Fischer's exact test.
However, both biological and statistical significance were considered together.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
No test article precipitation and no relevant influence of the test article on the pH value or the osmolarity of the culture medium was observed (solvent control : 285 mOsm, pH 7.4 versus 298 mOsm and pH 7.4 at 1150 μg/mL) .

RANGE-FINDING/SCREENING STUDIES:
The applicable concentration range of the test article for the cytogenetic experiments was determined in a pre-test using the determination of cell numbers 24 h after start of treatment as indicator for cytotoxicity. The highest applied concentration in the pre-test on toxicity (1150 μg/mL = 10 mM) was chosen with regard to the current OECD Guideline for in vitro mammalian cytogenetic tests.

COMPARISON WITH HISTORICAL CONTROL DATA:
The aberration rates (aberrant cells exclusive gaps) of the cells after treatment with the test article (exp . I : 1 .0 %- 2.5 %; exp . II: 1 .5 % - 2 .5 %) were in the range of the solvent control values (exp . I and exp. II : 1 .0 % - 3 .0 %) and in the range of the historical control data : 0 .0 % - 4.0 %.

Remarks on result:

other: all strains/cell types tested

In the absence and the presence of S9 mix, in both experiments no reduction of the mitotic indices could be observed up to treatment concentrations of 10 mM (1150 μg/ml) . Evaluation of the cell numbers revealed slightly but dose related reductions of the cell numbers in experiment I in the absence of S9 mix (18 h : 62.7 % of control ; 28 h 76.6 % of control). In the presence of S9 mix no clearly dose related effect could be observed.

In both independent experiments, in the absence and presence of S9 mix the test article did not increase the frequency of cells carrying structural chromosome aberrations . The aberration rates (aberrant cells exclusive gaps) of the cells after treatment with the test article (exp. I : 1.0 %- 2.5 %; exp. II: 1.5 % - 2.5 %) were in the range of the solvent control values (exp. I and exp. II : 1.0 % - 3 0 %) and in the range of our historical control data : 0.0 % - 4.0 %.

In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test a rt icle (exp. I : 1.5 % - 5.0 %; exp. II : 2.5 % - 4 .0 %) as compared to the rates of the solvent controls (exp.I : 2.0 % - 2.5 %; exp. II: 2.0 % - 4 0 %).

In both experiments, EMS (600 μg/mL) and CPA (0.71 μg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

In conclusion, under the experimental conditions reported, the test article N-Formylmorpholin did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) .

Conclusions:

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in
vitro.

Executive summary:

Under the experimental conditions of this study, the test substance N-formylmorpholine does not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

AMES test:

In the key reverse gene mutation assay (BASF AG 1985; 40/1110180/84), the bacteria strains TA 1535, TA 1537, TA 98 and TA 100 of S. typhimurium and WP2 uvrA of E.coli were exposed to the test item (99.68 % a.i.) at concentrations of 0, 20, 100, 500, 2000, 2500 or 5000 µg/plate (3 plates/dose) in the presence and absence of mammalian metabolic activation applying the standard plate method in two independant experiments. The test item was completely soluble in water. Cytotoxicity was only observed in strain TA 1535 at 5000 µg/plate in the second of to experiments and assessed as artefact. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

CA test:

In a mammalian cell cytogenetics chromosome aberration assay (CCR, 1997) V79 cell cultures were exposed to the test item (99.9 % a.i.) at concentrations of 0.0, 37.5, 75.0, 150.0, 300.0, 600.0 or 1150 µg/mL with and without metabolic activation and the concentrations 150.0, 600.0 and 1150 µg/mL were examined for structural chromosome changes after 18 and 28 hours.

In both cytogenetic experiments in the absence and the presence of S9 mix no reduced mitotic indices were observed. In both independent experiments, neither a significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. In addition, no increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Positive control substances induced the expected increases in chromosome aberrations and thus confirmed the sensitivity of the test system.

HPRT assay:

In a mammalian cell gene mutation assay for the HPRT locus (BASF SE, 2012; 50M0173/97M001) CHO cells culturedin vitrowere exposed to the test item at concentrations of 0; 150; 200; 300; 400; 600; 800 or 1200 µg/mLin the presence and absence of mammalian metabolic activation (S9 -mix) in two independent experiments. No cytotoxicity was observed. There was no evidence of induced mutant colonies over background.

The positive controls induced the appropriate response.

Justification for selection of genetic toxicity endpoint
No study was selected since all studies were negative.

Short description of key information:
No potential for genetic toxicity of the test item was indicated in the Ames Test, no increase of structural chromosomal changes was noted in a chromosome aberration assay with V79 cells and there was no evidence of induced mutant colonies over background in the HPRT test.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. Based on the negative in vitro results of the Ames test, HPRT test and the CA assay, the test item has not to be classified and labelled mutagenic according to Regulation (EC) No 1272/2008.