4-methylmorpholine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a key reverse mutation test in bacteria (Ames test) according to a method equivalent to OECD guideline 471 (Stankowski, 1990), the test substance proved to be negative for mutagenicity with and without metabolic activation. This was confirmed in two supporting, reliable Ames studies.
In a read-across chromosome aberration test performed according to OECD guideline 473, the related substance N-ethylmorpholine tested negative with and without metabolic activation.
In a read-across mouse lymphoma test performed according to a method equivalent to OECD Guideline 476 (Myhr, 1980), the related substance 4 -ethylmorpholine tested negative with and without metabolic activation.
In a mammalian cell gene mutation assay in a rat hepatocyte primary culture performed according to a method equivalent to OECD Guideline 482 (San Sebastian JR, 1990), the test substance tested negative.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990-06-25 - 1990-06-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): 6398-24-20
- Lot No.: Project #90-005
- Substance type: Clear colorless liquid
- Physical state: Liquid
- Purity: responsibility of the sponsor
- Stability under test conditions: There was no apparent change in the physical state of the test article during the assay.
- Storage condition of test material: For the purposes of this study, the test article was stored at room temperature in the container received from the sponsor.

Target gene:

histidine locus

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: uvrB deletion mutation and a rfa mutation

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

other: uvrB deletion mutation and a rfa mutation

Metabolic activation:

with and without

Metabolic activation system:

aroclor 1254-induced male Sprague-Dawley rat liver homogenate

Test concentrations with justification for top dose:

preliminary toxicity prescreen: 50, 167, 500, 1670 and 5000 ug/plate in absence of S9
mutation assay: 167, 500, 1670, 5000, 7500 and 10000 ug/plate in the presence and absence of S9

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Remarks:

solvent or test substance without tester strains

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

in the absence of metabolic activation 10.0 ug/plate for TA1535 and TA100

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Remarks:

solvent or test substance without tester strains

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

in the absence of metabolic activation 150 ug/plate for TA1537

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Remarks:

solvent or test substance without tester strains

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

in the absence of metabolic activation 5.00 ug/plate for TA1538 and TA98

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Remarks:

solvent or test substance without tester strains

Positive controls:

yes

Positive control substance:

other: 2-anthramine, 2.50 ug/plate for all 5 tester strains

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicate

Evaluation criteria:

A positive result is defined as a statistically significant, dose-dependent increase in the number of histidine-independent revertants with at least one dose level inducing a revertant frequency that is two-fold the spontaneous solvent control value. If the test article does not induce a statistically significant, dose-dependent increase in revertant frequency but does induce a revertant frequency at one dose level that is two-fold the spontaneous control value, the results is considered equivocal. A negative result is defined as the absence of a statistical significant or dose-dependent increase in the number of histidine-independent revertants.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

evaluated in toxicity prescreen by treating duplicate cultures of strain TA100 in absence of S9; the substance was not toxic up to 5000 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

evaluated in toxicity prescreen by treating duplicate cultures of strain TA1538 in absence of S9; the substance was not toxic up to 5000 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

The test substance demonstrated to be negative in the Ames/Salmonella Plate Incorporation Assay under the conditions of the test, and according to the criteria of the protocol. The substance is considered not to be classified as mutagenic according to the CLP Regulation.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1978-04-07 - 1978-05-01

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

concentrations tested higher than recommended

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): RC - 106
- Substance type: clear colorless liquid
- Physical state: liquid
- Purity: no data
- Storage condition of test material: Room temperature, protected from light
- Other: solubility: approximately 1 g/mL; molecular weight 101.15 g/mole

Target gene:

Histidine locus

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced Sprague-Dawley rat liver S9 mix

Test concentrations with justification for top dose:

50 mg/plate, 10 mg/plate, 5.0 mg/plate, 1.0 mg/plate and 0.5 mg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 5.0 µg/plate for strains TA1535 and TA100

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without S9 100.0 µg/plate for strain TA1537

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without S9 5 µg/plate for strains TA1538 and TA98

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene at 5 µg/plate for strain TA1535

Remarks:

With S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 6-aminochrysene at 1.0 µg/plate for strain TA1537

Remarks:

With S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 10.0 µg/plate for strains TA1538, TA98 and TA100

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
The following were added, in order, to 2 mL of molten top agar contained in 13 x 100 mm tubes:
1) 0.1 mL of a log phase culture of the appropriate indicator strain
2) 0.1 mL of the appropriate concentration of test material
Each tube was prepared individually, immediately vortexed, and the contents poured over the surface of a minimal-glucose agar plate. Each plate was rotated to evenly distribute the top agar before it hardened. All plates were incubated for 48 hours at 37°C. After incubation, the plates were observed for revertant colonies and the colonies were counted and recorded.

NUMBER OF REPLICATIONS: triplicates

Evaluation criteria:

a. The spontaneous revertant levels for each strain when used in either the direct or activated plate assay must be within the acceptable limits as defined by the historical data,
b. All sterility controls must be negative,
c. All positive controls must demonstrate that the indicator strains are functional with known mutagens as evidenced by an increase of at least three times the number of revertant colonies per plate as the spontaneous revertant controls,
d. to be considered positive for mutagenic activity, the test material should exhibit a dose response effect (increasing numbers of revertant colonies with increased amounts of the test sample),
e. for strains TA1535, TA1537, TA1538, and TA98, the test sample should produce a positive dose response over the concentrations with the lowest increase in revertants/plate greater than or equal to 3x the solvent control value to be considered mutagenic, and
f. to be considered mutagenic for strain TA100, the test sample should produce a positive dose response over three concentrations with at least one dose producing an increase in revertants/plate greater than or equal to 3.5x the solvent control value.
All of these criteria must be met before the results of testing conducted with any test sample can be considered valid.

Statistics:

Least squares linear regression analysis was used to compute the "best fit" regression line of dose response on dose level for the test sample. These regression line of dose response on dose level for the test sample. These regression lines represent the strength of the dose response obtained against each bacterial indicator strain. For each response line, y = mx + b, the sample regression coefficient, m, was tested for a statistically significant deviation from the value zero (0) which could indicate mutagenic activity. The null hypothesis and the alternative hypothesis are stated as follows:
H0: m = 0, the data do not suggest mutagenic activity
HA: m>0, the data suggest increasing mutagenic activity with dose
Significance level = 0.05

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

see field 'Additional information on results'

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

see field 'Additional information on results'

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: A filter paper disc-agar overlay zone of inhibition method was used to give a preliminary indication of the toxicity of the test material for the test organism. Sterile filter paper discs (6 mm diameter) for the test organism. Sterile filter paper discs (6 mm diameter) were saturated with a range of 5 different concentrations of the test agent. The treated discs were then placed onto the surface of minimal glucose agar plates. Five mL of top agar were seeded with 0.1 mL of an overnight broth culture of either S. typhimurium TA 1537 or TA 100, vortexed, and poured over the surface of the plates. Each concentration of the test material was tested with both strains. Positive and negative control discs were likewise prepared. All plates were incubated for 48 hours at 37°C after the agar overlay had hardened.

After incubation, all plates were observed for zones of inhibition and the diameters of the zones were measured. The concentration of test material showing either no zone of inhibition or the smallest zone was then used the basis for selection of the doses to be used in the plate assay.
Size of zone of inhibition (in mm):
TA1537: 16.0 mm at 1000 mg/mL; 0.0 mm at 0.01, 0.1, 1.0, 10 and 100 mg/mL
TA100: 12.5 mm at 1000 mg/mL; 8.0 mm at 100 mg/mL; 0.0 mm at 0.01, 0.1, 1.0 and 10 mg/mL

Remarks on result:

other: all strains/cell types tested

Conclusions:

When tested under the conditions of this investigation, the substance did not induce a significant increase in the number of point mutations observed in any of the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, or TA 100. While the linear regression analysis for TA 100 without metabolic activation inducated a significant dose response. none of the doses of RC-106 had induced an increase in revertants/plate of at least 3.5x the solvent control value. Therefore, it was concluded that the substance did not exhibit mutagenic activity in the Salmonella typhimurium indicator strains even after the addition of a metabolic activating system.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

only 4 S. typhimurium strains used, only 2-AA used as positive control substance in the presence of S9-mix

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only 4 S. typhimurium strains used, only 2-AA used as positive control substance in the presence of S9-mix

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): 4-Methylmorpholine
- Physical state: colorless liquid
- Analytical purity: 99.44 %
- Lot/batch No.: T 8
- Stability under test conditions: The stability of the test substance throughout the study will be verified analytically by reanalysis.
- Storage condition of test material: room temperature

Target gene:

His- operon

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

The S9 fractions (1 volume) were prepared from the liver of Aroclor 1254 (20 g/100 ml in peanut oil, ip)-induced male SD rats and mixed with cofactors (9 volumes)

Test concentrations with justification for top dose:

- First experiment: standard plate test with and without S9-mix: 0; 20; 100; 500; 2500 and 5000 µg/plate.
- Second experiment: preincubation test with and without S9-mix: 0; 20; 100; 500; 2500 and 5000 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: the test substance was soluble in water

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

other: the Ames test is regularly conducted in the testing facility

Positive controls:

yes

Positive control substance:

other: with S-9 mix: all strains 2.5 µg 2-aminoanthracene; without S-9 mix: strains TA100, TA1535: 5 µg N-methyl-N'-nitro-N-nitrosoguanidine; TA98: 10 µg 4-nitro-o-phenylendiamine; TA1537: 100 µg 9-aminoacridine.

Remarks:

DMSO was used as vehicle for all positive control substances

Details on test system and experimental conditions:

1) FIRST EXPERIMENT

METHOD OF APPLICATION: standard plate test

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

1) SECOND EXPERIMENT
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; the titer was determined only in the experiments with S-9 mix both without test substance (vehicle only) and after adding the two highest amounts of substance and in the presence of histidine.

Evaluation criteria:

The test chemical is considered positive in this assay if the following criteria are met: a dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system.

Statistics:

Mean revertants per plate were calculated.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

An increase in the number of his revertants was not observed both in the standard plate test and in the preincubation test either without S-9 mix or after the addition of a metabolizing system.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

No bacteriotoxic effect (reduced his background growth) was observed.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No test substance precipitation was found.

Remarks on result:

other: all strains/cell types tested

First experiment: Standard plate test

Dose (µg/plate)	TA1535		TA100		TA1537		TA98
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
0	15±3	16±4	120±15	124±4	8±1	10±2	25±4
20	17±3	17±4	130±6	134±24	5±1	11±2	25±3	35±2
100	14±3	15±2	127±14	144±3	8±1	10±2	23±1	41±3
500	15±2	15±3	120±13	139±8	8±3	9±3	23±2	34±25
2500	13±2	17±3	114±7	115±58	7±3	9±0	25±3	35±2
5000	14±3	16±4	128±9	114±15	7±1	18±5	34±4	36±7
2 -AA	-	145±30	-	737±64	-	104±81	-	726±28
MNNG	785±18	-	786±91	-	-	-	-	-
AAC	-	-	-	-	579±32	-	-	-
NOPD	-	-	-	-	-	-	1100±101	-
ENNG	-	-	-	-	-	-	-	-

Mean ± SD

Second experiment: Preincubation-test

Dose (µg/plate)	TA1535		TA100		TA1537		TA98
500	13±1	15±3	97±6	132±18	7±3	9±1	22±6	33±6
2500	15±3	16±3	107±12	126±17	9±2	9±3	23±4	29±5
5000	13±1	16±2	111±15	120±14	6±2	10±2	20±2	26±5
2-AA	-	110±9	-	646±34	-	107±9	-	658±51
MNNG	711±12	-	741±65	-	-	-	-	-
AAC	-	-	-	-	653±189	-	-	-
NOPD	-	-	-	-	-	-	+69±43	-
ENNG	-	-	-	-	-	-	-	-

Mean ± SD

2-AA: 2-aminoanthracene

MNNG; N-methyl-N-nitro-N-nitrosoguanidine

ENNG; N-ethyl-N-nitro-N-nitrosoguanidine

NOPD: 4-nitro-o-phenylendiamine

AAC: 9-aminoacridine chloride monohydrate

Conclusions:

The test substance demonstrated to be negative in the Ames/Salmonella Plate Incorporation Assay under the conditions of the test, and according to the criteria of the protocol.

Reference 4

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990-07-25 - 1990-08-31

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The study is not a standard genetic toxicity endpoint study.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): 6398-24-20
- Substance type: clear colorless liquid
- Physical state: liquid
- Purity: responsibility of the sponsor; recorded in the sponsor's file
- Storage condition of test material: the substance was stored at room temperature in the container received from the sponsor
- Stability: no apparent change in the physical state during storage

Species / strain / cell type:

hepatocytes: rat hepatocytes in primary culture from fisher 344 rats

Details on mammalian cell type (if applicable):

- Type and identity of media: Williams' Medium E (WME) supplemented with 10% v/v calf serum
- Properly maintained: yes

Metabolic activation:

not applicable

Test concentrations with justification for top dose:

0.5, 5, 25, 50, 100, 500, 750, 1000, 2500 and 5000 ug/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Williams' Medium E

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-acetamidofluorene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Cell exposure: 1 x 1E05 viable hepatocytes were inoculated into 12 well cluster dishes containing 15 mm diameter Thermanox plastic coverslips in WME containing 10% calf serum. The hepatocytes were allowed to attach for approximately 2 hours in a 37°C CO2 incubator. The cultures were rinsed and serum-free medium containing test compound and 10 uC/mL of 3H-thymidine. After exposure, the cultures were washed three times with 3 mL volumes of phosphate buffered saline by aspiration.
- Cell fixation: The cells on coverslips were swelled in 1% sodium citrate for 10-15 minutes and fixed in three 10-minute changes of 100% ethanol:glacial acetic acid (3:1). The fixed cultures were then washed twice with approximately 2 mL volumes of dH2O. The coverslips were air dried and mounted cell surface up on glass slides with permount. Slides were dipped in NTB-2 photographic emulsion in the dark, allowed to dry overnight and stored at 4°C in light-proof slide boxes containing desiccant for one week.
- Staining: After seven days of exposure time, autoradiographs were developed in D19 at approximately 15°C for 4 minutes, washed in deionized water with 5 mL glacial acetic acid for 30 seconds, immersed in Fixer for 10 minutes, washed in running tap water for 5 minutes, dried, stained in Harris Alum hematoxylin followed by a dip rinse in acid alcohol, rinsing in running tap water for 2-5 minutes and a dip rinse in ammonium water. The slides were then rinsed in running tap water for 2-5 minutes, dipped in 70% ethyl alcohol, followed by a 10-60 second dip in eosin solution. The slides were then rinsed in 3 separate baths of 95% ethyl alcohol for 2 minute intervals, followed by rinsing in 3 separate baths of 100% ethyl alcohol for 2 minute intervals. The slides were air dried, and coverslipped in permount. Excess emulsion was scrapped off.

NUMBER OF REPLICATIONS: triplicate

Evaluation criteria:

see below

Key result

Species / strain:

hepatocytes: rat hepatocytes in primary culture from fisher 344 rats

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

the dose levels of 100 ug/mL to 5000 ug/mL were determined to be cytotoxic and were not evaluated

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Net nuclear grain counts represent the difference between the nuclear count and the highest of three cytoplasmic grain counts. Each treatment is representative of triplicate cultures. Non of the treated cultures produced mean net nuclear grain counts that were substantially greater than the untreated control (WME). The negative and positive control values were within the acceptable range of the mean historical data.

Remarks on result:

other: all strains/cell types tested

Conclusions:

The test substance was considered negative in inducing unscheduled DNA repair in the primary hepatocytes under the experimental conditions of this assay.

Reference 5

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of 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:

guideline study with acceptable restrictions

Remarks:

read-across from related substance

Justification for type of information:

Read-across from structural analogue (4-ethylmorpholine). Justification for this read-across approach is included in section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

other: CHL/IU cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

observed at 1200 µg/mL after 24 hr exposure without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Based on read-across from structural analogue

Remarks:

no adverse effect is expected

Conclusions:

No reliable study on chromosome aberration potential of the target substance is available. Data generated with the related substance N-ethylmorpholine is used for endppoint coverage.
N-ethylmorpholine did not induce chromosomal aberrations in CHL/IU cells, with or without an exogenous metabolic activation system.
The same is assumed to be correct for the target substance.

Reference 6

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

Read-across GLP study performed according to OECD Guideline 473. The original study report is in Japanese language, although the figures and tables are in English. An English summary is available from the Japanese authorities and an extensive summary is present in the OECD HPV program files.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Guidelines for Screening Mutagenicity Testing of Chemicals (Chemical Substance Control Law of Japan)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Name of test material (as cited in study report): N-ethylmorpholine
- Molecular formula (if other than submission substance): C6 H13 N O
- Molecular weight (if other than submission substance): 115.18
- Smiles notation (if other than submission substance): CCN1CCOCC1
- InChl (if other than submission substance): InChI=1/C6H13NO/c1-2-7-3-5-8-6-4-7/h2-6H2,1H3
- Analytical purity: 99.84%
- Lot/batch No.: 2901P0
- Storage condition of test material: The solution was kept in the dark at 4 degree C before use. The solution was proved to be stable.

Species / strain / cell type:

mammalian cell line, other: CHL/IU cells

Details on mammalian cell type (if applicable):

no data

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9: rat liver induced with phenobarbital and 5,6-benzoflavone, which was purchased from Kikkoman Corp

Test concentrations with justification for top dose:

0, 150, 300, 600, 1200 ug/mL
The doses were based on the results of a cell growth inhibition test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: saline

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 1-methyl-3-nitro-1-nitrosoguanidine 2.5 ug/mL

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9 mix 10 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 6 hours (with and without S9) or 24 hours (without S9)

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: cell growth inhibition

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
Number of examined cells, type and number of abnormal cells, and number of polyploid cells were obtained for every dose group.
- Determination of endoreplication: no data

Statistics:

Multi-sample chi-square test was done and then Fischer's exact test was done (p<0.05, 0.01).

Key result

Species / strain:

other: CHL/IU cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

observed at 1200 ug/mL after 24 hr exposure without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

With the 6 hr short-term treatment, chromosomal aberrations or polyploidy were not induced, with or without S9 mix. Moreover, chromosomal aberrations or polyploidy were not induced after the 24 hr continuous treatment without S9 mix.
Cytotoxicity was not observed for 6 hr short-term treatment, with or without S9 mix, however it was observed at 1200 ug/mL for 24 hr continuous treatment without S9 mix.
There were no problems of the responses at each control group.

Conclusions:

N-ethylmorpholine did not induce chromosomal aberrations in CHL/IU cells, with or without an exogenous metabolic activation system.

Reference 7

Endpoint:

in vitro gene mutation study in mammalian cells

Type of 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:

comparable to guideline study with acceptable restrictions

Remarks:

read-across from structural analogue

Justification for type of information:

Based on read-across from structural analogue. A justification for this read-across approach is included in section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Based on read-across from structural analogue no adverse effect is expected

Conclusions:

No reliable in vitro gene mutation study in mammalian cells with the target substance is available. Data generated with the related substance N-ethylmorpholine is used for endpoint coverage.
An in vitro gene mutation study in mammalian cells is performed with N-ethylmorpholine. In the absence of S9, the mutant frequency in the treated cultures was not significantly altered from the negative control values, not even at the highly toxic dose of 5000 nl/ml. In the presence of S9, the mutant frequencies in the assayed cultures remained similar to the negative controls, despite the greater toxicity observed at concentration exceeding 1250 nl/ml. Any metabolic products formed in the presence of S9 were not detectably mutagenic over a wide range of toxic action.
The same is assumed to correct for the target substance.

Reference 8

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1980-09-08 - 1980-09-26

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study performed to a method similar to OECD TG476 without significant deviations. The spacing between selected concentrations is too large in some cases, especially in the case of the lowest dose and the next lowest dose in the absence of S9.n

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

spacing between selected concentrations in some cases too large

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): 4236-30-5
- Molecular formula (if other than submission substance): C6 H13 N O
- Molecular weight (if other than submission substance): 115.18
- Smiles notation (if other than submission substance): CCN1CCOCC1
- InChl (if other than submission substance): InChI=1/C6H13NO/c1-2-7-3-5-8-6-4-7/h2-6H2,1H3

- Substance type: clear pale yellow liquid
- Physical state: liquid
- Analytical purity: no data
- Impurities (identity and concentrations): no data
- Lot/batch No.: 4236-30-5
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data
- Storage condition of test material: no data

Target gene:

thymidine kinase (TK) locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Fischer's mouse leukemia medium supplemented with L-glutamine, sodium pyruvate and 10% v/v horse serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

Without S9: 78, 2500, 3000, 4000, 5000 nl/ml
With S9: 78, 156, 1250, 2000, 2500 nl/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

0.5 µl/ml without S9

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Remarks:

0.3 µl/ml with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium (cloning medium: growth medium + 0.35% agar)

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

SELECTION AGENT (mutation assays): 3 µg/ml TFT

NUMBER OF REPLICATIONS: single; solvent control in duplo

NUMBER OF CELLS EVALUATED: 3x10^6 cells

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

The minimum condition considered necessary to demonstrate mutagenesis for any given treatment is a mutant frequency that exceeds 150% of the concurrent background frequency by at least 10 x 10^-6. The background frequency is defined as the average mutant frequency of the solvent and untreated negative controls.
The observation of a mutant frequency that meets the minimum criterion for a single treated culture within a range of assayed concentrations is not sufficient evidence to evaluate a test material as a mutagen. The following test results must be obtained to reach this conclusion for either activation or nonactivation conditions:
- a dose-related or toxicity-related increase in mutant frequency should be observed, preferably for at least 3 doses.
- an increase in mutant frequency may be followed by only small or no further increases at higher concentrations or toxicities. However a decrease in mutant frequencies to values below the minimum criterion is not acceptable in a single assay for classifying the test material as a mutagen. If the mutagenic activity at lower concentrations or toxicities was large, a repeat assay will be performed to confirm the mutagenic activity.
- if an increase of about two times the minimum criterion or greater is observed for a single dose near the highest testable toxicity,the test material will be considered mutagenic. Smaller increases at a single dose near the highest testable toxicity will require confirmation by a repeat assay.

Statistics:

No

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

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: test material was not toxic to 24h cell growth at doses 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.

COMPARISON WITH HISTORICAL CONTROL DATA: no data

ADDITIONAL INFORMATION ON CYTOTOXICITY: concentrations up to 5000 nl/ml without S9 and 2500 nl/ml with S9 became highly toxic (but without causing a dose-related increase in mutant frequency).

Conclusions:

An in vitro gene mutation study in mammalian cells is performed with N-ethylmorpholine. In the absence of S9, the mutant frequency in the treated cultures was not significantly altered from the negative control values, not even at the highly toxic dose of 5000 nl/ml. In the presence of S9, the mutant frequencies in the assayed cultures remained similar to the negative controls, despite the greater toxicity observed at concentration exceeding 1250 nl/ml. Any metabolic products formed in the presence of S9 were not detectably mutagenic over a wide range of toxic action.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No in vivo genetic toxicity study is available.

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial reverse mutation assay:

Stankowski (1990) performed an Ames (plate incorporation assay) test with S typhimurium strains TA1535, TA1537, TA98, TA100 and TA1538 with and without metabolic activation.

Following test concentrations were applied: 167, 500, 1670, 5000, 7500 and 10000 µg/plate (in triplicate). Solvent control, negative control and positive controls were run in triplicate. There were no observed increases in mutation frequency with and without metabolic activation at the tested dose levels. Solvent, negative and positive controls were valid. The substance was not toxic up to 5000 ug/plate. This study is selected as key study. Two K2 supporting studies confirmed the negative mutagenic results with and without metabolic activation in the same S. typhimurium strains (Billhimer, 1978 and BASF, 1992). S. typhimurium strain TA1538 has not been tested by BASF (1992).

 

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro:

San Sebastian JR (1990) studied the gene mutation potential in rat hepatocytes (rat hepatocytes in primary culture from fisher 344 rats) without metabolic activation. Following doses were evaluated in triplicate: 0.5, 5, 25, 50, 100, 500, 750, 1000, 2500 and 5000 µg/mL. A solvent control (Williams' Medium E) and a positive control (2-acetamidofluorene) were scored as well. Under the conditions of this study, the test substance was not genotoxic in the hepatocyte primary culture/DNA repair test. Net nuclear grain counts represent the difference between the nuclear count and the highest of three cytoplasmic grain counts. None of the treated cultures produced mean net nuclear grain counts that were substantially greater than the untreated control (WME). The negative and positive control values were within the acceptable range of the mean historical data.

 

Cytogenicity test:

Data generated with the related substance N-ethylmorpholine is used for endpoint coverage in a read-across approach. A chromosome aberration test was performed with N-ethylmorpholine (NEM) with the mammalian cell line CHL/IU with and without metabolic activation. Following doses were tested in duplicate: 0, 150, 300, 600 and 1200 µg/m for 6 hours (with and without S9) or 24 hours (without S9). With the 6 hr short-term treatment, chromosomal aberrations or polyploidy were not induced, with or without S9 mix. Moreover, chromosomal aberrations or polyploidy were not induced after the 24 hr continuous treatment without S9 mix. Cytotoxicity was not observed for 6 hr short-term treatment, with or without S9 mix, however it was observed at 1200 ug/mL for 24 hr continuous treatment without S9 mix. There were no problems observed in the control groups.

 

Mammalian cell gene mutation assay:

Data generated with the related substance N-ethylmorpholine is used for endpoint coverage in a read-across approach.

Myhr (1980) performed a mouse lymphoma test with N-ethylmorpholine in L5178Y cells with and without metabolic activation. Following doses were tested: without S9: 78, 2500, 3000, 4000, 5000 nL/mL; with S9: 78, 156, 1250, 2000 and 2500 nL/mL. In the absence of S9, the mutant frequency in the treated cultures was not significantly altered from the negative control values, not even at the highly toxic dose of 5000 nl/ml. In the presence of S9, the mutant frequencies in the assayed cultures remained similar to the negative controls, despite the greater toxicity observed at a concentration exceeding 1250 nl/ml. Any metabolic products formed in the presence of S9 were not detectably mutagenic over a wide range of toxic action.

 

The justification for read-across within this endpoint can be found in section 13.

Justification for classification or non-classification

Based on the available data and according to the criteria of the CLP Regulation (EC) 1272/2008, NMM should not be classified for mutagenicity.