4-methylmorpholine 4-oxide, monohydrate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro:

Bacterial reverse mutation assay: The study is performed according to a method similar to OECD Guideline 471 in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA (Kato M, 1993). According to the results of the study, NMMO is not mutagenic in the Ames test with and without metabolic activation.

In an additional bacterial reverse mutation assay (Johnson FM, 1976), NMMO was not mutagenic with or without metabolic activation. However, in this assay only two relevant strains recommended in OECD Guideline 471 were used (S. typhimurium TA 1535 and TA 1537).

Chromosome aberration test:

No in vitro chromosome aberration test is available for NMMO, however an in vivo mammalian micronucleus test according to OECD Guideline 474 is available.

Mammalian cell gene mutation test:

The study is performed according to a method similar to OECD Guideline 490 in mouse lymphoma L5178Y cells (Cifone MA, 1981). The results of Mouse lymphoma forward mutation assay indicate that, under the conditions of this study, NMMO was concluded to be negative with and without metabolic activation.

Genome mutation:

The study is performed according to the method of Kakunaga ("Quantitative system for assay of malignant transformation by chemical carcinogens using a clone derived from BALB/3T3". Int. J. Cancer 12: 463-473, 1973). The results of the in vitro mammalian cell BALB/3T3 transformation assay indicate that, under the conditions of this study, NMMO was concluded to be negative (Rundell JO, 1981).

DNA damage and/or repair:

The test is performed in a rat hepatocyte primary culture of adult male F344 rats according to a method similar to OECD Guideline 482 (Tong C, 1982). The results of the hepatocyte primary culture/DNA Repair Assay indicate that, under the conditions of this study, NMMO was concluded to be negative.

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:

From April 14, 1981 to May 22, 1981

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 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

Positive controls are wrongly chosen. No colony sizing performed on negative and positive controls.

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): 4236-40-5 Order No. J-64
- Substance type: clear, nearly-colorless liquid
- Physical state: liquid

Target gene:

thymidine kinase

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).
- Properly maintained: yes
- Stocks are maintained in liquid nitrogen and laboratory cultures are periodically checked for the absence of mycoplasma contamination by culturing methods.
- 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 growth medium for three or more days before use.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced Fischer 344 or SD adult male rat liver S9 homogenate

Test concentrations with justification for top dose:

Dose range finding:
nonactivation and activation assay: 0.625, 1.250, 2.500, 5.000 and 10.000 uL/mL
Final test:
nonactivation and activation assay: 6.0, 8.0 and 10.0 µL/mL of culture medium

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The substance was miscible with deionized water up to 10µl/ml. Therefore, just prior to use, stock solutions were prepared by performing serial dilutions of the test material in water. The stock solutions were then diluted 1:10 into tubes of culture medium containing the cells to initiate the treatments.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

0.5 µL/mL (nonactivation assay)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Remarks:

0.3 µL/mL (activation assay)

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 (mostly 2 days)
- Selection time (if incubation with a selection agent): 10 days of incubation

SELECTION AGENT (mutation assays): Selection medium is cloning medium containing 100 µg/mL of BrdU or 3 µg/mL of TFT. Cloning medium consists of growth medium with the addition of agar to a final concentration of 0.35% to achieve a semisolid state.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

NUMBER OF CELLS EVALUATED
3 x 10^6 cells are seeded in selection medium and after 10 days mutant colonies are counted.

Evaluation criteria:

See field 'Any other information on materials and methods incl. tables.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

treatments up to a highly concentrated 10 µL/mL were moderately toxic with and without S9 microsomal activation

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

First trial (without S9): 0.00061 - 10 µl/ml tested. Test substance was non-toxic to moderately toxic, but not dose-related. Test substance was negative in this trial.
First trial (with S9): 0.0061 - 10 µl/ml tested. Test substance was highly toxic. No mutant analysis possible. Instead of Fisher 344 rat derived S9, which was used in the first trial, an S9 mix derived from SD rats was used in the next trials.
Second trial (without S9): 3 - 10 µl/ml tested. Still only moderate toxicity observed. Test substance was also negative in this trial.
Second trial (with S9): 3 - 10 µl/ml tested. Less toxicity observed than when tested without S9. Test substance was negative in this trial (only one slightly positive response).
Third trial (with S9): 3 - 10 µl/ml tested. Still nontoxic to moderate toxicity. Test substance was negative in this trial.

Percent relative growth (relative to controls) ranged from 62.7 to 106.6% in the activated trial, and from 26.8 to 77.6% in the non-activated trial. When compared to the untreated cultures, NMMO failed to induce a statistically significant number of mutant colonies in both the activated and non-activated trials. Cell survival and observed mutational frequencies, for both positive and negative controls in activated and non activated trials, fell within acceptable ranges for this assay based on historical data for this cell line and laboratory. NMMO is considered to be negative, or non-mutagenic, in this assay.

Conclusions:

The substance did not induce repeatable increases in the mutant frequency at the TK locus in L5178Y mouse lymphoma cells. Treatments up to the highly concentrated 10 µL/mL were only moderately toxic with and without S9 microsomal activation and did not induce significant increases in the mutant frequency. The substance was therefore considered to be inactive under conditions of moderate toxicity in the mouse lymphoma forward mutation assay.

Executive summary:

In a mammalian cell gene mutation assay conducted similar to OECD guideline 490, L5178Y mouse lymphoma cells cultured in vitro were exposed to 4-methylmorpholine 4-oxide (NMMO), dissolved in distilled water at concentrations of 6.0, 8.0 and 10.0 µl/mL in the absence and presence of mammalian metabolic activation.

NMMO was tested up to limit concentrations.

When compared to the untreated cultures, NMMO failed to induce a statistically significant number of mutant colonies in both the activated and non-activated trials. Cell survival and observed mutational frequencies, for both positive and negative controls in activated and non activated trials, fell within acceptable ranges for this assay based on historical data for this cell line and laboratory and therefore did induce the appropriate response.

There was no evidence or a concentration related positive response of induced mutant colonies over background.

NMMO is considered to be negative, or non-mutagenic, in this assay.

This study is classified as acceptable and satisfies the requirement for Test Guideline OECD 490 for in vitro mutagenicity (mammalian forward gene mutation) data.

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:

From April 20, 1993 to May 12, 1993

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:

duplicate plating not scientifically justified

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): N-methylmorpholine oxide, or NMMO
- Physical state: liquid
- Analytical purity: 60%
- Impurities (identity and concentrations): water 39%, N-methylmorpholine 1%
- Lot No.: 101-2-0692
- Stability under test conditions: stable
- Other: solubility: soluble in water more than 5%; molecular weight 117; boiling point 118.5°C

Target gene:

histidine (S. typhimurium) and tryptophan (E. coli)

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and 5,6-benzoflavone induced liver homogenate S9 fraction from male Sprague-Dawley rats

Test concentrations with justification for top dose:

313, 625, 1250, 2500, 5000 µg/plate

Vehicle / solvent:

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

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

furylfuramide

Remarks:

without S9 mix Migrated to IUCLID6: 0.01 µg/plate for TA100 and WP2uvrA, 0.1 µg/plate for TA98

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 mix Migrated to IUCLID6: 0.5 µg/plate for TA1535

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix Migrated to IUCLID6: 80 µg/plate for TA1537

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9 mix Migrated to IUCLID6: 5 µg/plate for TA98, TA100 and TA1537

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene: 2 µg/plate for TA1535; 10µg/plate for WP2uvrA

Remarks:

with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation method

0.1 mL of the test substance was placed in glass tube. Then 0.1 mL of the sub-culture, and 0.5 mL of S9 mix or 0.5 mL of 0.1M sodium phosphate buffer pH 7.4 was added to the test substance and incubated at 37°C for 20 minutes. The suspension was then thoroughly mixed with 2.0 mL of the top agar and overlaid onto the minimum glucose agar plate. After 48 hours' incubation at 37°C, revertant colonies were counted. Negative and positive controls were studied simultaneously. Test was taken on the two plates per concentration. Toxicity of the test substance was observed with a stereoscopic microscope.

NUMBER OF REPLICATIONS: duplicate cultures

Evaluation criteria:

The numbers of revertant colonies of all test substance groups were compared with those obtained from both negative control groups. The test substance was considered as positive if a reproducibility produced, a dose related and more than 2 fold increase in the number of revertant colonies occurred at the negative control value.

Key result

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

Positive controls validity:

valid

Key result

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

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

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

Key result

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

Positive controls validity:

valid

Key result

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

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS: not detected

RANGE-FINDING/SCREENING STUDIES: performed with all 5 strains with and without S9 mix at concentrations: 10, 50, 100, 500, 1000, 5000 µg/plate. Also positive controls and solvent control were tested. Result at all 5 strains: non-mutagenic. No cytotoxicity observed.

Conclusions:

Based on the present Ames test It is concluded that the substance is considered as negative for mutagenic potential with and without metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria performed equivalent/similar to OECD 471, four histidine-requiring strains (TA98, TA100, TA1535, TA1537) of Salmonella typhimurium and E. coli (WP2 uvr A), were exposed to 4-methylmorpholine 4-oxide (60%) at final concentrations of the test item of 313, 625, 1250, 2500 and 5000 µg/plate, both in the absence and in the presence of metabolic activation using a phenobarbital and 5,6-benzoflavone-induced rat liver post-mitochondrial fraction (S-9). All test item treatments in this study were performed using formulations prepared in water.

Vehicle and positive control treatments were included for all strains in both experiments.

Following these treatments, neither an evidence of genotoxicity nor of cytotoxicity was observed.

The mean numbers of revertant colonies fell within acceptable ranges for vehicle control treatments, and were within historical control range of the positive control treatments.

It was concluded that the test item did not induce mutation in four histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium or E. coli WP2 uvr A when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 µg/plate (the maximum recommended concentration according to current regulatory guidelines),in the absence and in the presence of a rat liver metabolic activation system (S-9).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Genetic toxicity in vivo:

DNA damage and/or repair: according to a method similar to OECD Guideline 486 in young adult Fischer F344 rats. The results of the in vivo hepatocyte primary culture/DNA repair test indicate that, under the conditions of this study, NMMO was concluded to be negative (SanSebastian JR, 1989).

Micronucleus assay in vivo:

In vivo micronucleus assay in ICR mice according to OECD Guideline 474. The substance was observed to be negative for genetic toxicity in vivo (Shambhu R, 2012).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 6, 1989 to October 18, 1989

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 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Deviations:

yes

Remarks:

No exposure time of 12-16 h was taken into account.

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Specific details on test material used for the study:

- Name of test material (as cited in study report): 89-009-1
- Substance type: Light yellow liquid
- Physical state: Liquid
- Purity: responsibility of the sponsor and recorded in the sponsor's file
- Stability: responsibility of the sponsor and recorded in the sponsor's file
- Storage condition of test material: At ambient temperature
- Other: specific gravity 1.1359 g/ml

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Kingston, NY
- Age at study initiation: twelve week old young adult Fischer 344 rats
- Weight at study initiation: 194-244 grams
- Fasting before study
- Housing: Animals were housed in accordance with the "Guide for the Care and Use of Laboratory Animals"
- Acclimation period: 20 days prior to initiation of the assay

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C ± 3°C
- Humidity (%): 30 to 70%
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: distilled water
- Amount of vehicle: 10 ml/kg

Details on exposure:

Dilutions were made just prior to use.

Duration of treatment / exposure:

3 hours

Frequency of treatment:

Single dose

Post exposure period:

Not applicable

Dose / conc.:

920 mg/kg bw/day (nominal)

Dose / conc.:

2 300 mg/kg bw/day (nominal)

Dose / conc.:

4 600 mg/kg bw/day (nominal)

Dose / conc.:

9 200 mg/kg bw/day (nominal)

No. of animals per sex per dose:

3

Control animals:

yes, concurrent vehicle

Positive control(s):

Dimethylnitrosamine, via oral gavage at 10 mg/kg.

Tissues and cell types examined:

The liver was excised and examined.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Sponsor recommended that the high dose be 1.0 x LD50 in rats (9200 mg/kg) as an estimate of the maximum tolerated dose. Three lower doses were also evaluated at 920, 2300 and 4600 mg/kg as well as the solvent control, dH2O and the positive control, DMN, at 10 mg/kg.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Eighteen male Fischer 344 rats were anesthetized with 50 mg/kg of sodium pentobarbital by intraperitoneal injection. The liver was excised and placed in 50 mL of warm serumless WME in a sterile culture dish. The liver was trimmed of fat, excess connective tissue and any sections of liver that still showed signs of blood. The liver was then transferred to a sterile (100 x 25 mm) culture dish containing 50 mL of warm Medium B. The liver was held by the connective tissue in the porta hepatis and the capsular membrane of the liver was opened and removed at several points on the dorsal surface with the aid of a scalpel and forceps. Cells were detached by gently combing the liver with a 3/4" camel's hair brush. Detachment was complete when only fibrous tissue remained. The cells were then aliquotted into a 50 mL centrifuge tube by pipetting gently with a sterile 10 mL polystyrene pipet. The volume of the tube was adjusted to 35-40 mL with WME supplemented with 10% v/v calf serum. The tube was allowed to stand in a vertical position for 10 minutes to flocculate the hepatocytes. Viability of the resuspended hepatocytes was measured by trypan blue dye exclusion.

DETAILS OF SLIDE PREPARATION: 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 2hours in a 37°C incubator. The cultures were rinsed with serum-free medium and refed with WME containing 10 uC/mL of 3H-thymidine and incubated. Four hours after exposure, the cultures were washed once with 2 mL WME and incubated overnight in 3 mL of WME supplemented with 0.25 mM unlabeled thymidine. Fourteen to sixteen hours after incubation, 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, the 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 and dried. They were then stained in Harris Alum hematoxylin followed by a dip rinse in acid alcohol, rinsed 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 acohol 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 scraped off.

METHOD OF ANALYSIS: Unscheduled DNA repair synthesis, evidenced by a net increase in black silver grains over the nucleus, is quantified by determining nuclear and cytoplasmic grain counts using an Artek 880 automated colony counter with microscopic/video camera attachment interfaced to an Apple II computer for data acquisition. A total of 450 cells/dose point (50 cells/coverslip, 3 coverslips/rat and 3 rats/dose point) were counted for autoradiographic UDS determination. Slides were coded before being counted. A correction coefficient was calculated by th following method. An area/grain ratio was obtained by visually scoring an area of each slide containing 3-5 nuclear grains and then obtaining an object area count with Artek Model 880 Colony Counter. Five such areas were scored, the differences summed and the mean obtained. This value serves as a correction coefficient. The cytoplasimc grain count was quantitated by randomly selecting the highest of three nuclear-sized areas adjacent to each nucleus. This value was subtracted from the uncorrected nuclear grain count to determine the net nuclear grain count. Both nuclei of binucleated cells were recorded separately. Replicative DNA synthesis was evidenced by nuclei blackened with grains too numerous to count. The data of the HPC/DNA Repair Assay are reported as mean grains/nucleus from the triplicate wells. The solvent control should have a net nuclear grain count of < or = 0 or the study will be repeated. Also the positive control, DMN, should yield a mean net nuclear grain count > or = 5.

Evaluation criteria:

The test article is reported positive when the minimum net grain count of 5 per nuclei is consistently observed in triplicate wells. Where possible a dose response profile should be observed. When an adequate dose response is not attained, the chemical may be classified as a "suspect" genotoxicant. To resolve the genotoxic potential of the chemical, the sponsor may choose to initiate a second experiment with dose levels closely bracketing the positive response which resulted in classifying the chemical as a "suspect" genotoxicant.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

None of the treated cultures produced mean net nuclear grain counts that were substantially greater than the solvent control. The positive control DMN gave a mean net nuclear grain count greater than the solvent control.

Conclusions:

The substance was negative in inducing unscheduled DNA synthesis in the liver of Fischer 344 rats in vivo at doses of 920, 2300, 4600 and 9200 mg/kg.

Executive summary:

In an in vivo unscheduled DNA synthesis assay in the liver, performed equivalent/similar to OECD TG 486, 4-methylmorpholine 4-oxide in distilled water was administered to Fischer 344 rats as a single dose via oral gavage at dose levels of 920, 2300, 4600 and 9200 mg/kg bw for 3 hours.

The test material was tested up to doses exceeding the limit dose of 2000 mg/kg bw.

The positive control dimethylnitrosamine (DMN at 10 mg/kg bw) gave a mean net nuclear grain count greater than the solvent control and therefore induced the appropriate response.

None of the treated cultures produced mean net nuclear grain counts that were substantially greater than the solvent control.

There was no evidence that unscheduled DNA synthesis as determined by radioactive tracer procedures was induced.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 28/02/2012 to 29/03/2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): NMMO (N-methyl morpholine oxide 50% solution)
- Substance type: light-yellow clear liquid
- Physical state: liquid
- Other: At BioReliance the substance was assigned the code number AD41ZH

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan, Frederick, MD
- Age at study initiation: 6 or 8 weeks old
- Weight at study initiation: dose range finding study: male mice: 29.5 - 34.9 g, female mice: 27.7 - 28.9 g; definitive micronucleus study: male mice: 32.2 - 35.8 g, female mice: 24.2 - 30.6 g
- Assigned to test groups randomly: yes: based on equalization of group mean body weights. At the time of randomization, the weight variation of animals did not exceed ± 20% from their group mean. Following randomization, the weight variation of animals did not exceed ± 20% from their group mean. Following randomization, animals were identified by sequentially numbered ear tags assigned to each animal during randomization process. The cage card contained the following information: the animal number, sex, study number, test article identification, treatment group number, dose level and route of administration. Cage cards were color coded as a function of treatment group.
- Fasting period before study: no
- Housing: mice of the same sex were housed up to five per rodent Micro-Barrier cage. Cages were placed on the racks equipped with an automatic watering system and Micro-VENT full ventilation, HEPA filtered system. Heat-treated Sani-Chip hardwood chips were used for bedding to absorb liquids.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 to 13 days after receipt

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): 10 changes of fresh HEPA-filtered air every hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Route of administration:

oral: gavage

Vehicle:

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

Details on exposure:

The test substance dose formulations were prepared fresh for each phase of the study prior to dose administration. The formulation (100 mg/mL) for the DRF study and all formulations (25, 50 and 100 mg/mL) for definitive study were prepared as follows:
- An appropriate amount of the test substance was weighed for each concentration separately
- An appropriate volume of the vehicle, 80% of the final volume was added to the respective containers
- Each formulation was vortexed for 2 minutes
- Remaining volume of the vehicle was added to achieve the final volume

All formulations appeared as clear solutions.

Duration of treatment / exposure:

No data

Frequency of treatment:

Single exposure

Post exposure period:

Dose Range Finding Study
In the dose range finding study, five male and five female mice were epxosed to NMMO at 2000 mg/kg. Mice were observed after dose administration and daily thereafter for 3 days for clinical signs of toxicity. Body weights were re

In-life evaluations:
Mice were observed after dose administration and throughout the course of the study for clinical signs of toxicity, which were recorded.

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

dose range finding study

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Vehicle control: 10
500 mg/kg: 5
1000 mg/kg: 5
2000 mg/kg: 10
Positive control: CP (50 mg/kg): 5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s):
- Route of administration: single oral gavage
- Doses / concentrations: 50 mg/kg, an aqueous dosing formulation of CP, at a concentration of 2.5 mg/mL was prepared fresh on the day of dose administration. An appropriate amount of CP was dissolved in appropriate volume of sterile water for injection.

Tissues and cell types examined:

Scoring for micronuclei (bone marrow evaluation):
To control for bias, bone marrow slides were coded using a random number table by an individual not involved with the scoring process. Using a fluorescent microscope and medium magnification (400 X; blue excitation filter in the range of 440-490 nm and barrier filter combination at 520 nm), an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion (1000X), the following cell populations and cellular components were evaluated and enumerated:
- Polychromatic erythrocytes (PCEs)
- Normochromatic erythrocytes (NCEs)
- Micronuclei (M)
- PCEs/ECs ratio

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: In the dose range finding study, five male and five female mice were exposed to NMMO at 2000 mg/kg, the maximum regulatory recommended dose level. In absence of mortality and severe signs of clinical toxicity, a dose of 2000 mg/kg was tested as high dose in the definitive micronucleus study. Two lower doses, one half and one fourth of the high dose, 500 and 1000 mg/kg were also tested.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Bone marrow collection and slide preparation:
At the scheduled bone marrow collection time, five mice per sex per treatment were euthanized by CO2 asphyxiation verified by toe pinch reflex. Immediately following euthanasia, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum.

DETAILS OF SLIDE PREPARATION:
The bone marrow cells were transferred to a labeled centrifuge tube containing approximately 1 mL fetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were re-suspended and a small drop of bone marrow suspension was spread onto a clean glass slide. Slides were labeled with experiment and animal number using led pencil. Two slides were prepared from each mouse. The slides were air dried and fixed in methanol. One set of slides was stained with a nucleic acid-specific stain, acridine orange, and was used in microscopic evaluation. The second set of slides was packaged for storage until the final report was issued.

METHOD OF ANALYSIS: In order to quantify the proliferation state of the bone marrow as an indicator of bone marrow toxicity, the proportion of polychromatic erythrocytes to total erythrocytes was determined for each mouse and treatment group (PCEs/ECs ratio). The proportion of polychromatic erythrocytes to total erythrocytes in test article animals should not be less than 20% of the control value.

Evaluation criteria:

The test substance is considered to induce a positive response if the incidence of micronucleated polychromatic erythrocytes at one or more doses is statistically elevated relative to the vehicle control (p < or = 0.05, Kastenbaum-Bowman tables).
In this study, the test substance is judged negative because no statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in the test substance groups relative to the concurrent negative (vehicle) groups is observed.
However, the results of the statistical analysis are not the only criteria in determination of the test substance potential to increase the incidence of micronucleated PCEs. The following would have been taken in considerations:
- Dose-dependent increase in the incidence of micronucleated PCEs. If there was evidence of a dose response with no evidence of a significant increase in any treated groups relative to the control group, the Sponsor would have been contacted, and possibility of additional testing would have been discussed, or the conclusions would have been made based on the initial results.
- Biological relevance. Values that were statistically significant but did not exceed the range of historical negative or vehicle controls would have been judged as not biologically significant and relevant.
- If criteria for either a positive or negative clastogenic response were not met, the results would have been judged as equivocal.

Statistics:

The incidence of micronucleated polychromatic erythrocytes per 2000 PCEs for each mouse and per 10000 PCEs for each treatment group was determined. Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution. All analyses were performed separately for each sex and sampling time.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

No mortality was observed in any of the treatment groups. All mice in the control substance groups and in NMMO groups at 500 and 1000 mg/kg appeared normal during the study period. Piloerection was observed in both sexes at 2000 mg/kg treatment group.

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Clinical signs of toxicity in test animals: No mortality was observed during the study. Piloerection was observed in male rats while female rats appeared normal during the study period. No appreciable changes in the group mean body weight were observed.

RESULTS OF DEFINITIVE STUDY
- No appreciable reductions in the ratio of polychromatic erythrocytes to total erythrocytes in the test substance groups relative to the respective vehicle control groups were observed, suggesting that NMMO did not markedly inhibit erythropoiesis.
- No statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in test substance groups relative to the respective vehicle control groups was observed in male or female mice at 24 or 48 hours after dose administration (binomial distribution, p>0.05, Kastenbaum-Bowman Tables).
- CP, the positive control, induced a statistically significant increase in the incidence of micronucleated PCEs (p< or =0.05, Kastenbaum-Bowman Tables) in both male and female mice. The number of micronucleated PCEs in the vehicle control groups did not exceed the historical vehicle control range. Based upon this, all criteria for a valid test were met.

Conclusions:

Under the conditions of the study, a single oral administration of NMMO (N-methyl morpholine oxide, 50% solution) at doses up to and including 2000 mg/kg did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow. Therefore, NMMO was concluded to be negative in the micronucleus test using male or female ICR mice.

Executive summary:

In an ICR mouse bone marrow micronucleus assay, Methyl morpholine 4 -oxide was administered to groups of 5 (positive control, 500 and 1000 mg/kg bw test material) to 10 (vehicle control, 2000 mg/kg bw test material) mice once via oral gavage. At study termination bone marrow slides were prepared and scored using a fluorescent microscope.

No mortality was observed during the study. Piloerection was observed in male rats while female rats appeared normal during the study period. No appreciable changes in the group mean body weight were observed.

No appreciable reductions in the ratio of polychromatic erythrocytes to total erythrocytes in the test substance groups relative to the respective purified water vehicle control groups were observed, suggesting that NMMO did not markedly inhibit erythropoiesis. There was no statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in test substance groups relative to the respective vehicle control groups observed in male or female mice at 24 or 48 hours after dose administration. The test material was tested up to the limit dose of 2000 mg/kg bw. Cyclophosphamide (CP), the positive control, induced a statistically significant increase in the incidence of micronucleated PCEs in both male and female mice. The number of micronucleated PCEs in the vehicle control groups did not exceed the historical vehicle control range. Based upon this, all criteria for a valid test were met.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

In the absence of any evidence for species specific effects or modes of action possibly observed effects would be regarded as relevant for humans.

Additional information

Additional information from genetic toxicity in vitro:

Genetic toxicity in vitro:

Bacterial reverse mutation assay:

Kato M(1993) performed an Ames (preincubation method) test with S typhimurium strains TA1535, TA1537, TA98 and TA100 and E. coli strain WP2 uvr A with and without metabolic activation.

Following test concentrations were applied: 313, 625, 1250, 2500, and 5000 µg/plate (in duplicate). Solvent control, negative control and positive controls were run in duplicate. 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 µg/plate. This study is selected as key study. A K2 supporting study confirmed the negative mutagenic results with and without metabolic activation in S. typhimurium strains TA1535, TA1537 and TA1538.

 

Mammalian cell gene mutation assay:

Cifone MA (1981) performed a mouse lymphoma test in L5178Y cells with and without metabolic activation. Following doses were tested: 6.0, 8.0 and 10.0 µL/mL of culture medium. The substance did not induce repeatable increases in the mutant frequency at the TK locus in L5178Y mouse lymphoma cells. Treatments up to the highly concentrated 10 µL/mL were only moderately toxic with and without S9 microsomal activation and did not induce significant increases in the mutant frequency. The substance was therefore considered to be non-mutagenic under conditions of moderate toxicity in the mouse lymphoma forward mutation assay.

 

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

Tong C (1982) studied the gene mutation potential in rat hepatocytes (rat hepatocytes in primary culture from fisher 344 rats) with metabolic activation. Following doses were evaluated in triplicate: 1%, 1E-01%, 1E-02%, 1E-03%, 1E-04% and 1E-05%. A solvent control (WMES (Williams medium E supplemented with 10% calf serum and 50 ug/mL gentamycin)) and a positive control (benzo(a)pyrene) were scored as well. Under the conditions of this study, the test substance was not genotoxic in the hepatocyte primary culture/DNA repair test. The mean net nuclear grain counts of the slides exposed at 1% and at lower concentrations did not exceed 5, therefore the substance was concluded to be not genotoxic to the hepatocytes in this HPC/DNA repair assay.

 

Genome mutation:

Rundell JO (1981) the studied genome mutation potential in BALB/3T3 cells. The concentration range of 52.6 mg/mL to 1.32 mg/mL (52.6, 26.3, 13.2, 6.58 and 1.32 mg/mL), corresponding to a survival range of approximately 20% to near 100% (estimated graphically) was chosen for the assay.

Twenty-four hours prior to treatment, a series of 60 mm dishes is seeded wit 1E04 cells/flask and incubated. At least 20 dishes are then treated for each of the following conditions: five preselected doses of test chemical; positive control; and solvent negative control if applicable. The dishes are incubated for a 24 hours exposure period; the cells are then washed and incubation is continued for approximately four weeks with refeeding twice a week. The assay is terminated by fixing the cell monolayers with methanol and staining with Giemsa. The stained dishes are examined by eye and by microscope to determine the number of foci of transformed cells.

The substance did not induce the appearance of a significant number of transformed foci over the concentration range of 52.6 mg/mL to 1.32 mg/mL. This concentration range correspond to approximately 20% to near 100% survival in the cytotoxicity test. Therefore, the test material is considered to be inactive in the Balb/3T3 In Vitro Transformation Assay.

 

Genetic toxicity in vivo:

DNA damage and/or repair:

San Sebastian JR (1989) performed an in vivo/in vitro hepatocyte primary culture/DNA repair test in male Fischer 344 rats.

Following test concentrations were applied: 920, 2300, 4600 and 9200 mg/kg.

None of the treated cultures produced mean net nuclear grain counts that were substantially greater than the solvent control. The positive control DMN gave a mean net nuclear grain count greater than the solvent control.

 

Micronucleus assay:

Shambhu R (2012) performed a micronucleus assay in ICR mice. Following test concentrations were applied: 500, 1000 or 2000 mg/kg. A concurrent vehicle (purified water) was used as negative control. Cyclophosphamide was used as positive control substance.

Under the conditions of the study, a single oral administration of NMMO (N-methyl morpholine oxide, 50% solution) at doses up to and including 2000 mg/kg did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow. Therefore, NMMO was concluded to be negative in the micronucleus assay using male or female ICR mice.

Justification for classification or non-classification

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