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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. Chromosome aberration test: No in vitro chromosome aberration test is available for NMMO, however an in vivo chromosome aberration test is available. Mammalian cell gene mutation test: The study is performed according to a method similar to OECD Guideline 476 in mouse lymphoma L5178Y cells. 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, T: 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 transformation assay indicate that, under the conditions of this study, NMMO was concluded to be negative. DNA damage and/or repair: The test is performed according to OECD guideline 482 in a rat hepatocyte primary culture of adult male F344 rats according to a method similar to OECD Guideline 482. The results of the hepatocyte primary culture/DNA Repair Assay indicate that, under the conditions of this study, NMMO was concluded to be negative. 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. Micronucleus assay: in vivo micronucleus assay in ICR mice according to OECD Guideline 474. The substance was observed to be negative for genetic toxicity in vivo.
Link to relevant study records
Reference
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:
other: Well documented GLP study performed according to a method similar to OECD Guideline 476. Positive controls used are specific for HPRT locus, rather than for TK locus. No colony sizing performed on negative and positive controls.
Qualifier:
equivalent or similar to
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
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. 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.
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 boserved 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.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

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.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

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 should not be classified for mutagenicity.