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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1981
Report Date:
1981

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- 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

Method

Target gene:
thymidine kinase
Species / strain
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.
Controlsopen allclose all
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.

Results and discussion

Test results
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 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.

Applicant's summary and conclusion

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 476, L5178Y mouse lymphoma cells cultured in vitro were exposed to NMMO, dissolved in distilled water at concentrations of 6.0, 8.0 and 10.0 µg/mL in the absence and presence of mammalian metabolic activation.

No induction of increases of the mutant frequency was observed.