Methyl carbamate

Genetic toxicity: in vitro

Administrative data

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 January 15, 1982 to February 5, 1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to method comparable to OECD Guideline 471.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1982

Report date:

1982

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Ames et al.: Mutation Research 31(6):347-364, 1975

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Methyl carbamate

EC Number:

209-939-2

EC Name:

Methyl carbamate

Cas Number:

598-55-0

Molecular formula:

C2H5NO2

IUPAC Name:

methyl carbamate

Method

Target gene:

TA 1537- uvrB rfa hisC3076 (Frame-shift)
TA 1538- uvrB rfa hisD3052 (Frame-shift)
TA 98- uvrB rfa hisD3052 (Frame-shift)
TA 1535- uvrB rfa hisG46 (Base-pair substitution)
TA 100- uvrB rfa hisG46 (Base-pair and frame shift)
WP-2 uvrA- uvrA tryp (Base-pair substitution)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 6

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S-9

Test concentrations with justification for top dose:

Stock solutions:
0.5, 1.58, 5, 15.8 and 50 mg/mL for plate incorporation tests
0 and 10 mg/mL for disc tests

Tested concentrations
0, 50, 158, 500, 1,581 and 5,000 μg/mL for plate incorporation tests
0 and 1,000 μg/mL for disc tests

Vehicle / solvent:

- Solvent vehicle: Dimethylsulfoxide (DMSO)
- Source: J. T. Baker Chemical Company, Phillipsburg, NJ
- Lot or control number: 115001106 and 020005286

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Controls 2

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Controls 3

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Controls 4

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Plate incorporation method and disc method (as impregnation on paper disc)

PLATE INCORPORATION METHOD
Base layer plates containing agar and minimal media were prepared in advance. On the day of the test, 0.1 mL sample at several dose levels was mixed with 0.1 mL bacterial cells, 0.5 mL S-9 mix or buffer, and trace amounts of histidine, biotin and tryptophan. This mixture was added to 2 mL of molten top agar at 45°C and poured on the base layer and allowed to solidify. Revertant colonies were counted after approximately 48 h incubation at 37°C. Three replicate plates were used for each dose level. For solvent controls, 0.1 mL of the solvent DMSO was used in place of test substance. Plates having less than 30 colonies were counted manually; other plates were counted with a New Brunswick Biotran II colony counter or manually.

DISC TEST
The disc test was performed in the same manner as the plate incorporation test except that the test substances were placed on paper discs as opposed to being incorporated in the top agar. For the solvent, 0.1 mL was placed on one-half inch discs. For the test substance, 0.1 mL of the 10 mg/mL concentration was placed on one-half inch discs. For the positive control mutagen, 10 μl of the appropriate concentration was placed on one-fourth inch discs. One disc was used per plate.

SOURCE:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100: Dr. B. N. Ames, University of California, Berkeley, California.
Escherichia coli WP-2 uvrA: Dr. B. Bridges, MRC Cell Mutation Unit, University of Sussex, Falmar, Brighton, England.

Evaluation criteria:

In the plate incorporation assay, if the mean number of revertants on plates at a given concentration is no more than twice the mean number on the solvent control plates, the test compound is considered non-mutagenic.

In the disc test, the presence of a ring of revertants around the disc indicates the test compound is mutagenic.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 6

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

No mutagenic effect of test substance was observed either in the presence or absence of metabolic activation system by plate incorporation and disc method.

Executive summary:

An in vitro bacterial reverse mutation assay was performed to test the potential of the test substance to cause gene mutation by method comparable to OECD Guideline 471. The assay was performed by 2 methods (plate incorporation and disc test) with 6 bacterial strains, Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538, and Escherichia coli WP-2 uvrA, in the presence and absence of Aroclor 1254-induced rat liver S-9. The test substance was tested at concentrations of 0, 50, 158, 500, 1,581 and 5,000 μg/mL for plate incorporation tests with three replicate plates per concentration and at concentrations of 0 and 1,000 μg/mL for disc tests with one replicate per concentration. Concurrent positive control mutagens and solvent controls were also used. Revertant colonies were counted after approximately 48 h incubation at 37°C. Plates having less than 30 colonies were counted manually; other plates were counted with a New Brunswick Biotran II colony counter or manually. The WP-2 uvrA was contaminated and therefore test was repeated for all strains. No dose level produced the mean number of revertants on plates more than twice the mean number on the solvent control plates in the plate incorporation assay. In addition, no plate containing a disc impregnated with test substance showed a ring of revertants around the disc. Positive control gave positive results, hence meeting validity criteria. Under the study conditions, no mutagenic effect of test substance was observed either in the presence or absence of metabolic activation system by plate incorporation and disc method (Allen JS, 1982).