2-nitrothiophene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1983

Report date:

1983

Materials and methods

Principles of method if other than guideline:

no guideline available

GLP compliance:

no

Type of assay:

other: Base-pair substitution, Fluctuation test and reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-nitrothiophene

EC Number:

210-190-9

EC Name:

2-nitrothiophene

Cas Number:

609-40-5

Molecular formula:

C4H3NO2S

IUPAC Name:

2-nitrothiophene

Test material form:

solid

Details on test material:

Supplier: Aldrich

Method

Species / strain

Species / strain 1

Species / strain / cell type:

other: Klebsiella pneumoniae

Details on mammalian cell type (if applicable):

ur- pro-

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 100

Metabolic activation:

without

Test concentrations with justification for top dose:

fluctuation test 0.02, 0.05, 0.1, 0.2, 0.5 mmol/L broth
plate-incorporation test 0, 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1 mmol/L top agar

Details on test system and experimental conditions:

Fluctuation test:
Fluctuation tests (Luria and Delbrück, 1943) were carried out as described previously (Voogd et al., 1979, 1980). As test organisms, a Klebsiella pneumoniae mutant, requiring uracil and proline as growth factors was used. In this fluctuation test, nutrient broth containing the substance under investigation and seeded with 100 bacteria/mL was divided into 105 portions of equal volume in sterile culture tubes. With Klebsiella pneumoniae the volume of each portion was 2.5 ml. After incubation during 20 h at 37°C the total number of streptomycin-resistant (including streptomycin-dependent) bacteria was determined in 100 portions by the pour-plate technique. Nutrient agar (pH 7.5) supplemented with streptomycin (100 µg/mL) was used. These plates were incubated for 3 days at 37°C. The number of bacteria present in the 5 remaining tubes was determined by using nutrient agar without streptomycin.
If mutants were present in more than 90 cultures, the number of mutants in the median culture was used to estimate the mutation frequency according to Lea and Coulson (1949). In this case the formula M = m/(N x V) was used in which m = number of mutations in the median culture.

Plate-incorporation test:
Furthermore, the plate-incorporation test developed by Ames et al. (1975) was used with Salmonella lyphimurium TA100. The strains were kindly provided by Dr. B.N. Ames. No metabolic activation was applied. On each plate 3 ml of top agar, in which the compound under investigation was present, was added to 20 ml of the selection medium. The compounds were dissolved in dimethyl sulfoxide.
With Salmonella typhimurium TA100 the investigated compound was considered to be mutagenic if the number of revertants on the selection plates was at least 1.5 X the number of revertants on the controi plates. Furthermore, some dose-response relationship should be apparent. To each plate (containing 20 mL of agar) 3 mL of top agar were added,. The test was done in triplicate.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Test results 2

Species / strain:

other: Klebsiella pneumoniae

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Applicant's summary and conclusion

Conclusions:

For 2 -nitrothiophene the lowest concentration that was mutagenic to Klebsiella pneumoniae in the fluctuation test was 0.05 mmole/L (50 µmol/L) broth and to Salmonella typhimurium TA100 in the plate-incorporation test it was 0.002 mmole/L (2 µmol/L) top agar, respectively.

Executive summary:

The mutagenic potential of the test item to induce base-pair substitution in bacteria was investigated in a fluctuation test with Klebsiella pneumonia ur- pro- and in a plate-incorporation test with Salmonella typhimurium TA100 without metabolic activation.

For 2 -nitrothiophene the lowest concentration that was mutagenic to Klebsiella pneumoniae in the fluctuation test was 0.05 mmole/L broth and to Salmonella typhimurium TA100 in the plate-incorporation test it was 0.002 mmole/L top agar, respectively.