2,4-Thiophenedicarbonitrile, 5-amino-3-methyl-

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:

23 January 2002 to 17 March 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

5-amino-3-methylthiophene-2,4-dicarbonitrile

EC Number:

610-868-8

Cas Number:

52603-48-2

Molecular formula:

C7N3H5S

IUPAC Name:

5-amino-3-methylthiophene-2,4-dicarbonitrile

Method

Target gene:

S. typhimurium: Histidine locus

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: TA 100 & TA1535 are sensitive to agents inducing base pair substitution. TA 1537 & TA98 are sensitive to agents inducing frame-shift mutations.

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: Sensitive to agents inducing base-pair substitution.

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 0, 50, 150, 500, 1500 and 5000 µg/plate
Concentration range in the main test (without metabolic activation): 0, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no data provided

The test material was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer. Analysis for concentration, homogeneity and stability of the test material formulations is not a requirement of the test guidelines and was, therefore, not determined. Prior to use, the solvent was dried using molecular sieves (sodium alumino-silicate) ie 2 mm pellets with a nominal pore diameter of 4 x 10^-4 microns.

Vehicle and positive controls were used in parallel with the test material. A solvent treatment group was used as the vehicle control.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: See "Any other information on materials and methods incl. tables" section below

Details on test system and experimental conditions:

The strains used in this assay were all mutants derived from Salmonella typhimurium LT2 and were those recommended for general screening.

TA100, TA1535, TA102: sensitive to agents inducing base-pair substitution

TA1537, TA98: sensitive to agents inducing frame-shift mutations

The strains were obtained from the University of California at Berkeley on culture discs on 4 August 1995 and were stored at -196°C in a Statebourne liquid nitrogen freezer, model SXR 34. Prior to the master strains being used, characterisation checks were carried out to determine the amino-acid requirement, presence of rfa, R factors, uvrB mutation and the spontaneous reversion rate.

In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot numbers 250427 06/06 and 250177 05/06) and incubated at 37°C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.


METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
Five concentrations of the test material (50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine supplemented, top agar, 0.1 ml of the test material formulation, vehicle or positive control and either 0.5 ml of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.

All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

SELECTION AGENT (mutation assays): NDA
SPINDLE INHIBITOR (cytogenetic assays): N/A
STAIN (for cytogenetic assays): N/A

NUMBER OF REPLICATIONS: Three

NUMBER OF CELLS EVALUATED: NDA

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS: N/A

OTHER:
Preliminary toxicity study: In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity of the test material. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The test was performed by mixing 0.1 ml of bacterial culture (TA100), 2 ml of molten, trace histidine supplemented, top agar, 0.1 ml of test material formulation, 0.5 ml of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel- Bonner Minimal agar (30 ml/plate). Ten doses of the test material and a vehicle control (dimethyl sulphoxide) were tested. In addition, 0.1 ml of the maximum concentration of the test material and 2 ml of molten, trace histidine supplemented, top agar was overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

Evaluation criteria:

The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pkM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 x 10^9 bacteria per ml.
Each mean positive control value should be at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels.
There should be no evidence of excessive contamination.

Statistics:

The test material may be considered positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression) significant increase in the revertant count in at least one strain of bacteria.

Results and discussion

Test results

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 102, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/plate (without metabolic activation only)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

TEST-SPECIFIC CONFOUNDING FACTORS
None. A filamentous black precipitate was observed at 5000 µg/plate with and without metabolic activation, this did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES:
The test material was toxic at 5000 µg/plate without S9-mix and non-toxic with S9-mix to the strain of Salmonella used (TA100). The test material formulation and the S9-mix used in this experiment were both shown to be effectively sterile.

COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The S9-mix used in both experiments was shown to be sterile.

Results for the negative controls were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Study.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test material caused a visible reduction in the growth of the bacterial background lawn or a substantial decrease in the frequency of revertant colonies in all of the tester strains at 5000 µg/plate.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Please see attachment "AMTC Ames results tables.pdf" for full results tables.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

The method was designed to meet the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test" and Method B13/14 of Commission Directive 2000/32/EC.

 

Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

 

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

The test material caused a visible reduction in the growth of the bacterial background lawn or a substantial decrease in the frequency of revertant colonies in all of the tester strains at 5000 µg/plate. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A filamentous black precipitate was observed at 5000 µg/plate, this did not prevent the scoring of revertant colonies.

 

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

 

The test material was considered to be non-mutagenic under the conditions of this test.