2-Butenoic acid, 4,4,4-trifluoro-3-(methylamino)-, ethyl ester, (2Z)-

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

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

ethyl (2Z)-4,4,4-trifluoro-3-(methylamino)but-2-enoate

EC Number:

801-277-8

Cas Number:

507448-65-9

Molecular formula:

C7H10F3NO2

IUPAC Name:

ethyl (2Z)-4,4,4-trifluoro-3-(methylamino)but-2-enoate

Method

Target gene:

urvA and uvrB

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat-liver post mitochrondrial supernatant (S9 fraction)

Test concentrations with justification for top dose:

Concentration range in the range finding test: 20.6 to 5000.0 µg/plate
Concentration ranges in the mutagenicity test: 312.5 to 5000.0 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulphoxide (DMSO)

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

solvent (DMSO)

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: 2-Aminoanthracene

Remarks:

with metabolic activation

Controls 2

Untreated negative controls:

yes

Remarks:

solvent DMSO)

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 4-Nitroquinoline (4-NQC)

Remarks:

without metabolic activation

Details on test system and experimental conditions:

Preparation of bacterial cultures:
Aliquots from frozen stocks were grown in liquid nutrient broth medium (NB-medium) for 8 hours then used for the experiment.

Controls of the genotype of the strains:
The characteristics of the strains were checked every two months. Histitidine-auxtorophy of the Salmonella strains was demonstrated by the requirement for L-histidine. The presence of the rfa character was assayed by the sensitivity for crystal-violet. The deletion of the uvrB gene was demonstrated by the sensitivity for UV-light. The Salmonella strains containing the R-factor (TA98 and TA100) were additionally checked for ampicillin resistance. The tryptophan-auxotrophy of E. coli WP2uvrA was demonstrated by the requirement for tryptophan. The absence of the uvrA gene was demonstrated by the sensitivity of the strain for UV-light. Furthermore, all strains were checked for their reversion properties with known mutagens (positive controls).

Solubilisation of the test substance:
CA 2455 A was dissolved in DMSO at room temperature. The test substance was soluble up to the concentration of 50 mg/ml. Lower concentrations of the test substance were obtained by serial dilution of the stock solution with DMSO. No precipitates or aggregates were noted.

Setting up of the plates:
Standard plate incorporation assay: 0.1ml of the overnight cultures were mixed with 2 ml of top agar, either 0.5 ml of 100mM sodium phosphate buffer (experiments without activation) or 0.5 ml of the activation mixture (experiments with activation) and 0.1 ml of a solution of the test substance, the positive control or the solvent as a negative control and poured on minimal agar in Petri dishes.

Preincubation assay: 0.1 ml of the overnight cultures were mixed with 0.5ml of the activation mixture (experiments with activation) and 0.1 ml of a solution of the test substance, the positive control or the solvent as a negative control and incubated for 30 min. at 37°C. Therafter 2 ml of top agar was added to the mixtures and they were poured on minimal agar in Petri dishes.

Each Petri dish contained about 20.0 ml of minimal agar (1.5% agar supplemented with 2% salts of the Vogel-Bonner Medium E and 2% glucose). The top agar was composed of 0.6% agar and 0.6% NaCl. In the experiment with Salmonella the top agar was supplemented with 10% of 0.5 mM L-histidine and 0.5mM d-biotin dissolved in water. In the experiment with E. coli it was supplemented with 10% of 0.5mM L-tryptophan dissolved in water.

Preliminary Range finding tests:
A range finding test was carried out with strains S. typhimurium TA 100 and E. coli WP2 uvrA with and without metabolic activation at six concentrations of the test substance and one negative control. The highest concentration applied was 5000 µg/plate. The five lower concentrations decreased by a factor of three. The plates were inverted and incubated for about 48 hours at 37 ± 1.5 °C in darkness. Thereafter, they were evaluated by counting the colonies and determining the background lawn. One plate per test substance concentration and negative control was used.

Mutagenicity tests:
The mutagenicity test was performed with the Salmonella typhimurium strains TA98, TA100, TA102, TA1535, TA1537 and Escherichia coli strain WP2 uvrA with and without metabolic activation. Each of the five concentrations of the test substance, a negative and positive control were tested, using three plates per test substance concentrations and controls. The highest concentration applied was determined by the preliminary range finding test and the four lower concentrations decreased by a factor of two. The plates were inverted and incubated for about 48 hours at 37 ± 1.5 °C in darkness. Thereafter, they were evaluated by counting the colonies and determining the background lawn.

Negative and positive controls:
The solvent alone was used as the negative control.
The positive controls (reference mutagens) with and without metabolic activation can be found in Table 1 and Table 2.

Scoring of the plates
Colonies were counted electronically using Artek Colony Counter (Fisher Scientific), or manually where minor agar damage or test chemical precipitates or strong colouration of the agar plates might have interfered with automatic counting. The results were sent on line to a computer. They were checked on a random basis by the operator. Observations indicating precipitates of the test substance in the top agar or a reduced or absent bacterial background lawn were registered additionally.

Evaluation criteria:

Assay acceptance criteria
A test is considered acceptable if the mean colony counts of the negative control values of all strains are within acceptable ranges and if the results of the positive controls meet the criteria for a positive response.

Criteria for a positive response
The test substance will be considered to be positive in the test system if one or both of the following conditions are met:
• At least a reproducible doubling of the mean number of revertants per plate above that of the negative control at any concentration for one or more of the following strains:
TA 98, TA 1535, TA 1537, E.coli WP2 uvrA.
• A reproducible increase of the mean number of revertants per plate for any concentration above that of the negative control by at least a factor of 1.5 for strains TA 100 or TA 102.
Generally a concentration-related effect should be demonstrable.

Statistics:

A statistical analysis was not performed.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Range finding tests:

Six concentrations of CA 2455 A ranging from 20.6 to 5000.0 µg/plate were tested with strain Salmonella typhimurium TA 100 and the strain Escherichia coli WP2 uvrA to determine the highest concentration to be used in the mutagenicity assay. The experiment was performed with and without metabolic activation.
Normal background growth was observed with both strains. The numbers of revertant colonies were not reduced. At the concentration of 5000.0 µg/plate the test substance precipitated in on the surface of the agar plates.
From the results obtained the highest concentration suitable for the mutagenicity test was selected to be 5000.0 µg/plate with and without metabolic activation.

In the confirmatory experiments performed out with and without metabolic activation, again after treatment of strains TA98, TA100, TA102, TA1535, TA 1537 and WP2uvrA with CA 2455 A, no increase in the incidence of either histidine- or tryptophan-prototrophic mutants was observed in comparison with the negative control.

In the mutagenicity tests normal background growth was observed with all stains at all concentrations. The numbers of revertant colonies were not reduced with increasing concentration. Therefore, the test substance exerted no toxic effect on the growth of the bacteria. At the concentration of 5000.0 µg/plate the test substance precipitated in on the surface of the agar plates.

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

Based on the result of these experiments and on standard evaluation criteria, it is concluded that CA 2455 A (Intermediate of CGA 276854) and it’s metabolites did not induce gene mutations in the strains of S. typhimurium and E. coli used.

Executive summary:

 

CA 2245 A (Intermediate of CGA 276854), identified as a brown liquid with a purity of 95.5%, batch no. 249-GE001/SU, was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimurium and in a tryptophan-requiring strain of Escherichia coli. The following strains were used: S. typhimurium TA 98, TA 100, TA 102, TA 1535, TA 1537 and E. coli WP2 uvrA. The test was performed with and without the addition of rat-liver post mitochrondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. The compound was dissolved in DMSO and tested at five concentrations in a range of 312.5 to 5000.0 µg/plate in the presence and absence of a metabolic activation system in the original mutagenicity test. In order to confirm the results, the experiment with metabolic activation was repeated on the strain of S. typhimurium at 78.1 to 1250 µg/plate and E. coli at 312.5 to 5000.0 µg/plate. In the repeat experiment without activation on strains TA 98, TA 100, TA 102, TA 1537 the concentrations of 62.5 to 1000.0 µg/plate were tested. The strains TA 1535 and E. coli were tested with the concentrations of 312.5 to 5000.0 µg/plate. Each strain was additionally tested in the presence and in the absence of a metabolic activation system with a suitable, known mutagen as positive control. 

The original experiment with and without metabolic activation and the confirmatory experiment without activation were performed as standard plate incorporation assay. The confirmatory experiment with metabolic activation was carried out as preincubation assay. 

In both experiments, performed with and without metabolic activation, none of the tested concentrations of CA 2245 A led to an increase in the incidence of either histidine- or tryptophan- prototrophic mutants by comparison with the negative control. 

Based on the result of these experiments and on standard evaluation criteria, it is concluded that CA 2245 A (Intermediate of CGA 276854) and it’s metabolites did not induce gene mutations in the strains of S. typhimurium and E. coli used.