3-Thiopheneacetic acid, 5-amino-4-cyano-2-(methoxycarbonyl)-, methyl ester

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:

2010

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

methyl 5-amino-4-cyano-3-(2-methoxy-2-oxoethyl)thiophene-2-carboxylate

Cas Number:

674773-12-7

Molecular formula:

C10H10N2O4S

IUPAC Name:

methyl 5-amino-4-cyano-3-(2-methoxy-2-oxoethyl)thiophene-2-carboxylate

Details on test material:

- Name of test material (as cited in study report):
- Molecular formula (if other than submission substance):
- Molecular weight (if other than submission substance):
- Smiles notation (if other than submission substance):
- InChl (if other than submission substance):
- Structural formula attached as image file (if other than submission substance): see Fig.
- Substance type: organic
- Physical state: solid
- Analytical purity:
- Impurities (identity and concentrations):
- Composition of test material, percentage of components:
- Isomers composition:
- Purity test date:
- Lot/batch No.: 7029-238
- Expiration date of the lot/batch:
- Radiochemical purity (if radiolabelling):
- Specific activity (if radiolabelling):
- Locations of the label (if radiolabelling):
- Expiration date of radiochemical substance (if radiolabelling):
- Stability under test conditions:
- Storage condition of test material:
- Other:

Method

Target gene:

histidine

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli, other: WP2 pKM101 and WP2 uvrA pKM101

Metabolic activation:

with and without

Metabolic activation system:

Arochlor induced rat liver S9

Test concentrations with justification for top dose:

1.6, 8, 40, 200, 1000 and 5000 microgramm/plate

Vehicle / solvent:

Dimethyl sulphoxide (DMSO)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

Remarks:

Migrated to IUCLID6: and 2-anthramine

Details on test system and experimental conditions:

-direct plating preincubation
-independent experiments-triplicates/concentrations

Evaluation criteria:

-genotoxicity (bacterial lawn and number of revertants)
-genotoxictity (number of revertants)

Statistics:

Statistical analysis using automated decision tree.
For positive control, Student t test versus vehicle control on square root data.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

An initial toxicity Range-Finder experiment was carried out in the absence and in the presence of S-9 in strain TA100 only, using final concentrations of test substance at 1.6, 8, 40, 200, 1000 and 5000 microgram/plate, plus negative (vehicle) and positive controls. Following these treatments, no evidence of toxicity was observed. These data were considered to be acceptable for mutation assessment and are presented as TA 100 mutagenicity data for Experiment 1.

Experiment 1 treatments of the remaining tester strains were performed in the absence and in the presence of S9 and retained the concentrations employed for the Range-finder Experiment. Following these treatments evidence of toxicity was observed in strcin TA1537 in the absence and presence of S-9 at 5000 mikrogram/plate, with some evidence of toxicity also apparent in strain TA98 in the absence of S-9 at 5000 microgram/plate.

Experiment 2 treatments of all tester strains were performed in the absence and in the presence of S-9. the maximum test concentration of 5000 mikrogram/plate was retained for all strains. Narrowed concentration intervals were employed covering the ranges 78.13-5000 microgramm/plate for strain TA 1537 or 156.3-5000 microgramm/plate for all remaining strains in order to examine more closely those concentrations of the test substance approaching the maximum test concentration and concidered therefore most likely providence of mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Following these treatments possible evidence of toxicity was observed in strain TA 1537 in the absence of S-9 only at 2500 and 5000 microgramm/plate.

No precipitation was observed on the test plates following incubation.

Negative vehicle and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies on negative control plates all fell within acceptable ranges and were sigificantly elevated by positive control treatments.

Although a statistically significant increase in revertant numbers was observed following test substance treatments of strain WP2 uvrA pKM101 in the absence of S-9 at 1250 microgramm/plate in Experiment 2 (Dunnett's test 1 % level), this was not concentration related or reproducible and was of sufficient magnitude to be considered as clear evidence of mutagenic activity in this assay system.

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

In conclusion the test substance did not induce mutation in four histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA 1535 and TA 1537) and two tryptophan-requiring strains of Escherichia coli (WP2 pKM101 and WP2 uvrA pKM101) when tested under the conditions of this study. These cnditions included treatments at concentrations up to 5000 microgram/plate in the absence and in the presence of rat iver metabolic activation system (S-9).