Registration Dossier

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:
29.1.-19.3.2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was carried out in accordance with internationally valid GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
See Overall remarks
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 3,4-Ethylenedioxythiophene
- Comercial name. EDOT
- Substance type: pure organic substance
- Physical state: liquid near colourless to pale yellow
- Analytical purity: > 99%
- Lot/batch No.: 091201
- Expiration date of the lot/batch: 21.1.2011
- Storage condition of test material: Keep in dark at room temperature. Stored in amber glass bottles under the argon atmosphere. Minimalize time for handling with substance without protective argon atmosphere as possible. Maximal time without protective atmosphere is 30 min.

Method

Target gene:
gene for synthesis histidine or tryptophan
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain characteristics:
other: histidine dependent strain
Metabolic activation:
with and without
Metabolic activation system:
post-mitochondrial fraction (S9)
Species / strain:
E. coli WP2 uvr A
Additional strain characteristics:
other: tryptophan dependent strain
Metabolic activation:
with and without
Metabolic activation system:
post-mitochondrial fraction (S9)
Test concentrations with justification for top dose:
15, 50, 150, 500, 1500 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: dimethylsulfoxide
- Justification for choice of solvent/vehicle: solubility of substance
Controlsopen allclose all
Negative controls:
yes
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Negative controls:
yes
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine
Negative controls:
yes
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Migrated to IUCLID6: hydrochloride monohydrate
Negative controls:
yes
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminofluorene
Negative controls:
yes
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Negative controls:
yes
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
other: N-methyl-N´-nitro-N-nitrosoguanidine
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

NUMBER OF REPLICATIONS: Two series of experiments were performed with each strain - without metabolic activation and with a supernatant of rat liver and a mixture of cofactors.
As the test substance is toxic in higher doses, any experiments with doses from 50 to 5000 µg per plate with 4 non-toxic doses sometimes could not be obtained. So, some third additional experiments were performed with doses 15-1500 µg per plate. That is the reason for what more than two experiments are performed in some strains.

DETERMINATION OF CYTOTOXICITY
- Method: total growth

Evaluation criteria:
The main criterion for evaluation of results was modified two-fold increase rule, which is compatible with the application of statistical methods (2, 3). After this rule the result is positive, if a reproducible dose-response effect occurs and/or a doubling of the ratio Rt/Rc is reached.

Statistics:
For the evaluation of results, the modified two-fold increase rule was used, which is compatible with the application of statistical methods (2, 3).

2. Dunkel V. C., Chu K.C. (1980): Evaluation of methods for analysis of microbial mutagenicity assays, in The Predictive Value of Short-Term Screening Tests in Carcinogenicity Evaluation, Elsevier North-Holland Biomedical Press, 231 - 240
3. Claxton L. D. et al. (1987): Guide for the Salmonella typhimurium/mammalian microsome tests for bacterial mutagenicity, Mutat. Res. 189, 83 - 91

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not determined
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not determined
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not determined
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
without metabolic activation, some signs of toxicity were observed in the highest dose
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not determined
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Slight precipitation occurred in all experiments in the bottom of the test tubes with top agar with the test substance in concentration of 5000 µg per plate. After shaking, the precipitation disappeared.


RANGE-FINDING/SCREENING STUDIES:
According to the guidelines, one toxic dose is allowed so the highest dose was left as maximum also in the first mutagenicity experiments. The starting dose was diluted according to guidelines (five different analysable concentrations with approximately half log (i.e. √10) intervals between test points). The maximum dose was partially toxic also in the first mutagenicity tests and, sometimes, toxicity occurred in the second highest dose (1500 µg per plate). Therefore, in the second mutagenicity experiments the maximum dose was lowered and another lowest dose was added according to the rules given above (15 µg per plate).

COMPARISON WITH HISTORICAL CONTROL DATA:
Spontaneous reversions, negative controls (solvent), and positive controls were compared with historical controls in our laboratory.

Any other information on results incl. tables

STUDY RESULTS

The results of experiments are summarized in the tables in FInal report. The tables contain the dose applied per plate in µg (Doses were applied to plates at a volume of 0.05 mL.), amount of S9per plate in µL, numbers of revertants on single plates, average number of revertants per plate x and its standard deviation sd, and ratio of revertants at tested dose (Rt) to number of revertants at negative control (Rc, dimethylsulfoxide).

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation

Under the above-described experimental design, the test substance 3,4-Ethylenedioxythiophene was nonmutagenic for all the Salmonella typhimurium as well as Escherichia coli strains both in experiments without as well as with metabolic activation.
Executive summary:

Test substance 3,4-Ethylenedioxythiophene was assayed for the mutagenicity by the Bacterial Reverse Mutation Test. The test was performed according to EU method B.13/14 Mutagenicity – Reverse mutation test using bacteria, which is analogous to the OECD Test Guideline No. 471.

Four  indicator Salmonella typhimurium strains TA 98, TA 100,  TA 1535 and TA 1537 and one indicator Escherichia coli WP2 uvrA strain were used. The test substance was dissolved in dimethylsulfoxide and assayed in doses of 50-5000 µg which were applied to plates in volume of 0.1 mL.

Two series of experiments were performed with each strain - without metabolic activation and with a supernatant of rat liver and a mixture of cofactors.

In the arrangement given above, the test substance 3,4-Ethylenedioxythiophene was nonmutagenic for all the used bacterial strains with as well as without metabolic activation.