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Toxicological information

Genetic toxicity: in vitro

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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)
Rationale for reliability incl. deficiencies:
other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Remarks:
Conducted according to guideline in effect at time of study conduct
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Remarks:
Conducted according to guideline in effect at time of study conduct
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Remarks:
Conducted according to guideline in effect at time of study conduct
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
trans-dichloroethylene
EC Number:
205-860-2
EC Name:
trans-dichloroethylene
Cas Number:
156-60-5
Molecular formula:
C2H2Cl2
IUPAC Name:
arsenic
Details on test material:
- Purity: 99.79%

Method

Target gene:
histidine
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S9
Test concentrations with justification for top dose:
Initial toxicity-mutation assay: 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate
Confirmatory mutagenicity assay: 15, 50, 150, 500, 1500 and 5000 μg per plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: The results from the solubility test showed that the test substance was insoluble in sterile water from approximately 10 to 50 mg/mL when prepared in the glass vessel, but formed a clear solution in DMSO at the maximum tested concentration of 500 mg/mL. Therefore, DMSO was selected as the solvent of choice for use in each assay.
Controls
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 60±2 minutes
- Expression time (cells in growth medium): 48 to 72 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: The condition of the bacterial background lawn was evaluated for evidence of test substance toxicity by using a dissecting microscope. Toxicity was scored relative to the vehicle control plate.
Evaluation criteria:
For the test substance to be evaluated as positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance. Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 2.0-times the mean vehicle control value. An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative if it was neither positive nor equivocal.
Statistics:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.

Results and discussion

Test resultsopen allclose all
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:
cytotoxicity
Remarks:
Toxicity was observed at 5000 μg per plate with all test conditions.
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Toxicity was observed at 5000 μg per plate with all test conditions.
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

In the initial toxicity-mutation assay, the maximum dose tested was 5000 μg per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 μL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. No precipitate was observed. Toxicity was observed at 5000 μg per plate with all test conditions except WP2 uvrA in the absence of S9 activation. This strain and activation condition was retested to confirm the lack of toxicity observed. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the retest and confirmatory mutagenicity assays was 5000 μg per plate.

In the retest of the initial toxicity-mutation assay, no positive mutagenic response was observed with tester strain WP2 uvrA in the absence of S9 activation. The dose levels tested were 15, 50, 150, 500, 1500 and 5000 μg per plate. Neither precipitate nor toxicity was observed.

In the confirmatory mutagenicity assay, no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The dose levels tested were 15, 50, 150, 500, 1500 and 5000 μg per plate. No precipitate was observed. Toxicity was observed at 5000 μg per plate with all test conditions.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
Under the conditions of this study, the test substance did not exhibit any mutagenic responses in either the presence or
absence of Aroclor-induced rat liver S9. Therefore, the test substance was concluded to be negative
Executive summary:

The test substance was tested in the Bacterial Reverse Mutation Assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9. The assay was performed in two phases, using the preincubation method. The first phase, the initial toxicity-mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test substance.

The results from the solubility test showed that the test substance was insoluble in sterile water from approximately 10 to 50 mg/mL when prepared in the glass vessel, but formed a clear solution in DMSO at the maximum tested concentration of 500 mg/mL. Therefore, DMSO was selected as the solvent of choice for use in each assay.

In the initial toxicity-mutation assay, the dose levels tested ranged from 1.5 to 5000 μg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. No precipitate was observed.

In the confirmatory mutagenicity assay, no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The dose levels tested were 15, 50, 150, 500, 1500 and 5000 μg per plate. No precipitate was observed. Toxicity was observed at 5000 μg per plate with all test conditions. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, the test substance did not exhibit any mutagenic responses in either the presence or absence of Aroclor-induced rat liver S9. Therefore, the test substance was concluded to be negative in this assay.