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EC number: 223-529-0 | CAS number: 3943-89-3
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 24 April 2016 to 12 May 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 016
- Report date:
- 2016
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Ethyl 3,4-dihydroxybenzoate
- EC Number:
- 223-529-0
- EC Name:
- Ethyl 3,4-dihydroxybenzoate
- Cas Number:
- 3943-89-3
- Molecular formula:
- C9H10O4
- IUPAC Name:
- ethyl 3,4-dihydroxybenzoate
- Test material form:
- solid: particulate/powder
Constituent 1
Method
- Target gene:
- His + (Salmonella typhimurium)
Trp + (E. coli)
Species / strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254 induced rat S9, characterized in TA100 with benzo-(a)-pyrene and 2-aminoanthracene
- Test concentrations with justification for top dose:
- 0, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
cytotoxicity at 5000 µg/plate - Vehicle / solvent:
- DMSO
Controls
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 2-nitrofluorene
- sodium azide
- methylmethanesulfonate
- other: ICR-191 and aminoanthracene
- Remarks:
- without metabolic activation TA1535 sodium azide TA1537 ICR-191 (exp 1)/2-nitrofluorene (exp 2) TA98 2-nitrofluorene TA100 methylmethanesulfonate WP2uvrA 4-nitroquinoline N-oxide with metabolic activation all strains 2-aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: exp 1 in agar plate incorporation; exp 2 in agar preincubation
- Cell density: 10E9 cells/ml
DURATION
- Preincubation period: 30 min at 37.0°C
- Exposure duration: 37.0 ± 1.0°C for 48 ± 4 h
SELECTION AGENT (mutation assays): histidine (Salmonella); tryptophan (E.coli)
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: decrease number of revertants, bacterial background lawn - Evaluation criteria:
- A test item is considered negative (not mutagenic) in the test if:
a)The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b)The negative response should be reproducible in at least one follow up experiment.
A test item is considered positive (mutagenic) in the test if:
a)The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b)In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment. - Statistics:
- NA
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 5000 µg/plate in both experiments
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 5000 µg/plate in both experiments
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 5000 µg/plate in both experiments
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 5000 µg/plate in both experiments
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Remarks:
- In absence of S9-mix in the second experiment the number of revertants was > historical control values
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 5000 µg/plate in both experiments
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- in experiment 1 in strain TA1537, fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. However, since no dose-relationship was observed, the reductions are not considered to be caused by toxicity of the test item. It is more likely these reductions are caused by incidental fluctuations in the number of revertant colonies.
Applicant's summary and conclusion
- Conclusions:
- The substance is not mutagenic in bacterial cells in presence and absence of metabolic activation
- Executive summary:
The substance was tested in the Ames test with Salmonella typhimurium TA100, TA1535, TA1537 and TA98 and with E.coli WP2uvrA in a plate incorporation and a preincubation test. In presence and absence of metabolic activation (rat S9 mix) no increase in the number of revertants was found. The highest concentration tested (5000 µg/plate) was cytotoxic.
Based on these results it is concluded that the substance is not mutagenic in bacterial cells in presence and absence of metabolic activation
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