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EC number: 274-986-8 | CAS number: 70892-34-1
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:
- 10-May-2012 to 31-May 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 012
- Report date:
- 2012
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Formaldehyde, reaction products with m-phenylenediamine, sodium sulfide (Na2S) and sulfur
- EC Number:
- 274-986-8
- EC Name:
- Formaldehyde, reaction products with m-phenylenediamine, sodium sulfide (Na2S) and sulfur
- Cas Number:
- 70892-34-1
- Molecular formula:
- not applicable
- IUPAC Name:
- Formaldehyde, reaction products with m-phenylenediamine, sodium sulfide (Na2S) and sulfur
- Details on test material:
- - Name of test material (as cited in study report): C.I. Leuco Sulphur Brown 37
- Substance type: Dark brown paste
- Physical state: Solid
- Storage condition of test material: At room temperature in the dark
Constituent 1
Method
- Target gene:
- - S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
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:
- Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
- Test concentrations with justification for top dose:
- Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate - Vehicle / solvent:
- - Vehicle used: DMSO
- Justification for choice of vehicle:
A homogeneous suspension could be obtained in DMSO. Test compound was stable in DMSO and DMSO has been accepted and approved by authorities and international guidelines.
Controlsopen allclose all
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- without S9
(650 µg/plate in DMSO for TA100)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 2-nitrofluorene
- Remarks:
- without S
(10 µg/plate in DMSO for TA98 and 15 µg/plate for TA1537)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- Remarks:
- without S9
(10 µg/plate in DMSO for WP2uvrA)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Remarks:
- without S9
(5 µg/plate in saline for TA1535)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene in DMSO for all tester strains
- Remarks:
- with S9
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48 hour
NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain.
NUMBER OF CELLS EVALUATED: 10E8 per plate
DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.
OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined. - Evaluation criteria:
- A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive if:
a) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
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 independently repeated experiment.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- valid
- Positive controls validity:
- not examined
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was only observed at the dose level of 5000 µg/plate in tester strain TA1535.
RANGE-FINDING/SCREENING STUDIES:
- No toxicity was observed up to and including the top dose of 5000 µg/plate
COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxicity was observed up to and including the top dose of 5000 µg/plate
Applicant's summary and conclusion
- Conclusions:
- Based on the results of this study it is concluded that C.I. Leuco Sulphur Brown 37 at concentrations up to 5000 µg/plate is mutagenic in the tester strains TA1537, TA98 and TA100 of the Salmonella typhimurium reverse mutation assay. C.I. Leuco Sulphur Brown 37 is not mutagenic in the other Salmonella typhimurium tester strain (TA1535) or Escherichia coli strain reverse mutation assay using strain WP2uvrA. The mutagenicity was confined only to incubations with metabolic activation.
- Executive summary:
In the presence of S9-mix, C.I. Leuco Sulphur Brown 37 induced dose related increases in three out of the five tester strains. The increases observed in the tester strains TA1537, TA98 and TA100 were above our laboratory historical control data range and 4.8 to 27-fold the concurrent control.
The tester strains TA1535 and WP2uvrA in the absence and presence of S9-mix and the tester strains TA1537, TA98 and TA100 in the absence of S9-mix showed negative responses over the entire dose range, i.e. no dose-related, increase in the number of revertants.
Since 4.8 to 27-fold, dose related increases were observed in three tester strains in the presence of S9-mix, these increases are considered biologically relevant, C.I. Leuco Sulphur Brown 37 is mutagenic in the presence of S9-mix.
In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
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