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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
Study period:
From 09 July 2014 and 03 September 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The reliability is rated 1 because the study followed the standard guideline of reference (OECD 471), which describes a procedure designed to evaluate this endpoint, the results were reviewed for reliability and assessed as valid, and the study was conducted under GLP condition.

Data source

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

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 (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
{[(2,4 and 2,5 and 2,6)-dimethylphenyl and 2-ethylphenyl]amino}{[(2,4 and 2,5 and 2,6)-dimethylphenyl and 2-ethylphenyl]amino}methaniminium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate
EC Number:
941-492-5
Cas Number:
1821464-52-1
Molecular formula:
C35H36N6SO3
IUPAC Name:
{[(2,4 and 2,5 and 2,6)-dimethylphenyl and 2-ethylphenyl]amino}{[(2,4 and 2,5 and 2,6)-dimethylphenyl and 2-ethylphenyl]amino}methaniminium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate
Constituent 2
Reference substance name:
{[(2,4 and 2,5 and 2,6)-dimethylphenyl and 2-ethylphenyl]amino}{[(2,4 and 2,5 and 2,6)-dimethylphenyl and 2-ethylphenyl]amino}methaniminium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate
IUPAC Name:
{[(2,4 and 2,5 and 2,6)-dimethylphenyl and 2-ethylphenyl]amino}{[(2,4 and 2,5 and 2,6)-dimethylphenyl and 2-ethylphenyl]amino}methaniminium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Sepisol Fast Yellow MG-F
- Physical state : powder
- Stability under test conditions: stable
- Storage condition of test material: room temperature - non hygroscopic.

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 from liver of rodent treated with Aroclor
Test concentrations with justification for top dose:
C1 : 0.015 mg/plate
C2 : 0.045 mg/plate
C3 : 0.15 mg/plate
C4 : 0.45 mg/plate
C5 : 1.5 mg/plate

Concentration were prepared by serial dilutions in the selected solvent from a stock solution of 30 mg/ml.
Vehicle / solvent:
- Solvent used: ethanol (96%)
- Justification for choice of solvent: The ethanol (96%) was chosen as the test item was soluble in it at a concentration of 50.0 mg/mL
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-Anthramine
Remarks:
The positive controls used are different for tests with and without metabolic activation or for different tester strains (see the "section Any other information on materials and methods incl.tables" for more details)
Details on test system and experimental conditions:
METHOD OF APPLICATION: under the direct incorporation of agar medium plate and the pre-incubation procedures

DURATION
- Preincubation period: 30 min at 37°C when the pre-incubation procedure is used.
- Exposure duration: 48-72 hr at 37°C
- Expression time (cells in growth medium): counting completed at the end of the exposure period in each plate

NUMBER OF REPLICATIONS: Triplicate, in parallel with negative and positive controls and the solvent of test material.

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertant colonies or a clearing or diminution of the background lawn.
Evaluation criteria:
A positive result involved taking into account a dose-response effect in the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system.
The result is considered positive whenever the number of revertants of the test item treated plates is higher than those observed in the solvent treated plates, according to the following criteria:
- TA98 strain : 2 fold higher
- TA100 strain: 2 fold higher
- TA1535 strain : 3 fold higher
- TA1537 strain : 3 fold higher
- WP2 (pkM101) : 2 fold higher

The biological relevance of the results was also considered.
Statistics:
no statistic was used

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at the highest dose
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA:
The mean solvent control and reference item control counts complied with the laboratory historical data for each strain

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

Under these conditions, the test item, Sepisol Fast Yellow MG-F does not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102, without or with metabolic activation, according to the OECD Guidelines n°471 and the method B13/B14 of the directive 2000/32/CE.
Executive summary:

Five doses (15 μg/plate to 1 500 μg/plate) of the solutions obtained from the test item, Sepisol Fast Yellow MG-F, have been tested for their capacity to induce reverse mutation in five Salmonella typhimurium strains. This study was performed in the absence and presence of metabolic activation. Two independent assays were carried out.

For assay n°1, five doses (15 μg/plate to 1 500 μg/plate) of the solutions obtained from the test item, Sepisol Fast Yellow MG-F were put in contact with the strains in the absence and presence of a metabolic activation system (S9-mix 10 % (v/v)).

For assay n° 2, five doses (15 μg/plate to 1 500 μg/plate) of the solution obtained from the test item, Sepisol Fast Yellow MG-F were put in contact with the strains in the absence of metabolic activation and with pre-incubation in the presence of metabolic activation system (S9-mix 10 % (v/v)).

For the two assays, negative and positive controls were carried out in parallel. Positive controls induced a significant increase in the number of revertant colonies compared to negative controls. There is no significant difference between the number of spontaneous reversions, the number of reversions obtained in the positive controls (without and with metabolic activation), and the mean of corresponding experimental “historical” values obtained in the laboratory (Table 11).

These results validate the two tests.

There is no evidence of any increase in the number of revertant colonies in the presence of the various concentration of the test item (1 500, 450, 150, 45 et 15 μg/plate), without and with metabolic activation, in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102.

Therefore, under these conditions, the test item, Sepisol Fast Yellow MG-F - Batch N°407940 (LEMI code : GAG020714) provided by BIMA 83 do not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102, without or with metabolic activation, according to the OECD Guidelines n°471 and the method B13/B14 of the directive 2000/32/CE.

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