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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 Jan 2017 - 19 Jan 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
1998
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium bis[2,4-dihydro-4-[[2-hydroxy-5-(methylsulphonyl)-4-nitrophenyl]azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-)
EC Number:
276-075-0
EC Name:
Sodium bis[2,4-dihydro-4-[[2-hydroxy-5-(methylsulphonyl)-4-nitrophenyl]azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-)
Cas Number:
71839-91-3
Molecular formula:
C34 H26 Cr N10 O12 S2 . Na
IUPAC Name:
sodium bis[2,4-dihydro-4-[[2-hydroxy-5-(methylsulphonyl)-4-nitrophenyl]azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-)

Method

Target gene:
his, trp
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:
liver S9 mix from induced rats
Test concentrations with justification for top dose:
0; 33; 100; 333; 1000; 2600 and 5200 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see below
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 37°C for 48 – 72 hours in the dark,

NUMBER OF REPLICATIONS: three

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants (factor ≤ 0.6); clearing or diminution of the background lawn (= reduced his- or trp- background growth)


POSITIVE CONTROLS

With S9 mix
• 2-aminoanthracene (2-AA)
- 2.5 μg/plate, dissolved in DMSO for strains: TA 1535, TA 100, TA 1537, TA 98
- 60 μg/plate, dissolved in DMSO for strain: Escherichia coli WP2 uvrA

Without S9 mix
• N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)
- 5 μg/plate, dissolved in DMSO for strains: TA 1535, TA 100

• 4-nitro-o-phenylenediamine (NOPD)
- 10 μg/plate, dissolved in DMSO for strain: TA 98

• 9-aminoacridine (AAC)
- 100 μg/plate, dissolved in DMSO for strain: TA 1537

• 4-nitroquinoline-N-oxide (4-NQO)
- 5 μg/plate, dissolved in DMSO for strain: E. coli WP2 uvrA
Rationale for test conditions:
Mutagenicity was observed in the standard plate test. Therefore, the standard plate test was repeated instead of the prival modification.
Evaluation criteria:
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was found from about 333 μg/plate onward with and without S9 mix.

HISTORICAL CONTROL DATA
In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control substances with and without S9 mix induced a significant increase in the number of revertant colonies within or nearby the range of the historical positive control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A bacteriotoxic effect (reduced his- background growth, decrease in the number of his+ revertants) was occasionally observed in the standard plate test depending on the strain and test conditions from about 2600 μg/plate onward.

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of this study, the test substance is mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.
Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA in a reverse mutation assay. The dose range applied was 33 μg - 5200 μg/plate in a standard plate test (SPT) with and without metabolic activation (liver S9 mix from induced rats). Precipitation of the test substance was found from about 333 μg/plate onward with and without S9 mix. A bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 2600 μg/plate onward. A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test either with or without metabolic activation using tester strains TA 1535, TA 100 TA 1537 and E. coli WP2 uvrA. A relevant, reproducible and partly dose dependent increase in the number of his+ revertants exceeding a factor of 2 compared to the concurrent vehicle control was observed with TA 98 without and with metabolic activation.

TA 98 without S9 mix: Increase of revertants at concentrations of 1000, 2600 and 5200 μg/plate in the 1st and 2nd Experiment.

TA 98 with S9 mix: Increase of revertants at a concentration of 5200 μg/plate in the 1st and 2nd Experiment.

Under the experimental conditions of this study, the test substance is mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.