Sodium bis[2,4-dihydro-4-[[2-hydroxy-5-(methylsulphonyl)-4-nitrophenyl]azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-)

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:

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Test guideline 3

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

Test material information

Constituent 1

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 results

Test results 1

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

Test results 2

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.