2,4-dihydro-5-methyl-2-phenyl-4-(phenylazo)-3H-pyrazol-3-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to Prival .

Link to relevant study records

Reference

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:

2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study according to guideline and GLP regulation

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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:

a) Syrian golden hamster liver S9 mix b) rat liver S9 mix

Test concentrations with justification for top dose:

First plate incorporation test:
a: without metabolic activation:
50, 160, 500, 1600 and 5000 µg/plate
b: with metabolic activation (10 % rat liver):
50, 160, 500, 1600 and 5000 µg/plate

Second plate incorporation test:
a: without metabolic activation:
0.5, 1.6, 5, 16 50 and 160 µg/plate (all tester strains)
b: with metabolic activation (1 0 % rat liver):
1.6, 5, 16, 50, 160 and 500 µg/plate (TA 1535 and TA 98)
5, 16, 25, 50, 80, 160 and 500 µg/plate (WP2uvrA)
25, 50, 80, 120, 160, 500, 1600 and 5000 µg/plate (TA 1537)

Preincubation test:
a: without metabolic activation:
1.6, 5, 16, 50, 160 and 500 µg/plate (TA 1535, TA 1537, TA 98 and WP2uvrA)
5, 16, 50, 160, 500 and 1600 µg/plate (TA 100)
b: with metabolic activation (30 % Syrian golden hamster liver):
0.5, 1.6, 5, 16, 50 and 160 µg/plate (all tester strains)

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

congo red

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Preincubation period: 30 min
- Exposure duration: 48 h
- Expression time (cells in growth medium): 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: other: reduction in the number of spontaneously occurring colonies and visible thinning of the bacterial lawn

Evaluation criteria:

5.5.1 Criteria for a valid assay
The assay is considered valid if the following criteria are met:
- the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency
- the positive controls induce increases in the mutation frequency which are significant and within the laboratory's normal range
5.5.2 Criteria for a positive response
A test compound is classified as mutagenic if it has either of the following effects:
a) it produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
b) it induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn.

If the test substance does not achieve either of the above criteria, it is considered to show no evidence of mutagenic activity in this system.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation

The test item is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to Prival at the dose levels investigated.

Executive summary:

The test item was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium and with Escherichia coli WP2uvrA. Three independent mutagenicity studies were conducted, two as the standard plate test with the plate incorporation method and a modified preincubation test (Prival test).

The studies were performed in the absence and in the presence of a metabolizing system derived from a rat liver homogenate or a hamster liver homogenate. For all studies, the compound was suspended in DMSO, and each bacterial strain was exposed to 5 dose levels in the first plate incorporation test and to 6 to 8 dose levels in the second plate incorporation test. In the preincubation test each bacterial strain was exposed to 6 dose levels. Doses for the first plate incorporation test ranged from 50 to 5000 µg/plate.

Visible precipitation of the test compound on the plates was observed at concentrations of 160 µg/plate and above. Because of the high toxicity in the first plate incorporation test dose ranges for the second plate incorporation test were variable across bacterial strains to account for varying susceptibilities to cytotoxic effects: low doses ranged from 0.5 to 160 µg/plate, and high doses ranged from 25 to 5000 µg/plate. For the modified preincubation test dose levels from 0.5 to 160 µg/plate were chosen in the absence of metabolic activation with all tester strains. In the presence of S9-mix doses ranged from 1.6 to 500 µg/plate with the strains TA 1535, TA 1537, TA 98 and WP2uvrA. The strain TA 100 was tested in a dose range of 5 to 1600 µg/plate.

Control plates without mutagen showed that the number of spontaneous revertant colonies was within the laboratory's historical control. All positive control compounds gave the expected increase in the number of revertant colonies.

Toxicity:

In the first plate incorporation test high toxicity was observed without metabolic activation at concentrations of 50 µg/plate and above with all Salmonella typhimurium strains. With the strain Escherichia coli WP2uvrA toxicity was observed at dose levels of 160 µg/plate and above.

In the presence of metabolic activation the test compound proved to be not toxic to the bacterial strains TA 100 and WP2uvrA. With the strains TA 1535, TA 1537 and TA 98 toxic effects were observed at concentrations of 160 µglplate and above.

In the second plate incorporation test the toxic effects of the first plate incorporation test were reproduced with the Salmonella typhimurium strains. With the Escherichia coli strain toxicity was observed at concentrations of 50 µg/plate and above in the absence, and at dose levels of 160 µg/plate and above in the presence of metabolic activation. In the preincubation test toxicity was observed without metabolic activation at dose levels of 16 µg/plate and above with the Salmonella typhimurium strains. With the strain Escherichia coli WP2uvrA toxicity was observed at dose levels of 50 µg/plate and above. In the presence of hamster liver metabolic activation the test compound proved to be toxic to the bacterial strains TA 1535, TA 1537, TA 98 and WP2uvrA at concentrations of 160 µg/plate and above. Toxicity was also found with the strain TA 100 at a concentration of 500 µg/plate.

Mutagenicity:

Plate incorporation test:

In the absence of the metabolic activation system the test compound did not cause relevant increases in the number of revertants in any of the bacterial strains. Also in the presence of rat liver activation system (10 % (v/v)), treatment of the cells with the test item did not result in relevant and reproducible increases in the number of revertant colonies.

Preincubation test:

In the absence and in the presence of hamster liver S9-mix (30% (v/v)) using the preincubation method according to Prival the test item did not cause relevant increases in the number of revertant colonies with any of the tester strains.

Summarizing, it can be stated that the test item is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to Prival at the dose levels investigated.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The test item is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to Prival .

Justification for selection of genetic toxicity endpoint
only available study

Justification for classification or non-classification

not classified

The test item is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to Prival .