4-[(1,5-dihydro-3-methyl-5-oxo-1-phenyl-4H-pyrazol-4-ylidene)methyl]-2,4-dihydro-5-methyl-2-phenyl-3H-pyrazol-3-one

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:

06 Dec 2016 - 15 Dec 2016

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 OTS 798.5100 (Escherichia coli WP2 and WP2 UVRA Reverse Mutation Test)

Version / remarks:

1998

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-[(1,5-dihydro-3-methyl-5-oxo-1-phenyl-4H-pyrazol-4-ylidene)methyl]-2,4-dihydro-5-methyl-2-phenyl-3H-pyrazol-3-one

EC Number:

225-184-1

EC Name:

4-[(1,5-dihydro-3-methyl-5-oxo-1-phenyl-4H-pyrazol-4-ylidene)methyl]-2,4-dihydro-5-methyl-2-phenyl-3H-pyrazol-3-one

Cas Number:

4702-90-3

Molecular formula:

C21H18N4O2

IUPAC Name:

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

Method

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:

phenobarbital and β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate were generally selected as maximum test dose at least in the 1st Experiment. No bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed in the standard plate test up to the highest concentration.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, ethanol was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Controls

Untreated negative controls:

yes

Remarks:

Additional plates were treated with soft agar, S9 mix, buffer, vehicle and the test substance but without the addition of tester strains (sterility control)

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 medium; in agar (plate incorporation); preincubation

DURATION
- Exposure duration: 48 – 72 hours

NUMBER OF REPLICATIONS: 3 test plates per dose or per control

DETERMINATION OF CYTOTOXICITY
- 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, strains: TA 1535, TA 100, TA 1537, TA 98
- 60 μg/plate, dissolved in DMSO, strain: Escherichia coli WP2 uvrA
Without S9 mix
• N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)
- 5 μg/plate, dissolved in DMSO, strains: TA 1535, TA 100
• 4-nitro-o-phenylenediamine (NOPD),
- 10 μg/plate, dissolved in DMSO, strain: TA 98
• 9-aminoacridine (AAC)
- 100 μg/plate, dissolved in DMSO, strain: TA 1537
• 4-nitroquinoline-N-oxide (4-NQO)
- 5 μg/plate, dissolved in DMSO, strain: E. coli WP2 uvrA

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

Species / strain:

other: 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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was found from about 333 μg/plate onward in the standard plate test and from about 1000 μg/plate in the preincubation test both with and without S9 mix.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed in the standard plate test up to the highest concentration. In the preincubation assay bacteriotoxicity (reduced his- background growth, decrease in the number of his+ revertants) was observed using the tester strain TA 1535 and TA 1537 without S9 mix at a concentration of 5000 μg/plate.

Any other information on results incl. tables

SUMMARY OF RESULTS

Standard Plate Test:

	Mean revertants per plate
Strain	TA 1535		TA 100		TA 1537		TA 98		WP2uvrA
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Ethanol	10.3	14.0	85.3	102.7	6.0	9.3	26.0	31.3	20.0	18.3
33	12.0	9.3	89.3	100.7	8.0	7.3	23.3	26.7	16.7	25.3
100	11.3	13.3	82.3	92.0	7.7	9.7	24.7	23.7	14.7	19.3
333	10.7	14.0	94.0	92.0	7.3	10.0	20.0	30.0	18.0	20.3
1000	9.7	10.3	77.3	93.7	7.3	7.0	18.7	35.0	15.3	30.0
2500	12.7	10.0	72.7	92.3	11.0	7.7	22.7	43.3	14.7	24.0
5000	10.0	10.0	76.3	78.3	4.0	9.3	18.3	22.3	19.3	19.0
positive control	5005.7	210.7	2840.0	2030.7	1252.7	154.3	585.7	1914.3	1570.3	117.0

Preincubation Test:

	Mean revertants per plate
Strain	TA 1535		TA 100		TA 1537		TA 98		WP2uvrA
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Ethanol	10.3	9.7	78.3	108.0	5.3	7.0	20.3	34.3	27.7	27.3
33	15.3	15.7	88.7	107.0	6.3	6.3	23.3	30.0	22.3	33.3
100	6.7	6.7	93.7	99.3	7.7	7.0	19.7	36.7	28.7	21.7
333	13.7	9.0	88.0	101.0	6.7	9.3	20.0	33.3	24.3	27.3
1000	12.3	10.3	110.3	106.3	7.0	6.3	16.0	25.3	20.3	24.0
2500	10.7	8.7	90.3	81.3	6.7	4.7	29.3	26.3	21.3	29.7
5000	6.0	10.3	85.0	81.7	0.3	10.0	23.3	29.0	25.0	19.3
positive control	2743.0	215.7	2331.7	2443.7	1058.0	144.3	542.3	2177.0	311.7	102.7

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions of this study, the test substance is not 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, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. The strains used were TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA with a dose range of 33 μg - 5000 μg/plate in both standard plate test (SPT) and preincubation test (PIT) each performed 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 at a concentration of 5000 μg/plate. 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 or in the preincubation test without S9 mix or after the addition of a metabolizing system. Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.