4-[(1-butyl-5-cyano-1,6-dihydro-2-hydroxy-4-methyl-6-oxo-3-pyridyl)azo]-N-(2-ethylhexyl)benzenesulphonamide

Genetic toxicity: in vitro

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:

04 Jun 2014 - 13 Jun 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-compliant guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 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-butyl-5-cyano-1,6-dihydro-2-hydroxy-4-methyl-6-oxo-3-pyridyl)azo]-N-(2-ethylhexyl)benzenesulphonamide

EC Number:

259-571-1

EC Name:

4-[(1-butyl-5-cyano-1,6-dihydro-2-hydroxy-4-methyl-6-oxo-3-pyridyl)azo]-N-(2-ethylhexyl)benzenesulphonamide

Cas Number:

55290-62-5

Molecular formula:

C25H35N5O4S

IUPAC Name:

4-[(1-butyl-5-cyano-2-hydroxy-4-methyl-6-oxo-1,6-dihydropyridin-3-yl)diazenyl]-N-(2-ethylhexyl)benzenesulfonamide

Details on test material:

- Physical state: Solid, yellow
- Storage condition of test material: Room temperature (protect against humidity)

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/β-naphthoflavone induced rat liver S9 fraction and uninduced hamster liver S9 fraction

Test concentrations with justification for top dose:

1st Experiment (standard plate test with and without S9 mix), all strains: 0; 33; 100; 333; 1000; 2600 and 5200 μg/plate
2nd Experiment (prival preincubation test with and without S9 mix), all strains: 0; 33; 100; 333; 1000; 2600 and 5200 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in ultrapure water, DMSO 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:

Sterility control: Additional plates were treated with soft agar, S9 mix, buffer, vehicle or the test substance but without the addition of tester strains

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see below

Remarks:

with and without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation (with Prival modification)

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 48 – 72 hours in the dark at 37°C

NUMBER OF REPLICATIONS: three plates per dose level

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants (factor ≤ 0.6) and clearing or diminution of the background lawn

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 mutati
on 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 historical negative control data range under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test substance was found from about 333 μg/plate onward with and without S9 mix.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A weak bacteriotoxic effect (slight decrease in the number of his+ revertants) was occasionally observed in the standard plate test depending on the strain from about 1 000 μg/plate onward only without S9 mix. In the preincubation assay bacteriotoxicity (decrease in the number of his+ or trp+ revertants) was occasionally observed depending on the strain and test conditions from about 2 600 μg/plate onward.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Experimental Result

Standard plate test

		Mean revertants
	Dose (µg/plate)	TA 1535		TA 100		TA 1537		TA 98		E. coli
		without S9	with S9	without S9	with S9	without S9	with S9	without S9	with S9	without S9	with S9
DMSO	-	11.3	11.7	44.0	57.7	5.7	6.7	26.0	30.7	64.0	82.7
Test item	33	9.3	11.7	48.0	56.0	3.0	6.3	17.0	33.0	54.7	73.7
	100	10.3	7.7	46.7	56.0	6.0	8.3	17.7	27.7	64.3	80.3
	333	7.7	9.3	45.3	60.0	3.7	6.7	27.0	32.0	52.7	83.7
	1000	5.3	7.7	50.0	69.0	4.0	5.3	17.0	23.7	59.3	79.7
	2600	6.3	8.0	53.0	54.7	5.3	6.3	21.0	23.3	46.7	83.3
	5200	6.7	10.0	58.0	61.7	3.0	6.7	15.7	27.0	49.3	71.3
positive control*		6482.3	291.0	5819.7	1532.0	1983.7	137.3	349.0	1295.7	862.7	201.0

Prival preincubation test

		Mean revertants
	Dose (µg/plate)	TA 1535		TA 100		TA 1537		TA 98		E. coli
		without S9	with S9	without S9	with S9	without S9	with S9	without S9	with S9	without S9	with S9
DMSO	-	12.0	12.3	72.7	67.3	9.7	12.3	23.7	38.3	75.0	73.0
Test item	33	12.7	15.0	73.0	79.3	13.0	13.0	21.7	34.7	71.3	85.0
	100	15.0	15.3	60.7	73.3	11.3	13.3	20.7	35.3	69.3	90.0
	333	11.7	13.3	74.3	74.7	10.7	10.7	21.7	33.7	62.7	77.0
	1000	11.0	12.0	70.0	66.7	10.7	11.0	19.0	27.7	70.3	68.0
	2600	12.0	9.7	48.7	70.0	8.0	10.0	21.3	22.7	41.7	59.7
	5200	6.0	5.3	49.3	71.0	2.3	5.0	15.3	18.7	35.3	66.3
positive control*		523.7	222.0	1304.7	893.7	1047.7	116.0	505.0	990.0	1014.7	240.3
Congo Red	210								530.3

*for details on positive conrol substances see above

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the experimental conditions of this study, the test substance is not mutagenic in the standard plate test or in the prival preincubation test in the absence and the presence of metabolic activation.

Executive summary:

In an Ames test following OECD guideline 471 and in compliance with GLP, 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. Tester strains used were Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA. The dose range used was 33 μg - 5200 μg/plate in both the standard plate test and the Prival preincubation test. Both assays were performed either with or without S9 mix. 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 1000 μg/plate onward. A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the prival preincubation test either without S9 mix or after the addition of a metabolizing system. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the standard plate test or in the prival preincubation test in the absence and the presence of metabolic activation.