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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was not mutagenic in the Ames test in Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvrA, tested both in the absence and presence of S9 mix and with Prival modification. The substance was further neither mutagenic nor clastogenic in mammalian cells.

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:

04 Jun 2014 - 13 Jun 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-compliant guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

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

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.

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.

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'.

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Bacterial reverse mutation assay

In an Ames test with Prival modification 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 (S. typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA). The dose range used was 33 - 5200 μg/plate in both the standard plate test and the Prival preincubation test in the presence of absence of 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. The results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study. In this study with and without S9 mix, the number of revertant colonies in the negative controls was within or nearby the range of the historical negative control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within or above the range of the historical positive control data. 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 (BASF, 2014).

In a supporting study also compliant with OECD guideline 471 and GLP but without Prival modification, negative results were reported as well, thus confirming the results in the key study (BASF, 1995).

HPRT

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. The assay was performed according to GLP and followed OECD guideline 476. Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). The concentrations used ranged from 0 to 100 µg/ml. According to the results of this study, the test substance did not relevantly increase the number of mutant colonies in two independently performed experiments, either without S9 mix or after the addition of a metabolizing system. The mutant frequencies at any concentration were close to the range of the concurrent vehicle control values and within the range of the historical negative control data. A statistically significant dose-dependent increase in mutant colonies was detected in the 1st Experiment in the absence of metabolic activation. However, all mutant rates obtained were well within the range of our historical negative control data and a similar increase was not observed in the parallel experiment, therefore, this finding can be regarded as biologically irrelevant. The mutation frequencies of the vehicle control groups were within our historical negative control data range including all vehicles used in our laboratory and, thus, fulfilled the acceptance criteria of this study. The increase in the frequencies of mutant colonies induced by the positive control substances EMS and DMBA clearly demonstrated the sensitivity of the test method and/or of the metabolic activity of the S9 mix employed. The values were within the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study. Thus, in the absence and the presence of metabolic activation, the test item is not a mutagenic substance in the HPRT locus assay using CHO cells under the experimental conditions chosen.

Micronuclelus Test

The test substance was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity). The assay was performed under GLP and followed OECD guideline 487. Three independent experiments were carried out, with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). A sample of at least 1000 cells for each culture were analyzed for micronuclei, i.e. 2000 cells for each test group. The test article did not lead to a biologically relevant increase in the number of micronucleated cells either without S9 mix or after the addition of a metabolizing system in three experiments performed independently of each other. The frequencies of micronuclei after test substance treatment were close to the range of the concurrent vehicle control values at both exposure times and clearly within the range of the historical negative control data. The single statistically significant increase in micronucleated cells observed in the 1st Experiment without metabolic activation has to be regarded as biologically irrelevant under the circumstance that this value was not reproducible at the next higher concentration and clearly within the historical negative control data range. The number of micronucleated cells in the vehicle control groups were within the historical negative control data range and, thus, fulfilled the acceptance criteria of this study. The increase in the frequencies of micronuclei induced by the positive control substances EMS and CPP clearly demonstrated the sensitivity of the test system and of the metabolic activity of the S9 mix employed. The values were within the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study. Thus, under the experimental conditions chosen here, the conclusion is drawn that the test item has not the potential to induce micronuclei (clastogenic and/or aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

Justification for selection of genetic toxicity endpoint
GLP compliant guideline study

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available experimental test data is reliable and suitable for the purpose of classification under Directive 67/548/EEC. Based on the data, classification for genetic toxicity is not warranted under Directive 67/548/EEC.

 

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. Based on the data, classification for genetic toxicity is not warranted under Regulation (EC) No.1272/2008.