Reaction mass of sodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a bacterial reverse mutation assay (AMES test) according to OECD Guideline 471 (BASF Colors&Effects, 2017), the test substance showed a mutagenic potential.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-08-25 to 2016-09-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997-07-21

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(EC) No 440/2008, 2008-05-30

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998-08

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No. of test material: N01-131001
- Expiration date of the lot/batch: 2023-03-25

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature


OTHER SPECIFICS: solid, dark brown

Target gene:

his/trp

Species / strain / cell type:

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

Remarks:

uvrA

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and beta naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

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. However, this maximum dose was tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 µL/plate might also be tested in repeat experiments for further clarification/substantiation.
In this study, due to the purity of the test substance 5.3 mg/plate was used as top dose in the 1st Experiment.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in 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

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA)

Remarks:

With S9 mix: 2.5 µg/plate, TA 1535, TA 100, TA 1537, TA 98; 60 µg/plate, Escherichia coli WP2 uvrA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)

Remarks:

Without S9 mix: 5 µg/plate, TA 1535, TA 100

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine (NOPD) (

Remarks:

Without S9 mix: 10 µg/plate, TA 98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without S9 mix: 100 µg/plate, TA 1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without S9 mix: 5 µg/plate, E. coli WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar

DURATION
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity detected by a decrease in the number of revertants (factor < 0.6) and/or clearing or diminution of the background lawn (= reduced his- or trp- background growth)

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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 with and without S9 mix.

Table 1 SPT - Without metabolic activation

Strain	Test group	Dose (µg/plate)	Mean revertants per plate	Standard deviation	Factor
TA 1535	DMSO Test item           MNNG	- 33 100 333 1000 2650 5300 5.0	9.7 9.3 17.0 20.0 25.3 0.0 0.0 5185.0	2.1 2.9 1.0 1.7 2.3 0.0 0.0 420.1	- 1.0 1.8 2.1 P 2.6 P 0.0 B P 0.0 B P 536.4
TA 100	DMSO Test item           MNNG	- 33 100 333 1000 2650 5300 5.0	94.0 196.0 441.0 924.0 1506.7 64.0 0.0 3672.3	3.0 28.2 49.2 194.2 78.9 16.8 0.0 226.3	- 2.1 4.7 9.8 P 16.0 P 0.7 P B 0.0 P B 39.0
TA 1537	DMSO Test item           AAC	- 33 100 333 1000 2650 5300 100	9.0 15.0 47.0 80.7 27.7 0.0 0.0 2104.3	1.7 4.4 10.4 11.0 6.8 0.0 0.0 217.9	- 1.7 5.2 9.0 P 3.1 P 0.0 P B 0.0 P B 233.8
TA 98	DMSO Test item           NOPD	- 33 100 333 1000 2650 5300 10	18.0 48.3 93.0 200.3 376.7 616.7 1120.7 955.3	2.0 3.5 9.0 4.0 17.0 167.0 207.3 16.7	- 2.7 5.2 11.1 P 20.9 P 34.3 P 62.3 P 53.1
E.coli	DMSO Test item           4-NQO	- 33 100 333 1000 2650 5300 5	22.7 18.7 19.3 19.7 18.08.2017 15.0 15.7 1440.7	8.1 4.9 2.5 4.0 2.6 4.4 8.1 107.7	- 0.8 0.9 0.9 P 0.8 P 0.7 P 0.7 P 63.6

 

 

Table 2: SPT - With metabolic activation

Strain	Test group	Dose (µg/plate)	Mean revertants per plate	Standard deviation	Factor
TA 1535	DMSO Test item           2-AA	- 33 100 333 1000 2650 5300 2.5	9.7 13.7 10.3 16.3 20.7 5.3 0.0 136.3	3.1 3.1 4.2 1.2 3.2 0.6 0.0 10.2	- 1.4 1.1 1.7 P 2.1 P 0.6 P 0.0 P B 14.1
TA 100	DMSO Test item           2-AA	- 33 100 333 1000 2650 5300 2.5	82.7 147.7 269.7 873.3 1189.3 1140.0 26.7 2322.7	7.5 9.3 11.5 166.3 22.0 136.9 23.7 210.7	- 1.8 3.3 10.6 P 14.4 P 13.8 P 0.3 P B 28.1
TA 1537	DMSO Test item           2-AA	- 33 100 333 1000 2650 5300 2.5	9.7 15.3 18.7 79.0 141.7 6.7 0.0 212.0	2.3 4.7 3.1 4.6 19.9 1.5 0.0 15.6	- 1.6 1.9 8.2 P 14.7 P 0.7 P 0.0 P B 21.9
TA 98	DMSO Test item           2-AA	- 33 100 333 1000 2650 5300 2.5	32.7 58.3 116.7 165.3 276.0 504.0 604.0 2476.7	6.7 6.0 13.7 15.8 17.3 68.4 146.9 59.7	- 1.8 3.6 5.1 P 8.4 P 15.4 P 18.5 P 75.8
E.coli	DMSO Test item           2-AA	- 33 100 333 1000 2650 5300 60	31.0 37.0 35.0 22.7 24.7 19.7 18.0 94.7	7.5 9.6 2.0 3.1 2.1 4.6 7.0 2.1	- 1.2 1.1 0.7 P 0.8 P 0.6 P 0.6 P 3.1

 

Table 3: Decreased revertant numbers were observed at following concentrations (µg/plate)

Experiment	S9	TA 1535	TA 100	TA 1537	TA 98	E.coli
1^st-SPT	Without	2650-5300	5300	2650-5300	-	-
	With	2650-5300	5300	5300	-	2650-5300
2^nd-SPT	Without	2650	-	2650	-	n.t.
	With	-	-	2650	-	n.t.

 

- = no adverse effect observed

n.t. = not tested

 

 

Table 4: Reduced background growth was observed at following concentrations (µg/plate):

Experiment	S9	TA 1535	TA 100	TA 1537	TA 98	E.coli
1^st-SPT	Without	2650-5300	2650-5300	2650-5300	-	-
	With	5300	5300	5300	-	-
2^nd-SPT	Without	2650	2650	2650	-	n.t.
	With	-	-	2650	-	n.t.

 

- = no adverse effect observed

n.t. = not tested

 

 

 

 

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a reverse gene mutation assay (AMES test) in bacteria according to OECD Guideline 471 (BASF Colors&Effects, 2017), 4 strains of S. typhimurium (TA98, TA100, TA1535, TA1537) and E.coli WP2 uvrA were exposed to the test substance in DMSO at concentrations of 0, 33, 100, 333, 1000, 2650, and 5300 μg/plate in the presence and absence of mammalian metabolic activation (phenobarbital and β-naphthoflavone induced rat liver S9 mix). A concentration dependent increased number of his+ revertants in TA98, TA 100 and TA1535 was observed with and without metabolic activation. The adequate positive controls induced the appropriate responses in the corresponding strains. Cytotoxicity was observed. Test substance precipitation was found from about 333 μg/plate onward with and without S9 mix. There was evidence for a concentration related positive response of induced mutant colonies over background. However, for a conclusive justification for classification or non-classification, further studies in mammalian cells are necessary.

Justification for classification or non-classification

The available experimental test data with the test substance are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.

In performed Ames assay with the test substance yielded a positive result. A result obtained in a single bacterial reverse mutation test is not sufficient for classification and labelling. As a result the test substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.