Reaction mass of Chromate(1-), [2-(3-chlorophenyl)-2,4-dihydro-4-[[2-hydroxy-5-(methylsulfonyl)phenyl]azo]-5-methyl-3H-pyrazol-3-onato(2-)][methyl [7-hydroxy-8-[[2-hydroxy-5-(methylsulfonyl)phenyl]azo]-1-naphthalenyl]carbamato(2-)]-, sodium and sodium bis[2-(3-chlorophenyl)-2,4-dihydro-4-[[2-hydroxy-5-mesylphenyl]azo]-5-methyl-3H-pyrazol-3-onato(2-)]chromate(1-) and sodium bis[methyl [7-hydroxy-8-[[2-hydroxy-5-mesylphenyl]azo]-1-naphthyl]carbamato(2-)]chromate(1-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an bacterial reverse mutation assay according to OECD Guideline 471 (with Prival modification) (BASF Colors&Effects, 2017), the test substance showed no 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:

2017-01-09 to 2017-02-24

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:

yes

Remarks:

incl. Prival Modification, according to OECD Guideline 471, Paragraph 16

Principles of method if other than guideline:

Prival Modification
- Short description of test conditions: The Prival preincubation test is a modification of the standard Ames reverse mutation assay (1), in which flavin mononucleotide (FMN), liver S9 mix from uninduced hamsters and a preincubation step are used to facilitate azo reduction and the detection of the resulting mutagenic aromatic amines.

(1) Prival, M.J.; Mitchell, V.D.:
Analysis of a method for testing azo dyes for mutagenicity in Salmonella typhimurium in the presence of flavin monoucleotide and hamster liver S9. Mut. Res., 97, 103 - 116 (1982)

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: 001-140902, Test substance No.: 16/0083-1
- Expiration date of the lot/batch: 2019-06-07

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

OTHER SPECIFICS: solid, 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/beta-naphthoflavone induced rat liver S9 fraction (STP)

Test concentrations with justification for top dose:

1st (STP) and 2nd (Prival) experiment:
0, 33, 100, 333, 1000, 2600, and 5200 µg/plate
3rd experiment (Prival):
0, 3.3, 10, 33, 100, 333, and 1000 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. 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 5.2 mg/plate was used as top dose.

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 rat liver 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: 2-aminoanthracene (2-AA)

Remarks:

With hamster liver S9 mix: 10 µg/plate, TA 1535, TA 100, TA 1537, TA 98, Escherichia coli WP2 uvrA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

congo red

Remarks:

With hamster liver S9 mix: 210 µg/plate, TA 98

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 (SPT); preincubation (Prival)

DURATION
- Preincubation period: 30 min (Prival)
- Exposure duration: 48 - 72 hours (SPT, Prival)

NUMBER OF REPLICATIONS: 3

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

Evaluation criteria:

Mutagenicity
Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments. In general, six doses of the test substance were tested with a maximum of 5 mg/plate, and triplicate plating was used for all test groups at least in the 1st Experiment. Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments were based on the findings of the 1st Experiment.

Toxicity
Toxicity detected by a
• decrease in the number of revertants (factor < 0.6)
• clearing or diminution of the background lawn (= reduced his- or trp- background growth)
was recorded for all test groups both with and without S9 mix in all experiments and indicated in the tables. Single values with a factor < 0.6 were not detected as toxicity in low dose groups.

Solubility
If precipitation of the test material was observed, it would be recorded and indicated in the tables. As long as precipitation did not interfere with the colony scoring, 5 mg/plate was generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose at least in the 1st Experiment even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate might also be tested in repeat experiments for further clarification/substantiation.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5200 µg/plate, without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 1000 µgplate

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:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 1000 µg/plate

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:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 333 µg/plate, witout metabolic activation; ≥ 2600 µg/plate with metabolic activation

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:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 1000 µg/plate

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:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 333 µg/plate without metabolic activation; ≥ 1000 µg/plate with metabolic activation

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:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 2600 µg/plate

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:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 1000 µg/plate without metabolic activation; ≥ 5200 µg/plate with metabolic activation

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 and 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 1000 µg/plate onward with and without S9 mix.

Remarks on result:

other: SPT

Table 1: STP 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 2600 5200 5	9.7 10.3 12.0 10.0 5.7 7.7 4.7 5852.7	2.1 2.1 5.7 2.6 1.5 0.6 2.1 151.5	- 1.1 1.2 1.0 0.6 P 0.8 P 0.5 P B 605.4
TA 100	DMSO Test item           MNNG	- 33 100 333 1000 2600 5200 5	87.7 101.0 100.0 93.7 59.7 11.0 0.0 4626.7	15.9 3.6 13.1 13.6 3.8 3.6 0.0 117.8	- 1.2 1.1 1.1 0.7 P 0.1 P 0.0 P B 52.8
TA 1537	DMSO Test item           AAC	- 33 100 333 1000 2600 5200 100	5.7 9.0 5.7 6.0 1.3 4.3 1.3 1167.0	2.1 0.0 2.5 6.1 0.6 2.5 0.6 281.5	- 1.6 1.0 1.1 0.2 P 0.8 P 0.2 P B 205.9
TA 98	DMSO Test item           NOPD	- 33 100 333 1000 2600 5200 10	16.0 16.7 14.7 14.5 8.3 9.3 4.3 670.3	1.0 0.6 3.1 6.4 1.5 3.1 2.1 83.5	- 1.0 0.9 0.9 0.5 P 0.6 P 0.3 P B 41.9
E. coli	DMSO Test item           4-NQO	- 33 100 333 1000 2600 5200 5	25.0 25.0 26.7 24.3 12.7 20.3 24.0 1206.0	2.0 5.3 8.6 5.1 2.5 6.7 5.6 5.3	- 1.0 1.1 1.0 0.5 P 0.8 P 1.0 P 48.2

 

Key to plate postfix codes

B:    Reduced background growth

P:    Precipitation

 

 

Table 2: STP 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 2600 5200 2.5	8.3 9.3 8.7 7.3 10.7 7.7 6.3 294.3	1.5 1.5 2.1 3.8 2.1 1.2 0.6 22.0	- 1.1 1.0 0.9 1.3 P 0.9 P 0.8 P 35.3
TA 100	DMSO Test item           2-AA	- 33 100 333 1000 2600 5200 2.5	107.0 121.7 116.0 104.0 108.0 52.3 3.7 2883.3	10.5 22.8 3.6 9.5 31.2 6.4 2.1 57.0	- 1.1 1.1 1.0 1.0 P 0.5 P 0.0 P 26.9
TA 1537	DMSO Test item           2-AA	- 33 100 333 1000 2600 5200 2.5	7.3 5.7 4.7 5.7 4.7 3.3 4.3 185.3	4.2 2.1 2.1 1.2 1.5 0.6 1.2 61.8	- 0.8 0.6 0.8 0.6 P 0.5 P 0.6 P 25.3
TA 98	DMSO Test item           2-AA	- 33 100 333 1000 2600 5200 2.5	33.7 35.3 35.3 25.7 14.7 10.7 7.0 2431.3	2.3 5.0 4.6 2.3 3.2 2.9 2.0 44.4	- 1.0 1.0 0.8 0.4 P 0.3 P 0.2 P 72.2
E. coli	DMSO Test item           2-AA	- 33 100 333 1000 2600 5200 60	25.0 21.0 21.3 14.7 20.7 20.0 18.3 205.7	7.2 1.7 2.1 1.5 4.0 3.5 6.0 19.9	- 0.8 0.9 0.6 0.8 P 0.8 P 0.7 P 8.2

 

Key to plate postfix codes

P:    Precipitation

 

 Table 3: Prival preincubation test 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 2600 5200 5	14.7 11.3 14.7 14.7 4.0 0.0 0.0 2440.3	1.2 2.5 1.5 5.1 0.0 0.0 0.0 127.4	- 0.8 1.0 1.0 0.3 P 0.0 B P 0.0 B P 166.4
TA 100	DMSO Test item           MNNG	- 33 100 333 1000 2600 5200 5	125.0 115.3 117.0 104.7 69.7 11.3 0.0 1567.7	20.9 11.5 19.3 10.3 44.7 3.2 0.0 246.7	- 0.9 0.9 0.8 0.6 P 0.1 P 0.0 P B 12.5
TA 1537	DMSO Test item           AAC	- 33 100 333 1000 2600 5200 100	9.3 6.0 7.0 4.7 4.3 0.0 0.0 462.7	4.9 3.6 3.6 2.1 1.5 0.0 0.0 225.2	- 0.6 0.8 0.5 B 0.5 P B 0.0 P B 0.0 P B 49.6
TA 98	DMSO Test item           NOPD	- 33 100 333 1000 2600 5200 10	18.7 16.0 13.3 7.0 4.7 0.0 0.0 455.3	7.0 4.4 3.2 3.5 1.5 0.0 0.0 74.8	- 0.9 0.7 0.4 0.3 P 0.0 P B 0.0 P B 24.4
E. coli	DMSO Test item           4-NQO	- 33 100 333 1000 2600 5200 5	16.7 19.0 26.3 21.7 11.7 15.7 11.0 321.3	2.1 7.9 9.6 8.6 5.5 3.2 2.6 45.1	- 1.1 1.6 1.3 0.7 P 0.9 P 0.7 P 19.3

 

Key to plate postfix codes

B:    Reduced background growth

P:    Precipitation

 

Table 4: Prival preincubation test 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 2600 5200 10	9.3 8.7 7.0 9.0 6.0 2.7 0.0 268.3	2.3 2.3 1.0 3.5 1.0 0.6 0.0 27.1	- 0.9 0.8 1.0 0.6 P 0.3 P 0.0 P B 28.8
TA 100	DMSO Test item           2-AA	- 33 100 333 1000 2600 5200 10	99.3 113.3 112.7 119.3 96.0 113.7 7.0 1035.7	10.1 11.4 15.3 10.6 8.7 1.5 4.0 115.4	- 1.1 1.1 1.2 1.0 P 1.1 P 0.1 P B 10.4
TA 1537	DMSO Test item           2-AA	- 33 100 333 1000 2600 5200 10	11.0 14.0 6.0 9.7 9.3 0.0 0.0 399.3	5.3 2.6 4.4 5.9 3.5 0.0 0.0 27.4	- 1.3 0.5 0.9 0.8 P 0.0 P 0.0 P B 36.3
TA 98	DMSO Test item           2-AA CoR	- 33 100 333 1000 2600 5200 10 210	25.7 25.0 21.3 26.0 8.3 2.0 0.0 635.7 269.3	3.1 6.2 3.2 7.8 2.3 1.0 0.0 99.9 79.2	- 1.0 0.8 1.0 0.3 P 0.1 P 0.0 P B 24.8 10.5
E. coli	DMSO Test item           2-AA	- 33 100 333 1000 2600 5200 10	20.3 16.7 11.3 31.0 15.7 15.7 14.3 536.7	5.7 3.2 1.5 1.7 4.7 6.5 0.6 180.4	- 0.8 0.6 1.5 0.8 P 0.8 P 0.7 P 26.4

 

Key to plate postfix codes

B:    Reduced background growth

P:    Precipitation

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Additional information

In a reverse gene mutation assay in bacteria according to OECD Guideline 471 (incl. Prival modification) (BASF Colors&Effects, 2017), 4 strains of S. typhimurium (TA98, TA100, TA1535, TA1537) and E.coli WP2 uvrA were exposed to ≥ 96 % the test substance in DMSO at concentrations of 0, 33, 100, 333, 1000, 2600, and 5200 μg/plate in the presence and absence of mammalian metabolic activation (standard plate test, SPT: phenobarbital and β-naphthoflavone induced rat liver S9 mix; Prival: uninduced hamster liver S9 fraction).  The modified Bacterial Reverse Mutation Test according to Prival et al. facilitates azo reduction and is therefore the most appropriate method for the investigation of azo-dyes and diazo compounds.

The test substance was tested up to a limit concentration of 5200 µg/plate. No increased number of revertant colonies was observed. The adequate positive controls with and without metabolic activation induced the appropriate responses in the corresponding strains. Negative controls were viable. A bacteriotoxic effect was observed depending on the strain and test conditions from about 333 μg/plate onward.

Under the experimental conditions chosen here, it is concluded that the test substance is not mutagenic in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Justification for classification or non-classification

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

Based on available data on genetic toxicity, the test item is not considered to be classified for genetic toxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.