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-)

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:

2017-01-09 to 2017-02-24

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

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

Test material

Test material information

Constituent 1

Reference substance name:

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-)

EC Number:

915-704-1

Molecular formula:

C36H28ClCrN7O10S2.Na / C34H26Cl2CrN8O8S2.Na / C38H30CrN6O12S2.Na

IUPAC Name:

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-)

Test material form:

solid

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

Method

Target gene:

his/trp

Species / strain

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.

Controls

Controls 1

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

Controls 2

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

Controls 3

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

Controls 4

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

Controls 5

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

Controls 6

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

Controls 7

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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

Test results 6

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

Test results 7

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

Test results 8

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

Test results 9

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

Any other information on results incl. tables

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