Reaction mass of Sodiumbis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and Sodium [1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and Sodiumbis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-)

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Sodiumbis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and Sodium [1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and Sodiumbis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-)

EC Number:

915-756-5

Molecular formula:

C32H18NaCrN6O8

IUPAC Name:

Reaction mass of Sodiumbis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and Sodium [1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and Sodiumbis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-)

Details on test material:

- Name of test material (as cited in study report): Eukesolar Black ER Liquid - dried
- Physical state: powder
- Analytical purity: 99.11 area-% (HPLC, 260 nm) 99.71 area-% (HPLC, 375 nm) (For details see analytical report-No.: 15L00121)
- Lot/batch No.: Dye powder sample 14/103 from Material no. 52629782; batch no. M-R/G
- Expiration date of the lot/batch: June 09, 2016
- Stability under test conditions: The stability of the test item under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Storage condition of test material: Room temperature

Method

Target gene:

HIS/TRP

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

33 μg - 6300 μg/plate (SPT, all tester strains)
3.3 μg - 1000 μg/plate (Prival; TA 100, TA1537 and TA 98)

Vehicle / solvent:

- Vehicle 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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

congo red

other: 2-aminoanthracene (2-AA), N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylenediamine

Remarks:

S9-mix from rats and hamsters was used

Details on test system and experimental conditions:

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

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

Acceptance criteria
Generally, the experiment was considered valid if the following criteria were met:
• The number of revertant colonies in the negative controls was within the range of the
historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in
the number of revertant colonies within the range of the historical positive control data or
above.
• Fresh bacterial culture containing approximately 10E9 cells per mL were used.

Assessment 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 mutation 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
range under all experimental conditions in at least two experiments carried out
independently of each other.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

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

Test results 2

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

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

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

Test results 4

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

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

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

Any other information on results incl. tables

MUTAGENICITY

Standard plate test

Tests without S9 mix

TA 1535: No relevant increase in the number of his+ revertants.

TA 100: Increase in the number of his+ revertants at 333 and 1000 μg/plate (factors 2.1 and 2.1, respectively).

TA 1537: Increase in the number of his+ revertants at 333 and 1000 μg/plate (factors 3.7 and 4.3, respectively).

TA 98: Increase in the number of his+ revertants at 33, 100, 333, 1000 and 3150 μg/plate (factors 4.0, 7.8, 12.8, 18.7 and 23.8,

respectively).

E. coli WP2 uvrA: No relevant increase in the number of trp+ revertants.

Tests with S9 mix

TA 1535: No relevant increase in the number of his+ revertants.

TA 100: Increase in the number of his+ revertants at 100, 333 and 1000 μg/plate (factors 2.2, 2.5 and 2.3, respectively).

TA 1537: Slight increase in the number of his+ revertants at 333 μg/plate (Factor 2.9).

TA 98: Increase in the number of his+ revertants at 33, 100, 333, 1000 and 3150 μg/plate (factors 5.0, 7.3, 13.1, 13.7 and 17.1,

respectively).

E. coli WP2 uvrA: No relevant increase in the number of trp+ revertants.

Prival preincubation test

Tests without S9 mix

TA 100: Increase in the number of his+ revertants at 333 and 1000 μg/plate (factors 1.9 and 2.6, respectively).

TA 1537: Increase in the number of his+ revertants at 333 and 1000 μg/plate (factors 6.0 and 7.8, respectively).

TA 98: Increase in the number of his+ revertants at 10, 33, 100, 333 and 1000 μg/plate (factors 3.3, 5.0, 8.7, 12.5 and 33.6,

respectively).

Tests with S9 mix

TA 100: Increase in the number of his+ revertants at 333 and 1000 μg/plate (factors 2.1 and 2.1, respectively).

TA 1537: Increase in the number of his+ revertants at 333 and 1000 μg/plate (factors 4.0 and 4.1, respectively).

TA 98: Increase in the number of his+ revertants at 33, 100, 333 and 1000 μg/plate (factors 2.1, 4.3, 8.9 and 14.5, respectively).

TOXICITY

A bacteriotoxic effect (decrease in the number of his+ or trp+ revertants) was observed in the standard plate test depending on the strain and test conditions from about 1000 μg/plate onward.

In the prival preincubation assay bacteriotoxicity (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was not observed up to the highest required concentration.

SOLUBILITY

Test substance precipitation was found from 333 μg/plate onward with and without S9 mix.

DISCUSSION

According to the results of the present study the test substance led to an evident and dose dependent increase in the number of his+ revertants with the tester strains TA 100, TA 1537 and TA 98, all with and without S9 mix. The increase of revertants was reproducible in two experiments (standard plate and prival preincubation test) carried out independently of each other. Based on the recent assessment criteria the test substance has to be considered positive.

Besides, 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 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 the range of the historical positive control data or above.

CONCLUSION

Thus, under the experimental conditions chosen here, it is concluded that Eukesolar Black ER Liquid - dried is a potent mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.