Cobaltate(3-), bis[6-amino-5-[(2-hydroxy-3,5-dinitrophenyl)azo]-1-naphthalenesulfonato(3-)]-, sodium

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The complete read across justification is detailed in section 13; source study has reliability 1.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Similar Substance 01

IUPAC Name:

Similar Substance 01

Test material form:

solid

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 (rat and hamster)

Test concentrations with justification for top dose:

33 μg - 5 200 μg/plate (SPT)
33 μg - 5 200μg/plate (Prival)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: [DMSO]
- Justification for choice of solvent/vehicle: DMSO was a suitable vehicle

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, N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylenediamine

Details on test system and experimental conditions:

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

Standard plate test
The experimental procedure of the standard plate test (plate incorporation method) was based on the method of Ames et al.

Salmonella typhimurium
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 ml agar (0.8 % w/v agar + 0.6% w/v NaCl) and 10 ml amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) were kept in a water bath at about 42 - 45 °C, and the remaining components were added in the following order:
0.1 ml test solution or vehicle (negative control)
0.1 ml fresh bacterial culture
0.5 ml S9 mix (with metabolic activation)
or
0.5 ml phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds.
After incubation at 37 °C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments
Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.

Escherichia coli
Test tubes containing 2-ml portions of soft agar (overlay agar), which consists of 100 ml agar (0.8 % w/v agar + 0.6% w/v NaCl) and 10 ml amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 ml test solution or vehicle (negative control)
0.1 ml fresh bacterial culture
0.5 ml S9 mix (with metabolic activation)
or
0.5 ml phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds.
After incubation at 37 °C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments
Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.

Prival Preincubation Test
The experimental procedure is based on the method described by Yahagi et al. and Matsushima et al. and has been modified further to include reductive conditions by Prival et al.
0.1 ml test solution or vehicle (negative control), 0.1 ml bacterial suspension and 0.5 ml S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) were incubated at 30 °C for 30 minutes using a shaker. Subsequently, 2 ml soft agar which consists of 100 ml agar (0.8% w/v agar + 0.6% w/v NaCl) and 10 ml amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin or 0.5 mM tryptophan) were added. After mixing, the samples were poured onto the Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.
Composition of the minimal glucose agar:
980 ml purified water
20 ml Vogel-Bonner E medium
15 g Difco bacto agar
5 g D-glucose, monohydrate
After incubation at 37 °C for 48 – 72 hours in the dark, the bacterial colonies were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perseptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.


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 10^9 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

Species / strain:

S. typhimurium TA 1537

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 2

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 3

Species / strain:

S. typhimurium TA 100

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

Species / strain:

S. typhimurium TA 98

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 5

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

Additional information on results:

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

SOLUBILITY
Test substance precipitation was found from about 2600 μg/plate onward with and without S9 mix.

CONTROL
The number of revertant colonies in the negative controls was within or marginally below the range of the historical negative control data for each tester strain, with or without metabolic activation.
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.

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions, test substance resulted as not a mutagenic in the bacterial reverse mutation test in absence and presence of metabolic activation.

Executive summary:

Method

Test substance was tested for its mutagenic potential based on the ability to induce point mutations in bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay (Ames standard plate test and Prival preincubation test, OECD guideline 471). The modified bacterial reverse mutation test according to Prival facilitates azo reduction and is therefore the most approriate method for the investigation of azo-dyes and diazo compounds.

Tested strains were TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA in a dose range of 33 to 5200 μg/plate, for both SPT and Prival with and without metabolic activation.

Results

Precipitation of the test substance was found from about 2600 μg/plate onward with and without S9 mix.

Toxicity in bacteria was occasionally observed depending on the strain and test conditions from about 1000 μg/plate onward.

The results of negative as well as positive controls fulfilled the acceptance criteria of this study.

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 Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.