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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Direct Blue 080 was tested for bacteria gene mutation and resulted as non mutagenic for all the Salmonella typhimurium as well as Escherichia coli strains with as well as without metabolic activation.

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:
28/06/2016-07/11/2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
For toxicity experiment, the starting suspension (5000 µg/0.1 mL) was diluted to concentration series 10 - 5000 µg per plate. The concentration row was tested for the toxicity in strain TA 98 without metabolic activation.
No precipitation occured in any concentration. In the highest concentration (5000 µg/0.1 mL) top agar was dark coloured, but evaluation was possible. No toxicity was observed in any dose. The concentration of 5000 µg/0.1 mL was then used as maximum in the first mutagenicity experiments
Vehicle / solvent:
Water
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
Details on test system and experimental conditions:
Test procedure:
The first experiments and toxicity test: 100 uL of the test substance of required concentration, 100 uL of 16-18 h culture of tester strain of density 108-109 CFU/mL, 0.5 mL relevant buffer and 30 (100) uL of S9 post mitochondrial fraction (in case of test with metabolic activation) were added to the 2 mL of molten top agar (with trace of histidine or tryptophan) kept in a test tube at 45 +/- 3 °C. After shaking the mixture was poured into a minimal glucose agar plate.
The second experiments: 0.5 mL of relevant buffer, 100 L of the test substance of required concentration, 100 uL of 16-18 h culture of tester strain of density 108-109 CFU/mL and 30 (100) uL of S9 post mitochondrial fraction in experiment with metabolic activation were mixed and shaken at 37 +/- 1 °C for 30 minutes. Then, 2 mL of molten top agar (with trace of histidine or tryptophan) was added and the mixture was poured into a minimal glucose agar plate.
Petri dishes prepared one or the other way were incubated of 48 - 72 h at 37 +/- 1°C, the number of revertant colonies on the plate was counted manually with exception of positive controls, which were counted by an AccuCount 1000.
For an adequate estimate of variation, triplicate plating was used at each dose level except in the toxicity test with strain TA 98, where test substance was tested in duplo.
Selection of doses/toxicity: 2.0 mL of water for injection were added to 100 mg
of the test substance in a test tube to reach the maximum dose recommended in guidelines - 5000 µg per plate (per 0.1 mL). The test substance was not dissolved at the highest concentration of application form, but it dissolved in top agar, because no precipitation was observed in Petri dishes. The other concentrations should be dissolved – sponsor declared solubility in water 26.12 gL-1 (i.e. 2612 μg per 0.1 mL).
Statistics:
Spontaneous reversions in negative and positive controls were compared with historical controls in the laboratory
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Under the above-described experimental design, the test substance, Direct Blue 080, was non mutagenic for all the Salmonella typhimurium as well as Escherichia coli strains with as well as without metabolic activation.
Pre-incubation had no influence on study results.
Executive summary:

The test substance Direct Blue 080 was assayed for the mutagenicity by the Bacterial Reverse Mutation Test. The performed test was based on EU method B.13/14 Mutagenicity – Reverse mutation test using bacteria,which is analogous to the OECD Test Guideline No. 471.

Four indicator Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and one indicator Escherichia coli WP2 uvrA strainwere used. The test substance was diluted in water for injection and assayed in doses of 50 - 5000mg per plate, which were applied to plates in volume of 0.1 mL.                     

Experiments were performed without as well as with metabolic activation with a supernatant of rat liver and a mixture of cofactors. The first experiments were performed without and the second experiments with 30 minutes of pre-incubation at 37±1°C and shaking.

Although, some questionable results occurred in some experiments, in the arrangement given above, the test substance, Direct Blue 080, was non-mutagenic for all the used tester strainswithout as well as with metabolic activation.

Pre-incubation had no influence on study results.

Additional information

Direct Blue 080 was tested for bacteria gene mutation and resulted as non mutagenic for all the Salmonella typhimurium as well as Escherichia coli strains with as well as without metabolic activation.

Despite the alert on benzidine functional group contained in the molecule, the substance has been tested in itself and evaluated in a weight of evidence approach based on similar substances of the same benzidine-congener-based dyes copper complex and the fact that under reduction, no release of benzidine has been measured over 10 ppm.

Short description of key information:

Non genotoxic

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

A mutation means a permanent change in the amount or structure of the genetic material in a cell. The term ‘mutation’ applies both to heritable genetic changes that may be manifested at the phenotypic level and to the underlying DNA modifications when known (including specific base pair changes and chromosomal translocations). The term ‘mutagenic’ and ‘mutagen’ will be used for agents giving rise to an increased occurrence of mutations in populations of cells and/or organisms. The more general terms ‘genotoxic’ and ‘genotoxicity’ apply to agents or processes which alter the structure, information content, or segregation of DNA, including those which cause DNA damage by interfering with normal replication processes, or which in a non- physiological manner (temporarily) alter its replication. Genotoxicity test results are usually taken as indicators for mutagenic effects.

Regarding Direct Blue 080 no classification for genetic toxicity is warranted under Regulation 1272/2008.