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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

For C.I. Direct Blue 264 no mutagenic or clastogenic effects were observed in a bacterial reverse mutation assay, in an in vitro gene mutation assay (HPRT) and in an in vitro micronucleus test.

Link to relevant study records

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-GLP
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
non GLP; content not given
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine biosynthesis
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Negative control
C.I. Direct Blue 264: 5000, 1581, 500, 158, 50, 16 µg/plate
Positive controls:
Na-azide: 10 µg/plate (TA1535)
Nitrofurantoin: 0.2 µg{plate (TA100)
4-nitro-1,2-phenylene diamine: 10 µg/plate (TA1537); 0.5 µg/plate (TA98)
Cumene: 50 µg/plate (TA102)
2-Aminoanthracene: 3 µg/plate

Due to precipitation of the substance, doses ranging from 100 to 3200µg/tube were chosen for the repeat tests.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
cumene hydroperoxide
other: nitrofurantoin, 4-nitro-1,2-phenylene diamine, 2-aminoantracene
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
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
Remarks on result:
other: all strains/cell types tested

No bacteriotoxic effects up to 158 µg/plate; total bacterial count remained unchanged an no growth inhibition was seen.

At doses above 158 µg/plate strain-specific cytotoxicity.

Compound precipitation started at 500 µg/plate; and 5000 µg/plate could not be used for evaluation.

Conclusions:
Interpretation of results: negative
Executive summary:

No evidence of mutagenic activity in plate incorporation test and preincubation test in the presence and absence of S9 mix.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD TG 487 In vitro mammalian cell micronucleus test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
Micronucleus test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix; Aroclor 1254-induced male Sprague-Dawley rats.
Test concentrations with justification for top dose:
The 4 hours treatment was conducted with concentrations of 2.5, 5, 10, 25, 50, 100 and 200 µg/mL without and with S9 mix.

In the independent repeat test, the treatment time in the experiment without S9 mix was extended to 24 hours with concentrations of 1, 2.5, 5, 10, 25, 50 and 100 µg/mL.
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: vinblastine sulfate salt
Species / strain:
Chinese hamster lung fibroblasts (V79)
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
Remarks on result:
other: all strains/cell types tested

The micronucleus test showed no biologically relevant increase in the frequencies of micronucleus containing V79 cells treated with C.I. Direct Blue 264 in the absence ( 4 hours or 24 hours treatment) or in the presence of S9 mix.

Conclusions:
Interpretation of results: negative
Executive summary:

Evaluation of the data does not indicate that C.I. Direct Blue 264 is a mutagen in the micronucleus test in vitro, when tested up to cytotoxic and precipitating concentrations in the absence or presence of metabolic activation.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
The study was performed to investigate the potential of C.I. Direct Blue 264 to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT locus in V79 cells
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Experiment I
Exposure period: 4 hours (with and without S9 mix)
Concentrations without S9 mix: 125, 250, 500, 750 and 1000(p) µg/mL
Concentrations with S9 mix: 15.6, 31.3, 62.5(p), 125(p) and 250(p) µg/mL

Experiment II
Exposure period: 4 hours (with and without S9 mix)
Concentrations without S9 mix: 3.9, 7.8, 15.6 and 31 µg/mL
Concentrations with S9 mix: 15.6, 31.3, 62.5(p), 125(p) and 250(p) µg/mL

p = precipitation visible to the unaided eye
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Species / strain:
Chinese hamster lung fibroblasts (V79)
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
Remarks on result:
other: all strains/cell types tested

The study was performed to investigate the potential of C.I. Direct Blue 264 to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation (S9 mix) and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The highest concentration of the pre-experiment (2000 μg/mL) was limited by the solubility properties of C.I. Direct Blue 264 in DMSO and aqueous medium. The maximum concentration of the main experiments was limited by cytotoxic effects and precipitation of the test item.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported C.I. Direct Blue 264 did not induce gene mutations at the HPRT locus in V79 cells. Therefore, C.I. Direct Blue 264 is considered to be non-mutagenic in this HPRT assay.

 

Conclusions:
Interpretation of results (migrated information):
negative
Executive summary:

The study was performed to investigate the potential of C.I. Direct Blue 264 to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation (S9 mix) and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The highest concentration of the pre-experiment (2000 μg/mL) was limited by the solubility properties of C.I. Direct Blue 264 in DMSO and aqueous medium. The maximum concentration of the main experiments was limited by cytotoxic effects and precipitation of the test item.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported C.I. Direct Blue 264 did not induce gene mutations at the HPRT locus in V79 cells. Therefore, C.I. Direct Blue 264 is considered to be non-mutagenic in this HPRT assay.

 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

No evidence of mutagenic activity was observed in a reliable bacterial reverse mutation assay using 5 bacterial strains (TA 1537, TA1535, TA102, TA100, TA98) similar to OECD TG 471, but not conducted according to GLP.

No evidence of mutagenic activity was reported in a gene mutation assay in Chinese Hamster V79 cells (HPRT) conducted according to OECD TG 474 and GLP.

No clastogenic or aneugenic activity was observed in an in vitro mammalian cell micronucleus test conducted according to OECD TG 487 and GLP.


Justification for selection of genetic toxicity endpoint
For C.I. Direct Blue 264 no mutagenic or clastogenic effects were observed in a bacterial reverse mutation assay, in an in vitro gene mutation assay (HPRT) and in an in vitro micronucleus test.

Justification for classification or non-classification

For C.I. Direct Blue 264 no mutagenic or clastogenic effects were observed in a bacterial reverse mutation assay, in an in vitro gene mutation assay (HPRT) and in an in vitro micronucleus test.

According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.