Tetrasodium tripotassium (.my.-((2,2'-((3-imino-6-sulfonatophenyl-(N-methyl)-imino)bis((6-chloro-1,3,5-triazin-4,2-diyl)imino(2-hydroxy-5-sulfonato-3,1-phenylen)azo(phenylmethylen)azo))bis(4-sulfonatobenzoato))-dicuprate(7-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

1992

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 mix

Test concentrations with justification for top dose:

- Standard plate test: 0; 36; 180; 900; 4500 and 9000 µg/plate
- Pre-incubation test: 0; 36; 180; 900; 4500 and 9000 µg/plate

Vehicle / solvent:

Water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

other: see Remarks

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 20 min (for preincubation test only)
- Exposure duration: 48-72 h
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays):

SPINDLE INHIBITOR (cytogenetic assays):

STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS:

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:

NUMBER OF CELLS EVALUATED:

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:

DETERMINATION OF CYTOTOXICITY
- Method:
— decrease in the number of revertants
— clearing or diminution of the background lawn (= reduced his or trp background growth)
— reduction in the titer
- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):

- OTHER:

Evaluation criteria:

Acceptance criteria
Generally, the experiment is to be considered valid if the following criteria are met:
• The number of revertant colonies in the negative controls was within the normal range of the historical control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control articles both with and without S-9 mix induced a significant increase in the number of revertant colonies within the range of the historical control data.
• The titer of viable bacteria was >10E9/ml.

Evaluation criteria
The test chemical is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e, about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system.

A test substance is generally considered nonmutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Key result

Species / strain:

other: All tested strains

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

Conclusions:

Under the conditions of the study, the test substance was therefore not mutagenic in the Salmonella typhimurium/ Escherichia coli reverse mutation assay.

Executive summary:

A study was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471 and EU Method B13/B14, in compliance with GLP. The tested strains were Salmonella typhimurium TA 1535, TA 100, TA 1537 and TA 98, and Escherichia coli strain WP2 uvrA. A standard plate and a pre-incubation test were run, both in dose range of 36 to 9000 µg test substance / plate, with and without Arochlor-induced rat liver S9 mix. No precipitation of the test substance and no bacterio-toxic effect were found. An increase in the number of his+ or trp+ revertants was not observed in either of the assays, either with or without S9 mix. Under the conditions of the study, the test substance was therefore not mutagenic in the Salmonella typhimurium/ Escherichia coli reverse mutation assay (Engelhardt, 2000).

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990/1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9-Mix

Test concentrations with justification for top dose:

A preliminary cytotoxicity test was performed to determine the concentrations to be used in the mutagenicity assay.
Cytotoxicity was characterized by the percentages of mitotic suppression in comparison with the controls by counting 1000 cells per concentration and duplicate. The experimental conditions in this pre-experimental phase were the same as described below for the mutagenicity assay.
Treatment in the pre-experimental phase was performed with the following concentrations:
without S9 mix:
24 h: 0.01; 0.03; 0.10; 0.30; 0.60; 1.00; 2.50; 5.00 mg/ml
48 h: 0.10; 0.30; 0.60; 1.00; 2.50; 5.00 mg/ml
with S9 mix:
24 h: 0.01; 0.03; 0.10; 0.30; 0.60; 1.00; 2.50; 5.00 mg/ml
48 h: 0.10; 0.30; 0.60; 1.00; 2.50; 5.00 mg/ml

DOSE SELECTION
Based an the results from this pre-experimental phase, several concentrations to be evaluated in the chromosomal aberration assay were chosen. The highest concentration should suppress if possible mitotic activity (% cells in mitosis) by approximately 20 % - 50 %, but not so great a reduction that insufficient
scorable cells could be found.
Because the experimental conditions of the pre-experimental phase met the requirements for the evaluation of chromosomal aberrations one (48 h) and three (24 h) concentrations of this preexperimental phase were chosen for scoring.
With the highest dose level 2.5 or 5.0 mg/ml, the mitotic activity of the lymphocytes was reduced at each fixation intervals except interval 48 h (without S9 mix). Cultures treated with higher dose levels as indicated for the respective intervals could not be evaluated due to insufficient scorable cells. Higher concentrations than 5.0 mg/ml were not applied.

Vehicle / solvent:

serum free culture medium

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in suspension
- Cell density at seeding (if applicable):

DURATION
- Preincubation period: 48 h
- Exposure duration: 4 and 24 h
- Expression time (cells in growth medium): 24 and 48 h
- Selection time (if incubation with a selection agent): 3 h
- Fixation time (start of exposure up to fixation or harvest of cells):


SPINDLE INHIBITOR (cytogenetic assays): colcemide

STAIN (for cytogenetic assays): giemsa

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:

NUMBER OF CELLS EVALUATED:

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 100

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:
- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):

- OTHER:

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY.
The chromosomal aberration assay is considered acceptable if it meets the following criteria:
a) the numbers of chromosomal aberrations found in the negative and/or solvent control cultures should reasonably fall within the laboratory historical control data range.
b) the positive control substances should produce a significant increase in the frequencies of aberrations.

EVALUATION OF RESULTS
The test article will be classified as mutagenic if it induces a significantly increased aberration rate with at least one of the concentrations tested as compared with the negative control.
A test article which produces no significant positive response at any test point will be regarded as non-mutagenic in this assay.
A statistical significance can be confirmed by means of a chisquare test.

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the chi-square test. Evaluation was performed only for cells carrying aberrations exclusive gaps.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(>= 2500 µg/ml)

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

The test article did not induce structural chromosome aberrations as determined by the chromosomal aberration test in human lymphocytes in vitro.
Therefore, Reactive Blue 1463 is considered to be non-mutagenic in this chromosomal aberration test.

Executive summary:

The test article was assessed for its potential to induce structural chromosome aberrations in human lymphocytes in vitro.

Preparation of chromosomes was done 24 h (low, medium and high dose), and 48 h (high dose) after start of treatment with the test article. The treatment interval was 4 h. In each experimental group two parallel cultures were used. Per culture 100 metaphases were scored for structural chromosomal aberrations.

The following dose levels were evaluated:

- without S9 mix:

24 h: 0.1; 1.0; 2.5 mg/ml

48 h: 2.5 mg/ml

- with S9 mix:

   24 h: 0.1; 0.3; 1.0; 2.5 mg/ml

48 h: 5.0 mg/ml

The concentration of the test article applied had been determined using the mitotic index as indicator for toxicity response.

Treatment of the cells with the highest concentrations reduced the mitotic index at fixation intervals 24 (with and without S9 mix) and 48 h (with S9 mix).

There was no biologically relevant increase in cells with aberrations after treatment with the test article either with or without metabolic activation by S9 mix at both fixation intervals.

Appropriate reference mutagens were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

CONCLUSION

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article did not induce structural chromosome aberrations as determined by the

chromosomal aberration test in human lymphocytes in vitro.

Therefore, Reactive Blue 1463 is considered to be non-mutagenic in this chromosomal aberration test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A study was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471 in compliance with GLP. The tested strains were Salmonella typhimurium TA 1535, TA 100, TA 1537 and TA 98. A standard plate and a pre-incubation test were run, both in dose range of 20 to 5000 µg test substance / plate, with and without Arochlor-induced rat liver S9 mix. No precipitation of the test substance and no bacterio-toxic effect were found. An increase in the number of his+ revertants was not observed in either of the assays, either with or without S9 mix. Under the conditions of the study, the test substance was therefore not mutagenic in the Salmonella typhimurium mutation assay (Engelhardt, 1990).

A second study was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471 and EU Method B13/B14, in compliance with GLP. The tested strains were Salmonella typhimurium TA 1535, TA 100, TA 1537 and TA 98, and Escherichia coli strain WP2 uvrA. A standard plate and a pre-incubation test were run, both in dose range of 36 to 9000 µg test substance / plate, with and without Arochlor-induced rat liver S9 mix. No precipitation of the test substance and no bacterio-toxic effect were found. An increase in the number of his+ or trp+ revertants was not observed in either of the assays, either with or without S9 mix. Under the conditions of the study, the test substance was therefore not mutagenic in the Salmonella typhimurium/ Escherichia coli reverse mutation assay (Engelhardt, 2000).

The test article was assessed for its potential to induce structural chromosome aberrations in human lymphocytes in vitro.

Preparation of chromosomes was done 24 h (low, medium and high dose), and 48 h (high dose) after start of treatment with the test article. The treatment interval was 4 h. In each experimental group two parallel cultures were used. Per culture 100 metaphases were scored for structural chromosomal aberrations.

The following dose levels were evaluated: - without S9 mix: 24 h: 0.1; 1.0; 2.5 mg/ml; 48 h: 2.5 mg/ml

- with S9 mix:    24 h: 0.1; 0.3; 1.0; 2.5 mg/ml; 48 h: 5.0 mg/ml

The concentration of the test article applied had been determined using the mitotic index as indicator for toxicity response. Treatment of the cells with the highest concentrations reduced the mitotic index at fixation intervals 24 (with and without S9 mix) and 48 h (with S9 mix). There was no biologically relevant increase in cells with aberrations after treatment with the test article either with or without metabolic activation by S9 mix at both fixation intervals.

Appropriate reference mutagens were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article did not induce structural chromosome aberrations as determined by the

chromosomal aberration test in human lymphocytes in vitro. Therefore, Reactive Blue 1463 is considered to be non-mutagenic in this chromosomal aberration test.

Justification for classification or non-classification

The assays for mutagenicity and cytogenicity were negative. Hence classification in not needed according to CLP (EC 1272/2008) criteria.