Reaction mass of copper complex of [(2,6-difluoroheterocycl-4-yl)amino]hydroxy{[2-hydroxy-3-sulfonato-5-(vinylsulfonyl)phenyl]diazenyl}naphthalene sulfonic acid, dialkali salt and copper complex of [(2,6-difluoroheterocycl-4-yl)amino]-hydroxy{[2-hydroxy-3-sulfonato-5-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl]diazenyl}naphthalene sulfonic acid, trialkali salt

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genetic toxicity of the test substance was assessed using an Ames test according to OECD Guideline 471, and an in vitro chromosomal aberration test in Chinese hamster lungs cells in accordance with OECD Guideline 473. The test substance was found to be non-genotoxic, with an without addition of metabolic activation. Cytotoxicity was observed in both tests. In addition, an in vitro Unscheduled DNA Synthesis Assay (UDS) was performed with a structural analogue to detect potential gene mutations in mammalian cells

Link to relevant study records

Reference

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:

04 Nov 2002 to 2 May 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

mammalian cell line, other: Chin. Hamster, V79-Zellen

Details on mammalian cell type (if applicable):

cell bank of 'Genetic Toxicology', Aventis Pharma Germany GmbH, Pro Tox, cell line V79 of Chinese hamster lung fibroblasts. Cultured in minimal essential medium (MEM) with Earles's salts and L-glutamine

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix; Aroclor induced rat liver

Test concentrations with justification for top dose:

First experiment:
Concentration range (without and with metabolic activation):625, 1250 and 2500 µg/ml

Second experiment/part II, without metabolic activation: 250, 500 and 750 µg/ml

Second experiment:
Concentration range in the main test (with metabolic activation): 625, 1250 and 2500 µg/ml
Concentration range in the main test (without metabolic activation): 125, 250 and 500 µg/ml

Vehicle / solvent:

cell culture medium (MEM)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

ethylmethanesulphonate

Remarks:

None

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

cyclophosphamide

Remarks:

None

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
-3h and 20 h (Experiment 1, with and without S9-mix)
- 20 h (Experiment 2, part II, without S9 mix)
- 3 and 28 h (Experiment 2, with s9 mix)
- 28 h (Experiment 2, without S9 mix)

SPINDLE INHIBITOR (cytogenetic assays): Colcemide (approx. 0.05 µg/mL /culture medium)
STAIN (for cytogenetic assays): 2 % (w/v) orcein solution

NUMBER OF REPLICATIONS: Two

NUMBER OF CELLS EVALUATED: 1000 cells of each cell culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes

Evaluation criteria:

The test substance is classified as clastogenic
(a) If it induces a statistically significant increase in the number of phases with aberrations (without gaps) with one or more of the concentrations tested as compared with the solvent controls.
(b) If there is a concentration-related increase in the number of phases with aberrations (Without gaps).
The test substance is classified as non-clastogenic if the tests are negative both with and without metabolic activation.

Statistics:

The biometry of the results was performed with a one side Fischer's exact test

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

( 2500 µg/ml)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

other: as specified above

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(>= 250 µg/ml)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No effects
- Effects of osmolality: No effects


RANGE-FINDING/SCREENING STUDIES:
Evaluation of the solubility of the suspension in cell culture medium showed that 5000 µg/mL was the highest practicable concentration and produced no precipitate which was visible to the naked eye. Microscopical precipitation was observed with all concentrations investigated.

Preliminary toxicity study: Was carried out using a maximum concentration of 5000 µg/mL and a range of lower dose levels down to 625 µg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

Observations:
Precipitations with and without S9-mix at all tested
concentrations

Remarks on result:

other: other: main test 3 and 20 hours

Remarks:

Migrated from field 'Test system'.

The sensitivity of the test system and efficacy of the S9-mix was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control substances.

Mutation results:

In the first and in the second main experiment the mitotic index was reduced (indication of toxicity) after treatment in the absence and the presence of S9-mix.

No relevant reproducible enhancement of metaphases with aberrations outside the range of the solvent control was found with any of the concentrations used, either with or without metabolic activation by S9-mix.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the test conditions, the test substance was not clastogenic in the in vitro chromosome aberration assay with V79 Chinese hamster lung cells.

Executive summary:

A study was conducted to investigate the potential of test substance to induce chromosome aberrations in V 79 cells of the Chinese hamster lung in vitro according to OECD Guideline 473, EPA OPPTS 870.5375, EU Method B.10. and Japanese Substances Control Law (JSCL) in compliance with GLP.

Two experiments with duplicate cultures were used for each concentration. The test substance was suspended in the test medium and tested at the following concentrations:

First experiment with 3/20 h treatment time:

without S9-mix: 625, 1250 and 2500 µg/mL

with S9-mix: 625,1250 and 2500 µg/mL

Second experiment with 20 h treatment time:

without S9-mix: 250, 500, and 750 µg/mL

Second experiment 3/28h treatment time with s9 mix: 624, 1250 and 2500 µg/mL

Second experiment 28/28 h treatment time without s9 mix: 1250, 250 and 500 µg/mL

The concentration ranges were based on the results of preliminary testing for solubility and toxicity. In the presence and absence of S9-mix an indication of cytotoxicity as reduction of mitotic index was observed.

Precipitation of the test compound was not visible to the naked eye but was observed in all microscopic determinations with all concentrations investigated.

Appropriate reference mutagens used as positive controls showed a significant increase in chromosome aberrations, thus indicating the sensitivity of the assay, and the efficacy of the S9-mix. No relevant reproducible enhancement of metaphases with aberrations outside the range of the solvent control was found with any of the concentration used with or without metabolic activation.

These data were found statistically significant enhanced in the Fisher's exact-test. Beside this no dose-dependency was observed.

Test substance did not induce chromosome aberrations in V79 Chinese hamster cells, either in the presence or in the absence of a metabolic activation.

Under the test conditions, the test substance was not clastogenic in the in vitro chromosome aberration assay with V79 Chinese hamster lung cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Ames test

A study was conducted to determine the mutagenic potential of test substance according to OECD Guideline 471 in compliance with GLP. S. typhimurium strains TA 100, TA 1535, TA 1537, TA 98 and TA 102 and Escherichia coli WP2uvrA pKM101 were used in the mutagenicity assay. Control plates without mutagen showed that the number of spontaneous revertant colonies was within the historical control range although slightly higher. All the positive control compounds showed the expected increase in the number of revertant colonies. Toxicity was observed with strain TA 1537 at the highest concentration in the absence and in the presence of metabolic activation. No mutagenicity was observed with or with metabolic activation at any other concentration with any strains. Under the test conditions, the test substance is considered not to be mutagenic in these bacterial test systems in the presence or absence of exogenous metabolic activation.

Chromosomal aberration test

A study was conducted to investigate the potential of test substance to induce chromosome aberrations in V 79 cells of the Chinese hamster lung in vitro according to OECD Guideline 473 in compliance with GLP. Two experiments with duplicate cultures were used for each concentration (up to 2500 µg/ml). Appropriate reference mutagens used as positive controls showed a significant increase in chromosome aberrations. No relevant reproducible enhancement of metaphases with aberrations outside the range of the solvent control was found with any of the concentration used with or without metabolic activation. Under the test conditions, the test substance was not clastogenic in the in vitro chromosome aberration assay with V79 Chinese hamster lung cells.

UDS assay

A study was conducted to investigate unscheduled DNA synthesis (UDS) in mammalian cells in vitro according to OECD 482 and GLP. Primary rat hepatocyte cultures were exposed to a structural analogue of Reactive Red F00-0124 in cell culture medium at concentrations of 100,30, 10, 3, 1, 0.3 and 0.1 ug/ml for 16-20 hours. The test item was tested up to the limit concentration, 5000 ug/ml in a range finding study. However, microscopic examination of the hepatocyte cultures showed dark blue incorporation of the test substance in all hepatocyte nuclei at the concentrations of 5000, 3000, 1000 and 300 ug/ml as a sign of heavy cytotoxicity and a very low survival rate. Therefore these concentrations were excluded from the evaluation of genotoxicity. The positive controls induced the appropriate response indicating the appropriate level of sensitivity. There was no evidence that unscheduled DNA synthesis (as determined by radioactive tracer procedures) was induced indicating the test item is not genotoxic in this in vitro test.

Justification for selection of genetic toxicity endpoint
GLP guideline study

Justification for classification or non-classification

Under the test conditions, the test substance is considered not to be mutagenic in an adequate testing battery in the presence or absence of exogenous metabolic activation.