2,2'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[N-(2,4-dimethylphenyl)-3-oxobutyramide]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item did not exert mutagenic activity in the reverse bacterial mutation assay (plate incorporation assay and pre-incubation assay according to Prival) with and without metabolic activation.

The test item was tested for DNA-damaging effects on rat hepatocytes in vitro. The investigations were performed with concentrations of 2, 10, 50 and 250 µg/ml. Testing of higher concentrations was not possible, because higher concentrations caused strong precipitations which rendered the microscopical evaluation of the specimens impossible. There were no marked differences in the number of silver grains per nucleus in the vehicle control and in the cultures treated with the various concentrations of the test item. The results with the positive control substance were within the normal range.

The test item was tested for DNA-damaging effects on human fibroblasts in vitro. The investigations were performed with concentrations of 2, 10, 50 and 250 µg/ml. Testing of higher concentrations was not possible, because higher concentrations caused strong precipitations which rendered the microscopical evaluation of the specimens impossible. There were no marked differences in the number of silver grains per nucleus in the vehicle control and in the cultures treated with the various concentrations of the test item. The results with the positive control substance were within the normal range.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 14 JUL 2006 to 25 JUL 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD TG 471), with Prival modification for azo-dyes

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

in accordance with German Chemikaliengesetz and Directive 88/320/EEC

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 (experiment I); hamster liver S9 (experiment II)

Test concentrations with justification for top dose:

Experiment I: 3, 10, 33, 100, 333, 1000, 2500, 5000 µg/plate
Experiment II: 33, 100, 333, 1000, 2500, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility properties of the solvent and its relative non-toxicity to the bacteria

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (TA 1535 and TA 100), 4-Nitro-o-phenylene-diamine (TA 1537 and TA 98), methyl methane sulfonate (WP2 uvrA)

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation (rat liver S9 mix)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (TA 1535, TA 100, TA 1537, WP2 uvrA), congo red (TA 98)

Remarks:

with metabolic activation (hamster liver S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Experiment I: plate incorporation assay with induced rat liver S9 mix (induction with phenobarbital/ß-naphthoflavone)
Experiment II: preincubation assay with non-induced hamster liver S9 mix

DURATION
- Preincubation period: Experiment II: 30° C for 30 up to 35 (strains TA 98, TA 100, WP2 uvrA) minutes
- Exposure duration: at least 48 hours

NUMBER OF REPLICATIONS: 3 plates per strain and dose level, including the controls

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, WP2 uvrA) or thrice (strains TA 1535, TA 1537) the colony count of the corresponding solvent colony is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the thresshold is regarded as an indication of mutagenic potential if reproduced in an independent second experiment. however, whenever the colony counts remain within the historical range of negative annd solvent controls such as an increase is not considered biologically relevant.

Statistics:

Arithmetic means and standard deviation of the counted colonies were calculated.

Species / strain:

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

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: of the test item occured at concentrations of 1000 µg/plate and more. Nevertheless the colonies could be counted manually.

COMPARISON WITH HISTORICAL CONTROL DATA: there were no biologically relevant deviations

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

The test item showed no mutagenic activity in both experiments (plate incorporation assay, preincubation assay) each with and without metabolic activation.

Conclusions:

Interpretation of results (migrated information):
negative

The test item did not exert mutagenic activity in the reverse bacterial mutation assay (plate incorporation assay and pre-incubation assay according to Prival) with and without metabolic activation.

Executive summary:

Mutagenic activity of the test item was investigated in Salmonella typhimurium strains TA 1535, TA 1537, TA98 and TA100 as well as Escherichia coli strain WP2 uvrA with (induced rat liver S9 mix) and without metabolic activation at concentrations of 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate using the plate incorporation assay. Due to the test items characteristic as an azo-dye the test was also conducted using the Prival modification, i.e. testing the above mentioned bacterial strains in the preincubation assay with uninduced hamster liver S9 mix for metabolic activation. This test was performed using the concentrations 33, 100, 333, 1000, 2500 and 5000 µg/plate.

The test item did not reveal any mutagenic activity under the conditions tested. The appropriate reference mutagenes showed distinct positive mutagenic effects.

Reference 2

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 25 MAR 1985 to 29 APR 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: comparable to guideline study with acceptable restrictions (e.g. no replication of study results, purity and compositions of test item not given)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Deviations:

not applicable

GLP compliance:

not specified

Type of assay:

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Species / strain / cell type:

hepatocytes: rat

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Test concentrations with justification for top dose:

2, 10, 50, 250 µg/ml

Vehicle / solvent:

ethyl alcohol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Species / strain:

hepatocytes: rat

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At the concentrations of 500 and 1000 µg/ml strong precipitations of the test substance rendered impossible any microscopical evaluation of the specimens in the pretest. Therefore, 250 µg/ml was chosen as the highest concentration.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

The test item did not increase the mean value of silver grains per nucleus in comparison to controls. The positive control substance (100 mM) yielded a marked increase in the mean value of silver grains per nucleus.

Conclusions:

Interpretation of results:
negative

There was no evidence of induction of DNA damage by the test item under the given experimental conditions.

Executive summary:

The test item was tested for DNA-damaging effects on rat hepatocytes in vitro. The investigations were performed with concentrations of 2, 10, 50 and 250 µg/ml. Testing of higher concentrations was not possible, because higher concentrations caused strong precipitations which rendered the microscopical evaluation of the specimens impossible. There were no marked differences in the number of silver grains per nucleus in the vehicle control and in the cultures treated with the various concentrations of the test item. The results with the positive control substance were within the normal range.

Reference 3

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 16 APR 1985 to 7 MAY 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: comparable to guideline study with acceptable restrictions (e.g. no replication of study results, purity and composition of test item not given)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Deviations:

not applicable

GLP compliance:

not specified

Type of assay:

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Species / strain / cell type:

other: human fibroblasts (CRL 1121)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Test concentrations with justification for top dose:

2, 10, 50, 250 µg/ml

Vehicle / solvent:

ethyl alcohol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Species / strain:

other: human fibroblasts (CRL 1121)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At the concentrations of 500 and 1000 µg/ml strong precipitations of the test substance rendered impossible any microscopical evaluation of the specimens in the pretest. Therefore, 250 µg/ml was chosen as the highest concentration.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

The test item did not increase the mean value of silver grains per nucleus in comparison to controls. The positive control substance (5 µM) yielded a marked increase in the mean value of silver grains per nucleus.

Conclusions:

Interpretation of results (migrated information):
negative

There was no evidence of induction of DNA damage by the test item under the given experimental conditions.

Executive summary:

The test item was tested for DNA-damaging effects on human fibroblasts in vitro. The investigations were performed with concentrations of 2, 10, 50 and 250 µg/ml. Testing of higher concentrations was not possible, because higher concentrations caused strong precipitations which rendered the microscopical evaluation of the specimens impossible. There were no marked differences in the number of silver grains per nucleus in the vehicle control and in the cultures treated with the various concentrations of the test item. The results with the positive control substance were within the normal range.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

No classification

The test item did no show any potential for mutagenicity or DNA damage.