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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

20-Mar-2001 to 27-May-2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study on structural analogue

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.18 (DNA Damage and Repair - Unscheduled DNA Synthesis - Mammalian Cells In Vitro)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5550 - Unscheduled DNA Synthesis in Mammalian Cells in Culture

GLP compliance:

yes

Type of assay:

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

Test material

Test material information

Constituent 1

Test material form:

other: solid

Details on test material:

see below

Method

Species / strain

Species / strain / cell type:

hepatocytes: primary, male rats

Metabolic activation:

not specified

Test concentrations with justification for top dose:

5000.0,3000.0, 1000.0, 300.0, 100.0,30.0, 10.0, 3.0, 1.0, 0.3 and 0.1 ug/ml

Vehicle / solvent:

not necessary because the test substance was dissolved/suspended directly into the test system (cell culture medium)

Controls

Untreated negative controls:

yes

Remarks:

untreated control

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Approximately 5 x 105 cells were seeded in a 35 mm culture dish, allowed to attach for approx. 2 hours and exposed to the test and control compound for 16 - 20 hours at 37 degrees C.
- Selection time (if incubation with a selection agent): After incubation the test substance was replaced by a 0.5 % neutral red solution, which was allowed to incubate for approx. four hours.

Survival was determined by photometric measurement of the neutral red extinction.

Evaluation criteria:

Criteria for a positive response:
-If the net nuclear grain number was at least five grains higher than the corresponding solvent control value, the results for that dose were considered significant.
-The test compound is classified as genotoxic if it reproducibly induces with one of the test compound concentrations a significant increase in ['H] thymidine incorporation in comparision with the solvent control.
- The test compound is classified as genotoxic if there is a reproducible significant concentration related increase in ['H] thymidine incorporation in cornparision with the solvent control.
-In the final instance, however the interpretation of the results were based on scientific judgement.

Results and discussion

Test results

Species / strain:

hepatocytes: primary male rat

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 300 µg/ml

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

In a preliminary experiment for solubility, the test was studied with respect to its solubility in hepatocyte attachment medium. The highest concentration at which no visible precipitation was observed, was 5000.0 ug/ml. Based on these results 5000.0 ug/ml was determined as maximum dose level for the main assays and ten lower concentrations were included in the treatment series. In both experiments cytotoxicity could not be determined with the neutral red assay, as death cells incorporated the dark blue test compound which disturbed the photometric measurement of neutral red. Microscopic examination of the hepatocyte cultures showed dark blue incorporation of the test substance in all hepatocyte nuclei at the concentrations of 5000.0, 3000.0, 1000.0 and 300.0 ug/ml as a sign of heavy cytotoxicity and a very low survival rate. Therefore these concentrations were excluded from the evaluation of genotoxicity. A dose range of seven concentrations from 0.1 to 100.0 ug/ml was used.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Teh test item is not genotoxic as determined by the in vitro UDS test in primary rat hepatocytes.

Executive summary:

In an unscheduled DNA synthesis assay, primary rat hepatocyte cultures were exposed to the test item in cell culture medium at concentrations of 5000.0, 3000.0, 1000.0, 300.0, 100.0,30.0, 10.0, 3.0, 1.0, 0.3 and 0.1 ug/ml for 16-20 hours.  

The test item was tested up to the limit concentration, 5000 ug/ml. However, microscopic examination of the hepatocyte cultures in a preliminary range finding experiment showed dark blue incorporation of the test substance in all hepatocyte nuclei at the concentrations of 5000.0, 3000.0, 1000.0 and 300.0 ug/ml as a sign of heavy cytotoxicity and a very low survival rate. Therefore these concentrations were excluded from the evaluation of genotoxicity. A dose range of seven concentrations from 0.1 to 100.0 ug/ml was used. The positive controls induced the appropriate response. There was no evidence that unscheduled DNA synthesis, as determined by radioactive tracer procedures was induced.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 482 for DNA damage and/or repair.