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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
17. November 2003 to 12. February 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to OECD 473 and GLP guideline. The registered substance oxidizes in the presence of water and air-borne oxygen rapidly to Indigo (CAS No. 482-89-3) and potassium hydroxide

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
TG III.2 Chromosome aberration test with mammalian cells in culture
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Indigo aus Indigo-Küpe

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Source: cell bank of "Genetic Toxicology", Aventis Pharma Deutschland GmbH, ProTox
Cell Culture Medium: Mininam essential medium with Earle's salts and L-glutamine
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix rat liver induced with Aroclor 1254
Test concentrations with justification for top dose:
20.5, 41, 82, 164, 327.9, 655.7, 1311.4, 2622.7 µg/ml
Vehicle / solvent:
cell culture medium
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation
Details on test system and experimental conditions:
Exposure period (with metabolic activation): 3 hours
Exposure period (without metabolic activation): 3 hours

Fixation time:
20 h; 28 h
Evaluation criteria:
Non-clastogenic:
- number of induced structural chromosome aberrations in all evaluated dose groups is in the range of historical control data
- no significant increase in number of structural chromosome aberrations observed

Clastogenic:
- number of induced structural chromosome aberrations in all evaluated dose groups is above the range of historical control data and
- either a concentration-related or a significant increase in number of structural chromosome aberrations observed
Statistics:
one-sided Fisher's exact test

Results and discussion

Test results
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:
not applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Test substance is not clastogenic
Executive summary:

The potential of the test item to induce chomosome aberrations was investigated in V79 cells of the Chinese hamster lung in vitro.

The test item was suspended in cell culture medium and the the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (without and with metabolic activation) up to 10 mM (= 2622.7 µg/ml). Both independent experiments were performed in duplicate for concentrations and incubation/expression intervals given below. Evaluations were made for concentrations ranging from little to maximum (50% survival) toxicity:

First experiment

with 3/20 h treatment/sampling time

without S9 mix: 20.5, 41, 82, 164, 327.9, 655.7, 1311.4, 2622.7 µg/ml

with S9 mix: 20.5, 41, 82, 164, 327.9, 655.7, 1311.4, 2622.7 µg/ml test item

Second experiment with 20/20 h treatment/sampling time

without S9 mix: 20.5, 41, 82, 164, 327.9, 655.7, 1311.4, 2622.7 µg/ml

Second experiment with 28/28 h treatment/sampling time

without S9 mix: 20.5, 41, 82, 164, 327.9 µg/ml

Second experiment with 3/28 h treatment/sampling time

with S9 mix: 20.5, 41, 82, 164, 327.9, 655.7, 1311.4 µg/ml

After treatment time macroscopic precipitation of the test compound was observed in a dose range of 237.9 µg/ml and above and microscopic precipitation of the test compound in a dose range of 20.5 µg/ml and above.

There were no biologically significant increases in the number of cells showing structural chromosome aberrations, either in the absence or in the presence of metabolic activation, up to and including cytotoxic concentrations. There were no statistical differences between treatment and control groups and no dose-response relationships were noted.

There was no increase in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation.

Appropriate reference mutagens used as positive controls showed a significant increase in chomosome aberrations, thus indicating the sensivty of the assay, and the efficacy of the S9 -mix.

In conclusion, Indigo aus Indigo-Küpe tested up to cytotoxic concentrations, both with and without metabolic activation, did not induce chromosome aberrations in V79 Chinese Hamster lung cells. Therefore, Indigo aus Indigo-Küpe is considered

not clastogenic in this test system.