Disodium [2,2'-bi-1H-indole]-3,3'-diolate

Genetic toxicity: in vitro

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 sodium hydroxide.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

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

Test material information

Constituent 1

Reference substance name:

Indigo

IUPAC Name:

Indigo

Details on test material:

- Name of test material (as cited in study report): Indigo aus Indigo-Küpe
- Molecular formula (if other than submission substance): C16H10N2O2
- Molecular weight (if other than submission substance): 262.27 g/mol
- Smiles notation (if other than submission substance): O=C3c4ccccc4NC3=C2Nc1ccccc1C2=O
- InChl (if other than submission substance): InChI=1/C16H10N2O2/c19-15-9-5-1-3-7-11(9)17-13(15)14-16(20)10-6-2-4-8-12(10)18-14/h1-8,17-18H
- Structural formula attached as image file (if other than submission substance): see Fig. 1
- Substance type: active substance
- Physical state: solid
- Certificate of analysis: CTS 20031162-SA
- Analytical purity: 99.4%
- Impurities (identity and concentrations): Indol 0.03%, Aniline 0.01%, N-Methylaniline <0.01%
- Purity test date: 14. Oct to 07 Nov. 2003
- Lot/batch No.: V2143/1
- Expiration date of the lot/batch: October 2008
- Storage condition of test material: at approximately 20°C in fume cupboard

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

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Controls 2

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.