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

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Acceptable, well-documented publication which meets basic scientific principles. The registered substance oxidizes in the presence of water and air-borne oxygen rapidly to Indigo (CAS No. 482-89-3) and sodium hydroxide. The study was only conducted in males. According to OECD test guideline 474 the test has only to be conducted in one gender “If at the time of the study there are data available from studies in the same species and using the same route of exposure that demonstrate that there are no substantial differences between sexes in toxicity, then testing in a single sex will be sufficient.“ As listed in the Indigo dossier, there are several studies performed with male and female animals. In none of these studies, any gender differences were noted. Specifically for mice, which was the test species in the in-vivo MNT, there exists a 95-week study in male and female mice. Also in this study, no gender difference was observed. It was hence fully in line with OECD guideline 474 to perform the study in males only.

Data source

Reference

Reference Type:

publication

Title:

Testing natural indigo for genotoxicity

Author:

Hesbert A, Bottin MC, de Ceaurriz J, Protois JC, Cavelier C

Year:

1984

Bibliographic source:

Toxicol Lett. 1984 Apr;21(1):119-25

Materials and methods

Principles of method if other than guideline:

Schmid W. The micronucleus test for cytogeneticanalysis, in Hollaender (Ed.) Chemical Mutagenesis, Principles ans Methods for their Detection, Vol. 4, Plenum, New York, 1976, p.31-53
Salamone M, Heddle J, Stuart E and Katz M. Studies on 3 model agents mitomycin C, cyclophosphamide and dimethylbenzanthracene, Mutation Res. 74:347-356, 1980

GLP compliance:

not specified

Type of assay:

micronucleus assay

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): C.I. vat blue 1
- Cemical name: 2-(1,3-dihydro-3-oxo-2H-indol-2-ylidene)-1,2-dihydro-3H-indol-3-one
- Molecular formula (if other than submission substance): C16H10N2O2
- Molecular weight (if other than submission substance): 262.2628
- 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 ingredient
- Physical state: solid
-Supplier: Ciba Geigy

Test animals

Species:

mouse

Strain:

Swiss

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 25 g

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 10% gum arabic aqueous solution
- Concentration of test material in vehicle: 4, 20, 40, 80 mg/mL
- Amount of vehicle (if gavage or dermal): 25 mL/kg bw
- Type and concentration of dispersant aid (if powder): suspension homogenized by ultrasonic homogenizer

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Indigo was suspended at contentrations of 4, 20, 40, 80 mg/mL in 10% gum arabic aqueous solution by means of an ultrasonic homogenizer

Duration of treatment / exposure:

twice with an interval of 24 hours

Frequency of treatment:

2-times

Post exposure period:

30 and 54 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
100, 500, 1000, 2000 mg/kg bw
Basis:
other: nominal in vehicle

No. of animals per sex per dose:

10 males

Control animals:

yes

Positive control(s):

cyclophosphamide
- Route of administration: intraperitoneal
- Doses / concentrations: 25 mg/kg

Examinations

Tissues and cell types examined:

bone marrow erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
maximum feasible dose

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
2 administrations with an interval of 24 hours
Sampling 30 and 54 hours after first administration

DETAILS OF SLIDE PREPARATION:
Bone marrow was flushed into a tube containing fetal bovine serum
Air dried smears were stained with May-Grünwald-Giemsa

METHOD OF ANALYSIS:
1000 polychromatic erythrocytes were scored by 2 people for incidence of micronuclei

Evaluation criteria:

not stated

Statistics:

Mann-Whitney-Wilcoxon test

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Indigo given by oral gavage as two equal dosages of 0.1, 1 and 1.2 g/kg body weight, did not induce the number or polychromatic erythrocytes with micronuclei. Indigo is therefore considered not to be clastogenic in the in vivo micronucleus assay in mice.

Executive summary:

The clastogenic potential of natural indigo was investigated in the micronucleus test in the bone marrow of male mice. The test compound was administered twice with an interval of 24 h, the animals were killed 30 h and 54 h after the first treatment. When the test compound was given by oral gavage as two equal dosages of 0.1, 1 and 1.2 g/kg body weight, no statistically significant increase in the percentage of polychromatic erythrocytes with micronuclei was observed for any group treated with natural indigo.