Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented publication which meets basic scientific principles

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

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.
GLP compliance:
not specified
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): C.I. vat blue 1
- Substance type: active ingredient
- Physical state: solid
-Supplier: Ciba Geigy

Test animals

Species:
mouse
Strain:
Swiss
Sex:
male
Details on test animals 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.