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

Repeated dose toxicity: dermal

Administrative data

Endpoint:

repeated dose toxicity: dermal

Remarks:

combined repeated dose and carcinogenicity

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

15 August 1963 to 7 June 1965

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented publication/study report 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.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1967

Report date:

1967

Reference 2

Reference Type:

publication

Title:

Toxicology of Indigo - a review

Author:

Ferber KH

Year:

1987

Bibliographic source:

J. Environ. Pathol. Toxicol. Oncol. 7, 73 -84 (1987)

Materials and methods

Principles of method if other than guideline:

U.S. FDA

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-(1,3-dihydro-3-oxo-2H-indol-2-ylidene)-1,2-dihydro-3H-indol-3-one

EC Number:

207-586-9

EC Name:

2-(1,3-dihydro-3-oxo-2H-indol-2-ylidene)-1,2-dihydro-3H-indol-3-one

Cas Number:

482-89-3

Molecular formula:

C16H10N2O2

IUPAC Name:

2,2'-biindole-3,3'(1H,1'H)-dione

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): D&C Blue 6
- Substance type: active ingredient
- Physical state: solid - dark blue powder
- Analytical purity: Sample A: 95%
Sample B + D: 97%
- Certified No.: Sample A: W4500
Sample B + D: W6182
- Lot/batch No.: K7024

Test animals

Species:

mouse

Strain:

Swiss Webster

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Pied Piper Farms
- Age at study initiation: -
- Weight at study initiation: -
- Fasting period before study: -
- Housing: 5/cage
- Diet: ad libitum
- Water: tap water ad libitum

Administration / exposure

Type of coverage:

open

Vehicle:

other: spectrograde benzene

Details on exposure:

TEST SITE
- Area of exposure: midscapular region
- Time intervals for shavings or clipplings: regular to keep skin relatively free of hair


REMOVAL OF TEST SUBSTANCE - No


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
- Concentration (if solution): 1%
- Constant volume or concentration used: yes

The dose levels wer corrected for purity of the dye content: for preparation of the 1% indigo formulation calculated for 100% indigo, a 1.05% or 1.03% formulation of the test substance as supplied had to be prepared for 95% and 97% purity of the delivered batches, respectively.

Sample A: Week 1 - 6
Sample B: Week 7 - 68
Sample D: Week 69 - 95


VEHICLE
- Justification for use and choice of vehicle (if other than water): TS is insoluble in water
- Amount(s) applied (volume or weight with unit): 0.1 mL

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

NA

Duration of treatment / exposure:

up to 95 weeks - application of formulation once a week

Frequency of treatment:

weekly

Doses / concentrations

Remarks:

Doses / Concentrations:
1 mg/animal
Basis:
other: calculated as 100% pure dye nominal per animal

No. of animals per sex per dose:

Negative Control: 100
Positive Control: 100
Dose Group: 50

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Details on study design:

About 1 to 5 animals from each group, incl. moribund mice, were sacrificed at monthly intervals following 2 to 36 applications. These resulted in a total of 28 or 30 interim killed animals from each control group and 16 mice from the test substance group.
3 to 5 mice of the surviving animals of each sex from each group were sacrificed after 74 applications. The remaining surviving mice were sacrificed after 95 applications.

Positive control:

NA

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly


DERMAL IRRITATION (if dermal study): No data


BODY WEIGHT: Yes
- Time schedule for examinations: Week 1 - 16: weekly (except negative control)
Week 27 - 52: biweekly
Week 53 - end: monthly

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
- Preserved tissues: application site, brain, pituitary, thyroid, thymus, lungs, liver, spleen, kidneys, adrenals, stomach, pancreas, small and large intestines, urinary bladder, gonads, lymph nodes (axillary), gross lesions
- Microscopic examination:
- animals sacrified in first 9 months:: application site all animals + liver from 9 to 13 mice/group
- animals sacrified at 75 weeks: application site, liver, lung from 10 mice/group
- terminal sacrifice: application site, liver, lung from 29, 26, 13 mice from the negative, vehicle control and Blue 6 groups, respectively
- most tissue masses, macroscopic lesion

Statistics:

Survival by life-table technique

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Dermal irritation:

not specified

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
The appearence and behavior of the mice was generaly comparable with that of controls. Clinical signs observed were either incidental findings or due to the vehicle and also observed in the control group. Survival of animals was high during the first year.

BODY WEIGHT AND WEIGHT GAIN
-

GROSS PATHOLOGY
Macroscopic findings in the D&C Blue 6-treated group were also seen in the vehicle control group and either incidental findings or due to the benzene application.

HISTOPATHOLOGY
Dermal application of a 1% solution of D&C Blue 6 in benzene for 95 weeks did not produce any distinctive histologic alteration which exceeded the range established in the sectons from the vehicle control mice.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

1 other: mg/animal/day

Sex:

male/female

Basis for effect level:

other: clinical signs; mortality; survival; body weight; gross pathology; histopathology

2

Dose descriptor:

NOAEL

Effect level:

ca. 25 other: mg/kg bw/week

Sex:

male/female

Basis for effect level:

other: based on assumed mean body weight of 40 g

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

No evidence was found that in mice repeated dermal application of 1 mg/animal, once per week for up to 95 weeks produced any effect attributable to the test substance. Lesions and tumors seen in treated mice were comparable to those in vehicle control animals.
Hence the NOAEL for repeated dermal administration of indigo is about 25 mg/kg bw/week based on assumed mean body weight of 40 g.

Executive summary:

The test substance was applied as a 1% solution (W/V) in spectro-grade benzene at a dose of 1 mg of test material (once/week) for up to 95 weeks, to 50 male and 50 female Swiss-Webster mice. 100 males and 100 females were used as negative controls (no treatment). 100 males and 100 females received application of the vehicle (benzene).

Autopsies were performed on all died or sacrificed animals in the absence of marked autolysis. Microscopic examination of the skin (application site) were performed from all animals died or sacrificed in the first 9 months and of the liver from 9 to 13 mice/group. Microscopic examination of the lungs, liver and skin (application site) from 10 negative controls, 10 vehicle controls, and 10 compound-treated animals were performed at 75 weeks. At the terminal sacrifice (95 weeks), sections of lung, liver and skin from 29 negative controls, 26 vehicle controls and 13 compound-treated animals were examined microscopically. Histopathology was also performed on most tissues and on grossly abnormal organs of the animals.

 

No evidence was found that in mice repeated dermal application of 1 mg/animal, once per week for up to 95 weeks produced any effect attributable to the test substance. Lesions and tumors seen in treated mice were comparable to those in vehicle control animals.