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

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

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

Adequacy of study:

key study

Study period:

February 1963 to February 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:

Chronic dietary study in the rat according to US FDA. Throughout the 2-year study, observations were made daily for mortality and weekly for gross signs of toxicity. Hematological values were determined and urinalyses made at 1, 3, 6, 12, 18 and 24 months. Autopsies were performed on all animals which died during the study. At 12 months, five males and 5 females from each sacrificed animal in the control and high dose groups were examined microscopically. At the termination of the study, histopathology was performed on all preserved tissues from 10 male and 10 female rats in the control group and on an equal number in the high dosage group.

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

Details on test material:

- Name of test material (as cited in study report): D&C Blue 6
- 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 - 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:

rat

Strain:

other: Charles River caesarean-derived albino rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles river
- Weight at study initiation: males: 70 to 127 g; females: 70 to 137 g
- Fasting period before study: no
- Housing: single
- Diet: Purina Lab Chow ad libitum
- Water: ad libitum
- Acclimation period: no data

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly during the 1st year; biweekly during the 2nd year
- Mixing appropriate amounts with (Type of food): Purina Lab Chow

The test item was incorporated into the diet on a weight/weight basis and thoroughly mixed in a twin-shell blender to provide thee apropriate dietary levels. The dose levels wer corrected for purity: 100% indigo

Sample A: Day 1 through 21st week
Sample B: 22nd week through 63rd week
Sample D: 64th week through 104th week (end of study period)

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

NA

Duration of treatment / exposure:

2 years

Frequency of treatment:

continuous

Doses / concentrations

Remarks:

Doses / Concentrations:
0.25%, 1%, 3%
Basis:
nominal in diet

No. of animals per sex per dose:

Control group: 80
Treated groups: 25

Control animals:

yes, plain diet

Details on study design:

The dose levels used in this study were selected based on a 6-week range-finding study in male rats (see MF 9/33)

Positive control:

NA

Examinations

Observations and examinations performed and frequency:

Mortality: daily
Gross signs of toxicity: weekly
Body weights, food consumption, clinical signs (physical appearence, behaviour), masses (incidence, appearence and location):
- Week 1 to 26: weekly
- Week 27 to end of 1st year: biweekly
- 2nd year: every four weeks


HAEMATOLOGY: Yes
- Time schedule for collection of blood: 1, 3, 6, 12, 18, 24 months
- How many animals: 5 rats/sex/group
- Parameters examined: total and differential leukocyte counts, hematocrit, hemoglobin


URINALYSIS: Yes
- Time schedule for collection of urine: 1, 3, 6, 12, 18, 24 months
- How many animals: 5 rats/sex/group pooled
- Parameters examined: appearence, pH, specific gravity, glucose, acetone, protein, bilirubin, occult blood, microscopic examination of sediment

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Animals + Schedule: All animals died intercurrently
12 months: 5 rats/sex/group
end of study: all surviving rats
- Organ weights: heart, liver, spleen, kidneys, testes, thyroids, adrenals
- Preservation: brain, pituitary, thyroid, heart, lungs, liver, spleen, kidneys, adrenals, stomach, pancreas, small and large intestines, mesenteric lymph node, urinary bladder, gonads, sternum, rib junction, gross lesions
HISTOPATHOLOGY: Yes
- Control and high dose rats: all preserved tissues - 12 months: all animals; 24 months: 10 males + 10 females
- Low and mid dose rats: pituitary, thyroid, liver, spleen, kidney, adrenal - 5 males + 5 females
all gross findings
- Few intercurrent rats: all preserved tissues

Statistics:

growth: 0 to 12 months
total food consumption: 1 to 13 weeks; 14 to 26 weeks, 28 to 52 weeks
survival: 12 and 24 months (life-table technique)
terminal body weights, organ weights, organ/body weight ratios: 12 and 24 months
hematology

F-test or analysis of variance at a 5% propability level
Prior F-test: homogeneity of variances - Bartlett

Homogeneous --> F-test --> significantly different: Scheffe
Heteogeneous --> Fisher-Behrens modified "t"-test

see Ostle B. Statistics in Research, Ames, Iowa, Iowa State College Press 1956
Snedecor GW. Statistical Methods, Ames, Iowa, Iowa State College Press 1956
Rao CR. Biometrics 14,1,1958
Sachs R. Toxicol. Appl. Pharmacol. 1,203,1959

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

not examined

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

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
Appearance and behavior of the test rats were generally comparable to those of the controls.
No significant difference in survival rates were noted.

TISSUE MASSES
Males: Control: 7/80; low dose: 1/25; mid dose: 4/25; high dose: 2/25
Females: Control: 34/80; low dose: 8/25; mid dose: 10/25; high dose: 7/25

BODY WEIGHT AND WEIGHT GAIN
Growth suppression was noted for rats at 3% with a trend (not statistically significant) for dose dependency.
Terminal body weights of high dose females at 12 months were lower than controls. This finding was not negatively assocoated by any other clinical or pathological relevant finding and therefore considered not to be of toxicological relevance.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
At the 3% level, food consumption in females was significantly lower than controls for the first six months but comparable to controls during the remainder of the study.

HAEMATOLOGY
Significantly lower hematocrit and hemoglobin values in comparison with controls were obtained at all time intervals except 24 months for the males fed 3%. No such effect was noted in the females. These findings were considered to be incidental findings.

URINALYSIS
Bilirubinuria was observed at 24 months in males and females at 1% and females at 3%

ORGAN WEIGHTS
12 months: lower heart weights, higher relative liver weights in 3% males
lower terminal bw in 3% females --> higher relative heart and liver weights
24 months: lower terminal bw in 3% males --> higher relative weights for testes
higher relative thyroid weight in 0.25% males
higher relative liver weights in 1% and 3% females
lower kidney weights in 3% females

No histopathologic findings correlated with these altered organ weights. These findings were considered to be incidental findings, partly related to the lower terminal body weight.

GROSS PATHOLOGY
At 2 years the kidneys of some mid and high dose rats showed a slight gray-green or blue coloration. This finding is most likely a staining effect of the test item and not related to any functional disorder or microscopical visible alteration. It is therefore considered to be of no toxicological relevance.

HISTOPATHOLOGY: NON-NEOPLASTIC
12 months: slight and variable alteration in kidney tubules in 3% males - not observed at 24 months
24 months: no findings

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

3 other: % in diet

Sex:

male/female

Basis for effect level:

other: overall effects clinical signs; mortality; body weight; food consumption; compound intake; haematology; clinical chemistry; urinalysis; gross pathology; organ weights; histopathology

2

Dose descriptor:

NOEL

Effect level:

ca. 1 200 mg/kg bw/day (nominal)

Sex:

male/female

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Administration of dietary concentrations of 0, 0.25, 1 and 3% D&C Blue No 6 for 2 years to rats was tolerated up to 3% (1200 mg/kg bw/day) of the test substance in their diets without serious effects.
Consequently, the NOEL in this study is 3% indigo in the diet.

Executive summary:

There are no repeated dose toxicity tests with the notified substance. The registered substance is the reduced form of the test item used in this study. Indigo Vat potassium salt oxidizes rapidly to indigo in the pressence of water and airborne oxygen.
The following assessment was performed based on data from repeated dose toxicity tests with Indigo (CAS-Nr.
482-89-3), commercial name: D&C Blue No. 6, certified by U.S. FDA (for coloring surgical sutures). 

On the basis of the data of the 6 -week range finding study, a 2 -year chronic feeding study of the test substance was carried out with three groups of 25 male and 25 female Charles River adult albino rats and a control group of 80 males and 80 females.
The dietary concentrations of 0, 0.25, 1.0 and 3.0% corresponded to the mean daily test substance intake of
about 0, 100, 400, 1200 mg/kg bw/day in males and in females.

Throughout the 2-year study, observations were made daily for mortality and weekly for gross signs of toxicity. Hematological values were determined and urinalyses made at 1, 3, 6, 12, 18 and 24 months. Necropsies were performed on all animals which died during the study. At 12 months, five males and 5 females from each sacrificed animal in the control and high dose groups were examined microscopically. At the termination of the study, histopathology was performed on all preserved tissues from 10 male and 10 female rats in the control group and on an equal number in the high dosage group.

Clinical laboratory investigations revealed statistically significant bilirubinuria at 24 months in males and females at 1% and females at 3%. However, there were no corresponding  histopathologic findings. Furthermore, there were no relevant changes in organ weights, gross or microscopic pathology in rats given up to 2100 mg/kg bw/day orally in the diet for 2 years.
The study appeared to demonstrate that after a period of adjustment to the higher dosage levels, the rats were able to tolerate up to of this substance in their diets without serious effects. NOEL for repeated dose toxicity is considered to be 1200 mg/kg/day.