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Repeated dose toxicity: oral

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Administrative data

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
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

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1967
Report Date:
1967
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.
GLP compliance:
no
Limit test:
no

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): 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:
rat
Strain:
other: Charles River caesarean-derived albino rats
Sex:
male/female
Details on test animals 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

open allclose all
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
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:

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 colouring 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 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. Haematological 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 five 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 histopathological findings. Furthermore, there were no relevant changes in organ weights, gross or microscopic pathology in rats given up to 1200 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 3% of this substance in their diets without serious adverse effects. The NOEL for repeated dose toxicity is considered to be 3% in food corresponding to about 1200 mg/kg/day.