Registration Dossier

Administrative data

Description of key information

There were no adverse effects noted up to the highest dose levels tested

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
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.
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
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
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
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
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
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
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
Critical effects observed:
not specified
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.


Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 200 mg/kg bw/day
Study duration:
chronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
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.
Principles of method if other than guideline:
U.S. FDA
GLP compliance:
no
Limit test:
no
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
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
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
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
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.
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
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
Critical effects observed:
not specified
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
25 mg/kg bw/day
Study duration:
chronic
Species:
mouse

Additional information

The following assessment was performed based on data from repeated dose toxicity tests with a FDA certified Indigo batch, commercial name: D&C Blue No. 6, which had to comply with the substance definition for certified batches.

Based on the specification for Indigo for use in medical devices as sutures as given under section 21CFR74.3106, the test substance definition for the certified batches used in these studies is the following:

Composition of D&C Blue No. 6 as given under section 21CFR74.3106

Constituent

Concentration range

Remarks

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

EC no.: 207-586-9

>= 95.0 % (w/w)

Total colour, not less than 95 %

Aniline

EC no.: 200-539-3

> 0.0 — <= 3.0 % (w/w)

Volatile matter at 135 °C (275 °F), not more than 3 %.

N-Methylaniline

EC no.: 202-870-9

> 0.0 — <= 3.0 % (w/w)

Volatile matter at 135 °C (275 °F), not more than 3 %

Isatin

> 0.0 — <= 0.3 % (w/w)

 

Anthranilic acid

> 0.0 — <= 0.3 % (w/w)

 

Indirubin

> 0.0 — <= 1.0 % (w/w)

 

Lead (as Pb)

> 0.0 — <= 10 ppm

 

Arsenic (as As)

> 0.0 — <= 3 ppm

 

Mercury (as Hg)

> 0.0 — <= 1 ppm

 

Indigo Küpe is instable in an aqueous phase at neutral pH or as solid material on air and oxidises rapidly to Indigo (EC number 207-586-9).However, due to the production process of Indigo Küpe, it contains a distinctly lower amount of aniline and methyl-aniline Hence, the samples of the mentioned studies provide a worst-case scenario with respect to aniline and methyl-aniline and it is feasible to use these studies to assess the respective endpoints covered by these studies.

These studies consist of a 6-week repeat dose study in male rats conducted atdietaryconcentrationsofup to 3% (2565 mg/kg bw/day),a 2-year chronic feeding studyin male and female rats atdietary concentrations ofup to 3% (1200 mg/kg bw/day),13-weekrange-finding study and 2-yearfeeding studyinmale andfemale beagle dogsatdietary concentrations ofup to 3% (750mg/kg bw/day.

In the 6-week range finding study in male rats at 0, 0.1, 0.23, 0.55, 1.29 and 3% (mean daily test substance intake of about 0, 90, 199, 485, 1152, and 2565 mg/kg bw/day, calculated on an assumed body weight of 250 g and food consumption of 7% body weight) no adverse in-life effects were noted. Except for a faint colour retention in subcutaneous and peritoneal fat, no consistent gross alterations were found in the tissues or viscera. At microscopic examination, mild degenerative changes in the central zone of the liver lobules were seen in rats receiving 3.0% (2565 mg/kg bw/day) in the diet. Organ weights and ratios to body weight, physical appearance and behaviour of the test rats were comparable to controls

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.

In the 13 -week range finding study, one male and one female adult purebred beagle dogs were fed the test substance for 13 weeks at the 1.0% to 3.0% level in a basal laboratory diet of Wayne Dog Feed. The dosage was increased by 1.0% at 2-week intervals until the 3.0% level was reached. The dietary concentrations of 1.0 and 3.0 % corresponded to the mean daily test substance intake of about 250, and 750 mg/kg bw/day, calculated on an assumed body weight of 10 kg and daily total food consumption of 250 g. No toxicological relevant findings were observed after 13 weeks administration of 3% (ca. 750 mg/kg bw/day) indigo in the diet.

Based on the results of the 13-week feeding study, 3 groups of 3 male and 3 female young adult purebred beagle dogs and a control group of 10 males and 10 females were fed a basal diet of Wayne Dog Feed containing the test substance at dosage levels of 0, 0.25, 1 and 3% for two years. These dietary corresponded to the mean daily test substance intake of about 0, 62.5, 250, and 750 mg/kg bw/day, calculated on an assumed body weight of 10 kg and daily total food consumption of 250 g. The dogs were observed daily for signs of toxic or pharmacologic effects. Haematological and biochemical studies and urinalyses were performed at the start and at 1, 3, 6, 12, 18 and 24 months. All survivors were sacrificed at 24 months for necropsies. Chronic exposure to the test substance resulted in no toxicologically significant effects on survival rates, behaviour, body weights or weight gains, organ weights, or in haematology, clinical chemistry, or urinalysis parameters. No gross pathological or histopathological findings related to the test substance exposures were observed up to 3% test substance in the diet (750 mg/kg bw/day). Based on the results of the study, 750 mg/kg bw/day is considered to be the no-observed-effect-level (NOEL) for the test substance in dogs of both sexes.

Justification for classification or non-classification

Test substance is practically non-toxic, no classification necessary.