Registration Dossier

Administrative data

Description of key information

There were no adverse effects noted up to the highest dose levels tested, with Indigo containing up to 3% aniline and methylaniline as a sum.

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:
experimental study
Adequacy of study:
supporting study
Study period:
15 February 1963 to 11 February 1965
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented publication/study report which meets basic scientific principles
Principles of method if other than guideline:
U.S. FDA - The dogs were observed daily for signs of toxic or pharmacologic effects. Hematological 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.
GLP compliance:
no
Limit test:
no
Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: adult
- Housing: single
- Diet: ground Wayne Dog Meal ad libitum
- Water: tap ad libitum
- Acclimation period: at least 4 weeks
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): biweekly
- Mixing appropriate amounts with (Type of food): ground Wayne Dog Meal

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: date first used: 1963-02-15
Sample B: date first used: 1963-07-12
Sample C: date first used: 1963-11-22
Sample D: date first used: 1964-01-15
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
NA
Duration of treatment / exposure:
104 weeks
Frequency of treatment:
continuous
Remarks:
Doses / Concentrations:
0.25%, 1%, 3%
Basis:
nominal in diet
No. of animals per sex per dose:
Control group: 10
Treated groups: 3
Control animals:
yes, plain diet
Details on study design:
The dose levels used in this study were selected based on a 13-week range-finding study in dogs (see MF 9/37)
Positive control:
NA
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS (appearence, behavior, elimination, signs of toxic or pharmaceutical effects)
- Time schedule: daily


BODY WEIGHT: Yes
- Time schedule for examinations: weekly


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated from the consumption and body weight gain data: Yes


HAEMATOLOGY: Yes
- Time schedule for collection of blood: initially and at 1, 3, 6, 12, 18, 24 months
- How many animals: all
- Parameters examined: total and differential leukocyte counts, hematocrit, hemoglobin, sedimentation rate


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: initially and at 1, 3, 6, 12, 18, 24 months
- How many animals: all
- Parameters examined: bromsulphalein liver function tests, blood urea nitrogen


URINALYSIS: Yes
- Time schedule for collection of urine: initially and at 1, 3, 6, 12, 18, 24 months
- Metabolism cages used for collection of urine: No data
- Parameters examined: appearence, pH, specific gravity, glucose, acetone, protein, bilirubin, occult blood, microscopic examination of sediment
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Organ weights: heart, liver, spleen, kidneys, testes, thyroids, adrenals
- Tissues preserved: brain, 3 levels of spinal cord, pituitary, thyroid, thymus, heart (incl. cororary arteries), lungs, liver, gall bladder, spleen, kidneys, adrenals, stomach, pancreas, small and large intestines, urinary bladder, gonads, prostate, uterus, skin, bone (rib ends), gross lesions

HISTOPATHOLOGY: Yes
- Control and high dose dogs: all preserved tissues
Statistics:
NA
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
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:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
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
One female at 1% died at the beginning of the 34th week; a swcond female at this level appeared depressed and gaunt in the 69th week. These findings were due to hookworms and the surviving animal responded to s.c. treatment with DNP. In this and 2 further dogs at 1% and 4 dogs at 3%, diarrhea or soft feces were seen occassionally.

BODY WEIGHT AND WEIGHT GAIN
Most of the dogs maintained or gained body weight. Each one dog in the low, mid, and high dose level lost 0.9, 0.8, and 1.3 kg bw, respectively.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
-

HAEMATOLOGY
No test item-related findings were noted. One female at 1% had elevated sedimentation rates and one male and female at 3% minimally elevated leukocyte counts.

CLINICAL CHEMISTRY
-

URINALYSIS
-

NEUROBEHAVIOUR
-

ORGAN WEIGHTS
-

GROSS PATHOLOGY
Scattered abnormal gross pathological findings did not appear to be compound related - the female died early had 15 heart worms in its heart.

HISTOPATHOLOGY: NON-NEOPLASTIC
-
Dose descriptor:
NOAEL
Effect level:
3 other: % in food
Sex:
male/female
Basis for effect level:
other: clinical signs; mortality; body weight; food consumption; compound intake; haematology; clinical chemistry; urinalysis; gross pathology; organ weights; histopathology
Dose descriptor:
NOAEL
Effect level:
ca. 750 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: calculated on an assumed body weight of 10 kg and daily total food consumption of 250 g
Critical effects observed:
not specified
Conclusions:
No toxicological relevant findings were observed after 2 years of administration of up to 3% (ca. 750 mg/kg bw/day) indigo in the diet. The oral NOAEL in male and female beagle dogs is approximately 750 mg/kg bw/day.
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).

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, 1.0 and 3.0 percent by weight for two years. The dietary concentrations of 0, 0.25, 1.0 and 3.0% corresponded to the mean daily test substance intake of about 0, 62.5, 250, and 750 mg/kg bw/d, 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. Heamatological 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, behavior, body weights or weight gains, organ weights, or in heamatology, clinical chemistry, or urinalysis parameters. No gross pathological or histopathological findings related to the test substance exposures were observed up to 3.0% test substance in the diet (equivalent to about 750 mg/kg bw/d, calculated on an assumed body weight of 10 kg and daily total food consumption of 250 g). Based on the results of the study, 750 mg/kg bw is considered to be the no-observed-effect-level (NOEL) for the test substance in dogs of both sexes.

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:
experimental study
Adequacy of study:
key study
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
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 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

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

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

 

This substance definition complies very well with the ones given for manufactured and imported Indigo in section 1.2 of the registration dossier. According to the sameness criteria defined and agreed on by the Indigo SIEF, only Indigo that has less than 1 % aniline and methylaniline is covered by this registration.Hence, the samples of the mentioned studies correspond to the registered substance 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.