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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

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

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

 

Vat Indigo Potassium Salt 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 Vat Indigo, it contains a distinctly lower amount of aniline and methyl-aniline (see section 1.2 Impurities). 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 at dietary concentrations of up to 3% (2565 mg/kg bw/day),a 2-year chronic feeding study in male and female rats at dietary concentrations of up to 3% (1200 mg/kg bw/day),13-week range-finding study and 2-year feeding study in male and female beagle dogs at dietary concentrations of up 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.