The evaluations contained in this publication were prepared by the
    Joint FAO/WHO Expert Committee on Food Additives which met in Rome,
    4-13 June 19741

    World Health Organization     Geneva     1975


    1  Eighteenth Report of the Joint FAO/WHO Expert Committee on
    Food Additives, Wld Hlth Org. techn. Rep. Ser., 1974, No. 557.
    FAO Nutrition Meetings Report Series, 1974, No. 54.



         This compound has been evaluated for acceptable daily intake by
    the Joint FAO/WHO Expert Committee on Food Additives (see Annex 1,
    Refs Nos 10 and 20) in 1964 and 1969.

         Since the previous evaluation additional data have become
    available and are summarized and discussed in the following monograph.
    The previously published monographs have been expanded and are
    reproduced in their entirety below.



         Microchemical methods have shown that filterable indigotine in
    the blood plasma of rabbits is excreted through the glomoruli. The
    filterable fraction is about 15% while 75% of the colour is excreted
    by the tubules. The lumina of the tubules and Bowman's capsules were
    injected with solutions of indigotine. 10 mg% was the lowest
    concentration which left detectable traces of the colour in the
    kidneys. When an increased dose of colour is injected and the interval
    between the injection and the excision of the kidney shortened, the
    colour can be detected in the intracapsular spaces. The tubular
    excretion of the indigotine resembles quantitatively that of phenol
    red (Kempton et al., 1937).

         After intravenous injection of 35S-labelled dye in the rats, 63%
    of the radioactivity appeared in the urine in six hours and 10% in the
    bile. The metabolites isatin-5-sulfuric acid and 5-sulfoanthranilic
    acid appeared in the urine after two hours. After oral administration,
    only 3% of the radioactivity appeared in urine in three days,
    suggesting poor absorption from the alimentary tract. Faeces contained
    60-80% of the oral dose. The faecal content was due to lack of
    absorption, not biliary excretion (Lethco & Webb, 1966).


    Special studies on mutagenicity

         The colour was tested for mutagenic effect in a concentration of
    0.5 g/100 ml in cultures of Escherichia coli. No mutagenic effect
    was found (Lück & Rickerl, 1960).

    Special studies on metabolites

         Isatin-5-sulfonic acid, one of the metabolites of the colour, was
    fed to groups of three-week-old rats, 10 male and 10 female per group,
    at levels of 0, 0.25, 0.5, 1 and 2% for the period of 13 weeks
    followed by gross and histopathology examinations. The "no-effect"
    level of the compound was considered to be 2% (FDA, 1969).

    Special studies on teratogenicity


         Groups of 20 pregnant rats received 0, 25, 75 and 250 mg/kg/day
    of indigotine by gavage from day six to 15 of gestation. A positive
    control group received retinoic acid (7.5 mg/kg/day). No changes in
    behaviour, appearance or body weight gain were noted in relation to
    the test substance. At sacrifice on day 20 no effect was seen on
    maternal or fetal parameters nor were any terata observed in
    offsprings related to colour administration (Anonymous, 1972a).


         Groups of 10 pregnant rabbits received 0, 25, 75 and
    250 mg/kg/day of indigotine by gavage from day six to 18 of gestation
    and were sacrificed on day 29. A positive control group received
    150 mg/kg/day thalidomide. No adverse effects were noted on behaviour,
    appearance and weight gain of dams, nor on maternal or fetal
    parameters. No unusual terata related to the colour were seen on
    offsprings (Anonymous, 1972b).

    Acute toxicity

    Animal    Route     mg/kg bw       Reference

    Mice      Oral       2 500         USFDA, 1969

    Mice      s.c.         405         USFDA, 1969

    Rat       Oral       2 000         Lu & Lavalleé, 1964

    Rats      i.v.          93         USFDA, 1969

         Five rats were given subcutaneous injections twice daily for
    three days and killed on the fourth day. An aqueous solution at a
    level of 250 mg/kg bw was used. No oestrogenic activity (normal
    uterine weight) was detected but the animals lost weight (Graham &
    Allmark, 1959).

         In experiments with guinea-pigs it was found that this colour had
    no sensitization activity (Bär & Griepentrog, 1960).

    Short-term studies


         Two groups, consisting of two male and two female beagles were
    given 1.0% and 2% indigotine in the diet for a two-year feeding trial.
    One male and one female served as controls. Two dogs on the high level
    died at 19 weeks; they were replaced and another dog was added to the
    control group. Two more on the high level died, at 21 and 40 weeks;
    one on the low level died at 36 weeks; and one control died at 34
    weeks. Deaths were due to virus infections. There were no clinical
    signs, gross lesions or microscopic pathology attributable to the dye
    (Kansen et al., 1966).


         Four groups of three male and three female weanling large white
    pigs were given 0, 150, 450 and 1350 mg/kg/day of indigotine in their
    diet for 90 days. There was no effect on growth, urine and serum
    analysis or organ weights. A slightly reduced haemoglobin level and
    red cell count was found after both 45 and 90 days in the blood of two
    of the three males given 1350 mg/kg. Histological examination revealed
    liver abscesses in one male fed indigotine at 150 mg/kg/day but this
    could not be attributed to the treatment given. No other abnormalities
    were seen (Gaunt et al., 1969).

    Long-term studies


         Groups of 25 male and 25 female mice received weekly for 104
    weeks subcutaneous injections of 2.5 mg indigotine as a 1% aqueous
    solution, the control group of 50 receiving 0.25 ml physiological
    saline. Many mice died from acute convulsions immediately after
    injection of the test dye but otherwise no deleterious effects
    attributable to the subcutaneous injections were noted. Tumours were
    randomly distributed among test and control groups (Hanson et al.,

         Groups of 30 male and 30 female mice (Charles River CD1 strain)
    were fed diets containing 0.2, 0.4, 0.8 or 1.6% indigotine for 80
    weeks, with a group of 60 males and 60 females serving as controls.
    The treatment had no effect on the death rate, body weight gain, organ
    weights or the results of the histopathological examination, including
    the incidence of tumours. There was a slight anaemia in mice given
    diets containing 0.8 or 1.6% indigotine.

         It is concluded that the feeding of indigotine to mice at dietary
    levels of up to 1.6% does not exert any carcinogenic effect. The no-
    untoward-effect level was 0.4% of the diet equivalent to an intake of
    approximately 550 mg/kg/day (Hooson et al., 1974).


         A group of 20 male and 20 female rats received the dye as 1%
    solution for two years. Twenty males and twenty females were controls.
    No adverse effects were observed on growth, reproduction and survival
    and no specific gross or microscopic pathology was noted (organs not
    stated). There was no difference in tumour incidence between the
    groups (Oettel et al., 1965).

         Groups of 24 rats, equally divided by sex, were fed the colour at
    0, 0.5, 1.0, 2.0 and 5.0% for two years. The growth of males was
    significantly inhibited at 2.0 and 5.0%. There was no change in
    mortality, organ weights or haematology, nor any gross or microscopic
    pathology related to treatment (Hansen et al., 1966). Groups of 80 and
    100 rats were injected weekly with a 2% aqueous solution or an
    equivalent volume of saline solution for two years. Survival of test
    animals did not differ from controls. Fourteen of 80 injected rats had
    a fibrosarcoma at the site of injection and one saline injected rat
    developed a fibroma at the injection site. No other effects were
    observed (Hansen et al., 1966).

         Twenty rats were given first 1 ml 2% solution subcutaneously
    later only 0.5 ml of a 0.5% solution. Fifty-five injections were
    administered over a period of seven months. Control groups received
    injections of 50% glucose or 0.9% sodium chloride. All animals were
    observed for lifespan. No local tumours and only one auxiliary tumour
    in the test group were found (Oettel et al., 1965).


         Several species have been investigated in short-term studies.

         One long-term study in mice has now been completed thus meeting
    the request of the Committee. The metabolic studies on this colour are
    fairly complete and the two long-term studies in the rat do not point
    to any significant toxic effects. A 13-week study on the major
    metabolite revealed no toxic effects.


    Level causing no toxicological effect

         Rat: 1% (= 10 000 ppm) in the diet equivalent to 500 mg/kg bw.

    Estimate of acceptable daily intake for man

         0-5 mg/kg bw


    Anonymous (1972a) International Research and Development Corporation.
         Unpublished report submitted to WHO

    Anonymous (1972b) International Research and Development Corporation.
         Unpublished report submitted to WHO

    Bär, F. & Griepentrog, F. (1960) Med. u. Ernahr., 1, 99

    Graham, R. C. B. & Allmark, M. G. (1959) Toxicol. appl. Pharmacol., 1,

    Hansen, W. H. et al. (1966) Toxicol. appl. Pharmacol., 8, 29

    Hooson, J. et al. (1974) Fd. Cosmet. Toxicol., in press

    Kempton, R. R., Bott, P. A. & Richards, A. N. (1937) Amer. J. Anat.,
         61, 505

    Gaunt, I. F. et al. (1969) Fd. Cosmet. Toxicol., 7, 17

    Lethco, E. J. & Webb, J. M. (1966) J. Pharmacol. exp. Ther., 154, 384

    Lu, F. C. & Lavalleé, A. (1964) Canad. pharm. J., 97, 30

    Lück, H. & Rickerl, E. (1960) Z. Lebensmitt.- Untersuch., 112, 157

    Oettel, H. et al. (1965) Arch. für Toxicol., 21, 9

    United States Food and Drug Administration (1969) Unpublished report.

    See Also:
       Toxicological Abbreviations
       Indigotine  (FAO Nutrition Meetings Report Series 46a)
       INDIGOTINE (JECFA Evaluation)