FAO Nutrition Meetings Report Series 
    No. 46A WHO/FOOD ADD/70.36

    The content of this document is the result of the deliberations of the
    Joint FAO/WHO Expert Committee on Food Additives which met in Rome,
    27 May - 4 June 19691

    Food and Agriculture Organization of the United Nations

    World Health Organization

    1 Thirteenth report of the Joint FAO/WHO Expert Committee on Food
    Additives, FAO Nutrition Meetings Report Series, in press;
    Wld Hlth Org. techn.  Rep. Ser., in press.


    Biological Data

    Biochemical aspects

    Microchemical methods have shown that filterable indigotine in the
    blood plasma of rabbits is excreted through the glomeruli. The
    filterable fraction is about 15 per cent. while 75 per cent. of the
    colour is excreted by the tubules. The lumina of the tubules and
    Bowman's capsules were injected with solutions of indigotine. Ten mg
    per cent. 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 per
    cent. of the radioactivity appeared in the urine in six hours and 10
    per cent. in the bile. The metabolites isatin-5-sulfuric acid and
    5-sulfoanthranilic acid appeared in the urine after two hours. After
    oral administration, only three per cent. of the radioactivity
    appeared in urine in three days, suggesting poor absorption from the
    alimentary tract. Faeces contained 60-80 per cent.of the oral dose.
    The faecal content was due to lack of absorption, not biliary
    excretion (Lethco & Webb, 1966).

    Acute toxicity


    Animal   Route                LD50            Reference
                             mg/kg body-weight

    Mice     Oral                >2 500           US FDA, 1969

    Mice     Subcutaneous           405           US FDA, 1949

    Rat      Oral                 2 000           Lu & Lavallée, 1964

    Rats     Intravenous             93           US FDA, 1969

    Five rats were given subcutaneous twice daily injections for three
    days and killed on the fourth day. An aqueous solution at a level of
    250 mg/kg body-weight 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).

    Special studies

    The colour was tested for mutagenic effect in a concentration of 0.5
    g/1  0.5 g/100 ml in cultures of Escherichia coli. No mutagenic
    effect was found. (Lück & Rickerl, 1960).

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

    Short-term studies

    Rat. Twenty rats were given first 1 ml two per cent. solution
    subcutaneous later only 0.5 ml of a 0.5 per cent. solution. Fifty-five
    injections were administered over a period of seven months. Control
    groups received injections of 50 per cent. glucose or 0.9 per cent.
    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).

    Dog. Two groups, consisting of two male and two female beagles were
    given 1.0 per cent. and two per cent. 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 (Hansen et al., 1966).

    Long-term studies

    Mouse. Groups of 25 male and 25 female mice received weekly for 104
    weeks subcutaneous injections of 2.5 mg indigotine as a one per cent.
    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
    (Hansen et al., 1966).

    Rat. A group of 20 male and 20 female rats received the dye as one
    per cent. solution for two years. Twenty males and 20 females were

    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 per cent. for two years. The growth of males was
    significantly inhibited at 2.0 and 5.0 per cent. 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 two per cent.
    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).


    The production of a high percentage of local sarcomata at the site of
    subcutaneous injections in rats led in the past to considerable
    discussion and consequently to extensive studies on this colour and
    the special condition of the experiment and does not constitute
    evidence of carcinogenicity by the oral route.

    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 thirteen-week study on the major metabolite revealed no
    toxic effects. Human metabolic studies would be useful.


    Level causing no toxicological effect in the rat

    One per cent. (= 10 000 ppm) in the diet equivalent to 500 mg/kg body

    Estimate of acceptable daily intake for man

                                      mg/kg body-weight

    Temporary acceptance                   0 -2.5

    Further work required by June 1974

    Two-year study in a non-rodent mammalian species


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

    Graham, R. C. B. & Allmark, M. G. (1959) Toxicol. appl. Pharmacol.,
    1, 144

    Hansen. W. H., Fitzhugh, O. G., Nelson, A. A. & Davis, K. J. (1966)
    Toxicol. appl. Pharmacol., 8, 29

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

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

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

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

    Oettel, H., Frohberg, H., Nothdurft, H. & Wilhelm, G. (1965) Arch.
    für Toxikol., 21, 9

    United States Food and Drug Administration (1969) Unpublished report

    See Also:
       Toxicological Abbreviations
       Indigotine (WHO Food Additives Series 6)
       INDIGOTINE (JECFA Evaluation)