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

    The colour was administered orally to rats as a two per cent. aqueous
    solution at a level of 200 mg per rat. Almost the entire amount was
    excreted unchanged in the faeces within 40 h after administration. In
    a later investigation, the presence of the colour in the bile was
    observed in rats, rabbits and dogs after oral administration. In the
    case of the dog, the amount did not exceed five per cent. of the dose
    administered (Hess & Fitzhugh, 1953; 1954; 1955). Administration of an
    aqueous solution of the colour by stomach tube resulted in 89 per
    cent. excretion in the faeces; none was found in the urine. After
    subcutaneous injection of 80-100 mg some 79 per cent. was excreted; 77
    per cent. appeared in the faeces and 2.5 per cent. in the urine
    (Imperial Chemical Industries, 1958).

    Acute toxicity


    Animal    Route                LD50            Reference
                              mg/kg body weight 

    Mouse     Subcutaneous         4 600           Gross, 1961

    Rat       Oral                > 2 000           Lu & Lavallée, 1964

    Groups of five young rats were given injections of the colour
    subcutaneously twice daily for three days. The rats were killed on the
    fourth day. The colour was administered in aqueous solution at a level
    of 250 mg/kg body weight each injection. No oestrogenic activity was
    detected. No other abnormalities were found (Graham & Allmark, 1959),

    Special studies

    o-Sulfobenzaldehyde, one of the components of Brilliant Blue FCF, was
    fed to groups of three-week-old rats, 10 males 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 o-sulfobenzaldehyde was considered to be two per
    cent. (United States Food and Drug Administration, 1969).

    Short-term studies

    Mouse: Daily injections of 2 mg of colour over 30 days were well
    tolerated by mice. Some animals showed swelling of the liver and
    spleen after receiving a total of 4 mg (Gross, 1961). Mice fed 1200 mg
    of the dye ever 19 days showed no damage (Gross, 1961).

    Rat: Subcutaneous injection of 1 ml of 0.8 per cent. aqueous
    solution twice weekly produced histological changes suggestive of
    subsequent sarcoma formation unassociated with chemical carcinogenic
    potential (Grasso & Golberg, 1966).

    Dog: Studies were conducted on beagle dogs fed diets containing one
    per cent. and two per cent. of the colour for one year. No gross or
    microscopic changes due to treatment were seen (Hansen et al., 1966).

    Cat: Parenteral injection into cats of a five per cent. aqueous
    solution, 1.0 g on the first day and 0.1 g from the ninth to the
    eighteenth day, did not produce methaemoglobinaemia or Heinz bodies
    (Gross, 1965).

    Mouse: A group of 57 male and 43 female mice were given 1 mg colour
    per day in the diet. Observations extended over 500-700 days. No
    evidence of carcinogenic action was found (Waterman & Lignac, 1958).
    Subcutaneous injections of 10 doses of 4 mg followed by 50 doses of 6
    mg showed no tumour production after 78 weeks (Imperial Chemical
    Industries, 1962).

    Rat: The colour was fed in a concentration of four per cent. of the
    diet to five male and five female rats for 600 days. Gross staining of
    the glandular stomach and some granular deposits in the stomach but no
    tumours were observed (Willheim & Ivy, 1953).

    Eighty-five rats were fed a diet containing 0.1 per cent. of the
    colour for their life span.  The daily intake was 10-15 mg. No tumours
    were found (Klinke, 1955). Groups of 24 weanling Osborne-Mendel rats,
    evenly divided by sex, were fed the colour at levels of 0, 0.5 per
    cent., 1.0 per cent., 2.0 per cent. and 5.0 per cent. for two years.
    No outward effects were observed during the experiment. Gross and
    microscopic examination revealed only incidental lesions, unrelated to
    treatment, with no effect on tumour production. The statistical
    evaluation revealed no changes in organ weights, mortality, or growth.
    Haematology was normal (Hansen et al., 1964). Four groups of 15 male
    and 15 female rats were given diets containing, 0, 0.03 per cent., 0.3
    per cent. and 3.0 per cent. of the colour for 75 weeks. No depression
    of growth, food consumption or food efficiency was found. Mortality
    was increased in the three per cent. group due to respiratory
    infection unrelated to the administration of the compound.
    Haematological findings were normal (Mannell et al., 1962).

    Subcutaneous injection of various preparations of the colour have been
    reported to produce fibrosarcomas at the site of the injection (Nelson
    & Hagan. 1953; Gross, 1961; Hansen et al., 1966). However, no tumours
    were produced by a course of subcutaneous injections of 0.5 ml of a
    four per cent. solution in isotonic saline (Mannell & Grice, 1964).

    Eighty-four male and 84 female Wistar rats were given twice-weekly 10
    doses of 20 mg per rat as a two per cent. solution followed by 50
    doses of 30g per rat as a six per cent, solution. One hundred and
    nineteen of the 168 rats developed sarcomas at the site of injection.
    Only seven animals survived two years. Males had no other tumours but
    females had six mammary, one ovarian, one uterine tumour and two
    hepatomas. Twenty-six controls survived two years out of 48 and the
    females developed one mammary, one ovarian and four uterine
    tumours (Imperial Chemical Industries, 1962). Six male and six female
    Slonacker rats were treated the same way as Wistar rats. All died
    within 420 days, tumours developing at the injection site in five
    males and three females. Out of 12 controls, 10 were alive at 400
    days, No tumours developed in controls (Imperial Chemical Industries,


    The production of a high percentage of local sarcomata at the site of
    subcutaneous injection in rats has led in the past to considerable
    discussion and consequently to extensive studies on this colour. The
    production of these sarcomata is considered to be related to the
    physico-chemical properties of the colour and special condition of the
    experiment and does not constitute evidence of carcinogenicity by the
    oral route.

    Biochemical studies have shown that the colour is poorly absorbed and
    is almost completely excreted in the faeces after parenteral
    administration. Extensive long-term studies in two species are
    available. In addition a 13-week study in rats with
    o-sulfobenzaldehyde, one of the components of commercial Brilliant
    Blue FCF, has been carried out. Oral feeding produced no pathological
    changes at the highest levels used in adequate experiments.
    Biochemical studies on metabolism using modern techniques are


    Level causing no toxicological effect in the rat

    Five per cent, in the diet (= 50 000 ppm) equivalent to 2500 mg/kg
    body weight/day.

    Estimate of acceptable daily intake for man

                                   mg/kg body weight/day
    Unconditional acceptance                             


    Graham, R. C. B. & Allmark, M. G. (1959) Toxic. Appl.Pharmac., 1,

    Grasso, P. & GoIberg L. (1966) Fd. Cosmet. Toxicol., 4, 269

    Gross, E. (1961) Z. Krebsforsch., 64, 287

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

    Hess, S. M. & Fitzhugh, O. G. (1953) Fed. Proc., 12, 330

    Hess, S. M. & Fitzhugh, O. G. (1954) Fed. Proc., 13, 365

    Hess, S. M. & Fitzhugh, O. G. (1955) J. Pharmacol. exp. Ther., 114,

    Imperial Chemical Industries (1958) Unpublished report

    Imperial Chemical Industries (1962) Unpublished report

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

    Mannell, W. A., Grice, H. C. & Allmark, M. G. (1962) J. Pharm.
    Pharmacol., 14, 378

    Mannell, W. A. & Grice, H. C. (1964) J. Pharm. Pharmacol., 16, 56

    Nelson, A. A. & Hagan, E. C. (1953) Fed. Proc., 12, 397

    United States Food and Drug Administration (1969) Unpublished report
    Willheim, R. & Ivy, A. C. (1953) Gastroenterology, 23, 1

    Waterman N. & Lignac, G. O. E. (1958) Acta. physiol. pharmacol.
    neerl., 7, 35

    See Also:
       Toxicological Abbreviations
       BRILLIANT BLUE FCF (JECFA Evaluation)