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

    After single oral doses of 2-20 mg in corn oil solution to rats, five
    to seven per cent. of the administered colour was recovered in the
    faeces in 48 h. When the dose was reduced to 0.5 mg no colour was
    found. When administered as a 0.5 per cent. dry mixture, 26.3 per
    cent. was found in the faeces. Rabbits and dogs showed similar
    results. Small amounts of 1-amino-2-naphthyl sulphate were identified
    in the urine of rats. This demonstrates that the colour is reduced at
    the azo linkage in the animal body followed by conjugation to give the
    0-glucuronide and ethereal sulphate derivatives of 1-amino-2-naphthol.

    After a single dose small amounts of the colour were found in the body
    fat. Repeated administration resulted in gradual reduction of the
    concentration of the colour in the fat and finally it disappeared
    altogether after seven to 10 days.

    In vitro incubation of emulsions of the colour with the intestinal
    contents of the rat, rabbit and dog resulted in destruction of the
    colour by the bacterial flora (Radomski, 1961).

    Acute toxicity

    In male mice the LD50 was > 4 g/kg body weight (Hazelton
    Laboratory, 1954).

    Short-term studies

    Dog: Nineteen beagle dogs received 0, 50 and 200 mg of the colour
    per kg body weight per day by capsule, six days per week for 76 or 104
    weeks. Clinical studies did not show differences between control group
    and the group with 50 mg/kg/day. The animals of the group of 200
    mg/kg/day showed in the beginning decreased activity and periods of
    poor appetite. The animals progressed in about 26 weeks and seemed
    comparable with the control animals in their appearance and behaviour.
    A decrease in cell volume, haemoglobin and total erythrocyte count was
    found in this group after 26 weeks. Five of the six animals of the 200
    mg/kg/day showed an increase in total counts of leucocytes. No
    biochemical values and urine analyses showed abnormalities. Four dogs
    of the 50 mg group and three control groups were sacrificed after 76
    weeks. The other dogs were sacrificed after 104 weeks. No significant
    gross or microscopic pathological abnormalities were found in the
    control animals and the animals of the 50 mg/kg/day. The organs,
    liver, kidneys and spleen of the animals with 200 mg/kg/day were
    significantly increased in weight. Histologically no abnormalities
    were found, except an evidence of haemopoietic hyperplasia (Paynter &
    Scala. 1964).

    Long-term studies

    Mouse. Groups of 50 male and 50 female mice were fed diets
    containing 0, 0.01 per cent., 0.03 per cent., 0.1 per cent., 0.3 per
    cent., 1.0 per cent. or 3.0 per cent. colour for periods up to 80
    weeks. Morbidity and mortality were increased considerably at and
    above the 0.3 per cent. level and tests were discontinued. At the 0.1
    per cent. level, there were increases in the mortality rate in both
    sexes and degenerative changes appeared in the livers of female mice.
    No untoward effects were found in mice on the 0.03 per cent. level.
    Dietary levels up to 0.1 per cent. had no significant effect on the
    type, incidence or time of appearance of tumours (Sharratt et al.,

    Another group of 50 male and 50 female mice were injected
    subcutaneously with a 10 per cent. suspension of the colour on
    alternate sites for 35 weeks. The next 15 injections were made at
    three-week intervals. A control group received only the vehicle.
    Females, but not males, showed an increased incidence of malignant
    tumours and they also appeared earlier. Adenocarcinoma of the lung and
    lymphosarcoma were most frequently found although their incidence was
    comparable in test and control groups on the oral regime. No tumours
    developed at the site of injection (Sharratt et al., 1966).

    Groups of 20 male and 20 female mice were fed on diets containing O,
    0.5 per cent. and 0.25 per cent. of colour for 24 months. The food
    intake and growth rate of female mice showed no significant
    differences from controls. Male mice showed a decreased food
    consumption and a corresponding decrease in growth rate at the 0.25
    per cent. level. There were deaths in various groups but not
    attributable to the ingestion of the dye. There were no significant
    haematological changes after one year. The terminal weights of five
    major organs were within normal range. Many of the mice showed a
    thickened bladder wall with patchy or total epithelial hyperplasia,
    and this occurred in all test groups but not in controls. (As male
    mouse at the highest level had a papillary carcinoma together with
    pigmented stones. The nature and extent of the bladder lesions are
    similar to that reported with mice fed 0.01 per cent. of
    4-ethylsulfonyl-naphthalene-1-sulfonamide (Dacre, 1965).

    Rat: Seven hundred and eighty rats received dietary levels of O,
    0.05, 0.1, 0.5, 1.0, 3.0 and 5.0 per cent. of the colour for two
    years. The animals of the groups with three and five per cent. showed
    a significant inhibition of the growth. These animals were killed
    after 31 weeks. The animals of the 0.05 and 0.1 per cent. exhibited
    normal appearance and behaviour. Some rats of 0.5 per cent. and of
    higher levels developed subcutaneous oedema-like swelling of the head,
    neck, thoracic region and forelimbs. Hydrothorax was revealed in a
    number of these animals.

    At 31 weeks clinical studies were performed and in the female animals
    at the five per cent. level the plasma-protein, haemoglobin and
    haematocrit values were significantly lowered. The male animals showed
    only the decreased plasma-protein levels. The ratio liver weight/body
    weight of the animals of the 0.5 per cent. and higher was increased.
    The histopathological examination of the organs of the animals of 0.05
    and 0.1 per cent. did not show abnormalities. At 0.5, 1.0, 3.0 and 5.0
    per cent. cardiac changes consisting of interstitial oedema and
    fibroblastic cell proliferation were found.

    Two groups of 50 male and 50 female rats were given once a week
    subcutaneously respectively 0.2 ml tricaprylin and 0.2 ml tricaprylin
    containing 20 mg of the colour for two years. The injections were
    given alternated in the axillary and inguinal regions. These
    injections gave small subcutaneous deposits or nodules located at the
    injection sites. During the experiments the nodules persisted but
    there was no evidence of active enlargement. These nodules showed
    histologically the encapsulated injected material with a thin fibrous
    wall. No tumours were found (Paynter & Scala, 1964).

    Groups of 20 male and 20 female rats were fed diets containing 0, 0.05
    per cent. and 0.25 per cent. colour for 24 months. Food intake and
    growth rate of male rats only showed a difference from controls, being
    reduced together with decreased growth rate at the 0.05 per cent.
    level. Mortality was not related to administration of the colour.
    Organ weights of five major organs were comparable in all groups.
    Haematology was normal after 12 months. Hyperplasia of the bladder
    epithelium, either partly or total, was noted at all levels tested
    together with thickening of bladder wall. The lesions were reminiscent
    of those occurring with 0.01 per cent. of 4-ethylsulforyl
    naphthalene-1-sulforanide in the diet (Dacre, 1965),


    Two long-term studios in mice and rats give discrepant findings in
    regard to bladder pathology with closely related dosage levels.
    Papillary carcinoma were produced but the presence of bladder stones
    made it difficult to judge whether the colour itself possesses weak
    carcinogenic activity. On the other hand there is evidence of
    metabolic conversion to a 1-amino-2-naphthylsulfate belonging to the
    group of substances known to induce bladder tumours when implanted in
    paraffin wax pellots. The induction of remote malignant tumours in
    female mice is noteworthy. The many unexplained findings observed
    require further study in other strains to confirm or refute them and
    to explain the difference between oral and parenteral effects.


    This colour should not be used as a food additive.


    Dacre, J. C. (1965) Proc. Univ. Otago med. Sch, 43, 31

    Paynter, O. E. & Scala, R. A. (1964) unpublished report by Hazleton

    Radomski, J. L. (1961) J. Pharmacol. exp. Ther., 134, 100

    Sharratt, M., Frazer. A. C. & Paranjoti, I. S. (1966) Fd. Cosmet.
    Toxicol., 4, 493

    See Also:
       Toxicological Abbreviations