The data contained in this document were examined by the
    Joint FAO/WHO Expert Committee on Food Additives*
    Geneva, 18-27 April 1977

    Food and Agriculture Organization of the United Nations
    World Health Organization

    * Twenty-first Report of the Joint FAO/WHO Expert Committee on Food
    Additives, Geneva, 1977, WHO Technical Report Series No. 617





         No information is available on the metabolic fate of Chocolate
    Brown HT beyond a report describing in vitro decolorization of the
    colour by rat liver homogenates.


    Acute toxicity


                                LD50 with 95%
    Species   Sex      Route    confidence limits    Reference
                                (mg/kg bw)

    Mouse     Male     i.p.     300 (268-336)        Hall and Lee, 1966

              Female   oral     >2000                Hall and Lee, 1966

                       i.p.     220 (169-286)        Hall and Lee, 1966

    Rat       Male )
              Female)  i.p.     375 (317-444)        Hall and Lee, 1966

              Male )
              Female)  oral     >2000                Hall and Lee, 1966

    Remark: the colouring was administered in aqueous solutions.

    Short-term studies


         The Chocolate Brown HT (purity minimal 85%) was given to groups
    of 12 male and 12 female rats (strain Porton) at levels of 0.5, 1.0
    and 2.0% for a period of 12 weeks. No adverse effect on the appearance
    or condition of the animals were found. Growth retardation, not
    associated with a diminished food intake, was evident in males at the
    1 and 2% dietary levels. Haematological examinations conducted at week
    6 and 12 and liver and kidney function tests (increased urinary

    glutamic-oxalo acetic transeminase activity) made at the end of
    the experiment revealed no departure from normality apart from
    non-significant reductions of red cell count and haematocrit in males
    at the 2% level.

         Furthermore a mild degree of renal dysfunction in both sexes at
    the two highest dietary levels were found. In the 2% level significant
    increases in the relative weights/body weight ratio of the brain and
    adrenals in males, the spleen and kidneys in both sexes and ovaries
    were found.

         In the gross and histological studies, brown pigmentation was
    evident in the Kupffer cells of the liver, the proximal convoluted
    tubules of the kidney and the lymph nodes especially of the small
    intestine. The intensity of this effect was proportional to the
    dietary level administered and the pigment was only very occasionally
    present, and in trace amounts, in animals of the 0.5% group.
    Otherwise, the types and incidence of pathological changes were
    comparable between control and test groups. In the group with 0.5% of
    Chocolate Brown HT in the diet for 12 weeks no adverse effects were
    seen (Hall and Lee, 1966).

         Chocolate Brown HT was fed to rats (Carworth Farm) at dietary
    levels of 0.0, 0.02, 0.06, 0.20, 0.60, 1.0 and 2% for 90 days. No
    adverse effect was observed in respect of appearance, behaviour or
    survival of animals. Although body weight gain in treated rats did not
    differ significantly from that of the controls, when adjustment was
    made for the total food intake, the slight growth retardation observed
    in males at the 1 and 2% levels and in females at the highest level
    became significant. Haematological examination revealed slight but
    significant decreases in haemoglobin, red cell count and haematocrit
    in male rats on the highest dietary level. In the biochemical studies,
    reductions in the blood urea levels occurred in both sexes and were
    significant in all groups except at the 0.06 and 0.6% levels.
    Increases in total serum protein were seen in males on the 0.6 and 1%
    levels. The absolute weights of the heart, kidneys, liver, spleen and
    testes remained unaffected at all levels.

         There was no evidence of pathological damage at any dietary level
    of the colouring administered but pigment was seen at the two highest
    levels in certain intestinal cells, lymph nodes and cells of the
    proximal convoluted tubes of the kidney (Chambers et al., 1966).


         Groups of three male and three female pigs (Large White strain)
    were dosed with Chocolate Brown HT at dose levels of 0, 5, 20 or
    100 mg/kg body weight/day for 13 weeks. At the beginning of the
    experiment the animals were 10 weeks old. The administration of the
    colouring had no adverse effect on mortality, growth, organ weights

    and urine composition. The haemoglobin levels in all three treatment
    groups of male pigs at week 13 were significantly below the control
    values. However, these findings were inconsistent with other blood
    parameters and tissue pathology.

         The incidence of histopathological lesions are comparable between
    the control and treated groups (details are not available) (Hendy et
    al., 1975).

    Long-term studies


         Groups of 48 male and 48 female mice (strain TF1) were given
    diets containing 0, 0.01, 0.1 or 0.5% Chocolate Brown HT (purity
    minimal 85%) for 80 weeks. A slightly reduced body weight gain and a
    lower heart weight in males of the 0.5% group was found. At the same
    level at week 77 the packed cell volume and total leucocyte count
    values in females were lower than those of the controls. However, the
    relationship of these findings with the treatment was questionable.
    There was no statistically significant difference in mortality between
    the treated and control groups. A brown coloration of the internal
    organs seen at the highest dose-level was due to the Chocolate Brown
    HT feeding.

         Besides an increased incidence of leucocyte infiltration in the
    liver in the females of the 0.5% group an increase in cystic ovaries
    was seen. The distribution of tumours was similar in all groups and
    from this study it was concluded that no evidence of a carcinogenic
    effect was found (Drake et al., 1975).


         Groups of 48 male and 48 female rats (Wistar strain) were given
    diets containing 0 (control), 500, 2000 or 10 000 ppm Chocolate Brown
    HT (purity minimal 85%) for two years. These treatments had no adverse
    effect on the bodyweight gain, food or water consumption, haematology,
    renal function, serum constituents and organ weights. The mortality
    was only slightly increased in the males with the highest dose level
    of the dye. The results of the histopathological examination showed no
    adverse effects except that in the mammary glands a non-significant
    ("dose-related") increase of the occurrence of fibroadenosis was seen.
    The incidence of tumours in the treated animals was not different from
    the control animals (Carpanini et al., 1975).


    Carpanini, F. M. B., Butterworth, K. R., Gaunt, I. F., Kiss, I. S.,
    Grasso, P. and Gangolli, S. D. (1975) Long-term toxicity studies on
    Chocolate Brown HT in rats, BIBRA Research Report No. 19/1975
    (unpublished report)

    Chambers, P. L., Hunter, C. G. and Stevenson, D. E. (1966) Short-term
    study of Chocolate Brown HT in rats, Fd. Cosmet. Toxicol., 4,

    Drake, J. J. P., Butterworth, K. R., Gaunt, I. F. and Hardy, J. (1975)
    Long-term toxicity studies of Chocolate Brown HT in mice, BIBRA
    Research Report No. 16/1975 (unpublished report)

    Fore, H., Walker, R. and Golberg, L. (1967) Fd. Cosmet. Toxicol.,
    5, 459

    Hall, D. E. and Lee, F. S. (1966) Acute (mouse and rat) and short-term
    (rat) toxicity studies on Chocolate Brown HT, Fd. Cosmet. Toxicol.,
    4, 143-149

    Hendy, R. J., Butterworth, K. R., Gaunt, I. F., Hooson, J. and Grasso,
    P. (1975) Short-term toxicity study of Chocolate Brown HT in pigs,
    BIBRA Research Report No. 17/1975 (unpublished report)

    Walker, R. (1970) The metabolism of azo compounds. A review of the
    literature, Fd. Cosmet. Toxicol., 8, 659

    See Also:
       Toxicological Abbreviations