INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
WORLD HEALTH ORGANIZATION
SUMMARY OF TOXICOLOGICAL DATA OF CERTAIN FOOD ADDITIVES
WHO FOOD ADDITIVES SERIES NO. 12
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
CHOCOLATE BROWN HT
EVALUATION FOR ACCEPTABLE DAILY INTAKE
BIOLOGICAL DATA
BIOCHEMICAL ASPECTS
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.
TOXICOLOGICAL STUDIES
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
Rat
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).
Pig
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
Mouse
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).
Rat
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).
REFERENCES
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,
151-155
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