INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
WORLD HEALTH ORGANIZATION
TOXICOLOGICAL EVALUATION OF SOME
FOOD COLOURS, EMULSIFIERS, STABILIZERS,
ANTI-CAKING AGENTS AND CERTAIN
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.
CITRUS RED 2
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).
In male mice the LD50 was > 4 g/kg body weight (Hazelton
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 &
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