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.
Adult rats were given by gavage or s.c. an aqueous solution of Wool
Green BS. Of 68 mg given orally only 0.16 mg appeared in the urine and
18.4 mg in the faeces, recovery being 29 per cent. After s.c.
administration of 30-56 mg, some 7-12 mg appeared in the urine and
2-18 mg in the faeces, only 38 per cent. being recovered (Daniel,
1959). Feeding pigs and calves with milk containing 25 mg of the
colour per litre, no absorption of the colour was found. All was
excreted in the faeces within five days and one to two weeks
respectively (Dalgaard-Mikkelsen & Rasmussen, 1962).
Animal Route LD50 Reference
per kg body weight
Rat oral 2.0 g Lu & Lavallée, 1964
Rat. Twenty rats were given 10 ml of milk daily containing 0.15 mg
of the colour/litre for three months. Another 20 rats were given 10 ml
of milk containing 30 mg of the colour/litre for five weeks. No
abnormalities were observed in food intake and growth. No pathological
changes were found. The gastric and intestinal mucosa of the animals
on the highest dose level showed a green colour. No colour was found
in the muscles, liver and kidneys (Dalgaard-Mikkelsen & Rasmussen,
1962). Four male and four female rats were given 50 ml of a 0.1 per
cent aqueous solution daily for 91 days. Only the faeces were
discoloured, not the urine. No ill-effects were observed (ICI, 1953).
Mouse. Thirty female Schofield mice were given injections three
times weekly, each mouse being given 10 doses of 4 mg (in 0.2 ml
solution) followed by 500 doses of 6 mg (in 0.2 ml). A maximum total
of 340 mg/mouse was given in 226 days. Thirty control mice were
injected with distilled water. After 78 weeks no tumours appeared at
the injection site in any of the mice of which 20 survived in each
group (ICI, 1964).
Rat. The colour was fed at a level of two per cent. in the diet of
20 male and 20 female Wistar rats for two years. Twenty-four of the
animals so treated were alive at the end of this time. No tumours were
observed and deaths occurring the second year were attributable to
lung infection which occurs also in control animals (ICI, 1964).
Two samples of the colour were used containing respectively 0.02 per
cent. of ether extractable material and 0.4 per cent. of ether
extractable material. Ten doses of 20 mg per rat (two per cent.
solution) were followed by 50 doses of 30 mg per rat (six per cent.
solution). The maximum total dose in each case was 1.7 gm per rat.
Twenty-four male and 24 female Wistar rats were tested with each
sample making a total of 96 animals. In five rats local sarcomata
developed at the site of injection. Fifty-four were still living at
two years. Five of the females developed mammary and two uterine
tumours. No subcutaneous sarcomata were seen in control rats (24 male
and 24 female rats were dosed with distilled water in parallel as
controls). Twenty-six out of 48 control rats were still alive at the
end of two years. In the female controls, one mammary, one ovarian and
four uterine tumours were found (ICI, 1964).
Forty rats were given a diet containing two per cent. of the colour
for 730 days. The total intake was 146 g/animal. Sixteen animals died
within 730 days. No tumours were found (ICI, 1964).
Six male and six female Slonaker rats were also given the same doses
as above and controls injected with distilled water were included as
before. Of the 12 rats dosed all were alive at 500 days, nine at 600
days and one at 700 days. No tumours developed at the injection site
in any of these rats. Three mammary tumours were found in the tested
group. One female control rat was found to have a mammary tumour (ICI,
A group of 20 young rats, 10 male and 10 females were given weekly
subcutaneous injections of 0.5 ml of a four per cent. solution of the
colour in isotonic saline. Each rat received 20 mg of the colour per
week. Another group was given isotonic saline as control. Totally 45
injections, i.e. 900 mg of the colour, were given. The experiment was
terminated after 71 weeks. The mortality was as in the control group.
No tumours were found (Mannell & Grice, 1964).
Thirty-five Wistar rats, 15 males and 20 females, aged two months,
were given the colour in a dose level of 10 000 ppm during the first
two months. After this period the dose level was increased to 20 000
ppm and given to the animals for their life-span. Fifteen animals
(five male and 10 female) were used as control. The average life-span
of the test animals was 21 months and for the control animals 24
months. The last animal died in both groups at an age of 31 months.
The colour was found to have no effect on behaviour of the animals,
growth, reproduction and the offspring. No histopathological
abnormalities were found except a benign tumour in the small intestine
of a female animal in the test group (Truhaut, 1964).
Sixty rats, half male and half female, age two months, were given a
diet containing 20 000 ppm for their life-span. Thirty animals were
used as controls. The average life-span of the test animals was 30
months, the control animals 24 months. The last animal of the two
groups died after 42 and 31 months respectively. No influence of the
colour on growth, blood picture, reproduction and the offspring was
found. One female animal in the test group had a benign tumour in the
small intestine and in two female control animals, a benign mammary
tumour was found (Truhaut, 1964).
Fifty Wistar rats, half male and half female, age two months, were
given 20 000 ppm, of the colour for their life-span. These animals
received a subcutaneous injection of 1 ml of a one per cent aqueous
solution each week. Twenty-five rats, 13 male and 12 female, were used
as controls. These animals received subcutaneously 1 ml of distilled
water weekly. The average life-span of the treated and control animals
was 29 months and the last animal died after 36 months in both groups.
No influence of the colour on growth, blood picture, reproduction and
on the offspring was noticed. In three female test animals and in one
female control animal a subcutaneous tumour was found (Truhaut, 1964).
One hundred Wistar rats, half male and half female, age two months,
were given subcutaneous injections of 1 ml of a three per cent. colour
solution in saline (each injection contained about 30 mg) twice weekly
for 30 weeks. The total amount given was about 1.8 g. Fifty control
animals, half male and half female were given subcutaneous injections
of 1 ml saline in the same way. The animals were observed for their
life-span (about two years). In the control animals, no tumours were
found on the site of injection while in three test animals, two male
and one female sarcomas were induced (Truhaut, 1964).
This colour has been studied in adequate long-term feeding tests in
rats and also parenterally in mice. Metabolic information is lacking.
Level causing no toxicological effect in the rat
Two per cent. (= 20 000 ppm) in the diet equivalent to 1000 mg/kg body
Estimate of acceptable daily intake for man
Temporary acceptance mg/kg body weight
0 - 25
Further work required by June 1974
Metabolic studies in several species, preferably including man and two
year studies in a non-rodent mammalian species.
Dalgaard-Mikkelsen, S. W. & Rasmussen, F. (1962) 16th International
Dairy Congress, 465
Daniel, J. W. (1959) Unpublished Report submitted by Imperial Chemical
Imperial Chemical Industries (1953) Unpublished Report
Imperial Chemical Industries (1964) Unpublished Report
Lu, F. C, & Lavallée, A. (1964) Canad. pharm. J., 97, 30
Mannell, W. A. & Grice, H. C. (1964) J. Pharm. Pharmacol., 16, 56