INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
WORLD HEALTH ORGANIZATION
TOXICOLOGICAL EVALUATION OF SOME
FOOD COLOURS, ENZYMES, FLAVOUR
ENHANCERS, THICKENING AGENTS, AND
CERTAIN FOOD ADDITIVES
WHO FOOD ADDITIVES SERIES 6
The evaluations contained in this publication were prepared by the
Joint FAO/WHO Expert Committee on Food Additives which met in Rome,
4-13 June 19741
World Health Organization Geneva 1975
1 Eighteenth Report of the Joint FAO/WHO Expert Committee on
Food Additives, Wld Hlth Org. techn. Rep. Ser., 1974, No. 557.
FAO Nutrition Meetings Report Series, 1974, No. 54.
GREEN S
Explanation
This compound has been evaluated for acceptable daily intake by
the Joint FAO/WHO Expert Committee on Food Additives (see Annex 1,
Refs Nos 10 and 20) in 1964 and 1969 under the name of Wool Green BS.
Since the previous evaluation additional data have become
available and are summarized and discussed in the following monograph.
The previously published monographs have been expanded and are
reproduced in their entirety below.
BIOLOGICAL DATA
BIOCHEMICAL ASPECTS
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%. 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% 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).
TOXICOLOGICAL STUDIES
Acute toxicity
Animal Route LD50 Reference
per kg bw
Rat Oral 2.0 g Lu & Lavalleé, 1964
Short-term studies
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%
aqueous solution daily for 91 days. Only the faeces were
discoloured, not the urine. No ill-effects were observed (ICI, 1953).
Long-term studies
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 2% 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%
of ether extractable material and 0.4% of ether extractable material.
Ten doses of 20 mg per rat (2% solution) were followed by 50 doses of
30 mg per rat (6% solution). The maximum total dose in each case was
1.7 mg 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 2% 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, 1964).
A group of 20 young rats, 10 male and 10 female were given weekly
subcutaneous injections of 0.5 ml of a 4% 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 male and 20 female, aged two months
were given the colour in a dose level of 10 000 ppm (1%) during the
first two months. After this period the dose level was increased to
20 000 ppm (2%) and given to the animals for their life-span. Fifteen
animals (five male and 10 females) 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, aged two months, were
given a diet containing 20 000 ppm (2%) 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, aged two months,
were given 20 000 ppm (2%) of the colour for their life-span. These
animals received a subcutaneous injection of 1 ml of a 1% 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, aged two
months, were given subcutaneous injections of 1 ml of a 3% 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).
Comments:
This colour has been studied in adequate long-term tests in rats
but metabolic information is lacking and the short studies in the pig
are not adequate for evaluation. No adequate reproduction and
embryotoxicity including teratology studies are available although
they have been requested previously.
Metabolic studies in several species preferably including man.
Adequate studies in a second rodent species and a nonrodent species.
Reproduction and embryotoxicity including teratology studies.
EVALUATION
The previous temporary ADI has been withdrawn.
REFERENCES
Dalgaard-Mikkelsen, S. W. & Rasmussen, F. (1962) 16th International
Dairy Congress, 465
Daniel, J. W. (1959) Unpublished Report submitted by Imperial Chemical
Industries
Imperial Chemical Industries (1953) Unpublished Report
Imperial Chemical Industries (1964) Unpublished Report
Lu, F. C. & Lavalleé, A. (1964) Canad. pharm. J., 97, 30
Mannell, W. A. & Grice, H. C. (1964) J. Pharm. Pharmacol., 16, 56
Truhaut, R. (1964) Unpublished report submitted to WHO