INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY WORLD HEALTH ORGANIZATION TOXICOLOGICAL EVALUATION OF SOME FOOD COLOURS, EMULSIFIERS, STABILIZERS, ANTI-CAKING AGENTS AND CERTAIN OTHER SUBSTANCES 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. WOOLGREEN BS 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 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). Acute toxicity Animal Route LD50 Reference per kg body weight Rat oral 2.0 g Lu & Lavallée, 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 per cent 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 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, 1964). 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). Comments This colour has been studied in adequate long-term feeding tests in rats and also parenterally in mice. Metabolic information is lacking. EVALUATION Level causing no toxicological effect in the rat Two per cent. (= 20 000 ppm) in the diet equivalent to 1000 mg/kg body weight/day. 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. 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, & Lavallée, A. (1964) Canad. pharm. J., 97, 30 Mannell, W. A. & Grice, H. C. (1964) J. Pharm. Pharmacol., 16, 56
See Also: Toxicological Abbreviations