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. CITRUS RED 2 Biological Data Biochemical aspects 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). Acute toxicity In male mice the LD50 was > 4 g/kg body weight (Hazelton Laboratory, 1954). Short-term studies 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 & Scala. 1964). Long-term studies 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., 1966). 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), Comments 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. EVALUATION This colour should not be used as a food additive. REFERENCES Dacre, J. C. (1965) Proc. Univ. Otago med. Sch, 43, 31 Paynter, O. E. & Scala, R. A. (1964) unpublished report by Hazleton Laboratories Radomski, J. L. (1961) J. Pharmacol. exp. Ther., 134, 100 Sharratt, M., Frazer. A. C. & Paranjoti, I. S. (1966) Fd. Cosmet. Toxicol., 4, 493
See Also: Toxicological Abbreviations