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. INDIGOTINE Biological Data Biochemical aspects Microchemical methods have shown that filterable indigotine in the blood plasma of rabbits is excreted through the glomeruli. The filterable fraction is about 15 per cent. while 75 per cent. of the colour is excreted by the tubules. The lumina of the tubules and Bowman's capsules were injected with solutions of indigotine. Ten mg per cent. was the lowest concentration which left detectable traces of the colour in the kidneys. When an increased dose of colour is injected and the interval between the injection and the excision of the kidney shortened, the colour can, be detected in the intracapsular spaces. The tubular excretion of the indigotine resembles quantitatively that of phenol red (Kempton et al., 1937). After intravenous injection of 35S-labelled dye in the rats, 63 per cent. of the radioactivity appeared in the urine in six hours and 10 per cent. in the bile. The metabolites isatin-5-sulfuric acid and 5-sulfoanthranilic acid appeared in the urine after two hours. After oral administration, only three per cent. of the radioactivity appeared in urine in three days, suggesting poor absorption from the alimentary tract. Faeces contained 60-80 per cent.of the oral dose. The faecal content was due to lack of absorption, not biliary excretion (Lethco & Webb, 1966). Acute toxicity Animal Route LD50 Reference mg/kg body-weight Mice Oral >2 500 US FDA, 1969 Mice Subcutaneous 405 US FDA, 1949 Rat Oral 2 000 Lu & Lavallée, 1964 Rats Intravenous 93 US FDA, 1969 Five rats were given subcutaneous twice daily injections for three days and killed on the fourth day. An aqueous solution at a level of 250 mg/kg body-weight was used. No oestrogenic activity (normal uterine weight) was detected but the animals lost weight (Graham & Allmark, 1959). In experiments with guinea-pigs it was found that this colour had no sensitization activity (Bär & Griepentrog, 1960). Special studies The colour was tested for mutagenic effect in a concentration of 0.5 g/1 0.5 g/100 ml in cultures of Escherichia coli. No mutagenic effect was found. (Lück & Rickerl, 1960). Isatin-5-sulphonic acid, one of the metabolites of the colour, was fed to groups of three-week-old rats, 10 male and 10 females per group, at levels of 0, 0.25, 0.5, 1 and 2 per cent. for a period of 13 weeks followed by gross and histopathology examinations. The "no-effect" level of the compound was considered to be two per cent. (FDA, 1969). Short-term studies Rat. Twenty rats were given first 1 ml two per cent. solution subcutaneous later only 0.5 ml of a 0.5 per cent. solution. Fifty-five injections were administered over a period of seven months. Control groups received injections of 50 per cent. glucose or 0.9 per cent. sodium chloride. All animals were observed for lifespan. No local tumours and only one auxiliary tumour in the test group were found (Oettel et al., 1965). Dog. Two groups, consisting of two male and two female beagles were given 1.0 per cent. and two per cent. Indigotine in the diet for a two-year feeding trial. One male and one female served as controls. Two dogs on the high level died at 19 weeks; they were replaced and another dog was added to the control group. Two more on the high level died, at 21 and 40 weeks; one on the low level died at 36 weeks; and one control died at 34 weeks. Deaths were due to virus infections. There were no clinical signs, gross lesions or microscopic pathology attributable to the dye (Hansen et al., 1966). Long-term studies Mouse. Groups of 25 male and 25 female mice received weekly for 104 weeks subcutaneous injections of 2.5 mg indigotine as a one per cent. aqueous solution, the control group of 50 receiving 0.25 ml physiological saline. Many mice died from acute convulsions immediately after injection of the test dye but otherwise no deleterious effects attributable to the subcutaneous injections were noted. Tumours were randomly distributed among test and control groups (Hansen et al., 1966). Rat. A group of 20 male and 20 female rats received the dye as one per cent. solution for two years. Twenty males and 20 females were controls. No adverse effects were observed on growth, reproduction and survival and no specific gross or microscopic pathology was noted (organs not stated). There was no difference in tumour incidence between the groups (Oettel et al., 1965). Groups of 24 rats, equally divided by sex, were fed the colour at 0, 0.5, 1.0, 2.0 and 5.0 per cent. for two years. The growth of males was significantly inhibited at 2.0 and 5.0 per cent. There was no change in mortality, organ weights or haematology, nor any gross or microscopic pathology related to treatment (Hansen et al., 1966). Groups of 80 and 100 rats were injected weekly with a two per cent. aqueous solution or an equivalent volume of saline solution for two years. Survival of test animals did not differ from controls. Fourteen of 80 injected rats had a fibrosarcoma at the site of injection and one saline injected rat developed a fibroma at the injection site. No other effects were observed (Hansen et al., 1966). Comments The production of a high percentage of local sarcomata at the site of subcutaneous injections in rats led in the past to considerable discussion and consequently to extensive studies on this colour and the special condition of the experiment and does not constitute evidence of carcinogenicity by the oral route. The metabolic studies on this colour are fairly complete and the two long-term studies in the rat do not point to any significant toxic effects. A thirteen-week study on the major metabolite revealed no toxic effects. Human metabolic studies would be useful. EVALUATION Level causing no toxicological effect in the rat One per cent. (= 10 000 ppm) in the diet equivalent to 500 mg/kg body weight/day. Estimate of acceptable daily intake for man mg/kg body-weight Temporary acceptance 0 -2.5 Further work required by June 1974 Two-year study in a non-rodent mammalian species REFERENCES Bär, F. & Griepentrog, F. (1960) Med. u. Ernähr., 1, 99 Graham, R. C. B. & Allmark, M. G. (1959) Toxicol. appl. Pharmacol., 1, 144 Hansen. W. H., Fitzhugh, O. G., Nelson, A. A. & Davis, K. J. (1966) Toxicol. appl. Pharmacol., 8, 29 Kempton, R. T., Bott, P. A. & Richards, A. N. (1937) Amer. J. Anat., 61, 505 Lethco, E. J. & Webb, J. M. (1966) J. Pharmacol. exp. Ther., 154, 384 Lu, F. C. & Lavallée, A. (1964) Canad. pharm. J., 97, 30 Lück, H. & Rickerl, E. (1960) Z. Lebensmitt.-Untersuch., 112, 157 Oettel, H., Frohberg, H., Nothdurft, H. & Wilhelm, G. (1965) Arch. für Toxikol., 21, 9 United States Food and Drug Administration (1969) Unpublished report
See Also: Toxicological Abbreviations Indigotine (WHO Food Additives Series 6) INDIGOTINE (JECFA Evaluation)