WORLD HEALTH ORGANIZATION WHO Food Additives Series 1972, No. 1 TOXICOLOGICAL EVALUATION OF SOME ENZYMES, MODIFIED STARCHES AND CERTAIN OTHER SUBSTANCES The evaluations contained in this publication were prepared by the Joint FAO/WHO Expert Committee on Food Additives which met in Rome, 16-24 June 19711 World Health Organization Geneva 1972 1 Fifteenth Report of the Joint FAO/WHO Expert Committee on Food Additives, Wld Hlth Org. techn. Rep. Ser., 1972, No. 488; FAO Nutrition Meetings Report Series, 1972, No. 50. The monographs contained in the present volume are also issued by the Food and Agriculture Organization of the United Nations, Rome, as FAO Nutrition Meetings Report Series, No. 50A (c) FAO and WHO 1972 CARAMEL COLOURS (AMMONIA PROCESS) Biological data Biochemical aspects Caramel refers to a large number of poorly-defined and complex products formed from various carbohydrates generally by heating with any of a wide range of acids, bases and salts, under varying conditions of temperature and pressure. It might be argued that caramel can be considered as a natural constituent of the diet as it can be formed when certain foods are cooked or when sucrose is heated. Toxicological discrimination is unwarranted between such caramel and caramels produced commercially from food-grade carbohydrates, with the exception of caramels prepared by processes using ammonia or ammonium salts. The production of violent hysteria and convulsions in cattle and sheep fed ammoniated sugar-containing feed supplements (nitrogen content 4-6 per cent.), at 6-25 per cent. of their rations led to the discovery of the presence of about 20 per cent. of pyrazines and 10 per cent. of imidazoles in these ammonium-treated molasses (WHO, 1970). 4-methylimidazole has been shown to be the most likely toxic component being a convulsant to rabbits, mice and chicks at oral doses of 360 mg/kg body-weight (Nishie et al., 1969). The pyrazines, on the other hand, are mild CNS depressants and weak anticonvulsants (Heyns, 1971). Analysis of food grade caramel colours, however, showed that only 0.002-0.02 per cent. of 4-methylimidazole is present in commercial products (Heyns, 1970). Commercial caramel colours of undefined origin contain 50-500 ppm 4-methylimidazole (Heyns, 1971) while other examinations have shown ranges of 100-700 ppm (Battelle Memorial Institute, 1971). It has been shown that the yields of imidazole compounds increased linearly with the increment of molar ratio of ammonia to glucose (Komoto, 1962). Further analyses for 4-methylimidazole were being carried out on a large variety of caramel colours (Nishie et al., 1971). Generally caramel colours contain 50 per cent. digestible carbohydrate, 25 per cent. non-digestible carbohydrate and 25 per cent. of melanoidins also found in roasted coffee, broiled meats and baked cereal products. In groups of 2 to 4 rats, the absorption of the colour-giving components of caramel was determined by faecal extraction. Recoveries varied widely for the 10 or 20 per cent. caramel solutions examined despite pretreatment for 100 days before testing. About one-third of the colour-giving components appeared to be absorbed but no conclusions could be drawn regarding the absorption of colourless components (Haldi & Wynn, 1951). Acute toxicity Animal Route LD50 Reference (mg/kg body-weight) rat oral > 2.3 ml = 1 900 Foote et al., 1958 oral > 25 ml = 17 500 Chacharonis, 1960 oral > 30 ml = 20 400 Chacharonis, 1963 No abnormalities were detected after observation of animals for 14 days following administration of 12 different caramel colour products mostly based on ammonia or ammonium sulphate catalysts (Foote et al., 1956; Chacharonis, 1960; Chacharonis, 1963). A single dose of up to 10 g/kg body-weight in mice and 15 g/kg in rabbits of caramels produced by the ammonia catalyser closed pan process or sodium hydroxide process did not cause convulsions or other signs of distress (Sharratt, 1971). Short-term studies Rat Groups of 5 male and 5 female rats were given 1 ml/kg bodyweight of concentrated caramel colour for 21 days. Some diarrhoea was induced in all animals but no other abnormalities were noted. Gross and histopathology revealed no significant changes due to administration of the test compound (Foote et al., 1958). Groups of 5 rats received either 10 or 20 per cent. caramel solution equivalent to about 10 or 20 g/kg body-weight as sole source of fluid for 127 days. Only dark faeces and very mild diarrhoea were noted. No adverse effects were noted regarding general health, body-weight, food and fluid consumption, haematology, gross and histopathology (Haldi & Wynn, 1951). Six groups of 5 male and 5 female weanling rats received 0 or 10 per cent. caramel solution as their sole fluid source for 100, 200 or 300 days respectively. No adverse effects were noted regarding growth, food and fluid intake, haematology, gross and histopathology (Haldi & Wynn, 1951). Groups of 16 male and 16 female rats received either 0 or 10 per cent. caramel solution for 100 days and groups of 5 rats received 20 per cent. caramel solution for 100 days. At the lower test level there were no observable adnormalities as regards growth, food consumption, haematology, gross and histopathology. Only growth and haematology were examined at the higher test level (Haldi & Wynn, 1951). Three groups of 20 male and female rats received either 0 or 11-14 g/kg body-weight of caramel solutions for 100 days. Growth and food intake did not differ significantly between test and control animals. Gross and histopathology showed no abnormal findings related to administration of the test compound (Haldi, 1958). Four groups of 10 male and 10 female rats received 0, 0.1, 1.0 and 10 per cent. of caramel colour in their diet for 12 weeks. No adverse effects were noted on growth, food consumption, urinalysis, haematology, gross and histopathology related to administration of the caramel colour (Prier, 1960). Groups of 10 male and 10 female rats received 0, 5 or 10 g/kg caramel colour in their diet for 3 months. Weight gain was normal in all groups. Food consumption, haematology and urinalysis were comparable. Gross and histopathology showed no test-related adverse findings (Chacharonis, 1960). Four groups of 10 male and 10 female rats received 0, 5, 10 and 20 per cent. of two different caramel colours in their diet for 90 days. In addition, a paired feeding study involving 5 male rats in two groups was run for 23 days with one sample at the 20 per cent. level, and there was no difference in the rate of growth. The only effects attributable to treatment were a mild depression in growth of male rats at the 10 and 20 per cent. level due to impalatability of the test diet. No other adverse findings were noted in growth, behaviour, mortality, haematology, urinalysis, gross pathology, organ weights, and histopathology (Kay & Calandra, 1962). Four groups of 10 male and 10 female rats received either 0 or 10 per cent. of 3 different caramel colours in their diet for 90 days. Weight gains showed slight reduction compared with controls but food consumption was normal for all groups. No abnormalities were noted regarding haematology, urinalysis, gross and histopathology (Chacharonis, 1963). Four groups of 15 male and 15 female rats received 0, 5, 10 and 20 per cent. of caramel colour in their diet for 90 days. No adverse effects were noted on appearance, behaviour, survival, bodyweights, food intake, haematology, blood chemistry, urinalysis, organ weights, gross and histopathology (Oser, 1963). Four groups of 10 male and 10 female rats received 0, 0.015, 0.3 and 3.0 per cent. of caramel colour in their diet for 90 days. No differences between test and control animals were noted regarding body-weight, food consumption, haematology, urinalysis, gross or histopathology (Neese, 1964). Four groups of rats received 0, 4, 8 and 16 per cent. caramel colour in their diet for 3 months. No convulsions or other behaviour abnormality or signs of neurological damage were seen. No macroscopic or microscopic pathological abnormalities were found in the CNS. Other results are still to come (Sharratt, 1971). Dog Four groups of 3 male and 3 female adult beagles received 0, 6, 12.5 and 25 per cent. of caramel colour in their diet 5 days per week for 90 days. No significant adverse effects were noted due to the test compound on growth behaviour, food consumption, mortality, liver function, kidney function, haematology, urine analysis, gross and histopathology (Kay & Calandra, 1962). Long-term studies No formal studies are available but in the course of a reproduction study using 25 pairs of 1 male and 1 female rat given 0 or 0.8 g/kg body-weight caramel colour in their drinking fluid as part of a beverage tested, several pairs survived for 2 years or longer. No deleterious effects on growth were noted. The data are rather incomplete (Bachmann et al., 1946). Reproduction studies Rat Fifteen male and 15 female rats were given 0 or 10 per cent. caramel solution as their sole fluid source until day 100 and were then mated. The F1 generation was weaned and given again 0 or 10 per cent. caramel solution until day 100. It showed no adverse effects as regards litter number, haematology, growth, food consumption, gross and histopathology (Haldi & Wynn, 1951). Comments A large variety of caramel colours has been tested in short-term studies in rats and one variety has been tested in dogs. No satisfactory long-term or multigeneration studies are available. The acute and short-term studies considered reveal that at high levels of intake there is no evidence of any convulsions or other adverse neurological effects or of histopathological damage. Therefore, the acute neurological effects produced by high doses of 4-methylimidazole would not appear to be of major concern when caramel colours containing only small amounts of this contaminant are used in food. The analytical evidence suggests the presence of 4-methylimidazole in the range of 50-700 ppm in caramel colours depending upon the process of manufacture; 200 ppm is taken as an average low value for 4-methylimidazole content. Since the effects of chronic ingestion of 4-methylimidazole are unknown, long-term studies on caramel colours produced by the ammonium sulphate process are required. EVALUATION Level causing no significant toxicological effect in the rat Twenty per cent. (200 000 ppm) in the diet equivalent to 10 000 mg/kg body-weight/day. Estimate of acceptable daily intake for man mg/kg body-weight Temporary acceptance1 0-100 Further work required by 1974 Long-term studies on caramel colours prepared by the ammonia or ammonium sulphate process containing several levels of 4-methylimidazole. REFERENCES Battelle Memorial Institute (1971) Unpublished report dated 4.5.71 Bachmann, G,, Haldi, J,, Wynn. W. & Ensor, C. (1946) J. Nutr., 32, 85 Chacharonis, P. (1960) Unpublished report No. S.A. 54219 of Scientific Associates Inc. Chacharonis, P. (1963) Unpublished report No. S.A. 79105 of Scientific Associates Inc. FAO/WHO (1970) Wld Hlth Org. techn. Rep. Ser., 445 Foote, W. L., Robinson, R. F. & Davidson, R. S. (1958) Unpublished report of Battelle Memorial Institute Haldi, J. & Wynn, W. (1951) Unpublished report of Emery University Haidi, J. (1958) Unpublished report of Emory University Heyns, K. (1970) Unpublished report of Technical Caramel Committee Heyns, K. (1971) Unpublished summary report Kay, J. H. & Calandra, J. C. (1962) Unpublished report of Industrial Bio-test Laboratories Inc. Komoto, M. (1962) J. Agric. Chem., 36, 305 1 Single strength colour equivalents containing not more than 200 ppm of 4-methylimidazole. Neese, P. O. (1964) Unpublished report of Wisconsin Alumni Research Foundation Nishie, K., Waiss, A. C. & Keyl, A. C. (1969) Toxicol. Appl. Pharmacol., 14, 301 Nishie, K., Waiss, A. C. & Keyl, A. C. (1971) Toxicol Appl. Pharmacol., 17, 1 Oser, B. L. (1963) Unpublished report No. 83911 of Food and Drug Research Laboratories Prier, R. F. (1960) Unpublished report No. 9070599 of Wisconsin Alumni Research Foundation Sharratt, M. (1971) Unpublished report
See Also: Toxicological Abbreviations