FAO Nutrition Meetings Report Series No. 40A,B,C WHO/Food Add./67.29 TOXICOLOGICAL EVALUATION OF SOME ANTIMICROBIALS, ANTIOXIDANTS, EMULSIFIERS, STABILIZERS, FLOUR-TREATMENT AGENTS, ACIDS AND BASES The content of this document is the result of the deliberations of the Joint FAO/WHO Expert Committee on Food Additives which met at Rome, 13-20 December, 19651 Geneva, 11-18 October, 19662 1 Ninth Report of the Joint FAO/WHO Expert Committee on Food Additives, FAO Nutrition Meetings Report Series, 1966 No. 40; Wld Hlth Org. techn. Rep. Ser., 1966, 339 2 Tenth Report of the Joint FAO/WHO Expert Committee on Food Additives, FAO Nutrition Meetings Report Series, 1967, in press; Food and Agriculture Organization of the United Nations World Health Organization 1967 AZODICARBONAMIDE Synonym Azobisformamide Chemical name Azodicarbonamide Empirical formula C2H4O2N4 Structural formulaMolecular weight 116.10 Definition Azodicarbonamide, after drying, contains not less than 98.6 per cent C2H4O2N4. Description Azodicarbonamide occurs as a yellow to orange red, odourless, crystalline powder. Uses As a strengthening agent for flour in a mixture usually containing 1 part by weight of azodicarbonamide with 9 parts by weight of a mixture of starch and tricalcium phosphate. Biological Data Biochemical aspects When azodicarbonamide reacts with flour, it behaves as a hydrogen acceptor and it is rapidly and completely converted into biurea, which is stable under baking conditions. Reaction between azodicarbonamide and flour only occurs on wetting. Forty-five minutes after treatment of a flour with 8.25 ppm of azodicarbonamide, less than 0.1 ppm of azodicarbonamide could be detected in the dough. When 14C-labelled azodicarbonamide was used for breadmaking, the activity remained in the bread and there was no liberation of labelled carbon dioxide. Biurea labelled with 14C was unaffected by pepsin or trypsin in vitro. When administered orally to rats in doses of 33 mg, labelled biurea was recovered quantitatively within 120 hours from faeces and urine. Absorption from the intestinal tract was about 20 per cent. No radioactivity was detectable in the blood after 24 hours; no tissues were found to accumulate biurea (Joiner et al., 1963; Oser et al., 1965). Special studies Study of the amino-acid pattern of gluten obtained from flours that were either untreated or treated with azodicarbonamide at 11 ppm or 110 ppm revealed no significant changes (Oser et al., 1965). No alteration in the amounts of thiamine, riboflavin, or niacin present in natural or enriched flour or bread made from such flours, was observed after treatment with 11 ppm of azodicarbonamide (Joiner et al., 1963; Oser et al., 1965). Acute toxicity Mouse. Single doses of 0, 1, 2, 4 and 6 g/kg body-weight were administered orally to groups of 5 mice. No adverse effects were observed (Joiner, 1965). Single doses of 0, 250 and 500 mg/kg body-weight were administered intraperitoneally to groups of 5 mice. Moderate depression and some diarrhoea, but no deaths, occurred. Single doses of 0, 750, 1000 and 1250 mg/kg body-weight were administered to groups of 5 mice intraperitoneally. Diarrhoea, cyanosis, dyspnoea and depression were observed, with 1, 4, 4 deaths in the three groups at 48 hours respectively (Oser, 1965). Rat. Single doses of O, 1, 3 and 6 g/kg body-weight were administered to groups of 5 rats by intragastric intubation. No ill effects were observed (Joiner, 1965). Short-term studies Rat. Doses of 0, l00 and 1000 mg/kg body-weight were administered daily by intragastric intubation to groups of 10 rats for a period of 8 weeks. No adverse effect was observed (Joiner, 1965). Dog. A daily dose, of 60 mg/kg body-weight was administered in the diet for 8 weeks to a dog, without demonstrable ill effect on growth rate, haematology and general health (Joiner, 1965). Rabbit. Intradermal and patch tests for skin sensitivity to azodicarbonamide were carried out on groups of 10 rabbits. Azodicarbonamide showed no activity as a primary skin irritant. No other effect was observed (United States Testing, 1952). Man. Despite large-scale industrial and bakery use of azodicarbonamide no dermatological problems associated with such use, have been reported. Studies on Biurea Short-term studies Rat. Two test groups of 25 male and 25 female rats and a control group of 10 male and 10 female rats were fed diets to which was added 0, 5 or 10 per cent. biurea, for a period of one year. The rate of weight gain of the male rats was slightly depressed during the first 12 weeks in the test groups, but recovered later. The appearance, behaviour, food intake and utilization, morbidity and mortality in the test and control groups were not significantly different. Haematological studies and blood biochemistry showed no abnormalities. No abnormalities were seen at autopsy, relative organ weights were within normal limits; histological examination of major organs showed no difference between test and control groups (Oser et al., 1965). Dog. Four mongrel dogs (2 male and 2 female) were fed a diet containing 5 per cent. (2 dogs) and 10 per cent. (2 dogs) of biurea, respectively. Some difficulty was experienced in persuading the animals to eat this diet. The 10 per cent. group thrived poorly and died at 20 weeks; the 5 per cent. group survived and was sacrificed at 11 months. The only pathological change noted was the presence of massive biurea calculi in the kidneys. No significant changes were seen in haematological studies, nor in the gross or microscopical study of the main organs (Oser et al., 1965). Groups of 4 mongrel dogs were fed diets containing 77 per cent. of bread to which was added 0, 750, 2370 or 7500 ppm of biurea, for 2 years. Weight gain and food intake showed no significant difference between control and test groups. Detailed study of blood formation, methaemoglobin levels and histopathological examination of major organs showed no differences between control and test groups, except for a greyish brown colour of the liver in two dogs, renal haemorrhage in one dog receiving 750 ppm of biurea, and dilated renal pelves in one dog receiving the highest dose (7500 ppm) of biurea (Oser et al., 1965). Long-term studies Rat. Groups of 25 male and 25 female weanling rats were fed diets containing 83 per cent. of bread to which was added 0, 750, 2370 or 7500 ppm of biurea, respectively, corresponding to biurea levels in the diet equivalent to 0, 311, 983, or 3110 ppm. The first generation was studied for a life span (two years); 10 males and 10 females were selected at random from the F1 generation and kept on the diet and observed for 28 weeks; the F2 generation was studied similarly and the F3 generation for 14 weeks. The appearance, behaviour, rate of weight gain and food intake in each group of all generations showed no significant differences. Reproduction and lactation were normal. Morbidity and mortality were similar in all groups and showed no dose-related variations. Haematological studies (including methaemoglobin levels) and gross and microscopic examination of all the main organs revealed no significant difference between test and control groups, except for one enlarged kidney in a female rat receiving a high dose of biurea. A detailed assessment of tumour incidence was made and nothing significant was found (Oser et al., 1965). Studies on flour overtreated with azodicarbonamide, and bread baked from it Short-term studies Rat.Two groups of 10 rats were fed daily for 8 weeks by gastric tube with untreated flour and with flour treated with azodicarbonamide (120 ppm). No adverse effect was observed on the rate of weight gain, blood formation, or the gross or microscopical appearance of the main organs (Oser et al., 1965). Dog. A group of 4 mongrel dogs was fed bread made from flour treated with azodicarbonamide (100 ppm). The animals were observed for two years. No abnormalities were found in weight gain, food intake, appearance, behaviour, and morbidity. At autopsy no macroscopic or microscopic change of any significance was found in the main organs (Oser et al., 1965). Long-term studies Rat. Flour treated with azodicarbonamide (100 ppm) was made into bread and this was fed to 25 male and 25 female weanling rats; bread made from the untreated flour was fed to a control group of 25 male and 25 female rats. The animals were observed over the life-span (2 years). Appearance, -behaviour, weight gain, food intake, reproduction and lactation, morbidity and mortality rate were not significantly different. Detailed studies on the blood and main organs revealed no macroscopic-or microscopic abnormalities. Tumour incidence was examined in detail and no alteration in the numbers or patterns of tumours found was observed (Oser et al., 1965) Comments Azodicarbonamide has been extensively studied and the theoretical point with regard to the possible effect of unconverted azodicarbonamide was covered by experiments using overtreated flour or bread made from it. The evidence strongly supports the view that azodicarbonamide is rapidly and completely converted to biurea on wetting and that this substance is stable in bread. Biurea itself is metabolically inert, has low toxicity and does not present any carcinogenic hazard. Azodicarbonamide has been adequately studied in several species and is similarly free from carcinogenic hazard. Long-term studies in mice are in progress (Frazer, 1966). Evaluation Acceptable level of treatment Flour: 0-45 ppm REFERENCES Frazer, A. C. (1966) University of Birmingham work in progress Joiner, R. R., Vidal, F. D. & Markes, H. C. (1963) Cereal Chemistry, 40, 539 Joiner, R. R., Unpublished report submitted by Wallace & Tiernan, 1965 Oser, B. L., Oser, M. & Morgareidge, K. (1965) Toxicol. appl. Pharmacol., 7, 445 United States Testing Company (1952) Unpublished report
See Also: Toxicological Abbreviations Azodicarbonamide (ICSC) AZODICARBONAMIDE (JECFA Evaluation) Azodicarbonamide (CICADS 16, 1999)