FAO Nutrition Meetings
    Report Series No. 40A,B,C
    WHO/Food Add./67.29


    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


    Synonym                      Azobisformamide

    Chemical name                Azodicarbonamide

    Empirical formula            C2H4O2N4

    Structural formula


    Molecular weight             116.10

    Definition                   Azodicarbonamide, after drying, contains
                                 not less than 98.6 per cent

    Description                  Azodicarbonamide occurs as a yellow to
                                 orange red, odourless, crystalline

    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.,

    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)


         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).


    Acceptable level of treatment

                        Flour:             0-45 ppm


    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 (CICADS 16, 1999)