The data contained in this document were examined by the
    Joint FAO/WHO Expert Committee on Food Additives*
    Rome, 3-12 April 1978

    Food and Agriculture Organization of the United Nations
    World Health Organization

    * Twenty-second Report of the Joint FAO/WHO Expert Committee on Food
    Additives, Geneva, 1978, WHO Technical Report Series No. 631



         This colour was evaluated at the Joint FAO/WHO Expert Committee
    on Food Additives in 1974. Since the previous evaluation, additional
    data have become available and are summarized below.


    Acute toxicity

         Acute testing of azorubine administered by various routes has
    resulted in the findings summarized below:


         Species        Route     LD50          References

         Rat            i.p.      1 g/kg        Gaunt et al., 1967
         Mouse          i.p.      0.8 g/kg      Gaunt et al., 1967
         Mouse          i.v.      0.8 g/kg      Deutsche Forschung, 1957
         Mouse          Oral      >8 g/kg       Gaunt et al., 1967
         Rat            Oral      >10 g/kg      Gaunt et al., 1967

    Short-term studies


         Sixteen Carworth Farm E strain rats of each sex were placed into
    groups which were fed 0.0, 0.05, 0.10, 0.50, and 1.0% azorubine for
    90 days. Feeding of this colour at these levels produced no
    deleterious effects on body weight, food consumption, haematology,
    renal, or hepatic function parameters. Females at the 1.0% dietary
    level were found to have elevated renal weight, but no untoward
    pathology was found upon examination of this organ. No 
    non-spontaneous, compound-induced tumours were found and no abnormal 
    gross pathology was observed. A no-effect level of 0.5% (250 mg/kg/day) 
    has been established for rats in a 90-day study, based upon the 
    elevated female renal weights (Gaunt et al., 1967).

         Sprague-Dawley rats (10/sex/experimental group; 20/sex/control
    group) were fed 0, 2, 4, 6 or 8% azorubine in the basal ration of
    Wayne Lablox for nine weeks. At levels of 6.0% or greater, the toxic
    effect elicited by this colour was seen to be reduction in body weight
    gain of animals in these groups. No other toxic manifestations were
    noted. This equates to a no-effect level of 2000 mg/kg (Holmes et al.,


         Three male and three female Pitman-Moore crossed Palouse strain
    miniature pigs per group were administered azorubine at levels of 0,
    250, 500 and 1000 mg/kg/day admixed with a basal diet composed of
    Hi-lean Rearers Pencils for 90 days. No untoward toxicology or
    pathology was noted at the conclusion of this study and no significant
    differences between control and treated animals were detected. A
    no-effect level of 1000 mg/kg/day was assigned based upon the results
    of this study (Gaunt et al., 1969).

    Long-term studies


         Thirty mice (15/sex) were administered azorubine subcutaneously
    for 52 weeks. The initial dose consisted of 0.1 cc of a 3% solution of
    the colour in arachis oil two times per week which was increased to 6%
    at the end of six months. Control mice received the arachis oil
    diluent alone in subcutaneous injections. Following the 52-week
    administration period, at which time each animal had received 468 mg,
    the animals were allowed to survive as long as possible. At the end of
    89 weeks after the initiation of treatment, one male and 11 female
    mice had expired. Seven of the females had been found to develop
    lymphomas while no subcutaneous sarcomas or hepatomas were observed.
    The lymphosarcomas observed were also seen to develop spontaneously in
    control animals and no toxicological significance was imparted to
    those observed. The conclusions drawn by the authors was that
    azorubine was non-carcinogenic in mice (Bonser et al., 1956).

         Azorubine was administered to ASH/CSI strain male and female mice
    (30/sex/group) for 80 weeks at levels of 0.01, 0.05, 0.25, or 1.25% of
    the diet. A control group of 60 animals per sex were fed only the
    basal ration of Oxoid pasteurized diet supplement with 80 ppm vitamin
    K3 and water ad libitum. The feeding of diets containing the colour
    additive had no effect on the behaviour, body weight or organ weight
    of the animals entered into the study. Female mice fed 1.25% were
    found to possess significantly lowered (p <0.001) haemoglobin levels
    at weeks 12 and 52 of the study, and, at week 52, males fed 0.25 and
    1.25% dye were found to have these same values decreased. The 1.25%
    females were also found to have a decreased packed cell volume at week
    52. No abnormal tumour distribution which could be considered to be
    compound related was detected. The minimum toxic effect level seen was
    1.25% with the symptoms being mild anaemia at week 80. A no-effect
    level of 0.25% (375 mg/kg/day) was ascribed to azorubine fed to mice
    over a period of 80 weeks (Mason et al., 1974).


         Azorubine was fed at levels of 0.0, 0.35, 0.8 and 2.0% of the
    diet to Sprague-Dawley rats (30 male and 30 female per group; 50 rats
    of each sex in the control group) in a multigeneration reproduction
    study. No deleterious effects were seen in the reproductive parameters
    assessed (fertility, viability, and lactation indices). No effects on
    body weight gain were observed although, with each successive
    generation, there was a trend toward increased dye consumption, this
    being indicative of increased food consumption. Thus, azorubine had no
    adverse effects on viability and reproductive abilities of rats when
    fed at levels of up to 2% for a study which included three in utero
    exposures of the subsequent generations, as seen in the F0, F1a,b,
    F2a,b, and F3a,b generations (Holmes et al., 1977a).

         Thirty male and 30 female Sprague-Dawley rats delivered following
    two generations of parental in utero (F3b) exposures to azorubine
    were placed into groups to receive 0.35, 0.8, and 2.0% of this colour
    additive for one year. The control group consisted of 50 animals of
    each sex. No adverse, dye-related effects on body weight gain were
    observed. A statistically significant increase (p <0.01) in
    bronchitis and tracheal irritation was found in male rats fed
    azorubine at a level of 2.0% of the diet. Urinalysis, other
    haematological values, and gross and histopathological findings were
    within normal limits. A no-effect level of 0.8% (400 mg/kg/day) was
    assigned for azorubine in rats, although the authors believed that the
    true value would have been higher (Holmes et al., 1977b).

    Miscellaneous studies

         Female New Zealand white rabbits were administered azorubine on
    days 6 through 18 of gestation by oral intubation at levels of 0
    (47 animals), 40 mg/kg/day (15 animals), 120 mg/kg/day (15 animals)
    and 400 mg/kg/day (20 animals) in a teratology study. Thalidomide
    (150 mg/kg/day) was administered to 15 rabbits as a positive control.
    Of the dye-treated animals, no effect was seen on body weight gain. A
    statistically non-significant increase in the number of spontaneous
    deaths among dams of the high dose group was found to be present.
    There was also a decrease in the implantation efficiencies of all
    females to which azorubine had been administered. This, however, was
    not deemed to be compound related in that implantation was assumed
    to have occurred prior to the initiation of the dye administration.
    No signs of toxicity or foetal abnormalities were found, thereby
    indicating that azorubine, at the levels administered, is 
    non-teratogenic (Smith et al., 1972a).

         Female Long-Evans rats were administered azorubine at levels
    of 100 mg/kg/day (22 animals), 300 mg/kg/day (24 animals), and
    1000 mg/kg/day (22 animals) on days 6-15 of gestation by oral

    intubation. Sixty-six rats served as control animals receiving the
    methylcellulose (0.5%) vehicle, and 22 animals were dosed with
    30 mg/kg/day of trypan blue as a positive control. No embryotoxic or
    teratogenic effects were seen in the animals administered azorubine
    (Smith et al., 1972b).

         In vitro assays were conducted by inclusion of azorubine
    (0.4 mg/mg tissue) in enzyme activity trials in an attempt to
    determine the effects of the dye on succinic oxidase system of rat
    liver homogenates. The results indicated that this dye inhibited the
    oxidative activity of this enzyme approximately 40% (Sikorska and
    Krauze, 1962).

         Administration of azorubine at doses up to 10 mg/kg produced no
    alterations of the blood pressure of anaesthetized dogs and rabbits
    (Vrbovsky and Selecky, 1959).

         Commercial azorubine preparations were fed to bull calves at
    levels of 1, 5, and 10 g/100 kg of skim milk powder. No dye was
    detected in any of the meat samples analysed, although the time from
    administration to analysis is not known (DeMoor et al., 1969).


    Smith, J. M., Kasner, J. A. and Cannelongo, B. (1972a) Carmoisine,
    Segment II. Rabbit Teratology Study. Bio/dynamics, Inc. Project No.
    71R-721F submitted to Inter-Industry Color Committee: Cosmetic,
    Toiletry and Fragrance Association. Unpublished report

    Smith, J. M., Kasner, J. A. and Andresen, W. (1972b) Carmoisine,
    Segment II. Rat Teratology Study. Bio/dynamics, Inc. Project No.
    71R-719F submitted to Inter-Industry Color Committee: Cosmetic,
    Toiletry and Fragrance Association. Unpublished report

    Bonser, G. M., Clayson, D. B. and Jull, I. W. (1956) The induction of
    tumors of the subcutaneous tissues, liver, and intestine in the mouse
    by certain dyestuffs and their intermediates, Brit. J. Cancer, 10,

    DeMoor, H., Hendrickx, H., Casteels, M., Eekhout, W. and Buysse, F.
    (1969) Azorubine in commercial products and its presence in meat,
    Meded. Rijksfac. Landbouwwetensch, 34(4), 1067-1079 (NETH)

    Deutsche Forschungsgemeinschoft-Farbstoff-Kommission (1957)
    Mitteilung, 6, 2. Auflage Toxikologische Daten von Farbstaffen
    und ihre zullassung fur Lebensmittel in verschiedenen Landern,
    p. 38, Franz Steiner Verlag GMBH, Wiesbaden

    Gaunt, I. F., Farmer, M., Grasso, P. and Gangolli, S. D. (1967) Acute
    (mouse and rat) and short-term (rat) toxicity studies on carmoisine,
    Food Cosmet. Toxicol., 5, 179-185

    Gaunt, I. F., Grasso, P., Kiss, I. S. and Gangolli, S. D. (1969)
    Short-term toxicity study on carmoisine in the miniature pig,
    Food Cosmet. Toxicol., 7, 1-7

    Holmes, P., Pritchard, A. B., Kirschman, J. C., Sefecka, R. and Ford,
    A. (1977a) Multigeneration reproduction studies with carmoisine in
    rats, submitted for publication

    Holmes, P. A., Pritchard, A. B., Kirschman, J. C., Sefecka, R. and
    Ford, A. (1977b) A one year feeding study in rats, submitted for

    Mason, P. L., Gaunt, I. F., Butterworth, K. R., Hardy, J., Kiss, I. S.
    and Grasso, P. (1974) Long-term toxicity studies of carmoisine in
    mice, Food Cosmet. Toxicol., 12, 601-607

    Sikorska, E. A. and Krauze, S. (1962) Wplywniektorych barwnikow
    zywnosciowych i kosmetycznych na aktywnosc oksydazy bursztynianowej,
    Roczniki Pzh., t XIII, No. 5, 457-466

    Vrbovsky, L. and Selecky, F. V. (1959) Niektore vitalne a ine farbiva
    ovplyvnujuce krvny tlak, Bratisl. lek. Listy, 39, 737-752

    See Also:
       Toxicological Abbreviations
       Azorubine (WHO Food Additives Series 6)
       Azorubine (WHO Food Additives Series 18)
       AZORUBINE (JECFA Evaluation)