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    INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

    WORLD HEALTH ORGANIZATION



    TOXICOLOGICAL EVALUATION OF SOME
    FOOD COLOURS, ENZYMES, FLAVOUR
    ENHANCERS, THICKENING AGENTS, AND
    CERTAIN FOOD ADDITIVES



    WHO FOOD ADDITIVES SERIES 6







    The evaluations contained in this publication were prepared by the
    Joint FAO/WHO Expert Committee on Food Additives which met in Rome,
    4-13 June 19741


    World Health Organization     Geneva     1975






              

    1  Eighteenth Report of the Joint FAO/WHO Expert Committee on
    Food Additives, Wld Hlth Org. techn. Rep. Ser., 1974, No. 557.
    FAO Nutrition Meetings Report Series, 1974, No. 54.

    ALLURA RED AC

    BIOLOGICAL DATA

    BIOCHEMICAL ASPECTS

         Orally administered dye is rapidly absorbed as evidenced by
    coloration of eyes and skin within two hours after intubation of 5 g
    into dogs (Anonymous, 1965b). Five rats were fed 5.19% dye in the diet
    and the excretion products determined in urine and faeces. About 29%
    of the total dye consumed was recovered unchanged from the faeces,
    less than 0.1% unchanged dye was found in the urine. Thus 71% appears
    to have been metabolized but it was found impossible to identify any
    metabolites chemically either in urine or faeces. It is suggested that
    azo reduction by gut flora will form principally 2-methoxy-5 methyl-
    aniline-4-sulfonic acid (cresidine-4-sulfonic acid)

    CHEMICAL STRUCTURE 1

    and 1-amino-2-naphthol-6-sulfonic acid

    CHEMICAL STRUCTURE 2

    Some of the cresidine sulfonic acid may also be further acetylated.
    All these metabolites are likely to be excreted in the urine and very
    little is likely to be excreted in the bile (Anonymous, 1970d).

    TOXICOLOGICAL STUDIES

    Special studies on reproduction and teratogenicity

    Rat

         Groups of 10 male and 20 female rats received 0%, 0.37%, 1.39% or
    5.19% dye in their diet through two parental (F1A was the P2
    generation) and two filial generations. Mating occurred after 27 weeks
    on either control or test diet both for the P1 and P2 generation.

    The fertility indices were low for the controls and test animals in
    the F1A and F1B generation as well as in the low test level F2A and
    all test levels of F2B generation. Growth was suppressed slightly for
    the lowest test level F1B and high test level F1A + F1B pups as well
    as for the high test level F2A and F2B pups. Other indices, litter
    size, pup weight at 24 hours, were comparable in each group in each
    generation. No consistent pathological changes were noted in the P1,
    P2, F1A, F1B and F2B generations. No evidence was seen of
    teratogenic or embryotoxic effects regarding implantation sites,
    resorption sites, and live fetuses indices. No difference from
    controls was noted with regard to appearance, anatomy and structure of
    test fetuses (Anonymous, 1969).

         Groups of 24, 19, 20, 21 and 16 pregnant rats received
    respectively 0, 15, 30, 100 and 200 mg/kg of the dye by gavage daily
    during pregnancy days 0-19. Dams were delivered by section on day 20
    of pregnancy. Preliminary gross observations indicate no dye-induced
    effects in terms of early or late deaths, resorptions per litter,
    pre-implantation loss, number of fetuses per litter and average fetus
    weight (Anonymous, 1974b).

    Rabbit

         In three groups of 14 rabbits, Allura Red was given in doses of
    0, 200 and 700 mg/kg bw by gavage from day 6 to 18 of pregnancy. There
    were no indications of a compound-related effect with regard to
    appearance and behaviour, body weight or in gross necropsy findings
    for the maternal does. No adverse effects on implantation and litter
    data were noted nor any fetal abnormalities (Anonymous, 1974a).

    Acute toxicity
                                                                        

                                      LD50
    Animal     Route                  (mg/kg bw)      Reference
                                                                        

    Rat        oral (gavage)          > 10 000        Anonymous, 1965a

    Rabbit     dermal                 > 10 000        Anonymous, 1967

    Dog        oral (intubation)      > 5 000         Anonymous, 1965b
                                                                        

         Tests for dermal irritation on intact and abraded rabbit skin
    showed no gross irritation at levels of 0.316, 1.0, 3.16 and 10 g/kg
    bw but resulted in persistent skin staining (Anonymous, 1967). Daily
    application at rates of 0.5 g/kg, five days a week for 15 applications
    on abraded and 65 applications on intact skin, of 0.1% and 1%
    solutions in water or as a hydrophilic ointment revealed no compound-

    related effect on general appearance, body weight, clinical laboratory
    studies, gross and microscopic pathology. No dermal irritation was
    noted except that produced by the hydrophilic ointment base
    (Anonymous, 1968).

    Short-term studies

    Rat

         Groups of 10 male and 10 female rats were fed diets containing 0,
    0.37, 0.72, 1.39, 2.69 and 5.19% dye for six weeks. No evidence of
    compound-related effects was noted as regards body weight, food
    consumption, survival, organ weights, gross and microscopic pathology.
    Haematology and urinalysis were normal and no evidence of Heinz body
    formation was noted (Anonymous, 1966a).

    Dog

         Four groups of one male and one female beagle dog received 0,
    125, 250 and 500 mg/kg daily in capsule form. No adverse effects were
    noted on body weight, food consumption, survival, organ weights, gross
    and histopathology, haematology and clinical chemistry. At the highest
    level there were slight ill-defined hepatic parenchymal changes in
    both dogs (Anonymous, 1966b).

         Groups of four male and four female beagle dogs received 0, 0.37,
    1.39 and 5.19% in their diet for 104 weeks. All animals remained
    normal regarding appearance, behaviour, haematology and clinical
    chemistry findings, gross and histopathology. Both faeces and urine
    were coloured at all test levels. At 52 weeks the adrenal cortical
    cells of the high level groups showed some vacuolation and brown
    pigment deposition in the Kupffer cells of females at the two lower
    test levels. These changes had disappeared by 104 weeks and special
    histological examination of the eyes revealed no adverse changes
    (Anonymous, 1970b).

    Long-term studies

    Mouse

         A control group of 50 male and 50 female mice, a positive control
    group of 25 male and 25 female mice and a test group of 50 male and 50
    female mice were treated with 0.1 ml of either distilled water, 5%
    test solution or 10 g 3,4-benzpyrene in acetone twice weekly for 20
    months. The results in the positive control group showed the mouse
    strain used to be sensitive to benzpyrene carcinogenesis. Survival,
    gross and histopathology of major organs were comparable in the
    negative controls and test animals. Histology of the skin revealed
    comparable incidence and degrees of severity of acanthosis,
    hyperheratosis and dermatitis for the negative control and test groups
    (Anonymous, 1970c).

    Rat

         Groups of 30 male and 30 female rats received in their diet 0,
    0.37, 1.39 and 5.19% dye for 92 weeks. Moderate growth depression
    occurred at the 5.19% level in both sexes. No other compound-related
    effects were noted as regards appearance, behaviour, survival, organ
    weights, clinical laboratory studies or gross and histopathology. No
    evidence of Heinz body formation was noted apart from a slight
    tendency to anaemia (Anonymous, 1970a).

    Comments:

         There is only very limited information available on the
    metabolism of this dye and further metabolic investigations using
    radio-labelled materials are planned. The available long-term study in
    rats showed no adverse effects except moderate growth depression at
    the highest level tested but was considered to be inadequate because
    of the poor survival and small number of animals examined terminally.

         The studies on reproduction and embryotoxicity including
    teratogenicity revealed no significant adverse effects in the two
    species investigated. A two-year study in a non-rodent species showed
    no compound-related toxic effects. An adequate life span study in the
    rat and the results of the planned studies on the metabolism of this
    colour are needed.

    EVALUATION

         Not possible on the data provided.

    REFERENCES

    Anonymous (1965a) Unpublished Report No. 165-114 dated 7 December 1965
         from Hazelton Laboratories Inc., submitted by Allied Chemical
         Corporation

    Anonymous (1965b) Unpublished Report dated 23 November 1965 from
         Hazelton Laboratories Inc., submitted by Allied Chemical
         Corporation

    Anonymous (1966a) Unpublished Report No. 165-112 dated 18 November
         1966 from Hazelton Laboratories Inc., submitted by Allied
         Chemical Corporation

    Anonymous (1966b) Unpublished Report No. 165-116 from Hazelton
         Laboratories Inc., submitted by Allied Chemical Corporation

    Anonymous (1967) Unpublished Report No. 165-119 dated 16 October 1967
         from Hazelton Laboratories Inc., submitted by Allied Chemical
         Corporation

    Anonymous (1968) Unpublished Report No. 165-120 dated 20 December 1968
         from Hazelton Laboratories Inc., submitted by Allied Chemical
         Corporation

    Anonymous (1969) Unpublished Report No. 165-125 dated 29 May 1965 from
         Hazelton Laboratories Inc., submitted by Allied Chemical
         Corporation

    Anonymous (1970a) Unpublished Report No. 165-121 dated 2 March 1970
         from Hazelton Laboratories Inc., submitted by Allied Chemical
         Corporation

    Anonymous (1970b) Unpublished Report No. 165-122 dated 2 March 1970
         from Hazelton Laboratories Inc., submitted by Allied Chemical
         Corporation

    Anonymous (1970c) Unpublished Report No. 165-123 dated 2 March 1970
         from Hazelton Laboratories Inc., submitted by Allied Chemical
         Corporation

    Anonymous (1970d) Unpublished Report No. 21855 dated 11 February 1970
         from Buffalo Research Laboratory, submitted by Allied Chemical
         Corporation

    Anonymous (1974a) Unpublished Report No. 165-142 dated 15 June 1974
         from Hazelton Laboratories Inc., submitted by Allied Chemical
         Corporation

    Anonymous (1974b) Unpublished Report submitted by the United States
         Food and Drug Administration


    See Also:
       Toxicological Abbreviations
       Allura red AC (WHO Food Additives Series 15)
       ALLURA RED AC (JECFA Evaluation)