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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.

    ORANGE RN

    BIOLOGICAL DATA

    BIOCHEMICAL ASPECTS

         The dye was administered in aqueous suspension to male rabbits
    (2-3 kg) by stomach tube at a level of 0.5 g/kg. The urine was
    analysed every 24 hours for three days after dosing. The identified
    metabolites and their proportions in 24 hours expressed as a
    percentage of the amount of dye administered were as follows (Daniel,
    1959):

         p-aminophenol                           3%

         aniline                                 0.9%

         1-amino-2-naphthol-6 sulfonic acid      42%

    When rabbits were fed 0.5 g per kg bw of the colour the following
    metabolites could be identified in 48 hours urine; total p-aminophenol
    (63%), p-aminophenylglucuronide (by difference 40%), aniline (0.9%),
    O-aminophenol (3%), 1-amino-2-naphthol-6 sulfonic acid (42% - 24
    hours).

         Reduction of the colouring occurred in vitro with bacteria
    (Streptococcus faecalis) isolated from intestinal contents (Walker,
    1968). This like any other azo dye is probably reduced in the gut by
    bacterial azo reductases (Walker, 1970).

    TOXICOLOGICAL STUDIES

    Special studies on the effect of erythrocytes

         Administration of 160 and 320 mg/kg bw/day to pigs for 10 days
    induced the formation of methaemoglobin and decreased the red cell
    life span from 54 days to 24 and 15 days respectively. In the order
    of decreasing potency nitrosobenzene, O-aminophenol, 1-amino-2-
    naphthol-6 sulfonic acid and p-aminophenol induced the formation of
    methaemoglobin when incubated with erythrocytes from pigs and humans.
    Pig erythrocytes were slightly more sensitive than erythrocytes
    obtained from humans, except to the action of nitrosobenzene, where
    human erythrocytes were more sensitive. 1-amino-2-naphthol-6 sulfonic
    acid was about three times as potent as p-aminophenol (Würtzen et al.,
    1972).

    Special studies on metabolites

         After intravenous injection of 7.8 mg Orange RN per kg bw to
    female pigs the following metabolites were identified in 24-hour
    urine: Orange RN (31%), 4'-hydroxy-l-phenylazo-2-naphthol-6 sulfonic
    acid (3%), total p-aminophenol (34%), total O-aminophenol (4%).
    1-amino-2-naphthol-6 sulfonic acid was present, but not determined.
    When the urine collection was expanded to 72 hours the excretion of
    p-aminophenol accounted for the rest of the dye. The excretion pattern
    for p-aminophenol suggests that Orange RN is partly excreted in the
    bile and thereafter undergoes azoreduction in the gut. After
    administration of the Orange RN (78 mg per kg bw) to female pigs by
    stomach tube the following metabolites were identified in the urine:
    total coloured metabolies (Orange RN and 4'-hydroxy-l-phenylazo-2-
    naphthol-6 sulfonic acid) (0.4%), total p-aminophenol (52%), total
    O-aminophenol (6%), aniline (0.3%). 1-amino-2-naphthol-6 sulfonic acid
    was present, but not determined (Larsen & Tarding, 1974).

    Acute toxicity
                                                                   

                                  LD50
    Animal         Route          mg/kg bw            Reference
                                                                   

    Rats           Oral           7 500               Dacre, 1969

    Mice           Oral           7 500               Dacre, 1969
                                                                   

    Short-term studies

    Rat

         Orange RN was fed to five groups of 15 male and 15 female rats at
    dietary levels of 0 (0%), 60 (0.006%), 600 (0.06%), 1200 (0.12%), or
    6000 (0.6%) ppm for over three months. At 6000 ppm (0.6%), there was
    marked Heinz body production, methaemoglobinaemia and reticulocytosis
    together with enlargement of the spleen and increased splenic iron. At
    1200 ppm (0.12%), these effects were present but less severe and at
    600 ppm (0.06%) the effects were of borderline significance. There was
    also an increased water intake and decreased renal concentrating
    ability in the rats at the highest dosage level (Gaunt et al., 1970).

    Pig

         Four groups of three male and three female Danish Landrace pigs 
    were given either 0, 10, 40 or 160 mg/kg bw/day of Orange RN in
    their diet for 110 days. In the 160 mg group hepatosis with liver
    enlargement, fibrosis and bile-duct proliferation was observed.

    Proliferation of the cells of the bile-ductule epithelium was found 
    in all test groups and the intensity of proliferation was dose- 
    related. At the highest dose level macrotic anaemia, haemoglobinemia
    and increase in ASAT and LD serum levels were observed. Heinz-body
    formation was marked in the groups fed 160 and 40 mg/kg bw. There was
    corresponding haemosiderosis of the spleen, liver and kidneys at these
    levels together with significant increases in the relative weight of
    the spleen and liver. In the highest dose group there was also focal
    liver necrosis in half the pigs (Olsen et al., 1973 and 1973a).

    Long-term studies

    Mouse

         Three groups of 20 male and 20 female mice were given in their
    diets 0, 0.05% or 0.25% of the dye for 20 months. More than 50% of the
    mice survived for this period after which they were sacrificed,
    autopsies performed and histological examinations undertaken. There
    were no significant differences in the mean organ weights of the
    different groups except that the mice on the 0.25% dose level showed
    elevated relative spleen weights. The food intake and growth rate of
    all the animals fed at the 0.05% and 0.25% levels showed no marked
    differences from the control animals. Mice on the 0.25% diet showed
    increased eosinophiland monocyte counts after 15 months (Dacre, 1969).

    Rat

         Three groups of 20 male and 20 female rats were given in their
    diets 0, 0.05% or 0.25% of the dye for two years. More than 50% of the
    rats survived for this period after which they were sacrificed,
    autopsies performed and histological examinations undertaken. There
    were no significant differences in the mean organ weights of the
    different groups except that the rats on the 0.25% dose level showed
    elevated relative spleen weights. The female rats on the 0.05% diet
    showed a decreased food consumption with a corresponding decrease in
    growth rate. There were no significant haematological abnormalities,
    no gross pathological changes and no consistent histopathological
    changes (Dacre, 1969).

    Comments:

         Available short and long-term studies in pigs, rats and mice
    demonstrate adverse effects in the haemopoietic system (Heinz bodies)
    and the liver. Evidence of haemolytic anaemia in rats and pigs
    reinforces the significance of the observations of splenomegaly
    reported in the available long-term studies in the rat and mouse.

         In addition bile duct proliferation has been recorded which was
    present in a dose-related manner. More information is needed on the
    metabolism of this colour, on reproduction, and possible embryotoxic
    including teratological effects.

    EVALUATION

         Not possible on the data available.

    REFERENCES

    Dacre, J. C. (1969) Proc. Univ. Otago med. Sch., 47, 3

    Daniel, J. W. (1962) Toxicol. appl. Pharmacol., 4, 572

    Gaunt, I. F. et al. (1971) Fd. Cosmet Toxicol., 9, 619

    Larsen, J. C. & Tarding, F. (1974) To be published in Acta
         Pharmacologica et Toxicologica

    Olsen, O. et al. (1973) Toxicology, 1, 249

    Olsen, P. & Hansen, E. (1973a) Acta pharmacol, toxicol., 32, 314

    Walker, R. (1968) Ph.D. Thesis, University of Reading

    Walker, R. (1970) Fd. Cosmet Toxicol., 8, 659

    Würtzen, G., Larsen, J. C. & Tarding, F. (1972) 8th International
         Congress on Clinical Chemistry, Copenhagen


    See Also:
       Toxicological Abbreviations
       Orange RN (WHO Food Additives Series 12)
       ORANGE RN (JECFA Evaluation)