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    INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

    WORLD HEALTH ORGANIZATION



    SUMMARY OF TOXICOLOGICAL DATA OF CERTAIN FOOD ADDITIVES



    WHO FOOD ADDITIVES SERIES NO. 12






    The data contained in this document were examined by the
    Joint FAO/WHO Expert Committee on Food Additives*
    Geneva, 18-27 April 1977




    Food and Agriculture Organization of the United Nations
    World Health Organization



    * Twenty-first Report of the Joint FAO/WHO Expert Committee on Food
    Additives, Geneva, 1977, WHO Technical Report Series No. 617

    ORANGE RN

    EVALUATION FOR ACCEPTABLE DAILY INTAKE

    BIOLOGICAL DATA

    BIOCHEMICAL ASPECTS

    Metabolism

         The dye was administered in aqueous suspension to male rabbits
    (2-3 kg weight) 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:
    p-aminophenol, 3%, aniline, 0.9% and 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-aminophenyl-glucuronide (by difference 40%),
    aniline (0.9%), O-aminophenol (3%), 1-amino-2-naphthol-6 sulfonic acid
    (42% - 24 hours) (Daniel, 1962).

         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 azo-reduction 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 metabolites (Orange RN and 4'-hydroxy-l-phenylazo-2-
    naphthol-6 sulfonic acid) (0.4%), total p-aminophenol (52%), total
    O-aminophenol (6%), aniline (O.3%). 1-amino-2-naphthol-6 sulfonic acid
    was present, but not determined (Larsen and Tarding, 1976).

         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).

         After gavage of 66 mg 35S-labelled Orange RN (labelled in the
    naphtholsulfonic acid moity) per kg bw to six male rats (weighing
    approximately 300 g) 11.5% (8.6-14.1) of the radioactivity was
    recovered in the urine in 48 hours, while 44% (33-55) was found in the
    faeces. Of the radioactivity excreted in urine 1.5% (0.3-3.4) was
    unreduced dye.

         After i.v. administration of 6 mg 35S-Orange RN per kg bw to
    four male rats 33% (24-50) of the radioactivity was found in urine
    within 48 hours, of which 15% (11.2-18.2) was unreduced Orange RN.
    Twenty-seven per cent. (24-33) of the activity was recovered in the
    faeces (Larsen and Tarding, 1977).

         After gavage of 80 mg 35S-labelled Orange RN (labelled in the
    naphtholsulfonic acid moity) per kg bw to four male rabbits (weighing
    3.5 kg) 52% (26-69) of the dose was recovered in the urine in 24
    hours. Of this only 1.5% of the dose (0.5-2.2) was unreduced dye.
    Three per cent. (1.2-4.9) of the dose was found in the faeces after 24
    hours.

         When 15 mg of 35S-labelled Orange RN per kg bw was given i.v. to
    two rabbits, 60% (68 and 51% respectively) of the dose was recovered
    in the urine within 24 hours, of which 37% was unreduced Orange RN.
    9.5% (10.0 and 9.0% respectively) of the dose was found in the faeces.

         The presence of radioactivity in the bile at sacrifice after 24
    hours indicated entero-hepatic circulation both after oral and i.v.
    administration (Larsen and Tarding, 1977).

         Using 35S-labelled ANSA* the absorption and excretion as well as
    the localization in the liver cell of ANSA was investigated in rats
    and rabbits. The results after p.o. administration shows that the
    rabbit absorbs and excretes into the urine a significant larger amount
    of ANSA (30-40%) than the rat (6-13%). The results after i.v.
    administration shows that some 60% of the injected dose is excreted in
    the urine in both species. In the rat 15-20% are recovered from the
    faeces, while only traces (0.1-0.5%) are found in faeces of rabbits. A
    significant amount of ANSA was retained in the blood and the liver of
    both species 24 and 48 hours after i.v. administration. After
    differential centrifugation of liver homogenates the majority of ANSA
    was found in the 9000 g supernatant (Larsen and Tarding, 1977).

    TOXICOLOGICAL STUDIES

    Special studies of the effect on 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.

              

    *  1-amino-2-naphthol-6-sulfonic acid.

    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).

    Acute toxicity

                                                          

    Animal    Route          LD50 mg/kg       Reference
                             mg/kg bw
                                                          
    Rat       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, 60, 600, 1200, or 6000 ppm for over three
    months. At 6000 ppm, there was marked Heinz body production,
    methaemoglobinaemia and reticulocytosis together with enlargement of
    the spleen and increased splenic iron. At 1200 ppm, these effects were
    present but less severe and at 600 ppm 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., 1971).

    Cat

         A daily oral dose of 200 mg/kg for six days induced increase in
    Heinz bodies (Wingler, 1953).

    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-duct epithelium was found in
    all test groups and the intensity of proliferation was dose-related.
    At the highest dose level macrotic anaemia, haemoglobinaemia 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).

         The induction and persistence of haematological changes and
    hepatic lesions were studied in a time-sequence experiment where four
    groups of SPF Danish Landrace pigs were given Orange RN (monosodium
    salt of 1-phenylazo-2-naphthol-6-sulfonic acid) 160 mg/kg bw in a
    balanced powdered diet for 7, 14, 28 and 56 days respectively,
    followed by a restitution period of 82 days on normal diet. A fifth
    group served as control. Serial liver biopsies were taken during the
    experiment for histological evaluation. Anaemia with Heinz bodies was
    induced after Orange RN for 28 days. Increased number of reticulocytes
    and Heinz bodies already after seven days. A marked bile duct/ductule
    proliferation was seen after Orange RN for 28 and especially 56 days.
    Only slight proliferation after seven or 14 days. The decrease in RBC
    disappeared after seven days recovery, but the increased value for
    reticulocytes and Heinz bodies could still be observed after 21 and
    30-40 days respectively. The hepatic lesions persisted after 82 days
    of restitution but the intensity was reduced. Large individual
    variations in reversibility could be noted among the pigs (Hasselager
    and Hansen, 1977).

    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 eosinophil 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).

    REFERENCES

    Dacre, J. C. (1969) Acute and chronic toxicity studies on Orange RN,
    Proc. Univ. Otaga med. Sch., 47, 3

    Daniel, J. W. (1962) The excretion and metabolism of edible food
    colors, Toxicol. Appl. Pharmacol., 4, 572-594

    Gaunt, I. F., Brantom, P. G., Kiss, I. S., Grasso, P and Gangolli,
    S. D. (1971) Short-term toxicity of Orange RN in rats, Food Cosmet.
    Toxicol., Vol. 9, iss. 5, pp. 619-630

    Larsen, J. C. and Tarding, F. (1976) Acta pharmacol. et toxicol.,
    39, 525-535

    Larsen, J. C. and Tarding, F. (1977) Abstract. 19th Meeting of the
    European Society of Toxicology, in Copenhagen, June 19-22

    Hasselager, E. and Hansen, E. (1977) Abstract of data to be published

    Olsen, P., Wuertzen, G., Hansen, E., Carstensen, J. and Poulsen, E.
    (1973) Short-term peroral toxicity of the food color Orange RN in
    pigs, Toxicology., 1 (3), 249-260

    Olsen, P. and Hansen, E. (1973a) Bile duct proliferation in pigs fed
    the food color Orange RN, Acta. Pharmacol. et Toxicol., 32, 314

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

    Walker, R. (1970) The metabolism of A20 compounds: A review of the
    literature, Fd. Cosmet. Toxicol., Vol. 8, pp. 659-676

    Wingler, A. (1953) Dyes in foodstuff dyeing. The problem of exposure
    to cancer. 2. Krebsforsch., 59, 134-155

    World Health Organization (1974) Food Additive Series, No. 6, Geneva
    Switzerland

    Wuertzen, G., Larsen, J. C. and Tarding, F. (1972) Formation of
    hemiglobin in vivo and in vitro after administration of a food
    additive Orange RN, Scand. J. Clin. Lab. Invest. Suppl., 29(126),
    13-20


    See Also:
       Toxicological Abbreviations
       Orange RN (WHO Food Additives Series 6)
       ORANGE RN (JECFA Evaluation)