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    AZORUBINE

    Explanation

         This colour was evaluated for acceptable daily intake by the
    Joint FAO/WHO Expert Committee on Food Additives in 1974 and 1978 (see
    Annex I, Refs. 34 and 48), and tentative specifications and a
    toxicological monograph were prepared in 1977 and 1978 respectively
    (see Annex I, Refs. 45 and 49). Since the previous evaluation,
    additional data had become available and are summarized and discussed
    in the following monograph. The previous monograph has been expanded
    and is reproduced in its entirety below.

    BIOLOGICAL DATA

    BIOCHEMICAL ASPECTS

    Absorption, distribution, excretion and metabolism

         Male Swiss albino mice (CD-1) (three to six per group) were given
    single doses of (14C)azorubine (5 µCi/mmol) by stomach tube
    (200 mg/kg, 6 µCi) or i.v. injection (200 mg/kg, 0.7 µCi). The plasma
    and tissue kinetics of the compound were studied by monitoring the
    decay of radioactivity in plasma, gastrointestinal tract, liver,
    kidney, lung, testes, spleen and gall bladder, 5, 10, 15, 30 minutes
    and 1, 2, 4, 8, 16, 32 and 96 hours after dosing. The faeces and urine
    of mice placed in individual metabolic cages were collected between
    four and 96 hours after dosing. After oral administration, peak levels
    of radioactivity occurred in plasma (0.08%/ml) and in the liver, lung,
    testes and spleen eight hours after dosing. Radioactivity was almost
    completely excreted in faeces (74%) and urine (19%) within 16-32 hours
    of oral dosing. After i.v. injection of (14C)azorubine, most of the
    radioactivity (76%) was excreted 24 hours after dosing in faeces (64%)
    and urine (12%). The plasma 14C-radioactivity decay curve after i.v.
    administration indicated a very rapid distribution of the compound
    into the tissues (t1/2 = 10 minutes) and an efficient excretion
    mostly through the gastrointestinal tract (92%) which was complete 48
    hours after dosing (Galli et el., 1981).

         Rats were injected intravenously with approximately 1 mg of the
    dye. The bile was collected for six hours and analysed. The recovery
    of the dye was an average of 38% (30-40%) of the administered quantity
    (Ryan & Wright, 1961). This like any other azo dye is probably reduced
    in the gut by bacterial azo reductases (Walker, 1970). The absorption,
    distribution and excretion of the red azo dye azorubine were studied
    in male Sprague-Dawley rats. (14C)azorubine (5 mCi/mmol) was
    administered to groups of at least three animals in a dose of
    200 mg/kg (25 µCi) by gavage or in the same dose (200 mg/kg, 3 µCi) by
    intravenous injection and radioactivity was measured in blood, tissue,
    faeces and urine at different times after dosing (5, 10 and 30

    minutes and 1, 2, 4, 8, 16, 32, 64 and 96 hours after azorubine
    administration). After oral administration of the dye, no
    radioactivity was detected in the brain, adipose tissue, muscle,
    testes, spleen or lung, and recovery of the administered radioactivity
    in faeces and urine was almost complete by 32 hours (82% and 8%
    respectively). The radioactivity profile of the blood indicates rapid
    but poor absorption of (14C)azorubine, a maximum radioactivity
    content, corresponding to 0.01% of the dose per ml of blood, being
    reached within 10 minutes. The decay curve for 14C-radioactivity in
    the blood after i.v. injection of (14C)azorubine indicated rapid
    distribution to the tissues and could be described in terms of a two-
    compartment mathematical study. The highest levels of radioactivity
    occurred in the gastrointestinal tract and liver after the injection,
    but after 24 hours no radioactivity was detectable in these or other
    tissues. All the radioactivity was recovered in the faeces and urine
    in the 24 hours following i.v. injection, the 79% of the dose present
    in the faeces indicating active excretion of the dye and its
    metabolites in the bile and poor reabsorption from the intestine. The
    bioavailability of (14C)carmoisine, calculated from the blood-
    radioactivity curves after oral and i.v. administration, was less than
    10% (Galli et el., 1982a).

         The absorption, metabolism and excretion of orally administered
    (14C)-labelled azorubine (32 mCi/mmol) have been studied in male and
    female Wistar albino rats, male MF-1 mice and male Dunkin-Hartley
    guinea-pigs. Following administration of a single oral dose of either
    0.5 mg/kg or 50 mg/kg (20 µCi/kg), the majority of the radioactivity
    was excreted in the urine and faeces in the first 24 hours: 18% and
    73% in rats, 17% and 66% in mice, and 37% and 45% in guinea-pigs
    respectively. Less than 0.03% of the dose was eliminated as CO2.
    Substantially all of the dose was recovered in the excrete within 
    72 hours, the majority being accounted for in the faeces. Although the
    male and female rat and the mouse excreted a similar proportion of the
    dose in the urine, the proportion of the radioactivity found in the
    urine of the guinea-pig was significantly greater than that of the
    other species at both dose levels. Pretreating male rats with
    unlabelled colouring in the diet (0.05% w/w) for 28 days to provide an
    intake of approximately 50 mg/kg/day prior to dosing with
    14C-labelled colouring (50 mg/kg), had no effect on the route of
    excretion or the time taken to eliminate all of the label, although
    there was evidence that the proportion of the metabolites extracted
    from the faeces was different from the corresponding untreated
    animals. Following a single dose of 14C-labelled colouring to non-
    pretreated rats, mice and guinea-pigs or rats given repeated doses of
    unlabelled colouring (50 mg/kg/day for 28 days), no marked
    accumulation of radioactivity in any tissue was found at 72 hours.
    Pregnant rats eliminated a single oral dose of 14C-labelled colouring
    (50 mg/kg at day 8 of pregnancy) at a similar rate to non-pregnant
    females, and the concentration of radioactivity in the foetuses was

    similar to that in the other tissues. Examination of urine by high-
    performance liquid chromatography showed that between 60% and 80% of
    the radioactivity in the urine was associated with naphthionic acid in
    the urine of all three species. A further 10% and 20% of the
    radioactivity in the urine co-eluted with 2-amino-1-naphthol-4-
    sulfonic acid (2-ANS). The third component, accounting for less than
    5% of the radioactivity in the rat and mouse but 16% in the guinea-pig
    co-chromatographed with 1,2-NQS (1,2-naphthoquinone-4-sulfonate) and
    the fourth, which accounted for between 2% and 5% of the radioactivity
    in the urine, was not identified. Naphthionic acid was also found in
    the faeces of all three species; however, no 2-ANS or 1,2-NQS was
    detected. Five unidentified metabolites were found in the faeces of
    all three species, the proportions of which varied between species.
    Two of these metabolites were hydrolysed by combined ß-glucuronidase
    and sulfatase treatment. No significant absorption of radioactivity
    during a one-hour period was found from isolated loops of small
    intestine of the rat, mouse or guinea-pig containing either 50, 500 or
    5000 ppm (0.005, 0.05 or 0.5%) carmoisine as measured by total
    recovery of injected radioactivity. Less than 0.03% of the
    administered radioactivity in the 50 mg/kg dose was recovered in the
    bile during one hour and only between 0.04% and 0.7% during five hours
    (Phillips et al., 1982).

         (14C)azorubine was administered to rats at the dose of 200 mg/kg
    bw (25 µCi) by gavage. Separation of radioactive compounds in faeces
    and urine of animals was carried out by HPLC with a UV and a
    radioactivity detector. In addition to unmodified carmoisine, five
    radioactive compounds were present. The main peak showed both the
    retention time and UV spectrum of authentic naphthionic acid.
    Metabolic patterns similar to those observed in vivo were found by
    incubation of 14C-carmoisine under anaerobic conditions with a
    bacterial suspension isolated from human faeces and from the
    intestinal contents of rats (Marinovich et al., 1983).

    Effects on enzymes and other biochemical parameters

         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 the succinic oxidase system of rat
    liver homogenates. The results indicated that this dye inhibited the
    oxidative activity of this enzyme by approximately 40% (Sikorska &
    Krauze, 1962).

    TOXICOLOGICAL STUDIES

    Special studies on carcinogenicity

         Two batches of textile grade azorubine (71.4% dye, 7.39% water,
    11.70% NaC1, 5.70% Na2SO4, 3.72% NaHCO3 for the first 11 months
    and 67.30% dye, 7.48% water, 7.85% NaC1, 12.20% Na2SO4, 5.16%
    NaHCO3 for the final 13 months) were used to conduct a
    carcinogenicity bioassay in mice and rats. Groups of mice (50 males
    and females per group) were fed diets containing textile grade
    azorubine to study potential carcinogenicity effects at levels of 0,
    3000 or 6000 ppm (0, 0.3 or 0.6%) for 103-104 weeks. Throughout the
    study, mean body weights of dosed female mice were comparable with
    those of the controls, while the mean body weight of high-dose male
    mice was slightly lower than that of the controls. No other compound-
    related clinical signs were observed. There was no significant
    decrease in survival between any of the groups of male or female mice.
    Results of histopathological examination indicated that administration
    of azorubine to male and female mice under the conditions of the
    bioassay was not associated with an increased incidence of any rumour
    type. However, although not dose related, non-neoplastic lesions as
    lymphoid hyperplasia of the spleen, haematopoiesis in the liver and
    lymphoid hyperplasia of the submucosa of the urinary bladder were
    observed in female mice (Anon., 1982).

         Textile grade azorubine was administered in diets containing 0,
    6000 or 12 500 ppm (0, 0.6 or 1.25%) of the dye for 103-104 weeks to
    groups of 50 male F344 rats, and 0, 12 500 or 25 000 ppm (0, 1.25 or
    2.5%) to groups of 50 female F344 rats. Control animals (90) were
    shared with feeding study of CI Acid Orange 10 and FD and C Yellow 
    No. 6, which were conducted concurrently. Mean body weights of dosed 
    rats of either sex were comparable with those of the controls 
    throughout most of the study. No compound-related clinical signs were 
    observed. The survival of the low-dose group of male rats was 
    significantly greater than that of the controls (P = 0.046) or of the 
    high-dose group (P <0.001). No significant differences were observed 
    between the control and high-dose groups of male rats or between any 
    groups of female rats. Endometrial stromal polyps of the uterus were 
    observed in high-dose female rats at an incidence significantly higher 
    (P = 0.008) than that seen in the controls (controls: 9/87, 10%; low 
    dose: 11/50, 22%; high dose: 14/50, 28%). However, the observed incidence 
    of polyps in the dosed groups was similar to the historical rate in 
    untreated female F344 rats (65/286, 23%; range 10-37%). Hence, the 
    increased incidence of this lesion is not regarded as being associated 
    with the administration of azorubine. The various non-neoplastic lesions
    represented among both control and dosed animals have been encountered
    previously as spontaneous occurrences in aging laboratory rats. An
    increased incidence of adrenal cortical focal hyperplasia,
    characterized by focal collections of basophilic, eosinophilic or
    vacuolated cells, was seen in high-dose rats of both sexes (males:

    5/89, 6%; 6/49, 12%; 8/50, 16%; females: 7/86, 8%; 7/50, 14%; 18/50,
    36%). Results of histopathological examination indicated that
    azorubine was not carcinogenic to male or female F344 rats under the
    conditions of this bioassay (Anon., 1982).

    Special studies on Heinz bodies

         Four cats were given 5% aqueous solution in doses of 1 g on the
    first day and 0.1 g on the ninth and eighteenth days. A negative test
    for Heinz bodies was obtained (Deutsche Forsch., 1957).

    Special studies on mutagenicity

         The colour was tested for mutagenic action in a concentration of
    0.5 g/100 ml in cultures of Escherichia coli. No mutagenic effect
    was found (Lück & Rickerl, 1960). Testing of azorubine for
    mutagenicity with Salmonella typhimurium TA-1538 50 µg/plate showed
    that the azo dye is not mutagenic in the absence or presence of a
    liver enzyme preparation (Garner & Nutman, 1977). This colour was
    tested for cytotoxic activity and for mutagenic effect in a
    concentration of 0, 1, 2, 20, 500 and 1000 µg/plate/108 bacteria in
    cultures of different strains of Salmonella typhimurium TA-1535,
    TA-1538, TA-100 and TA-98 either in the presence or absence of liver
    microsomal fraction. No mutagenic effect was found (Viola & Nosotti,
    1978). Azorubine 5 mg/ml did not induce mitotic gene conversion in
    Saccharomyces cerevisiae BZ 34 when treated either in stationary-
    phase or log-phase culture without microsomal activation. Under these
    treatment conditions neither significant cell killing nor inhibition
    of cell division was observed (Sankaranarayanan & Murthy, 1979). No
    evidence of mutagenic potential of azorubine (5 mg/ml) was obtained in
    two different bacterial test systems, Escherichia coli WPZ and
    Salmonella typhimurium TA-1538, either in the presence or absence of
    liver microsomal fraction (S-9 mix) (Haveland-Smith & Combes, 1980).

    Special studies on placental transfer

         Three or six pregnant Sprague-Dawley rats per group received
    (14C)azorubine (200 mg/kg, 25 µCi) by gavage on days 16-19 of
    gestation. Animals were killed on day 19 of gestation, two, 16 and 
    64 hours after dosing, and blood, maternal tissues, amniotic fluids,
    placentae, maternal uterus, foetal membranes and foetuses were
    analysed for radioactivity. No evidence for transplacental transfer of
    (14C)azorubine or its metabolites was obtained. More than 90% of the
    radioactivity was excreted in faeces and urine within 64 hours. In a
    similar experiment no significant differences in maternal body weight,
    food intake of dams, number of foetuses, litter size and foetal weight
    were observed in treated (200 mg/kg, 25 µCi) and control animals when
    the dye was administered by gavage on day 11 of gestation, and the
    animals were killed on day 19 of gestation (Galli et el., 1982b).

    Special studies on reproduction

    Rat

         Twenty-five male and 25 female rats received 1% azorubine in
    their drinking-water for 180 days, giving approximately 55 g dye per
    animal. A similar group of 50 rats acted as controls. Weight gain,
    mortality and general condition were similar in both groups. After
    seven months the animals were mated and an F1 generation produced.
    After weaning the pups were put again on 1% azorubine and after four
    months mated to produce an F2 generation. No abnormalities regarding
    litter number or fertility were noted. After 200 days on 1% azorubine,
    the F2 generation was kept on normal diet and water for two years. No
    adverse effects were seen on mortality or tumour incidence (Hecht,
    1966).

    Special studies on sensitizing effects

         In experiments on guinea-pigs, it was found that this colour had
    no sensitizing activity (Bär & Griepentrog, 1960).

    Special studies on teratogenicity

         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 el., 1972b).

         Female New Zealand white rabbits were administered azorubine on
    days 6 through 18 of gestation by oral intubation at a level 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).

    Acute toxicity

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

                                                                        

    Animal         Route        LD50        Reference
                              (g/kg bw)
                                                                        

    Mouse          i.p.          0.8        Gaunt et al., 1967
                   i.v.          0.8        Deutsche Forsch., 1957
                   oral        > 8.0        Gaunt at al., 1967

    Rat            i.p.          1.0        Gaunt et al., 1967
                   oral        >10.0        Gaunt et al., 1967
                                                                        

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

    Short-term studies

    Rat

         Three weanling rats were given a 0.1% solution of carmoisine to
    drink for 28 days (daily consumption approximately 15 mg). No toxic
    effects were noted (Goldblatt & Frodsham, 1952). Sixteen Carworth Farm
    E strain rats of each sex were placed into groups which were fed 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 a 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., 1978a).

    Pig

         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

    Mouse

         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 the 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 were that
    azorubine was non-carcinogenic in mice (Bonser et el., 1956).

         Azorubine was administered to ASH/CS1 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 was fed only the basal
    ration of Oxoid pasteurized diet supplemented with 80 ppm (0.008%)
    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 a decreased packed
    cell volume. No abnormal rumour 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 at., 1974).

    Rat

         Ten rats were given the colour in the drinking-water in a
    concentration of 1% for 209 days. The daily intake was 1.2 g/kg bw and
    the total amount administered was 51 g per animal. The observation
    period was 919 days. No tumours were found (Deutsche Forsch., 1957).
    Ten rats were given 1% of the colour in the drinking-water for 250
    days. The daily intake was 7.94 g/kg bw and the total intake 52 g per
    animal. The observation period was 545 days. No tumours were found
    (Deutsche Forsch., 1957). Ten rats were given a diet containing 0.2%
    of the colour for 417 days. The daily intake was approximately
    0.1 mg/kg bw and the total intake was 11 g per animal. The observation
    period was 838 days. No tumours were found (Deutsche Forsch., 1957). A
    group of 10 rats were given twice weekly subcutaneous injections of
    0.5 ml of a 1% solution (= 5 mg) of the colour for one year. The
    animals were kept under observation for over 938 days. One axillary
    tumour was observed in one animal (Deutsche Forsch., 1957). In a
    repeat experiment another group of 10 rats was given twice weekly
    subcutaneously 0.5 ml of a 1% solution (= 5 mg) of the colour for one
    year. No tumours were formed after 521 days, each animal having
    received 0.5 g (Deutsche Forsch., 1957).

         Azorubine was fed at levels of 0, 0.35, 0.8 and 2.0% of the diet
    to Sprague-Dawley rats (30 males and 30 females 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 up to 2% in 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., 1978a). 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, gross
    pathological 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 el., 1978b).

         Groups of 114 (control) and 66 (treated) Wistar rats of each sex
    were given a diet to provide intakes of 0 (control), 100, 400 or
    1200 mg azorubine/kg/day for nine weeks (F0 generation). Diet
    composition was:

                   Dye content                 89.5%
                   Volatile matter              4.7%
                   Sodium chloride              4.7%
                   Sodium sulfate               1.7%

    These animals were mated and the females allowed to rear the resulting
    offspring, with the treatment continuing throughout mating, pregnancy
    and lactation. Young were randomly selected to provide groups of 90
    (control) and 54 (treated) of each sex. These were given the same
    treatment as their parents for up to 110 weeks for the females or 115
    weeks for the males (F1 generation). The appearance of the rats was
    normal apart from an external contamination of the fur, colour in the
    urine and dark faeces. Animals of both generations given 1200 mg
    azorubine/kg/day were slightly lighter than the controls, despite a
    small increase in food intake. There was an increased water intake by
    these same animals and, on the basis of periodic renal function tests,
    a tendency to excrete larger volumes of urine. The ability of the
    kidney to concentrate urine under condition of dehydration was not
    impaired. Haematological analysis on 20 animals of each sex at 3, 6,
    12, 18 and 24 months and on all survivors at the end of the study
    revealed isolated statistically significant differences between the
    treated and control rats, but these were not consistent between the
    sexes or with time and were not considered to be related to the
    treatment. There were increases in caecum weight at the highest dose
    level, but no other changes in organ weights that were due to
    treatment. Investigations of kidney function using 20 animals of each
    sex at 3, 6, 9, 12, 18 and 24 months did not reveal any changes that
    could be related to azorubine treatment. There were low concentrations
    of glucose in serum collected from the survivors of both sexes given
    1200 mg azorubine/kg/day and females given 400 mg/kg. In the absence
    of any associated findings, this could not be firmly related to
    treatment.

         There were a few high-dose males with bladder hyperplasia
    possibly due to irritant metabolites in the urine. There was an
    increase in the number of high-dose females with adrenal blood/fibrin
    cysts and five high-dose females with intimal hyperplasia/medial
    hypertrophy of the pancreatic blood vessels. The incidences of most
    rumours were similar in treated and control rats. There was a small
    increase in the number of adrenal phaeochromocytoma in the high-dose
    males, but the incidence was well within the background for the same
    strain of rat (Stevenson et al., 1982).

    Comments

         Following administration of a single dose of (14C)azorubine to
    male and female rats, mice and guinea-pigs, the majority of the
    radioactivity was excreted in the urine and faeces in the first 
    24 hours. Substantially all of the dose was recovered in the exereta
    within 72 hours, the majority being accounted for in the faeces
    (60-75%). Following a single dose of 14C-labelled colouring to non-
    pretreated rats, mice and guinea-pigs or rats given repeated doses of
    unlabelled colouring, no marked accumulation of radioactivity in any
    tissue was found. Pregnant rats eliminated a single oral dose of
    14C-labelled colouring at a similar rate to non-pregnant females and
    no evidence for transplacental transfer of (14C)azorubine or its
    metabolites was obtained. Naphthionic acid was found in the urine
    (60-80% of the radioactivity) and faeces of all three species. A
    further 10-20% of the radioactivity in the urine was co-chromato-
    graphed with 2-amino-1-naphthol-4-sulfonic acid and less than 5% of
    the radioactivity in the rat and mouse, but 16% in the guinea-pig
    co-chromatographed with 1,2-naphthoquinone-4-sulfonate. Five
    unidentified metabolites were found in the faeces of all three
    species. No evidence of mutagenic potential of azorubine was obtained
    in different bacterial tests systems in the presence of absence of
    liver microsomal fraction. A carcinogenic bioassay of textile grade
    azorubine was conducted in rats and mice fed up to 1250 and 900 mg/kg
    bw, respectively. Under the conditions of this bioassay, textile grade
    azorubine was not carcinogenic for rats or mice of either sex.
    Reproduction studies including teratogenicity did not reveal any
    compound-related adverse effect. A long-term study carried out in the
    mouse indicates a no-effect level of 0.25% (375 mg/kg/day). A
    multigeneration reproduction study in the rat did not show adverse
    effects in the reproductive parameters assessed, in body weight gain,
    in urine and haematological values, in gross pathological and
    histopathological findings. An adequate one-year long-term study in
    rats exposed in utero to azorubine indicates that there were not
    changes that could be related to treatment. With the exception of the
    higher caecum weights at the highest dose, the remaining organ weights
    were not influenced by azorubine. The histopathology did not reveal
    any significant differences between the control and treated animals.
    There was no evidence of a treatment-related increase in the total
    number of animals with benign and malignant rumours. It is concluded
    that azorubine is not carcinogenic and that the no-untoward-effect
    level in this study was 400 mg/kg bw per day of azorubine.

    EVALUATION

    Level causing no toxicological effect

    Mouse:    0.25% (2500 ppm) in the diet, equivalent to 375 mg/kg bw.
    Rat  :    0.8% (8000 ppm) in the diet, equivalent to 400 mg/kg bw.
    Pig  :    0.1% (1000 ppm) in the diet, equivalent to 400 mg/kg bw.

    Estimate of acceptable daily intake for man

    0-4.0 mg/kg bw.

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    See Also:
       Toxicological Abbreviations
       Azorubine (WHO Food Additives Series 6)
       Azorubine (WHO Food Additives Series 13)
       AZORUBINE (JECFA Evaluation)