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    PONCEAU 4R

    Explanation

         This colour was evaluated for acceptable daily intake by the
    Joint FAO/WHO Expert Committee on Food Additives in 1964, 1969, 1974,
    1978 and 1981 (see Annex I, Refs. 8, 19, 34, 48 and 57). Toxicological
    monographs were issued in 1964, 1969, 1974 and 1978 (see Annex I,
    Refs. 10, 20, 35 and 49). Since the previous evaluation, additional
    data have become available and are summarized and discussed in the
    following monograph. The previously published monographs have been
    expanded and are reproduced in their entirety below.

    BIOLOGICAL DATA

    BIOCHEMICAL ASPECTS

    Absorption, distribution, excretion and metabolism

         Ponceau 4R is reduced in vitro by rat caecal contents (Walker,
    1968), but Ryan & Wright (1961) showed that rats excreted 30-45% of an
    intravenous dose unchanged in the bile within six hours. The bile was
    coloured in rats and mice given intraperitoneally doses of the dye
    (Gaunt et al., 1967). The absorption, metabolism and excretion of
    orally administered 14C-labelled Ponceau 4R has been studied in male
    and female Wistar albino rats, male CD-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 bw), substantially all of the dose
    was excreted in urine and faeces within 72 hours, with the majority
    being accounted for in the faeces (90%) and less than 0.003% of the
    higher dose eliminated as 14CO2. In all three species studied,
    naphthionic acid was the major urinary metabolite, whereas naphthionic
    acid, 7-hydroxy-8-amino-naphthalene-1,3-disulfonic acid and unchanged
    dye were found in faeces. Pretreating male rats with unlabelled
    colouring in the diet (50 mg/kg/day) for 28 days prior to dosing with
    14C-labelled colouring (20 µCi/kg) had no effect on the route of
    excretion or the time taken to eliminate all of the label. Following a
    single dose of 14C-labelled colouring to previously untreated 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. Although pregnant rats
    eliminated a single oral dose of 14C-labelled colouring at a similar
    rate to non-pregnant females, some retention of radioactivity in the
    foetuses was found (0.3-0.5%). No significant absorption of Ponceau 4R
    was detected using isolated loops of small intestine of the rat,
    containing concentrations of colouring of 50, 500 or 5000 ppm (0.005,
    0.05 or 0.5%). Some absorption was seen from isolated loops of mouse
    intestine at the lowest concentration, and from the guinea-pig at the
    two higher concentrations. Less than 0.1% of the administered
    radioactivity in the 50 mg/kg dose was recovered in the bile during 
    one hour and between 0.6% and 1.3% during five hours. Two radioactive

    components were present in the urine of all three species. The major
    component (70% of the radioactivity in the rat) present in the
    methanol extract co-chromatographed with naphthionic acid in three
    systems. The other component had a similar chromatographic mobility to
    7-hydroxy-8-amino-naphthalene-1,3-disulfonic acid. A third (minor)
    radioactive component seen in mouse urine co-chromatographed with
    Ponceau 4R. Thin-layer radio-chromatography of faecal extracts from
    rats, mice and guinea-pigs given 50 mg 14C-labelled Ponceau 4R/kg
    showed that three radioactive compounds were present. The compounds
    co-chromatographed with naphthionic acid, 7-hydroxy-8-amino-
    naphthalene-1,3-disulfonic acid and unchanged Ponceau 4R. In all three
    species approximately 25-35% of the administered dose of colouring
    (due correction being made for recovery of radioactivity (80%)) was
    excreted unchanged in the faeces during 24 hours (Phillips et al.,
    1982).

    TOXICOLOGICAL STUDIES

    Special studies on mutagenicity

         This colour was tested for mutagenic effect in a concentration of
    0.5 g/100 ml in cultures of Escherichia coli. No mutagenic effect
    was found (Lück & Rickerl, 1960). The mutagenicity of Ponceau 4R has
    been tested in vitro using mutant strains of Bacillus subtilis
    with (rec+) and without (rec-) the ability to repair DNA by
    recombination mechanisms. No increase in lethatity of bacteria was
    observed which indicates that Ponceau 4R does not react with DNA (Kada
    et al., 1972). Cultured rat foetal hepatocytes were used to test 11
    different food colours for cytotoxicity. Ponceau 4R was rated as
    moderately toxic (Sako et al., 1980).

    Special studies on reproduction

         Ponceau 4R was given in the diet of Wistar rats over three
    generations at concentrations to provide 0, 50, 500 or 1250 mg/kg/day.
    Animals of both sexes were used in groups of 36 for treated and 60 for
    control groups. The parent generation (F0) were given Ponceau 4R from
    the time they were weanlings and the succeeding generations were
    exposed throughout their lifetimes, including in utero, since
    treatment continued during the gestation and lactation periods. The
    parental generation was bred twice to provide the F1a and F1b
    generations. The former were used to provide the subsequent generation
    and animals for a long-term study. The second breeding provided data
    covering in utero and post-partum development. The foetuses of the
    F1b, F2 and F3 generations were examined for skeletal development
    whereas those allowed to litter were monitored over the first 21 days
    of life for survival and developmental milestones. All animals were
    killed and subjected to a post-mortem examination which, for a
    selection of animals from each generation, included weighing of organs
    and, for the F3 generation, a histological examination of the
    tissues.

    During the study, eight females and two males involving all three
    generations of adult rats (F0, F1a and F2) died or were killed
    because of sickness. The post-mortem examinations of these animals,
    the majority of which were females in the later stages of pregnancy,
    did not reveal any findings that could be associated with treatment. A
    general observation made throughout the study was the pink coloration
    of the fur of animals exposed to Ponceau 4R and softer more yellow-
    coloured faeces from those given 500 and 1250 mg/kg/day Ponceau 4R. A
    yellow coloration was also seen in the caecal contents of the treated
    animals and was attributed to the presence of a yellow metabolite.

         The clinical observations and records of body weight, food intake
    and water intake during the three pre-mating feeding periods did not
    show any changes that could be associated with treatment. The various
    post-mortem examinations showed a pink colouring of the
    gastrointestinal tract at all dose levels. There were enlarged caeca
    in animals of the two higher dose levels after prolonged feeding but
    not in weanling animals, and lower liver weights in F2 and F3
    offspring of the same dose levels. These findings were not considered
    to represent an adverse effect. Examination of the uterine contents of
    pregnant females of the F0, F1a and F2 generations revealed no
    treatment-related differences in the incidence of pre- and post-
    implantation losses or of the weight and appearance of the foetuses.
    The only finding from the examination of the stained skeletons of
    these young indicated a slightly more advanced development in those
    from the treated groups. The postnatal development of the offspring
    from females allowed to litter, as judged by survival, body weight and
    developmental milestones, was not affected by treatment. There were no
    differences between the untreated animals and those given 1250 mg
    Ponceau 4R/kg/day in the histological examination of the tissues from
    the F3 generation. It is concluded that this study has shown a lack
    of any adverse effects of Ponceau 4R fed up to 1250 mg/kg/day over
    three generations. The reproductive performance of the adults, the
    in utero development of the young and the post-partum development of
    the pups were normal (Stevenson et al., 1980).

    Special studies on sensitization

         In experiments with guinea-pigs it was found that this colour had
    no sensitization (Bär & Griepentrog, 1960). A negative test for Heinz
    bodies was obtained after administering this colour in a 5% aqueous
    solution by stomach tube to four cats (DFG, 1957).

    Special studies on teratogenicity

         Groups of unspecified numbers of NMRI mice received daily doses
    of 7.5, 30 or 100 mg/kg bw Ponceau 4R by gavage either from days 0 to
    7 or days 6 to 18 of pregnancy. The foetuses were removed on day 18
    for detailed examinations. No effect of treatment with the dye could
    be observed with regard to number of implantations, frequency of
    foetal death and resorptions, gross malformations, skeletal or 

    internal malformations, or retarded growth as judged by foetal weight
    (Larsson, 1975). An embryotoxicity study on Ponceau 4R was carried out
    in an unspecified number of SPF Wistar rats. The substance was given
    by gavage from days 1 to 20 of the gestation period in doses of 0,
    1000, 2000 and 4000 mg/kg bw per day, dissolved in distilled water.
    The foetuses were removed on day 21. No effect due to the treatment
    with the dye was observed with regard to the number of corpora luteae
    (CL), of implantations, of foetuses dead or alive, with regard to
    gross malformations, to skeletal and internal malformations, or to the
    weight of the foetuses (Meyer & Hansen, 1975). Pregnant Wistar rats
    were fed a diet containing 0, 0.01, 0.1 or 1% Ponceau 4R throughout
    the gestation period. Observation of the foetuses at term revealed no
    deleterious effects due to the treatment with the dye in regard to
    embryonic death or intrauterine growth. Gross skeletal and visceral
    abnormalities were not generally noted except for a decrease in the
    number of ossified phalanges of the hind limb in the 0.01% dye-treated
    group. In the postnatal study, survival index, body weight gain,
    skeletal development and external differentiation were similar between
    the dye-treated groups and the controls. However, the 1% dye-treated
    group showed an increased incidence of dilated renal pelvises in the
    offspring observed at eight weeks after birth. In order to re-examine
    this problem, pregnant rats of the same strain were fed a diet
    containing 0, 0.01 or 1% Ponceau 4R throughout the gestation period
    and were allowed to deliver their young. Kidneys of the offspring were
    examined macroscopically at four days, three weeks, 10 weeks and 22
    weeks after birth. Dilated renal pelvises were observed in each group
    including the controls, but no significant difference in incidence was
    shown between the dye-treated groups and the controls. Histological
    examination of the kidneys with enlarged pelvises showed no remarkable
    pathological changes. No other kidney anomalies were found in the
    dye-treated groups. In conclusion, Ponceau 4R is considered to show no
    adverse effects on prenatal and postnatal development in rats (Kihara
    et al., 1977).

    Acute toxicity
                                                                      

    Animal      Route          LD50              Reference
                            (mg/kg bw)
                                                                      

    Mouse       Oral         8 000               Gaunt et al., 1967
                i.p.        >1 750 approx.       Gaunt et al., 1967

    Rat         Oral        >8 000               Gaunt et al., 1967
                i.p.         2 000 approx.       DFG, 1957

    Female      i.p.         2 600               Gaunt et al., 1967

    Male        i.p.           600               Gaunt et al., 1967
                i.v.         1 000               DFG, 1957
                                                                      

    Short-term studies

    Rat

         Groups of 16 male and 16 female rats were fed diets containing 0,
    0.5, 1 and 2% dye for 90 days. No adverse effects were seen in
    appearance, behaviour, growth, food consumption, haematological
    indices, SGPT and SGOT serum levels except at the 2% level, when
    females had slightly increased transaminase values, red-cell counts
    and haemoglobin concentration. Renal function tests and organ weights
    were normal. Gross pathology and histopathology showed no difference
    between the test groups (Gaunt et al., 1967). Eleven rats were given
    1% of the colour in their drinking-water for 216 days and observed for
    791 days. Two animals died during the experiment; one had a sarcoma of
    the liver (DFG, 1957).

    Pig

         Four groups of three large white pigs of each sex were fed 0
    (control), 100, 300 or 900 mg Ponceau 4R/kg bw per day for three
    months. One female from the highest dose level died on day 23, but the
    death was attributed to an enteric infection. No differences between
    treated and control pigs were detected in growth, composition of urine
    and serum, organ weights or histopathology. There was a slight
    reduction in the number of erythrocytes at week 6 in males given
    900 mg/kg/day (Gaunt et al., 1969).

    Long-term studies

    Mouse

         Groups of 30 mice of each sex (Ash-CS1 strain) were fed on diets
    containing 0.01, 0.05, 0.25 and 1.25% Ponceau 4R for 82 weeks, with a
    group of 60 mice of each sex serving as controls. There was evidence
    that the colouring was converted to a yellow metabolite in the
    gastrointestinal tract. The feeding of Ponceau 4R had no adverse
    effect on mortality, body weight gain, organ weight or the incidence
    of tumours. A mild anaemia was present in the first six months of the
    study in mice given 0.25 or 1.25% Ponceau 4R. There was an increased
    incidence of foamy reticuloendothelial cells in the liver at the 1.25%
    level and an increased incidence of glomerulonephrosis at this and the
    0.25% level. This study shows that the colouring has no carcinogenic
    potential in mice when fed at dietary concentrations up to 1.25%. The
    no-untoward-effect level was 0.05% (Mason et al., 1974).

    Rat

         Ten rats were given 0.2% of the colour in their diet for 417
    days. The total intake was 11 g/animal. Observation extended for 1011
    days. One rat died. No tumours were found (DFG, 1957). In a similar
    experiment the dye was given to 11 rats as a 1% solution in the
    drinking-water, the daily dose being 1 g/kg for a period of 216 days.
    Total ingestion amounted to 52 g/animal. Animals were observed for 791
    days. One rat developed a sarcoma in the liver; two others died (DFG,
    1957)

         Four groups of 10 male and 10 female rats were given diets
    containing 0, 0.03, 0.3 and 3% of the colour for 64 weeks. No effect
    was noted on mortality. Females at the highest level had a lower food
    consumption throughout the experiment than the controls with a
    significant decrease in body weight at 16 and 64 weeks. In females the
    relative weights of heart, liver and kidney were increased. No effects
    were found on histopathology and as regards haemoglobin levels
    (Allmark et al., 1957).

         Seventy-five rats were fed the colour at a level of 0.1% of the
    diet. No rumours were observed. Similar results were obtained with 10
    rats fed at 0.2% of the diet. Feeding extended for life span (DFG,
    1957). Thirteen rats were given twice weekly s.c. injections of 0.5 ml
    of 1% of the colour for 365 days. Observation extended for 857 days.
    Five animals died during the experiment. No tumours were found (DFG,
    1957).

         Groups of 66 male and female Wistar rats were given diets to
    provide 50, 500 or 1250 mg Ponceau 4R/kg/day for nine weeks and 114
    males and females were given basal diet to serve as controls. Each
    female was paired with a male of the same dose group for 13 days and
    treatment continued during pregnancy and pup-rearing periods. Young
    were selected on the basis of one male and one female per litter to
    give group sizes of 54 and 96 of each sex for the treated and control
    groups respectively. The selection was made so that the treatment of
    the young was the same as their parents and it continued until only
    approximately 20% of the animals survived. This resulted in a maximum
    treatment duration of 114 weeks for the males and 118 weeks for the
    females. The fur of all treated animals was coloured pink due to
    contamination with Ponceau 4R, but, apart from this finding, none of
    the observations on the parental animals, including those of fertility
    and pup rearing, could be related to treatment.

         The young exposed to 50, 500 or 1250 mg Ponceau 4R/kg/day for
    their life span showed no difference of survival. There was a slightly
    lower weight gain in males and females given 1250 mg/kg that was not
    due to a lower food intake. The animals given 500 or 1250 mg/kg/day
    had soft and unformed faeces thought to be associated with an increase
    in the weight of the wall of the caecum and its contents and

    elimination of water with the faeces. This water loss was in turn
    considered to be responsible for an increase in water consumption in
    the same animals. There were no changes that could be related to
    treatment in the haematological examinations at 3, 6, 12, 18 and 24
    months and at the end of the study, and none that could be considered
    to represent an adverse effect of treatment in the renal concentration
    tests or measurement of cell excretion and urinary pH at 3, 6, 12, 18
    and 24 months. At 18 and 24 months, the incidence of females with high
    concentrations of protein in the urine was greater in the group given
    1250 mg/kg/day than in the controls.

         Kidney weight was elevated, not dose related, in the males given
    the lowest (+123%) and the highest (+128%) levels of treatment with no
    comparative effect in the females and no histological findings that
    were associated with this increase in weight. Testes weight was
    elevated at the two highest dose levels (+114 and +133% respectively).
    With the exception of the higher caecum and kidney weights, the
    remaining organ weights were not influenced by Ponceau 4R. The
    histopathology did not reveal any differences between the control and
    treated animals that could be related to treatment and neither did the
    analysis of serum and plasma of animals killed at the end of the
    study. The types and incidence of tumours were those expected for this
    strain of rat and there was no evidence of a treatment-related
    increase in the number of animals with each finding or in the total
    number of animals with benign and malignant tumours. It is concluded
    that Ponceau 4R was not carcinogenic to rats fed 1250 mg/kg/day for
    the bulk of their life span. The no-untoward-effect level was
    500 mg/kg/day (Stevenson et al., 1981).

    OBSERVATIONS IN MAN

         In a series of 51 patients showing signs of allergy, the authors
    showed that 16% reacted to an oral dose of Ponceau 4R. This reaction
    rate was no more than was seen with other synthetic colours and was
    less than with aspirin or a natural colour (Annatto extract). No
    evidence was presented to show if the colour was the sensitizer or if
    it was a cross-reaction with some other material (Mikkelson et al.,
    1978). Patch tests were carried out on 17 different colouring matters
    including Ponceau 4R using 50 patients who had been given a
    presumptive diagnosis of possible allergic contact dermatitis to
    coloured dyes. None of the patients tested demonstrated sensitization
    to Ponceau 4R (Rapaport, 1980).

    Comments

         Metabolism studies indicate that Ponceau 4R is rapidly absorbed
    from the gastrointestinal tract and undergoes extensive metabolism
    with the formation of naphthionic acid and 7-hydroxy-8-amino-
    naphthalene-1,3-disulfonic acid. Following a single dose of

    14C-labelled colouring to previously untreated rats, mice and guinea-
    pigs or rats given repeated doses of unlabelled colouring for 28 days,
    no marked accumulation of radioactivity in any tissue was found.
    Pregnant rats eliminated a single oral dose of 14C-Ponceau 4R at a
    similar rate to non-pregnant females. Following administration of a
    single oral dose to male and female rats, mice and guinea-pigs,
    substantially all of the dose was excreted in urine and faeces within
    72 hours, with the majority being accounted for in the faeces (90%). A
    three-generation reproduction study in the rat indicates that the
    clinical observations, body weight, food and water intake during the
    feeding periods did not show any changes that could be associated with
    treatment. Examination of the uterine contents of pregnant females and
    the F0, F1a and F2 generations revealed no treatment-related
    differences in the incidence of pre- and post-implantation losses or
    of the weight and appearance of the foetuses. The reproductive
    performance of the adults, the in utero development of the young and
    the post-partum development of the pups were normal. It is concluded
    that this study has shown an absence of any adverse effects of Ponceau
    4R in rats fed up to 1250 mg/kg bw per day over three generations.
    Teratogenicity studies did not reveal any compound-related adverse
    effects. No mutagenic or cytotoxic effect was found when the colour
    was tested in vitro experiments. An adequate long-term study has
    been carried out in the rat. There were no changes that could be
    related to treatment. The histopathology did not reveal any
    differences between the control and treated animals and neither did
    the analysis of serum and plasma of animals killed at the end of the
    study.

    EVALUATION

    Level causing no toxicological effect

         Mouse: 0.25% (2500 ppm) in the diet, equivalent to 375 mg/kg bw.

    Estimate of acceptable daily intake for man

         0-4.0 mg/kg bw.

    REFERENCES

    Allmark, M. G., Mannell, W. A. & Grice, H. C. (1957) Chronic toxicity
         studies of food colours. III - Observations on the toxicity of
         malachite green, new coccine and nigrosine in rats, J. Pharm.
         Pharmacol., 9, 622-628

    Bär, F. & Griepentrog, F. (1960) Die Allergenwirkung von Fremden
         Stoffen in der Lebensmitteln, Med. u. Ernähr., 1, 99-104

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

    Gaunt, I. F. et al. (1967) Acute (mouse and rat) and short-term (rat)
         toxicity studies on Ponceau 4R, Food Cosmet. Toxicol., 5,
         187-194

    Gaunt, I. F. et al. (1969) Short-term toxicity on Ponceau 4R in the
         pig, Food Cosmet. Toxicol., 7, 443-449

    Kada, T., Tutikawa, K. & Sadate, Y. (1972) In vitro and host-
         mediated "rec-assay" procedures for screening chemical mutagens
         and phloxine, a mutagenic red dye detected, Mutation Res.,
         16, 165

    Kihara, T., Yasuda, Y. & Tanimura, T. (1977) Effects on pre- and post-
         natal offspring of pregnant rats fed Food Red No. 102, Abstract,
         Teratology, 111-112 (December)

    Larsson, K. S. (1975) A teratologic study with the dyes Amaranth and
         Ponceau 4R in mice, Toxicology, 4, 75-82

    Lück, H. & Rickerl, E. (1960) Food additives and mutative effects. VI
         Report, Z. Lebensmitt.-Untersuch., 112, 157-174

    Mason, P. L. et al. (1974) Long-term toxicity study of Ponceau 4R in
         mice. Unpublished report No. 1/1974, submitted to WHO by the
         British Industrial Biological Research Association

    Meyer, O. & Hansen, E. V. (1975) A study of the embryotoxicity of the
         food colour Ponceau 4R in rats, Toxicology, 5, 201-207

    Mikkelson, H., Larsen, J. C. & Tarding, F. (1978) Hypersensitivity
         reactions to food colours with special reference to the natural
         colour Annatto extract (butter colour), Archs Toxicol., Suppl.
         1, 141

    Phillips, J. C., Bex, C. S. & Gaunt, I. F. (1982) The metabolic
         disposition of 14C-labelled Ponceau 4R in the rat, mouse and
         guinea-pig, Fd Chem. Toxicol., 20, 499-505

    Rapaport, M. J. (1980) Patch testing of colour additives, Contact
         Dermatitis, 6, 231

    Ryan, A. J. & Wright, S. E. (1961) The excretion of some azo dyes in
         rat bile, J. Pharm. Pharmacol., 13, 492-495

    Sako, F. et al. (1980) Cytotoxicity of food dyes on cultured fetal rat
         hepatocytes, Toxicol. Appl. Pharmacol., 54, 285-292

    Stevenson, B. I. et al. (1980) Multigeneration toxicity study in rats
         with Ponceau 4R, Vol. I. Unpublished report No. 1/205/80, from
         the British Industrial Biological Research Association, submitted
         to WHO by the UK Colours Steering Group

    Stevenson, B. I. et al. (1981) Long-term study in rats with Ponceau 4R
         using animals exposed in utero, Vol. I. Unpublished report No.
         293/2/81, from the British Industrial Biological Research
         Association, submitted to WHO by the UK Colours Steering Group

    Walker, R. (1968) Ph.D. Thesis, University of Reading - cited by Gaunt
         et al. (1969)
    


    See Also:
       Toxicological Abbreviations
       Ponceau 4R  (FAO Nutrition Meetings Report Series 46a)
       Ponceau 4R (WHO Food Additives Series 6)
       Ponceau 4R (WHO Food Additives Series 13)
       PONCEAU 4R (JECFA Evaluation)