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.



         This compound has been evaluated for acceptable daily intake by
    the Joint FAO/WHO Expert Committee on Food Additives (see Annex 1,
    Ref. Nos. 10 and 20) in 1964 and 1969.

         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.



         The metabolic behaviour and excretory pattern for erythrosine
    have been studied in adult rats. The colour was given by stomach tube
    in log-spaced doses from 0.5-500 mg per kg bw. In five days the
    recovery in the excreta was 102%. After an intravenous application of
    3 mg per kg bw the urine and bile for the initial two to four hours
    was collected, an average of 55% (50.4-58.0%) of the administered
    quantity was found in the bile. In the urine 1.3% (0.8-1.8%). No
    glucuronic acid conjugation is found.

         This colour was found to be largely excreted in the faeces by
    rats (55-72%) and despite the presence of two groups capable of
    undergoing conjugations, no colour could be identified in the urine. A
    small amount of the colour (0.4-1.7%) was excreted in the bile
    (Daniel, 1962).

         Consideration has been given to the possibility that iodide may
    be liberated from erythrosine and may disturb thyroid function. In the
    rat, erythrosine is metabolically stable and 100% of the amount is
    ingested and excreted with its iodine content intact after
    administration of 500 mg/kg (Webb et al., 1962). Protein-bound and
    total blood iodine levels were elevated in rats given erythrosine by
    stomach tube twice weekly in a chronic study (Bowie et al., 1966).
    However the elevated PBIs (protein-bound iodine) were due to
    interference by erythrosine in PBI determinations rather than thyroid
    dysfunction in rats and gerbils (USFDA, 1969). In man, oral
    administration of 16 mg of erythrosine daily for 10 days resulted in
    an increase of protein-bound iodine in the serum from 6-11 µg/100 ml
    after 15 to 20 days, followed by a sharp decline in iodine levels in
    the next 10 days with gradual return to the initial value in three
    months (Anderson et al., 1964). Erythrosine could be an adventitious
    source of iodide (Vought et al., 1972).

         Large doses of erythrosine labelled with l131 given orally to
    rats inhibited uptake of l131 by the thyroid of treated animals. Daily
    doses over 1 mg are necessary for this effect (Marignan et al., 1965).

         When cherries coloured with erythrosine are stored in plain cans,
    fluorescein is readily formed by interaction of the tin-iron couple
    present. This does not occur in lacquered cans. The production of
    fluorescein (with 4 I atoms) from erythrosine occurs in presence of
    metallic iron and/or tin and free organic acid (result of
    electrochemical reduction in the can) (Dickinson & Raven, 1962).

         It was found that this colour in a concentration of 200-400 mg/l,
    inhibited the action of pepsin but had no effect on lipase activity
    (Diemair & Hausser, 1951).

         It was also found that this colour had in vivo as well as
    in vitro haemolytic effect. In the in vivo studies the mouse was
    used (Waliszowski, 1952).

         Erythrosine was administered to rats in doses of 5, 10, 15 and
    50 mg per rat weighing 200-250 g, twice weekly for six months.
    Haemoglobin was reduced at three months, also the red cell count. The
    cholesterol levels of males were depressed. Excretion of the dye was
    mainly in the faeces and predominantly unchanged (Bowie et al., 1966).


    Special studies on mutagenicity

         This colour was tested for mutagenic activity and showed a very
    slight but statistically significant mutagenic effect on Escherichia
    coli in concentrations of 0.5 g/100 ml. It was found that xanthene
    molecule itself was the causative factor and that the substituent
    groups only modify the effect (Lück et al., 1963; Lück & Rickerl,

    Special studies on reproduction


         Groups of 20 female and 10 male rats were placed on dietary
    levels of 0, 1.25, 12.5, 37.5 and 125.0 mg/kg of erythrosine and used
    to carry out a three-generation reproduction study. No adverse effects
    were noted upon fertility, litter size, litter viability or post-
    partum development (Anonymous, 1974).

    Special studies on teratogenicity


         Groups of 15 to 19 pregnant rats were given orally 25, 85 and
    250 mg/kg per day by gavage from days 6 to 15 of gestation and were
    sacrificed on day 20. Control groups of 16 to 17 pregnant rats and
    positive control groups of 21 rats (given 200 mg sodium salicylate/
    kg/day) were used. Body weight, mortality of dams and general
    appearance showed no adverse effects. There were no specific effects
    due to colour on fetal mortality, fetal weight, external and internal
    fetal development and skeletal development. The species was sensitive
    to salicylate (Anonymous, 1972a).


         Groups of 12 pregnant does were given orally in gelatin capsules
    12.5, 40 or 125 mg/kg bw of colour from day 6 to 18 and were
    sacrificed on day 29. 10-14 does acted as controls and 12-14 does as
    positive control given thalidomide. No adverse effects were seen due
    to the colour on body weight gain, mortality of dams, fetal survival,
    fetal body weights, internal and external fetal development and
    skeletal development. Thalidomide gave the expected result (Anonymous,

    Acute toxicity

    Animal      Route     mg/kg bw     Reference

    Mouse       Oral         6 800     BIBRA, 1973
                i.v            370     Waliszewski, 1952, DFG, 1957

    Rat         i.p.           300     DFG, 1957
                Oral           360     BIBRA, 1973
                           > 2 000     DFG, 1957
                             7 100     BIBRA, 1973
                             1 895     Lu. & Lavallee, 1964

                             1 840     Hansen et al., 1973
    Rabbit      i.p.           350     BIBRA, 1973
                i.v.           200     DFG, 1957

    Gerbil      Oral         1 930     USFDA, 1969

         A group of five young rats were given subcutaneous injections
    twice dally for three days. The rats were killed on the fourth day.
    The colour was administered in aqueous solution at a level of 250 mg
    per kg bw each injection. No oestrogenic activity (normal uterine
    weight) was detected (Graham & Allmark, 1959).


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

    Short-term studies


         In a 90-day study on five groups of 15 male and 15 female rats
    erythrosine was given in the diets at 0.25%, 0.5%, 1% and 2%. No
    adverse effects were noted as regards body weight, food intake,
    haematology, blood and urine analysis which were related to
    administration of test substance. Organ weights were normal except
    that absolute and relative caecal enlargement was seen at all levels
    tested. It was dose-related but histology was normal. Absolute and
    relative thyroid weight was increased at the 2% level. Histopathology
    showed no abnormalities except pigment deposition in renal tubules in
    females only at the 2% level but in males at all levels again in a
    dose-related manner. The pigment was identified as protein-bound
    erythrosine. In addition total PBI was raised at all levels in a dose-
    related manner, protein-bound erythrosine in serum behaved similarly
    and non-protein bound iodine also increased with dose levels.
    Thyroxine iodine however remained unchanged and 1131 uptake was
    reduced (BIBRA, 1973).


         Four groups of three male and three female pigs were given 0,
    167, 500 and 1500 mg/kg/day in their diet for 90 days. No obvious
    adverse effects were noted but detailed reports are not yet available
    (BIBRA, 1973).

    Long-term studies


         A total of 122 male and female mice produced by mixed breeding
    from five different strains were given a diet containing 1 mg per
    animal per day of the colour. Mice at the age of 50-100 days were
    used. A number of the animals were sacrificed after an observation
    period of 500 days and the survivors of the rest after 700 days. A
    total of 168 mice was used as control. Positive control groups which
    were given O-aminoazotoluene and dimethylaminoazobenzene were also

    included. In these groups the formation of liver tumours was noted
    after approximately 200 days. The incidence of tumours in mice
    receiving the colour was not significantly greater than in the
    controls (Waterman & Lignac, 1958). Chronic feeding studies were
    conducted with mice. Seventy mice were fed at 1 and 2%. Because of the
    small number of animals surviving the experiment and the small number
    of tumours found, no effect of tumour formation could be attributed to
    the colour (USFDA, 1964).


         Groups of 24 weanling rats, evenly divided by sex, were fed this
    colour at 0, 0.5, 1.0, 2.0 and 5.0% for two years. Slight growth
    depression was observed in the animals at the 5% level, and those
    above 0.5% had distended caecums but microscopically the distended
    caecums showed normal histology. The statistical evaluation of the rat
    study revealed no changes in organ weights at the highest level. There
    was some diarrhoea at the 5% level. There was no difference in
    survival (USFDA, 1964). Forty rats were fed 1% of the colour for two
    years. Six animals died between time. No tumours were found (DFG,

         The colour was fed at a level of 4% of the diet to five male and
    five female rats for periods up to 18 months. Gross staining was
    observed in the glandular stomach and small intestine and granular
    deposits in the stomach, small intestine and colon. Hepatic cirrhosis
    was noted in one case out of four rats living up to 12 months. Fifty
    control animals observed for 20 months or more failed to develop
    tumours, or hepatic cirrhosis (Willheim & Ivy, 1953).

         Groups of 12 male and 12 female weanling Osborne-Mendel rats were
    fed 0, 0.5, 1.0, 2.0 and 5.0% erythrosine in their diet for two years.
    Growth depression was observed in rats given 5%. The relative spleen
    weight was depressed in all male test groups and in females at the 5%
    level. Slight caecal enlargement was noted at 1% and increased with
    dose but the histology of the enlarged caeca was normal. No other
    gross or histopathological findings related to colour administration
    were noted (Hansen et al., 1973).

         Groups of 25 male and 25 female 100-day-old rats and a group of
    50 male and 50 female controls were fed 0, 0.5, 1.0, 2.0 and 4.0%
    erythrosine in their diet for 86 weeks. Other groups of 25 male and 25
    female rats aged 100 days were intubated twice a week for 85 weeks
    with erythrosine at 0, 100, 235, 750 and 1500 mg/kg bw. After this
    treatment animals were kept on normal diets until two years. Body
    weight decreases were seen at 2 and 4%. Elevated PBI, due to
    interference by erythrosine with PBI determination rather than thyroid
    dysfunction, were seen. Thyroxine-iodine levels were not affected.
    There were no other haematological differences and no anaemia was
    seen. No adverse gross pathology was noted; histopathology had not
    shown any colour related abnormalities (Hansen et al., 1973a).

         Feeding with 1% of the colour for two years did not influence the
    body weight or fertility of Wistar rats. There was no pathological
    damage and no significant difference in tumour incidence from control
    rats (Oettel et al., 1965).

         Twenty rats were subject to weekly subcutaneous injections of
    1 ml of a 5% aqueous solution for 596 days. The total quantity of
    colour administered was 2.65 g/animal. Seven rats survived 300 days or
    more. No tumours were observed (Umeda, 1956).

         Eighteen rats were injected subcutaneously with aqueous solutions
    of erythrosine at 12 mg/animal once per week for two years. No tumours
    either at the injection sites or in other parts of the body were
    observed (Hansen et al., 1973).


         Groups of gerbils, 30/test and 60/control, evenly divided by sex
    twice a week for 19 months with 0, 200, 750 and 900 mg/kg (those on
    900 mg received 1200 mg for the first three months). Body weight
    decreases were seen at all feeding levels (only females at 1%).
    Elevated PBIs, due to interference by erythrosine with PBI
    determination, were seen. No other haematological differences were
    seen. No adverse gross pathology was noted. Histopathology has not
    been completed (USFDA, 1969).


         Two-year feeding studies were conducted with groups of three male
    and three female beagles at levels of 0, 0.5, 1.0 and 2.0% in the
    diet. All dogs survived the study. No gross or microscopic pathology
    related to colour administration was seen (Hansen et al., 1973).


         There are adequate long-term studies on several species available
    as well as some information.

         The observed elevation of PBI levels does not appear to have any
    toxicological significance in relation to the thyroid activity. More
    extensive metabolic studies in other species preferably including man
    and of the mechanism underlying the effect on plasma-bound iodine are


    Level causing no toxicological effect

         Rat: 0.5% (= 5000 ppm) in the diet equivalent to 250 mg/kg bw.

    Estimate of acceptable daily intake for man

         0-2.5 mg/kg bw



         Metabolic studies, preferably including man.


    Anderson, C. J., Keiding, N. R. & Nicholson, A. B. (1964) Scand.
          J. Clin. Lab. Inrest., 16, 549

    Anonymous (1972a) Industrial Biotest Laboratory, Unpublished report
         submitted to WHO

    Anonymous (1972b) Industrial Biotest Laboratory, Unpublished report
         submitted to WHO

    Anonymous (1974) Industrial Biotest Laboratories, Unpublished report
         submitted to WHO

    Bär, F. & Griepentrog, F. (1960) Med. u. Ernahr, 1, 99

    BIBRA (1967) Fd Cosmet. Toxicol., 5, 100

    Bowie, W. C., Wallace, W. C. & Lindstrom, H. V. (1966) Fed. Proc., 25,

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

    Deutsche Forschungsgemeinschaft, Farbstoff Kommission (1957)
         Mitteilung 6

    Dickinson, D. & Raven, T. W. (1962) J. Sci. Food Agric., 13, 650

    Diemair, W. & Hausser, H. (1951) Z. Lebensmitt-Untersuch., 92, 1965

    Graham, R. C. B. & Allmark, M. G. (1959) Toxicol. appl. Pharmacol., 1,

    Grasso, P. & Golberg, L. (1966) Fd. Cosmet. Toxicol., 4, 269

    Hansen, W. H. et al. (1973) Fd. Cosmet. Toxicol., 11(4), 527

    Hansen, W. H. et al. (1973a) Fd. Cosmet. Toxicol., 11(4), 535

    Lu, F. C. & Lavallee, A. (1964) Canad. pharm. J., 97, 30

    Lück, H. & Rickerl, E. (1960) Z. Lebensmitt-Untersuch., 122, 157

    Lück, H., Wallnofer, P. & Bach, H. (1963) Path. et Microbiol. (Basel),
         26, 206

    Marignan, R., Boucard, M. & Gelis, C. (1965) Trav. Soc. Pharm.
         Montpellier, 24, 127

    Nelson, A. A. & Hagan, E. C. (1953) Fed. Proc., 12, 397

    Umeda, M. (1956) Gann, 47, 51

    United States Food and Drug Administration (1969) Unpublished report

    Vought, R. L., Brown, F. A. & Wolff, J. (1972) J. Clin. Endocr.
         Metal., 34, 747

    Waliszewski, T. (1952) Acta Pol. pharm., 9, 127

    Waterman, N. & Lignac, G. O. E., as summarized by Genderen, H. van
         (1958) Acta Physiol. pharmacol, neerl., 7, 35

    Webb, J. M., Fonda, M. & Brouwer, E. A. (1962) J. Pharmacol. exp.
         Ther., 137, 141

    Willheim, R. & Ivy, A. C. (1953) Gastroenterology, 23, 1

    See Also:
       Toxicological Abbreviations
       Erythrosine  (FAO Nutrition Meetings Report Series 46a)
       Erythrosine (WHO Food Additives Series 19)
       Erythrosine (WHO Food Additives Series 21)
       Erythrosine (WHO Food Additives Series 24)
       Erythrosine (WHO Food Additives Series 28)
       Erythrosine (WHO Food Additives Series 44)
       ERYTHROSINE (JECFA Evaluation)