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    INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

    WORLD HEALTH ORGANIZATION



    TOXICOLOGICAL EVALUATION OF SOME
    FOOD COLOURS, EMULSIFIERS, STABILIZERS,
    ANTI-CAKING AGENTS AND CERTAIN
    OTHER SUBSTANCES



    FAO Nutrition Meetings Report Series 
    No. 46A WHO/FOOD ADD/70.36




    The content of this document is the result of the deliberations of the
    Joint FAO/WHO Expert Committee on Food Additives which met in Rome,
    27 May - 4 June 19691





    Food and Agriculture Organization of the United Nations

    World Health Organization



                   
    1 Thirteenth report of the Joint FAO/WHO Expert Committee on Food
    Additives, FAO Nutrition Meetings Report Series, in press;
    Wld Hlth Org. techn.  Rep. Ser., in press.


    ERYTHROSINE

    Biological Data

    Biochemical aspects

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

    This colour was found to be largely excreted in the faeces by rats
    (55-72 per cent.) 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 per cent.) 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 per cent. 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
    disfunction 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 15-20 days, followed by a sharp decline in iodine levels in the
    next 10 days with gradual return to the initial value in three months
    (Andersen et al., 1964).

    Large doses of erythrosine labelled with I131 given orally to rats
    inhibited uptake of I131 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
    electro-chemical reduction in the can) (Dickinson & Raven, 1962).

    It was found that this colour in a concentration of 200-400 mg/litre,
    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
    (Waliszewski, 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

    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, 1960).

    Acute toxicity

                                                                   

    Animal    Route         LD50              Reference
                       mg/kg body weight 
                                                                   

    Mouse     I.v.            370             Waliszewski, 1952
                                              DFG, 1957

              I.p.            300             DFG, 1957

    Rat       Oral         >2 000             DFG, 1957
                                              Lu & Lavallée, 1964 
                                              USFDA, 1969

    Rabbit    I.v.            200             DPG, 1957

    Gerbil    Oral          1 930             USFDA, 1969
                                                                   

    A group of five young rats were given subcutaneous injections twice
    daily 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 body weight each injection. No oestrogenic activity (normal uterine
    weight) was detected (Graham & Allmark, 1959).

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

    Short-term studies

    Rat: 1 ml of a 0.8 per cent. solution, when injected subcutaneously
    twice weekly did not produce any changes likely to lead to sarcomata
    formation (Grasso & Golberg, 1966).

    Dog: Two-year feeding studies were conducted with three male and
    three female beagles at levels of 0, 0.5, 1.0 and 2.0 per cent. All
    dogs survived the study. No gross or microscopic pathology related to
    the colour was seen (USFDA, 1969).

    Long-term studies

    Mouse: 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 dimethylaminoasobenzene 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 one and two per cent. 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).

    Rat: 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 five per cent. level,
    and those above 0.5 per cent. 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 five per
    cent. level. There was no difference in survival (USFDA, 1964). Forty
    rats were fed one per cent. of the colour for two years. Six animals
    died between time. No tumours were found (DFG, 1957).

    The colour was fed at a level of four per cent. 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 100-day-old rats, 50/test level and 100/control, evenly
    divided by sex, were fed 0, 0.5, 1.0, 2.0 and 4.0 per cent.
    erythrosine for 24 months. Groups of 15 male and 15 female 100-day-old
    rats were intubated twice a week for 19 months with erythrosine at 0,
    100, 235, 750 and 1500 mg/kg. Body weight decreases were seen at two
    and four per cent. Elevated PBIs, due to interference by erythrosine
    in PSI determination rather than thyroid dysfunction, were seen. There
    were no other haematological differences. No adverse gross pathology
    was noted; histopathology has not been completed (USFDA, 1969).

    Feeding with one per cent, 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 subjected to weekly subcutaneous injections of 1 ml
    of a five per cent. 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 1 ml, of a two per cent. or
    three per cent. solution per week for periods of 94-99 weeks. No
    tumours either at the site of the injections or in the other parts of
    the body were observed (Nelson & Hagan, 1953).

    Gerbils: Groups of gerbils, 30/test level and 60/control, evenly
    divided by sex, were fed erythrosine at 0, 1.0, 2.0 and 4.0 per cent.
    for 24 months. Groups of gerbils, 40/test level and 60/control, evenly
    divided by sex, were intubated 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 one per cent.). Elevated PBIs, due to interference by
    erythrosine with PSI determination, were seen. No other haematological
    differences were seen. No adverse gross pathology was noted.
    Histopathology has not been completed (USFDA, 1969).

    Comments

    The long-term studies in two species are adequate. Most of the colour
    is excreted in the faeces and some of the absorbed colour is excreted
    via the bile. Elevation of protein-bound iodine levels has been
    observed although no toxicological significance in relation to thyroid
    activity can be assigned to this observation at present. Conversion to
    fluorescein, a nephrotoxic compound is possible and should be avoided.

    EVALUATION

    Level causing no toxicological effect in the rat

    0.5 per cent. (= 5000 ppm) in the diet equivalent to 250 mg/kg body
    weight per day.

    Estimate of acceptable daily intake for man

                                          mg/kg body weight

    Temporary acceptance                        0-1.25

    Further work required by June 1972

    Studies on the metabolism in several species and preferably in man and
    elucidation of the mechanism underlying the effect of this colour on
    plasmabound iodine levels.

    REFERENCES

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

    BIBRA (1967) Fd Cosmet. Toxicol., 5, 109

    Bowie, W. C., Wallace, W. C. & Lindstrom, H. V. (1966) Fed. Proc.,
    25,  556

    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, 144

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

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

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

    Lück, H., Wallnöfer, P. & Bach, H. (1963) Path. et Microbial.
    (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

    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 (WHO Food Additives Series 6)
       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)