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    WHO Food Additives Series, 1972, No. 4




    EVALUATION OF MERCURY, LEAD, CADMIUM
    AND THE FOOD ADDITIVES AMARANTH,
    DIETHYLPYROCARBONATE, AND OCTYL GALLATE




    The evaluations contained in this publication were prepared by the
    Joint FAO/WHO Expert Committee on Food Additives which met in Geneva,
    4-12 April 19721






    World Health Organization
    Geneva
    1972





                   

    1 Sixteenth Report of the Joint FAO/WHO Expert Committee on Food
    Additives, Wld Hlth Org. techn. Rep. Ser., 1972, No. 505; FAO
    Nutrition Meetings Report Series, 1972, No. 51.

    AMARANTH

    Biological data

    Biochemical aspects

    Adult rats were given intravenous injections of the colour.  The bile
    was collected for six hours and an average of 53% (43-79%) of the
    quantity of the colour administered was found (Ryan & Wright, 1961).

    Six adult rats were given a single oral dose of 100 mg as aqueous
    solution per animal.  Only 0.45% of the dose administered was found in
    the faeces collected over a period of 48 hours.  A single oral dose of
    50 mg per animal was administered to four rats.  Only 2.8% was
    absorbed from the gastro-intestinal tract; the metabolites in the
    urine and bile were predominantly products resulting from the
    reductive fission of the azo-linkage, such as 1-amino4-naphthalene
    sulfonic acid and 1-amino-2-hydroxy-3, 6-naphthalene disulfonic acid.
    The former compound was found also in the faeces.  The liver enzyme
    that reduces azo-linkages plays little part in the metabolism, as was
    shown in experiments in which the colour was given by intrasplenic
    infusion.  Reduction of the compound is therefore most probably
    effected by the intestinal bacteria (Radomski & Mellinger, 1962).

    After a prolonged administration of a daily dose of 50 mg/rat the
    vitamin A content of the liver showed a threefold to fourfold
    decrease. The glutathione content of the liver and spleen was
    increased (Galea et al., 1962).

    Acute toxicity

                                                                     

    Animal    Route          LD50             References
                        per kg body-weight
                                                                     

    Rat       I.p.           1 g              Deutsche Forsch., 1957

    Rat       I.v.           1 g              Deutsche Forsch., 1957
                                                                     

    Special studies

    The colour was tested for mutagenic action in a concentration of
    0.5 g/100 mi in cultures of Escherichia coli.  No mutagenic effect
    was found (Rück & Rickerl, 1960).

    Short-term studies

    Rat

    A group of five young rats were given the colour subcutaneously 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 estrogenic activity (normal uterine
    weight) was detected in comparison with a control group.  No other
    abnormalities were found (Graham & Allmark, 1959).

    Guinea-pig

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

    Cat

    The test for Heinz bodies was negative after injecting four cats with
    a 5% aqueous solution of this colour at a level of 1 g on the first
    day and 0.1 g on the ninth and on the eighteenth day. (Deutsche
    Forsch., 1957).

    Long-term studies

    Mouse

    Twenty mice were fed 15-20 mg of this colour five days a week for
    periods up to 477 days.  Autopsies were conducted on 18 of the mice.
    No lesions were observed in the one liver examined (Cook et al.,
    1940).

    Feeding studies were-conducted with C3Hf and C57Bl mice; 100 of each
    strain were fed at 1.0 and 2.0% and 200 of each strain served as
    controls.  No tumours were observed in the mice of either strain
    (U.S., FDA, 1964).

    Two groups of 100 mice received either 0 or 0.01 g amaranth paste 
    (= 0.004 g amaranth) by gavage daily.  One drop of either
    9,10-dimethyl-2-benzanthracene or 3,4 benzypyrene was applied once per
    week to interscapular skin.  Papillomata appeared 3.5 weeks earlier in
    test animals and in a greater number of animals.  A larger percentage
    became malignant in test animals (Baigusheva, 1968).

    Three groups of 15 male and 15 female rats were given diets containing
    0.03%, 0.3% and 1.5% of the colour for 64 weeks.  The mortality of the
    rats was the same as that in a similar control group.  At a level of
    1.5% a significant decrease in growth rate was found in female rats
    but not in male rats.  This was attributed to an effect on food
    utilization rather than on food consumption.  Female rats fed the
    colour at 0.3% and 1.5% showed an increase in liver weight.  At the
    higher concentration there was also an increase in kidney weight.  No

    influence on food intake, histo pathology and blood-picture and no
    significant difference in tumour incidence was found (Mannel et al.,
    1958).

    Ten rats were fed the colour at a level of 0.2% in the diet.  Each
    animal received an average of 0.1 g/kg body-weight per day for 417
    days.  The total intake of the colour was 11 g/animal.  The
    observation period was 830 days.  One intestinal carcinoma was
    observed (Deutsche Forsch., 1957).

    No tumours were noted in 11 rats subjected to the subcutaneous
    injection of 0.5 ml of 1% solution twice weekly for 365 days.  The
    observation period was 879 days.  The total dose administered was 
    0.5 g/animal (Deutsche Forsch., 1957).

    At a level of 11% in the diet the colour was fed to five male and five
    female rats for periods up to 18 months.  Gross staining of the
    glandular stomach and small intestine was observed.  Granular deposits
    were noted in the stomach, small intestine and in some cases in the
    colon.  A lymphosarcoma was observed.  No tumours occurred in 50
    control animals surviving 20 months or more (Willheim & Ivy, 1953).

    This colour was injected subcutaneously into 18 rats of both sexes for
    94 to 99 weeks.  In general 1 ml of a 2-3% solution was injected
    weekly for 693 days.  No tumours were observed (Nelson & Hagan, 1953).

    Groups of 24 weanling rats were fed the colour of 0.5%, 1.0%, 2.0% and
    5.0% in the diet.  A similar group served as controls.  The animals on
    5.0% showed slight growth inhibition.  Gross and microscopic
    examinations revealed a questionable increase in the number of mammary
    tumours; two tumours were observed in the control group and 3, 3, 6
    and 4 tumours respectively in the groups receiving the colour.
    Additional feeding studies for two years were carried out to repeat
    this result with Osborne-Mendel and Spragul-Dawley rats at 0.0, 1.0,
    and 2.0% with 100 animals of each strain.  There was no statistically
    significant influence on the formation of tumours (U.S., FDA, 1964).

    One group of 50 outbred rats was fed a diet containing amaranth paste
    (40% amaranth) for 25 months.  A control group of 35 rats was used.
    Dietary levels ranged from 0.8% to 1.6% amaranth.  Tumours of the
    peritoneum and intestine began to appear in 18 survivors at 19 months.
    By 25 months a total of 11 tumours was found.  No tumours were seen in
    controls.  All tumours were histologically malignant (Baigusheva,
    1968).

    Two groups of 50 outbred male rats were fed on a diet containing
    either 0% or 2% amaranth for 33 months, that is until all animals
    died.  A small depression of body-weight, compared with controls was
    observed but this was not statistically significant.  The 15 tumours
    found (in 13 rats) included 3 lymphosarcomas, 4 sarcomas, 1
    adenofibroma, 3 intestinal carcinomas, 1 hepatoma and 3 skin
    carcinomas.  The first tumour appeared after six months, most of the
    others at 21-23 months.  The author reported that not one of the 50

    control rats developed any kind of tumour during the whole course of
    the experiment (Andrianova, 1970).

    Dog

    A seven year toxicity study was carried out on female beagles. Five
    dogs were fed 2% of the colour in the diet, three dogs were used as
    controls.  No histopathological or other abnormalities were found
    (U.S., FDA, 1964).

    Reproduction and teratogenicity studies

    Rat

    Groups of one male and four female rats were fed diets supplying 1.5
    and 15 mg/kg body-weight/day of amaranth.  There were two control
    groups of a similar size.  Each group was mated three times, at 4-5
    months, 7-8 months and 10-12 months after the start of the study.  The
    following were studied: female fertility, gestation period, numbers of
    live and dead pups born and numbers surviving for four days and one
    month.  Abnormalities in the pups were noted.  Similar observations
    were carried out on first (F1) and second (F2) generations of rats
    fed on the same diets as their parents.  The authors considered that
    their results showed amaranth decreased fertility, increased the
    number of stillbirths, produced deformities in the young and reduced
    survival of the young (Shtenberg & Gavrilenko, 1971).

    Groups of 13-15 pregnant rats were given 0, 7.5, 15, 30, 100 or 200
    mg/kg/day amaranth by stomach tube from day 0-19 and sacrificed on day
    20.  No adverse effects were noted on implantation.  The percentage of
    dead foetuses increased in a dose-related manner.  At 200 mg/kg/day
    there was a foetotoxic effect.  Resorptions increased from 15 mg/kg
    upwards with total resorption of occasional litters at 100 and 200
    mg/kg/day.  No teratogenic abnormality related to amaranth was seen.
    No obvious effect was notod on foetal sex (Collins & McLaughlin,
    1972).

    Eight groups of 16-22 pregnant rats were given 0, 15, 50 or 150
    mg/kg/day of amaranth by stomach tube on days 6-15 of pregnancy and
    Caesarean section was performed on day 20.  No compound-related
    adverse effects were noted in terms of implantation, foetal mortality,
    foetal weight or reproductive performance in any of the experimental
    groups as compared to the control animals.  No evidence of
    compound-induced teratogenic effects was noted (Industrial BioTest
    Laboratories, 1972a).

    In another study five groups of four pregnant Charles River rats
    received from gestation day 6-15 inclusive, 0, 15, 150, 450 and 1500
    mg/kg body-weight amaranth per day by gavage.  Females were killed on
    day 20.  No abnormal effects were seen as regards maternal weight
    gain, litter size, average foetal weight and number of resorptions. 
    No gross abnormalities related to the compound were seen (Burnett,
    1972).

    Rabbit

    Eight groups of 10-14 pregnant rabbits were given 0, 1.5, 5.0 and 15.0
    mg/kg/day of amaranth by capsule on days 6-16 of pregnancy and
    sacrificed on day 29 by Caesarean section.  No evidence of
    compound-induced teratogenic effects were noted, nor were significant
    effects seen in terms of implantation, pup weight, young born alive
    and total numbers of resorptions.  Average numbers of early
    resorptions per litter revealed an increase which approaches
    statistical significance (P =.05) for the 1.5 and 15.0 mg/kg groups
    while for the 5.0 mg/kg group the increase is significant (Industrial
    Bio-Test Laboratories, 1972b).

    Comments on experimental studies reported

    Several long-term studies have been carried out in rats but only two
    studies extended over their lifespan.  Long-term studies have also
    been done in mice and dogs.  Many aspects of the newer studies were
    difficult to interpret particuarly with regard to the carcinogenic
    potential of amaranth and its adverse effects on the rat foetus and
    reproduction.  However, the most recent studies gave somewhat
    conflicting results with regard to feototoxicity although none of them
    produced any evidence of teratogenic effects related to amaranth
    administration.  It was not possible to establish the reason for this
    difference as there were divergencies in the conduct of these tests in
    relation to time and route of administration.  Considerable
    controversy still exists over the correct route of administration in
    teratogenicity tests and there is evidence that variations in this
    parameter could lead to observable effects which are not
    compound-related.  Such factors made the interpretation of the
    experimental data very difficult.  The teratogenic study in rabbits
    was similarly not clear-cut in its results.

    EVALUATION

    The new information from reproduction and other studies was considered
    in conjunction with the fact that further relevant studies are in
    progress at present.  The Committee was unable to make a final
    re-evaluation of the colour at this stage.  However, the unconditional
    ADI previously established has been changed to a temporary ADI, with
    appropriate adjustment.

                                                      mg/kg body-weight

    Temporary acceptable daily intake for man              0-0.75

    Further work required

    Results of various studies now in progress to be submitted within
    three years.

    REFERENCES

    Andrianova, M. M. (1970) Vop. Pitan., 29 (5), 61

    Baigusheva, M. M. (1968) Vop. Pitan., 27 (4), 46

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

    Burnett, C. (1972) Unpublished report submitted to WHO

    Collies, T. F. X. & McLaughlin, J. R. (1972) Unpublished report to
    FDA submitted to WHO

    Cook, J. W. et al. (1940) Amer. J. Cancer, 40, 62

    Deutsche Forschungsgemeinschaft, Bad-Godesberg, Federal Republic of
    Germany, Farbstoff Kommission (1957) Mitteilung 6

    Galea, V., Ariesan, M. & Luputiu, G. (1962) Parmacia (Buc), 10, 531

    Graham, R. C. B. & Allmark, M. G. (1959) Toxicol. appl. Pharmacol., 1,
    144

    Industrial Biotest Labs. Inc. (1972a) Report No. B700 submitted to WHO

    Industrial Biotest Labs. Inc. (1972b) Report No. J701 submitted to WHO

    Mannel, W. A. et al. (1958) J. Pharm. Pharmacol., 10, 625

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

    Radomski, J. L. & Mellinger, T. J. (1962) J. Pharmacol. exp. Ther.,
    136, 259

    Rück, H. & Rickerl, E. (1960) Z. Lebensm.-Untersuch., 112, 157

    Ryan, A. J. & Wright, S. E. (1961) J. Pharm. Pharmacol., 13, 492

    Shtenberg, A. I. & Gavrilenko, E. V. (1970) Vop. Pitan., 29 (2), 66

    U.S. Food and Drug Administration, Unpublished report submitted to
    WHO, April 1964

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


    See Also:
       Toxicological Abbreviations
       Amaranth (WHO Food Additives Series 8)
       Amaranth (WHO Food Additives Series 13)
       Amaranth (WHO Food Additives Series 19)
       AMARANTH (JECFA Evaluation)
       Amaranth (IARC Summary & Evaluation, Volume 8, 1975)