Toxicological evaluation of some food
    additives including anticaking agents,
    antimicrobials, antioxidants, emulsifiers
    and thickening agents


    The evaluations contained in this publication
    were prepared by the Joint FAO/WHO Expert
    Committee on Food Additives which met in Geneva,
    25 June - 4 July 19731

    World Health Organization


    1    Seventeenth Report of the Joint FAO/WHO Expert Committee on
    Food Additives, Wld Hlth Org. techn. Rep. Ser., 1974, No. 539;
    FAO Nutrition Meetings Report Series, 1974, No. 53.



         These compounds have been evaluated for acceptable daily intake
    by the Joint FAO WHO Expert Committee on Food Additives (see Annex 1,
    Ref. No. 13) in 1965.

         The previously published monograph has been revised and is
    reproduced in its entirety below.



         14C-labelled CaNa2EDTA, when fed to rats in doses of 50 mg kg
    bw,  was absorbed only to an extent of 2 to 4%; 80 to 90% of the dose
    appeared in the faeces within 24 hours, and absorption was still
    apparent at 48 hours. At the low pH of the stomach the calcium chelate
    is dissociated with subsequent precipitation of the free acid and this
    is only slowly redissolved in the intestines (Foreman et al., 1953).

         Experiments in man also revealed poor absorption; only 2.5% of a
    3 g dose given was excreted in the urine (Srbova & Teisinger, 1957).
    These authors also confirmed the dissociation of the calcium chelate
    in the stomach. When 200 mg CaNa2EDTA was introduced into the
    duodenum of rats the authors found an absorption rate of 6.5 to 26%. 
    A dose of 1.5 mg of 14C-labelled CaNa2EDTA given in a gelatine
    capsule to normal healthy men was absorbed to an extent of 5% (Foreman
    & Trujillo, 1954).

         In feeding experiments, in rats receiving disodium EDTA at
    dietary levels of 0.5, 1.0 and 5.0%, the faeces contained 99.4, 98.2
    and 97.5% of the excreted material (Yang, 1964).

         Similar experiments conducted also in rats gave essentially the
    same results. Thirty-two hours after a single dose of 95 mg disodium
    EDTA/rat, 93% was recovered from the colon. After doses of 47.5, 95.0
    and 142.5 mg disodium EDTA the amount of EDTA recovered in the urine
    was directly proportional to the dose given, suggesting that EDTA was
    absorbed from the gastrointestinal tract by passive diffusion. The
    motility of the intestine was not affected by the compound (Chan,

         After parenteral administration to rats, 95 to 98% of injected
    14C-labelled CaNa2EDTA appeared in the urine within six hours. All
    the material passed through the body unchanged. Peak plasma levels
    were found approximately 50 minutes after administration. Less than

    0.1% of the material was oxidized to 14CO2, and no organs
    concentrated the substance. After i.v. injection, CaNa2EDTA passed
    rapidly out of the vascular system to mix with approximately 90% of
    the body water, but did not pass into the red blood cells and was
    cleared through the kidney by tubular excretion as well as by
    glomerular filtration (Foreman et al., 1953). The same was also found
    in man using 14C-labelled CaNa2EDTA. Three thousand milligrams were
    given i.v. to two subjects and were almost entirely excreted within 12
    to 16 hours (Srbova & Teisinger, 1957).

         The maximum radioactivity in the urine after application of
    14C-labelled CaNa2EDTA to the skin was only 10 ppm (0.001%) (Foreman
    & Trujillo, 1954).

         In biological systems, Ca ion will usually, be most accessible
    o EDTA. In general, zinc seems to be next most accessible. About
    80% of the zinc of liver is freely available to EDTA. The over-all
    availability of the other physiologically important metals is probably
    in the order: Cu >Fe >Mn >Co (Chenoweth, 1961). EDTA removes about
    1.4% of the total iron from ferritin at pH 7.4 to form an iron chelate
    (Westerfield, 1961). Transfer of Fe from Fe-transferrin to EDTA
    in vitro occurs at a rate of less than 1% in 24 hours. In vivo
    studies in rabbits demonstrated transfer of iron only from FeEDTA to
    transferrin and not the reverse. It appeared that tissue iron became
    available to chelating agents including EDTA only when an excess
    of iron was present (Cleton et al., 1963). Equal distribution
    between a mixture of EDTA and siderophilin was obtained only at
    EDTA:siderophilin ratios of 20-25:1 (Rubin, 1961). Human iron
    deficiency anaemia was successfully treated with FeEDTA although 84%
    of labelled FeEDTA was excreted in the faeces and none appeared in the
    urine. Red cells, however, contained labelled Fe and reticulocytosis
    occurred. Since FeEDTA administered i.v. was almost quantitatively
    excreted in the urine, it was concluded that FeEDTA was degraded prior
    to absorption, when given orally (Lapinleimu & Wegelius, 1959).
    Rabbits absorbed about 10% of oral FeEDTA, and the rest was excreted
    in the faeces, while anaemic rats absorbed 50% of 6 mg/kg bw oral
    FeEDTA but only 25% FeSO4 (Rubin & Princiotto, 1960). Addition of 1%
    Na2EDTA to a diet containing more than optimal amounts of iron and
    calcium lowered the absorption and storage of iron in rats and
    increased the amount present in plasma and urine. The metabolism of
    calcium, however, was apparently unaffected (Larsen et al., 1960). A
    diet containing 0.15 mg of iron, 4.26 of calcium and 1 mg of EDTA per
    rat (equivalent to 100 ppm (0.01%) in the diet) for 83 days had no
    influence on calcium and iron metabolism, e.g. the iron content of
    liver and plasma (Hawkins et al., 1962).

         CaNa2EDTA increased the excretion of zinc (Perry & Perry, 1959),
    and was active in increasing the availability of zinc in soybean
    containing diets to poults (Kratzer et al., 1959). CaNa2EDTA enhanced
    the excretion of Co, Hg, Mn, Ni, Pb, Tl and W (Foreman, 1961). The

    treatment of heavy metal poisoning with CaEDTA has become so well
    established that its use for more commonly seen metal poisonings, e.g.
    lead, is no longer reported in the literature (Foreman, 1961). EDTA
    could not prevent the accumulation of 90Sr, 106Ru, 141Ba and 226Ra
    in the skeleton. 91Y, 239Pu and 238U responded fairly well to EDTA,
    the excretion being accelerated (Catsch, 1961).

         EDTA had a lowering effect on serum cholesterol level when given
    orally or i.v. It may have acted by decreasing the capacity of serum
    to transport cholesterol (Gould, 1961). Disodium EDTA had a pyridoxin-
    like effect on the tryptophan metabolism of patients with porphyria or
    scleroderma, due to a partial correction of imbalance of polyvalent
    cations (Lelievre & Batz, 1961).

         In vitro, 0.0033 M EDTA inhibited the respiration of liver
    homogenates and of isolated mitochondria of liver and kidney (Lelievre
    & Batz, 1961). The acetylation of sulfanilamide by a liver extract was
    also inhibited (Lelievre, 1960). EDTA stimulated glucuronide synthesis
    in rat liver, kidney and intestines but inhibited the process in
    guinea-pig liver (Pogell & Leloir, 1961; Miettinen & Leskinen, 1962).
    Of the heavy metal-containing enzymes, EDTA at a concentration of
    about 10-3 M inhibited aldehyde oxidase and homogentisinicase.
    Succinic dehydrogenase, xanthine oxidase, NADH-cytochrome reductase
    and ceruloplasmin (oxidation of p-phenylenediamine) were not inhibited
    (Westerfield, 1961). Disodium EDTA was found to be a strong inhibitor
    for sigma-aminolevulinic acid dehydrogenase, 5.5 × 10-6 M
    causing 50% inhibition (Gibson et al., 1955). The i.p. injection of
    4.2 mmol/kg bw (equivalent to 1722 mg/kg bw) CaNa2EDTA caused in rats
    an inhibition of the alkaline phosphatase of liver, prostate and serum
    up to four days depending on the dose administered; zinc restored the
    activity (Nigrovic, 1964).

         In vitro, EDTA inhibited blood coagulation by chelating Ca2+.
    The complete coagulation inhibition of human blood required
    0.65-1.0 mg/ml. The i.v. injection of 79-200 mg EDTA/rabbit had no
    effect on blood coagulation (Dyckerhoff et al., 1942).

         I.v. injections of Na2EDTA and CaNa2EDTA had some
    pharmacological effect on the blood pressure of cats; 0-20 mg/kg bw
    CaNa2EDTA (as Ca) produce a slight rise; 20-50 mg/kg, a biphasic
    response; and 50 mg/kg, a clear depression (Marquardt & Schumacher,

         One per cent. Na2EDTA enhances the absorption of 14C-labelled
    acidic, neutral and basic compounds (mannitol, inulin, decamethenium,
    sulfanilic acid and EDTA itself) from isolated segments of rat
    intestine, probably due to an increased permeability of the intestinal
    wall (Schanker & Johnson, 1961).


    Special studies on embryotoxicity

         Disodium EDTA injected at levels of 3.4, 1.7 and 0.35 mg/egg gave
    40, 50 and 85% hatch, respectively. At the highest level, some embryos
    which failed to hatch showed anomalies (McLaughlin & Scott, 1964).

    Acute toxicity

    (a)  Disodium EDTA

    Animal         Route       (mg/kg bw)         References

    Rat            oral        2 000-2 200        Yang, 1964

    Rabbit         oral        2 300              Shibata, 1956

                   i.v.        47a                Shibata, 1956

    a    Dose depending on the rate of infusion.

    (b)  Ca-disodium EDTA

    Animal     Route   (mg/kg bw)          References

    Rat        oral    10 000 ± 740        Oser et al,, 1963

    Rabbit     oral    7 000 approx.       Oser et al., 1963

               i.p.    500 approx.         Bauer et al., 1952

    Dog        oral    12 000 approx.      Oser et al., 1963

         The oral LD50 in rats is not affected by the presence of food in
    the stomach or by pre-existing deficiency in Ca, Fe, Cu or Mn (Oser et
    al., 1963).

         Oral doses of over 250 mg/animal cause diarrhoea in rats (Foreman
    et al., 1953).

         There are many reports in the literature on kidney damage by
    parenteral over-dosage of CaEDTA. A review was given by Lachnit
    (1961). Lesions simulating "versene nephrosis" in man have also been
    produced in rats. Disodium EDTA in doses of 400-500 mg i.p. for 21
    days caused severe hydropic degeneration of the proximal convoluted
    tubules of the kidneys. CaNa2EDTA produced only minimal focal
    hydropic changes in 58% of animals, disappearing almost two weeks
    after stopping the injections (Reuber & Schmieller, 1962).

    Short-term studies


         Groups of five male rats received 250 or 500 mg/kg bw CaNa2EDTA
    i.p. daily for three to 21 days and some were observed for an
    additional two weeks. Weight gain was satisfactory and histology of
    lung, thymus, kidney, liver, spleen, adrenal, small gut and heart was
    normal except for mild to moderate renal hydropic change with focal
    subcapsular swelling and proliferation in glomerular loops at the
    500 mg level. There was very slight involvement with complete recovery
    at the 250 mg level. Lesions were not more severe with simultaneous
    cortisone administration (Reuber & Schmieller, 1962).

         Groups of three male and three female rats were fed for four
    months on a low mineral diet containing one-half the usual portion of
    salt mixture (i.e. 1.25% instead of 2.50%) with the addition of 0% and
    1.5% CaNa2EDTA. The test group showed a reduced weight gain, but
    there was no distinct difference in general condition of the animals
    (Yang, 1964).

         In another experiment three groups of eight to 13 male and
    female rats were fed a low-mineral diet containing 0, 0.5 and 1% of
    CaNa2EDTA for 205 days. No significant differences from the controls
    were shown regarding weight gain, mortality, gross pathology of the
    organs and histopathology of liver, kidney and spleen except a very
    slight dilatation of hepatic sinusoids. Blood coagulation time, total
    bone ash and blood calcium level were unaffected. No significant
    erosion of molars was noted. Basal metabolism was in the normal range
    (Chan, 1964).

         Rats were fed for 44 to 52 weeks on a diet containing 0.5%
    disodium EDTA without any deleterious effect on weight gain, appetite,
    activity and appearance (Krum, 1948). In another experiment three
    groups of 10 to 13 males and females were fed a low-mineral diet
    (0.54% Ca and 0.013% Fe) with the addition of 0, 0.5 and 1% disodium
    EDTA for 205 days. At the 1% level some abnormal symptoms were
    observed: growth retardation of the males, lowered erythrocyte and
    leucocyte counts, a prolonged blood coagulation time, slightly but
    significantly raised blood calcium level, a significantly lower ash
    content of the bone, considerable erosion of the molars and diarrhoea.

    Gross and histological examination of the major organs revealed
    nothing abnormal. Rats fed for 220 days on an adequate mineral diet
    containing 1% disodium EDTA showed no evidence of dental erosion
    (Chan, 1964).

         Groups of six rats were maintained for 12 weeks on diets
    containing 0.5, 1 and 5% disodium EDTA. No deaths occurred and there
    were no toxic symptoms except diarrhoea and lowered food consumption
    at the 5% level. Mating in each group was carried out when the animals
    were 100 days old. Mating was repeated 10 days after weaning the first
    litters. Parent generation rats of 0, 0.5 and 1% levels gave birth to
    normal first and second litters. The animals given 5% failed to
    produce litters (Yang, 1964). To elucidate possible teratogenic
    effects, daily doses of 20-40 mg/rat EDTA were injected i.m. into
    pregnant rats at days six to nine, 10 to 15 and 16 to the end of
    pregnancy. A dose of 40 mg was lethal within four days but 20 mg was
    well tolerated, allowing normal fetal development; 40 mg injected
    during days six to eight or 10 to 15 produced some dead or malformed
    fetuses, especially polydactyly, double tail, generalized oedema or
    circumscribed head oedema (Tuchmann-Duplessis & Merciar-Parot, 1956).

         Groups of five male rats were given 250, 400 or 500 mg/kg bw
    disodium EDTA i.p. daily for three to 31 days; some groups were
    observed for another two weeks. At the 500 mg level all rats became
    lethargic and died within nine days, the kidneys being pale and
    swollen, with moderate dilatation of bowel and subserosal
    haemorrhages. Histological examination of a number of organs showed
    lesions only in the kidneys. Animals at the 400 mg level died within
    14 days, kidney and bowel symptoms being similar to the 500 mg level.
    One rat at the 250 mg dose level showed haemorrhage of the thymus. All
    three groups showed varying degrees of hydropic necrosis of the renal
    proximal convoluted tubules with epithelial sloughing: recovery
    occurred in all groups after withdrawal of disodium EDTA (Reuber &
    Schmieller, 1962).


         Eight groups of three rabbits were given either 0.1, 1, 10 or
    20 mg/kg bw disodium EDTA i.v., or 50, 100, 500 or 1000 mg/kg bw
    orally for one month. All animals on the highest oral test level
    exhibited severe diarrhoea and died. In the other groups body weight,
    haemogram, urinary nitrogen and urobilinogen were unaffected.
    Histopathological examination of a number of organs showed
    degenerative changes in the liver, kidney, parathyroid and endocrine
    organs and oedema in muscle, brain and heart at all levels of
    treatment (Shibata, 1956).


         Four groups of one male and three female mongrels were fed diets
    containing 0, 50, 100 and 250 mg/kg bw CaNa2EDTA daily for 12 months.
    All appeared in good health, without significant change in blood
    cells, haemoglobin and urine (pH, albumin, sugar, sediment). Blood
    sugar, non-protein nitrogen and prothrombin time remained normal.
    Radiographs of ribs and of long bones showed no adverse changes at the
    250 mg level. All dogs survived for one year.Gross and microscopic
    findings were normal (Oser et al., 1963).

    Long-term studies


         In a two-year study five groups totalling 33 rats were fed 0,
    0.5, 1 and 5% disodium EDTA. The 5% group showed diarrhoea and
    consumed less food than the rats in other groups. No significant
    effects on weight gain were noted nor were blood coagulation time, red
    blood cell counts or bone ash adversely affected. The mortality of the
    animals could not be correlated with the level of disodium EDTA. The
    highest mortality rate occurred in the control group. Gross and
    microscopic examination of various organs revealed no significant
    differences between the groups (Yang, 1964).

         Four groups of 25 male and 25 female rats were fed diets
    containing 0, 50, 125 and 250 mg/kg bw CaNa2EDTA for two years.
    Feeding was carried on through four successive generations. Rats were
    mated after 12 weeks' feeding and allowed to lactate for three weeks
    with one week's rest before producing a second litter. Ten male and
    10 female rats of each group (F1 generation) and similar F2 and F3
    generation groups were allowed to produce two litters. Of the second
    litters of the F1, F2 and F3 generations only the control and the
    250 mg/kg bw groups were kept until the end of two-years' study on the
    F0 generation. This scheme permitted terminal observation to be made
    on rats receiving test diets for 0, 0.5, 1, 1.5 or 2 years in the F3,
    F2, F1 and F0 generations, respectively.  No significant
    abnormalities in appearance and behaviour were noted during the 12
    weeks of the post weaning period in all generations. The feeding
    experiment showed no statistically significant differences in weight
    gain, food efficiency, haemopoiesis, blood sugar, non-protein
    nitrogen, serum calcium, urine, organ weights and histopathology of
    liver, kidney, spleen, heart, adrenals, thyroid and gonads. Fertility,
    lactation and weaning were not adversely affected for each mating.
    Mortality and tumour incidence were unrelated to dosage level. The
    prothrombin time was normal. There was no evidence of any chelate
    effect on calcification of bone and teeth. Liver xanthine oxidase and
    blood carbonic anhydrase activities were unchanged (Oser et al., 1963)


         CaNa2EDTA is very poorly absorbed from the gut. The compound is
    metabolically inert and no cumulation in the body has been found. A
    vast clinical experience in its use in the treatment of metal
    poisoning has demonstrated its safety in man. Long-term feeding
    studies in rats and dogs gave no evidence of interference with mineral
    metabolism in either species. Adverse effects on mineral metabolism
    and nephrotoxicity were only seen after parenteral administration of
    high doses.

         The long-term studies with Na2EDTA are difficult to assess
    because of the small number of animals and the high mortality rate
    in all groups. Metabolic studies and feeding studies demonstrate that
    the use of CaNa2EDTA is preferable to that of Na2EDTA, because of
    the effect of the latter in sequestering calcium. Under certain
    circumstances, necessitating an accurate complexing of ions other than
    calcium, it may be used provided no excess of Na2EDTA remains.


    Level causing no toxicological effect

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

    Estimate of acceptable daily intake for man

         0-2.5* mg/kg bw


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    Foreman, H. (1961) Fed. Proc., 20 (Suppl. 10), 191


    *    Calculated as CaNa2EDTA. No excess of Na2EDTA should remain in

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    See Also:
       Toxicological Abbreviations