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    SODIUM IRON EDTA

    First draft prepared by
    Dr I.C. Munro
    CanTox Inc., Mississauga, Ontario
    Canada

    1  EXPLANATION

         The Committee was asked to comment on the safety of sodium iron
    (III) ethylenediaminetetraacetate (sodium iron EDTA, sodium iron
    edetate, NaFeEDTA, NaFe(III)EDTA) as a dietary supplement for use in
    supervised food fortification programmes in populations in which
    iron-deficiency anaemia is endemic.  The Committee was informed that
    use of iron in this form would be restricted to this specific
    application and would be supervised.

         Sodium iron EDTA has not previously been evaluated by the Joint
    FAO/WHO Expert Committee on Food Additives.  However, disodium and
    calcium disodium EDTA were evaluated at the seventeenth meeting
    (Annex 1 reference 32). An ADI of 2.5 mg EDTA CaNa2EDTA/kg body
    weight/day was established.  Sodium iron EDTA was placed on the
    agenda to provide an assessment of its safety for use in supervised
    food fortification programmes in populations in which iron
    deficiency anaemia is endemic.

         With respect to iron, a provisional maximum tolerable daily
    intake of 0.8 mg/kg/bw was established by the Committee at the
    twenty-seventh meeting (Annex 1, reference 62). The view stated at
    the twenty-sixth meeting (Annex 1, reference 59), that the tolerable
    daily intake should not be used as a guide for fortifying processed
    foods, was reiterated.  This monograph discusses the safety of
    NaFeEDTA for food fortification in developing countries.

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

         The biochemistry of EDTA metal complexes is inextricably tied
    to their chemical properties. An understanding of these chemical
    properties is essential in interpreting the biochemistry and
    toxicology of EDTA metal complexes. A brief discussion of the
    chemical properties of EDTA metal complexes is presented here to
    facilitate understanding of the material that follows.

         Ethylenediaminetetraacetic acid (EDTA) is a hexadentate
    chelator capable of combining stoichiometrically with virtually
    every metal in the periodic table (Chaberck and Martell, 1959). With
    divalent or trivalent metal ions a neutral or anionic metal chelate
    results.  The metal is largely prevented from reacting with
    competing anions and its solubility is greatly increased.  The
    effectiveness of EDTA as a chelate for a particular metal ion is
    given by its stability constant with the metal ion.  Chelation
    potential is affected by pH, the molar ratio of chelate to metal
    ion, and the presence of competing metal ions capable of forming
    complexes with EDTA (Plumb  et al., 1950; Martell, 1960; Hart,
    1984). The stability constants for different metal-EDTA complexes
    vary considerably and any metal which is capable of forming a strong
    complex with EDTA will at least partially displace another metal.

         Of the nutritionally important metals, Fe3+ has the highest
    stability constant (log k of 25.1), followed by Cu2+ with 18.4,
    Zn2+ with 16.1, Fe2+ with 14.6, Ca2+ with 10.6, mg2+ with 8.7
    and Na+ with 1.7 (West and Sykes, 1960). The situation is somewhat
    complicated by each metal having an optimum pH for chelate formation
    ranging from pH 1 for Fe3+, to pH 3 for Cu2+, pH 4 for Zn2+, pH
    5 for Fe2+, pH 7.5 for Ca2+, and pH 10 for mg2+ (West and
    Sykes, 1960).  When NaFeEDTA is ingested with foods, the Fe3+ ion
    would be expected to remain firmly bound to the EDTA moiety during
    passage through the gastric juice, but could be exchanged for
    Cu2+, Zn2+, Fe2+ or Ca2+ in the duodenum. Similarly when
    Na2EDTA and Na2CaEDTA are consumed with foods, the Na+ and Ca2+
    ions would be predominantly exchanged in the gastric juice for Fe3+
    ions, which could again in turn be exchanged for Cu2+, Zn2+, Fe2+
    or Ca2+ further down the gastrointestinal tract. The extent to
    which the metal EDTA complexes form is dependent on the pH and the
    concentration of the competing metals as well as competing ligands.
    The lower stability constant and higher pH optimum of the mg-EDTA
    chelate make reaction with this metal less likely.

         An appreciation of the chelating properties of EDTA with
    respect to iron provide the basis of our understanding of the
    observed effects of EDTA on food iron absorption.  Ferric food iron
    is poorly absorbed by human beings because it is precipitated from
    solution above pH 3.5 unless suitable complexing agents are present.

    It may therefore be partially insoluble in the upper small intestine
    where most nonhaem iron is absorbed (Conrad and Schade, 1968;
    MacPhail  et al., 1981).

         When EDTA is present in a meal, iron (primarily Fe3+) remains
    complexed with EDTA under the acidic conditions prevailing in the
    stomach.  The chelate holds the iron in solution as the pH rises in
    the upper small intestine, but the strength of the complex is
    progressively reduced allowing at least partial exchange with other
    metals and the release of some of the iron for absorption.  There is
    convincing evidence that iron chelated by EDTA (NaFeEDTA) is
    available for absorption via the physiologically regulated pathways
    responsible for iron uptake (Candela  et al., 1984). The results of
    absorption studies with NaFeEDTA indicate that iron is dissociated
    from the EDTA moiety prior to absorption. The results of these
    studies are summarized in Section 2.1.1.

    2.1.1  Absorption and excretion

    2.1.1.1  Absorption and excretion of Fe from NaFeEDTA - Injection
             studies

         When NaFeEDTA is injected intravenously into rats most of the
    iron (70-90%) is lost through the urine within 24 hours (Najarajan
     et al., 1964; Anghileri, 1967).  A small proportion enters the
    physiological iron pool destined primarily for haemoglobin synthesis
    probably because of the slow release of iron to the iron transport
    protein, transferrin, in the circulation (Bates  et al., 1967). 
    After intramuscular or intraperitoneal injection a greater
    proportion of the iron is available for physiological exchange with
    compartments in the bone marrow and liver.  The longer contact time
    between transferrin and EDTA, allows for greater transfer of iron
    from the chelate to the physiological transport protein (Rubin
     et al., 1970).

         FeEDTA administered intravenously to humans was almost
    quantitatively excreted in urine (Lapinleimu and Wegelius, 1959).

    2.1.1.2  Absorption and excretion of Fe from NaFeEDTA - oral studies

         Human iron deficiency anaemia was successfully treated with
    FeEDTA given orally with 84% of labelled FeEDTA excreted in the
    faeces and none in the urine.  Red cells, however, contained
    labelled Fe and reticulocytosis occurred (Will and Vilter, 1954).

         Studies carried out in swine using a doubly labelled
    Na55Fe[2-14C]EDTA preparation demonstrated rapid transfer of 55Fe
    to the plasma with a peak at 1 hour and subsequent incorporation of
    4.6% of the administered dose into circulating haemoglobin (Candela
     et al., 1984).  A small fraction (0.3%) of the 55Fe administered
    was excreted in the urine. In contrast to the 55Fe only a small

    percentage of the 14C could be detected in the plasma at any time. 
    Absorption occurred over an extended period (5-20 hours).  A total
    of about 5% of the 14C labelled EDTA was eventually absorbed in the
    duodenum and jejunum and quantitatively excreted in the urine.

         In a parallel experiment when 5 mg Fe as Na59FeEDTA was given
    to six fasting human volunteers, mean radioiron absorption as
    measured by red blood cell utilization was 12.0%.  Only 0.3% of the
    administered dose of iron was excreted in the urine.  The studies
    demonstrate that Fe and EDTA are absorbed independently when
    NaFeEDTA is administered by mouth (Candela  et al., 1984).

         Similar conclusions were reached in an earlier human absorption
    study carried out by MacPhail  et al. (1981).  Na59FeEDTA was
    administered to human volunteers.  Between 3 and 25% of the 59Fe
    was absorbed, but less than 1% of the administered 59Fe appeared in
    the urine over the subsequent 24 hours.  All 59Fe absorbed in the
    form of the intact Na59FeEDTA complex would be expected to be
    excreted in the urine within 24 hours (based on the results of
    Nagarajan  et al., 1964 and Anghileri, 1967) demonstrating again
    that most of the iron is released from the EDTA complex before
    absorption.  Similar conclusions have been reached with another iron
    chelator (nitrilotriacetic acid), the properties of which have been
    studied extensively in experimental animals (Simpson and Peters,
    1984).

    2.1.1.3  Absorption and excretion of EDTA from EDTA metal chelates

    Rats

         14C-labelled CaNa2EDTA, when fed to rats at 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 (EDTA), and this is only slowly redissolved in the intestine
    (Foreman  et al., 1953).

         In feeding experiments in rats receiving disodium EDTA at
    dietary levels of 0.5, 1.0 or 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, 1964).

         When 200 mg CaNa2EDTA was introduced into the duodenum of
    rats an absorption rate of 6.5 to 26% was observed (Srbrova and
    Teisinger, 1957).

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

    Humans

         Experiments in humans also revealed poor absorption; only 2.5%
    of a 3 g dose given was excreted in the urine (Srbrova and
    Teisinger, 1957).  These authors also confirmed the dissociation of
    the calcium chelate in the stomach.  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 and Trujillo,
    1954).

         The absorption of the EDTA moiety from orally administered
    NaFeEDTA has not been measured directly in humans.  However
    physicochemical considerations indicate that EDTA absorption from
    NaFeEDTA should be similar to that from other metal complexes, such
    as CaNa2EDTA and CrEDTA.  As described above, poor absorption of
    the intact NaFeEDTA can be inferred from the measurements of urinary
    radioiron excretion after the oral administration of Na59FeEDTA
    made by MacPhail and coworkers in 1981.

         Similar results have been obtained with a tightly bound
    chelate, 51CrEDTA from which any released metal is very poorly
    absorbed (Bjarnason  et al., 1983; Aabakken and Osnes, 1990).  Only
    1-5% of a dose of 51CrEDTA given in a fasting state is absorbed by
    the healthy intestinal mucosa.  In the presence of disorders of the
    gastrointestinal tract the absorption may be doubled.  The 
    51CrEDTA that is absorbed appears to be taken up through
    intercellular junctions as the intact complex.  The amount absorbed
    has been used as a measure of the integrity of the bowel mucosa.

         In summary, most of the iron in NaFeEDTA is released to the
    physiological mucosal uptake system before absorption.  Only a very
    small fraction of the NaFeEDTA complex (less than 1%) is absorbed
    intact and this is completely excreted in the urine.  An additional
    small fraction (less than 5%) of the EDTA moiety is absorbed,
    presumably bound to other metals in the gastrointestinal tract, and
    is also completely eliminated in the urine.

    2.1.1.4  Bioavailability of iron from NaFeEDTA

         The results of iron absorption studies comparing the
    bioavailability of iron from FeSO4 and NaFeEDTA fortified foods are
    listed in Table 1. For purposes of comparison the individual
    absorption values have been standardized to a reference absorption

    of 40% to remove the influence of varying iron requirements in
    different subjects.  A reference absorption value of 40% is assumed
    to represent borderline iron deficiency (Hallberg  et al., 1978). 
    The bioavailability of iron from FeSO4 varies over a wide range
    and correlates with the relative proportions of enhancers and
    inhibitors known to be present in the meals.

         Enhanced bioavailability was most marked in meals with poor
    FeSO4 bioavailability (FeSO4 absorptions less than 4%).  Between
    2.1 to 2.9 times as much iron was absorbed under such circumstances. 
    This point is exemplified by the results of the study by Viteri
     et al. (1978) (see Table 1) in which iron absorption from a
    NaFeEDTA-fortified meal of beans, maize, and coffee was 2.7 times
    greater than that from the same meal containing FeSO4 (Viteri
     et al., 1978).

         In contrast, the absorption of iron from NaFeEDTA eaten with
    identical meals varies only two to three fold.  More iron was
    absorbed from the meals containing NaFeEDTA in all but one case in
    which Na2EDTA and FeSO4 were eaten with sugar cane syrup (see
    Table 1).

         The absorption of Fe from NaFeEDTA has been studied in a wide
    variety of meals.  Comparisons with iron absorption from simple iron
    salts have not always been made.  However, some studies provide
    useful information about the suitability of three staple food items
    as potential vehicles for fortification with NaFeEDTA.  This
    information is summarized in Table 2.  Some studies listed in Table
    1 have been included under the appropriate categories (all values
    are corrected to 40% reference absorption, or a serum ferritin of
    27 Ág/l).  It is evident that approximately 10% of the fortification
    iron added would be absorbed by iron deficient individuals if these
    staple foods were used as the vehicle for delivering the
    fortificant.


        Table 1.  Comparison of iron absorption from meals of different iron bioavailability
              fortified with ferrous sulfate or NaFeEDTA; Standardized Iron Absorption (%)a

                                                                                                                

           Components of Meal              A          B       Ratio    Reference
                                         FeSO4    NaFeEDTA     B/A

                                                                                                                

    1.   Rice Milk                        1.7       4.5        2.6     Viteri  et al., 1978
    2.   Beans, Maize, Coffee             2.0       5.3        2.7     Viteri  et al., 1978
    3.   Egyptian flat breadb             2.1       5.3        2.5     el Guindi  et al., 1988
    4.   Bran                             2.7       7.8        2.9     MacPhail  et al., 1981
    5.   Beans, Plantain, Rice,           3.1       7.0        2.3     Layrisse and Martinez-Torres, 1977
         Maize, Soyc
    6.   Rice                             3.9      11.5        2.9     MacPhail and Bothwell, unpublished, 1992
    7.   Maize Meal                       4.0       8.2        2.1     MaPhail  et al., 1981
    8.   Beans, Plantain, Rice,           4.2       7.4        1.8     Layrisse and Martinez-Torres, 1977
         Maize, Soy, Orange Juicec
    9.   Beans, Plantain, Rice,           4.3       9.6        2.2     Layrisse and Martinez-Torres, 1977
         Maize, Soy, Meatc
    10.  Potato                           5.9       7.3        1.2     Lamparelli  et al., 1987
    11.  Wheat                            6.2      14.6        2.3     Martinez-Torres  et al., 1979
    12.  Milk                            10.2      16.8        1.6     Layrisse and Martinez-Torres, 1977
    13.  Sweet Manioc                    14.1      16.6        1.2     Martinez-Torres  et al., 1979
    14.  Sugar cane Syrupc               33.1      10.8        0.3     Martinez-Torres  et al., 1979
                                                                                                                

    a.  Geometric means standarized to a reference (Ferrous ascorbate) absorption of 40%
    b.  A mixture of FeSO4 and Na2EDTA was used in this study.
    c.  Comparison between FeSO4 and NaFeEDTA not in the same individuals.
    

        Table 2.  Percentage iron absorption from meals containing
              NaFe(III)EDTA

                                                                       

    Vehicle     No. of     Standardized          References
                Studies    Iron Absorption
                           (Range)

                                                                       

    Wheat          4       10.1 (5.3 - 14.6)     Martinez-Torres  et al.,
                                                 1979 and el Guindi  et al.,
                                                 1988

    Maize          7       9.1 (7.6 - 12.0)      Martinez-Torres  et al.,
                                                 1979 and MacPhail  et al.,
                                                 1981

    Cassava        3       13.5 (11.0 - 16.4)    Martinez-Torres  et al.,
                                                 1979
                                                                       
    
    2.1.1.5  Effect of NaFeEDTA on bioavailability of intrinsic food
             iron

         Conclusions drawn from much of the experimental work on food
    iron absorption and iron fortification are based on the observation
    that soluble iron added to a meal and the intrinsic nonhaem food
    iron behave as a common pool, which is equally susceptible to
    enhancers and inhibitors of iron absorption present in the meal
    (Cook  et al., 1972; Hallberg and Bjorn-Rasmussen, 1972);  NaFeEDTA
    shares this property.  When Na59FeEDTA was added to meals
    containing foods labelled intrinsically with 55Fe, the ratio
    between the proportions of iron absorbed from the two sources was
    close to unity (Layrisse and Martinez-Torres, 1977; Matrinez-Torres
     et al., 1979; MacPhail  et al., 1981), with the exception of one
    study in which Na59FeEDTA fortified sugar was sprinkled onto
    55Fe-labelled maize immediately before it was eaten (MacPhail
     et al., 1981). These results indicate that the Na59FeEDTA
    equilibrates with the common pool, since without such equilibration,
    the amount of food iron absorbed would be much lower than the amount
    absorbed from NaFeEDTA (MacPhail  et al., 1981).  Inadequate mixing
    of the NaFeEDTA-fortified sugar with the maize meal probably
    accounted for the lack of equilibration in the one inconsistent
    study reported by MacPhail  et al. (1981).  These results reveal
    another important property of NaFeEDTA.  Equilibration of NaFeEDTA
    with the common pool iron improves the bioavailability of the
    intrinsic food iron as well.  Therefore NaFeEDTA improves iron

    balance by supplying iron in a form less affected by dietary
    inhibitors, but also improves the absorption of nonhaem iron in the
    meal derived from other sources.

         This point is further illustrated by the results of a number of
    studies which demonstrate that the positive effects of EDTA on iron
    absorption are shared by other elements of the common pool, such as
    another iron salt added to the meal.  When FeSO4 and NaFeEDTA were
    fed to humans on separate days in the same type of meal (maize
    porridge), iron absorption from the NaFeEDTA fortified meal was
    significantly better. However the iron from FeSO4 was as well
    absorbed as that from NaFeEDTA when they were fed together in the
    same meal (MacPhail  et al., 1981; Martinez-Torres  et al., 1979). 
    More direct evidence of reciprocal exchange between food iron and
    iron added as NaFeEDTA was provided by experiments in which subjects
    were given maize porridge fortified with equimolar quantities of
    59FeSO4 and Na55FeEDTA (McPhail  et al., 1981).  The ratio
    between the two isotopes was almost the same in the meal and the
    urine.  This implies that exchange of iron between FeSO4 and
    NaFeEDTA must occur before absorption of the chelate, since only the
    small amount of iron (less than 1%) absorbed as the intact chelate
    would subsequently appear in the urine (for explanation see section
    2.1).

    2.1.1.6  The effect of Na2EDTA on iron absorption

         Na2EDTA is widely used as a food additive to prevent
    oxidative damage by free metals.  Since Na2EDTA readily chelates
    iron in the gut to form NaFeEDTA, its effect on iron absorption is
    of interest.  In a recent study (el Guindi  et al., 1988) Na2EDTA
    was added, together with an equimolar quantity of iron as FeSO4, to
    bread with a high concentration of phytate (an inhibitor of iron
    absorption).  The combination was associated with a 2.6x enhancement
    in iron absorption when compared with results with FeSO4 used
    alone.  Mean percentage iron absorption was approximately equivalent
    to that reported in other similar studies using NaFeEDTA.  It is
    evident that the same effect on iron absorption can be achieved in
    meals containing compounds that inhibit iron absorption by adding
    Na2EDTA and a soluble iron salt as is the case for adding
    NaFeEDTA.

         The effects of Na2EDTA on iron absorption appear to be
    influenced by the molar ratio of EDTA to iron.  Earlier work
    suggested that increasing the molar ratio of Na2EDTA to Fe was
    associated with a progressive reduction in iron absorption (Cook and
    Monsen, 1976).

         These observations have been extended recently: iron absorption
    from a series of rice meals containing Na2EDTA and iron in a molar
    ratio of 1:1 was compared to rice containing Na2EDTA and iron in
    molar ratios (EDTA:Fe) ranging from 0:1 to 4:1.  Statistically
    significant enhancement of absorption occurred at ratios of Fe:EDTA
    between 1:4 and 1:1.  The enhancing effect of EDTA on iron
    absorption appeared to be maximal at a molar ratio (EDTA:Fe) of
    approximately 1:2, not 1:1 as previously assumed.  At this molar
    ratio over three times as much iron was absorbed from the EDTA
    containing meal as was the case for the control meal containing no
    EDTA (MacPhail and Bothwell, unpublished data, 1992).

    2.1.2  Distribution

         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 systems 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 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 (Srbrova and Teisinger,
    1957). These results indicate that intact CaNa2EDTA, and presumably
    other EDTA metal complexes are rapidly excreted and do not
    accumulate.

    2.1.3  Biotransformation

         Neither the iron nor the EDTA moiety of NaFeEDTA undergoes
    biotransformation. Evidence for this conclusion comes from studies
    discussed in the previous section which indicated that both EDTA and
    iron are excreted unchanged following ingestion of NaFeEDTA.

    2.1.4  Influence of EDTA compounds on the biochemistry of metals

         EDTA removes about 1.4% of the total iron from ferritin at pH
    7.4 to form an iron chelate (Westerfeld, 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).

         Addition of 1% Na2EDTA to a diet containing more than optimal
    amounts of iron and calcium lowered the absorption and storage or
    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/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).

         Copper absorption and retention were improved at 500 mg EDTA/kg
    but not at 200 mg or 1 000 mg EDTA/kg. Apart from a very small
    increase in urinary copper excretion, dietary EDTA had no influence
    on copper metabolism (Hurrell  et al., 1993).

         CaNa2EDTA increased the excretion of zinc (Perry and 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, Ti and W 
    (Foreman, 1961).  The treatment of heavy metal poisoning with
    CaNa2EDTA 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).

         Food fortification with NaFeEDTA may be expected to increase Zn
    and Cu absorption and retention but not Ca nor Mg.  A diet
    containing RDA quantities of each metal (800 mg Ca and, 350 mg,
    10 mg Zn, and 2 mg Cu) which was fortified with 10 mg Fe as NaFeEDTA
    would contain a 1.5 molar excess of EDTA over Zn, an 8-fold molar
    excess of EDTA over Cu, but 80 times less EDTA than Ca and 50 times
    less EDTA than mg on a molar basis.  The small quantity of chelate
    with respect to Ca and mg would be unlikely to have any detrimental
    effect.  Both NaFeEDTA and NaEDTA may increase the absorption and
    retention of Zn and Cu when added to low bioavailability diets. 
    This conclusion is supported by experiments with turkey poults
    (Kratzer  et al., 1959), chicks (Scott and Ziegler, 1963) and rats
    (Forbes, 1961) which have demonstrated that Zn bioavailability and
    animal growth is improved when Na2EDTA is added at 150-300 mg/kg
    to animal rations based on soybean protein isolate. The enhancing
    effect of EDTA on zinc absorption in these studies can be explained
    by a combination of two factors. Firstly, EDTA forms soluble
    chelates with Zn from which the metal is potentially absorbable, and
    secondly, Zn is prevented from forming non-absorbable complexes with
    phytic acid.  EDTA does not enhance Zn absorption when absorption
    inhibitors are absent from the meal as evidenced by the observation
    that Na2EDTA (1 000 mg/kg) improved Zn absorption in rats fed a
    casein-based diet with added phytic acid, but had no effect in the
    absence of phytic acid (Oberleas  et al., 1966).

         Other chelating substances can also enhance Zn absorption from
    low bioavailability diets. Vohra and Kratzer compared the growth
    promoting effect of chelates with stability constants (log k) for Zn
    varying from 5.3 to 18.8 in turkey poults fed zinc deficient diets
    based on soy protein isolate. They found that
    ethylenediaminediacetic acid-dipropionic acid, hydroxyethyl-EDTA,
    and EDTA (stability constants 14.5, 14.5 and 16.1, respectively)
    were the most effective (Vohra & Kratzer, 1964).

         These earlier observations were made with Na2EDTA.  However,
    NaFeEDTA has been shown to have similar properties in a recent
    study.   Zinc, copper, and calcium balances were performed in rats
    fed low Zn (6.1 mg/kg) soybean based diets containing 36 mg/kg added
    Fe as either ferrous sulfate or NaFeEDTA.  In some experimental
    groups additional Na2EDTA was added to the diet containing
    NaFeEDTA to give dietary EDTA levels of 200, 500 and 1 000 mg/kg. 
    Changing the iron compound in the diet from ferrous sulfate to
    NaFeEDTA at a level of 200 mg/kg increased apparent Zn absorption,
    urinary Zn excretion and Zn retention significantly (p < 0.05), but
    caused no changes in Cu nor Ca absorption or excretion. Increasing
    the dietary EDTA level to 500 mg/kg (molar ratio EDTA:Zn, 19:1) and
    1 000 mg/kg (molar ratio EDTA:Zn, 38:1) further increased both Zn
    absorption and urinary Zn excretion. At the highest dietary EDTA
    level (1 000 mg/kg), Zn retention was significantly higher than with
    no dietary EDTA, but lower than with  500 mg/kg EDTA.  This resulted
    from an increase in urinary excretion of Zn to 15.6% of intake. 
    Similar results were obtained with a Zn-sufficient (30 mg/kg)
    soybean diets, but more EDTA was required to achieve optimal ratios
    for improved absorption.

         These studies demonstrate that an 11-fold molar excess of EDTA
    over Cu increased Cu absorption and retention but that neither a 4.5
    nor 23-fold molar excess had a significant effect.  A human diet
    containing the RDA for Zn and Cu which was fortified with 10 mg Fe
    as NaFeEDTA would be expected to contain a 1.5 molar excess of EDTA
    over Zn and an 8-fold molar excess of EDTA over Cu.  NaFeEDTA
    fortification would therefore be expected to have very little effect
    on Zn and Cu balance.  A small beneficial effect could occur in
    meals containing little Zn or Cu or large quantities of phytate
    (Hurrell  et al., 1993).

         The applicability of the observations made in experimental
    animals to human nutrition has been confirmed by recent observations
    made by Hurrell's group.  The metabolism of Zn and Ca was studied
    using a stable isotope technique in 10 adult women fed a breakfast
    meal of bread rolls made from 100 g high extraction wheat flour and
    fortified with 5 mg Fe as FeSO4 or NaFeEDTA.  The test meals 
    contained a 3.3 molar excess of EDTA over Zn but some 10-fold less
    EDTA than Ca.  Changing the Fe fortification compound from ferrous
    sulfate to NaFeEDTA significantly increased 70Zn absorption
    (p<0.05) from this meal from 20.9% to 33.5%.  Urinary 70Zn

    excretion also rose from 0.3% to 0.9%. Calcium metabolism was
    similar with the two different iron compounds (Davidsson  et al,
    1993).

         Earlier studies using less precise methodology have led to
    similar conclusions.  Adding NaFeEDTA to a low bioavailability
    Guatemalan meal did not influence Zn absorption by human subjects.
    However, as these workers measured Zn absorption based on plasma Zn
    concentrations after ingesting 25 mg Zn with a meal, the molar
    concentration of EDTA was some 10-fold less than that of Zn and an
    improvement in Zn absorption would not be expected (Solomons  et al.
    (1979).

         Finally, no significant changes in plasma Zn concentration were
    observed in field studies in which NaFeEDTA was used as a food
    fortificant over a two year period (Viteri  et al., 1983, Ballot
     et al., 1989b).

    2.1.5  Effects on enzymes and other biochemical parameters

         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 and Betz, 1961).

          In vitro, 0.0033 M EDTA inhibited the respiration of liver
    homogenates and of isolated mitochondria of liver and kidney
    (Lelievre and Betz, 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 and Leloir, 1961;
    Miettinen and Leskinen, 1962).  Of the heavy metal-containing
    enzymes, EDTA at a concentration of about 10-3 M inhibited aldehyde
    oxidase and homogentisinase.  Succinic dehydrogenase, xanthine
    oxidase, NADH-cytochrome reductase and ceruloplasmin (oxidation of
    p-phenylenediamine) were not inhibited (Westerfeld, 1961).  Disodium
    EDTA was found to be a strong inhibitor for delta-aminolevulinic
    acid dehydrogenase, 5.5 x 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 Ca. 
    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 and
    Schumacher, 1957).

         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 and Johnson, 1961).

    2.2  Toxicological studies

    2.2.1  Acute toxicity studies

         The results of acute toxicity studies with disodium EDTA are
    summarized in Table 3.

    Table 3. Results of acute toxicity studies with disodium EDTA.

                                                              

    Animal       Route         LD50           References
                            (mg/kg bw)
                                                              

    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

         The results of acute toxicity studies with Ca-disodium EDTA are
    summarized in Table 4.

    Table 4. Results of acute toxicity studies with Ca-disodium EDTA.

                                                              

    Animal       Route         LD50           References
                            (mg/kg bw)
                                                              

    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 Lechnit
    (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 and
    Schmieller, 1962).

    2.2.2  Short-term toxicity studies

    2.2.2.1  Rats

         Groups of five male rats received 250 or 500 mg/kg bw/dy
    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 and 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).

         Groups of five male rats were given 250, 400 or 500 mg/kg bw/dy
    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 50 mg
    level.  One rat at the 250 mg dose level showed haemorrhage of the
    thymus.  All three groups showed varying degrees of hydrophic
    necrosis of the renal proximal convoluted tubules with epithelial
    sloughing: recovery occurred in all groups after withdrawal of
    disodium EDTA (Reuber and Schmieller, 1962).

    2.2.2.2  Rabbits

         Eight groups of three rabbits were given either 0.1, 1, 10 or
    20 mg/kg bw/dy disodium EDTA i.v., or 50, 100, 500 or 1 000 mg/kg
    bw/dy orally for one month.  All animals on the highest oral test
    level exhibited severe diarrhoea and died.  In the other groups body
    weight, haemograms, 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).

    2.2.2.3  Dogs

         Four groups of one male and three female mongrels were fed
    diets containing 0, 50, 100 and 200 mg/kg bw/dy 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).

    2.2.3  Long-term toxicity/carcinogenicity studies

    2.2.3.1  Mice

         Groups of 50 male and 50 female B6C3F1 mice received trisodium
    EDTA (Na3EDTA) in the diet at concentrations of 3 750 or 7 500 ppm
    for 103 weeks, followed by one week during which standard diet
    without EDTA was fed. A control group consisting of 20 mice of each
    sex received the standard diet. Food was available  ad libitum and
    fresh food was provided three times per week.

         Animals were examined for signs of toxicity twice per day, and
    were weighed and palpated for masses regularly (schedule not
    stated). Gross and microscopic pathological examinations were
    performed on animals found dead or moribund and on those sacrificed
    at the end of the study. Microscopic examinations were conducted on
    the following tissues and organs: skin, lymph nodes, mammary gland,
    salivary gland, bone marrow, trachea, lungs and bronchi, heart,
    thyroid, parathyroids, oesophagus, stomach, small intestine, liver,
    gallbladder, pancreas, spleen, kidneys, adrenals, urinary bladder,
    prostate or uterus, testis or ovary, brain and pituitary.

         Survival rates were comparable among treated and control
    animals of both sexes. No treatment-related clinical signs of
    toxicity were noted during the study. Body weight gain was decreased
    in high-dose males during the second year of the study (no
    statistical analysis). From the graphical representation of the
    data, it appears that the body weights in the high-dose group were
    approximately 10% below that of controls during the last nine months
    of the study. In females, average body weights in treated groups
    were consistently lower than the average control body weight for
    most of the study period, however, the differences among the three
    groups were very slight. No tumours or non-neoplastic lesions
    attributable to treatment were observed (NCI, 1977).

    2.2.3.2  Rats

         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 (0.5% 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 systems 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).

         In a two-year study, five groups of 33 rats each 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/dy CaNa2EDTA for two years. 
    Feeding was carried on through four successive generations.  Rat
    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 F1, F2, and F3 generations only the
    control and the 250 mg/kg bw/dy 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,
    haematopoiesis, 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).

         Groups of 50 male and 50 female Fisher F344 rats received
    trisodium EDTA (Na3EDTA) in the diet at concentrations of 3 750 or
    7 500 ppm for 103 weeks, followed by one week during which standard
    diet without EDTA was fed (NCI, 1977). A control group consisting of
    20 rats of each sex received the standard diet of Wayne Lab Blox
    Meal. Food was available  ad libitum and fresh food was provided
    three times per week.

         Animals were examined for signs of toxicity twice per day, and
    were weighed and palpated for masses regularly (schedule not
    stated). Gross and microscopic pathological examinations were
    performed on animals found dead or moribund and on those sacrificed
    at the end of the study. Microscopic examinations were conducted on

    the following tissues and organs: skin, lymph nodes, mammary gland,
    salivary gland, bone marrow, trachea, lungs and bronchi, heart,
    thyroid, parathyroids, oesophagus, stomach, small intestine, liver,
    gallbladder, pancreas, spleen, kidneys, adrenals, urinary bladder,
    prostate or uterus, testis or ovary, brain and pituitary.

         Survival was comparable among control and treated groups of
    male rats. There was a significant dose-related increase in survival
    in treated groups of females compared to controls. Body weights were
    comparable among treated and control groups, and there were no
    clinical signs of toxicity in treated animals. No tumours or
    non-neoplastic lesions attributable to treatment were observed (NCI,
    1977).

    2.2.4  Reproduction studies

    2.2.4.1  Rats

         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 EDTA/rat 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 and Mercier-Parot, 1956).

         In a four generation study, groups of rats received CaNa2EDTA
    at doses of 50, 125 or 250 mg/kg/day via the diet.  No reproductive
    or teratogenic effects were observed in any of the three generations
    of offspring (Oser  et al., 1963).  This study is discussed in
    greater detail in Section 2.2.3 of this monograph.

         Groups of pregnant Sprague-Dawley rats were fed Na2EDTA in
    standard diet at levels of 2 or 3% from day 1 to 21 of gestation. 
    Another group of pregnant rats received 3% Na2EDTA in standard
    diet from day 6 to 14 of gestation.  A third group received 3%
    Na2EDTA and 1 000 ppm zinc in the diet from day 6 to 21 of
    gestation.  Controls received standard diet, which contained 100 ppm
    zinc.  The number of mated animals per group ranged from 5 to 16. on
    day 21 of gestation fetuses were removed, fixed in Bouin's solution
    and stored in 70% ethanol. Fetuses were examined under a dissecting

    microscope for gross external abnormalities. Razor cut sections were
    examined for abnormalities of the eye and head. In rats fed 2% EDTA
    during pregnancy, litter size was normal and fetuses were alive.
    Gross congenital malformations were apparent in 7% of the treated in
    fetuses, compared to 0% in controls. In rats fed 3% EDTA during
    pregnancy, almost half of the implantation sites had dead fetuses or
    resorptions. Full term young were significantly smaller than
    controls and 100% of them were malformed. Maternal toxicity as
    manifested by diarrhoea was observed in rats fed 2 or 3% EDTA in the
    diet. Malformations included severe brain malformations, cleft
    palate, malformed digits, clubbed legs and malformed tails. The
    detrimental effects of EDTA were prevented by supplementation of the
    diet with 1 000 ppm zinc. These findings suggest that the
    teratogenic effects observed in rats fed EDTA at very high levels in
    the diet are due to zinc deficiency (Swenerton and Hurley, 1971).

         Groups of pregnant CD rats were treated with Na2EDTA via the
    diet at a dose of 954 mg/kg/day (3% in the diet; 42 rats), by
    gastric intubation at doses of 1 250 mg/kg/day (split dose of
    625 mg/kg twice/day; 22 rats) or 1 500 mg/kg/day (split dose of
    750 mg/kg twice/day; 8 rats), or by subcutaneous injection at a dose
    of 375 mg/kg/day (25 rats). Animals were dosed on gestation day 7
    through 14. The number of control animals for each exposure route
    were: diet, 38; gavage, 20; subcutaneous injection, 14. Fetuses were
    removed at day 21 of gestation. One third of the fetuses from each
    litter (including all stunted fetuses and those with external
    malformations) were dissected and examined for visceral
    abnormalities. All fetuses surviving to the time of sacrifice were
    fixed and examined for skeletal malformations. Maternal toxicity as
    evidence by decreased food consumption, diarrhoea and diminished
    weight gain was observed in groups treated by all three dose routes.
    In the dietary exposure group, there were no maternal deaths, but
    there was a significant increase in fetal death and 71% of the
    fetuses were malformed. In the group administered 625 mg/kg/day by
    gavage, only 64% of the dams survived treatment. In those surviving,
    the number of fetal resorptions was similar to controls and 20.5% of
    the fetuses were malformed. Seven out of eight of the dams
    administered 750 mg/kg/day by gavage failed to survive. In the group
    administered EDTA by subcutaneous injection, 76% of the dams
    survived, the number of resorptions was significantly increased
    above control levels and the proportion of malformed fetuses was
    similar to controls. The types of malformations were consistent with
    those observed by Swenerton and Hurley, although these former
    workers only evaluated external malformations. The results of this
    study indicate that the route of exposure to EDTA is an important
    factor in determining its lethality and teratogenicity (Kimmel,
    1977).

         Groups of 20 pregnant CD rats were administered EDTA,
    Na2EDTA, Na3EDTA, Na4EDTA or CaNa2EDTA by gavage at a total
    dose of 1 000 mg EDTA/kg/day in two divided doses per day during
    gestation day 7 through 14. All fetuses were subjected to gross
    examination. One third were sliced and examined for visceral
    abnormalities and the other two thirds were dissected, processed and
    examined for skeletal abnormalities. The incidence of diarrhoea was
    increased in all treated groups. Food intake was decreased in
    treated groups as was weight gain during the treatment period.
    Litter size and fetal mortality were unaffected by treatment in all
    groups. No treatment-related teratogenic effects were observed in
    any group (Schardein  et al., 1981).

    2.2.5  Special studies on embryotoxicity

    2.2.5.1  Chickens

         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 and
    Scott, 1964).

    2.2.6  Special studies on genotoxicity

         Na3EDTA was tested for mutagenicity in the L5178Y tk+/tk-
    mouse lymphoma cell forward mutation assay. Two experiments were
    conducted with S9, and three without S9, using EDTA concentrations
    of up to 5 000 ug/ml. No mutagenicity was observed with or without
    S9 (McGregor  et al., 1988).

         Na3EDTA was tested for mutagenicity in  Salmonella
     typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 as well
    as in  Escherichia coli WP  uvrA, in the presence and absence of
    S9. Concentrations of up to 1 mg/plate were tested. No evidence of
    mutagenicity was found in either of these bacterial systems, by four
    independent laboratories (Dunkel  et al., 1985).

    2.2.7  Special studies on skin sensitization

         Groups of 10 Hartley guinea-pigs received topical application
    of Na3EDTA, ethylene diamine (EDA) or epoxy resin (positive
    control) four times over 10 days to a shaved and depilated area on
    the back. Following a two week recovery period, animals received a
    challenge on the clipped flank. Animals originally treated with EDTA
    were not sensitized to EDTA. Animals originally treated with EDA
    were sensitized to EDA, but not to EDTA. The results of this study
    indicate a lack of sensitizing potential of EDTA and a lack of
    cross-sensitization between EDA and EDTA (Henck  et al. 1985).

    2.3  Observations in humans

         Three comprehensive field trials have been carried out using
    NaFeEDTA as an iron fortificant in fish sauce (Garby and Areekul,
    1974), off-white sugar (Viteri  et al., 1983) curry powder (Ballot
     et al., 1989b).  The salient features of these trials are listed
    in Table 5. All three trials were preceded by some estimate of the
    iron status of the population and care was taken to establish the
    acceptability and bioavailability of iron from the chosen vehicle
    prior to the trials (Garby and Areekul, 1974, Viteri et.al, 1983,
    Lamparelli et.al, 1987, Ballot et.al, 1989a).  The choice of food
    vehicle in each case reflected the dietary habits of the population.

    2.3.1  NaFeEDTA fortified fish sauce

         Fortified fish sauce was provided for a period of one year to
    the population of a Thai village.  The packed red cell volume (PCV)
    values before and after the fortification program showed a
    significant increase as compared to a control village supplied with
    unfortified fish sauce.  The biggest mean change (+4.7) was seen in
    a sub-group of women who were anaemic at the start of the trial
    (initial PCV < 33).  Although a similar sub-group of women in the
    control group also improved during the year (mean change +2.1) the
    increase in PCV in the fortified group was significantly greater. 
    The same pattern was seen in both men and children with low initial
    PCV values.

         In terms of iron nutrition the increase of 4.7 PCV units over
    initial values represents an increase of about 187 mg iron, in total
    body iron or an increase in daily absorption of about 0.5 mg over
    the duration of the trial (1 year).  This is 64% of the expected
    increase in body iron of 0.8 mg/day calculated on the basis of an
    anticipated absorption of 8% and an assumed daily intake of 10 ml
    fortified fish sauce (10 mg Fe).  Iron stores were not measured in
    this trial and the calculation does not take into account any
    absorbed iron which may have been laid down in stores.  The
    calculated value therefore would be an underestimate of the total
    amount of iron actually absorbed.  Nevertheless it illustrates that
    fortification with NaFeEDTA is a highly effective method for
    improving iron status.

         Overall this trial demonstrated that fortification of fish
    sauce at modest levels using NaFeEDTA is feasible, and that it can
    produce a significant improvement in iron status as assessed by a
    single simple criterion (PCV) (Garby and Areekul, 1974).

    2.3.2  NaFeEDTA fortified sugar

         The design of this trial makes interpretation of the results
    difficult.  The analysis is based on the comparative changes in iron
    status observed in four communities.  Three (#13, #14, #16) were
    test sites, and one was a control site (#15).  The initial iron
    status of individuals drawn from test community #14 was
    significantly worse than that of individuals from the other test
    communities and the control community (#15).  Unfortunately
    compliance was poor in this community and also in test community
    #13.  Furthermore seventy percent of the families in test
    communities number 13 and 14 used fortified sugar for only half of
    the time.  The remaining 30% used it for 80% of the time.  Finally,
    subjects with severe anaemia were given therapeutic iron to improve
    their iron status prior to the trial.    Despite the presence of these
    confounding factors, the haemoglobin values rose in both males and
    females after 20 months of fortification, although the values did
    not reach statistical significance.  Only the children (5-12 years)
    in communities #13 and #16 showed a significant improvement in
    haemoglobin levels when compared to children in the control
    community #15 (+2.2▒1.7 and +2.2▒1.5 respectively vs +1.6▒1.2 g/dl). 
    The greater benefit observed in children may have resulted from the
    fact that sugar consumption was greater in children than in adults
    when considered relative to body weight.  Mean serum ferritin which
    is a measure of the size of iron stores increased in each of the
    test communities, but not in the control community.  In conclusion
    it should be noted that the relatively modest improvement in iron
    status noted in this trial may also have been due, in part, to the
    fact that the fortification level was considerably less than in the
    other two trials (4.3 vs 10-15 and 7.7 mg/person/day) (Viteri
     et al., 1983).


        Table 5. Outline of field trials using NaFeEDTA to fortify various
             food vehicles

                                                                                                                                              

    References                  Garby and Areekul, 1974            Viteri,  et al., 1983               Ballot,  et al., 1989a,b

                                                                                                                                              

    Geographical region         Thailand                           Central America                    South Africa

    Population studied          Two rural villages                 4 rural Guatemalan                 Urban Indian community in a
                                                                   communities                        municipal housing estate

    Design of trial             Controlled (one village)           Controlled (community #15)         Controlled (random allocation
                                not blinded                        not blinded                        by families) double-blinded

    Sample studies              Test village (284) control         #13 - 186 #14 - 306                263 Families (672 subjects)
                                village (330)                      #15 - 234 #16 - 296 severe         129 control families 134 fortified
                                                                   anaemics treated prior to          families Hb < 9 g/dl excluded
                                                                   trial

    Food vehicle                Fish-sauce (salt substitute)       Off-white sugar distribution:      Masala (curry powder) distributed
                                30 g NaCl/l, 10 mg Fe/l            sold to store keepers. Purchased   directly to families monthly free
                                distributed by village             by participants (poor compliance   of charge
                                head-man as required               #13 and #14)

    Cons. of food vehicle       10 - 15 ml/person/day              33 g/person/day; children          5.5 g/person/day
                                                                   highest consumption

    Fe absorption               8%                                 8%                                 10%

    Level of fortification      1 mg Fe/ml                         13mg Fe/100g                       1.4 mg Fe/g
    and intake                  10 - 15 mg Fe/person/day           4.29 mg Fe/person/day              7.7 mg Fe/person/day

    Acceptability               No changes                         Barely perceptible yellowing       Slight darkening of food
                                                                                                                                              

    Table 5 (contd).

                                                                                                                                              

    References                  Garby and Areekul, 1974            Viteri,  et al., 1983               Ballot,  et al., 1989a,b

                                                                                                                                              

    Duration of trial           12 months                          20 months                          24 months

    % Abnormal iron status      30 - 50 of population anaemic;     Low     Low   Low                          Females     Males
    prior to trial              34% initial PCV below normal       Comm    PCV   Sat
                                                                   Ferr                               IDA       24          4
                                                                   #13      31    34   52             ID        53         24
                                                                   #14      43    58   72
                                                                   #15      35    12   37
                                                                   #16      21    23   34

    Measurements taken          Packed cell volume (PCV)           Haemoglobin, PCV, %Sat, FEP,       Haemoglobin, %Sat, Serum Ferritin
                                                                   Serum Ferritin, Cu, Zn
                                                                                                                                              
    IDA = Iron Deficiency anaemia; ID = Iron Deficiency; % Sat = % Saturation of Transferrin; FEP =
    Free Erythrocyte Protoporphyrin; PCV = Packed Cell Volume; Comm = Community
    

    2.3.3  NaFeEDTA fortified masala

         The design of the most recent fortification trial differed from
    those of earlier studies in that it was conducted in a single
    community with families randomly assigned to control and test
    groups.  The groups were matched for iron status.  It was also
    double-blinded and care was taken to ensure that cross-over between
    groups did not occur.  Fortified or unfortified masala was
    distributed directly to each family.  In addition to evaluating
    fortification the usual indices of improving iron status (increasing
    haematocrit or haemoglobin and ferritin) in each individual by using
    a composite of haemoglobin concentration, percent saturation of
    transferrin, and serum ferritin concentration, an attempt was made
    to estimate the total body iron (in mg) in each individual by using
    a composite of haemoglobin concentration, percent saturation of
    transferrin and serum ferritin concentration (Cook,  et al, 1986). 
    This comprehensive index of iron nutrition made it possible to
    compare subjects with wide variations in iron status and thus to
    assess both the beneficial and potentially adverse effects of
    additional iron i.e. development of iron overload (Ballot,  et al.,
    1989a, b).

         Significant improvement in body iron as assessed by the index
    was detectable in the group of women receiving fortified masala
    after one year of the program (Ballot,  et al., 1989a, b).  This
    improvement continued during the second year when the rise in
    haemoglobin concentration became significantly greater than in the
    control group.  The prevalence of iron deficiency dropped
    dramatically in the women receiving fortified masala.  Iron
    deficiency anaemia was detected in 22% of individuals at the start
    of the study, but only to 4.9% after two years.  The most
    significant improvement in iron status was noted in women who
    entered the trial with iron deficiency (especially in those with
    anaemia).  They showed an increase in calculated body iron of 505 mg
    which is equivalent to the absorption of an additional 0.7 mg
    iron/day.  The latter figure is close to the predicted improvement
    in iron balance of 0.8 mg daily based on isotopic absorption studies
    using NaFeEDTA fortified masala (Lamparelli  et al., 1987).

         In iron-replete males the rise in calculated body iron was
    modest and only reached significance in alcohol abusers receiving
    fortified masala. This suggests that iron-replete males are unlikely
    to accumulate excessive amounts of iron under these fortification
    conditions.

    3.  COMMENTS

         The Committee was concerned about over-fortification or misuse
    of this product and did not recommend its availability for general
    use by individuals.

         The Committee noted that sodium iron EDTA dissociates in the
    intestine, and iron in this form is approximately twice as
    bioavailable as iron in the form of iron sulfate.  The available
    studies indicated that only a fraction, if any, of the iron EDTA
    chelate is absorbed as such, that EDTA from sodium iron EDTA is only
    poorly absorbed and that the majority is excreted in the faeces. 
    The portion that is absorbed (<5%) is rapidly excreted in urine. 
    The proposed supplementation programme would result in intakes of
    iron and EDTA of approximately 0.2 and 1.34 mg/kg bw/day,
    respectively.

    4.  EVALUATION

         Based on previous evaluations of both iron and EDTA and the
    available bioavailability and metabolism data, the Committee
    provisionally concluded that use of sodium iron EDTA meeting the
    tentative specifications prepared at the present meeting in
    supervised food fortification programmes in iron-deficient
    populations does not present a safety problem.  The Committee
    requested that additional studies be conducted to assess the site of
    deposition of iron administered in this form and further studies to
    assess the metabolic fate of sodium iron EDTA following long-term
    administration.

         The Committee emphasized that its evaluation pertains only to
    the use of sodium iron EDTA as a dietary supplement to be used under
    supervision and expressed its concern about the potential for over-
    fortification because of the enhanced bioavailability of iron in
    this form.

         The Committee developed new tentative specifications for sodium
    iron (III) ethylenediaminetetraacetate (NaFeEDTA).  In preparing the
    specifications, the Committee was aware that food-grade NaFeEDTA is
    not commercially available.  However, the Committee was advised,
    that this substance is being evaluated for its usefulness in
    fortifying the diet in areas of the world where iron deficiency in
    the population is endemic, prepared specifications, which it
    believed would assist in the evaluation.  The Committee obtained
    analytical data and other information on fertilizer-grade NaFeEDTA,
    which is widely available, and considered this together with the
    existing specifications for disodium ethylenediaminetetra-acetate
    and calcium disodium ethylenediaminetetraacetate in formulating the
    specifications.  Because further information on assay and purity
    data for food-grade material and on analytical methodology is still
    needed, the specification was designated as tentative.

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    See Also:
       Toxicological Abbreviations