Prepared by the Fifty-third meeting of the Joint FAO/WHO
    Expert Committee on Food Additives (JECFA)

    World Health Organization, Geneva, 2000
    IPCS - International Programme on Chemical Safety


    First draft prepared by Dr J.B. Greig

    Joint Food Safety & Standards Group, Department of Health, London,
    United Kingdom

    Biological data
         Toxicological studies
              Acute toxicity
              Short-term studies of toxicity
              Developmental toxicity
              Special study of nutrition
         Observations in humans


         The sodium iron salt of ethylenediamine tetraacetate (EDTA) was
    evaluated by the Committee at its forty-first meeting (Annex 1,
    reference  107), which provisionally concluded that sodium iron EDTA
    that met the tentative specifications prepared at the meeting would
    not present a safety problem when used in supervised food
    fortification programmes in iron-deficient populations. The Committee
    requested the results of additional studies to assess the site of
    deposition of iron administered in this form and to assess the
    metabolic fate of sodium iron EDTA after long-term administration. The
    Committee emphasized that its evaluation applied 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
    of food because of the enhanced bioavailability of iron in this form.

         Several studies were submitted for consideration at the present
    meeting in response to the Committee's request.


    2.1  Toxicological studies

    2.1.1  Acute toxicity

         A summary report of the results of determinations of the LD50 of
    sodium iron EDTA in rats and mice was submitted (Sichuan Provincial
    Sanitary and Anti-epidemic Station, 1998).

    2.1.2  Short-term studies of toxicity


         The experiment described below was conducted according to the
    OECD principles of GLP. After a pilot experiment, groups of 40 male
    Sprague-Dawley Crl:CD(R) BR rats aged five to six weeks were fed
    diets containing iron in the form of sodium ferric EDTA or ferrous
    sulfate heptahydrate (FeSO4) to provide iron at concentrations of 35,
    70, or 140 mg/kg of diet. The mean daily intakes of iron at the low,
    intermediate, and high doses were 2.8, 5.7, and 12 mg/kg bw per day
    with FeSO4 and 2.8, 5.7, and 11 mg/kg bw per day with sodium iron
    EDTA. The animals were fed the diet for 31 days, at which time 20
    animals in each group were killed, while the remaining 20 were
    continued on the diet for a further 30 days. A group of 10 untreated
    male rats were killed at the start of the experiment to establish the
    baseline levels of the parameters that were to be measured. The
    toxicolo-gical end-points that were studied included clinical signs,
    body weights, organ weights, food consumption, food conversion
    efficiency, haematological and clinical chemical end-points, and the
    pathological appearancce of the liver, spleen, and any gross lesions.
    The distribution of iron was evaluated by determining iron and total
    iron-binding capacity in plasma and non-haem iron in the liver,
    spleen, and kidneys.

         No deaths occurred during the experiment, and the clinical signs
    observed are commonly seen in laboratory rats. Occasional
    statistically significant changes in food consumption were not
    obviously related to treatment. Food conversion efficiency was
    variable but showed some small, statistically significant changes
    during the first four weeks. The iron content of the diets was within
    10% of the intended value in 25 of the 26 batches prepared for both
    the pilot and main experiments, but the content of the 26th batch was
    13% lower than the intended 70 mg/kg of iron.

         Administration of either FeSO4 or sodium iron EDTA resulted in
    increased non-haem iron concentrations in the liver, spleen, and
    kidney when compared with the baseline values after 31 or 61 days of
    feeding, and the changes were related to dose except in the spleen at
    61 days. The non-haem iron concentra-tion in these organs resulting
    from administration of sodium iron EDTA was always either
    significantly lower or not significantly different from the
    concentration resulting from administration of FeSO4. The plasma iron
    concentration was statistically significantly higher after 31 days of
    the low dose of sodium iron EDTA than after the intermediate dose, but
    no other significant difference was seen at 31 or 61 days. There were
    no statistically significant differences in the total iron-binding
    capacity of blood plasma after 32 days of feeding, and the
    statistically significant differences seen at 62 days were minor and
    unrelated to administered dose or the iron compound. After 31 days of
    feeding, there were no statistically significant differences in

    terminal body weight or absolute or relative organ weights in treated
    animals. At 61 days, the absolute and relative thymus weights of rats
    given the low dose of sodium iron EDTA were significantly higher than
    those at the low dose of FeSO4 or at the other doses of sodium iron
    EDTA. No other significant changes in body weight or organ weight were

         The haematological examinations showed a statistically
    significant difference in the mean corpuscular haemoglobin
    concentration at 32 days only between rats given sodium iron EDTA and
    those given the low dose of FeSO4, but the difference was less than
    1.5% and no difference was seen between individual groups treated with
    sodium iron EDTA or at 62 days. At 32 days, animals given the high
    dose of sodium iron EDTA also had a statistically significantly
    increased eosinophil count, but it did not appear to be related to
    treatment or dose. No significant changes were found in any group at
    62 days. Some inconsistent changes were seen in clinical chemical
    end-points in blood plasma collected on days 32 and 62. The activity
    of alkaline phosphatase was greater in all groups at day 32 than at
    day 62 and was greater in the FeSO4-treated groups than in those
    given sodium iron EDTA; although statistically significant differences
    were seen, there was no evidence of a dose-response relationship and
    the effect had disappeared by 62 days. A statistically significant
    increase in total bilirubin occurred at 32 days in the group given the
    intermediate dose of FeSO4, but no effect was seen at 62 days.
    Statistically significant differences in sodium ion concentration at
    32 and 62 days and in chloride ion concentration at 32 days did not
    appear to be related to biologically significant changes. An
    apparently dose-related decrease in calcium ion concentration in both
    treated groups at 62 days was considered by the study authors to be
    associated with observed changes in albumin and hence total protein
    concentrations. Whereas liver damage might have been a cause of the
    decreased plasma albumin concentration, the absence of any other
    indicators of liver damage (such as liver-derived plasma enzyme
    activities and histological changes) led the study authors to conclude
    that the changes were of no toxicological significance.

         Histopathological examination of sections of the liver and spleen
    stained with haematoxylin and eosin did not reveal any dose-or
    treatment-related changes. Such observations as were made were
    considered to be unremarkable and occurred either sporadically or with
    a uniform distribution across all groups. At 32 days, the accumulation
    of iron did not result in a positive Prussian blue reaction in stained
    sections of the liver. A general increase was seen in the spleen in
    all groups, but the reaction tended to be confined to the red pulp and
    was classified as very slight to slight, although increased staining
    tended to be found in spleens from the groups fed the doses providing
    high concentrations of iron. At 62 days, the Prussian blue staining of
    the liver sections did not suggest a dose-or treatment-related effect.

    The changes seen in the spleens of all animals generally suggested a
    dose-and time-related increase in staining, with no evidence of a
    difference between the groups fed FeSO4 and sodium iron EDTA (Appel,

         A summary report of a 90-day study in rats given sodium iron EDTA
    in the diet was submitted (Su  et al., 1999).

    2.1.3  Genotoxicity

         A summary report of the results of tests for genotoxicity  in
     vitro and  in vivo was submitted (Sichuan Provincial Sanitary and
    Anti-epidemic Station, 1998).

        Table 1. Results of assays for the genotoxicity of sodium iron ethylenediamine
    tetraacetic acid (EDTA)

    End-point        Test object                  Concentration    Result

    Reverse          S. typhimurium TA97a,        Not reported     Negativea
    mutation         TA98, TA100, TA102,
                     TA1535, TA1537, TA1538

    Gene mutation    Tk locus in mouse            0.03-325 mg/ml   Positive and
                     lymphoma L5178Y cells        as Fe            cytotoxic with S9;
                                                                   negative without S9

    From Dunkel et al. (1999); sodium iron EDTA of 98% purity was used.
    S9, 9000 × g supernatant of rat liver
    2.1.4  Developmental toxicity

         A summary report of the results of a study of teratogenicity in
    rats of sodium iron EDTA was submitted (Sichuan Provincial Sanitary
    and Anti-epidemic Station, 1998).

    2.1.5  Special study of nutrition


         Eight groups of eight weanling male Sprague-Dawley rats weighing
    about 45 g were randomized to receive a zinc-sufficient diet
    containing zinc at 30 mg/kg of diet or a zinc-deficient diet
    containing zinc at 6.1 mg/kg of diet. Three of the groups in each of
    these two blocks were fed a diet containing sodium iron EDTA, while
    the diet of the other group contained FeSO4. The iron content of all

    diets was 50.1 mg/kg, but two groups fed sodium iron EDTA in each
    block received a further dose of 300 or 800 mg/kg diet. After
    receiving the diets for 18 days, the animals were transferred to
    metabolism cages for three days to allow study of the balance of
    calcium, copper, and zinc. Animals that did not eat normally during
    this period were removed from the analysis, and, on day 21, all
    animals were killed and the right femur removed for analysis of
    calcium and zinc.

         The rats given the zinc-sufficient diets ate significantly more
    food, gained more weight, and had a higher femur zinc content than
    those given the zinc-deficient diets. Changing from FeSO4 to sodium
    iron EDTA had no significant effect on the zinc-sufficient rats but
    resulted in a significant increase in food intake and body-weight
    gain. Increasing the EDTA content of the diet significantly but
    inconsistently reduced the femur zinc content of the zinc-sufficient
    rats, while in the zinc-deficient rats increasing the EDTA content
    reduced both food intake and body-weight gain with no significant
    change in femur calcium or zinc content.

         Zinc absorption (intake minus faecal excretion) and retention
    (intake minus (faecal excretion plus urinary excretion)) were both
    greater in the rats given the zinc-deficient diet. Fortification with
    sodium iron EDTA increased both parameters and also urinary zinc
    excretion, although only the latter appeared to be related to the dose
    of EDTA. The dietary intake of zinc, the nature of the iron compound
    used to supplement the diet, and the concentration of EDTA had no
    marked effect on the absorption or excretion of either copper or
    calcium except for a small but significant dose-related effect of EDTA
    concentration on urinary calcium excretion (Hurrell et al., 1994).

    2.1.6  Observations in humans

         The effect of various ratios of disodium EDTA to FeSO4 on the
    absorption of isotopically labelled FeSO4 was studied in 127 women,
    some of whom had depleted iron stores and some of whom were also
    anaemic. At molar ratios of EDTA:Fe of 1-0.25, the absorption of iron
    from a meal with low iron bioavaila-bility was increased (MacPhail et
    al., 1994).

         In a random, cross-over study, 10 healthy women aged 23-49 and
    weighing 40-70 kg received diets fortified with 10 mg Fe from either
    FeSO4 or sodium iron EDTA for 14 days, starting after a period of
    menstruation and with an interval of about four weeks. Other than the
    addition of sugar, butter, or jam to match the caloric requirements of
    each subject, each received a basal diet, and all of the water that
    was drunk was ultra-pure. The first five days of each arm of the study
    were used for dietary adaptation, and then a six-day chemical balance
    study was carried out in the course of which stable isotope-labelled
    calcium and zinc were administered.

         Significant enhancement of apparent zinc absorption was seen with
    sodium iron EDTA as compared with FeSO4 (34 ± 17% (SD) and 21 ±
    4.4%). Urinary zinc excretion was also significantly increased, but
    the difference in zinc retention was not significant. Absorption of
    isotopic calcium was not affected by the iron compound used, although
    a small but statistically significant increase in urinary excretion of
    isotopic calcium was confirmed in the chemical balance study. Overall,
    no significant negative effect of sodium iron EDTA on the uptake and
    excretion of zinc was observed (Davidsson et al., 1994).

         After an overnight fast, test meals containing 5 mg of Fe as
    either FeSO4 or sodium iron EDTA and a radioisotopic 59Mn tracer
    were administered to one man and nine women, one of whom was
    post-menopausal. The activity of 59Mn excreted in urine was measured
    in seven consecutive 24-h samples, and whole-body retention of 59Mn
    was measured on days 0, 1, 2, 3, 4, 5, 6, 10, 14, 17, 21, 27, and 28.
    On day 28, the second arm of the cross-over study was started with
    feeding of the other iron compound and a second tracer dose of 59Mn
    in a similar protocol. No statistically significant difference in
    manganese retention or urinary excretion was seen with the two iron
    compounds (Davidsson et al., 1998).

         A double-blind intervention study was carried out in Guatemala
    over 32 months to test the efficacy in controlling iron deficiency of
    the addition of sodium iron EDTA to sugar. The study was carried out
    on a community basis and had been planned to involve two highland and
    two lowland communities of populations ranging from 1144 to 1726, with
    one community in each area as controls. It had been planned to obtain
    an age-and sex-stratified sample of 318 persons from each community
    for detailed dietary, parasitological, haematological, and biochemical
    studies. The detection of a high prevalence of anaemia in one of the
    planned control communities, however, resulted in its conversion to a
    treatment community. Additionally, although the initial response rates
    in the communities were 54-78%, application of exclusion criteria
    resulted in failure to achieve the target basal numbers. There were
    additional losses of subjects during the study. The sugar supply of
    the treated communities, which was already supplemented with vitamin
    A, was further fortified with sodium iron EDTA at 1 g/kg sugar,
    equivalent to an Fe concentration of 130 mg/kg sugar. Dietary surveys
    during the study showed that the mean (± SD) individual sugar intake
    ranged from 35 ± 16 to 38 ± 24 g/day.

         Biochemical measures of indicators of iron status--haemoglobin
    concentration, saturation of serum iron-binding capacity, free
    erythrocyte protoporphyin concentration, serum ferritin concentration,
    and iron store mass--were reported from the basal and final surveys.
    The changes observed indicated an improvement in iron status in the
    treated populations, with a reduction in iron-deficiency

    erythropoiesis. The authors reported that forti-fication of sugar with
    sodium iron EDTA resulting in the absorption of iron at 0.95-3.1
    mg/day had no adverse effects on people of each sex of various ages
    (Viteri et al., 1995).


         A study specifically designed to address the Committee's concerns
    involved feeding male rats diets containing iron in two forms, FeSO4
    and sodium iron EDTA, each at three doses, for 62 days. The dietary
    concentrations provided iron intakes of 2.8, 5.7, and 11 mg/kg bw per
    day (sodium iron EDTA) or 12 mg/kg bw per day (FeSO4). Generally,
    there was a dose-related increase in the amount of non-haem iron
    stored in liver, spleen, and kidney, but smaller or equal amounts of
    iron were taken up from the sodium iron EDTA-containing diet in
    comparison with that containing FeSO4. There was no evidence that the
    total iron-binding capacity of blood plasma was altered by treatment
    with sodium iron EDTA. The Committee therefore concluded that there
    was no evidence that administra-tion of iron in the form of sodium
    iron EDTA would result in greater uptake of iron than from an
    equivalent dietary concentration of FeSO4 once the nutritional
    requirement for iron is satisfied. There was no evidence of adverse
    effects at the highest daily intake of iron, 11 mg/kg bw, which is 55
    times greater than the proposed daily human intake of 0.2 mg/kg bw in
    food fortification programmes.

         Short-term studies in rats and humans have shown no adverse
    effects of dietary intake of sodium iron EDTA on the balance of other
    minerals such as calcium, copper, manganese, and zinc. The results of
    an intervention study in iron-deficient populations in Guatemala
    demonstrated the efficacy of a diet supplemented with sodium iron EDTA
    in reducing the prevalence of iron deficiency in humans.


         The Committee considered that the data submitted satisfied its
    concerns about the use of sodium iron EDTA in food fortification
    programmes. It was aware of the results of studies of the acute
    toxicity, mutagenicity, teratogenicity, and 90-day toxicity in rats of
    sodium iron EDTA. Full reports of these studies were not available,
    but the information was not considered necessary for evaluating the
    safety of this compound. The Committee also received an assessment of
    the potential intake of sodium iron EDTA by consumers in the United
    States that would result from fortification of foodstuffs. The
    Committee was of the view that this assessment was not relevant to any
    proposed use of sodium iron EDTA as a food fortifier in areas of iron

         The Committee concluded that sodium iron EDTA could be considered
    safe for use in supervised food fortification programmes, when public
    health officials had determined the need for iron supplementation of
    the diet of a population. Such programmes would provide daily iron
    intakes of approximately 0.2 mg/kg bw.


    Appel, M.J. (1999) A feeding study to examine the disposition and
    accumulation of iron fed as sodium iron EDTA in rats. Unpublished
    report No. V99.426 from TNO Nutrition and Food Research Institute,
    Zeist, The Netherlands. Submitted to WHO by the International Life
    Sciences Institute.

    Davidsson, L., Kastenmayer, P. & Hurrell, R.F. (1994) Sodium iron EDTA
    [NaFe(III)EDTA] as a food fortificant: The effect on the absorption
    and retention of zinc and calcium in women.  Am. J. Clin. Nutr., 60,

    Davidsson, L., Almgren, A. & Hurrell, R.F. (1998) Sodium iron EDTA
    [NaFe(III)EDTA] as a food fortificant does not influence absorption
    and urinary excretion of manganese in healthy adults.  J. Nutr., 128,

    Dunkel, V.C., San, R.H.C., Seifried, H.E. & Whittaker, P. (1999)
    Genotoxicity of iron compounds in  Salmonella typhimurium and L5178Y
    mouse lymphoma cells.  Environ. Mol. Mutag., 33, 28-41.

    Hurrell, R.F., Ribas, S. & Davidsson, L. (1994) NaFe3+EDTA as a food
    fortificant: Influence on zinc, calcium and copper metabolism in the
    rat.  Br. J. Nutr., 71, 85-93.

    MacPhail, A.P., Patel, R.C., Bothwell, T.H. & Lamparell, R.D. (1994)
    EDTA and the absorption of iron from food.  Am. J. Clin. Nutr., 59,

    Sichuan Provincial Sanitary and Anti-epidemic Station (1998) Acute
    toxicity, mutagenicity and teratogenicity of NaFeEDTA in rats and
    mice. Translation of unpublished report submitted to WHO by J. Chen.

    Su, Y.L., Chen, Z.L., Zhang, J. & Jiang, Z.C. (1999) A 90-day rat
    study on subchronic toxicity of NaFeEDTA. Translation of unpublished
    report submitted to WHO by J. Chen.

    Viteri, F.E., Alvarez, E., Batres, R., Torún, B., Pineda, O., Mejia,
    L.A. & Sylvi, J. (1995) Fortification of sugar with iron sodium
    ethylenediaminotetracetate (FeNaEDTA) improves iron status in
    semirural Guatemalan populations.  Am. J. Clin. Nutr., 61, 1153.

    See Also:
       Toxicological Abbreviations