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    PHOSPHORIC ACID AND PHOSPHATE SALTS

    Explanation

         These compounds have been evaluated for acceptable daily intake
    by the Joint FAO/WHO Expert Committee on Food Additives in 1961, 1963,
    1964, 1965, 1969 and 1970 (see Annex I, Refs. 6, 7, 9, 13, 20 and 23).
    A toxicological monograph was published in 1974 (see Annex I, Ref.
    33).

         Since the previous evaluation, additional data have become
    available and are summarized and discussed in the following monograph.
    The previously published monograph has been revised, and deals with
    the following compounds:

         phosphoric acid
         calcium phosphate (mono-, di-, and tri-basic)
         sodium phosphate (mono-, di-, and tri-basic)
         disodium diphosphate
         tetrasodium diphosphate
         pentapotassium triphosphate
         pentasodium triphosphate
         sodium polyphosphate (Graham's salt)
         sodium hexametaphosphate
         sodium potassium polyphosphate (Tammann's salt)
         Kurrol's salt (KPO3)n
         sodium tripolyphosphate
         disodium phosphate
         magnesium phosphate (mono-, di-, and tri-basic)
         potassium phosphate (mono-, di-, and tri-basic)
         bone phosphate
         ammonium phosphate, dibasic
         ammonium polyphosphate
         calcium polyphosphate
         calcium pyrophosphate
         potassium polyphosphate
         potassium pyrophosphate

         The toxicity of these phosphate compounds is in part due to
    separate problems arising from the various cations and to the
    phosphate anions in general. Most of the available toxicity data has
    been developed with sodium salts. However, it is unlikely that effects
    of the potassium and ammonium moieties of the salts, at the levels
    used in foods, would be of concern and the effect of these salts would
    not be significantly different from sodium salts. In the case of
    calcium and magnesium salts, these salts provide a source of calcium,
    magnesium and phosphate ions. Problems relating to calcium, magnesium
    and phosphate imbalances in the diet are of concern and are considered
    in this monograph. They are primarily due to excess phosphorus in the

    diet, and deficiencies in calcium and magnesium. Thus the present
    monograph is applicable to all the inorganic phosphates listed. A
    separate monograph has been prepared for aluminium phosphates.

    BIOLOGICAL DATA

    BIOCHEMICAL ASPECTS

         Several reviews of phosphate metabolism and toxicity are
    available (Ellinger, 1972; FASEB, 1975; FASEB, 1981; Roe, 1979).

         Phosphorus is an essential constituent of the human organism, not
    only in the bones and teeth, but also in many enzyme systems.
    Phosphorus compounds play an important role in carbohydrate, fat and
    protein metabolism.

    (a)  Phosphoric acid, phosphates and orthophosphates

         Free orthophosphate is the major form in which phosphorus is
    absorbed from the diet. The amount and rate at which other phosphates
    are available for absorption depends on their enzymic hydrolysis to
    orthophosphates. The level of inorganic phosphate in the blood is
    stabilized by exchange with the mineral deposit in the skeleton
    through the action of parathyroid hormone. This hormone inhibits
    tubular reabsorption of phosphates by the kidney and brings about
    demineralization of bone tissue through the action of osteoclasts. The
    amount of parathyroid hormone that enters the circulation is probably
    regulated by the calcium level of the blood. Intestinal absorption
    depends on requirements and is therefore limited. Excretion takes
    place mainly in the faeces as calcium phosphate so that the continuous
    use of excessive amounts of sodium phosphate and phosphoric acid may
    cause a loss of calcium. As a result of physiological regulating
    mechanisms, man and animals can tolerate large variations in phosphate
    intake without the balance being upset.

         Some investigators have considered that the formation in the
    intestinal tract of insoluble salts of phosphate with calcium, iron
    and other metal ions might result in decreased absorption of such
    minerals. From studies dealing with this aspect (Lang, 1959; van Esch
    et al., 1957; Lauersen, 1953; van Genderen, 1961) it is concluded that
    moderate dose levels of phosphates do not impair absorption as shown
    by results from carcass analyses or haemoglobin determinations. Doses
    of 2-4 g of phosphate act as weak saline cathartics.

         Phosphate supplementation of the diet of rodents has been shown
    to lead to reduction in the incidence of dental caries and different
    phosphates have different powers in reducing the cariogenic potential
    of the carbohydrates in a diet (van Reen & Ostrom, 1962; Konig et al.,
    1961; McClure, 1960). Phosphate supplements seem to exert their
    cariostatic effect on the tooth surface either directly during eating
    or by excretion in the saliva (Anon., 1968a,b).

         Little specific toxicological information on potassium
    monophosphates is available. There is no reason to consider that the
    potassium salts, in the amounts that could be used as food additives,
    behave differently from the sodium salts and are therefore dealt with
    together.

    (b)  Disodium phosphate and tetrasodium diphosphate

         In the animal body, diphosphate is formed from adenosyl
    triphosphate (ATP) in many enzymatic reactions. It is either utilized
    by entering phosphorolytic reactions, or it is hydrolysed by an
    inorganic diposphatase to monophosphate (Long, 1961). Ingested
    diphosphate is readily converted to monophosphate (Fourman, 1959;
    Mattenheimer, 1958); no diphosphate was found in faeces or urine of
    rats treated with diets containing up to 5% tetrasodium diphosphate.
    In these experiments diphosphate was almost completely absorbed by the
    gut and excreted as monophosphate in the urine.

         The pyrophosphates are formed by the loss of 1 molecule of water
    from 2 molecules of an orthophosphate. The pyrophosphates (e.g.
    Ca2P2O7) are readily absorbed intact but then are completely
    hydrolysed to orthophosphate (Schreier & Noller, 1955). Achlorhydria
    may have an effect on the metabolism and toxicity of the condensed
    phosphates. It has been estimated that the half-life for hydrolysis of
    pyrophosphate at pH 2 and 39░C (normal gastric conditions) is 400
    hours (Mahoney & Hendricks, 1978).

    (c)  Triphosphates and polyphosphates

         Several studies indicate that polyphosphates can be hydrolysed
    in vivo by enzymes with the formation of monophosphates. The
    localization of different polyphosphates in the nuclei of animal
    cells has been demonstrated (Grossmann & Lang, 1962). Injected
    hexametaphosphate is more slowly degraded than tripolyphosphate
    (Gosselin et al., 1952), and the higly polymerized Tammann's salt (KNa
    polyphosphate) is even more slowly eliminated from the blood after
    i.v. injection than is Graham's salt (Gotte, 1953). When administered
    parenterally a small part of these products may escape in the urine as
    oligophosphates (Gosselin et al., 1952; Gotte, 1953). The higher
    polyphosphates are probably not absorbed as such in the intestinal
    tract. After hydrolysis into smaller units, absorption takes place.
    The larger the molecule, the less the speed of hydrolysis and
    absorption, as shown by studies using P32-labelled polyphosphate
    (Ebel, 1958).

         After giving hexametaphosphate to rats and rabbits by stomach
    tube, no more than trace amounts of labile phosphate were found in
    the urine (Gosselin et al., 1952). The oral administration of

    radiolabelled Tammann's salt did not give rise to radioactivity in the
    blood (Gotte, 1953). With Graham's salt and Kurrol's salt,* 10-30% was
    absorbed as monophosphate and small amounts of oligophosphates were
    found in the urine (Lang et al., 1955; Lang, 1958). In experiments in
    rats with labelled tripolyphosphates and Graham's salts these polymers
    were not absorbed as such, but were taken up after hydrolysis into
    monophosphate and diphosphate. In a period of 18 hours only 40% of the
    dose of Graham's salt was hydrolysed and absorbed. The bacterial flora
    of the intestinal tract may contribute to the hydrolysis of the
    polyphosphates (Schreier & Noller, 1955). In other experiments,
    radiolabelled Kurrol's salt was given orally to rats. About half the
    radioactivity was recovered from the faeces, mainly as polymeric
    phosphate, and only a small percentage of the dose was found in the
    urine, in this case in the form of monophosphate.

         Ring as well as linear polyphosphates including sodium trimeta,
    sodium tetrameta, sodium tripoly and sodium hexametaphosphate
    (Graham's salt) were shown to be hydrolysed in in vitro rat and
    porcine small intestine, while incubation in simulated intestinal
    fluid gave only slight hydrolysis. Trimeta, tetrameta, tripoly and
    hexameta were reduced by 12-36%, 5%, 76-80% and 10%, respectively in
    in vitro rat intestine in 1 hour, by 9-11%, 4-5%, 30% and 10-19%,
    respectively in in vitro porcine intestine in 1 hour and by 0-4%,
    0-1%, 17-19% and 2-3%, respectively in 12 hours in simulated
    intestinal fluid. The results indicate that simulated digestive juice
    contains little polyphosphatase activity. This enzyme is present in
    the intestinal lumen (Ivey & Shaver, 1975).

         It is noted that, for practical reasons, in the studies cited
    high dosages were given to the animals. The efficiency of hydrolysis
    and absorption may be greater at low-dose levels, such as were used in
    the short-term and long-term feeding experiments. In some of these
    (van Esch et al., 1957) the "monophosphate action", as demonstrated by
    the production of nephrocalcinosis, was not much smaller than when the
    same dose level was administered by the addition of monophosphate to
    the food. In another study, this applied only to tripolyphosphate,
    while Graham's salt had definitely less effect on the kidney (Hahn et
    al., 1958).

              

    *    Kurrol's salt is a polymer of high molecular weight obtained by
         fusion of monopotassium monophosphates. The formula is
         (KPO3)n.H2O, where n = 400 to 5000.

         The possibility of the intermediate formation of small amounts of
    trimetaphosphate in the hydrolysis of polyphosphates has been
    considered (Mattenheimer, 1958). At present, the only known method of
    production of sodium polyphosphate is by the fusion process. In this
    process metaphosphates are also formed in amounts up to 8% and their
    presence is technically unavoidable. It is of interest to note
    that these metaphosphates (sodium trimetaphosphate and sodium
    tetrametaphosphate) have been tested in short-term experiments in rats
    and dogs in conjunction with polyphosphates (Hodge, 1956). The
    metaphosphates are also hydrolysed to monophosphates. No specific
    action of these metaphosphates different from that of the other
    phosphates has been observed, and it is concluded that the presence of
    these impuritites does not present a hazard. It is also noted that the
    preparation of sodium polyphosphates used in the toxicological studies
    mentioned always contained metaphosphates in amounts up to 8%.

         It has been considered by many authors that the ingestion of
    polyphosphate in the food may result in a loss of minerals (Ca, Fe,
    Cu, Mg) which are bound to the polyphosphate and are lost in the
    faeces with unhydrolysed polyphosphate. For this reason, in most of
    the toxicological studies cited, particular attention has been paid to
    the mineral composition of the carcass and to the possible development
    of anaemia.

         The experimental results available indicate that such an
    action, if it occurs at all, is not significant. Anaemia is not a
    characteristic feature of treatment with high-dose levels of
    polyphosphate and hexametaphosphate had no effect on iron utilization
    by rats (Chapman & Campbell, 1957).

         The use of polyphosphates for the prevention of scale formation
    in lead pipe water systems may lead to excessive lead levels in
    drinking-water (Buydens, 1957).

    (d)  Calcium and magnesium phosphates

         Calcium phosphates are insoluble in water and constitute the
    following series: calcium phosphate (monobasic) which is used as
    acidulant and mineral supplement; calcium phosphate (dibasic) is used
    as dietary supplement in doses of 1 g orally; calcium phosphate
    (tribasic) is used as gastric antacid in doses of 1 g orally; and bone
    phosphate. Metabolically they behave as sources of calcium and
    phosphate ions. Magnesium phosphates are mostly insoluble in water and
    form the following series: magnesium phosphate (monobasic); magnesium
    phosphate (dibasic) which is used as laxative; magnesium phosphate
    (tribasic) is used as antacid in doses of 1 g orally. Metabolically
    they behave as sources of magnesium and phosphate ions. The phosphate
    moiety of these compounds need not be considered separately from other
    monophosphates from the toxicological point of view.

    TOXICOLOGICAL STUDIES

    Special studies on mutagenicity

         Monocalcium phosphate, monopotassium phosphate and monosodium
    phosphate were not mutagenic in an in vitro assay using
    Saccharomyces cerevisiae strain D4 and Salmonella typhimurium
    strains TA-1535, TA-1537 and TA-1538 with and without the addition of
    mammalian metabolic activation preparation (Litton Bionetics, Inc.,
    1975a,b,c).

         Sodium acid pyrophosphate was not mutagenic in a number of tests
    including: (1) host-mediated assay in mice using either Salmonella
    typhimurium TA-1530 or mitotic recombination frequency in
    Saccharomyces cerevisiae D3; (2) in an in vitro assay using
    S. typhimurium strains TA-1535, TA-1536 or TA-1537 and TA-1538,
    with and without the addition of mammalian metabolic activation
    preparations; (3) dominant lethal test in rats; and (4) translocation
    test in rats (Newell et al., 1974).

         Tetrasodium pyrophosphate was not mutagenic in an in vitro
    assay using S. cerevisiae strain D4, and S. typhimurium strains
    TA-1535, TA-1537 and TA-1538, with and without the addition of
    mammalian metabolic activation preparations (Food and Drug Research
    Lab., Inc., 1975d).

         Sodium tripolyphosphate was not mutagenic in a number of tests
    including: (1) host-mediated assay in mice using S. typhimurium
    strains TA-1530 and G-46 or mitotic recombination frequency in
    S. cerevisiae D3; (2) in an in vitro assay using S. typhimurium
    strains TA-1530 and G-46, and S. cerevisiae D3, with and without
    activation; (3) a cytogenetic study in vivo of rat bone marrow cells
    and in vitro of human lung cells in tissue culture; and (4) a
    dominant lethal study in rats (Litton Bionetics, Inc., 1974).

         Sodium hexametaphosphate was not mutagenic in an in vitro assay
    using S. typhimurium strains TA-1535, TA-1536 and TA-11537, with and
    without the addition of mammalian metabolic activation preparation
    (Litton Bionetics, 1975c).

    Special studies on teratogenicity and reproduction

         Verrett et al. (1980) reported that monocalcium phosphate,
    tricalcium phosphate, and monopotassium phosphate were not teratogenic
    in the developing chicken embryo. Monosodium phosphate, tetrasodium
    pyrophosphate, and sodium hexametaphosphate were found to produce
    teratogenic effects when injected during the period of rapid
    organogenesis in chicken embryos. Several different types of anomalies
    were observed, mostly structural.

         Teratology studies using rats, mice, rabbits or hamsters were
    performed on the following phosphates: monocalcium phosphate, sodium
    acid pyrophosphate, sodium tripolyphosphate, sodium hexametaphosphate
    and tetrasodium phosphate. The dose levels reported were the maximum
    levels tested.

         For studies in mice, groups each of approximately 24 pregnant
    albino CD-1 mice were dosed by oral intubation with the test substance
    from day 6 through day 16 of gestation. Body weights were recorded on
    days 0, 6, 11, 15 and 17 of gestation. On day 17, all dams were
    subjected to caesarean section and the number of implantation sites,
    resorption sites and live and dead foetuses recorded. The body weight
    of the live pups was also taken. The urinogenital tract level of each
    dam was examined for abdominal abnormality. All foetuses were examined
    for the presence of external congenital abnormalities. One-third of
    the foetuses were examined for visceral abnormalities and the
    remaining two-thirds for skeletal abnormalities.

         For studies in rats, groups each of approximately 24 pregnant
    female albino rats (Wistar-derived stock) were dosed daily by oral
    intubation from day 6 of gestation through day 15. Body weights were
    recorded on days 0, 6, 11, 15 and 20. On day 20, all dams were
    subjected to caesarean section, and observations for dams and foetuses
    similar to those described in the mouse study were carried out.

         For studies in hamsters, groups each of approximately 22-25
    pregnant female golden hamsters were dosed daily by oral intubation
    with the test compound from day 6 of gestation through day 10. Body
    weights were recorded on days 0, 6, 8, 10 and 14. On day 14, all
    animals were subjected to caesarean section, and observations for dams
    and foetuses, similar to those observed in the mouse study, were
    carried out.

         For studies in rabbits, groups each of approximately 20-22
    pregnant Dutch-belted rabbits, were dosed by oral intubation with the
    test compound from day 6 through day 18. Body weights were recorded on
    days 0, 6, 12, 18 and 29 of gestation. On day 29, all does were
    subjected to caesarean section, and observations, similar to those
    described in the mouse study, were carried out.

    (a)  Phosphoric acid, phosphates and orthophosphate

         Monosodium phosphate showed no maternal toxicity or teratogenic
    effects at dose levels up to 370 mg/kg bw in mice and 410 mg/kg bw in
    rats (Food and Drug Research Lab., Inc., 1975a).

         Monopotassium phosphate showed no maternal toxicity or
    teratogenic effects at dose levels up to 320 mg/kg bw in mice and
    282 mg/kg bw in rats (Food and Drug Research Lab., Inc., 1975b).

         Sodium acid pyrophosphate showed no maternal toxicity or
    teratogenic effects at dose levels up to 335 mg/kg bw in mice,
    169 mg/kg bw in rats, 166 mg/kg bw in hamsters and 128 mg/kg bw in
    rabbits (Food and Drug Research Lab., Inc., 1973a).

    (b)  Tetrasodium diphosphate

         Tetrasodium diphosphate showed no maternal toxicity or
    teratogenic effects at dose levels up to 130 mg/kg bw in mice and
    138 mg/kg bw in rats (Food and Drug Research Lab., Inc., 1975c).

    (c)  Triphosphates and polyphosphates

         Sodium hexametaphosphate showed no maternal toxicity or
    teratogenic effects at dose levels up to 370 mg/kg bw in mice and
    240 mg/kg bw in rats (Food and Drug Research Lab., Inc., 1975d).

         Sodium tripolyphosphates showed no maternal toxicity or
    teratogenic effects at dose levels up to 238 mg/kg bw in mice,
    170 mg/kg bw in rats, 250 mg/kg bw in rabbits and 141 mg/kg bw in
    hamsters (Food and Drug Research Lab., Inc., 1973b).

    (d)  Calcium phosphate

         Monocalcium phosphate showed no maternal toxicity or teratogenic
    effects at dose levels up to 465 mg/kg bw in mice and 410 mg/kg bw in
    rats (Food and Drug Research Lab., Inc., 1973c).


    Acute toxicity
        (a)  Phosphoric acid, phosphates and orthophosphates
                                                                                      

                                                  Approx.
         Compound            Animal    Route       LD50              Reference
                                                (mg/kg bw)
                                                                                      

    NaH2PO4                Guinea-pig   Oral     2 000 MLD     Eichler, 1950

    Monosodium phosphate   Mouse        Oral     3 700         Food and Drug Research
                           Rat          Oral     4 100         Lab., Inc., 1975a

    Monopotassium          Mouse        Oral     3 200         Food and Drug Research
    phosphate              Rat          Oral     2 820         Lab., Inc., 1975b

    Sodium acid            Mouse        Oral     3 350         Food and Drug Research
    pyrophosphate          Rat          Oral     1 690         Lab., Inc., 1975a
                           Hamster      Oral     1 660
                                                                                      

    (b)  Tetrasodium diphosphate
                                                                                      

                                                  Approx.
         Compound          Animal      Route       LD50              Reference
                                                (mg/kg bw)
                                                                                      

    Na4P2O7                Mouse        Oral       1 300        Food and Drug Research
                           Rat          Oral       1 380        Lab., Inc., 1975c
                                                                                      

    (c)  Triphosphates and polyphosphates
                                                                                      

                                                  Approx.
         Compound          Animal      Route       LD50              Reference
                                                (mg/kg bw)
                                                                                      

    Sodium triphosphate    Mouse        Oral       2 380        Food and Drug Research
                           Rat          Oral       1 700        Lab., Inc., 1973b
                           Rabbit       Oral       2 500

    1/3 Kurrol's salt      Rat          Oral       4 000        van Esch et al., 1957
    and 2/3 tetra- and
    disodium diphosphate
    (water soluble,        Rat          i.v.          18        van Esch et al., 1957
    neutral)

    Sodium                 Mouse        Oral       3 700        Food and Drug Research
    hexametaphosphate      Rat          Oral       2 400        Lab., Inc., 1975d
                                                                                      

    (d)  Calcium phosphate
                                                                                      

                                                  Approx.
         Compound          Animal      Route       LD50              Reference
                                                (mg/kg bw)
                                                                                      

    Monocalcium            Mouse        Oral       4 600        Food and Drug Research
    phosphate              Rat          Oral       2 170        Lab., Inc., 1973c
                                                                                      
    
    Short-term studies

    (a)  Phosphoric acid, phosphates and orthophosphates

    Rat

         There are many reports of short-term studies to determine the
    effects of the addition of monophosphates to the diet of rats (Maynard
    et al., 1957; Selye & Bois, 1956; MacKay & Oliver, 1935; Behrens &
    Seelkopf, 1932; McFarlane, 1941; van Esch et al., 1957; Sanderson,
    1959). Pathological effects in the parathyroids, kidneys and bones
    have been observed in mature male rats fed a diet containing an
    excessively high level (8%) of sodium orthophosphate for 7 months or
    until the animal succumbed (Saxton & Ellis, 1941). Histological and
    histochemical changes in the kidneys have been found in rats fed for
    24-72 hours on a diet containing an excess of inorganic phosphate
    (10% disodium acid phosphate)(Craig, 1957).

         Three groups of 12 rats each were fed diets containing added
    dibasic potassium phosphate so that the calcium and phosphorus
    concentrations in the experimental diets were as follows:

                                                                 

          Diet                   Calcium %         Phosphorus %
                                                                 

    Control                        0.56                0.42
    "normal orthophosphate"        0.47                0.43
    "high orthophosphate"          0.50                1.30
                                                                 

         The experiment was conducted in 3 stages, with experimental
    observations made when animals had consumed the test diets for 50,
    60 and 150 days. No adverse physiological effects were observed
    clinically at autopsy or on histological examination. All the data
    obtained from this study indicated that there was probably adequate
    absorption and utilization of calcium, phosphorus and iron with both
    high and normal levels of monophosphate (Dymsza et al., 1959).

         Reports of short-term studies do not provide for a
    differentiation between the action of the mono-, di- and trisodium or
    potassium salts; several authors have used "neutral mixtures", e.g. of
    mono- and disodium monophosphates. There is no reason to expect a
    specific action on the part of 1 of these 3 monophosphates, the
    relevant factor being the phosphate content and the acidity of the
    food mixture as a whole. On high-dose levels, hypertrophy of the
    parathyroid glands has been observed. A more important and more
    sensitive criterion for the deleterious action of phosphate overdosage
    is the appearance of calcification in soft tissues, especially in the

    kidney, stomach and aorta. Kidney calcification may be observed in a
    few weeks or months, depending on the dose level. The pathology of
    calcification and necrosis of the tubular epithelium in the kidneys
    (nephrocalcinosis) has been studied in detail (MacKay & Oliver, 1935;
    McFarlane, 1941; Sanderson, 1959; Fourman, 1959).

         It is difficult to indicate a border-line between those levels
    that do not produce nephrocalcinosis and those that produce early
    signs of such changes, because: (1) even on diets to which no
    phosphate has been added, rats, in apparently healthy condition, may
    have a few isolated areas of renal calcification; (2) the composition
    of the diet (amount of calcium, acid-base balance, vitamin D) has an
    important influence on the appearance of renal calcification.

         There are numerous reports of experimental phosphate-containing
    diets that do not produce kidney damage by excessive calcification,
    e.g. the Sherman diet (0.47-0.51%) (Lang, 1959; Hahn & Seifen, 1959;
    van Esch et al., 1957), the diet used by MacKay & Oliver (1935)
    (0.62% P) and the commerical "Purina A" diet (0.90% P) (Lang, 1959).

         Early calcification has been observed in rats on a Sherman diet
    to which 1% of a 2:3 mixture of NaH2PO4 and Na2HPO4 was added,
    bringing the P content to 0.71% (van Esch et al., 1957). Similar
    effects were observed with the addition of a phosphate mixture
    resulting in a P content of 0.89% (Hahn & Seifen, 1959), and with
    levels of phosphate in the diet corresponding to a P content varying
    from 1.25% to 2.85% (Lang, 1959; MacKay & Oliver, 1935; Eichler, 1950;
    McFarlane, 1941; van Esch et al., 1957; Haldi et al., 1939).

         In another study (Dymaza et al., 1959), however, a diet to which
    K2HPO4 had been added and containing 1.3% P and 0.5% Ca did not
    produce nephrocalcinosis in a group of mice within a period of 150
    days, although the weight of the kidneys was increased. Also food and
    protein efficiency were diminished as compared with animals on the
    control diet. These effects may have resulted from the large amount of
    salts added to the diet in these experiments.

         Diets containing various levels of calcium, phosphorus and
    magnesium were reported to produce uroliths in female Wistar rats. The
    incidence and severity of calcium phosphate uroliths were reduced by
    increasing the magnesium concentration in the diet from 0.2% to 0.8%
    and by increasing the calcium-to-phosphorus ratio to greater than 1
    (Chow et al., 1980).

         Diets containing 0.8% calcium and 0.9% phosphorus caused calcium
    phosphate deposits in the soft tissues of guinea-pigs (Hogan et al.,
    1950). When the phosphorus content of the diet was reduced to 0.5%,
    the number of animals with deposits was reduced from 90% to 10%.
    Further work (House & Hogan, 1955) demonstrated that with sufficient
    magnesium and potassium in the diet the calcium phosphate deposition

    could be significantly reduced. It has been pointed out that these 2
    studies may explain the wide variations in the levels of phosphate
    reported to cause toxicological effects (Ellinger, 1972). Thus,
    "optimum ratios of calcium to phosphorus, or of magnesium and
    potassium to phosphorus most likely explain why the diets of some
    studies required higher levels of phosphates in order to demonstrate
    adverse physiological effects".

    Sheep

         Wether sheep (5 per treatment) were fed on a basal diet to which
    urea had been added as a nitrogen source. Comparisons were made
    between this diet and diets which in addition contained either 11 g of
    dicalcium phosphate or 6.25 g of ammonium polyphosphate per day.
    Addition of either form of supplementary P caused increased appetite,
    daily weight gain, and apparent N retention. These utilization
    experiments were conducted over 1-week periods and it was concluded
    that ammonium polyphosphate was a satisfactory form of dietary P for
    ruminants (Fishwick, 1974).

    (b)  Disodium phosphate and tetrasodium diphosphate

    Rat

         In a series of successive experiments (Hahn & Seifen, 1959; Hahn
    at al., 1958), Na4P2O7 was added in concentrations of 1.8%, 3% and
    5% to a modified Sherman diet and fed to groups of 34-36 young rats
    for 6 months. The studies also included control groups and groups
    receiving the same levels of sodium monophosphate. With 3% and 5%
    diphosphate diets growth was significantly decreased and at both these
    concentrations nephrocalcinosis appeared as the main toxic effect. The
    degree of damage to the kidneys was about the same as that observed in
    the corresponding monophosphate groups.

         With the 1.8% diphosphate and monophosphate diets, normal growth
    occurred, but a slight yet statistically significant increase in
    kidney weight was noted. Microscopic examination revealed kidney
    calcification in some of the animals, both in the diphosphate and
    monophosphate groups. This was more extensive than the calcification
    occasionally found in the control animals. In an additional
    experiment, 1.1% of diphosphate and of monophosphate were used (Hahn,
    1961). There was a slight growth retardation in the first part of the
    experiment. After 39 weeks a slight degree of kidney calcification was
    noted and this was the same for both phosphates (Hahn, 1961).

         In a recent series of experiments (Datta et al., 1962), Sherman
    diets containing 1%, 2.5% and 5% Na4P2O7 were fed for 16 weeks to
    groups of 20 male and female rats weighing between 90 and 115 g; a
    similar group received a diet containing 5% monophosphate. In the

    sodium phosphate groups, growth was normal up to the 2.5% level;
    kidney weight was increased at the 2.5% level (females) and above;
    kidney function was (concentration test) decreased at the 2.5% level
    (males) and above. Kidney damage (calcification, degeneration and
    necrosis) was observed in a greater percentage of rats in the 1%
    group than in the controls. At the higher concentration of sodium
    diphosphate more severe kidney damage occurred and, in addition, some
    of the animals had hypertrophy and haemorrhages of the stomach. The
    latter abnormality was not found in the 5% monophosphate group.

    (c)  Triphosphates and polyphosphates

    Rat

         Groups of 5 male rats were fed for a period of 1 month on diets
    containing 0.2%, 2% and 10% sodium hexametaphosphate or 0.2%, 2% and
    10% sodium tripolyphosphate. Control groups were given the standard
    diet, or diets with the addition of 10% sodium chloride or 5% disodium
    phosphate (Hodge, 1956).

         With 10% of either of the polyphosphate preparations and also
    with 10% sodium chloride in the diet, growth retardation occurred, but
    none of the rats died. Increased kidney weights and tubular necrosis
    were, however, observed. With 2% of polyphosphate in the diet, growth
    was normal, but in the kidneys inflammatory changes were found which
    were different from the tubular necrosis observed in the 10% groups.
    With 0.2% of polyphosphate in the diet, normal kidneys were seen. In
    another series of experiments (Hahn & Seifen, 1959; Hahn et al., 1958;
    Hahn et al., 1956), 3% and 5% of sodium tripolyphosphate (pH 9.5 in 1%
    solution) and 1.8%, 3% and 5% of Graham's salt (pH 5) were added to a
    modified Sherman diet, which was fed during 24 weeks to groups of 36
    male and 36 female rats. Growth retardation was exhibited by the rats
    in the 5% polyphosphate groups. With 3% of either preparation, a
    temporary growth inhibition was observed, and with 1.8% of Graham's
    salt (male animals) growth was normal. Nephrocalcinosis was observed
    in the 3% and 5% groups. It was noted that the degree of damage with
    Graham's salt was less than that in control groups treated with the
    same concentrations of orthophosphate; with tripolyphosphate, however,
    kidney damage was practically identical with that exhibited by the
    animals in the orthophosphate group. In the animals on a diet
    containing 1.8% Graham's salt, calcification in the kidneys was slight
    or absent and the kidney weights were normal (Hahn & Seifen, 1959).

         In a further group of experiments (van Esch et al., 1957; van
    Genderen, 1958), Kurrol's salt was used in a commercial preparation
    consisting of 1/3 Kurrol's salt and 2/3 of a mixture of disodium and
    tetrasodium diphosphate (Na2H2P2O7 and Na4P2O7). Kurrol's
    salt is practically insoluble in water, but the mixture with 
    diphosphate can be dissolved and a 1% solution had a pH of 7.6. Groups
    of 10 male and 10 female rats were fed for a period of 12 weeks on a

    Sherman diet to which 0.5%, 1%, 2.5% and 5% of the preparation had
    been added. Normal growth was observed in the groups treated with the
    0.5%, 1% and 2.5% concentrations of the polyphosphate mixture, but in
    those receiving the 5% concentrations, growth retardation was
    exhibited. Kidney weights were normal in the 0.5% group, slightly
    increased (males significantly) in the 1% group and further increased
    in the 2.5% and 5% groups. The histopathological examination revealed
    that in the kidneys of the animals of the 5% group, definite
    nephrocalcinosis had occurred, with extensive damage to the tubular
    tissue. Calcification was also observed in other tissues. In the 2.5%
    group, a less extensive nephrocalcification with lymphocyte
    infiltrations were found. In the 0.5% group, kidney structure was
    normal. The results obtained with this polyphosphate preparation were
    practically identical, qualitatively and quantitatively, with the
    results of a similar experiment made with a neutral mixture of
    NaH2PO4 and Na2HPO4 carried out at a later date in the same
    laboratory (Hahn et al., 1958; Gotte, 1953).

         In other experiments, groups of 12 male rats were treated with
    diets to which 0.9% and 3.5% sodium hexametaphosphate had been added
    (corresponding to 0.46% and 1.20% P). Other groups received the
    control diet alone (0.4% P and 0.5% Ca), or with addition of potassium
    monophosphate. To the experimental diets different amounts of salts
    were added to replace cornstarch in order to equalize the levels of
    major minerals; this resulted in a rather high salt concentration.
    The duration of treatment was up to 150 days. With 3.5% added
    hexametaphosphate growth and food and protein efficiency were poorest.
    The kidneys of the animals fed the high level of hexametaphosphate
    were significantly heavier than those of the control rats. This was
    perhaps a manifestation of the high salt load on the kidneys. No
    histopathological abnormalities were observed in kidney sections from
    animals taken from any of the groups (Dymsza et al., 1959).

    Dog

         Sodium tripolyphosphate (Na5P3O10) and sodium hexameta-
    phosphate were fed to 1 dog each in a dose of 0.1 g/kg/day for
    1 month; 2 other dogs received daily doses which increased from
    1.0 g/kg at the beginning to 4.0 g/kg at the end of a 5-month period.

         The dog treated with the starting dose of 10 g/kg/day of
    hexametaphosphate began to lose weight when the daily dose reached
    2.5 g/kg, while the one receiving gradually increasing doses of
    tripolyphosphate lost weight only when its diet contained
    4.0 g/kg/day. In other respects (urinalysis, haematology, organ
    weights) the animals were normal, with the exception of an enlarged
    heart, due to hypertrophy of the left ventricle, in the dog receiving
    gradually increasing doses of sodium tripolyphosphate. In addition,
    tubular damage to the kidneys was observed in both dogs on the higher
    dose regime. In the tissues of the dogs fed 0.1 g/kg/day no changes
    were found that could be attributed to the treatment (Hodge, 1956).

    Other species

         Ammonium polyphosphate, when substituted for 50% or 100% of the
    phosphorus supplied by defluorinated rock phosphate in corn-soybean
    meal diets for growing-finishing pigs, was found not to influence
    daily feed intake, rate of gain, or feed:gain ratio over a 13-week
    period when compared with controls (Clawson & Armstrong, 1981).
    Similar results were observed by Kornegay (1972) in pigs, by Jensen &
    Edwards (1980) in chickens, and by Fishwick (1974) in wether sheep.

    (d)  Calcium and magnesium phosphates

         Groups of weanling Sprague-Dawley rats were fed 0.06% or 0.10%
    magnesium during phosphate depletion for 14 days. It was found that:
    (1) the hypomagnesaemia of phosphate depletion depends on the intake
    of magnesium; (2) the hypercalcaemia and hypercalciuria of phosphate
    depletion are not caused by changes in magnesium homeostasis; (3) low
    dietary magnesium during phosphate depletion may cause a defect in
    soft tissue utilization of P in the growing rat. Animals at the higher
    magnesium level had a significantly lower soft tissue content of P
    compared to those at the lower level (Brautbar et al., 1979).

         Groups of 3-week-old male Sprague-Dawley rats were maintained on
    diets containing either 0.03% or 0.44% phosphorus for periods up to 8
    weeks. Mg balance became negative during the first day of phosphate
    depletion. Mg absorption increased by the third week but Mg balance
    remained negative throughout the 8 weeks. Mg levels in muscle, kidney,
    and liver tissues were not affected but Mg was lost from bone. Bone
    calcium content was not affected (Kreusser et al., 1978).

    Long-term studies

    (a)  Phosphoric acid, phosphates and orthophosphates

    Rat

         Ellinger (1972) cited work done by Bonting in 1952 where over 700
    rats were fed diets containing up to 0.75% phosphoric acid. Effects on
    reproduction were looked for through 3 generations; no adverse effects
    were observed. The study was reported to include examination of the
    blood, tissues, mineral balance, nitrogen retention, and the acidic
    conditions of the digestive tract.

         Three successive generations of rats were fed diets containing
    0.4% and 0.75% of phosphoric acid for 90 weeks. No harmful effects on
    growth or reproduction could be observed. No significant differences
    were noted in the blood picture in comparison with control rats and
    there was no other pathological finding which could be attributed to

    the diets. There was no acidosis or any changes in the calcium
    metabolism. The dental attrition was somewhat more marked than that in
    the control rats (Lang, 1959). No other long-term studies on
    monophosphates have been found in the literature.

    (b)  Disodium phosphate and tetrasodium diphosphate

    Rat

         No specific studies with diphosphates have been made, but in 1
    series of experiments a mixed preparation was used which consisted of
    2/3 Na2H2P2O7 and 1/3 Kurrol's salt. Concentrations of 0.5%, 1%
    and 5% were added to a Sherman diet and given to groups of 10 male and
    10 female rats. From these animals a second and third generation were
    produced, during which the treatment with phosphates was continued.
    Growth and fertility and average life span were normal and the
    life span was not significantly reduced up to the 2.5% level.
    Nephrocalcinosis occurred at the 1% level and above. At 0.5% no
    abnormalities were observed that were not also present in control
    animals. At none of the concentrations did tumours appear with higher
    frequency than in the controls (van Esch et al., 1957).

    (c)  Triphosphates and polyphosphates

    Rat

         To a Sherman diet containing 0.47% P, a mixture of 1/3 Kurrol's
    salt and 2/3 diphosphate was added in concentrations of 0.5%, 1%, 2.5%
    and 5% and fed to groups of 30 male and 10 female rats from weaning to
    end of their life span (van Esch et al., 1957). Two successive
    generations of offspring were produced on these diets. Significant
    growth inhibition was observed in the 5% group of both first and
    second generations. In other groups, growth was normal. Fertility was
    normal in the 0.5%, 1% and 2.5% groups, but much decreased in the 5%
    group. Haematology of the 0.5%, 1% and 2.5% groups showed a decreased
    number of erythrocytes in the 2.5% group, second generation only. In
    the 0.5% group, no kidney damage attributable to the polyphosphate
    treatment was observed, but in the groups having higher intakes renal
    calcification occurred in a degree increasing with the dose level.

         In another series of feeding studies (Hodge, 1960a), diets
    containing 0.05%, 0.5% and 5% sodium tripolyphosphates were given for
    2 years to groups of 50 male and 50 female weanling rats. Only when 5%
    polyphosphate was added to the diet was growth reduced; the reduction
    was significant in males but slight and delayed in females. A smaller
    number of rats survived in the 5% groups than in the other groups. A
    low grade of anaemia was sometimes observed in the 5% groups only.
    Increased kidney weights were noted in the 5% group; pathological
    changes which could be ascribed to treatment were not observed in
    the 0.5% and 0.05% groups. In the control groups and the 0.5%

    tripolyphosphate group, reproduction studies were carried out over 3
    generations involving the production of 2 litters in each generation.
    Reproduction was normal and no changes in the offspring were observed.

         A long-term study (Hodge, 1960b) of the same design was made with
    sodium hexametaphosphate also at concentrations of 0.05%, 0.5% and 5%
    in the diet. Growth retardation occurred only in the 5% groups.
    Mortality was high in all groups but had no relation to the amount of
    hexametaphosphate in the diet. Periodic blood examination gave
    haematological values. Kidney weights were increased in the 5% group
    and calcification was present. Rats given the 0.5% diet did not have
    significant changes in the kidneys. Reproduction studies for 3
    generations in the 0.5% group revealed normal performance in every
    respect.

    Special studies on calcium and phosphorus metabolism with reference
    to calcium and phosphorus and magnesium ratios in the diet

         Diets containing various levels of calcium, phosphorus and
    magnesium were reported to produce uroliths in female Wistar rats. The
    incidence and severity of calcium phosphate uroliths were reduced by
    increasing the magnesium concentration in the diet from 0.2% to 0.8%
    and by increasing the calcium-to-phosphorus ratio to greater than 1
    (Chow et al., 1980).

         Diets containing 0.8% calcium and 0.9% phosphorus caused calcium
    phosphate deposits in the soft tissues of guinea-pigs (Hogan et al.,
    1950). When the phosphorus content of the diet was reduced to 0.5%,
    the number of animals with deposits was reduced from 90% to 10%.
    Further work (House & Hogan, 1955) demonstrated that with sufficient
    magnesium and potassium in the diet, the calcium phosphate deposition
    could be significantly reduced.

         Groups of weanling Sprague-Dawley rats were fed 0.06% or 0.10%
    magnesium during phosphate depletion for 14 days. It was found that:
    (1) the hypomagnesaemia of phosphate depletion depends on the intake
    of magnesium; (2) the hypercalcaemia and hypercalciuria of phosphate
    depletion are not caused by changes in magnesium homeostasis; (3) low
    dietary magnesium during phosphate depletion may cause a defect in
    soft tissue utilization of P in the growing rats. Animals at the
    highest magnesium level had a significantly lower soft tissue content
    of P compared to those at the lower level (Brautbar et al., 1979).

         Groups of 3-week-old male Sprague-Dawley rats were maintained on
    diets containing either 0.03% or 0.44% phosphorus for periods up to 8
    weeks. Mg balance became negative during the first day of phosphate
    depletion. Mg absorption increased by the third week but Mg balance
    remained negative throughout the 8 weeks. Mg levels in muscle, kidney,
    and liver tissues were not affected (Kreusser et al., 1978).

         Although many studies on Ca/P metabolism have been carried out
    with rats, the pig was selected by DeLuca et al. (1976) as a more
    appropriate animal model. Groups of adult pigs were fed diets
    containing 0.65% Ca and 1, 2 or 3 times this amount of P for 6 months.
    Ca:P ratios of 1:2 or 1:3 resulted in decreased weight gain, slight
    hypocalcaemia, significant hyperphosphataemia, increased PTH, and an
    increased rate of bone turnover. At the 1:3 ratio, total kidney
    calcium and phosphorus were increased; rib ash and femoral bone
    formation were decreased.

         Wild-caught cinnamon ringtail monkeys were maintained for up to
    88 months on high phosphorus-containing diets (Ca:P ratios of up to
    1:4). Only minor bone changes were observable with a microscope; these
    changes were not detectable by conventional means (Anderson et al.,
    1977). This finding prompted the authors to suggest that primates may
    tolerate a greater phosphate load than lower species without incurring
    bone loss. They also suggested that findings in lower species must be
    re-evaluated with respect to the hypothesis that high dietary
    phosphate levels are a significant etiological factor in human senile
    osteoporosis.

         High phosphate intakes result in reduced urinary excretion of
    calcium (FASEB, 1981); however, Spencer et al. (1975) demonstrated
    that 47Ca absorption in humans was not influenced by high phosphate
    intakes.

         Bell et al. (1977) fed 8 adult volunteers a diet containing
    0.7 g Ca and 2.1 g P per day for 4 weeks after a 1-month control
    period. Intestinal distress, soft stools, or mild diarrhoea resulted.
    These symptoms subsided in 6 but occurred intermittently throughout
    the 4 weeks in the other 2 subjects. The high phosphorus diet
    increased serum and urinary P and decreased serum and urinary Ca.

         Parfitt & Kleerekoper (1980) reported that the dietary intake of
    phosphate by human adults is rarely great enough to raise plasma
    phosphate levels appreciably.

         FASEB (1981) reviewed the literature but could not reach any
    conclusion on the optimal Ca:P ratio or whether this ratio is of any
    dietary significance in man:

         "High phosphorus intakes in some animal species result in bone
    loss even with normally adequate intakes of calcium. The Ca:P ratio at
    which bone loss occurs varies with the absolute intakes of the
    individual elements; a ratio of 1:2 prevents bone loss when calcium
    intakes are low, whereas bone loss occurs even at a ratio of 2:1 when
    intakes are high. Various expert groups have recommended a Ca:P ratio
    of 1:1 to 2:1 for man, but there is insufficient evidence at present
    to support the establishment of a firm ratio."

         It was noted that the Ca:P ratio in bone is about 2:1 and that
    self-selection of phosphorus in animals approaches this ratio (1.8:1)
    during rapid growth and favours phosphorus in adulthood (1:1.2). Thus,
    children would be more susceptible to excessive dietary phosphate
    intakes.

    OBSERVATIONS IN MAN

         Studies on 15 students, who drank 2000-4000 mg of phosphoric acid
    in fruit juices every day for 10 days, and on 2 males who received
    3900 mg of phosphoric acid every day for 14 days, revealed no
    observable change in urine composition indicative of a disturbed
    metabolism (Lauersen, 1953). The long-continued daily intake of
    5000-7000 mg of NaH2PO4 (corresponding to 1500 mg of P) did not
    produce adverse effects (Lang, 1959). Similarly a daily intake of
    6000 mg of NaH2PO4.2H2O was tolerated for 15 days without
    difficulty (Lauersen, 1953).

         Thomas (1972) administered orthophosphates to some 37 renal
    calculi patients for periods of up to 8 years with usually favourable
    results. Two groups of anionic inhibitors of mineralization were found
    in the urine and the more potent of these was found to be present at a
    lower concentration in the urine of affected patients when compared
    with normal controls. Alkaline phosphate (K2HPO4) was found to cause
    increased concentrations of these mineralization inhibitors in urine
    (acidic and neutral phosphates increased only the less potent
    inhibitor).

         In a study in which the daily basal diet of 4 men contained
    450 mg calcium and 1400 mg phosphorus, supplementation with 750 mg
    phosphorus as phosphoric acid for 1 week resulted in a slight decrease
    in urinary excretion of calcium. When the treatment was continued for
    12 weeks, there was a further decrease in urinary calcium excretion
    (Malm, 1953). In another study, groups of 6 women previously on a
    basal diet containing 300 mg calcium and 800 mg phosphorus, received a
    diet containing 1500 mg calcium and 1400 mg phosphorus, or 1500 mg
    calcium and 800 mg phosphorus. The high level of phosphorus in the
    first diet resulted in decreased calcium utilization when compared to
    the second group (Leichsenring et al., 1951).

    Comments

         Metabolically, the phosphate salts provide a source of the
    various cations and the phosphate ion. Of greatest concern is the
    toxicity arising from calcium, magnesium and phosphate imbalance in
    the diet. Phosphate salts were not mutagenic in a number of test
    systems. Teratogenic effects have not been observed in mammalian test
    systems.

         Numerous animal studies have shown that excessive dietary
    phosphorus causes an increase of plasma phosphorus and a decrease in
    serum calcium. The resulting hypocalcaemia stimulates secretion of PTH
    which in turn increases the rate of bone resorption and decreases
    calcium excretion. These homeostatic adjustments to high dietary
    phosphorus may result in bone loss and calcification of soft tissues
    in animals.

         The dose levels of phosphate producing nephrocalcinosis were not
    consistent among the various rat feeding studies. However, the rat is
    exquisitely susceptible to calcification and hydronephrosis upon
    exposure to acids forming calcium chelates or complexes. The lowest
    dose levels that produce nephrocalcinosis overlap the higher dose
    levels failing to do so. However, this may be related to other dietary
    imbalances, such as the level of magnesium in the diet. There is still
    uncertainty on the optimal Ca:P ratio and whether this ratio is of any
    dietary significance in man.

         The lowest level of phosphate that produced nephrocalcinosis in
    the rat (1% P in the diet) is used as the basis for the evaluation
    and, by extrapolation based on the daily food intake of 2800 calories,
    this gives a dose level of 6600 mg P per day as the best estimate of
    the lowest level that might conceivably cause nephrocalcinosis in man.
    The usual calculation for provision of a margin of safety is probably
    not suitable for food additives that are also nutrients. Ingested
    phosphates from natural sources should be considered together with
    that from food additives sources. Since phosphorus (as phosphates) is
    an essential nutrient and an unavoidable constituent of food, it is
    not feasible or appropriate to give a range of values from zero to a
    maximum.

    EVALUATION

    Estimate of maximum tolerable daily intake for man

    70 mg/kg bw.*

              

    *    This figure represents the maximum tolerable daily intake (MTDI)
         of phosphates. It is not an ADI. The MTDI is expressed as
         phosphorus and it applies to the sum of phosphates naturally
         present in food and the additives listed below. It also applies
         to diets that are nutritionally adequate in respect of calcium.
         However, if the calcium intake were high, the intake of phosphate
         could be proportionately higher, and the reverse relationship
         would also apply.

         Phosphates and polyphosphates evaluated for food additive use
         (see WHO Technical Report Series, No. 683, 1982): phosphoric
         acid; ammonium phosphate, dibasic; bone phosphate; calcium
         phosphate (mono-, di- and tri-basic); magnesium phosphate (mono-,
         di- and tri-basic); sodium phosphate (mono-, di- and tri-basic);
         sodium aluminium phosphate, basic; sodium aluminium phosphate,
         acidic; disodium dihydrogen diphosphate; tetrasodium diphosphate;
         pentasodium triphosphate; pentapotassium triphosphate; sodium
         hexametaphosphate; ammonium polyphosphate; calcium polyphosphate;
         potassium polyphosphate; sodium polyphosphate; sodium potassium
         polyphosphate; sodium tripolyphosphate; calcium pyrophosphate;
         tetrapotassium pyrophosphate.

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    See Also:
       Toxicological Abbreviations