542. Phosphoric acid and phosphate salts (WHO Food Additives Series 17)

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).

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).

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

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.

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).

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

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).

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