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MSM
Percentage Absorption of EDTA
Toxicological evaluation of
some food
additives including anticaking
agents,
antimicrobials, antioxidants,
emulsifiers
and thickening agents
WHO FOOD
ADDITIVES SERIES NO. 5
The evaluations
contained in this publication
were prepared by the Joint FAO/WHO
Expert
Committee on Food Additives which met
in Geneva,
25 June - 4 July 19731
World Health Organization
Geneva
1974
1
Seventeenth Report of the Joint FAO/WHO Expert Committee
on
Food Additives, Wld Hlth Org. techn.
Rep. Ser., 1974, No. 539;
FAO Nutrition Meetings Report
Series, 1974, No. 53.
ETHYLENEDIAMINETETRAACETATE, DISODIUM AND CALCIUM
DISODIUM SALTS
Explanation
These
compounds have been evaluated for acceptable daily intake
by the Joint FAO WHO Expert Committee
on Food Additives (see Annex 1,
Ref. No. 13) in 1965.
The
previously published monograph has been revised and is
reproduced in its entirety below.
BIOLOGICAL DATA
BIOCHEMICAL ASPECTS
14C-labelled
CaNa2EDTA, when fed to rats in doses of 50 mg
kg
bw, was absorbed only to an
extent of 2 to 4%; 80 to 90% of the dose
appeared in the faeces within 24
hours, and absorption was still
apparent at 48 hours. At the low pH of
the stomach the calcium chelate
is dissociated with subsequent
precipitation of the free acid and this
is only slowly redissolved in the
intestines (Foreman et al., 1953).
Experiments in man also revealed poor absorption; only
2.5% of a
3 g dose given was excreted in the
urine (Srbova & Teisinger, 1957).
These authors also confirmed the
dissociation of the calcium chelate
in the stomach. When 200 mg CaNa2EDTA
was introduced into the
duodenum of rats the authors found an
absorption rate of 6.5 to 26%.
A dose of 1.5 mg of 14C-labelled
CaNa2EDTA given in a gelatine
capsule to normal healthy men was
absorbed to an extent of 5% (Foreman
& Trujillo, 1954).
In
feeding experiments, in rats receiving disodium EDTA at
dietary levels of 0.5, 1.0 and 5.0%,
the faeces contained 99.4, 98.2
and 97.5% of the excreted material
(Yang, 1964).
Similar
experiments conducted also in rats gave essentially the
same results. Thirty-two hours after a
single dose of 95 mg disodium
EDTA/rat, 93% was recovered from the
colon. After doses of 47.5, 95.0
and 142.5 mg disodium EDTA the amount
of EDTA recovered in the urine
was directly proportional to the dose
given, suggesting that EDTA was
absorbed from the gastrointestinal
tract by passive diffusion. The
motility of the intestine was not
affected by the compound (Chan,
1964).
After
parenteral [into the blood directly
-- a vein] administration to rats, 95 to 98% of
injected
14C-labelled CaNa2EDTA
appeared in the urine within six hours. All
the material passed through the body
unchanged. Peak plasma levels
were found approximately 50 minutes
after administration. Less than
0.1% of the material was oxidized to
14CO2, and no organs
concentrated the substance. After i.v.
injection, CaNa2EDTA passed
rapidly out of the vascular system to
mix with approximately 90% of
the body water, but did not pass into
the red blood cells and was
cleared through the kidney by tubular
excretion as well as by
glomerular filtration (Foreman et al.,
1953). The same was also found
in man using 14C-labelled
CaNa2EDTA. Three thousand milligrams were
given i.v. to two subjects and were
almost entirely excreted within 12
to 16 hours (Srbova & Teisinger,
1957).
The
maximum radioactivity in the urine after application of
14C-labelled CaNa2EDTA
to the skin was only 10 ppm (0.001%) (Foreman
& Trujillo, 1954).
In
biological systems, Ca ion will usually, be most
accessible
o EDTA. In general, zinc seems to be
next most accessible. About
80% of the zinc of liver is freely
available to EDTA. The over-all
availability of the other
physiologically important metals is probably
in the order: Cu >Fe >Mn >Co
(Chenoweth, 1961). EDTA removes about
1.4% of the total iron from ferritin
at pH 7.4 to form an iron chelate
(Westerfield, 1961). Transfer of Fe
from Fe-transferrin to EDTA
in vitro occurs at a rate of
less than 1% in 24 hours. In vivo
studies in rabbits demonstrated
transfer of iron only from FeEDTA to
transferrin and not the reverse. It
appeared that tissue iron became
available to chelating agents
including EDTA only when an excess
of iron was present (Cleton et al.,
1963). Equal distribution
between a mixture of EDTA and
siderophilin was obtained only at
EDTA:siderophilin ratios of 20-25:1
(Rubin, 1961). Human iron
deficiency anaemia was successfully
treated with FeEDTA although 84%
of labelled FeEDTA was excreted in the
faeces and none appeared in the
urine. Red cells, however, contained
labelled Fe and reticulocytosis
occurred. Since FeEDTA administered
i.v. was almost quantitatively
excreted in the urine, it was
concluded that FeEDTA was degraded prior
to absorption, when given orally (Lapinleimu
& Wegelius, 1959).
Rabbits absorbed about 10% of oral
FeEDTA, and the rest was excreted
in the faeces, while anaemic rats
absorbed 50% of 6 mg/kg bw oral
FeEDTA but only 25% FeSO4
(Rubin & Princiotto, 1960). Addition of 1%
Na2EDTA to a diet
containing more than optimal amounts of iron and
calcium lowered the absorption and
storage of iron in rats and
increased the amount present in plasma
and urine. The metabolism of
calcium, however, was apparently
unaffected (Larsen et al., 1960). A
diet containing 0.15 mg of iron, 4.26
of calcium and 1 mg of EDTA per
rat (equivalent to 100 ppm (0.01%) in
the diet) for 83 days had no
influence on calcium and iron
metabolism, e.g. the iron content of
liver and plasma (Hawkins et al.,
1962).
CaNa2EDTA
increased the excretion of zinc (Perry & Perry, 1959),
and was active in increasing the
availability of zinc in soybean
containing diets to poults (Kratzer et
al., 1959). CaNa2EDTA enhanced
the excretion of Co, Hg, Mn, Ni, Pb,
Tl and W (Foreman, 1961). The
treatment of heavy metal poisoning
with CaEDTA has become so well
established that its use for more
commonly seen metal poisonings, e.g.
lead, is no longer reported in the
literature (Foreman, 1961). EDTA
could not prevent the accumulation of
90Sr, 106Ru, 141Ba and
226Ra
in the skeleton. 91Y,
239Pu and 238U responded fairly well to
EDTA,
the excretion being accelerated (Catsch,
1961).
EDTA had
a lowering effect on serum cholesterol level when given
orally or i.v. It may have acted by
decreasing the capacity of serum
to transport cholesterol (Gould,
1961). Disodium EDTA had a pyridoxin-
like effect on the tryptophan
metabolism of patients with porphyria or
scleroderma, due to a partial
correction of imbalance of polyvalent
cations (Lelievre & Batz, 1961).
In
vitro, 0.0033 M EDTA inhibited the respiration
of liver
homogenates and of isolated
mitochondria of liver and kidney (Lelievre
& Batz, 1961). The acetylation of
sulfanilamide by a liver extract was
also inhibited (Lelievre, 1960). EDTA
stimulated glucuronide synthesis
in rat liver, kidney and intestines
but inhibited the process in
guinea-pig liver (Pogell & Leloir,
1961; Miettinen & Leskinen, 1962).
Of the heavy metal-containing enzymes,
EDTA at a concentration of
about 10-3 M
inhibited aldehyde oxidase and homogentisinicase.
Succinic dehydrogenase, xanthine
oxidase, NADH-cytochrome reductase
and ceruloplasmin (oxidation of p-phenylenediamine)
were not inhibited
(Westerfield, 1961). Disodium EDTA was
found to be a strong inhibitor
for sigma-aminolevulinic acid
dehydrogenase, 5.5 × 10-6 M
causing 50% inhibition (Gibson et al.,
1955). The i.p. injection of
4.2 mmol/kg bw (equivalent to 1722
mg/kg bw) CaNa2EDTA caused in rats
an inhibition of the alkaline
phosphatase of liver, prostate and serum
up to four days depending on the dose
administered; zinc restored the
activity (Nigrovic, 1964).
In
vitro, EDTA inhibited blood coagulation by chelating
Ca2+.
The complete coagulation inhibition of
human blood required
0.65-1.0 mg/ml. The i.v. injection of
79-200 mg EDTA/rabbit had no
effect on blood coagulation (Dyckerhoff
et al., 1942).
I.v.
injections of Na2EDTA and CaNa2EDTA
had some
pharmacological effect on the blood
pressure of cats; 0-20 mg/kg bw
CaNa2EDTA (as Ca) produce a
slight rise; 20-50 mg/kg, a biphasic
response; and 50 mg/kg, a clear
depression (Marquardt & Schumacher,
1957).
One per
cent. Na2EDTA enhances the absorption of
14C-labelled
acidic, neutral and basic compounds (mannitol,
inulin, decamethenium,
sulfanilic acid and EDTA itself) from
isolated segments of rat
intestine, probably due to an
increased permeability of the intestinal
wall (Schanker & Johnson, 1961).
TOXICOLOGICAL STUDIES
Special studies on embryotoxicity
Disodium
EDTA injected at levels of 3.4, 1.7 and 0.35 mg/egg gave
40, 50 and 85% hatch, respectively. At
the highest level, some embryos
which failed to hatch showed anomalies
(McLaughlin & Scott, 1964).
Acute toxicity
(a) Disodium EDTA
LD50
Animal
Route (mg/kg bw)
References
Rat
oral 2 000-2
200 Yang, 1964
Rabbit
oral 2 300
Shibata, 1956
i.v. 47a
Shibata, 1956
a Dose
depending on the rate of infusion.
(b) Ca-disodium EDTA
LD50
Animal Route
(mg/kg bw)
References
Rat
oral 10 000 ± 740
Oser et al,, 1963
Rabbit oral
7 000 approx. Oser et
al., 1963
i.p. 500 approx.
Bauer et al., 1952
Dog
oral 12 000 approx.
Oser et al., 1963
The oral
LD50 in rats is not affected by the presence
of food in
the stomach or by pre-existing
deficiency in Ca, Fe, Cu or Mn (Oser et
al., 1963).
Oral
doses of over 250 mg/animal cause diarrhoea in rats
(Foreman
et al., 1953).
There
are many reports in the literature on kidney damage by
parenteral over-dosage of CaEDTA. A
review was given by Lachnit
(1961). Lesions simulating "versene
nephrosis" in man have also been
produced in rats. Disodium EDTA in
doses of 400-500 mg i.p. for 21
days caused severe hydropic
degeneration of the proximal convoluted
tubules of the kidneys. CaNa2EDTA
produced only minimal focal
hydropic changes in 58% of animals,
disappearing almost two weeks
after stopping the injections (Reuber
& Schmieller, 1962).
Short-term studies
Rat
Groups
of five male rats received 250 or 500 mg/kg bw CaNa2EDTA
i.p. daily for three to 21 days and
some were observed for an
additional two weeks. Weight gain was
satisfactory and histology of
lung, thymus, kidney, liver, spleen,
adrenal, small gut and heart was
normal except for mild to moderate
renal hydropic change with focal
subcapsular swelling and proliferation
in glomerular loops at the
500 mg level. There was very slight
involvement with complete recovery
at the 250 mg level. Lesions were not
more severe with simultaneous
cortisone administration (Reuber &
Schmieller, 1962).
Groups
of three male and three female rats were fed for four
months on a low mineral diet
containing one-half the usual portion of
salt mixture (i.e. 1.25% instead of
2.50%) with the addition of 0% and
1.5% CaNa2EDTA. The test
group showed a reduced weight gain, but
there was no distinct difference in
general condition of the animals
(Yang, 1964).
In
another experiment three groups of eight to 13 male and
female rats were fed a low-mineral
diet containing 0, 0.5 and 1% of
CaNa2EDTA for 205 days. No
significant differences from the controls
were shown regarding weight gain,
mortality, gross pathology of the
organs and histopathology of liver,
kidney and spleen except a very
slight dilatation of hepatic
sinusoids. Blood coagulation time, total
bone ash and blood calcium level were
unaffected. No significant
erosion of molars was noted. Basal
metabolism was in the normal range
(Chan, 1964).
Rats
were fed for 44 to 52 weeks on a diet containing 0.5%
disodium EDTA without any deleterious
effect on weight gain, appetite,
activity and appearance (Krum, 1948).
In another experiment three
groups of 10 to 13 males and females
were fed a low-mineral diet
(0.54% Ca and 0.013% Fe) with the
addition of 0, 0.5 and 1% disodium
EDTA for 205 days. At the 1% level
some abnormal symptoms were
observed: growth retardation of the
males, lowered erythrocyte and
leucocyte counts, a prolonged blood
coagulation time, slightly but
significantly raised blood calcium
level, a significantly lower ash
content of the bone, considerable
erosion of the molars and diarrhoea.
Gross and histological examination of
the major organs revealed
nothing abnormal. Rats fed for 220
days on an adequate mineral diet
containing 1% disodium EDTA showed no
evidence of dental erosion
(Chan, 1964).
Groups
of six rats were maintained for 12 weeks on diets
containing 0.5, 1 and 5% disodium
EDTA. No deaths occurred and there
were no toxic symptoms except
diarrhoea and lowered food consumption
at the 5% level. Mating in each group
was carried out when the animals
were 100 days old. Mating was repeated
10 days after weaning the first
litters. Parent generation rats of 0,
0.5 and 1% levels gave birth to
normal first and second litters. The
animals given 5% failed to
produce litters (Yang, 1964). To
elucidate possible teratogenic
effects, daily doses of 20-40 mg/rat
EDTA were injected i.m. into
pregnant rats at days six to nine, 10
to 15 and 16 to the end of
pregnancy. A dose of 40 mg was lethal
within four days but 20 mg was
well tolerated, allowing normal fetal
development; 40 mg injected
during days six to eight or 10 to 15
produced some dead or malformed
fetuses, especially polydactyly,
double tail, generalized oedema or
circumscribed head oedema (Tuchmann-Duplessis
& Merciar-Parot, 1956).
Groups
of five male rats were given 250, 400 or 500 mg/kg bw
disodium EDTA i.p. daily for three to
31 days; some groups were
observed for another two weeks. At the
500 mg level all rats became
lethargic and died within nine days,
the kidneys being pale and
swollen, with moderate dilatation of
bowel and subserosal
haemorrhages. Histological examination
of a number of organs showed
lesions only in the kidneys. Animals
at the 400 mg level died within
14 days, kidney and bowel symptoms
being similar to the 500 mg level.
One rat at the 250 mg dose level
showed haemorrhage of the thymus. All
three groups showed varying degrees of
hydropic necrosis of the renal
proximal convoluted tubules with
epithelial sloughing: recovery
occurred in all groups after
withdrawal of disodium EDTA (Reuber &
Schmieller, 1962).
Rabbit
Eight
groups of three rabbits were given either 0.1, 1, 10 or
20 mg/kg bw disodium EDTA i.v., or 50,
100, 500 or 1000 mg/kg bw
orally for one month. All animals on
the highest oral test level
exhibited severe diarrhoea and died.
In the other groups body weight,
haemogram, urinary nitrogen and
urobilinogen were unaffected.
Histopathological examination of a
number of organs showed
degenerative changes in the liver,
kidney, parathyroid and endocrine
organs and oedema in muscle, brain and
heart at all levels of
treatment (Shibata, 1956).
Dog
Four
groups of one male and three female mongrels were fed
diets
containing 0, 50, 100 and 250 mg/kg bw
CaNa2EDTA daily for 12 months.
All appeared in good health, without
significant change in blood
cells, haemoglobin and urine (pH,
albumin, sugar, sediment). Blood
sugar, non-protein nitrogen and
prothrombin time remained normal.
Radiographs of ribs and of long bones
showed no adverse changes at the
250 mg level. All dogs survived for
one year.Gross and microscopic
findings were normal (Oser et al.,
1963).
Long-term studies
Rat
In a
two-year study five groups totalling 33 rats were fed 0,
0.5, 1 and 5% disodium EDTA. The 5%
group showed diarrhoea and
consumed less food than the rats in
other groups. No significant
effects on weight gain were noted nor
were blood coagulation time, red
blood cell counts or bone ash
adversely affected. The mortality of the
animals could not be correlated with
the level of disodium EDTA. The
highest mortality rate occurred in the
control group. Gross and
microscopic examination of various
organs revealed no significant
differences between the groups (Yang,
1964).
Four
groups of 25 male and 25 female rats were fed diets
containing 0, 50, 125 and 250 mg/kg bw
CaNa2EDTA for two years.
Feeding was carried on through four
successive generations. Rats were
mated after 12 weeks' feeding and
allowed to lactate for three weeks
with one week's rest before producing
a second litter. Ten male and
10 female rats of each group (F1
generation) and similar F2 and F3
generation groups were allowed to
produce two litters. Of the second
litters of the F1, F2
and F3 generations only the control and the
250 mg/kg bw groups were kept until
the end of two-years' study on the
F0 generation. This scheme
permitted terminal observation to be made
on rats receiving test diets for 0,
0.5, 1, 1.5 or 2 years in the F3,
F2, F1 and F0
generations, respectively. No significant
abnormalities in appearance and
behaviour were noted during the 12
weeks of the post weaning period in
all generations. The feeding
experiment showed no statistically
significant differences in weight
gain, food efficiency, haemopoiesis,
blood sugar, non-protein
nitrogen, serum calcium, urine, organ
weights and histopathology of
liver, kidney, spleen, heart,
adrenals, thyroid and gonads. Fertility,
lactation and weaning were not
adversely affected for each mating.
Mortality and tumour incidence were
unrelated to dosage level. The
prothrombin time was normal. There was
no evidence of any chelate
effect on calcification of bone and
teeth. Liver xanthine oxidase and
blood carbonic anhydrase activities
were unchanged (Oser et al., 1963)
Comments:
CaNa2EDTA
is very poorly absorbed from the gut. The compound is
metabolically inert and no cumulation
in the body has been found. A
vast clinical experience in its use in
the treatment of metal
poisoning has demonstrated its safety
in man. Long-term feeding
studies in rats and dogs gave no
evidence of interference with mineral
metabolism in either species. Adverse
effects on mineral metabolism
and nephrotoxicity were only seen
after parenteral administration of
high doses.
The
long-term studies with Na2EDTA are difficult
to assess
because of the small number of animals
and the high mortality rate
in all groups. Metabolic studies and
feeding studies demonstrate that
the use of CaNa2EDTA is
preferable to that of Na2EDTA, because of
the effect of the latter in
sequestering calcium. Under certain
circumstances, necessitating an
accurate complexing of ions other than
calcium, it may be used provided no
excess of Na2EDTA remains.
EVALUATION
Level causing no toxicological
effect
Rat:
5000 ppm (0.5%) in the diet equivalent to 250 mg/kg bw.
Estimate of acceptable daily intake
for man
0-2.5*
mg/kg bw
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Chenoweth, M. B. (1961) Fed. Proc.,
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Cleton, F., Turnbull, A. & Finch, C.
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Dyckerhoff, H., Marx, R. & Ludwig, B.
(1942) Z. ges. exp. Med., 110,
412
Foreman, H. (1961) Fed. Proc.,
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* Calculated as CaNa2EDTA.
No excess of Na2EDTA should remain in
foods.
Foreman, H., Vier, M. & Magee, M.
(1953) J. biol. Chem., 203, 1045
Foreman, H. & Trujillo, T. T. (1954)
J. lab. clin. Med., 43, 566
Gibson, K. D., Neuberger, A. & Scott,
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618
Gould, R. G. (1961) Fed. Proc.,
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J. clin. Invest., 38, 1452
Pogell, B. M. & Leloir, L. F. (1961)
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