World Health Organisation on EDTA plus long term safety studies

[Guest guest]

027. EDTA, calcium disodium (FAO Nutrition Meetings Report Series 40abc) FAO

Nutrition Meetings

Report Series No. 40A,B,C

WHO/Food Add./67.29

TOXICOLOGICAL EVALUATION OF SOME

ANTIMICROBIALS, ANTIOXIDANTS, EMULSIFIERS,

STABILIZERS, FLOUR-TREATMENT AGENTS, ACIDS AND BASES

The content of this document is the result of the deliberations of the

Joint FAO/WHO Expert Committee on Food Additives which met at Rome,

13-20 December, 19651 Geneva, 11-18 October, 19662

1 Ninth Report of the Joint FAO/WHO Expert Committee on Food

Additives, FAO Nutrition Meetings Report Series, 1966 No. 40;

Wld Hlth Org. techn. Rep. Ser., 1966, 339

2 Tenth Report of the Joint FAO/WHO Expert Committee on Food

Additives, FAO Nutrition Meetings Report Series, 1967, in press;

Food and Agriculture Organization of the United Nations

World Health Organization

1967

CALCIUM DISODIUM ETHYLENEDIAMINETETRAACETATE

Synonyms Calcium Disodium EDTA; Calcium Disodium

Edetate

Chemical names Calcium disodium ethylenediaminetetraacetate;

Calcium disodium

(ethylenedinitrilo)tetraacetate

Empirical formula C10H12CaN2Na2O8.2H2O

Structural formula

Molecular weight 410.31

Definition Calcium disodium ethylenediaminetetraacetate,

on the anhydrous basis, contains not less

than 97 per cent. and not more than the

equivalent of 102 per cent.

C10H12CaN2Na2O8.

Description Calcium disodium ethylenediaminetetraacetate

occurs as white, odourless crystalline

granules or as a white to nearly white

powder, slightly hygroscopic with a faint

saline taste.

Use As a sequestrant.

Biological Data

Biochemical aspects of ethylenediaminetetraacetic acid (EDTA) and

its salts

14C-labelled CaNa2EDTA, when fed to rats in doses of 50 mg/kg

body-weight, was absorbed only to an extent of 2-4 per cent.; 80-90

per cent. 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 per

cent. 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

6.5-26 per cent. A dose of 1.5 mg of 14,C-labelled CaNa2EDTA given

in a gelatine capsule to normal healthy men was absorbed to an extent

of 5 per cent. (Foreman & Trujillo, 1954).

In feeding experiments, in rats receiving disodium EDTA at

dietary levels of 0.5, 1.0 and 5.0 per cent., the faeces contained

99.4, 98.2 and 97.5 per cent. of the excreted material (Yang, 1964;

Fellers et al., 1956)

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 per cent. 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 administration to rats, 95-98 per cent. of

injected 14C-labelled CaNa2EDTA appeared in the urine within 6

hours. All the material passed through the body unchanged. Peak plasm

levels were found approximately 50 minutes after administration. Less

than 0.1 per cent. 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

per cent, 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 2 subjects and were almost entirely excreted within

12-16 hours (Srbova & Teisinger, 1957).

The maximum radioactivity in the urine after application of

14C-labelled CaNa2EDTA to the skin was only 10 ppm (Foreman &

Trujillo, 1954).

In biological systems, Ca ion will usually be most accessible to

EDTA. In general, zinc seems to be next most accessible. About 80 per

cent. of the zinc or 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 per cent. 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 that 1 per cent. 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 beams 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 per cent. 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 per cent. of oral FeEDTA,

and the rest was excreted in the faeces, while anaemic rats absorbed

50 per cent. of 6 mg/kg body-weight oral FeEDTA but only 25 per cent.

FeSO4 (Rubin & Princiotto, 1960). Addition of 1 per cent. Na2 EDTA

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 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 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 ( & , 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 intravenously. 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; Mittinen & Leskinen, 1962).

Of the heavy metal-containing enzymes, EDTA at a concentration of

about 10-3M inhibited aldehyde oxidase and homogentisinicase.

Succinic dehydrogenase, xanthine oxidase, NADF-cytochrome reductase,

and ceruloplasmin (oxidation of p-phenylenediamine) were not inhibited

(Westerfield, 1961). Disodium EDTA was found to be a strong inhibitor

for delta-aminolevulinic acid dehydrogenase, 5.5 × 10-6 M causing

50 per cent. inhibition (Gibson et al., 1955). The i.p. injection of

4.2 mmol/kg body-weight (equivalent to 1722 mg/kg body-weight) CaNa2

EDTA caused in rats an inhibition of the alkaline phosphatase of

liver, prostate and serum up to 4 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

body-wight 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, decamethonium

sulfanilic acid and EDTA itself) from isolated segments of rat

intestine, probably due to an increased permeability of the intestinal

wall (Schanker & , 1961).

Acute toxicity

Animal Route LD50 References

mg/kg body-weight

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., 1964

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 jr. 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 per cent. of animals, disappearing almost 2

weeks after stopping the injections (Reuber & Schmieller, 1962).

Short-term studies

Rat. Groups of 5 male rats received 250 or 500 mg/kg

body-weight CaNa2EDTA i.p. daily for 3-21 days and some were observed

for an additional 2 weeks. Weight gain was satisfactory and histology

of lung, thymus, kidney, liver, spleen, adrenal, small gut and heart

was normal 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 3 male and 3 female rats were fed for 4 months or, low

mineral diet containing one-half the usual portion of salt

mixture(i.e. 1.25 per cent. instead of 2.50 per cent.) with the

addition of 0 per cent. and 1.5 per cent. 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, 3 groups of 8-13 male and female rats were

fed a low-mineral diet containing 0 per cent, 0.5 per cent. and 1 per

cent 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).

Dog. Four groups of 1 male and 3 female mongrels were fed diets

containing 0, 50, 100 and 250 mg/kg body-weight 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 1 year. Gross and

microscopic findings were normal (Oser et al., 1963).

Long-term studies

Rat. Four groups of 25 male and 25 female rats ware fed diets

containing 0, 50, 125 and 250 mg/kg body-weight CaNa2EDTA for 2

years. Feeding was carried on through 4 successive generations. Rats

were mated after 12 weeks' feeding and allowed to lactate for 3 weeks

with 1 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 2 litters. Of the second

litters of the F1, F2 and F3 generations only the control and the

250 mg/kg body-weight groups were kept until the end of 2 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 the one-year study in 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.

Evaluation

Level causing no toxicological effect

Rat. 50 000 ppm in the diet, equivalent to 250 mg/kg

body-weight/day.

Estimate of acceptable daily intakes for man

mg/kg bodyweight1

Unconditional acceptance 0-1.25

Conditional acceptance 1.25-2.5

REFERENCES

Bauer, R. O., Rullo, F. R., Spooner, G. & Woodman, E. (1952) Fed.

Proc., 11, 321

Catsch, A. (1961) Fed. Proc., 20 (Suppl. 10), 206

Chan, M. S. (1964) Food Cosmet. Toxicol., 2, 763-765

1 As calcium disodium salt

Chenoweth, M. B. (1961) Fed Proc., 20 (Suppl. 10), 125

Cleton F., Turnbull, A. & Finch, C. A. (1963) 42, 327

Dyckerhoff, H., Marx, R. & Ludwig, B, (1942) Z. ges. exp. Med.,

110, 412

Foreman, H. (1961) Fed. Proc., (Suppl. 10), 191

Foreman, H. & Trujillo, T. T. (1954) J. lab. clin. Med., 43, 566

Foreman, H., Vier, M. & Magee, M. (1953) J. biol. Chem., 203, 1045

Gibson, K. D., Neuberger, A. & , J. C. (1955) Biochem. J.,

61, 618

Gould, R. G. (1961) Fed. Proc., 20 (Suppl. 10), 252

Hawkins, W. W., Leonhard, V. G., Maxwell, J. E. & Rastogi, K. S.

(1962) Canad. J. Biochem., 40, 391

Kratzer, F. H., Allred, J. A., , P. N., Marshall, B. J. & Vohra,

P. (1959) J. Nutr., 68, 313

Lachnit, V. (1961) Arch. Gewerbepath. Gewerbehyg., 18, 495

Lapinleimu, K. & Wegelius, R. (1959) Antibiotic Med. Clin, Ther.

(Br. Edit.),6, 151

Larsen, B. A., Bidwell, R. G. S. & Hawkins, W. W. (1960) Canad. J.

Biochem., 38, 51

Lelièvre, P. (1960) C.R. Soc. Biol. (Paris), 154, 1890

Lelièvre, P. & Betz, E. H. (1961) C.R. Soc. Biol. (Paris), 155, 199

Marquardt, P. & Schumacher, H. (1957) Arzneimittelforsch., 7, 5

Miettinen, T. A. & Leskinen, E. (1962) Ans. Med. exp. Fenn., 40,

427

Nigrovic, V. (1964) Arch. exp. Pathol. Pharmacol., 249, 206

Oser, B. L., Oser, M. & Spencer, H. C. (1963) Toxincol. appl.

Pharmacol., 5, 142

, H. M. & , E. F. (1959) J. clin. Invest., 38, 1452

Pogell, B. M. & Leloir, L. F. (1961) J. biol. Chem., 236, 293

Reuber, M. D. & Schmieller, G. C. (1962) Arch. environ. Health, 5,

430

Rubin, M. (1961) Fed. Proc., (Suppl. 10) 149

Rubin, M. & Princiotto, J. V. (1960) Ann. N.Y. Acad. Sci., 88, 450

Schanker, L. S. & , J. M. (1961) Biochem. Pharmacol., 421

Shibata, S. (1956) Folio pharmacol. Jan., 52, 113

Srbrova, J. & Teisinger, J. (1957) Arch. Gewerbepathol., 15, 572

Westerfeld, W. W. (1961) Fed Proc., (Suppl. 10), 158

Yang, S. S. (1964) Food Cosmet. Toxicol., 2, 763

See Also:

Toxicological Abbreviations

[Guest guest]

> 027. EDTA, calcium disodium (FAO Nutrition Meetings Report Series 40abc) =

FAO Nutrition Meetings

> Report Series No. 40A,B,C

> WHO/Food Add./67.29

>

>

>

>

> TOXICOLOGICAL EVALUATION OF SOME

> ANTIMICROBIALS, ANTIOXIDANTS, EMULSIFIERS,

> STABILIZERS, FLOUR-TREATMENT AGENTS, ACIDS AND BASES

>

>

>

>

>

> The content of this document is the result of the deliberations of th=

e

> Joint FAO/WHO Expert Committee on Food Additives which met at Rome,

> 13-20 December, 19651 Geneva, 11-18 October, 19662

>

>

>

>

>

>

> 1 Ninth Report of the Joint FAO/WHO Expert Committee on Food

> Additives, FAO Nutrition Meetings Report Series, 1966 No. 40;

> Wld Hlth Org. techn. Rep. Ser., 1966, 339

>

> 2 Tenth Report of the Joint FAO/WHO Expert Committee on Food

> Additives, FAO Nutrition Meetings Report Series, 1967, in press;

>

>

> Food and Agriculture Organization of the United Nations

> World Health Organization

> 1967

>

>

> CALCIUM DISODIUM ETHYLENEDIAMINETETRAACETATE

>

> Synonyms Calcium Disodium EDTA; Calcium Disodium

> Edetate

>

> Chemical names Calcium disodium ethylenediaminetetraacetate=

;

>

> Calcium disodium

> (ethylenedinitrilo)tetraacetate

>

> Empirical formula C10H12CaN2Na2O8.2H2O

>

> Structural formula

>

>

>

> Molecular weight 410.31

>

> Definition Calcium disodium ethylenediaminetetraacetate=

,

> on the anhydrous basis, contains not less

> than 97 per cent. and not more than the

> equivalent of 102 per cent.

> C10H12CaN2Na2O8.

>

> Description Calcium disodium ethylenediaminetetraacetate=

> occurs as white, odourless crystalline

> granules or as a white to nearly white

> powder, slightly hygroscopic with a faint

> saline taste.

>

> Use As a sequestrant.

>

> Biological Data

>

> Biochemical aspects of ethylenediaminetetraacetic acid (EDTA) and

> its salts

>

> 14C-labelled CaNa2EDTA, when fed to rats in doses of 50 mg/kg

> body-weight, was absorbed only to an extent of 2-4 per cent.; 80-90

> per cent. 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 i=

n

> the intestines (Foreman et al., 1953).

>

> Experiments in man also revealed poor absorption; only 2.5 per

> cent. of a 3 g dose given was excreted in the urine (Srbova &

> Teisinger, 1957). These authors also confirmed the dissociation of th=

e

> calcium chelate in the stomach. When 200 mg CaNa2EDTA was introduced

> into the duodenum of rats, the authors found an absorption rate of

> 6.5-26 per cent. A dose of 1.5 mg of 14,C-labelled CaNa2EDTA given

> in a gelatine capsule to normal healthy men was absorbed to an extent=

> of 5 per cent. (Foreman & Trujillo, 1954).

>

> In feeding experiments, in rats receiving disodium EDTA at

> dietary levels of 0.5, 1.0 and 5.0 per cent., the faeces contained

> 99.4, 98.2 and 97.5 per cent. of the excreted material (Yang, 1964;

> Fellers et al., 1956)

>

> 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 per cent. 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 tha=

t

> 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 administration to rats, 95-98 per cent. of

> injected 14C-labelled CaNa2EDTA appeared in the urine within 6

> hours. All the material passed through the body unchanged. Peak plasm=

> levels were found approximately 50 minutes after administration. Less=

> than 0.1 per cent. 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 9=

0

> per cent, of the body water, but did not pass into the red blood cell=

s

> 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 2 subjects and were almost entirely excreted within

> 12-16 hours (Srbova & Teisinger, 1957).

>

> The maximum radioactivity in the urine after application of

> 14C-labelled CaNa2EDTA to the skin was only 10 ppm (Foreman &

> Trujillo, 1954).

>

> In biological systems, Ca ion will usually be most accessible to=

> EDTA. In general, zinc seems to be next most accessible. About 80 per=

> cent. of the zinc or liver is freely available to EDTA. The over-all

> availability of the other physiologically important metals is probabl=

y

> in the order: Cu>Fe>Mn >Co (Chenoweth, 1961). EDTA removes about

> 1.4 per cent. 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 that 1 per cent. 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 beams 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 per cent. 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 wa=

s

> degraded prior to absorption, when given orally (Lapinleimu &

> Wegelius, 1959). Rabbits absorbed about 10 per cent. of oral FeEDTA,

> and the rest was excreted in the faeces, while anaemic rats absorbed

> 50 per cent. of 6 mg/kg body-weight oral FeEDTA but only 25 per cent.=

> FeSO4 (Rubin & Princiotto, 1960). Addition of 1 per cent. Na2 EDTA

> 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 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 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 ( & , 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 intravenously. 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 (Lelievr=

e

> & Batz, 1961). The acetylation of sulfanilamide by a liver extract wa=

s

> also inhibited (Lelievre, 1960). EDTA stimulated glucuronide synthesi=

s

> in rat liver, kidney and intestines but inhibited the process in

> guinea-pig liver (Pogell & Leloir, 1961; Mittinen & Leskinen, 1962).

> Of the heavy metal-containing enzymes, EDTA at a concentration of

> about 10-3M inhibited aldehyde oxidase and homogentisinicase.

> Succinic dehydrogenase, xanthine oxidase, NADF-cytochrome reductase,

> and ceruloplasmin (oxidation of p-phenylenediamine) were not inhibite=

d

> (Westerfield, 1961). Disodium EDTA was found to be a strong inhibitor=

> for delta-aminolevulinic acid dehydrogenase, 5.5 × 10-6 M causing

> 50 per cent. inhibition (Gibson et al., 1955). The i.p. injection of

>

> 4.2 mmol/kg body-weight (equivalent to 1722 mg/kg body-weight) CaNa2

> EDTA caused in rats an inhibition of the alkaline phosphatase of

> liver, prostate and serum up to 4 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

> body-wight 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, decamethonium

> sulfanilic acid and EDTA itself) from isolated segments of rat

> intestine, probably due to an increased permeability of the intestina=

l

> wall (Schanker & , 1961).

>

> Acute toxicity

>

>

>

> Animal Route LD50 References

> mg/kg body-weight

>

>

> 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., 1964

>

>

> 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 e=

t

> al., 1963).

>

> Oral doses of over 250 mg/animal cause diarrhoea jr. 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 per cent. of animals, disappearing almost 2

> weeks after stopping the injections (Reuber & Schmieller, 1962).

>

> Short-term studies

>

> Rat. Groups of 5 male rats received 250 or 500 mg/kg

> body-weight CaNa2EDTA i.p. daily for 3-21 days and some were observed=

> for an additional 2 weeks. Weight gain was satisfactory and histolog=

y

> of lung, thymus, kidney, liver, spleen, adrenal, small gut and heart

> was normal 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 3 male and 3 female rats were fed for 4 months or, low=

> mineral diet containing one-half the usual portion of salt

> mixture(i.e. 1.25 per cent. instead of 2.50 per cent.) with the

> addition of 0 per cent. and 1.5 per cent. 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, 3 groups of 8-13 male and female rats wer=

e

> fed a low-mineral diet containing 0 per cent, 0.5 per cent. and 1 per=

> cent 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 significan=

t

> erosion of molars was noted. Basal metabolism was in the normal range=

> (Chan, 1964).

>

> Dog. Four groups of 1 male and 3 female mongrels were fed diets

> containing 0, 50, 100 and 250 mg/kg body-weight 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 1 year. Gross and

> microscopic findings were normal (Oser et al., 1963).

>

> Long-term studies

>

> Rat. Four groups of 25 male and 25 female rats ware fed diets

> containing 0, 50, 125 and 250 mg/kg body-weight CaNa2EDTA for 2

> years. Feeding was carried on through 4 successive generations. Rats

> were mated after 12 weeks' feeding and allowed to lactate for 3 weeks=

> with 1 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 2 litters. Of the second

> litters of the F1, F2 and F3 generations only the control and the

> 250 mg/kg body-weight groups were kept until the end of 2 years' stud=

y

> 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, an=

d

> 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 the one-year study in 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.

>

> Evaluation

>

> Level causing no toxicological effect

>

> Rat. 50 000 ppm in the diet, equivalent to 250 mg/kg

> body-weight/day.

>

> Estimate of acceptable daily intakes for man

>

> mg/kg bodyweight1

>

> Unconditional acceptance 0-1.25

> Conditional acceptance 1.25-2.5

>

> REFERENCES

>

> Bauer, R. O., Rullo, F. R., Spooner, G. & Woodman, E. (1952) Fed.

> Proc., 11, 321

>

> Catsch, A. (1961) Fed. Proc., 20 (Suppl. 10), 206

>

> Chan, M. S. (1964) Food Cosmet. Toxicol., 2, 763-765

>

>

>

>

> 1 As calcium disodium salt

>

>

> Chenoweth, M. B. (1961) Fed Proc., 20 (Suppl. 10), 125

>

> Cleton F., Turnbull, A. & Finch, C. A. (1963) 42, 327

>

> Dyckerhoff, H., Marx, R. & Ludwig, B, (1942) Z. ges. exp. Med.,

> 110, 412

>

> Foreman, H. (1961) Fed. Proc., (Suppl. 10), 191

>

> Foreman, H. & Trujillo, T. T. (1954) J. lab. clin. Med., 43, 566

>

> Foreman, H., Vier, M. & Magee, M. (1953) J. biol. Chem., 203, 1045

>

> Gibson, K. D., Neuberger, A. & , J. C. (1955) Biochem. J.,

> 61, 618

>

> Gould, R. G. (1961) Fed. Proc., 20 (Suppl. 10), 252

>

> Hawkins, W. W., Leonhard, V. G., Maxwell, J. E. & Rastogi, K. S.

> (1962) Canad. J. Biochem., 40, 391

>

> Kratzer, F. H., Allred, J. A., , P. N., Marshall, B. J. & Vohra,=

> P. (1959) J. Nutr., 68, 313

>

> Lachnit, V. (1961) Arch. Gewerbepath. Gewerbehyg., 18, 495

>

> Lapinleimu, K. & Wegelius, R. (1959) Antibiotic Med. Clin, Ther.

> (Br. Edit.),6, 151

>

> Larsen, B. A., Bidwell, R. G. S. & Hawkins, W. W. (1960) Canad. J.

> Biochem., 38, 51

>

> Lelièvre, P. (1960) C.R. Soc. Biol. (Paris), 154, 1890

>

> Lelièvre, P. & Betz, E. H. (1961) C.R. Soc. Biol. (Paris), 155, 199

>

> Marquardt, P. & Schumacher, H. (1957) Arzneimittelforsch., 7, 5

>

> Miettinen, T. A. & Leskinen, E. (1962) Ans. Med. exp. Fenn., 40,

> 427

>

> Nigrovic, V. (1964) Arch. exp. Pathol. Pharmacol., 249, 206

>

> Oser, B. L., Oser, M. & Spencer, H. C. (1963) Toxincol. appl.

> Pharmacol., 5, 142

>

> , H. M. & , E. F. (1959) J. clin. Invest., 38, 1452

>

> Pogell, B. M. & Leloir, L. F. (1961) J. biol. Chem., 236, 293

>

> Reuber, M. D. & Schmieller, G. C. (1962) Arch. environ. Health, 5,

> 430

>

> Rubin, M. (1961) Fed. Proc., (Suppl. 10) 149

>

> Rubin, M. & Princiotto, J. V. (1960) Ann. N.Y. Acad. Sci., 88, 450

>

> Schanker, L. S. & , J. M. (1961) Biochem. Pharmacol., 421

>

> Shibata, S. (1956) Folio pharmacol. Jan., 52, 113

>

> Srbrova, J. & Teisinger, J. (1957) Arch. Gewerbepathol., 15, 572

>

> Westerfeld, W. W. (1961) Fed Proc., (Suppl. 10), 158

>

> Yang, S. S. (1964) Food Cosmet. Toxicol., 2, 763

>

>

>

>

> See Also:

> Toxicological Abbreviations

>

>

>

>

>