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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Pre-GLP study; study meets generally accepted scientific principles
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
publication
Title:
Safety evaluation studies of Calcium EDTA
Author:
Oser BL, Oser M, Spencer HC
Year:
1963
Bibliographic source:
Toxicol. Appl. Pharmacol. 5, 142-162

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Two year feeding study in rats in combination with a 5-generation study (see also section 7.8.1)
GLP compliance:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium calcium edetate
EC Number:
200-529-9
EC Name:
Sodium calcium edetate
Cas Number:
62-33-9
Molecular formula:
C10H12CaN2O8.2Na
IUPAC Name:
calcium disodium 2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetate
Test material form:
other: dilution in water
Details on test material:
A 25% solution of Ca-EDTA was used.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
The rats were housed individually in raised-bottom cages, fresh water being available at all times.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
water
Details on oral exposure:
The basal diet for the rats was a mixture of natural foods supplemented with inorganic salts and vitamins. Its composition resembles the food consumption pattern of the United States population with respect to the ratios of milk, meat, and grain components. Vitamins and minerals were present at levels adequate for normal growth and development. The test material was added at such levels as to provide 50, 125, and 250 mg calcium EDTA (anhydrous basis) per kilogram body weight of rat per day. Because of the initially high and gradually diminishing ratio of food intake to body weight during the period between weaning and maturity, adjustments of the proportion of test material in the diet were made biweekly up to the eleventh week. Since the food intake of rats at this time is normally stabilized at approximately 50 g per kilogram body weight, the concentration of test material was kept constant for the remainder of the study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
In the studies reported, a 25% solution of calcium EDTA was used. Two samples were prepared and stored in polyethylene bottles
at room temperature. Portions for use in the experimental work were withdrawn as needed. Analytical studies demonstrated that these solutions
were stable throughout the period covered by this work.
Duration of treatment / exposure:
Two year
Frequency of treatment:
Daily through the diet
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 125 and 250 mg/kg bw
Basis:
nominal in diet
No. of animals per sex per dose:
25
Control animals:
yes
Details on study design:
One aspect of the extended study included reproduction and lactation experiments. Offspring bred from the dams were raised on their respective
parents' diets and carried through similar short-term studies as in the initial generation. Comparable growth, food intake, and clinical data were consequently assembled for the 12-week postweaning period in four successive generations (see section 7.8.1).

Survivors of the short-term experiment (approximately 23 rats of each sex per group) were continued without change of dietary treatment for the remainder of the 2-year period. Growth records were maintained, but food consumption records were discontinued. All rats were inspected daily for physical condition; the hematologic, blood chemical, and urinary examinations were repeated at 1, 1.5, and 2 years.

When the F0 rats reached 2 years on test, the entire study was terminated.

The rats selected from each generation for breeding were continued on their respective diets for a 12-week feeding period, as described for
the F0 generation. Following the weaning of the second litters in the descendant generation rats at the 50- and 125-mg/kg dosage levels were
sacrificed and examined grossly post mortem, but the control and highest dosage level groups were continued without change in dietary treatment until about the end of the 2-year study. This scheme permitted terminal observations to be made on rats receiving test diets for 0.5, 1, 1.5, or
2 years, in the F3, F2, F1 and F0 generations, respectively.

Positive control:
No

Examinations

Observations and examinations performed and frequency:
The animals were observed daily for physical condition and behavior. The body weights of all rats and the food intake of a representative sampling of 10 rats of each sex per group were recorded weekly. The efficiency of food utilization was calculated for the 12-week period which constituted the short-term phase of the experiment. At 6 and 12 weeks, the following determinations were made on one-fourth of the rats in each group: blood hemoglobin levels, red and white blood cell counts, differential white cell counts, prothrombin time, blood sugar and nonprotein nitrogen, and serum calcium
levels. Urine was examined for albumin and reducing sugar, and the sediment was examined microscopically.

Sacrifice and pathology:
Two rats of each sex per group were sacrificed for histopathologic examination at 12 weeks. Autopsies were conducted on all rats that died throughout the study (except where autolytic changes were too advanced) and on those sacrificed either during or at the end of the test period. Histopathologic examinations were made of the principal organs of the animals or where gross pathologic observations so indicated.

All animals that died, or were sacrificed at the specified periods were autopsied and weights were taken of livers, kidneys, spleens, hearts, adrenals, thyroids, and gonads. As stated above, representative animals in the F0 groups had been sacrificed at 12 weeks for histopathologic each dose level were sacrificed and examined for gross pathologic changes. At both periods livers and kidneys of rats at the lower dose levels were examined microscopically. At the end of the study, 15 or more major organs and tissues were likewise examined in 10 or more rats of each sex in the 250 mg/kg and control groups.
Other examinations:
Because the sequestering action of calcium EDTA suggested the possibility of interference with mineral metabolism, certain additional examinations were made. These included determination of the ash content of the tibias of rats in the highest dosage and control groups; microscopic examination of the jaws of representative animals (at 20 X magnification) for evidence of dental caries; xanthine oxidase determinations in the liver (because molybdenum is a component of this enzyme); and carbonic anhydrase determinations in serum (because zinc is a component of this enzyme).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Short term study
No significant abnormalities of appearance or behavior were noted in the postweaning test period either of the initial group of rats fed levels of 0, 50, 125, and 250 mg calcium EDTA per kilogram body weight or in the three descendant generations raised on the same diets.
The 12-week growth responses of the four generations (F0, F1 , F2 , and F3) revealed the fact that in almost every instance growth of the experimental groups was as good as or better than that of the control groups of the same sex and within the same generation. Except for the females at the two higher levels of dosage (125 and 250 mg/kg), in which case the gains of the test groups were greater than the controls, the differences in weight gain compared to that of the control groups were small and not statistically significant (P > 0.05). The differences noted among the several generations were not consistently related either to dosage or to the sequence of generations.
No evidence of interference with the hematopoietic process was observed. The hemoglobin levels, hematocrits, and red blood cell counts at all levels of dosage and in all of the generations were normal. The leucocyte counts showed no trend with increasing dietary level of the test material. In subsequent generations the ranges were lower but likewise unrelated to dosage. The levels for blood sugar, blood NPN, and serum calcium in all generations were essentially normal regardless of dietary treatment. The findings were also negative with respect to the urinary albumin and sugar, at all levels of dosage and in all generations. A normal assortment of microscopic elements (a few leucocytes, occasional squamous or round epithelial cells, and a moderate number of crystals) was seen, but not to any greater extent in the test groups than in the controls.
The only deaths in the short-term studies were in the F0 generation. Here three control males died between the 10th and 12th weeks, one male in the 50 mg/kg test group died in the 12th week, and one female in the 125 mg/kg group in the 8th week. No deaths occurred in the 250 mg/kg F0 group or at any dosage levels in the descendant generations.

Long term study
From the 2-year responses in the F0 generation it can be seen that up to the 76th week the growth responses within sexes at all levels were essentially equal. During the final half-year the average body weights varied somewhat more because of premortal losses and deaths, but no significant variations occurred in the intergroup relationships. It is clear, therefore, that ingestion of calcium EDTA up to 250 mg per kilogram body weight per day (corresponding to a dietary level of 5000 ppm) for four generations, had no adverse effect upon the growth of these rats. The hemoglobin, hematocrit, and red blood cell counts all fell within normal ranges up to 1 year. Following this there was a slight downward trend in hemoglobin and red cells with advancing age in all groups, including the controls, but there were no dose-related differences. The total and differential leucocyte counts likewise
disclosed no effects attributable to the test material. In all groups, including the controls, the proportion of polymorphonuclear neutrophiles
increased with advancing age, but even at the 2S0-mg dosage level the values were similar to those found in the controls.
Prothrombin times, determined at 78 and 104 weeks in both the control and 2S0-mg groups, were normal. The ranges for average blood sugar, nonprotein nitrogen, and serum calcium values of all groups over the entire 2-year period were not affected. Urinary findings were essentially normal
throughout, the only findings worthy of mention being the slight to moderate (and only occasionally marked) senile albuminuria in 1.5- to
2-year-old rats and the sporadic occurrence of oxalate crystals in all groups early in the test but not subsequently.
At 1.5 years survival in all groups ranged from 62 to 86%. Subsequent losses due to death or to sacrifice of moribund rats reduced the groups to approximately half the original size at the 2-year point. In the last quarter of the 2-year period deaths were somewhat more frequent than has been observed previously in these laboratories, possibly because the entire rat colony was moved to new quarters during this period. Comparison of the test and control groups revealed no significant effects of the dietary treatments on the longevity of the rats. That the small differences in mortality among the various groups were not the result of the dietary treatment is apparent from the response of the 250 mg group in which the average survival of the combined sexes was 61% compared to 45%, for the controls.
By virtue of their diverse character and sporadic distribution among the groups, the gross pathologic findings were considered not to be causally related to test dosage. Pulmonary changes were typical of the respiratory infection common in laboratory rats and their frequency in the test groups
was, for the most part, less than in the controls. Liver abnormalities also occurred at least as frequently in the control as in the test groups. Except for mammary tumors which are fairly common in females with a history of continuous breeding, the character and number of tumors observed indicated them to be of an incidental nature. They occurred with a frequency comparable to that usually seen in this colony.
No significant differences were found for the liver, kidneys, spleen, heart, adrenals, gonads, or thyroid glands.
Microscopically, no important aberrations were evident in the liver, kidneys, gastrointestinal tract, and tibias of the four rats in each group selected for sacrifice either at 12 weeks or at 1 year. Especially noteworthy is the fact that in the 250-mg/kg group, in which 13 organs and tissues of each rat were examined, the findings were consistently negative. In the histopathologic examinations of the F0 generation rats sacrificed at 2 years, principal attention was directed to the highest dose level and the control groups. In the examination of approximately 15 organs and tissues of ten males and ten females in each of those groups, a few organs revealed changes of such a nature and incidence as to suggest a possible relationship to dosage. Hence, the organs in which those deviations were found, namely, liver, pituitary, and adrenal glands, were subsequently examined in the 50- and 125-mg/kg groups and in additional animals of the 250-mg/kg and control groups.
The total incidence of liver pathology for the combined sexes was not significantly greater in the 250-mg/kg group (13/40) than in the controls (11/40). This comparison, as well as the similar incidence at the two lower dosages, is the basis for the conclusion that these hepatic changes are not related to dosage. In the anterior pituitaries, focal hyperplasia was seen occasionally and with equal or greater frequency in the control than in the test groups. Focal hyperplasia in the adrenal cortex and occasionally in the medulla, occurred with a frequency which was not correlated to increasing dosage. Thus, it may be concluded that these changes were not causally related to test dosage.
The remaining organs examined histologically included the kidneys, pancreas, heart, spleen, lungs, marrow, stomach, small and large intestines, gonads, thyroid, parathyroid, lymph nodes, spinal cord, and tibias. In none of these organs (including the few spleens of elevated weight) were significant morphological deviations observed.
The tests and examinations made to determine whether the chelating action of the test material interfered with various aspects of mineral metabolism proved negative. The tibias of rats sacrificed at the 12-week period were examined by the "line-test" procedure used in vitamin D bioassays and showed no evidence of abnormal calcification. At the end of the 2-year period, the ash content of the tibias in the control and 250-mgjkg groups were approximately the same, viz., 56.8 vs. 55.170 in the males and 54.2 vs. 52.3%, respectively, in the females. There was no difference in either the incidence or severity of dental caries in male or female rats fed 0.25% calcium EDTA in the preliminary experiments. Regardless of dose level, there were no detectable differences between the treated and control animals in the F0, F1 , and F2 generations with respect to the number or severity of the carious
lesions. All animals, including controls, showed marked caries activity.
The two metallo-enzymes whose activity was assayed were blood carbonic anhydrase and liver xanthine oxidase, their mineral components being zinc and molybdenum, respectively. F3 generation rats on test diets for at least 6 months were employed for these examinations. Blood was drawn by cardiac puncture and liver tissue was obtained at autopsy. The results revealed no significant effect on the levels of these enzymes.

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related changes up to and including the highest dose of 250 mg/kg bw

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Two-year feeding studies in rats showed no significant deviations from normal physiological responses nor any evidence of interference with mineral metabolism at levels of calcium EDTA up to 250 mg per kilogram body weight.
Executive summary:

In 2-year feeding studies with rats receiving diets containing calcium EDTA at levels to provide 50, 125, or 250 mg per kilogram body weight, no adverse effects on growth or food efficiency were observed. Hematologic examination, conducted periodically, and determination of prothrombin time, blood sugar, NPN, and serum calcium were likewise normal throughout the test period. At autopsy neither gross examination nor the weights of the major organs disclosed any significant differences between the test and control groups. The histopathologic findings likewise revealed no consistent or dose-related effects.

Observations on the incidence and severity of dental caries, and "line tests" of the tibias failed to suggest any evidence of an adverse effect on the calcification processes. Determinations of the xanthine oxidase content in liver tissue at autopsy, and on carbonic anhydrase content of the blood, revealed no significant changes resulting from chronic ingestion of calcium EDTA. The normal physiologic responses and behavior of the rats, even under the stresses of reproduction through successive generations, are consistent with the lack of effect of the chelating agent on these or other important metallo-enzyme systems.