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Description of key information

The following oral key studies were evaluated for the determination of DNEL values:

Study I: In the present subchronic toxicity study on rats, a NOAEL of 1670 mg/kg bw /day for Na2H2EDTA was found.

Study II: 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.

Study III: In a 1-year study with dogs no treatment-related changes occured up to and including the high dose level of 250 mg/kg bw (nominal) or 338 mg/kg bw (actual intake).

A study according to OECD 410 (sub-acute 28 days, dermal) on CDTA revealed no toxic effects up to a dose of 800 mg/kg bw.

All other repeated dose or reproductive toxicity studies were consideres as less reliable (weight of evidence or supporting study) and thus less relevant for the DNEL derivation.

The lowest NO(A)EL being found was 250 mg/kg bw.

Based on the results of the studies and according to the classification of similar soluble EDTA salts (e.g. EDTA-K2, EDTA-Na2) it is justified to classify as STOT RE 2 (H373).

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1970
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
A 13 weeks feeding study on rats was performed using 3 different dose groups and one control group. After 13 weeks 50% of the animals of each group were sacrificed and tissues examined for gross and histopathologic changes. The remaining animals were placed on control diet for 4 weeks. Thereafter animals were sacrificed and examined for gross and histopathologic changes.
GLP compliance:
no
Species:
rat
Strain:
other: Holtzman
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 120 ± 6 g
- Diet: Ground Purina Laboratory Chow
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
The top level (10.0% ) diet was prepared by adding the appropriate amount of Na2H2EDTA to the basic diet. This was then diluted with the basic diet to prepare the 5.0% diet. Lower concentrations were similarly prepared by dilution of the next highest concentration.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Remarks:
1; 5; 10% Na2H2EDTA
Basis:
nominal in diet

These concentrations in % can be converted in g/kg bw/day using the following calculations: week 1: food consumed = 116 g/week
10.0 % Na2H2EDTA = 11.6 g/week --> 1.65 g/day of EDTA consumed
1.65 g/day / 120 g bw = 0.0138 g/g bw/day = 13.8 g/kg bw/day

Analogously, the doses of EDTA for the 1.0 und 5.0% solutions can be calculated and give the following results:
5.0 % Na2H2EDTA --> 6.607 g/kg bw/day
1.0 % Na2H2EDTA --> 1.67 g/kg bw/day

The calculation were performed considering only the week 1 but can be repeated for the entire duration of the experiment (13 weeks).
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION :
- Mean food consumption g/animal
- Time schedule: weekly

WATER CONSUMPTION
- not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the 4th and 13th week
- Parameters checked: hematocrit, hemoglobin, total and differential white blood cell counts, prothrombin times

CLINICAL CHEMISTRY
- Time schedule for collection of blood: at the end of the 4th and 13th week
- Parameters checked: calcium level

URINALYSIS: Yes
- Time schedule for collection of urine: not specified
- Parameters checked: Calcium

OPHTHALMOSCOPIC EXAMINATION: No
CLINICAL CHEMISTRY: No
Sacrifice and pathology:
Fifty percent of the survivors were sacrificed and autopsied at the end of the 13-week period. Tissues were observed for gross and histopathologic changes (in kidney, liver, heart, testes).
The remaining animals were placed on control diets for 4 weeks and then sacrificed and examined for both gross and histopathologic changes.
Statistics:
Analysis of variance and Duncan multiple range test
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
1% dose group: nothing abnormal detected
5% dose group: 2/10 animals died; diarrhea starting at the third day
10% dose group: 6/10 animals died; animals were emaciated, diarrhea starting at the third day
Mortality:
mortality observed, treatment-related
Description (incidence):
5% dose group: 2/10 animals died; diarrhea starting at the third day
10% dose group: 6/10 animals died; animals were emaciated, diarrhea starting at the third day
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
1% dose group: no difference to the control group
5% and 10% dose group: statistically significant reduced body weight gain
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
1% dose group: normal food consumption
5% and 10% dose group: statistically significant lower food consumption than the control
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
1% dose group: nothing abnormal detected
5% dose group: twice the water consumption of the control
10% dose group: nothing abnormal detected
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
after 4 weeks: hematocrits and hemoglobins of the 10% dose group slightly depressed
after 13 weeks: nothing abnormal detected in any of the groups
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
serum calcium level did not differ from the control
Urinalysis findings:
no effects observed
Description (incidence and severity):
1% dose group: no difference to the control
5% and 10% dose group: ca. twice as much as in the control
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
1% dose group: nothing abnormal detected
5% and 10% dose group: pale livers
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Nothing abnormal detected
Key result
Dose descriptor:
NOAEL
Effect level:
1 670 mg/kg bw/day (nominal)
Sex:
male
Critical effects observed:
not specified
Conclusions:
In the present subchronic toxicity study on rats, a NOAEL of 1670 mg/kg bw /day for Na2H2EDTA was found.
Executive summary:

This subchronic toxicity study on rats showed that animals that received 5.0 or 10.0% EDTA consumed significantly less food than the controls and had lower average body weights. These were dose-related. Persistent diarrhea was observed in both these groups. The mortality rate among the rats receiving 10.0% EDTA diet was 60%.

The autopsies revealed no significant findings except for pale livers of 10.0% EDTA group.

In conclusion, it was found that EDTA was not well tolerated at dietary levels above 5.0% (calculated as 6607 mg/kg bw/day). A NOAEL of 1670 mg/kg bw /day for Na2H2EDTA was derived.

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
1970
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Repeated dose toxicity of K3EDTA can only be caused by the organic moiety of the substance as potassium cations are known to be not toxic. This moiety is also present in Na2EDTA. Alkali metal salts of edetic acid are known to rapidly dissociate under physiological conditions.Thus the repeated dose toxicity of Na2EDTA will be comparable and a read-across approach is justified.
Reason / purpose for cross-reference:
read-across source
Qualifier:
no guideline followed
Principles of method if other than guideline:
A 13 weeks feeding study on rats was performed using 3 different dose groups and one control group. After 13 weeks 50% of the animals of each group were sacrificed and tissues examined for gross and histopathologic changes. The remaining animals were placed on control diet for 4 weeks. Thereafter animals were sacrificed and examined for gross and histopathologic changes.
GLP compliance:
no
Species:
rat
Strain:
other: Holtzman
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 120 ± 6 g
- Diet: Ground Purina Laboratory Chow
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
The top level (10.0% ) diet was prepared by adding the appropriate amount of Na2H2EDTA to the basic diet. This was then diluted with the basic diet to prepare the 5.0% diet. Lower concentrations were similarly prepared by dilution of the next highest concentration.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Remarks:
1; 5; 10% Na2H2EDTA
Basis:
nominal in diet

These concentrations in % can be converted in g/kg bw/day using the following calculations: week 1: food consumed = 116 g/week
10.0 % Na2H2EDTA = 11.6 g/week --> 1.65 g/day of EDTA consumed
1.65 g/day / 120 g bw = 0.0138 g/g bw/day = 13.8 g/kg bw/day

Analogously, the doses of EDTA for the 1.0 und 5.0% solutions can be calculated and give the following results:
5.0 % Na2H2EDTA --> 6.607 g/kg bw/day
1.0 % Na2H2EDTA --> 1.67 g/kg bw/day

The calculation were performed considering only the week 1 but can be repeated for the entire duration of the experiment (13 weeks).
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION :
- Mean food consumption g/animal
- Time schedule: weekly

WATER CONSUMPTION
- not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the 4th and 13th week
- Parameters checked: hematocrit, hemoglobin, total and differential white blood cell counts, prothrombin times

CLINICAL CHEMISTRY
- Time schedule for collection of blood: at the end of the 4th and 13th week
- Parameters checked: calcium level

URINALYSIS: Yes
- Time schedule for collection of urine: not specified
- Parameters checked: Calcium

OPHTHALMOSCOPIC EXAMINATION: No
CLINICAL CHEMISTRY: No
Sacrifice and pathology:
Fifty percent of the survivors were sacrificed and autopsied at the end of the 13-week period. Tissues were observed for gross and histopathologic changes (in kidney, liver, heart, testes).
The remaining animals were placed on control diets for 4 weeks and then sacrificed and examined for both gross and histopathologic changes.
Statistics:
Analysis of variance and Duncan multiple range test
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
1% dose group: nothing abnormal detected
5% dose group: 2/10 animals died; diarrhea starting at the third day
10% dose group: 6/10 animals died; animals were emaciated, diarrhea starting at the third day
Mortality:
mortality observed, treatment-related
Description (incidence):
5% dose group: 2/10 animals died; diarrhea starting at the third day
10% dose group: 6/10 animals died; animals were emaciated, diarrhea starting at the third day
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
1% dose group: no difference to the control group
5% and 10% dose group: statistically significant reduced body weight gain
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
1% dose group: normal food consumption
5% and 10% dose group: statistically significant lower food consumption than the control
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
1% dose group: nothing abnormal detected
5% dose group: twice the water consumption of the control
10% dose group: nothing abnormal detected
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
after 4 weeks: hematocrits and hemoglobins of the 10% dose group slightly depressed
after 13 weeks: nothing abnormal detected in any of the groups
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
serum calcium level did not differ from the control
Urinalysis findings:
no effects observed
Description (incidence and severity):
1% dose group: no difference to the control
5% and 10% dose group: ca. twice as much as in the control
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
1% dose group: nothing abnormal detected
5% and 10% dose group: pale livers
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Nothing abnormal detected
Key result
Dose descriptor:
NOAEL
Effect level:
1 670 mg/kg bw/day (nominal)
Sex:
male
Critical effects observed:
not specified
Conclusions:
In the present subchronic toxicity study on rats, a NOAEL of 1670 mg/kg bw /day for Na2H2EDTA was found.
Executive summary:

This subchronic toxicity study on rats showed that animals that received 5.0 or 10.0% EDTA consumed significantly less food than the controls and had lower average body weights. These were dose-related. Persistent diarrhea was observed in both these groups. The mortality rate among the rats receiving 10.0% EDTA diet was 60%.

The autopsies revealed no significant findings except for pale livers of 10.0% EDTA group.

In conclusion, it was found that EDTA was not well tolerated at dietary levels >= 5.0% (calculated as 6607 mg/kg bw/day). A NOAEL of 1670 mg/kg bw /day for Na2H2EDTA was derived.

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1963
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
In a 2 year feeding study on Wistar rats including reproductive and lactation experiments in four successive generations groups of 25 male and 25 female animals were exposed to CaNa2EDTA at dietary levels providing daily doses of approximately 50, 125, and 250 mg/kg bw.
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
A 25% solution of the test item was used
Species:
rat
Strain:
other: FDRL (derived from Wistar strain)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Housing: individually
- Diet: "natural type diet" ad libitum
- Water: ad libitum
Route of administration:
oral: feed
Vehicle:
water
Details on oral exposure:
Test material was provided daily at 50, 125, and 250 mg/kg bw. 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:
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:
2 years
Frequency of treatment:
Daily through the diet
Remarks:
0, 50, 125 and 250 mg/kg bw
Basis:
nominal in diet
No. of animals per sex per dose:
25
Control animals:
yes, plain diet
Details on study design:
Survivors of the short-term experiment of 12 weeks (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.
Representative animals in the groups had been sacrificed at 12 weeks for histopathologic examination. Again at the end of one year, two males and two females at each dose level were sacrificed and examined for gross pathologic changes.
Positive control:
No
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 weighted 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).
Statistics:
Duncan multiple rank and multiple F test
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:
no effects observed
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.
Key result
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
Critical effects observed:
not specified
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 the 2 -year feeding studies with rats receiving diets containing calcium EDTA at levels 50, 125 or 250 mg/kg bw, no adverse effects on growth or food efficiency were observed. Hematologic examinations and determination of prothrombin time, blood sugar and serum calcium were normal through the test period. At autopsy neither gross examination nor the weight of the major organs disclosed any significant difference between test and control groups.

The normal physiologic responses and behavior of the rats are consistent with the lack of effect of calcium EDTA.

Endpoint:
chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1963
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Repeated dose toxicity of K3EDTA can only be caused by the organic moiety of the substance as potassium cations are known to be not toxic. This moiety is also present in CaNa2EDTA. Alkali metal salts of edetic acid are known to rapidly dissociate under physiological conditions. Considering that reproductive toxicity is a long-term effect also the complex of calcium with edetic acid assumed as dissociative. Thus the repeated dose toxicity of CaNa2EDTA will be comparable and a read-across approach is justified.
Reason / purpose for cross-reference:
read-across source
Qualifier:
no guideline followed
Principles of method if other than guideline:
In a 2 year feeding study on Wistar rats including reproductive and lactation experiments in four successive generations groups of 25 male and 25 female animals were exposed to CaNa2EDTA at dietary levels providing daily doses of approximately 50, 125, and 250 mg/kg bw.
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
A 25% solution of the test item was used
Species:
rat
Strain:
other: FDRL (derived from Wistar strain)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Housing: individually
- Diet: "natural type diet" ad libitum
- Water: ad libitum
Route of administration:
oral: feed
Vehicle:
water
Details on oral exposure:
Test material was provided daily at 50, 125, and 250 mg/kg bw. 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:
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:
2 years
Frequency of treatment:
Daily through the diet
Remarks:
0, 50, 125 and 250 mg/kg bw
Basis:
nominal in diet
No. of animals per sex per dose:
25
Control animals:
yes, plain diet
Details on study design:
Survivors of the short-term experiment of 12 weeks (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.
Representative animals in the groups had been sacrificed at 12 weeks for histopathologic examination. Again at the end of one year, two males and two females at each dose level were sacrificed and examined for gross pathologic changes.
Positive control:
No
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 weighted 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).
Statistics:
Duncan multiple rank and multiple F test
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:
no effects observed
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.
Key result
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
Critical effects observed:
not specified
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 the 2 -year feeding studies with rats receiving diets containing calcium EDTA at levels 50, 125 or 250 mg/kg bw, no adverse effects on growth or food efficiency were observed. Hematologic examinations and determination of prothrombin time, blood sugar and serum calcium were normal through the test period. At autopsy neither gross examination nor the weight of the major organs disclosed any significant difference between test and control groups.

The normal physiologic responses and behavior of the rats are consistent with the lack of effect of calcium EDTA.

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1963
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Dogs were exposed for 3 months
GLP compliance:
no
Specific details on test material used for the study:
A 25% solution of Ca-EDTA was used
Species:
dog
Strain:
other: Mongrel
Sex:
male/female
Details on test animals or test system and environmental conditions:
Sixteen healthy mongrel dogs about 6 months of age were obtained from a reliable commercial dealer. Prior to the experiment they were given a careful physical examination and allowed to become conditioned to the laboratory environment. During this period they were deparasitized and immunized against distemper, hepatitis, and leptospirosis. At regular intervals they were given "booster" injections of the vaccines and sera to provide continued protection against intercurrent disease.
Route of administration:
oral: feed
Vehicle:
water
Details on oral exposure:
The basal diet employed in the dog studies was a commercial kibbled dog meal (Purina) supplemented with 1% mixtures of water- and oil-soluble vitamins, respectively. Calcium EDTA was added to the test rations at levels designed to provide 50, 100, and 250 mg per kilogram of body weight per day on the basis of an assumed intake of 30 g of ration per kilogram body weight per day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
n 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:
12 months with interim examinations at 12 , 26 and 52 weeks.
Frequency of treatment:
Through feeding
Remarks:
0, 50, 100, 250 mg/kg bw (based on an assumed intake of 30 g of ration per kg bw per day)
Basis: nominal intake through diet
Remarks:
0, 58, 130, and 338 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
Four dogs per group (1 male / 3 females in the test groups, 2 males / 2 females in the control group)
Control animals:
yes
Details on study design:
Dogs were housed individually, rations and fresh water being supplied ad libitum.
Positive control:
No
Observations and examinations performed and frequency:
Records were kept of food intake for the first 4 weeks in order to establish the basis for the dietary level of the calcium EDTA necessary to furnish the required dosages per unit of body weight. The appearance and behavior of the dogs were checked daily and records of body weight were made weekly. Initially and at 12 weeks hematologic examinations were made and blood sugar and nonprotein nitrogen levels were determined. The animals were then continued without change in dietary treatment for the remainder of a I-year period. At 12, 26, and 52 weeks the urine of each dog was examined for sugar, albumin, and microscopic elements in the sediment. Observations were continued and hematologic and blood chemical tests were repeated at 26 and 52 weeks.
Sacrifice and pathology:
Toward the end of the I-year study, radiographic examinations were made of the rib cages and leg bones of the dogs receiving the highest dosages of test material, and also, at the end of the test period, of the femurs of all the dogs. Each animal was then sacrificed and autopsied; the major organs were weighed and preserved in formalin. Histopathologic examinations were made of the liver, kidneys, and pituitary of all the dogs. At the 250-mg/kg level, 13 additional organs (pancreas, stomach, small intestine, colon, spleen, heart, thyroid, lymph nodes, bladder, gonads, adrenals, marrow, and bone) were examined.
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
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:
not examined
Details on results:
All the dogs remained in apparent good health and showed normal liveliness and activity throughout the entire period of the test. During the first 12 weeks they gained weight but leveled off during the remainder of the period; in only a few cases small but insignificant weight losses occurred. There were no differences in weight response among the groups attributable to the dietary treatments. Food consumption figures during the first 4 weeks indicated that the quantities actually ingested were somewhat in excess of the amounts expected on the basis of an estimated food intake of 30 g per kilogram body weight. Thus the groups that were expected to receive 50, 100, and 250 mg of test material per kilogram of body weight, actually received on the average 58, 130, and 338 mg/kg, respectively.
The hematologic findings suggest that the dogs at all dosage levels were in a better state of health after one year of test feeding than they were initially. At the start, the hematocrit and hemoglobin levels were on the low normal side whereas many of the leucocyte counts (principally the polymorphonuclear neutrophiles) were elevated, but with the passage of time both parameters were reversed. The effects were similar in all groups and appear to indicate basic improvement in the physiological state of the dogs under the conditions of housing and feeding. At no period during the test did the urine examinations (pH, albumin, sugar, and microscopic elements in the sediment) or blood chemical analyses (sugar, nonprotein nitrogen, and prothrombin time) indicate any abnormalities.
Roentgenographs were taken of the chondrocostal junctions and the tibias of each dog just prior to the termination of the study to determine
whether prolonged ingestion of the sequestrant had interfered with calcification. They were examined without reference to the diets the dogs had
been receiving. No evidence of osteoporosis or other osseous changes was found.
Autopsies of the dogs sacrificed at the end of the year showed no gross pathologic changes. The relative weights of six organs (liver, kidneys,
spleen, heart, adrenals, and gonads) shown in Table 12, were normal. Microscopic examination of the tissues of the dogs revealed abnormal
findings in only two dogs, viz., a chronic pyelonephritis in a control animal and inflammatory cells in the portal area of the liver in a dog in the
100-mg/kg calcium EDTA group. All organs examined in the dogs at the 250-mg/kg level showed negative findings.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 338 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related changes up to and including the high dose level of 250 mg/kg bw (nominal) or 338 mg/kg bw (actual intake)
Critical effects observed:
not specified
Conclusions:
No treatment-related changes up to and including the high dose level of 250 mg/kg bw (nominal) or 338 mg/kg bw (actual intake).
Executive summary:

Groups of dogs were fed diets furnishing 0, 50, 100 and 250 mg/kg bw of calcium EDTA for 1 year. The hematologic findings suggest that dogs at all dosage levels were even better in health after 1 year test than initially. No deviations from normal or control values were noted in urine or blood chemistry. Examinations of rib cages and leg bones in the highest dosage group showed no evidence of osseous change. All dogs survived the 1 -year test period. No gross aberrations were seen at autopsy, and the weights of liver, kidneys, spleen, heart and gonads were normal. Histopathologic findings of the liver, kidneys and pituitary of the adrenals and 12 additional organs in the dogs of the highest dosage group were negative.

Endpoint:
chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1963
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Repeated dose toxicity of K3EDTA can only be caused by the organic moiety of the substance as potassium cations are known to be not toxic. This moiety is also present in CaNa2EDTA. Alkali metal salts of edetic acid are known to rapidly dissociate under physiological conditions. Considering that reproductive toxicity is a long-term effect also the complex of calcium with edetic acid assumed as dissociative. Thus the repeated dose toxicity of CaNa2EDTA will be comparable and a read-across approach is justified.
Reason / purpose for cross-reference:
read-across source
Qualifier:
no guideline followed
Principles of method if other than guideline:
Dogs were exposed for 3 months
GLP compliance:
no
Specific details on test material used for the study:
A 25% solution of Ca-EDTA was used
Species:
dog
Strain:
other: Mongrel
Sex:
male/female
Details on test animals or test system and environmental conditions:
Sixteen healthy mongrel dogs about 6 months of age were obtained from a reliable commercial dealer. Prior to the experiment they were given a careful physical examination and allowed to become conditioned to the laboratory environment. During this period they were deparasitized and immunized against distemper, hepatitis, and leptospirosis. At regular intervals they were given "booster" injections of the vaccines and sera to provide continued protection against intercurrent disease.
Route of administration:
oral: feed
Vehicle:
water
Details on oral exposure:
The basal diet employed in the dog studies was a commercial kibbled dog meal (Purina) supplemented with 1% mixtures of water- and oil-soluble vitamins, respectively. Calcium EDTA was added to the test rations at levels designed to provide 50, 100, and 250 mg per kilogram of body weight per day on the basis of an assumed intake of 30 g of ration per kilogram body weight per day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
n 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:
12 months with interim examinations at 12 , 26 and 52 weeks.
Frequency of treatment:
Through feeding
Remarks:
0, 50, 100, 250 mg/kg bw (based on an assumed intake of 30 g of ration per kg bw per day)
Basis: nominal intake through diet
Remarks:
0, 58, 130, and 338 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
Four dogs per group (1 male / 3 females in the test groups, 2 males / 2 females in the control group)
Control animals:
yes
Details on study design:
Dogs were housed individually, rations and fresh water being supplied ad libitum.
Positive control:
No
Observations and examinations performed and frequency:
Records were kept of food intake for the first 4 weeks in order to establish the basis for the dietary level of the calcium EDTA necessary to furnish the required dosages per unit of body weight. The appearance and behavior of the dogs were checked daily and records of body weight were made weekly. Initially and at 12 weeks hematologic examinations were made and blood sugar and nonprotein nitrogen levels were determined. The animals were then continued without change in dietary treatment for the remainder of a I-year period. At 12, 26, and 52 weeks the urine of each dog was examined for sugar, albumin, and microscopic elements in the sediment. Observations were continued and hematologic and blood chemical tests were repeated at 26 and 52 weeks.
Sacrifice and pathology:
Toward the end of the I-year study, radiographic examinations were made of the rib cages and leg bones of the dogs receiving the highest dosages of test material, and also, at the end of the test period, of the femurs of all the dogs. Each animal was then sacrificed and autopsied; the major organs were weighed and preserved in formalin. Histopathologic examinations were made of the liver, kidneys, and pituitary of all the dogs. At the 250-mg/kg level, 13 additional organs (pancreas, stomach, small intestine, colon, spleen, heart, thyroid, lymph nodes, bladder, gonads, adrenals, marrow, and bone) were examined.
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
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:
not examined
Details on results:
All the dogs remained in apparent good health and showed normal liveliness and activity throughout the entire period of the test. During the first 12 weeks they gained weight but leveled off during the remainder of the period; in only a few cases small but insignificant weight losses occurred. There were no differences in weight response among the groups attributable to the dietary treatments. Food consumption figures during the first 4 weeks indicated that the quantities actually ingested were somewhat in excess of the amounts expected on the basis of an estimated food intake of 30 g per kilogram body weight. Thus the groups that were expected to receive 50, 100, and 250 mg of test material per kilogram of body weight, actually received on the average 58, 130, and 338 mg/kg, respectively.
The hematologic findings suggest that the dogs at all dosage levels were in a better state of health after one year of test feeding than they were initially. At the start, the hematocrit and hemoglobin levels were on the low normal side whereas many of the leucocyte counts (principally the polymorphonuclear neutrophiles) were elevated, but with the passage of time both parameters were reversed. The effects were similar in all groups and appear to indicate basic improvement in the physiological state of the dogs under the conditions of housing and feeding. At no period during the test did the urine examinations (pH, albumin, sugar, and microscopic elements in the sediment) or blood chemical analyses (sugar, nonprotein nitrogen, and prothrombin time) indicate any abnormalities.
Roentgenographs were taken of the chondrocostal junctions and the tibias of each dog just prior to the termination of the study to determine
whether prolonged ingestion of the sequestrant had interfered with calcification. They were examined without reference to the diets the dogs had
been receiving. No evidence of osteoporosis or other osseous changes was found.
Autopsies of the dogs sacrificed at the end of the year showed no gross pathologic changes. The relative weights of six organs (liver, kidneys,
spleen, heart, adrenals, and gonads) shown in Table 12, were normal. Microscopic examination of the tissues of the dogs revealed abnormal
findings in only two dogs, viz., a chronic pyelonephritis in a control animal and inflammatory cells in the portal area of the liver in a dog in the
100-mg/kg calcium EDTA group. All organs examined in the dogs at the 250-mg/kg level showed negative findings.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 338 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related changes up to and including the high dose level of 250 mg/kg bw (nominal) or 338 mg/kg bw (actual intake)
Critical effects observed:
not specified
Conclusions:
No treatment-related changes up to and including the high dose level of 250 mg/kg bw (nominal) or 338 mg/kg bw (actual intake).
Executive summary:

Groups of dogs were fed diets furnishing 0, 50, 100 and 250 mg/kg bw of calcium EDTA for 1 year. The hematologic findings suggest that dogs at all dosage levels were even better in health after 1 year test than initially. No deviations from normal or control values were noted in urine or blood chemistry. Examinations of rib cages and leg bones in the highest dosage group showed no evidence of osseous change. All dogs survived the 1 -year test period. No gross aberrations were seen at autopsy, and the weights of liver, kidneys, spleen, heart and gonads were normal. Histopathologic findings of the liver, kidneys and pituitary of the adrenals and 12 additional organs in the dogs of the highest dosage group were negative.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
chronic
Species:
rat

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
GLP compliance:
yes (incl. QA statement)
Limit test:
yes
Specific details on test material used for the study:
The test item provided by the sponsor was in the powdered form. Hence, the test item was moistened with distilled water and a paste was made and applied on the clipped area of the rat skin.
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Total 50 Males + 50 Females (15 male + 15 females for DRF study), (20 males + 20 females for Main groups, 10 males + 10 females for recovery group and 5 males + 5 females for randomization purpose) were received.
Sex:
male/female
Type of coverage:
occlusive
Vehicle:
water
Details on exposure:
Required quantity of the test item was calculated based on individual animal’s recent body weight and then test item was applied on the clipped area (approximately 10% of the body surface area). After application of the test item that area of skin was covered with cotton gauze and held in place with non-irritating adhesive occlusive (crepe bandage). For control group only distilled water was applied and same procedure was carried out. The test item was held in contact with the skin for at least 6 hour and the patch was removed after completion of exposure period. At the end of the contact period, residual test item was washed using soaked absorbent cotton in distilled water and dried using absorbent cotton.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
The animals were treated with the test item for at least 6 hours daily for a minimum period of 14 days for DRF study and 28 days for main and recovery group animals. After Day 28, the test application for recovery group animals was stopped and these animals were observed for additional 14 days in order to detect delayed occurrence, if any, or recovery from toxic effects.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control group
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
400 mg/kg bw/day (nominal)
Dose / conc.:
800 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 males, 5 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
For the DRF study the following doses were applied: 0, 250, 500, 750, 1000 mg/kg bw.
The dose groups for the main study were choosen due to effects in the highest dose group of the DRF study.
Sacrifice and pathology:
Animals sacrificed by using over dose of CO2 and examined externally.
Clinical signs:
no effects observed
Description (incidence and severity):
No treatment related clinical signs were observed in either sex upto 1000 mg/kg body weight in Dose Range Finding (DRF) study and upto 800 mg/kg body weight in Main study.
Dermal irritation:
no effects observed
Description (incidence and severity):
Skin scoring did not reveal erythyma or oedema in any of the groups throughout the study period for both DRF as well as main study.
Mortality:
no mortality observed
Description (incidence):
No treatment related mortality/morbidity were noted in either sex upto 1000 mg/kg body weight in Dose Range Finding (DRF) study and upto 800 mg/kg body weight in Main study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean body weights for the DRF study showed a statistically significant decrease in the G4 and G5 male. Even in the % body weight change, a reduction was observed in the G5 males. Due to this result obtained, for the main study 1000 mg/kg body weight dose was not selected. Even though this reduction was obtained it can not be certainly said that it is test item related as the number of animals in these groups were 3 and even if one animal shows a reduction (which could be incidental), will give statistical significant result.
Mean body weights remained comparable among the main groups and also between the recovery groups for every time point recorded in both the sexes during and after the exposure period.
Nonetheless, a slight statistically significant increase was observed in % body weight change of G3 female at day 8 when compared to the control group animals (G1). But this difference was not seen till the end of the study period and hence the result can be regarded as inconsequential. Decrease in % body weight of G4R male was also observed at day 8 and day 28 however the same was not observed during the additional 14 days recovery period of these animals and hence this result does not affect the inference of the study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The feed consumed per animal per day remained statistically comparable among the experimental groups within each sex at every point of observation for DRF and the main study animals.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Eye examinations through ophthalmoscope did not reveal any abnormalities in the control or high-dose groups in either sex during the last week of treatment for DRF and Main group and recovery group animals.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The hematology parameters revealed a statistically significant increase in mean corpuscle haemoglobin concentration (MCHC) of G3 males when compared to the control animals. Platelet count was also found to be increased in G3 and G4 males in comparison to the control group however the values obtained were still within the biological range. In female only the Mean Corpuscular Hemoglobin (MCH) parameter decreased significantly for the G4 group animals when compared to the control animals. In view of the results available from the present study, the increased haemoglobin content in females does not seem to be a biologically significant outcome of test-item application. Female recovery group depicted a statistically significant increase in the platelet count, prothrombin time and activated prothrombin time.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Clinical biochemistry parameters revealed statiscally significant increase in the creatinine kinase enzyme level in the G2 male when compared to the G1 control goup animals, however in G2 group only one animal showed that increase and should be considered as incidental. In main group of females none of the parameters showed any change that was significant. However, G4 high dose recovery female group (G4R) showed an increase in the AST levels when compared to the control recovery group (G1R). Though a siginificant decrease has been observed in AST levels, the values obtained for recovery group are well within the biological range and in accordance with the liver histology thus has no impact on the results of the study. The high dose male recovery group (G4R) depicted a statistically significant decrease in the total protein as well as in the globulin level when compared to the male control recovery group (G1R), because of which the A/G ratio increased significantly. Even for both these parameters, the level of total protein as well as globulin falls under the biological range and the decrease has no biological significance to the outcome of test-item application.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
No statistically significant change was observed in the absolute organ weight of all animals of either sex. In the organ weight relative to body weight parameter, G3 (mid-dose) male group showed statistically significant increase in the adrenal when compared to the control group (G1). This observation was inconsistent with the dose levels and therefore not of great informative value, in the context of the current study. An increase in testes was also noted in the G4 recovery male (G4R) group in comparison with the control male recovery group (G1R). A statistically significant increase was also observed in the lungs of female (high dose) recovery group (G4R) when compared to the control recovery group (G1R). The differences in mean weight of lungs is not a cause for concern as far as test-item effects from this study are concerned.
Gross pathological findings:
no effects observed
Description (incidence and severity):
External examination of all male and female rats of control and all treated groups including recovery groups did not reveal any abnormality of pathological significance for both DRF as well as for main study. Internal examination of the rats of control and other treated groups did not reveal any abnormality of pathological significance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopic examination of control group and treatment group at 800mg/kg revealed varying degree of pathological changes in different organs. This includes. Liver: focal mild hepatocellular neutrophilic infiltration (female:G4:1/5), Lungs: multifocal minimal to mild, lymphocytic infiltration (female: G1: 1/5; G4: 2/5). Thyroid: focal minimal ultimobranchial cyst (male: 1/5).
The observed lesions in lungs, liver and thyroid in treated rats are of low occurrence rate and compared well with the rats of control groups in either sex. Such lesions usually develop in the different laboratory animals to certain extent, hence are considered to be spontaneous / incidental in nature. These lesions are also reported by previous workers during toxicological studies (Boorman et al., 1990; Greaves, 2007).
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No neoplastic histopathological findings were observed.
Other effects:
no effects observed
Details on results:
Based on the above-stated results and under the experimental conditions used in this study, it is hereby concluded that the No Observed Adverse Effect Level (NOAEL) of the test item, CDTA acid (Trade name: Goldmann CDTA HHQ) (CAS No.: 13291-61-7) is found to be 800 mg/kg body weight, when administred for 28 days to Wistar Rats with a 14 days recovery period.
Key result
Dose descriptor:
NOAEL
Effect level:
800 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
dermal irritation
food consumption and compound intake
haematology
histopathology: neoplastic
histopathology: non-neoplastic
mortality
ophthalmological examination
organ weights and organ / body weight ratios
Remarks on result:
other: no effects were observed that can be assigned to the toxicity of the test substance.
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
800 mg/kg bw/day (nominal)
Conclusions:
Based on the above-stated results and under the experimental conditions used in this study, it is hereby concluded that the No Observed Adverse Effect Level (NOAEL) of the test item, CDTA acid (Trade name: Goldmann CDTA HHQ) (CAS No.: 13291-61-7) is found to be 800 mg/kg body weight, when administred for 28 days to Wistar Rats with a 14 days recovery period.
Executive summary:

A total of 60 Wistar rats (30 males and 30 females) were randomly allocated to six different dose groups for Main study. Each group consisted of 5 animals/sex for main group and 5 animals/sex for recovery group. Main Study doses were selected based on the results of DRF study. The animals allocated to Group G1/G1R, G2, G3, and G4/G4R received 0, 200, 400 and 800 mg/kg body weight of CDTA acid (Trade name: Goldmann CDTA HHQ) for 28 consecutive days. The animals of G1 group were applied distilled water for 28 consecutive days.

Observations on the animals encompassed mortality/morbidity, onset of any clinical signs/symptoms, skin scoring for erythma and oedema, body weight, body weight changes, feed consumption, ophthalmological examination, hematology, clinical biochemistry, gross pathology and histopathology.

The results of this study reveal that CDTA acid does not cause mortality upto a dose of 800 mg/kg body weight when dermal application was done for 28 days. Further, no clinical signs or symptoms were observed in any animals of the study. While feed consumption remained comparable across groups through the course of the study, a difference in the percent change in body weights was noted between females of G1 and G3 groups and in G1R and G4R male, which, nevertheless, was found unrelated to test-item administration.

Ophtalmological profile was also found normal in the control and high dose groups in both the sex.

Clinical chemistry parameters tested herein were inclusive of serum biochemistry, electrolytes and hematology. The values largely remained unperturbed due to test-item application. The observed variation in different hematological and biochemical parameters at the end of treatment and recovery period, did not show dose dependency and further these variations are noted in either male or female animals. The observed values which varied significantly are well within biological range. Further, no abnormality of pathological significance is observed at histopathological observations which are consistent with these noted variations.

Examination of external features and gross pathology during necropsy revealed no noteworthy observations attributable to test-item administration.

Histopathological observations were made for G1 (vehicle control) and G4 (high-dose) animals and no treatment related changes showed up in any of the observed tissues at the tested dose (800 mg/kg body weight) in either sex.

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Based on the structural similarity and identical functional groups (amino groups at an alkyl chain) a read across approach is justified.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
GLP compliance:
yes (incl. QA statement)
Limit test:
yes
Specific details on test material used for the study:
The test item provided by the sponsor was in the powdered form. Hence, the test item was moistened with distilled water and a paste was made and applied on the clipped area of the rat skin.
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Total 50 Males + 50 Females (15 male + 15 females for DRF study), (20 males + 20 females for Main groups, 10 males + 10 females for recovery group and 5 males + 5 females for randomization purpose) were received.
Sex:
male/female
Type of coverage:
occlusive
Vehicle:
water
Details on exposure:
Required quantity of the test item was calculated based on individual animal’s recent body weight and then test item was applied on the clipped area (approximately 10% of the body surface area). After application of the test item that area of skin was covered with cotton gauze and held in place with non-irritating adhesive occlusive (crepe bandage). For control group only distilled water was applied and same procedure was carried out. The test item was held in contact with the skin for at least 6 hour and the patch was removed after completion of exposure period. At the end of the contact period, residual test item was washed using soaked absorbent cotton in distilled water and dried using absorbent cotton.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
The animals were treated with the test item for at least 6 hours daily for a minimum period of 14 days for DRF study and 28 days for main and recovery group animals. After Day 28, the test application for recovery group animals was stopped and these animals were observed for additional 14 days in order to detect delayed occurrence, if any, or recovery from toxic effects.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control group
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
400 mg/kg bw/day (nominal)
Dose / conc.:
800 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 males, 5 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
For the DRF study the following doses were applied: 0, 250, 500, 750, 1000 mg/kg bw.
The dose groups for the main study were choosen due to effects in the highest dose group of the DRF study.
Sacrifice and pathology:
Animals sacrificed by using over dose of CO2 and examined externally.
Clinical signs:
no effects observed
Description (incidence and severity):
No treatment related clinical signs were observed in either sex upto 1000 mg/kg body weight in Dose Range Finding (DRF) study and upto 800 mg/kg body weight in Main study.
Dermal irritation:
no effects observed
Description (incidence and severity):
Skin scoring did not reveal erythyma or oedema in any of the groups throughout the study period for both DRF as well as main study.
Mortality:
no mortality observed
Description (incidence):
No treatment related mortality/morbidity were noted in either sex upto 1000 mg/kg body weight in Dose Range Finding (DRF) study and upto 800 mg/kg body weight in Main study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean body weights for the DRF study showed a statistically significant decrease in the G4 and G5 male. Even in the % body weight change, a reduction was observed in the G5 males. Due to this result obtained, for the main study 1000 mg/kg body weight dose was not selected. Even though this reduction was obtained it can not be certainly said that it is test item related as the number of animals in these groups were 3 and even if one animal shows a reduction (which could be incidental), will give statistical significant result.
Mean body weights remained comparable among the main groups and also between the recovery groups for every time point recorded in both the sexes during and after the exposure period.
Nonetheless, a slight statistically significant increase was observed in % body weight change of G3 female at day 8 when compared to the control group animals (G1). But this difference was not seen till the end of the study period and hence the result can be regarded as inconsequential. Decrease in % body weight of G4R male was also observed at day 8 and day 28 however the same was not observed during the additional 14 days recovery period of these animals and hence this result does not affect the inference of the study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The feed consumed per animal per day remained statistically comparable among the experimental groups within each sex at every point of observation for DRF and the main study animals.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Eye examinations through ophthalmoscope did not reveal any abnormalities in the control or high-dose groups in either sex during the last week of treatment for DRF and Main group and recovery group animals.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The hematology parameters revealed a statistically significant increase in mean corpuscle haemoglobin concentration (MCHC) of G3 males when compared to the control animals. Platelet count was also found to be increased in G3 and G4 males in comparison to the control group however the values obtained were still within the biological range. In female only the Mean Corpuscular Hemoglobin (MCH) parameter decreased significantly for the G4 group animals when compared to the control animals. In view of the results available from the present study, the increased haemoglobin content in females does not seem to be a biologically significant outcome of test-item application. Female recovery group depicted a statistically significant increase in the platelet count, prothrombin time and activated prothrombin time.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Clinical biochemistry parameters revealed statiscally significant increase in the creatinine kinase enzyme level in the G2 male when compared to the G1 control goup animals, however in G2 group only one animal showed that increase and should be considered as incidental. In main group of females none of the parameters showed any change that was significant. However, G4 high dose recovery female group (G4R) showed an increase in the AST levels when compared to the control recovery group (G1R). Though a siginificant decrease has been observed in AST levels, the values obtained for recovery group are well within the biological range and in accordance with the liver histology thus has no impact on the results of the study. The high dose male recovery group (G4R) depicted a statistically significant decrease in the total protein as well as in the globulin level when compared to the male control recovery group (G1R), because of which the A/G ratio increased significantly. Even for both these parameters, the level of total protein as well as globulin falls under the biological range and the decrease has no biological significance to the outcome of test-item application.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
No statistically significant change was observed in the absolute organ weight of all animals of either sex. In the organ weight relative to body weight parameter, G3 (mid-dose) male group showed statistically significant increase in the adrenal when compared to the control group (G1). This observation was inconsistent with the dose levels and therefore not of great informative value, in the context of the current study. An increase in testes was also noted in the G4 recovery male (G4R) group in comparison with the control male recovery group (G1R). A statistically significant increase was also observed in the lungs of female (high dose) recovery group (G4R) when compared to the control recovery group (G1R). The differences in mean weight of lungs is not a cause for concern as far as test-item effects from this study are concerned.
Gross pathological findings:
no effects observed
Description (incidence and severity):
External examination of all male and female rats of control and all treated groups including recovery groups did not reveal any abnormality of pathological significance for both DRF as well as for main study. Internal examination of the rats of control and other treated groups did not reveal any abnormality of pathological significance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopic examination of control group and treatment group at 800mg/kg revealed varying degree of pathological changes in different organs. This includes. Liver: focal mild hepatocellular neutrophilic infiltration (female:G4:1/5), Lungs: multifocal minimal to mild, lymphocytic infiltration (female: G1: 1/5; G4: 2/5). Thyroid: focal minimal ultimobranchial cyst (male: 1/5).
The observed lesions in lungs, liver and thyroid in treated rats are of low occurrence rate and compared well with the rats of control groups in either sex. Such lesions usually develop in the different laboratory animals to certain extent, hence are considered to be spontaneous / incidental in nature. These lesions are also reported by previous workers during toxicological studies (Boorman et al., 1990; Greaves, 2007).
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No neoplastic histopathological findings were observed.
Other effects:
no effects observed
Details on results:
Based on the above-stated results and under the experimental conditions used in this study, it is hereby concluded that the No Observed Adverse Effect Level (NOAEL) of the test item, CDTA acid (Trade name: Goldmann CDTA HHQ) (CAS No.: 13291-61-7) is found to be 800 mg/kg body weight, when administred for 28 days to Wistar Rats with a 14 days recovery period.
Key result
Dose descriptor:
NOAEL
Effect level:
800 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
dermal irritation
food consumption and compound intake
haematology
histopathology: neoplastic
histopathology: non-neoplastic
mortality
ophthalmological examination
organ weights and organ / body weight ratios
Remarks on result:
other: no effects were observed that can be assigned to the toxicity of the test substance.
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
800 mg/kg bw/day (nominal)
Conclusions:
Based on the above-stated results and under the experimental conditions used in this study, it is hereby concluded that the No observed Adverse Effect Level (NOAEL) of the test item, CDTA acid (Trade name: Goldmann CDTA HHQ) (CAS No.: 13291-61-7) is found to be 800 mg/kg body weight, when administred for 28 days to Wistar Rats with a 14 days recovery period.

Based on different molecular weights of the test substance and the substance to be registered a weight correction is necessary.
Molecular weight CDTA (test substance): 346.3 g/mol
Molecular weight EDTA-K3 (substance to be registered): 405.5 g/mol
800 mg/kg body weight * 405.5 g/mol / 346.3 g/mol = 937 mg/kg body weight

Thus the corrected NOAEL for EDTA-K3 can be stated to be 937 mg/kg body weight.
Executive summary:

A total of 60 Wistar rats (30 males and 30 females) were randomly allocated to six different dose groups for Main study. Each group consisted of 5 animals/sex for main group and 5 animals/sex for recovery group. Main Study doses were selected based on the results of DRF study. The animals allocated to Group G1/G1R, G2, G3, and G4/G4R received 0, 200, 400 and 800 mg/kg body weight of CDTA acid (Trade name: Goldmann CDTA HHQ) for 28 consecutive days. The animals of G1 group were applied distilled water for 28 consecutive days.

Observations on the animals encompassed mortality/morbidity, onset of any clinical signs/symptoms, skin scoring for erythma and oedema, body weight, body weight changes, feed consumption, ophthalmological examination, hematology, clinical biochemistry, gross pathology and histopathology.

The results of this study reveal that CDTA acid does not cause mortality upto a dose of 800 mg/kg body weight when dermal application was done for 28 days. Further, no clinical signs or symptoms were observed in any animals of the study. While feed consumption remained comparable across groups through the course of the study, a difference in the percent change in body weights was noted between females of G1 and G3 groups and in G1R and G4R male, which, nevertheless, was found unrelated to test-item administration.

Ophtalmological profile was also found normal in the control and high dose groups in both the sex.

Clinical chemistry parameters tested herein were inclusive of serum biochemistry, electrolytes and hematology. The values largely remained unperturbed due to test-item application. The observed variation in different hematological and biochemical parameters at the end of treatment and recovery period, did not show dose dependency and further these variations are noted in either male or female animals. The observed values which varied significantly are well within biological range. Further, no abnormality of pathological significance is observed at histopathological observations which are consistent with these noted variations.

Examination of external features and gross pathology during necropsy revealed no noteworthy observations attributable to test-item administration.

Histopathological observations were made for G1 (vehicle control) and G4 (high-dose) animals and no treatment related changes showed up in any of the observed tissues at the tested dose (800 mg/kg body weight) in either sex.

Additional information

Justification for classification or non-classification