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Registration Dossier
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EC number: 236-308-9 | CAS number: 13291-61-7
Toxicological information
Endpoint summary
Administrative data
Description of key information
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.
A subchronic toxicity study (13 weeks) on rats receiving 1.0%,
5.0 and 10.0% Na2H2EDTA showed that animals that received 5.0 or 10.0% EDTA consumed significantly less food than the controls and had lower average body weights.The mortality rate among the rats receiving 10.0% EDTA diet was 60%. A NOAEL of 1670 mg/kg bw /day for Na2H2EDTA was derived.
A NOAEL >= 250 mg/kg bw /day was therefore assumed for the substance trans-cyclohexane-1,2-dinitrilotetraacetic acid (CAS 13291-61-7), structurally similar to the substances tested. Up to the dose tested, there was no adverse effect observed.
Key value for chemical safety assessment
- Toxic effect type:
- dose-dependent
Repeated dose toxicity: via oral route - systemic effects
Link to relevant study records
- 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:
- The substance disodium dihydrogen ethylenediaminetetraacetate (CAS 139-33-3) is structurally similar to the substance trans-cyclohexane-1,2-dinitrilotetraacetic acid (CAS 13291-61-7), therefore used for the read-across.
- 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:
- The substance Sodium calcium edetate (CAS 62-33-9) is structurally similar to the substance trans-cyclohexane-1,2-dinitrilotetraacetic acid (CAS 13291-61-7), therefore used for the read-across.
- 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:
- The substance Sodium calcium edetate (CAS 62-33-9) is structurally similar to the substance trans-cyclohexane-1,2-dinitrilotetraacetic acid (CAS 13291-61-7), therefore used for the read-across.
- 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:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 4 (not assignable)
- Rationale for reliability incl. deficiencies:
- documentation insufficient for assessment
- Qualifier:
- no guideline available
- Principles of method if other than guideline:
- A 1 month feeding study on rats was performed using 3 different dose groups. 10 days after the start of the study half of the animals of each group were sacrificed on completion of the feeding period the remaining animals were sacrificed for biochemical and morphological examinations. Additionally one group was treated with CaNa2EDTA.
- GLP compliance:
- not specified
- Species:
- rat
- Strain:
- not specified
- Sex:
- male/female
- Route of administration:
- oral: feed
- Vehicle:
- not specified
- Details on oral exposure:
- - TS was incorporated in the normal dietary constituents
- Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- 1 month
- Frequency of treatment:
- daily
- Dose / conc.:
- 500 mg/kg bw/day (nominal)
- Remarks:
- original data: 1.0% of the diet, assuming an conversion factor of 20
- Dose / conc.:
- 1 125 mg/kg bw/day (nominal)
- Remarks:
- original data: 2.25% of the diet, assuming an conversion factor of 20
- Dose / conc.:
- 2 500 mg/kg bw/day (nominal)
- Remarks:
- original data: 5% of the diet, assuming an conversion factor of 20
- No. of animals per sex per dose:
- 15
- Control animals:
- not specified
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- Some mortalities in the 2500 mg/kg bw/day dose group (no further data)
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Marked suppression of body weight in the 2500 mg/kg bw/day dose group (no further data)
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Reduction of total leucocytes and lymphocytes, increase of bound urine nitrogen and decrease of Ca in serum in the 2500 mg/kg bw/day dose group (no further data)
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Decrease of liver, spleen and thymus weight in the 2500 mg/kg bw dose group (no further data).
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- In the animals which were sacrificed at the 10th day of the study, some parakeratosis was detected in the oesophagus and forestomach in the 2500 mg/kg bw/day dose group.
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 1 125 mg/kg bw/day (nominal)
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- mortality
- Critical effects observed:
- not specified
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- supporting study
- Reliability:
- 4 (not assignable)
- Rationale for reliability incl. deficiencies:
- documentation insufficient for assessment
- Justification for type of information:
- The substance Disodium dihydrogen ethylenediaminetetraacetate (CAS 139-33-3) is structurally similar to the substance trans-cyclohexane-1,2-dinitrilotetraacetic acid (CAS 13291-61-7), therefore used for the read-across.
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- no guideline available
- Principles of method if other than guideline:
- A 1 month feeding study on rats was performed using 3 different dose groups. 10 days after the start of the study half of the animals of each group were sacrificed on completion of the feeding period the remaining animals were sacrificed for biochemical and morphological examinations. Additionally one group was treated with CaNa2EDTA.
- GLP compliance:
- not specified
- Species:
- rat
- Strain:
- not specified
- Sex:
- male/female
- Route of administration:
- oral: feed
- Vehicle:
- not specified
- Details on oral exposure:
- - TS was incorporated in the normal dietary constituents
- Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- 1 month
- Frequency of treatment:
- daily
- Dose / conc.:
- 500 mg/kg bw/day (nominal)
- Remarks:
- original data: 1.0% of the diet, assuming an conversion factor of 20
- Dose / conc.:
- 1 125 mg/kg bw/day (nominal)
- Remarks:
- original data: 2.25% of the diet, assuming an conversion factor of 20
- Dose / conc.:
- 2 500 mg/kg bw/day (nominal)
- Remarks:
- original data: 5% of the diet, assuming an conversion factor of 20
- No. of animals per sex per dose:
- 15
- Control animals:
- not specified
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- Some mortalities in the 2500 mg/kg bw/day dose group (no further data)
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Marked suppression of body weight in the 2500 mg/kg bw/day dose group (no further data)
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Reduction of total leucocytes and lymphocytes, increase of bound urine nitrogen and decrease of Ca in serum in the 2500 mg/kg bw/day dose group (no further data)
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Decrease of liver, spleen and thymus weight in the 2500 mg/kg bw dose group (no further data).
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- In the animals which were sacrificed at the 10th day of the study, some parakeratosis was detected in the oesophagus and forestomach in the 2500 mg/kg bw/day dose group.
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 1 125 mg/kg bw/day (nominal)
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- mortality
- Critical effects observed:
- not specified
- Endpoint:
- chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Principles of method if other than guideline:
- A bioassay for possible carcinogenicity was conducted by administering the test material in feed to B6C3F1 mice. The chemical was administered to 50 males and 50 females at low and high concentrations, for 103 weeks. Matched-control groups were composed of 20 males and 20 females. Animals were analysed for mortality, clinical signs, histopathological as well as gross pathological changes.
- GLP compliance:
- no
- Limit test:
- no
- Species:
- mouse
- Strain:
- B6C3F1
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, USA
- Weight at study initiation: 19-22 g
- Age at study initiation: 28 days
- Housing: five per cage in solid polycarbonate cages
- Diet: prepared from Wayne Lab Blox Meal (Allied Mills, Inc.) ad libitum
- Water: acidulated water ad libitum
- Acclimation period: 14 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25°C
- Humidity (%): 45-55%
- Air changes (per hr): 15 times per hour
- Photoperiod (hrs dark / hrs light): 16/8 - Route of administration:
- oral: feed
- Vehicle:
- unchanged (no vehicle)
- Details on oral exposure:
- DIET PREPARATION
- Rate of preparation of diet (frequency): 2 times/week
- Mixing appropriate amounts with (Type of food): Wayne Lab Blox Meal (Allied Mills, Inc.)
- Storage temperature of food: 4°C
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- - Analyses were performed, using FDA methods to determine the efficiency of the mixing procedure and the stability of the test chemical in feed. Recoveries were found to be 90.3 + / -1.4% of the theoretical value at 7500 ppm EDTA and 90.4 + / - 3.4% of the theoretical value at 3750 ppm. It was concluded from these results that the preparations contained reasonably accurate concentrations of EDTA and were mixed homogeneously, and that the chemical was stable in feed for at least a week.
- Duration of treatment / exposure:
- 103 weeks
- Frequency of treatment:
- daily
- Dose / conc.:
- 469 mg/kg bw/day (nominal)
- Remarks:
- original data: 3750 ppm; conversion factor according to EU risk assessment
- Dose / conc.:
- 938 mg/kg bw/day (nominal)
- Remarks:
- original data: 7500 ppm; conversion factor according to EU risk assessment
- No. of animals per sex per dose:
- 50 (except for the control, which consisted of only 20 animals)
- Control animals:
- yes, plain diet
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily
BODY WEIGHT: Yes
- Time schedule for examinations: approximately once a month - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes; all major organs, not specified but presumably all organs used for histopathology: skin, lymph nodes, mammary gland, salivary gland, bone marrow, trachea, lungs and bronchi, heart, thyroids, parathyroids, esophagus, stomach, small intestine, large intestine, liver, gallbladder, pancreas, spleen, kidneys, adrenals, urinary bladder, prostate or uterus, testis or ovary, brain, and pituitary.
HISTOPATHOLOGY: Yes;
skin, lymph nodes, mammary gland, salivary gland, bone marrow, trachea, lungs and bronchi, heart, thyroids, parathyroids, esophagus, stomach, small intestine, large intestine, liver, gallbladder, pancreas, spleen, kidneys, adrenals, urinary bladder, prostate or uterus, testis or ovary, brain, and pituitary. - Statistics:
- - Statistical tests of differences in survival between groups are compared using the method of Cox (1972) for two groups and an extension of this method by Tarone (1975) for more than two groups.
- Statistical analysis of the incidence of tumors was made using the Fisher exact test (Cox, 1970) to compare a control group to a group of treated animals at each dose. In addition, the Armitage and Cochran test for linear trend in proportions, with continuity correction (Armitage, 1971), was used. - Description (incidence and severity):
- Ataxia occurred in a low-dose male at 8 months, and ascites was noted in mice of both sexes during the second year of the study.
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- No statistically significant difference in survival between the different groups in both sexes. (males: 5/50 (10%) of the low-dose group, 2/50 (4%) of the high-dose group, and 1/20 (5%) of the control; females: 0/50 low-dose female mice, 1/20 (5%) of the control group and 3/50 (6%) of the high-dose group)
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- In male mice only the high-dose group showed throughout most of the test period a decrease in average body weight compared to the controls. In female mice average body weights of the treatment groups were depressed in a dose-related manner during the test period, although the effect was small. No individual data given. No information whether this effect is statistically relevant is available.
- Histopathological findings: neoplastic:
- effects observed, non-treatment-related
- Description (incidence and severity):
- A variety of neoplasms was observed in both treated and control animals. Each type observed has been encountered previously as a spontaneous lesion in the mouse. However, the incidence of neoplasms in all groups was high in the hematopoietic, endocrine, digestive, and respiratory systems. The incidence of neoplasms in other systems was variable. (See table 1 and 2)
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- >= 938 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- body weight and weight gain
- clinical signs
- gross pathology
- histopathology: non-neoplastic
- mortality
- Critical effects observed:
- not specified
- Endpoint:
- chronic toxicity: oral
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- 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:
- The substance Trisodium hydrogen ethylenediaminetetraacetate (CAS 150-38-9) is structurally similar to the substance trans-cyclohexane-1,2-dinitrilotetraacetic acid (CAS 13291-61-7), therefore used for the read-across.
- Reason / purpose for cross-reference:
- read-across source
- Principles of method if other than guideline:
- A bioassay for possible carcinogenicity was conducted by administering the test material in feed to B6C3F1 mice. The chemical was administered to 50 males and 50 females at low and high concentrations, for 103 weeks. Matched-control groups were composed of 20 males and 20 females. Animals were analysed for mortality, clinical signs, histopathological as well as gross pathological changes.
- GLP compliance:
- no
- Limit test:
- no
- Species:
- mouse
- Strain:
- B6C3F1
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, USA
- Weight at study initiation: 19-22 g
- Age at study initiation: 28 days
- Housing: five per cage in solid polycarbonate cages
- Diet: prepared from Wayne Lab Blox Meal (Allied Mills, Inc.) ad libitum
- Water: acidulated water ad libitum
- Acclimation period: 14 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25°C
- Humidity (%): 45-55%
- Air changes (per hr): 15 times per hour
- Photoperiod (hrs dark / hrs light): 16/8 - Route of administration:
- oral: feed
- Vehicle:
- unchanged (no vehicle)
- Details on oral exposure:
- DIET PREPARATION
- Rate of preparation of diet (frequency): 2 times/week
- Mixing appropriate amounts with (Type of food): Wayne Lab Blox Meal (Allied Mills, Inc.)
- Storage temperature of food: 4°C
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- - Analyses were performed, using FDA methods to determine the efficiency of the mixing procedure and the stability of the test chemical in feed. Recoveries were found to be 90.3 + / -1.4% of the theoretical value at 7500 ppm EDTA and 90.4 + / - 3.4% of the theoretical value at 3750 ppm. It was concluded from these results that the preparations contained reasonably accurate concentrations of EDTA and were mixed homogeneously, and that the chemical was stable in feed for at least a week.
- Duration of treatment / exposure:
- 103 weeks
- Frequency of treatment:
- daily
- Dose / conc.:
- 469 mg/kg bw/day (nominal)
- Remarks:
- original data: 3750 ppm; conversion factor according to EU risk assessment
- Dose / conc.:
- 938 mg/kg bw/day (nominal)
- Remarks:
- original data: 7500 ppm; conversion factor according to EU risk assessment
- No. of animals per sex per dose:
- 50 (except for the control, which consisted of only 20 animals)
- Control animals:
- yes, plain diet
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily
BODY WEIGHT: Yes
- Time schedule for examinations: approximately once a month - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes; all major organs, not specified but presumably all organs used for histopathology: skin, lymph nodes, mammary gland, salivary gland, bone marrow, trachea, lungs and bronchi, heart, thyroids, parathyroids, esophagus, stomach, small intestine, large intestine, liver, gallbladder, pancreas, spleen, kidneys, adrenals, urinary bladder, prostate or uterus, testis or ovary, brain, and pituitary.
HISTOPATHOLOGY: Yes;
skin, lymph nodes, mammary gland, salivary gland, bone marrow, trachea, lungs and bronchi, heart, thyroids, parathyroids, esophagus, stomach, small intestine, large intestine, liver, gallbladder, pancreas, spleen, kidneys, adrenals, urinary bladder, prostate or uterus, testis or ovary, brain, and pituitary. - Statistics:
- - Statistical tests of differences in survival between groups are compared using the method of Cox (1972) for two groups and an extension of this method by Tarone (1975) for more than two groups.
- Statistical analysis of the incidence of tumors was made using the Fisher exact test (Cox, 1970) to compare a control group to a group of treated animals at each dose. In addition, the Armitage and Cochran test for linear trend in proportions, with continuity correction (Armitage, 1971), was used. - Description (incidence and severity):
- Ataxia occurred in a low-dose male at 8 months, and ascites was noted in mice of both sexes during the second year of the study.
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- No statistically significant difference in survival between the different groups in both sexes. (males: 5/50 (10%) of the low-dose group, 2/50 (4%) of the high-dose group, and 1/20 (5%) of the control; females: 0/50 low-dose female mice, 1/20 (5%) of the control group and 3/50 (6%) of the high-dose group)
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- In male mice only the high-dose group showed throughout most of the test period a decrease in average body weight compared to the controls. In female mice average body weights of the treatment groups were depressed in a dose-related manner during the test period, although the effect was small. No individual data given. No information whether this effect is statistically relevant is available.
- Histopathological findings: neoplastic:
- effects observed, non-treatment-related
- Description (incidence and severity):
- A variety of neoplasms was observed in both treated and control animals. Each type observed has been encountered previously as a spontaneous lesion in the mouse. However, the incidence of neoplasms in all groups was high in the hematopoietic, endocrine, digestive, and respiratory systems. The incidence of neoplasms in other systems was variable. (See table 1 and 2)
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- >= 938 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- body weight and weight gain
- clinical signs
- gross pathology
- histopathology: non-neoplastic
- mortality
- Critical effects observed:
- not specified
- Endpoint:
- chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Principles of method if other than guideline:
- A study for possible carcinogenicity was conducted by administering the test material in feed to Fischer 344 rats. The chemical was administered to 50 males and 50 females at low and high concentrations, for 103 weeks. Matched-control groups were composed of 20 males and 20 females. Animals were analysed for mortality, clinical signs, histopathological as well as gross pathological changes.
- GLP compliance:
- no
- Limit test:
- no
- Species:
- rat
- Strain:
- Fischer 344
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Schmidt, Madison, Wisconsin, USA
- Weight at study initiation: 85-110 g
- Age at study initiation: 28 days
- Housing: four per cage in solid polycarbonate cages
- Diet: prepared from Wayne Lab Blox Meal (Allied Mills, Inc.) ad libitum
- Water: acidulated water ad libitum
- Acclimation period: 14 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25°C
- Humidity (%): 45-55%
- Air changes (per hr): 15 times per hour
- Photoperiod (hrs dark / hrs light): 16/8 - Route of administration:
- oral: feed
- Vehicle:
- unchanged (no vehicle)
- Details on oral exposure:
- DIET PREPARATION
- Rate of preparation of diet (frequency): 3 times/week
- Mixing appropriate amounts with (Type of food): Wayne Lab Blox Meal (Allied Mills, Inc.)
- Storage temperature of food: 4°C - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- - Analyses were performed, using FDA methods to determine the efficiency of the mixing procedure and the stability of the test chemical in feed. Recoveries were found to be 90.3 + / -1.4% of the theoretical value at 7500 ppm EDTA and 90.4 + / - 3.4% of the theoretical value at 3750 ppm. It was concluded from these results that the preparations contained reasonably accurate concentrations of EDTA and were mixed homogeneously, and that the chemical was stable in feed for at least a week.
- Duration of treatment / exposure:
- 103 weeks
- Frequency of treatment:
- daily
- Dose / conc.:
- 248 mg/kg bw/day (nominal)
- Remarks:
- original data: 3750 ppm (conversion according to EU risk assessment)
- Dose / conc.:
- 495 mg/kg bw/day (nominal)
- Remarks:
- original data: 7500 ppm (conversion according to EU risk assessment)
- No. of animals per sex per dose:
- 50 (except for the control, which consisted of only 20 animals)
- Control animals:
- yes, plain diet
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily
BODY WEIGHT: Yes
- Time schedule for examinations: approximately once a month
FOOD CONSUMPTION AND COMPOUND INTAKE: No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes; all major organs, not specified but presumably all organs used for histopathology: skin, lymph nodes, mammary gland, salivary gland, bone marrow, trachea, lungs and bronchi, heart, thyroids, parathyroids, esophagus, stomach, small intestine, large intestine, liver, gallbladder, pancreas, spleen, kidneys, adrenals, urinary bladder, prostate or uterus, testis or ovary, brain, and pituitary.
HISTOPATHOLOGY: Yes;
skin, lymph nodes, mammary gland, salivary gland, bone marrow, trachea, lungs and bronchi, heart, thyroids, parathyroids, esophagus, stomach, small intestine, large intestine, liver, gallbladder, pancreas, spleen, kidneys, adrenals, urinary bladder, prostate or uterus, testis or ovary, brain, and pituitary. - Statistics:
- - Statistical tests of differences in survival between groups are compared using the method of Cox (1972) for two groups and an extension of this method by Tarone (1975) for more than two groups.
- Statistical analysis of the incidence of tumors was made using the Fisher exact test (Cox, 1970) to compare a control group to a group of treated animals at each dose. In addition, the Armitage and Cochran test for linear trend in proportions, with continuity correction (Armitage, 1971), was used. - Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No significant signs were observed among test animals during the first year of the study. In the 6 months preceding termination of the test, corneal opacities, ascites, and urine stains, occurred in both treatment and control groups.
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- The male rats exhibited a negative dose-related trend in survival with the probability level of P = 0.103; the treated and control groups of male rats can thus be considered as comparable to each other in survival. The female rats also exhibited a negative dose-related trend in survival. Even though this effect was statistically significant, it is most likely a chance finding as treated animals did not exhibit any deviations from the control group in regard to clinical signs or pathology.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- Average body weights of treated male and female rats were comparable to those of the matched controls throughout the study.
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Inflammatory and degenerative changes were observed in about the same frequency in all groups. These lesions appeared to be related to age and no to the administration of the chemical.
- Histopathological findings: neoplastic:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The incidence of neoplasms was high in the reproductive and endocrine systems and lower in the hematopoietic, respiratory, integumentary, and digestive systems. No neoplasms were observed in the nervous, musculoskeletal, or urinary systems or in organs of special sense. A number of tumors occurred in other organ systems of both sexes, controls as well as treated animals. In some instances the incidence of tumors in the controls exceeded that of the treated animals. With the possible exception of endocrine tumors in the males, no clear association between the incidence of tumors, treatment, or sex could be established (see table 1 and 2).
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- >= 495 mg/kg bw/day (nominal)
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- clinical signs
- mortality
- Critical effects observed:
- not specified
- Endpoint:
- chronic toxicity: oral
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- 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:
- The substance Trisodium hydrogen ethylenediaminetetraacetate (CAS 150-38-9) is structurally similar to the substance trans-cyclohexane-1,2-dinitrilotetraacetic acid (CAS 13291-61-7), therefore used for the read-across.
- Reason / purpose for cross-reference:
- read-across source
- Principles of method if other than guideline:
- A study for possible carcinogenicity was conducted by administering the test material in feed to Fischer 344 rats. The chemical was administered to 50 males and 50 females at low and high concentrations, for 103 weeks. Matched-control groups were composed of 20 males and 20 females. Animals were analysed for mortality, clinical signs, histopathological as well as gross pathological changes.
- GLP compliance:
- no
- Limit test:
- no
- Species:
- rat
- Strain:
- Fischer 344
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Schmidt, Madison, Wisconsin, USA
- Weight at study initiation: 85-110 g
- Age at study initiation: 28 days
- Housing: four per cage in solid polycarbonate cages
- Diet: prepared from Wayne Lab Blox Meal (Allied Mills, Inc.) ad libitum
- Water: acidulated water ad libitum
- Acclimation period: 14 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25°C
- Humidity (%): 45-55%
- Air changes (per hr): 15 times per hour
- Photoperiod (hrs dark / hrs light): 16/8 - Route of administration:
- oral: feed
- Vehicle:
- unchanged (no vehicle)
- Details on oral exposure:
- DIET PREPARATION
- Rate of preparation of diet (frequency): 3 times/week
- Mixing appropriate amounts with (Type of food): Wayne Lab Blox Meal (Allied Mills, Inc.)
- Storage temperature of food: 4°C - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- - Analyses were performed, using FDA methods to determine the efficiency of the mixing procedure and the stability of the test chemical in feed. Recoveries were found to be 90.3 + / -1.4% of the theoretical value at 7500 ppm EDTA and 90.4 + / - 3.4% of the theoretical value at 3750 ppm. It was concluded from these results that the preparations contained reasonably accurate concentrations of EDTA and were mixed homogeneously, and that the chemical was stable in feed for at least a week.
- Duration of treatment / exposure:
- 103 weeks
- Frequency of treatment:
- daily
- Dose / conc.:
- 248 mg/kg bw/day (nominal)
- Remarks:
- original data: 3750 ppm (conversion according to EU risk assessment)
- Dose / conc.:
- 495 mg/kg bw/day (nominal)
- Remarks:
- original data: 7500 ppm (conversion according to EU risk assessment)
- No. of animals per sex per dose:
- 50 (except for the control, which consisted of only 20 animals)
- Control animals:
- yes, plain diet
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily
BODY WEIGHT: Yes
- Time schedule for examinations: approximately once a month
FOOD CONSUMPTION AND COMPOUND INTAKE: No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes; all major organs, not specified but presumably all organs used for histopathology: skin, lymph nodes, mammary gland, salivary gland, bone marrow, trachea, lungs and bronchi, heart, thyroids, parathyroids, esophagus, stomach, small intestine, large intestine, liver, gallbladder, pancreas, spleen, kidneys, adrenals, urinary bladder, prostate or uterus, testis or ovary, brain, and pituitary.
HISTOPATHOLOGY: Yes;
skin, lymph nodes, mammary gland, salivary gland, bone marrow, trachea, lungs and bronchi, heart, thyroids, parathyroids, esophagus, stomach, small intestine, large intestine, liver, gallbladder, pancreas, spleen, kidneys, adrenals, urinary bladder, prostate or uterus, testis or ovary, brain, and pituitary. - Statistics:
- - Statistical tests of differences in survival between groups are compared using the method of Cox (1972) for two groups and an extension of this method by Tarone (1975) for more than two groups.
- Statistical analysis of the incidence of tumors was made using the Fisher exact test (Cox, 1970) to compare a control group to a group of treated animals at each dose. In addition, the Armitage and Cochran test for linear trend in proportions, with continuity correction (Armitage, 1971), was used. - Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No significant signs were observed among test animals during the first year of the study. In the 6 months preceding termination of the test, corneal opacities, ascites, and urine stains, occurred in both treatment and control groups.
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- The male rats exhibited a negative dose-related trend in survival with the probability level of P = 0.103; the treated and control groups of male rats can thus be considered as comparable to each other in survival. The female rats also exhibited a negative dose-related trend in survival. Even though this effect was statistically significant, it is most likely a chance finding as treated animals did not exhibit any deviations from the control group in regard to clinical signs or pathology.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- Average body weights of treated male and female rats were comparable to those of the matched controls throughout the study.
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Inflammatory and degenerative changes were observed in about the same frequency in all groups. These lesions appeared to be related to age and no to the administration of the chemical.
- Histopathological findings: neoplastic:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The incidence of neoplasms was high in the reproductive and endocrine systems and lower in the hematopoietic, respiratory, integumentary, and digestive systems. No neoplasms were observed in the nervous, musculoskeletal, or urinary systems or in organs of special sense. A number of tumors occurred in other organ systems of both sexes, controls as well as treated animals. In some instances the incidence of tumors in the controls exceeded that of the treated animals. With the possible exception of endocrine tumors in the males, no clear association between the incidence of tumors, treatment, or sex could be established (see table 1 and 2).
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- >= 495 mg/kg bw/day (nominal)
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- clinical signs
- mortality
- Critical effects observed:
- not specified
Referenceopen allclose all
The group treated with CaNa2EDTA showed similar, but milder effects (no further data).
The group treated with CaNa2EDTA showed similar, but milder effects (no further data).
Table 1: Analyses of the Incidence of Primary Tumors at Specific Sites in Male Mice Fed EDTA Trisodium Salt in the Diet
| Morphology (p-value) | Control | 469 mg/kg bw/day | 938 mg/kg bw/day | |
| Hematopoietic System: Malignant Lymphoma | 2/20 (n.s.) | 7/46 (n.s.) | 7/48 (n.s.) | |
| Thyroid: C-cell Adenoma | 0/10 (n.s.) | 1/29 (n.s.) | 1/33 (n.s.) | |
| Pituitary: Chromophobe Adenoma | 1/13 (n.s.) | 0/19 (n.s.) | 1/26 (n.s.) | |
| Lung: Alveolar/Bronchiolar Adenoma and Carcinoma | 2/18 (0.096) | 2/50 (n.s.) | 3/49 (n.s.) | |
| Liver: Hepatocellular Adenoma and Neoplastic Nodule | 3/19 (n.s.) | 10/44 (n.s.) | 10/47 (n.s.) | |
Table 2: Analyses of the Incidence of Primary Tumors at Specific Sites in Female Mice Fed EDTA Trisodium Salt in the Diet
| Morphology (p-value) | Control | 469 mg/kg bw/day | 938 mg/kg bw/day |
| Hematopoietic System: Malignant Lymphoma | 5/19 (n.s.) | 11/49 (n.s.) | 12/47 (n.s.) |
| Thyroid: C-cell Adenoma | 1/12 (n.s.) | 3/33 (n.s.) | 1/34 (n.s.) |
| Pituitary: Chromophobe Adenoma | 2/12 (n.s.) | 6/34 (n.s.) | 4/29 (n.s.) |
| Lung: Alveolar/Bronchiolar Adenoma and Carcinoma | 0/19 (n.s.) | 3/47 (n.s.) | 3/49 (n.s.) |
| Liver: Hepatocellular Adenoma and Neoplastic Nodule | 0/19 (n.s.) | 1/46 (n.s.) | 1/47 (n.s.) |
n.s. = not significant |
Table 1: Analyses of the Incidence of Primary Tumors at Specific Sites in Male Mice Fed EDTA Trisodium Salt in the Diet
| Morphology (p-value) | Control | 469 mg/kg bw/day | 938 mg/kg bw/day | |
| Hematopoietic System: Malignant Lymphoma | 2/20 (n.s.) | 7/46 (n.s.) | 7/48 (n.s.) | |
| Thyroid: C-cell Adenoma | 0/10 (n.s.) | 1/29 (n.s.) | 1/33 (n.s.) | |
| Pituitary: Chromophobe Adenoma | 1/13 (n.s.) | 0/19 (n.s.) | 1/26 (n.s.) | |
| Lung: Alveolar/Bronchiolar Adenoma and Carcinoma | 2/18 (0.096) | 2/50 (n.s.) | 3/49 (n.s.) | |
| Liver: Hepatocellular Adenoma and Neoplastic Nodule | 3/19 (n.s.) | 10/44 (n.s.) | 10/47 (n.s.) | |
Table 2: Analyses of the Incidence of Primary Tumors at Specific Sites in Female Mice Fed EDTA Trisodium Salt in the Diet
| Morphology (p-value) | Control | 469 mg/kg bw/day | 938 mg/kg bw/day |
| Hematopoietic System: Malignant Lymphoma | 5/19 (n.s.) | 11/49 (n.s.) | 12/47 (n.s.) |
| Thyroid: C-cell Adenoma | 1/12 (n.s.) | 3/33 (n.s.) | 1/34 (n.s.) |
| Pituitary: Chromophobe Adenoma | 2/12 (n.s.) | 6/34 (n.s.) | 4/29 (n.s.) |
| Lung: Alveolar/Bronchiolar Adenoma and Carcinoma | 0/19 (n.s.) | 3/47 (n.s.) | 3/49 (n.s.) |
| Liver: Hepatocellular Adenoma and Neoplastic Nodule | 0/19 (n.s.) | 1/46 (n.s.) | 1/47 (n.s.) |
n.s. = not significant |
Table 1: Analyses of the Incidence of Primary Tumors at Specific Sites in Male Rats Fed EDTA Trisodium Salt in the Diet
| Morphology (p-value) | Control | 250 mg/kg bw/day | 500 mg/kg bw/day |
| Hematopoietic System: Leukemia, Malignant Lymphoma and Lymphocytic Leukemia | 3/20 (n.s.) | 4/50 (n.s.) | 4/50 (n.s.) |
| Adrenal: Pheochromocytoma | 2/20 (n.s.) | 5/49 (n.s.) | 4/50 (n.s.) |
| Thyroid: C-cell Adenoma | 0/17 (n.s.) | 6/45 (p = 0 0.08) | 3/38 (n.s.) |
| Pituitary: Chromophobe Adenoma | 0/18 (p = 0.089) | 3/47 (n.s.) | 5/44 (n.s.) |
| Lung: Alveolar/Bronchiolar Adenoma and Carcinoma | 1/18 (n.s.) | 2/50 (n.s.) | 3/49 (n.s.) |
| Liver: Hepatocellular Adenoma and Neoplastic Nodule | 0/20 (n.s.) | 1/48 (n.s.) | 1/50 (n.s.) |
| Testis: Interstitial-cell Tumor | 19/20 (n.s.) | 43/50 (n.s.) | 44/50 (n.s.) |
Table 2 Analyses of the Incidence of Primary Tumors at Specific Sites in Female Rats Fed EDTA Trisodium Salt in the Diet
| Morphology (p-value) | Control | 250 mg/kg bw/day | 500 mg/kg bw/day |
| Hematopoietic System: Leukemia, Malignant Lymphoma and Lymphocytic Leukemia | 1/20 (n.s.) | 8/50 (n.s.) | 0/50 (n.s.) |
| Adrenal: Pheochromocytoma | 1/20 (n.s.) | 1/49 (n.s.) | 1/48 (n.s.) |
| Thyroid: C-cell Adenoma | 0/11 (n.s.) | 0/36 (n.s.) | 1/37 (n.s.) |
| Pituitary: Chromophobe Adenoma | 6/19 (n.s.) | 10/48 (n.s.) | 11/50 (n.s.) |
| Lung: Alveolar/Bronchiolar Adenoma and Carcinoma | 0/20 (n.s.) | 3/48 (n.s.) | 2/48 (n.s.) |
| Liver: Neoplastic Nodule | 0/20 (n.s.) | 1/48 (n.s.) | 0/48 (n.s.) |
| Uterus: Endometrial Stromal Polyp | 5/20 (n.s.) | 6/50 (n.s.) | 7/50 (n.s.) |
| Mammary Gland: Fibroadenoma | 4/20 (n.s.) | 3/50 (n.s.) | 3/50 (n.s.) |
n.s. = not significant |
Table 1: Analyses of the Incidence of Primary Tumors at Specific Sites in Male Rats Fed EDTA Trisodium Salt in the Diet
| Morphology (p-value) | Control | 250 mg/kg bw/day | 500 mg/kg bw/day |
| Hematopoietic System: Leukemia, Malignant Lymphoma and Lymphocytic Leukemia | 3/20 (n.s.) | 4/50 (n.s.) | 4/50 (n.s.) |
| Adrenal: Pheochromocytoma | 2/20 (n.s.) | 5/49 (n.s.) | 4/50 (n.s.) |
| Thyroid: C-cell Adenoma | 0/17 (n.s.) | 6/45 (p = 0 0.08) | 3/38 (n.s.) |
| Pituitary: Chromophobe Adenoma | 0/18 (p = 0.089) | 3/47 (n.s.) | 5/44 (n.s.) |
| Lung: Alveolar/Bronchiolar Adenoma and Carcinoma | 1/18 (n.s.) | 2/50 (n.s.) | 3/49 (n.s.) |
| Liver: Hepatocellular Adenoma and Neoplastic Nodule | 0/20 (n.s.) | 1/48 (n.s.) | 1/50 (n.s.) |
| Testis: Interstitial-cell Tumor | 19/20 (n.s.) | 43/50 (n.s.) | 44/50 (n.s.) |
Table 2 Analyses of the Incidence of Primary Tumors at Specific Sites in Female Rats Fed EDTA Trisodium Salt in the Diet
| Morphology (p-value) | Control | 250 mg/kg bw/day | 500 mg/kg bw/day |
| Hematopoietic System: Leukemia, Malignant Lymphoma and Lymphocytic Leukemia | 1/20 (n.s.) | 8/50 (n.s.) | 0/50 (n.s.) |
| Adrenal: Pheochromocytoma | 1/20 (n.s.) | 1/49 (n.s.) | 1/48 (n.s.) |
| Thyroid: C-cell Adenoma | 0/11 (n.s.) | 0/36 (n.s.) | 1/37 (n.s.) |
| Pituitary: Chromophobe Adenoma | 6/19 (n.s.) | 10/48 (n.s.) | 11/50 (n.s.) |
| Lung: Alveolar/Bronchiolar Adenoma and Carcinoma | 0/20 (n.s.) | 3/48 (n.s.) | 2/48 (n.s.) |
| Liver: Neoplastic Nodule | 0/20 (n.s.) | 1/48 (n.s.) | 0/48 (n.s.) |
| Uterus: Endometrial Stromal Polyp | 5/20 (n.s.) | 6/50 (n.s.) | 7/50 (n.s.) |
| Mammary Gland: Fibroadenoma | 4/20 (n.s.) | 3/50 (n.s.) | 3/50 (n.s.) |
n.s. = not significant |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 250 mg/kg bw/day
- Study duration:
- chronic
- Species:
- rat
Additional information
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