trans-cyclohexane-1,2-dinitrilotetraacetic acid

Toxicity to reproduction

Administrative data

Endpoint:

multi-generation reproductive toxicity

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

Data source

Reference

Reference Type:

publication

Title:

Safety evaluation studies of Calcium EDTA

Author:

Oser BL, Oser M, Spencer HC

Year:

1963

Bibliographic source:

Toxicol Appl Pharmacol 5, 142-162

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

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

Test material

Test material information

Constituent 1

Reference substance name:

Sodium calcium edetate

EC Number:

200-529-9

EC Name:

Sodium calcium edetate

Cas Number:

62-33-9

Molecular formula:

C10H12CaN2O8.2Na

IUPAC Name:

calcium disodium 2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetate

Specific details on test material used for the study:

A 25% solution of the test item was used.

Test animals

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

Administration / exposure

Route of administration:

oral: feed

Vehicle:

water

Details on 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.

Details on mating procedure:

After approximately 13 weeks after the start of the exposure (when the rats were about 120 days of age and sexually mature) matings were set up with one male and two females per cage. As pregnancy was recognized (visually, by palpation, or by weight increments) the dam was transferred to an individual cage. If pregnancy was not established by the third week, the male was replaced. A female was regarded as infertile and matings were discontinued after two successive mating failures. Lactation was allowed to continue for 3 weeks, the pups being weighed at 4, 12, and 21 days.
After weaning, death, or destruction of their litters, the females were allowed a 1-week rest period before remating. In successive matings the males were rotated among females within their respective test groups.
Ten rats of each sex selected from as many litters as possible and representative of the average weight within the litters were assigned to the F1 generation groups. They were raised to maturity in accordance with the same program as the parent generation. Similarly, groups of rats from second litters of the F1 generation and, in turn, the F2 and F3 generations, were each carried through the production of two litters. When the F0 rats reached 2 years on test, the entire study was terminated.
The rats selected from each generation for breeding were continued on their respective diets for a 1 2-week feeding period, as described for the F0 generation. Following the weaning of the second litters in the descendant generation rats at the 50- and 125-mg/kg dosage levels were sacrificed and examined grossly post mortem, but the control and highest dosage level groups were continued without change in dietary treatment until about the end of the 2-year study.

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:

continuously

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 125, 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.

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

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

Examinations

Parental animals: Observations and examinations:

Blood hemoglobin levels, red and white blood cells counts, differential white cell counts, prothrombin time, blood sugar and nonprotein nitrogen, and serum calcium levels, urine for albumin and reducing sugar, and the sediment was examined microscopically.

Postmortem examinations (parental animals):

Histopathologic examinations of the principal organs or where gross pathologic observations so indicated. All animals that died, or were sacrificed at the specified periods were autopsied and weights were taken of livers, kidneys, spleens, hearts, adrenals, thyroids, and gonads.

Statistics:

Duncan multiple rank and multiple F test

Reproductive indices:

Fertility Index (FI): the proportion of matings resulting in pregnancy;
Gestation Index (GI): the proportion of pregnancies resulting in live litters

Offspring viability indices:

Viability Index (VI): the proportion of rats born that survive 4 days or longer;
Lactation Index (LI): the proportion of rats alive at 4 days that survive to weaning

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

At 1.5 years survival in all groups ranged from 62 to 86%. Within the last half year of the study deaths were more frequent, however this was not an effect of the treatment (average survival in the 250 mg/kg bw dose group: 61%; in the control 45%)

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Except for the females at the two higher levels of dosage (125 and 250 mg/kg).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

serum calcium level did not differ from the control

Description (incidence and severity):

No abnormalities observed in liver, kidney, heart and testes

Reproductive function / performance (P0)

Reproductive performance:

no effects observed

Description (incidence and severity):

Poor responses with respect to some of the criteria of reproductive performance occurred occasionally for no discernible reason. However, that they were not causally related to the test material may be inferred from the fact that they were not correlated with dosage or with the number of generations through which dosage continued.

Details on results (P0)

GROWTH
Growth in all groups and in all four generations proceeded at a normal rate, plateauing at about 1 year. In the F0 generation the growth responses within sexes at all levels were essentially equal up to the 76th week. 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. Growth data for the F1, F2, and F3 generation rats in the control and highest dosage test groups was as good as or better than that of the control group.

BLOOD PARAMETERS
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 blood cells with advancing age in all groups, including the controls, but there were no dose-related differences. The total and differential leukocyte counts likewise disclosed no effects attributable to the test material. Prothrombin times, determined at 78 and 104 weeks in both the responses in both the 250 mg/kg bw group and the control were in the normal range as well as blood sugar, nonprotein nitrogen and serum calcium levels.

URINARY ANALYSIS
Essentially normal.

REPRODUCTIVE PERFORMANCE: Poor responses with respect to some of the criteria of reproductive performance occurred occasionally for no discernible reason. However, that they were not causally related to the test material may be inferred from the fact that they were not correlated with dosage or with the number of generations through which dosage continued.

ORGAN WEIGHTS
No significant differences were found for the liver, kidneys, spleen, heart, adrenals, gonads or thyroid glands.

PATHOLOGY
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 correlated with occurred at least as frequently in the control as in the test groups. Except for mammary tumors which are fairly common in females with variance 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.

HISTOLOGY
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. In the 250-mg/kg bw 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 revealed changes in the anterior pituitaries (focal hyperplasia); adrenal cortex (focal hyperplasia); medulla (focal hyperplasia) and liver. However, they were not dose related.

RESULTS OF ADDITIONAL TESTS
- The tibias of rats sacrificed at the 12-week period 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-mg/kg groups were approximately the same.
- There was no difference in either the incidence or severity of dental caries
- There were no significant differences in the two metallo-enzymes blood carbonic anhydrase and liver xanthine oxidase

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 250 mg/kg bw/day

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

not examined

Haematological findings:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not specified

Histopathological findings:

not examined

Details on results (F1)

From the gestation indexes it can be seen that essentially all pregnant dams carried their litters to normal parturition. Survival in the immediate postpartum period was lower in the F0 generation than in subsequent generations. However, the differences in response due to dose levels were not statistically significant whereas those for generations were. The viability indexes in the F2 and F3 generations were significantly greater than in the F0 and F1 rats. The lactation indexes showed similar trends, those for the F1, F2, and F3 generations indicating better survival of weanlings than in the F0 generation. Although the mean weights at weaning (another indication of the efficiency of lactation) were not significantly different for the various generations or levels of dosage, the lowest average weight occurred in the F2 group at the highest dose level. However, the greatest number of pups per litter were nursed by the dams of that group.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 250 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

clinical signs

mortality

Results: F2 generation

General toxicity (F2)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Body weight and weight changes:

no effects observed

Haematological findings:

no effects observed

Urinalysis findings:

no effects observed

Developmental neurotoxicity (F2)

Behaviour (functional findings):

no effects observed

Details on results (F2)

- There was no difference between treated and control animals in either the incidence or severity of dental caries

Effect levels (F2)

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

> 250 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

haematology

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In 2 -year feeding studies with rats receiving diets containing calcium EDTA at levels to provide 50, 125, or 250 mg per kilogram body weight, no adverse effects on growth or food efficiency were observed. Hematologic examination, conducted periodically, and determination of prothrombin time, blood sugar, NPN, and serum calcium were likewise normal throughout the test period. Responses similar to those seen in the parent generation were observed in the rats of the three succeeding generations maintained on the same diet. Under the stresses of repeated pregnancies and lactation, no adverse effect of calcium EDTA was observed as measured by any of the usual indexes of reproduction or lactation efficiency. At autopsy neither gross examination nor the weights of the major organs disclosed any significant differences between the test and control groups. The histopathologic findings likewise revealed no consistent or dose-related effects.