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Toxicity to reproduction

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Administrative data

Endpoint:
multi-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Pre-GLP study; study meets generally accepted scientific principles

Data source

Reference
Reference Type:
publication
Title:
Safety Evaluation Studies of Calcium EDTA
Author:
Oser BL, Oser M. Spencer HC
Year:
1963
Bibliographic source:
Toxicology and Applied Pharmacology 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

Constituent 1
Chemical structure
Reference substance name:
Sodium calcium edetate
EC Number:
200-529-9
EC Name:
Sodium calcium edetate
Cas Number:
62-33-9
Molecular formula:
C10H12CaN2O8.2Na
IUPAC Name:
calcium disodium 2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetate

Test 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:
unchanged (no vehicle)
Details on exposure:
See section 7.5.1
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:
In the studies reported, a 25% solution of calcium EDTA was used. Two samples were prepared and stored in polyethylene bottles
at room temperature. Portions for use in the experimental work were withdrawn as needed. Analytical studies demonstrated that these solutions
were stable throughout the period covered by this work.
Duration of treatment / exposure:
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:
23
Control animals:
yes, plain diet
Positive control:
No

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations:

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

HEMATOLOGY
BLOOD CHEMICAL
URINARY EXAMINATIONS

See also section 7.5.1.
Oestrous cyclicity (parental animals):
Not determined
Sperm parameters (parental animals):
Not determined
Postmortem examinations (parental animals):
See also section 7.5.1.
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
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

See also section 7.5.1

MORTALITY
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%)

BEHAVIOR
No significant abnormalities or differences in behavior or appearance of the rats in any of the generations or among the various dose levels were observed.

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: see below

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)

Dose descriptor:
NOAEL
Effect level:
>= 250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no significant effects at 250 mg/kg bw/day

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: P1 (second parental generation)

Effect levels (P1)

Dose descriptor:
NOAEL
Effect level:
>= 250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no significant effects at 250 mg/kg bw

Target system / organ toxicity (P1)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
not examined
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)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no significant effects at 250 mg/kg bw

Target system / organ toxicity (F1)

Critical effects observed:
no

Results: F2 generation

Effect levels (F2)

Dose descriptor:
NOAEL
Generation:
F2
Effect level:
>= 250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No significant effects at 250 mg/kg bw/day

Target system / organ toxicity (F2)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

REPRODUCTIVE PERFORMANCE

- Sometimes poor performance (see Table 1) however these effects were not dose-related.

Table 1: Reproduction and lactation data for four generations of rats fed with CaNa2EDTA

Average litter size
Dose (mg/kg bw/day) Generation Total number of matings At birth At weaning Average weight of pups at weaning (g) F.I. G.I. V.I. L.I.
None F0 46 7.7 5.7 44.9 70 94 57 78
F1 20 8.6 7.5 47.5 85 100 92 89
F2 20 8.3 7.8 41.3 95 100 85 96
F3 20 8.4 8.7 37.4 75 100 88 90
50 F0 41 8.3 7.3 41 90 100 69 82
F1 20 5.8 5.7 46.5 65 92 76 89
F2 20 7.7 6.5 44.7 80 100 90 88
F3 20 9.8 9.2 39.7 95 95 96 97
125 F0 44 9.2 8.7 42 57 96 46 76
F1 18 6.4 6.5 47.6 78 93 66 95
F2 20 8.2 6.6 49.6 75 100 89 84
F3 20 8.1 6.6 46.1 95 84 76 87
250 F0 46 8.9 6.9 42.8 85 100 70 72
F1 19 5.5 6.3 45.3 58 100 67 93
F2 12 10.5 8.1 40.5 92 100 92 86
F3 20 6.8 6.3 49.4 70 93 79 93

F.I. = Fertility Index = (pregnancies/mating) x 100

G.I. = Gestation Index = (litters born/pregnancies) x 100

V.I. = Viability Index = (pups alive at 4 days/pups born) x 100

L.I. = Lactation Index = (pups weaned/pups alive at 4 days) x 100

Applicant's summary and conclusion

Conclusions:
There was no evidence for an influence of EDTA-CaNa2 on the fertility of rats.
Executive summary:

In 2 -year feeding studies with rats receiving diets containing calcium EDTA at levels to provide 50, 125, or 250 mg per kilogram body weight, no adverse effects on growth or food efficiency were observed. Hematologic examination, conducted periodically, and determination of prothrombin time, blood sugar, NPN, and serum calcium were likewise normal throughout the test period. 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.