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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
multi-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
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, B.L. et al.
Year:
1963
Bibliographic source:
Toxicology and Applied Pharmacology 5, 142-162 (1963)

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 doses of approximately 50, 125, and 250 mg/kg bw/day. 25 rats of each sex were assigned to 3 test groups and one control group. The rats were dosed for 12 weeks and 2 rats of each sex and each group were sacrificed for histopathological examinations. The remaining rats were kept on the diet until the end of the 2 year treatment period. After approximately 13 weeks, mating was set up with one male and two females per cage. After littering, lactation was allowed to continue for 3 weeks. 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 litter. When the F0 rats reached 2 years on test, the entire study was terminated.
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 material form:
solid

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 mating procedure:
Approximately 13 weeks after the start of the exposure (when the rats were about 120 days of age and sexually mature) mating 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 mating was 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 resting period before re-mating. In successive mating 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 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 bw/day 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
Duration of treatment / exposure:
2 years
Frequency of treatment:
continuously
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
125 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
No. of animals per sex per dose:
23
Control animals:
yes, plain diet

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes

BODY WEIGHT: Yes

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

HEMATOLOGY
BLOOD CHEMICAL
URINARY EXAMINATIONS


Postmortem examinations (offspring):
SACRIFICE
- Representative animals of the F0 generation were sacrificed 12 weeks after the start of the exposure. At the end of year one, two males and two females of each dose level were sacrificed and at the end of the study 10 or more rats of the control and the 250 mg/kg bw/day dose group.
WEIGHT
- of liver, kidneys, spleen, heart, adrenals, thyroids and gonads
HISTOPATHOLOGY
- the animals which died or were sacrificed during the study: liver and kidney at the lower dose levels
- the animals which were sacrificed at the end of the study: liver, anterior pituitaries, adrenals, kidneys, pancreas, heart, spleen, lungs, marrow, stomach, small and large intestines, gonads, thyroid, parathyroid, Iymph nodes, spinal cord, and tibias of the control and the 250 mg/kg bw dose group
ADDTIONAL EXAMINATIONS
- determination of the ash content of the tibials of rats in the highest dose group and control group
- microscopic examination of the jaws of representative animals for evidence of dental caries
- xanthine oxidase determination in the liver
- carbonic anhydrase determination in serum
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):
see "Additional other information on results"
Description (incidence and severity):
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.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
see "Additional other information on results"
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
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.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
In the histopathologic examinations of the P0 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.

Reproductive function / performance (P0)

Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Sometimes poor performance (see table 1) however they were not dose related.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
Remarks on result:
other: Highest dose tested

Target system / organ toxicity (P0)

Critical effects observed:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
Growth data for the F1 generation rats in the control and highest dosage test groups was as good as or better than that of the control group.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
see "Additional other information on results"
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
see "Additional other information on results"
Urinalysis findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No significant differences were found for the weights of liver, kidneys, spleen, heart, adrenals, gonads or thyroid glands.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
see "Additional other information on results"
Histopathological findings:
no effects observed
Description (incidence and severity):
see "Additional other information on results"

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed
Description (incidence and severity):
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.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
haematology
urinalysis
gross pathology
histopathology: non-neoplastic
Remarks on result:
other: Highest dose tested

Target system / organ toxicity (F1)

Critical effects observed:
not specified

Results: F2 generation

General toxicity (F2)

Clinical signs:
no effects observed
Description (incidence and severity):
Growth data for the F1 generation rats in the control and highest dosage test groups was as good as or better than that of the control group.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
see "Additional other information on results"
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
see "Additional other information on results"
Urinalysis findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No significant differences were found for the weights of liver, kidneys, spleen, heart, adrenals, gonads or thyroid glands.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
see "Additional other information on results"
Histopathological findings:
no effects observed
Description (incidence and severity):
see "Additional other information on results"

Developmental neurotoxicity (F2)

Behaviour (functional findings):
no effects observed
Description (incidence and severity):
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.

Effect levels (F2)

open allclose all
Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
>= 250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
haematology
urinalysis
gross pathology
histopathology: non-neoplastic
Remarks on result:
other: Highest dose tested
Key result
Dose descriptor:
NOAEL
Generation:
other: F3
Effect level:
>= 250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
haematology
urinalysis
gross pathology
histopathology: non-neoplastic
Remarks on result:
other: Highest dose tested

Target system / organ toxicity (F2)

Critical effects observed:
not specified

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

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/day dose group: 61%; in the control 45%)

GROWTH

Growth in all groups and in all four generations proceeded at a normal rate, plateauing at about 1 year. Growth data for 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/day group and the control were in the normal range as well as blood sugar, non-protein nitrogen and serum calcium levels.

GROSS 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/day group, in which 13 organs and tissues of each rat were examined, the findings were consistently negative.

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 bw/day 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

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