Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
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
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
Species:
rat
Strain:
other: FDRL (derived from Wistar strain)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Housing: individually
- Diet: "natural type diet" ad libitum
- Water: ad libitum
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
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
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.
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: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
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
Dose descriptor:
NOAEL
Effect level:
>= 250 mg/kg bw/day (nominal)
Sex:
male/female
Dose descriptor:
NOAEL
Effect level:
>= 250 mg/kg bw/day (nominal)
Sex:
male/female
Remarks on result:
other: Generation: F3 (migrated information)
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
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.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 250 mg/kg bw/day (nominal)
Sex:
male/female
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
>= 250 mg/kg bw/day (nominal)
Sex:
male/female
Reproductive effects observed:
not specified

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

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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
Study duration:
chronic
Species:
rat
Quality of whole database:
The results of the study were confirmed by other studies with metal-chelates (EDTA-MnNa2 and DTPA-FeNaH), see also read across document in section 13.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

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 EDTA-CaNa2 at dietary levels providing daily doses of approximately 50, 125, and 250 mg/kg bw (Oser et al., 1963). No significant differences in behavior or appearance nor adverse effects on the growth or on the longevity of the rats in any of the generations or among the various dose levels were reported. Evaluations of various tissues and organs (weight, histopathologic examinations) including gonads (testes) gave negative results even in the high dose group. Criteria for reproductive and lactational effects were evaluated as proportion of matings resulting in pregnancy (fertility index), proportion of pregnancies resulting in live litters (gestation index), proportion of pups that survive 4 days or longer (viability index), and proportion of rats alive at 4 days that survive to weaning. Poor responses with respect to some of the criteria of reproductive performance occurred occasionally but were not correlated with dosage or with the number of generations through which dosage continued. The overall data for two matings in the four successive generations did not give evidence for significant treatment related differences in either of these indexes. The authors concluded that the No Observed Adverse Effect Level of EDTA-CaNa2 was observed as measured by any of the usual indices of reproduction or lactation efficiency even under the stresses of repeated pregnancies and lactation. The NOAEL derived from this study is therefore at least 250 mg/kg bw/day for the parent and F1 to F3 generation.

Studies with EDTA-Na2H2 were not taken into consideration for setting a NOAEL because of methodological flaws. Studies with other metal-chelates (EDTA-MnNa2 and DTPA-FeNaH) showed effects on fertility only at very levels of 1500 mg/kg bw but not at 500 mg/kg bw. But although effcets on sperm were

seen at the highest dose of EDTA-MnNa2 or DTPA-FeNaH tested (1500 mg/kg bw), it did not result in effects on reproduction as there were no changes in reproductive performance in animals of these groups.


Short description of key information:
One fertility studies using EDTA-CaNa2 is available. Therefore this study together with studies of EDTA-Na2H2, EDTA-MnNa2 and DTPA-FeNaH were used for risk assessment (for read-across justification also refer to section 13). Data from a multigeneration study on rats with EDTA-CaNa2 did not give evidence for adverse effects on reproductive performance and outcome for doses of up to 250 mg/kg bw/day. The studies with EDTA-Na2H2 were not taken into considerationfor setting a NOAEL because of methodological flaws. The studies with EDTA-MnNa2 and DTPA-FeNaH showed effects only ate very high levels (1500 mg/kg bw). Hence the NOAEL for EDTA-CaNa2 is at least 250 mg/kg bw/day.

Justification for selection of Effect on fertility via oral route:
Pre-GLP study; study meets generally accepted scientific principles

Effects on developmental toxicity

Description of key information
Results are available on several EDTA-compounds and on DTPA-FeNaH. After repeated treatment of dams withseveral of the non-metal EDTA chelates during various periods of gestation and with the use of different routes of substance application (diet, gavage, s.c., i.m.) impaired embryo/fetal development and the induction of a pattern of gross malformations were observed during these investigations with the exception of one gavage study (Schardein et al ., 1981). Gross malformations, comprised cleft palate, severe brain deformities, eye defects, micro- or agnathia, syndactyly, clubbed legs and tail anomalies. These effects were almost exclusively exhibited in studies using maternally toxic dosage levels. With the exception of one oral (single-dose/gavage) study, during which no teratogenic effects were induced, the fetotoxic and teratogenic effects are occurring at exposure levels of approximately 1,000 mg/kg bw/day and above. This was confirmed by studies with EDTA-MnNa2 and DTPA-FeNaH that showed developmental toxicity only at 1500 mg/kg bw.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study meets scientifically accepted methods
Reason / purpose:
reference to other study
Principles of method if other than guideline:
EDTA and four of its salts, disodium, trisodium, calcium di-sodium, and tetrasodium edetate, were studied for teratogenic potential in rats. Equimolar doses based on 1000 mg/kg were given by gastric intubation on Days 7 to 14 of gestation. On day 21 of gestation the dams of each group were sacrificed and litter data for each dam collected.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
other: CD albino
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Weight at study initiation: mean: 241 g
- Diet: Purina Lab Chow ad libitum
- Water: tap water ad libitum


Route of administration:
oral: gavage
Vehicle:
other: 0.2 M phosphate buffer
Details on exposure:
pH of dosing solution: 6.9

Analytical verification of doses or concentrations:
no
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Proof of pregnancy: vaginal plug, sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
7 days (day 7 to day 14 of gestation)
Frequency of treatment:
equally divided doses twice daily
Duration of test:
21 days
Remarks:
Doses / Concentrations:
1374 mg/kg bw/ day (divided into two equal doses)
Basis:
actual ingested
No. of animals per sex per dose:
20
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: once a week

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21



Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: each fetus
- Gross inspection, slicing and visceral abnormalities: 1/3 of the litter
- Skeletal examinations: 2/3 of the litter
Statistics:
Means and standard errors were calculated for litter size, followed by analysis of variance. Pre- and postimplantation losses, embryonic viability, and fetal survival were evaluated by analysis of covariance. Fetal weights were also evaluated by analysis of covariance following calculation of mean weight/litter by sex, the values representing means and standard errors of mean litter weights. Significant variance by either analysis of variance or covariance was further evaluated by Dunnett's t test to locate the source of variance. Sex distribution was analyzed by partitioned Chi-square.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
- diarrhea in 10% of the animals: daily after application; it disappeared after the last day of dosing
- decreased food intake during treatment (see Table 1)
- reduced weight gain during treatment; recovery within the post treatment period
Dose descriptor:
LOAEL
Effect level:
1 340 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
>= 1 340 mg/kg bw/day (actual dose received)
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- no effects on litter size, sex ratio or mortality index (see Table 2)
- no drug related statistically significant increase of malformation could be observed in the treated animals.
(A total of 1084 pups from drug-treated dams were examined. These were compared to 237 pups from 19 dams treated with the vehicle and 278 pups from 20 untreated dams. In addition, 752 pups from dams treated with edetic acid and its salts together with 165 and 191 pups from the vehicle and untreated control groups, respectively, were cleared and examined for skeletal defects. Twenty-four pups from the drug-treated groups had abnormalities. These included 20 with bifid vertebrae, 1 with agenesis of the ribs, 2 with inhibition of osteogenesis of the skull or ribs, and 1 with malformed ribs.
There was no definitive pattern regarding treatment with a particular compound and the occurrence of anomalies. None of the pups in the vehicle control group had abnormalities while 8 untreated control pups exhibited some major defect. One untreated control fetus was stunted and had multiple abnormalities including eye defect, ectrodactyly, and a curly tail. Histological examination of the eyes revealed a cataract in one eye and a dysmorphic lens
and retina in the other. Five additional control pups had bifid vertebrae while 2 had malformed vertebrae or sternebrae.)
Dose descriptor:
NOAEL
Effect level:
>= 1 340 mg/kg bw/day (actual dose received)
Basis for effect level:
other: teratogenicity
Dose descriptor:
NOAEL
Effect level:
>= 1 340 mg/kg bw/day (actual dose received)
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOAEL
Effect level:
>= 1 340 mg/kg bw/day (actual dose received)
Basis for effect level:
other: fetotoxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Table 1: Food consumption and weight gain in rats treated with EDTA and EDTA salts on days 7 through 14 of gestation

Days Control (untreated) Vehicle Control 967 mg/kg bw/day EDTA 1243 mg/kg bw/day Na2 EDTA 1245 mg/kg bw/day Na3 EDTA 1340 mg/kg bw/day CaNa2 EDTA 1374 mg/kg bw/day Na4 EDTA
Mean food consumption (g/day)
0-7 20.7 21.4 19.9 20.4 19.9 20.4 19.0
7-14 22.3 22.5 18.4 17.5 19.1 20.9 18.5
14-21 24.7 25.3 26.7 27.2 25.7 26.5 25.6
Mean body weight gain (g)
0-7 31.9 35.1 37.6 35.6 33.2 37.2 26.9
7-14 28.5 26.1 16.5 13.7 20.4 25.1 18.3
14-21 102.7 93.3 104.4 100.2 98.4 108.1 94.2

Table 2: Reproductive data in dams receiving EDTA and EDTA salts on days 7 through 14 of gestation

Fetuses
Sex Body weight (mean g ± SE) Number
Treatment No of dams Litter size (mean ± SE) Post implantation loss (%) M F M F Dead live Resorbed
Control (untreated) 20 14.0 ± 0.4 4 52 48 5.3 ± 0.1 5.6 ± 0.1 1 278 12
Vehicle Control 19 12.5 ± 0.1 3 50 50 5.3 ± 0.1 5.7 ± 0.1 0 237 7
967 mg/kg bw/day EDTA 17 12.7 ± 0.6 3 49 51 5.5 ± 0.1 5.7 ± 0.1 0 216 6
1243 mg/kg bw/day Na2 EDTA 19 12.6 ± 0.4 1 56 44 5.4 ± 0.1 5.6 ± 0.1 0 202 3
1245 mg/kg bw/day Na3 EDTA 18 12.4 ± 1.0 8 48 52 5.4 ± 0.2 5.7 ± 0.1 0 210 11
1340 mg/kg bw/day CaNa2 EDTA 17 13.5 ± 0.4 2 52 48 5.3 ± 0.1 5.6 ± 0.1 0 230 4
1374 mg/kg bw/day Na4 EDTA 19 11.9 ± 0.7 4 47 52 5.2 ± 0.1 5.5 ± 0.1 0 226 10

2 dams had to be killed due to dosing errors

Conclusions:
No teratogenic effects occurred when pregnant rats were given an oral dose of 1340 mg EDTA-CaNa2/kg bw on days 7 to 14 of gestation; slight maternal toxicity was noted at this level.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Study duration:
subacute
Species:
rat
Quality of whole database:
The results of the study were confirmed by other studies with metal-chelates (EDTA-MnNa2 and DTPA-FeNaH).
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

EDTA and four of its salts were evaluated for their teratogenic potential in CD albino rats (Schardein et al., 1981). Groups of 20 females were treated by gavage during g.d. 7 to 14 with 1,000 mg EDTA/kg bw/day as well as with equimolar doses of disodium, trisodium, calcium disodium and tetrasodium edetate. The dose level had been selected from preliminary studies with edetic acid in which there had been some evidence of both maternal and fetotoxicity under the same experimental conditions. For the dams significant drug-related reactions including diarrhea and depression of activity were reported. The former occurred in all drug groups with highest incidences for tetrasodium edetate (90%) and edetic acid (80%) and lowest incidence for calcium disodium edetate (10%). Three dams died during treatment with disodium edetate. Besides slightly decreased food intake in all test groups, treatment with all of the test compounds caused reduced weight gain in the dams during the treatment period. The mortality index of offspring in all treated groups as measured by postimplantation loss was comparable to that of the vehicle and untreated control group. None of the test compounds significantly affected litter size at term or mean fetal body weight when compared to either control. Fetuses were examined for external, visceral and skeletal anomalies. Incidental findings of skeletal anomalies did not reveal a definitive pattern regarding treatment with a particular compound. The authors stated that under these experimental conditions no teratogenic effects were evidenced even at maternally toxic doses.

In a further developmental study pregnant Sprague-Dawley rats were exposed during various periods of gestation to purified diets adjusted to either 100 or 1,000 ppm zinc (provided as zinc carbonate) and containing 2 or 3% Na2EDTA corresponding to 1000 or 1500 mg/kg bw daily intake (Swenerton and Hurley, 1971). The groups of 8 to 16 females had been set on the control diet at least 5 days before breeding and mated to normal stock-fed males. The evaluation of treatment related effects to the dams was not indicated in this study, except for the report on moderate to severe diarrhea in all females that were fed diets containing Na2EDTA. While obviously complete reproductive failure occurred with the 3% Na2EDTA/100 ppm zinc diet fed during g.d. 0-21, with the 2% Na2EDTA/100 ppm zinc diet reproductive outcome was essentially comparable to that of controls, however with lower mean body weight of the pups and with 7% malformed of the fullterm fetuses. Exposure to the 3% Na2EDTA/100 ppm zinc diet during the period of g.d. 6-14, and 6-21 resulted in respectively 40% and 54% dead or absorbed fetuses, reduced number of dams with live pubs, clearly reduced mean fetal body weight and ratios of respectively 87% and 100% malformed living offspring. Gross malformations comprised cleft palate, severe brain deformities, eye defects, micro- or agnathia, syndactyly, clubbed legs and tail anomalies. The reported fetotoxic and teratogenic effects were similar to those from earlier experiments with zinc deficient diets administered to pregnant rats for various periods of during gestation (Hurley, 1966). In contrast, the live offspring of dams fed 3% Na2EDTA supplemented with 1,000 ppm zinc from g.d. 6-21 did not exhibit any malformations, and the mean number of live pups/litter and the mean fetal body weight were comparable to those of controls. The authors concluded from this study that Na2EDTA ingested during pregnancy was teratogenic, whereas supplementation with zinc prevented the detrimental effects of EDTA. It was suggested that the congenital anomalies caused by EDTA were due specifically to zinc deficiency. This was also supported by zinc analyses of fetuses (Hurley and Swenerton, 1966), where clearly lower zinc contents were found in fetuses from deficient mothers in comparison to those from zinc supplemented dams, indicating that the reported effects rather occur because of a direct lack of zinc in fetal tissues than from indirect effects of maternal metabolism on fetal development.

The toxic and teratogenic effects of Na2EDTA were studied in female CD rats following different routes of administration (dietary, gavage, s.c) during g.d. 7-14 (Kimmel, 1977). Dietary exposure to 3% Na2EDTA amounting to an average dose of 954 mg Na2EDTA/kg bw/day resulted in reduced food intake, severe diarrhea and severe weight loss in the dams during treatment and produced a significant proportion of fetal deaths (about 33% resorptions/litter), significantly lower average fetal weight and gross external, internal and skeletal malformations in about 71% of the survivors. Treatment with 1,500 or 1,250 mg Na2EDTA/ kg bw/day administered by gavage (respectively 625 mg/kg and 750 mg/kg twice daily) resulted in severe toxicity to the dams (7 out of 8 animals died in the 1,500 mg dose group), in particular 36% maternal deaths, significantly reduced weight gain, and diarrhea in the 1,250 mg dose group and a significantly higher proportion of (about 21%) malformed survivors. Treatment with 375 mg/kg bw administered subcutaneously produced signs of severe pain (vocalisations and shock) to the dams and resulted in 24% maternal deaths, significantly reduced food intake and maternal weight loss during the period of treatment. Fetal toxicity (about 32% resorptions/litter, significantly reduced fetal weight) and a rate of about 4% malformed survivors/litter were reported for this route of application.

EDTA-MnNa2 showed developmental effects only at 1500 mg/kg bw, whereas no developmental effects were observed at 1500 mg/kg bw DTPA-FeNaH. The effects observed following treatment with EDTA-MnNa2 consisted of a decreased number of females with live born pups, decreased number of (live) pups, increased postimplantation loss. No such effects were seen at 500 mg/kg bw. Because of the higher affinity of EDTA for Zn it can also be expected that at the high level of 1500 mg/kg bw, sufficient Mn will be exchanged for Zn and as such Zn-deficiency may also occur as has occurred with the 'empty' (non-metal containing) chelates. The affinity of EDTA for Ca is even lower.


Justification for selection of Effect on developmental toxicity: via oral route:
Study is available for EDTA-CaNa2 and study meets scientifically accepted methods

Justification for classification or non-classification

Effects on reproduction and effects on fetal development have only been observed at high levels, i.e. at levels of approximately 1000 mg/kg bw and above, and are considered to be due to zinc deficiency. Therefore, based on the results obtained in these studies and taking into account the provisions laid down in Council Directive 67/548/EEC and CLP, classification with regard to toxicity to reproduction is not required.