Tetrasodium ethylenediaminetetraacetate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Fertility studies using Na4EDTA are not available. Therefore studies with CaNa2EDTA or Na2EDTA were used for risk assessment. Data from a multigeneration study on rats with CaNa2EDTA did not give evidence for adverse effects on reproductive performance up to 250 mg/kg bw/day. For estimating a NOAEL other studies were not taken into consideration because of methodological flaws. Hence the NOAEL is 250 mg/kg bw/day.

Link to relevant study records

Reference

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

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

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:

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

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

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.

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 performance:

effects observed, non-treatment-related

Description (incidence and severity):

Sometimes poor performance (see table 1) however they were not dose related.

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

Critical effects observed:

not specified

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"

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.

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

Critical effects observed:

not specified

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"

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.

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

Critical effects observed:

not specified

Reproductive effects observed:

no

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

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

multigeneration reproduction toxicity study (Read-Across)

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 CaNa2EDTA at dietary levels providing doses of approximately 50, 125, and 250 mg/kg bw/day (Oser, 1963). No significant differences in behavior or appearance or 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 mating 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 mating in the four successive generations did not give evidence for significant treatment related differences in either of these indexes. No adverse effect of CaNa2EDTA 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 >= 250 mg/kg bw/day for the parent and F1 to F3 generation.

In a poorly documented summary of a reproduction study preliminary data on the effects of exposure of Wistar albino rats to diets containing 0.5, 1.0, and 5.0% Na2EDTA (according to about 300, 600, and 3000 mg/kg bw/day) were presented (Yang,1964). It was reported, that the parent generations of the two lower exposure groups gave birth to normal first and second litters, while those animals of the highest dose level failed to produce any litters, even though they had been mated for 2 months. No more details were given. Also data on the second generation were not available.

Additional information related to fertility was obtained from a further oral administration study (Muralidhara, 1991). Administration of 5, 10, and 15 mg Na2EDTA/kg bw/day to male adult Swiss albino mice for five consecutive days did not affect neither absolute or relative weights of epididymides and testes nor histoarchitecture of these two organs assayed at 1, 3, 5, and 7 weeks after treatment. Likewise, no effects were detected on caudal sperm counts, and there were no changes in the incidence of sperm head abnormalities or in the percentage of abnormal sperms. Furthermore treatment of male mice with 10 mg Na2EDTA/kg bw/day in distilled water for 5 consecutive days induced no increase in the incidence of post implantation embryonic deaths over a mating period of 8 weeks, except for a statistically insignificant about twofold increase during week 2 and 3 of mating. However, these results are not reliable due to insufficient documentation and methodological deficiencies.

Effects on developmental toxicity

Description of key information

After repeated treatment of dams 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, 1981). Gross malformations, comprised cleft palate, severe brain deformities, eye defects, micro- or agnathia, syndactyly, clubbed legs and tail anomalies. Since in most of these studies, EDTA or Na4EDTA had been administered at only one dose level, no oral NOAEL for either developmental toxicity or maternal toxicity could be established.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

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

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

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 bw/day 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 or test system 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: 3.9

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: co-housed
- If co-housed:
- 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

Dose / conc.:

1 374 mg/kg bw/day (actual dose received)

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^2.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

diarrhea in 90% of the animals: daily after application; it disappeared after the last day of dosing

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

reduced weight gain during treatment; recovery within the post treatment period

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

decreased food intake during treatment (see table 1)

Dose descriptor:

NOAEL

Effect level:

1 374 mg/kg bw/day (actual dose received)

Basis for effect level:

other: maternal toxicity

Abnormalities:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

see table 2

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

see table 2

External malformations:

no effects observed

Description (incidence and severity):

no test item related statistically significant increase of malformation could be observed in the treated animals

Skeletal malformations:

effects observed, non-treatment-related

Details on embryotoxic / teratogenic effects:

- no effects on mortality index (see table 2)
A total of 1084 pups from test item-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 test item-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.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 374 mg/kg bw/day (actual dose received)

Basis for effect level:

other: teratogenicity, embryotoxicity, fetotoxicity

Abnormalities:

no effects observed

Developmental effects observed:

no

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

Reference 2

Endpoint:

developmental toxicity

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

Qualifier:

no guideline followed

Principles of method if other than guideline:

In a developmental study, pregnant Sprague-Dawley rats were exposed during various periods of gestation to purified diets adjusted to either 100 or 1000 ppm zinc (provided as zinc carbonate) and containing 2 or 3% Na2EDTA corresponding to 1000 or 1500 mg/kg bw daily intake. On the last day of gestation fetuses were removed by caesarian section, fixed, stained and examined for gross abnormalities as well as brain and eye abnormalities. In addition, the number of implantation sites was counted.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 210 +/- 10 g
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

Route of administration:

oral: feed

Details on exposure:

- Diets were prepared by adding 2 or 3 g Na2EDTA to 100 g of the control ration (2 or 3% diets). Some of the 3% EDTA diet was supplied with additional 1000 ppm zinc

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: co-housed
- If co-oused:
- Length of co-habitation: 1 night
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

2% group: 21 days (day 0 of gestation to day 21)
3% groups for: 21 (day 0 of gestation to day 21); 8 (day 6 of gegastation to day 14) or 15 (day 6 of gegastation to day 21) days respectively
3% group + zinc: 15 days (day 6 of gegastation to day 21)

Frequency of treatment:

continuously

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

original data: 2% EDTA (conversion according to EU Risk Assessment)

Dose / conc.:

1 500 mg/kg bw/day

Remarks:

original data: 3% EDTA (conversion according to EU Risk Assessment)

No. of animals per sex per dose:

5 to 16 (see table 1)

Control animals:

yes, plain diet

Maternal examinations:

- cage side examination

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of implantations: Yes

Fetal examinations:

- Fetuses were examined for external congenital abnormalities
- Eye and brain were examined in razor cut sections
- No examination of internal abnormalities was performed

Description (incidence and severity):

All females had moderate to severe diarrhea.

Remarks on result:

not determinable

Abnormalities:

not specified

Details on embryotoxic / teratogenic effects:

- 2% EDTA group: all rats had living young at term; the litter size was normal; young were slightly smaller than controls; 7% of the fetuses were malformed
- 3% EDTA (day 0 - 21): reproduction was severely disturbed; none of the females had grossly visible implantation sites
- 3% EDTA (day 6- 14 and 6-21): almost all females had implantation sites; half of the sites had dead or resorbed fetuses; in the 6-21 day group were 100% of the fetuses malformed
3% EDTA + Zn: normal reproduction; none of the young were malformed
The malformations consisted of: cleft lip, cleft palate, hydroencephalus, anencephalus, exencephalus, hydranencephalus, curly, short or missing tail, fused or missing digits, clubbed legs micro- or agnathia, micro- or anophthalmia (see table 1).

Remarks on result:

not determinable

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 1: The effect of dietary EDTA supplementation on reproduction in rats

	Dietary EDTA		Rats (No.)			Implantation sites			Living young at term
Dietary zinc (ppm)	%	Period of ingestion	Mated	With implantation sites	With living young at term	Total No.	Dead or resorbed fetuses (%)	Affected (%)	Total No	Mean No. per litter	Mean weight of young (g)	Malformed (%)
100	0	0 (control)	11	11	11	132	6	6	124	11.4	5.3	0
	2	0 - 21	5	5	5	61	5	11	58	11.6	4.6	7
	3	0 - 21	8	0	0	0			0
		6 - 14	11	10	8	103	40	97	70	7	3.7	87
		6 - 21	16	16	11	182	54	100	183	5.5	1.8	100
1000	3	6 - 21	8	7	7	88	8	8	81	11.6	5	0

Reference 3

Endpoint:

developmental toxicity

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

Qualifier:

no guideline followed

Principles of method if other than guideline:

Toxic and teratogenic effects of EDTA were studied in the rat following administration via the diet, per gavage or subcutaneously. For every route of exposure appropriate control were used.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

other: CD

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charls River, Boston, USA
- Diet: 20 g/day
- Water: ad libitum

Route of administration:

oral: gavage

Vehicle:

other: phosphate buffer

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: co-housed
- If co-housed:
- Length of co-habitation: 1 night
- Proof of pregnancy: sperm in vaginal smear referred to as day 1 of pregnancy

Duration of treatment / exposure:

day 7 through 14 gestation

Frequency of treatment:

twice daily

Duration of test:

until day 21 of gestation

Dose / conc.:

625 mg/kg bw/day

Remarks:

total 1250 mg of EDTA/kg bw/day

Dose / conc.:

750 mg/kg bw/day

Remarks:

total 1500 mg of EDTA/kg bw/day

No. of animals per sex per dose:

Control: 20
Treated animals: 22 (low dose); 8 high dose

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: throughout the study

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule: until day 18 of gestation

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- no data on organs examined

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: No data
- Number of implantations: Yes
- Number of early resorptions:No data
- Number of late resorptions: No data

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: 1/3 per litter and all malformed fetuses
- Skeletal examinations: Yes: all fetuses alive and all malformed fetuses

Statistics:

Mann-Whitney U tests

Description (incidence and severity):

diarrhea

Mortality:

mortality observed, treatment-related

Description (incidence):

mortality (36.4% in the low dose group; 87.5% in the high dose group; see table 1)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

reduced weight gain

Remarks on result:

not determinable

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was an insignificant decrease in fetal weight in the EDTA group which may have been due to the larger proportion of normal fetuses in this group.

Description (incidence and severity):

The typical syndrome of gross malformations included cleft palate, micrognathia, microphthalmia, menigocoele, phocomelia, clubfoot and ectrodactyly, umbilical hernia, and short curly tail. One control fetus was stunted and had craniorachischisis, umbilical hernia, ablepheria, fused, wavy ribs, and sternebral dysplasia.

Description (incidence and severity):

The skeletons of fetuses exhibited extreme dysplasia including shortened, missing, or wavy ribs, misaligned and fusedcentra, as well as anomalies associated with external defects.

Description (incidence and severity):

Internal anomalies included great vessel anomalies, interventricular septal defects, small or missing lung lobes, missing thymus, small kidneys with associated hydronephrosis and hydroureter, and small undifferentiated gonads situated lateral to the kidneys.

Remarks on result:

not determinable

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 1: Toxic and Teratogenic Effects of EDTA Administered to the Rat by Gavage

	Control	EDTA	EDTA
Total dose/day (mg/kg)	-	1250	1500
Number of dams treated	20	22	8
Percentage of maternal deaths	0	36.4	87.5
Number of live dams with implants at term (%)	20 (100)	14 (100)	1
Mean number of implants/litter	12.4 ± 0.5	12.4 ± 0.5	1
Mean percentage of resorptions/litter	5.4 ± 1.6	6.9 ±  2.6	-
Mean percentage of malformed/litter	0.4 ± 0.4	20.5 ± 10.5*	-
Average fetal weight ± SE (g)	3.8 ± 0.05	3.48 ± 0.24	-
Maternal weight gain  ± SE (g)	28.1 ± 2.81	13.8 ± 4.79*	-
Average daily food consumption  ± SE (g)	18.4 ± 0.58	15.3 ±  0.82*	-

* = p < 0.05

Reference 4

Endpoint:

developmental toxicity

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

Qualifier:

no guideline followed

Principles of method if other than guideline:

Toxic and teratogenic effects of EDTA were studied in the rat following administration via the diet, per gavage or subcutaneously. For every route of exposure appropriate control were used.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

other: CD

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charls River, Boston, USA
- Diet: 20 g/day
- Water: ad libitum

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- 3% of the diet

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: co-housed
- If co-housed:
- Length of co-habitation: 1 night
- Proof of pregnancy: sperm in vaginal smear]referred to as day 1 of pregnancy
- Any other deviations from standard protocol:

Duration of treatment / exposure:

day 7 through 14 gestation

Frequency of treatment:

continuously

Duration of test:

until day 21 of gestation

Dose / conc.:

954 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Control 38
EDTA: 42

Control animals:

yes, plain diet

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: throughout the study

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule: until day 18 of gestation

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- no data on organs examined

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: No data
- Number of implantations: Yes
- Number of early resorptions:No data
- Number of late resorptions: No data

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: 1/3 per litter and all malformed fetuses
- Skeletal examinations: Yes: all fetuses alive and all malformed fetuses

Statistics:

Mann-Whitney U tests

Description (incidence and severity):

diarrhea

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

weight loss was observed (see table 1)

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

The average fetal weight for the EDTA group was significantly less than that for the control group.

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

significant increase of litter resorption

Description (incidence and severity):

The typical syndrome of gross malformations included cleft palate, micrognathia, microphthalmia, menigocoele, phocomelia, clubfoot and ectrodactyly, umbilical hernia, and short curly tail.Two control fetuses were malformed ; one had cleft palate, and the second had hydronephrosis and hydroureter.

Description (incidence and severity):

The skeletons of fetuses exhibited extreme dysplasia including shortened, missing, or wavy ribs, misaligned and fusedcentra, as well as anomalies associated with external defects

Description (incidence and severity):

Internal anomalies included great vessel anomalies, interventricular septal defects, small or missing lung lobes, missing thymus, small kidneys with associated hydronephrosis and hydroureter, and small undifferentiated gonads situated lateral to the kidneys.

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 1: Toxic and Teratogenic Effects of EDTA Administered to the Rat in the Diet

	Control	EDTA
Total dose/day (mg/kg bw/day)	-	954
Number of dams treated	38	42
Percentage of maternal deaths	0	0
Number of live dams with implants at term (%)	36 (92)	34 (81)
Mean number of implants/litter	11.7 ± 0.4	11.5 ± 0.3
Mean percentage of resorptions/litter	4.3 ± 1.3	33.4 ± 6.5 ***
Mean percentage of malformed/litter	0.4 ± 0.3	71.1 ± 7.9***
Average fetal weight ± SE (g)	3.82 ± 0.05	2.51 ± 0.13 ***
Maternal weight gain  ± SE (g)	35.6 ± 1.64	- 46.1 ± 2.78 ***
Average daily food consumption  ± SE (g)	19.2 ± 0.18	7.0 ± 0.38***

*** = p<0.001

Reference 5

Endpoint:

developmental toxicity

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

Qualifier:

no guideline followed

Principles of method if other than guideline:

Toxic and teratogenic effects of EDTA were studied in the rat following administration via the diet, per gavage or subcutaneously. For every route of exposure appropriate control were used.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

other: CD

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Boston, USA
- Diet: 20 g/day
- Water: ad libitum

Route of administration:

subcutaneous

Vehicle:

other: phosphate buffer

Details on exposure:

- EDTA was administered subcutaneously under the skin on the upper back; 0.75 mL/250 g

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: co-housed
- If co-housed:
- Length of co-habitation: 1 night
- Proof of pregnancy: sperm in vaginal smear referred to as day 1 of pregnancy

Duration of treatment / exposure:

day 7 through 14 gestation

Frequency of treatment:

daily

Duration of test:

until day 21 of gestation

Dose / conc.:

375 mg/kg bw/day

No. of animals per sex per dose:

Control: 14
EDTA: 25

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: throughout the study

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule: until day 18 of gestation

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- no data on organs examined

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: No data
- Number of implantations: Yes
- Number of early resorptions:No data
- Number of late resorptions: No data

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: 1/3 per litter and all malformed fetuses
- Skeletal examinations: Yes: all fetuses alive and all malformed fetuses

Statistics:

Mann-Whitney U tests

Description (incidence and severity):

diarrhea

Description (incidence):

see table 1

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

weight loss

Remarks on result:

not determinable

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

The average fetal weight for the EDTA group was significantly less than that for the control group.

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

significant increase of litter resorption (see table 1)

External malformations:

no effects observed

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

cervical ribs and fused and branched ribs

Visceral malformations:

no effects observed

Description (incidence and severity):

One control fetus had unilateral hydroureter.

Remarks on result:

not determinable

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 1: Toxic and Teratogenic Effects of EDTA Administered to the Rat Subcutaneously

	Control	EDTA
Total dose/day (mg/kg)	-	375
Number of dams treated	14	25
Percentage of maternal deaths	0	24
Number of live dams with implants at term (%)	13 (93)	15 (79)
Mean number of implants/litter	12.6 ± 0.5	11.9 ± 0.8
Mean percentage of resorptions/litter	1.3 ± 0.9	31.9 ± 10.5*
Mean percentage of malformed/litter	0.7 ± 0.7	4.3 ± 2.8
Average fetal weight ± SE (g)	3.77 ± 0.09	3.27 ± 0.12*
Maternal weight gain  ± SE (g)	31.1 ± 2.96	-1.7 ± 8.69***
Average daily food consumption  ± SE (g)	18.1 ± 0.48	11.5 ± 1.40***

Additional information

EDTA and four of its salts were evaluated for their teratogenic potential in CD albino rats (Schardein, 1981). Groups of 20 females were treated by gavage during g.d. 7 to 14 with 1000 mg EDTA/kg bw/day as well as with equimolar doses of disodium, trisodium, calcium disodium and tetrasodium edetate (dissolved and suspended in phosphate buffer with final pH values ranging from 3.9 to 9.2). 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 test item-related reactions including diarrhea and depression of activity were reported. The former occurred in all test item 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. 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 1000 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 1000 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 1500 or 1250 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 1500 mg dose group), in particular 36% maternal deaths, significantly reduced weight gain, and diarrhea in the 1250 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

Toxicity to reproduction: other studies

Additional information

Several in vitro tests for teratogenicity have been performed. In a study of Schmid (1985) 9.5 days old rat embryos were cultured in heat inactivated male rat serum containing Aroclor induced liver S9-mix and 10 - 300 µg/mL EDTA (unclear whether the free acid or a salt was tested). After 48 h incubation no effects on crown-rump and head length or degrees of differentiation were observed. There were also no malformations observed. In another study, no interference for adverse differentiation of murine neuroblast cells was found (clone N1E-115) (no information regarding the concentrations available) (Mummery, 1984). However, severe cytotoxicity was observed with an EC100 of 292 µg/mL and an NEC of 2.9 µg/mL. Flint (1984) conducted a teratogenic test in vitro with cell cultures of forelimb buds and midbrains of 13 day old rat embryos. He reported that up to 500 µg/mL EDTA had no effect on forelimb bud differentiation, but inhibited CNS differentiation at an IC50 of 2.8 µg/mL and an NEC of 1 µg/mL. 292 µg/mL EDTA also inhibited the cell differentiation to myotubes and ganglia in Oregon R Drosophila embryonic cultures (Bournias-Vardiabasis,1983). In an additional study by Flint (1984), 40 mg/kg bw of EDTA sodium salt were administered to rabbits on the 12th day of gestation. 16 h later embryos were removed and midbrain and forelimb buds cultured for 5 days. EDTA inhibited cell culture development by less than 20%. It was stated that EDTA interferes with the cell culture development. In a not very reliable study by Gassett (1977), 0.1 or 3% EDTA solution were dropped into the eye of 4 rabbits 6 times a day. The treatment was performed from sixth to the eighteenth day of gestation. Fetuses were obtained by cesarian section at day 29 of gestation. No teratogenic effect was reported for both EDTA concentrations, however at the 3% dose group the number of fetuses alive dropped from 23 in the 0.1% dose group to 8. In parallel the number of aborted or resorbed fetuses increased from 3 to 19.

Two additional reports on the treatment of pregnant woman with CaNa2EDTA are available. In one case a 8 months pregnant woman was treated for 7 days with 75 mg/kg bw/day CaNa2EDTA for lead poisoning (Angle 1964). 4 weeks later the mother delivered of normal infant (3.2 kg); developmental assessment of this boy at age 4 3/4 revealed nothing abnormal. In the other case a 7th month pregnant woman was treated with 0.5 g CaNa2EDTA/day intravenously 3 times a week for 4 weeks (Abendroth, 1971). The delivered infant was healthy and a follow up check at age of 4 also revealed nothing abnormal.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. No test substance specific adverse effects were seen on fertility, reproductive performance and developmental toxicity which justify a classification according to Regulation (EC) No 1272/2008.As a result the substance is not considered to be classified under Regulation (EU) No 1272/2008, as amended for the ninth time in Regulation (EU) No 2016/1179.