N-(2-hydroxyethyl)ethylenediaminetriacetic acid

Endpoint summary

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:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study meets generally accepted scientific principles For read-across justification refer to section 13.

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 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:

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

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

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: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

HEMATOLOGY
BLOOD CHEMICAL
URINARY EXAMINATIONS

Postmortem examinations (parental animals):

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 dose group.

WEIGHT
- of liver, kidneys, spleen, heart, adrenals, thyroids and gonads

HISTOPATHOLOGY
- in the animals which died or were sacrificed during the study: liver and kidney at the lower dose levels
- in 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.

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: no significant effects

Critical effects observed:

no

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No significant effects

Critical effects observed:

no

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No significant effects

Critical effects observed:

no

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No significant effects

Critical effects observed:

no

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 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

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

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.

Table 1: Reproduction and lactation data for four generations of rats fed withCaNa2EDTA

			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

RESULTS OF ADDITIONAL TESTS

- The tibias of rats sacrificed at the 12-week period showed no evidence of abnormal calcification.

- At the end of the 2-year period, the ash content of the tibias in the control and 250-mg/kg groups were approximately the same.

- There was no difference in either the incidence or severity of dental caries

- There were no significant differences in the two metallo-enzymes blood carbonic anhydrase and liver xanthine oxidase

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

chronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A study available on a structural analogue of the substance, EDTA-CaNa2 investigated effects on reproductive performance and lactation experiments in four successive generations of Wistar rats in a 2 year feeding study. 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/day (Oser et al., 1963). No significant differences in behaviour 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. Evaluation of various tissues and organs (weight, microscopic examination) including gonads (testes) gave negative results even in the high dose group. Reproductive and lactational indices evaluated included fertility index, gestation index viability index and proportion of rats alive at 4 days that survived 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 adverse effect level (NOAEL) of EDTA-CaNa2, as measured by any of the usual indices of reproduction or lactation efficiency derived from this study is >= 250 mg/kg/day for the parental and F1 to F3 generations.

 

A poorly documented summary of preliminary data from a reproduction study on the effects of exposure of Wistar rats to diets containing 0.5, 1.0, and 5.0% (approximately 300, 600, and 3,000 mg/kg/day) EDTA-Na2H2, another structural analogue of the substance, has been reported by Yang,1964. The parental generations of the two lower exposure groups reportedly 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 and data on the second generation were also not reported.

Additional information related to fertility may be obtained from another oral administration study (Muralidhara, 1991) in which administration of 5, 10, and 15 mg/kg EDTA-Na2H2 to male adult Swiss albino mice for five consecutive days did not affect neither absolute or relative weights of epididymides and testes nor the microscopic architecture of these two organs when examined 1, 3, 5, and 7 weeks after treatment. Similarly, 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. Treatment of male mice with 10 mg Na2EDTA/kg body weight 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 may not be reliable as they were obtained in the same study which reported invalid results in a micronucleus assay.

Short description of key information:

Fertility studies on the substance are not available. Studies are available on structural analogues of the substance, EDTA-CaNa2 and EDTA-Na2H2 (for read-across justification 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 at dose levels up to 250 mg/kg/day and the NOAEL was regarded as being 250 mg/kg/day.

Justification for selection of Effect on fertility via oral route:

Best documented of the studies available

Effects on developmental toxicity

Description of key information

Developmental toxicity studies on the substance are not available. Studies are available on structural analogues of the substance, sodium salts of EDTA (for read-across justification refer to Section 13). After repeated treatment of dams with EDTA-Na4 (and several other EDTA substances) during various periods of gestation and with the use of different routes of substance administration (diet, gavage, s.c.) impaired embryo/foetal 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 and occurred at exposure levels of approximately 1,000 mg/kg/day and above, and are considered to be related to zinc deficiency.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study meets scientifically accepted methods For read-across justification refer to section 13.

Reason / purpose for cross-reference:

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 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: 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^2.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
- diarrhea in 90% 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:

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

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

>= 1 374 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:

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

Basis for effect level:

other: teratogenicity

Dose descriptor:

NOAEL

Effect level:

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

Basis for effect level:

other: embryotoxicity

Dose descriptor:

NOAEL

Effect level:

>= 1 374 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

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

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 tetrasodiumedetate. 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 substance-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% EDTA-Na2H2 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 EDTA-Na2H2. While obviously complete reproductive failure occurred with the 3% EDTA-Na2H2/100 ppm zinc diet fed during g.d. 0 -21, with the 2% EDTA-Na2H2/100 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% EDTA-Na2H2/100 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% EDTA-Na2H2 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 EDTA-Na2H2 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 EDTA-Na2H2 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% EDTA-Na2H2 amounting to an average dose of 954 mg EDTA-Na2H2/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 EDTA-Na2H2/ 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.

These effects were explained by the high affinity of EDTA for Zn resulting in Zn-deficiency. .

Justification for selection of Effect on developmental toxicity: via oral route:

Best documented of the studies available using a suitable route of administration and various EDTA substances

Justification for classification or non-classification

A multigeneration study of reproductive toxicity revealed no functional changes in fertility or reproductive performance. Pre-natal developmental toxicity studies revealed no effects on developmental toxicity at dose levels that were not maternally toxic.

In accordance with Regulation (EC) No. 1272/2008 there were no effects observed sufficient to warrant classification.