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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
17 February to 12 March 1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study (with certain deviations), to GLP; on related material

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
treated to gestation day 15, not up to 1 day before birth
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): trisodium EDDS (under code)
- Molecular formula (if other than submission substance): C10-H13-N2-O8. 3Na
- Molecular weight (if other than submission substance): 358
- Smiles notation (if other than submission substance): [Na+].[Na+].[Na+].OC(=O)[C@H](CC([O-])=O)NCCNC(CC([O-])=O)C([O-])=O
- Substance type: technical product
- Physical state: clear yellow liquid
- Purity test date: no data
- Lot/batch No.: no data
- Expiration date of the lot/batch: no data
- Stability under test conditions: stable for greater than study period
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
other: Sprague-Dawley derived Crl:CD VAF/Plus
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, Michigan, USA
- Age at study initiation: 12.5 weeks
- Weight at study initiation: 229-302 g
- Fasting period before study: no data
- Housing: individually (except when mating) in stainless steel, wire-mesh cages
- Diet (e.g. ad libitum): conventional, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 10 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23-25
- Humidity (%): 40-45
- Air changes (per hr): "environmentally controlled"
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 17 February To: 12 March 1994

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): powdered diet (mixed with an aliquot of diet which was then added to the bulk of diet and mixed)
- Storage temperature of food: no data


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
No data in report, samples sent to sponsor for analysis
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: no data
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- Any other deviations from standard protocol: no
Duration of treatment / exposure:
gestation day 6-15
Frequency of treatment:
ad libitum (in diet)
Duration of test:
gestation day 0-20
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 2000, 8000 or 16000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0, 132, 551, or 944 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
34 females per group (4 animals/group were killed on gestation day 16 and blood sampled for levels of copper, zinc and iron)
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: based on a previous range-finding study
- Rationale for animal assignment (if not random): the order in which the animals were assigned corresponded to the day the copulatory plug was observed and the order of appearance on the breeding record. First mated female was assigned to the first group, the second mated female to the second group, and all the remaining animals were assigned in this manner until the required number of mated females had been placed into each group.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily throughout study; daily on gestational days 6 to 20.
- Cage side observations: mortality and signs of overt toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 6, 9, 10, 12, 16 and 20

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterus in detail (see below); thoracic and abdominal cavities and organs subjected to gross examination for morphological changes

OTHER: four rats in each group were killed on gestation day 16 and blood samples were analysed for zinc, copper and iron.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: foetal weights
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No
Statistics:
Analysis of variance, Bartlett's test for homogeneity of variance and t-tests to determine the significance of differences in body weights, food consumption, mean numbers of corpora lutea, total implantations, live foetuses, gravid uterine weight and mean foetal body weights. Male to female sex ratios and proportions of litters with malformations and developmental variations were compared using the Chi-square or Fisher's test for homogeneity of R x C contingency tables to determine the significance of differences. The proportions of resorbed and dead foetuses and postimplantational losses were compared using the Kruskal-Wallis test.
Indices:
- Pregnancy index = No. females pregnant/ No. females mating
- Preimplantation loss = (No. corpora lutea No. implantations)/No. corpora lutea
- Postimplantation loss = (No. implantations ¿ No. viable foetuses)/ No. implantations
Historical control data:
Historical control data is tabulated as an appendix to the report

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
No maternal deaths occurred and no treatment related gross changes were observed. A statistically significant reduction in food consumption and body weight gain was noted for the high-dose group, as compared with the control group, during the entire dosing period, gestation days 6-15. A dose-dependent reduction in blood zinc levels was seen (which reached statistical significance at 551 mg/kg bw/day and above). Iron and copper levels were decreased in the high-dose groups, but this did not reach statistical significance. Mean gravid uterine weight for the high-dose group was significantly reduced.

There was a statistically significant increase in postimplantational losses in the high-dose group, which selectively reduced the numbers of live male foetuses, thus affecting the sex ratio. The number of early resorptions was statistically significantly higher in the high-dose group.





Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
ca. 551 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Foetal body weights were significantly reduced in the high-dose group compared to the control group.

Gross malformations
A statistically significant increase in the following malformations was observed in the high dose group when compared with the incidence in the control group, (foetal incidence % control: foetal incidence % high-dose group): cleft palate (0:63), adactyly (0:33), microphthalmia (0.2:22), short lower jaw (0:21), microcephaly (0:3), anal atresia (0.2:8), malformed anal opening (0:4), omphalocele (0:3), gastroschisis (0:2.5), malformed genital tubercle (0:1.5), ectrodactyly (0:33), syndactyly (0:19), micromelia (0:11), short tail (0:5.3), bent tail (0:43), and short, bent tail (0:30). Some additional malformations were observed in the high-dose group at an incidence level above that in the control group but were not statistically significant. The numbers and type of malformations observed in the low- and mid-dose groups were similar to control levels.

In addition, subcutaneous white discolouration in the dorsal cervical and thoracic regions was observed only in the high-dose group (3.5% incidence, compared to none in the controls).

Soft tissue malformations:
Visceral examination revealed a statistically significant increase in the incidence of the following malformations in the high dose group as compared with the control group (foetal incidence % control: foetal incidence % high-dose group): short lower jaw (0:16), cleft palate (0:10), confirmed cleft palate (0:66), anophthalmia (0.4:5.2), confirmed microphthalmia (0:14), folded retina (0:14), malformed lens (0:7), internal hydrocephaly (0.4:21), malformed brain (0:3), syringomyelia (0:4), pulmonary hypoplasia and aplasia (both 0:82), malformed stomach (0:54), renal hypoplasia, malformed kidney (0:4), omphalocele (0:4), malformed intestine (0:5), malformed testes (0:7), malformed epididymis (0:5), malformed vas deferens (0:5), malpositioned testis (0:11), malformed ovary (0:17) and cavitation of the urinary and reproductive tract (0:4). Some additional visceral malformations were observed in the high-dose group at a frequency greater than the control group but were not statistically significant.

Skeletal malformations:
The incidence of several skeletal malformations occurred at a statistically significantly increased level for the high-dose group as compared with the control group, and consisted of the following (foetal incidence % control: foetal incidence % high-dose group): subcutaneous calcification (0:5), malformed skull bones (0:24), cleft palate (0:63), mandibular micrognathia (0:17), fused nasal bones (0:13), tail malformation (0.4:87), vertebral and associated rib malformation (0.4:34), bent scapula (0:66), bent clavicles (0:15), fused sternebrae (0:57), shortened rib (0:31), malformed ribs (0:5), and increased incidence of bent/malformed/absent limb bones and digits.

Additional skeletal malformations were observed in the high-dose group, but were not statistically significant.

There were instances of malformed bones observed in foetuses from the control, low-and mid-dose groups, however, the incidence level for each occurrence was one to two foetuses and no dose related increase was noted and hence these were of doubtful toxicological significance.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
ca. 551 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: embryotoxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In a GLP study, conducted according to EPA Guidelines (OPPTS 870.3700), a NOAEL of 551 mg/kg bw/day was derived for both maternal and developmental toxicity in rats given trisodium EDDS in the diet from day 6 to 15 of pregnancy.
Executive summary:

In a GLP study, conducted according to EPA Guidelines (OPPTS 870.3700), trisodium EDDS was assessed for its potential to cause maternal and developmental toxicity in Charles River rats. Groups of 34 pregnant female rats were given trisodium EDDS in the diet at 0, 2000, 8000 or 16000 ppm (about 0, 132, 551 or 944 mg/kg bw/day based on food consumption) from gestation days 6 to 15. Four rats in each group were killed on gestation day 16 and blood samples were analysed for zinc, copper and iron. The remaining animals were killed on gestation day 20 and the dams examined for gross abnormalities and the uterus was examined for viable foetuses, early and late resorptions, number of implantations and corpora lutea. Foetuses were weighed and examined for external malformations and variations. About one-half of the foetuses were examined for soft-tissue effects and the remaining foetuses were examined for skeletal alterations.

Evidence of maternal toxicity in the high-dose group was seen as reduced body weight gain and food consumption. A dose-dependent reduction in blood zinc levels was seen (which reached statistical significance at 551 mg/kg bw/day and above). Iron and copper levels were decreased in the high-dose groups, but this did not reach statistical significance. Mean gravid uterine weight for the high-dose group was significantly reduced. There was a statistically significant increase in postimplantational losses in the high-dose group, which selectively reduced the numbers of live male foetuses, thus affecting the sex ratio. The number of early resorptions was statistically significantly increased in the high-dose group. Foetuses in the high-dose group had a diverse range of external, visceral and/or skeletal malformations and developmental variations, affecting all the major organs and skeletal structures. The whole developmental period was therefore affected by the test substance.

In conclusion, a NOAEL of 551 mg/kg bw/day was derived for both maternal and developmental toxicity in rats given trisodium EDDS in the diet from day 6 to 15 of pregnancy.

[Data on trisodium EDDS is considered relevant to use for understanding the potential developmental toxicity of EDDS acid, and is acceptable for using as read-across information.]