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Effects on fertility

Description of key information
In a GLP study, conducted according to EPA Guidelines (OPPTS 870.3700), trisodium EDDS caused a dose-related and statistically significant reduction in plasma levels of copper and zinc, but no such reductions in plasma iron levels were seen. Maternal toxicity, evident as soft feces and a slight reduction in body weight gain, was seen at 1000 mg/kg bw/day (the highest tested dose), therefore 400 mg/kg bw/day considered the study NOAEL (Denny, 1996e).
Effect on fertility: via oral route
Dose descriptor:
NOAEL
700 mg/kg bw/day
Additional information

No relevant data on fertility effects in humans were identified.

In a GLP study conducted according to OECD Guideline 415, trisodium EDDS was assessed for its ability to induce reproductive and developmental toxicity in rats. Groups of 25 Spague-Dawley rats of each sex were given 0, 90, 250 or 700 mg/kg bw/day of the test substance (in water) by oral gavage for 70 days before mating, and throughout mating, pregnancy and weaning. Culling of the offspring occurred on day 4 after birth to standardize the litters, and the remaining offspring were killed on day 21 after delivery. The animals were observed for mortality, clinical signs of toxicity, body weight gain, feed consumption, changes in the estrus cycle, precoital index, mating index, fertility, gestation index, number and sex of offspring, litter size and viability, and lactation index. At sacrifice, gross necropsy of the thoracic, abdominal and pelvic viscera was carried out, the reproductive organs, brain and pituitary were weighed and sperm evaluations were performed. The females were examined for the number and distribution of implantation sites. Necropsies were performed on the pups culled at 4 days for external and internal abnormalities and the brains were sectioned to detect hydrocephaly. No significant systemic adverse effects were observed on the parental animals, and the reproductive and developmental parameters examined were unremarkable. Therefore the study NOAEL was considered to be 700 mg/kg bw/day (York, 1999).

Further insight into the potential of trisodium EDDS to effect the reproductive system can be obtained from a 90-day dietary study (see IUCLID Chapter 7.5.1 for further details). In thisGLP study, trisodium EDDS was fed to groups of rats (10/sex/dose) in the diet at about 0, 50, 300, 700 or 1000 mg/kg bw/day; further groups of 10 animals/sex were given the control or high-dose diets for 90 days, followed by a 28-day recovery period. The duration of the estrus cycle was determined in females over the last month of the study and sperm concentration, motility and morphology in males were also examined. At study termination, animals were examined both macroscopically and microscopically (including the epididymides, ovaries, uterus, vagina, seminal vesicles and testes), and absolute and relative organ weights determined (including the epididymides, ovaries, uterus, seminal vesicles and testes). No treatment-related effects on organ weights or length of estrus cycle were seen. In males at the top dose only, an increase in the numbers of abnormal sperm was reported (decrease in normal sperm morphololgy with a corresponding increase in head-only sperm), but by the end of the recovery period sperm morphology had returned to control levels in all but one male. No adverse effects on sperm concentration or motility were reported. Atypical residual bodies of minimal severity and incidence were noted in in the testes of the high-dose males (5/20), but these were not apparent following the 4-week recovery period. A NOAEL of 1000 mg/kg bw/day was established for trisodium EDDS based on a lack of (non-reversible) adverse effects seen on the reproductive system of rats in a 90 -day dietary study (Dotti et al. 1995).

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

No 2 -, 3- or multi-generation studies on EDDS acid, its simple salts or EDTA were identified.

However, In a good-quality early study (prior to GLP), calcium disodium EDTA dihydrate was assessed for the potential to cause adverse effects in a multigeneration reproductive toxicity study in Wistar rats. Groups of 25 rats of each sex (P generation) were fed 0, 50, 125 or 250 mg/kg bw/day in the diet from weaning (day 21) for up to 2 years. At 13 and 21 weeks of age, male rats were mated with females to provide two litters (F1 generation). At weaning of F1, 10 animals/sex were selected from as many litters as possible to provide the F2 generation. The F3 generation was similarly derived from F2 animals. All rats were fed the same diet as their parents throughout the study. The study was terminated 2 years after the first exposure of the P generation (i.e. the F1, F2 and F3 generations were terminated at 18, 12 and 6 months). No treatment-related adverse effects were observed on the fertility, gestation or lactation index, or on viability of the offspring in any of the three generations of offspring. No treatment-related deaths, or changes in body weight gain or food consumption were observed across the generations. There were no treatment-related differences in the levels of haemoglobin, haematocrit, blood sugar and non-protein nitrogen, urinary albumin and sugar, red and white blood cell counts, differential white cell count or prothrombin times among any of the generations. Gross pathology and microscopic examination of 15 organs and tissues, including the gonads, at study termination was unremarkable. Therefore, the NOAEL was considered to be 250 mg calcium disodium EDTA/kg bw/day, the highest tested dose. Based on the structural similarity of the two ethylenediamines, it is expected that trisodium EDDS is also unlikely to induce reproductive toxicological effects at similar dose levels (Oser et al. 1963).

Groups of Wistar rats (2 males and 4 females/group) were maintained on diets containing 0, 0.5, 1.0 or 5.0% disodium EDTA (approximately 0, 250, 500 or 2500 mg/kg bw/day, respectively) and allowed to mate upon reaching 100 days old. In order to obtain second litters, mating was repeated 10 days after weaning of the first litters. Normal first and second litters were reported from rats administered up to 500 mg/kg bw/day, while those animals of the highest dose failed to produce any litters, even after 2 months of mating. No more details were given in the brief summary, and data on the second generation were not available. Therefore, in this study, the NOAEL for reproductive effects was 500 mg/kg bw/day of disodium EDTA (Yang, 1952).

Additional information related to fertility can be obtained from a further study (Muralidhara and Narasimhamurthy, 1991). Oral administration of 5, 10 or 15 mg disodium EDTA/kg bw to male adult Swiss albino mice for five consecutive days had no effect on 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 disodium EDTA/kg bw for five consecutive days induced no increase in the incidence of postimplantation embryonic deaths over a mating period of 8 weeks, except for a statistically insignificant about two-fold increase during week 2 and 3 of mating.

[Due to their close structural similarity, data on EDTA and its simple salts are considered relevant to use for understanding the potential reproductive toxicity of EDDS acid, and is acceptable for using as read-across information.]

References (not included elsewhere in IUCLID dossier - need to move to reference list in CSR)

BIBRA (1964). Summaries of toxicological data. Toxicology of EDTA. Food and Cosmetics Toxicology 2, 763-7.

EU (2004a). European Union Risk Assessment Report (RAR); edetic acid (EDTA). Vol. 49. European Chemicals Bureau (ECB). Final report available at http://ecb.jrc.ec.europa.eu/DOCUMENTS/Existing-Chemicals/RISK_ASSESSMENT/REPORT/edtareport061.pdf.

EU (2004b). European Union Risk Assessment Report (RAR); tetrasodium ethylenediaminetetraacetate (Na4EDTA). Vol. 51. European Chemicals Bureau (ECB). Final report available at http://ecb.jrc.ec.europa.eu/DOCUMENTS/Existing-Chemicals/RISK_ASSESSMENT/REPORT/na4edtareport062.pdf

Muralidhara A and Narasimhamurthy K (1991). Assessment of in vivo mutagenic potency of ethylenediaminetetraacetic acid in albino mice. Food and Cosmetics Toxicology 29, 845-849 (cited in EU, 2004a,b).

Yang S-S (1952). Toxicological investigation ofethylenediaminetetraacetic acid in the rat. Thesis dated May 1952 (cited in BIBRA, 1964).


Short description of key information:
In a GLP study conducted according to OECD Guideline 415, no adverse affects on measures of fertility and development (or systemic toxicity) were seen in groups of rats administered trisodium EDDS by stomach tube at up to 700 mg/kg bw/day (considered the study NOAEL) in a one-generation reproduction study (York, 1999).

No 2-, 3- or multi-generation studies on EDDS acid, its simple salts, or EDTA were identified.

However, data from a multigeneration study on rats with calcium disodium EDTA gave no evidence for adverse effects on reproductive performance and outcome at up to 250 mg/kg bw/day, considered the study NOAEL (Oser et al. 1963). In a limited and briefly reported 2-generation reproductive toxicity study, normal first and second litters were reported from rats fed disodium EDTA in the diet at up to about 500 mg/kg bw/day (considered the study NOAEL), while those animals of the highest dose (2500 mg/kg bw/day) failed to produce any litters. Data on the second generation were not available (Yang, 1952).

Effects on developmental toxicity

Description of key information
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 (Denny, 1996a). 
In a GLP study, conducted according to EPA Guidelines (OPPTS 870.3700), NOAELs of 400 and 1000 mg/kg bw/day were derived for maternal and developmental toxicity, respectively, in groups of rats administered trisodium EDDS by oral gavage from day 6 to 15 of pregnancy. Some skeletal variations were seen in the high dose group, indicative of developmental delay (Denny, 1996b).
Effect on developmental toxicity: via oral route
Dose descriptor:
NOAEL
551 mg/kg bw/day
Additional information

No relevant data on developmental effects in humans were identified.

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 (Denny, 1996a).

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 36 pregnant female rats were given trisodium EDDS (in water) by oral gavage at 0, 50, 400 or 1000 mg/kg bw/day from gestation days 6 to 15. Six (satellite) 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 carcass weight, a decrease in food consumption during the treatment period, an increased incidence of soft stools and decreased defecation. There was no effect on blood copper, zinc or iron levels. No differences in mean implantation sites, mean number of live foetuses, mean postimplantation losses or mean foetal body weights were observed between the treated and control groups. The incidence of a particular skeletal variation in the high-dose group was statistically higher than that of the control group. Skeletal variations in this group included 25 presacral vertebrae in 14 foetuses from 4 litters, a finding which was absent in the control or the other treatment groups. Unossified sternebrae were noted in 72 foetuses from 24 litters in the high-dose group and an extra full rib was observed in 9 foetuses from 8 litters. The variation, 7th cervical rib, was observed in 4 and 7 foetuses in 4 litters each from the 400 and 1000 mg/kg bw/day groups, respectively, in comparison to a single affected foetus from the control group, however the incidence was not statistically significant. These variations were interpreted as evidence of developmental delay and not as permanent malformations. In conclusion, a NOAEL of 400 mg/kg bw/day was derived for maternal toxicity, and a NOAEL of 1000 mg/kg bw/day for developmental toxicity, in rats given trisodium EDDS by oral gavage from day 6 to 15 of pregnancy (Denny, 1996b).

In a GLP range-finding study, conducted to a protocol similar to OECD Guideline 414, trisodium EDDS given in the diet to pregnant female rats at 0, 2000, 8000, 16000, 24000 or 40000 ppm resulted in maternal toxicity at 16000 ppm and above. Therefore, a NOAEL for maternal toxicity of 8000 ppm, about 530 mg/kg bw/day, was determined. Due to the considerable reduction of food consumption at 24000 and 40000 ppm (and corresponding body weight losses at these high doses), developmental toxicity could not accurately be assessed, but did not occurr at non-maternally toxic levels (Denny, 1996c).

In a GLP range-finding study, conducted to a protocol similar to OECD Guideline 414, trisodium EDDS showed no evidence of maternal or developmental toxicity when administered by oral gavage to pregnant rats on gestation days 6-15 at up to 1000 mg/kg bw/day, considered the study NOAEL (Denny, 1996d).

[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.]

Toxicity to reproduction: other studies

Additional information

In a GLP study, conducted according to EPA Guidelines (OPPTS 870.3700), trisodium EDDS was assessed for its potential to cause maternal toxicity and alter plasma levels of copper, iron and zinc following oral administration in pregnant Charles River rats. Groups of 15 female rats were administered 0, 50, 400 or 1000 mg/kg bw/day of the test substance by oral gavage from gestation day 6 to 15. The animals were observed for signs of clinical toxicity throughout the study and at regular intervals body weights and food consumption were recorded. Blood samples were taken 2 and 4 h after the last dose on day 15 and analysed for plasma levels of copper, iron and zinc. The dams were examined for gross abnormalities of the major organs; no examination of the fetuses took place. No deaths occurred during the study. Soft feces were observed in five animals at the high-dose but no other clinical observations were considered to be treatment-related. Body weight gain was slightly reduced in a dose-dependent manner and food consumption was reduced for the first three days of dosing in the high-dose group. A statistically significant, dose-related reduction in plasma zinc levels was evident in all treatment groups compared to the controls at both the 2 and 4 h sampling times. Plasma copper levels were reduced in all treatment groups, reaching significance at 4 h in the low-dose group and at both time points in the mid- and high-dose groups. No statistically significant or dose-related reduction in plasma iron levels were seen. In conclusion, trisodium EDDS caused maternal toxicity, evident as soft feces and a slight reduction in body weight gain at 1000 mg/kg bw/day, and caused a dose-related, statistically significant reduction in plasma levels of copper and zinc when given orally to pregnant rats on days 6-15 of gestation. No statistically significant or dose-related reduction in plasma iron levels were seen (Denny, 1996e).

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

Justification for classification or non-classification

According to EU CLP and DSD regulations, EDDS acid and its trisodium salt do not meet the requirement for classification as developmental or reproductive toxicants, under the test conditions described.