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Effects on fertility

Additional information

PDTA-H4, a chelating agent, was associated with alterations in zinc homeostasis at the high dose level of 300 mg/kg/day, as evidenced by decreased serum zinc levels, increased urinary zinc excetion, clinical/gross observations of hind foot scaling, and histopathological findings such as hyperplasia, hyperkeratosis and/or parakeratosis of the esophagus, stomach and skin, and testicular changes (degeneration and/or atrophy of the seminiferous tubules, decreased or absent spermatids). All of these effects are known to be associated with zinc deficiency in rats. Four male rats in this group were sacrificed in moribund condition prior to the scheduled termination and had signs of marked zinc deficiency at necropsy. Decreases in feed consumption and body weights were noted in both males and females, with males being more severely affected (top dose males weighed 33% less than controls at termination). Accompanying the compromised zinc status at the top dose level were marked decreases in fertility and pup survival. No adverse effects of PDTA-H4 on systemic or reproductive toxicity were found in the 60 or 10 mg/kg/day groups. Although statistically identified increases in urinary zinc excretion were noted at 60 mg/kg/day (both sexes) and 10 mg/kg/day (females only), this was not considered an adverse effect, as serum zinc levels and other zinc-sensitive parameters were unaltered. The no-observable-adverse-effect-level (NOAEL) for systemic and reproductive effects in this study was 60 mg/kg/day.

With regard to the structurally-related substance EDTA, in a 2 -year feeding study on Wistar rats including reproductive and lactation experiments in four successive generations, groups of 25 male and 25 female animals were exposed to EDTA-CaNa2 at dietary levels providing daily doses of approximately 50, 125, and 250 mg/kg bw (Oser et al., 1963). No significant differences in 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. Evaluations of various tissues and organs (weight, histopathologic examinations) including gonads (testes) gave negative results even in the high dose group. Criteria for reproductive and lactational effects were evaluated as proportion of matings resulting in pregnancy (fertility index), proportion of pregnancies resulting in live litters (gestation index), proportion of pups that survive 4 days or longer (viability index), and proportion of rats alive at 4 days that survive to weaning. Poor responses with respect to some of the criteria of reproductive performance occurred occasionally but were not correlated with dosage or with the number of generations through which dosage continued. The overall data for two matings in the four successive generations did not give evidence for significant treatment related differences in either of these indexes. The authors concluded that no adverse effect of EDTA-CaNa2 was observed as measured by any of the usual indices of reproduction or lactation efficiency even under the stresses of repeated pregnancies and lactation. The NOAEL derived from this study is >= 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% EDTA-Na2H2 (corresponding to about 300, 600, and 3,000 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 were obtained from a further oral administration study (Muralidhara, 1991). Administration of 5, 10, and 15 mg EDTA-Na2H2 per kg bw 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 EDTA-Na2H2 per kg bw 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 as they were obtained in the same study which reported invalid results in the micronucleus assay.

PDTA-H4 is a chelating agent with a high affinity for metals such as Zinc, Manganese and Calcium. Zinc is one of the most abundant metals in the human body (2 -4g) and is present as a cofactor in a large number of enzymes (between 100 and 300), covering almost all classes of enzyme. As such deficiencies in zinc can produce a wide array of symptoms including both reproductive and developmental toxicity. If a dietary 2 -generation or multigeneration reproductive toxicity study were to be performed with PDTA-H4 in the absence of any supplementation of essential minerals such as zinc, then it is highly likely that the PDTA-H4 would complex with enough of the zinc in the diet leading to an insufficient zinc intake in the animals. This would lead to evidence of male reproductive toxicity (specifically degeneration of the testicular tissue and reduced fertility) and many of the symptoms of zinc deficiency such as alopecia, diarrhea, eye and skin lesions etc.. Such a study would therefore not provide evidence of the reproductive of PDTA but rather the toxicity associated with a deficiency in zinc. Studies conducted with a similar chelating agent, EDTA (see above) failed to produce any evidence of testicular degeneration following dosing via gavage or drinking water. However, it induced developmental toxic effects (see below at Developmental toxicity).

It is expected that PDTA -like other chelants such as EDTA- is poorly absorbed both orally and via dermal application and is unlikely to be absorbed significantly via inhalation due to its high particle size (>10 microns diameter) when in powdered form and low volatility when in solution. It is not metabolised and is excreted quickly. With the exception of being able to complex metal ions, chelating agents are of low chemical reactivity as evidenced by their lack of genotoxicity and skin sensitising potential. As such, it is unlikely that the chelating agent itself is a proximate toxicant, but rather that its ability to bind metal ions is responsible for observed toxicity (as indicated above). Therefore if one were to conduct a study where metal ions (for example zinc) were supplemented sufficiently it is unlikely that any systemic toxicity, including developmental or reproductive effects would be observed. This is supported by developmental toxicity studies conducted with EDTA using additional zinc (see below at Developmental toxicity).

With regard to the metal-chelates (non-empty chelates), a study with EDTA-MnNa2 (Wolterbeek, 2010) showed decreased sperm motility at the highest dose of EDTA-MnNa2 tested (1500 mg/kg bw); it did not result in effects on reproduction as there were no changes in reproductive performance in animals of these groups. A study with DTPA-FeNaH (yet another chelate; summary not included), also resulted in decreased sperm motility at the highest dose of 1500 mg/kg bw, and besides this, decreased epididymes weight and sperm reserve. NOAELs in these two studies were 500 mg/kg bw.

These studies showed that if effects were observed, they were observed at higher levels than those of the empty chelates, indicating that binding of zinc (and perhaps other essential metal ions) had been less; as such bioavailability of those metal ions was higher.

In conclusion, it is plausible that a standard 2 -generation study conducted with PDTA-FeNH4 would (again) identify evidence of reproductive toxicity at a level in excess of 1000 mg/kg bw, as was seen with EDTA-MnNa2 and DTPA-FeNaH. However, such toxicity would be due to an induced deficiency in zinc and any reproductive effects would be observed only in the presence of, and secondary to parental toxicity. Therefore it is considered inappropriate to propose any further testing on PDTA-FeNH4 since the likely outcome of the study can be predicted without using animals unnecessarily. Reproductive toxicity effects secondary to a zinc deficiency should not be considered relevant if it can be demonstrated that occupational or consumer exposure to PDTA-FeNH4 would not result in a deficit in an individuals zinc status. Based on the NOAELs of 500 mg/kg bw and the expected very low 'intake' of PDTA-FeNH4 by both workers and consumers, it is expected that there is a high margin of safety, and as such a very low risk. Since reproductive toxicity would require a zinc deficiency to be induced as a first step in the toxicity it is very unlikely that this would occur. This was also concluded by Heimbach et al. (2000) and in the RAR on EDTA-H4 and EDTA-Na4 (2004). Heimbach et al. (2000) concluded that EDTA compounds are not reproductive toxicants when fed with a nutrient sufficient diet or minimal diets supplemented with Zn. Because in this case animals were treated with PDTA-FeNH4 and not with an empty chelate such as EDTA-H4 or EDTA-Na4, binding of zinc will even be less.


Short description of key information:
No study is available on PDTA-FeNH4. However, several studies are available on related substances as PDTA-H4, EDTA-CaNa2, EDTA-Na2H2, and EDTA-MnNa2. The first three substances are considered to be 'empty' chelates (i.e. they may bind metal ions quickly); the last one, EDTA-MnNa2, is a metal chelate. This one will bind other metal ions less quickly. A one generation study with PDTA-H4 showed effects on reproduction at a level of 300 mg/kg bw. The NOAEL was 60 mg/kg bw. Reproduction toxicity studies using the structurally very related substance EDTA-FeNH4 are not available. Therefore studies with EDTA-CaNa2 or EDTA-Na2H2 have been used for comparison. Data from a multigeneration study on rats with EDTA-CaNa2 did not give evidence for adverse effects on reproductive performance and outcome for doses of up to 250 mg/kg bw/day. For estimating a NOAEL other studies were not taken into consideration because of methodological flaws. Hence the NOAEL >= 250 mg/kg bw/day for EDTA-CaNa2. Decreased sperm motility was seen at the highest dose of EDTA-MnNa2 tested (1500 mg/kg bw); it did not result in effects on reproduction as there were no changes in reproductive performance in animals of these groups.

Effects on developmental toxicity

Description of key information
No data available on PDTA-FeNH4.
With regard to the structurally related substance EDTA, 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 et al., 1981). The effects observed were generally observed at high doses and consisted of 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 had been administered at only at one dose level, no oral NOAEL for either developmental toxicity or maternal toxicity could be established. In a study with EDTA-MnNa2 developmental effects were seen at a high level of 1500 mg/kg bw only; DTPA-FeNaH induced no developmental effects at all at a level of 1500 mg/kg bw.
Additional information

No data are available on PDTA-FeNH4.

The structurally related substance 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 gestation days 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 EDTA-H4 in which there had been some evidence of both maternal and fetotoxicity under the same experimental conditions. For the dams significant drug-related reactions including diarrhea and depression of activity were reported. The former occurred in all groups with highest incidences for EDTA-Na4 (90%) and EDTA-H4 (80%) and lowest incidence for EDTA-CaNa2 (10%). Three dams died during treatment with EDTA-Na2H2. 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 1000 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 gestation days 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 gestation days 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 1000 ppm zinc from gestation days 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 Swencrton, 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 gestation days 7 -14 (Kimmel, 1977). Dietary exposure to 3% EDTA-Na2H2 amounting to an average dose of 954 mg EDTA-Na2H2 per 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 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 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.

With regard to the metal-chelates (non-empty chelates), a study with EDTA-MnNa2(Wolterbeek, 2010) showed parental toxicity and effects on pup development at the highest dose of EDTA-MnNa2 tested (1500 mg/kg bw) consisting of a decreased number of females with live pups, a decreased number of (live) pups, and increased implantation loss. The NOAEL in this study was 500 mg/kg bw. A study with DTPA-FeNaH (summary not included), did not result in developmental effects even not at the high dose of 1500 mg/kg bw; the NOAEL for parental toxicity was 500 mg/kg bw.

These studies showed that if effects were observed, they were observed at higher levels than those of the empty chelates, indicating that binding of zinc (and perhaps other essential metal ions) had been less; as such bioavailability of those metal ions was higher.

In summary, there is a significant body of literature on the effects of the developing fetus of deficiencies in nutrients such as zinc. From this it is very clear that zinc plays such an important role in so many of the processes involved in the growth and development of the fetus that a deficiency in this nutrient has serious consequences. Subsequently any substance capable of negatively affecting the zinc status of the mother is likely to have adverse effects on the developing fetus resulting in varying degrees of malformations. These malformations will also depend on the duration and severity of the deficiency.

As indicated above PDTA-FeNH4 is a chelating agent with a high affinity for metals such as Zinc, Manganese and Calcium. As such deficiencies in zinc can produce a wide array of symptoms including both reproductive and developmental toxicity. If a developmental toxicity study were to be performed with PDTA-FeNH4 in the absence of any supplementation of essential minerals such as zinc, then it is highly likely that the PDTA-FeNH4 at high doses would complex with enough of the zinc in the diet leading to an insufficient zinc intake in the animals. This would lead to evidence of developmental toxicity such as terata of the skeletal and viscera and many othe symptoms of zinc deficiency such as alopecia, diarrhea, eye and skin lesions etc.. Such a study would therefore not provide evidence of the developmental toxicity of PDTA-FeNH4 but rather the toxicity associated with a deficiency in zinc.

As indicated above, it is expected that PDTA -like other chelants such as EDTA- is poorly absorbed both orally and via dermal application and is unlikely to be absorbed significantly via inhalation due to its high particle size (>10 microns diameter) when in powdered form and low volatility when in solution. It is not metabolised and is excreted quickly. With the exception of being able to complex metal ions, chelating agents are of low chemical reactivity as evidenced by their lack of genotoxicity and skin sensitising potential. As such, it is unlikely that the chelating agent itself is a proximate toxicant, but rather that its ability to bind metal ions is responsible for observed toxicity (as indicated above). Therefore if one were to conduct a study where metal ions (for example zinc) were supplemented sufficiently it is unlikely that any systemic toxicity, including developmental or reproductive effects would be observed. This is supported by developmental toxicity studies conducted with EDTA using additional zinc (see above).

In conclusion, it is plausible that a standard developmental toxicity study conducted with PDTA-FeNH4 could identify evidence of developmental toxicity, although it should be noted that no developmental effects were seen in the case of DTPA-FeNaH (another iron-containing chelate) up to a dose of 1500 mg/kg bw. However, if induced, such toxicity would be due to an induced deficiency in zinc and any developmental effects would be observed only in the presence of, and secondary to parental toxicity. Therefore it is considered inappropriate to propose any further testing on PDTA-FeNH4 since the likely outcome of the study can be predicted without using animals unnecessarily. Developmental toxicity effects secondary to a zinc deficiency should not be considered relevant if it can be demonstrated that occupational or consumer exposure to PDTA-FeNH4 would not result in a deficit in an individuals zinc status. Based on an expected relatively high NOAEL (see results of EDTA-MnNa2 and DTPA-FeNaH) and the expected very low 'intake' of PDTA-FeNH4 (as described above) by both workers and consumers, it is expected that there is a high margin of safety, and as such a very low risk. Since developmental toxicity would require a zinc deficiency to be induced as a first step in the toxicity it is very unlikely that this would occur.

Justification for classification or non-classification

Based on the results obtained in the toxicity studies with various EDTA substances (metal, salt and acid) and taking into account the provisions laid down in Council Directive 67/548/EEC and CLP, a classification has not to be done with respect to toxicity to reproduction as effects were mainly seen at levels in excess of 1000 mg/kg bw.

Studies with EDTA-MnNa2 and DTPA-FeNaH (other metal chelates), showed effects on reproduction at a high level of 1500 mg/kg bw. At that level, effects on fertility (specifically male fertility) were observed for both substances, and effects on pup survival only for EDTA-MnNa2. However, such effects on reproduction were considered secondary to a zinc deficiency and occured in conjunction with clear systemic toxicity consistent with such a deficiency. In addition, it is plausible that a standard reproduction and developmental toxicity study conducted with PDTA-FeNH4 at high doses would identify evidence of reproduction and/or developmental toxicity, as observed with the EDTA substances, which would also be secondary to a zinc deficiency.

Because effects on reproduction of the metal chelates EDTA-MnNa2 and DTPA-FeNaH were seen at a level of 1500 mg/kg bw - which is in excess of 1000 mg/kg bw; which was also true for the empty EDTA's (EDTA-Na4 and EDTA-H4) - and no such effects were seen at the still very high level of 500 mg/kg bw, no classification for the metal chelate PDTA-FeNH4 is proposed.