Acetic acid, oxo-, sodium salt, reaction products with ethylenediamine and phenol, iron sodium salts

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route

Dose descriptor:

NOAEL

175 mg/kg bw/day

Additional information

In an extended OECD 422 study (with a 10-wk premating period) with FeNa-EDDHA, at the high dose of 300 mg/kg bw/day (which was gradually reduced to 175 mg/kg bw/day during the course of the study due to too high toxicity) there were notable effects on the ability of the females to give birth when they had strongly adverse reduced food consumption and body weights, and changes in haematology and clinical chemistry parameters. Parental toxicity was especially noted in pregnant females of the high FeNa-EDDHA group and it was finally decided to sacrifice the remainder of these females around gestation day 22. In the animals found dead or killed prematurely, microscopic findings consisted of renal tubular degeneration and necrosis, hepatocellular degeneration and necrosis with and without hemorrhage, increased pigmented macrophages within splenic red pulp and medullary sinuses of mesenteric lymph nodes (draining lymph nodes of the intestinal tract, oral dosing), decreased lymphoid tissue of the thymus (atrophy), and compensatory responses within bone marrow of progenitor cells. These were collectively interpreted to reflect kidney and liver toxicity due to oral dosing of FeNa-EDDHA with increased sensitivity especially in gestational females. These findings were not noted in the animals scheduled for necropsy on Day 92 (males) and Day 112 (females). Increased numbers of pigmented macrophages within draining lymph nodes of the intestines and within splenic red pulp with progenitor cell ratio alterations of the bone marrow were interpreted to be highly suggestive of red blood cell injury and increased turnover. The findings noted may be related to the intake of the test substance which affected the weight gain, nutrition and condition of these animals, and caused subsequent complications during parturition. Most of these effects were predicted on maternal condition alone and were considered secondary to maternal performance. There were slight reductions in litter and pup weights on PND13 in the mid dose FeNa-EDDHA group, but there were no other findings which were indicative of effects on fertility, gestation, development of the conceptus or survival in lactation following administration of the test material for 10 weeks prior to and during gestation. Thus, the NOAEL for systemic toxicity in parental animals was 25 mg FeNa-EDDHA/kg bw/day under the conditions of this study; the NOAEL for reproductive toxicity was at least 175 mg/kg bw/day, and for developmental toxicity (lower litter/pup weight) 25 mg/kg bw/day but this was noted in the presence of clear maternal toxicity. 

In this study, a 5th group was included which was treated with the structurally related FeNa-EDDHMA, for bridging purposes. The dose for this group was also gradually reduced from 300 to 175 mg/kg bw/day, and similar findings were noted although to a slightly lesser extent. The remaining females of this group were not euthanized but kept on study wthout further dosing (no dosing from Day 95 - end of gestation - until Day 112 - scheduled sacrifice).

FeNa-EDDHMA was earlier tested in a one-generation reproduction (OECD 415) toxicity study (NOTOX B.V., 1997). FeNa-EDDHMA was administered to 28 Wistar rats/sex/dose level by single oral gavage at dose levels of 50, 200 or 750 mg/kg bw/day. A concurrent control group was treated with the vehicle only. Treatment commenced 10 weeks prior to mating for males and 2 weeks prior to mating for females and continued for both sexes until at least the end of the lactation period. Pregnant females were allowed to litter normally. On day 4 of lactation, each litter was adjusted to 4 males and 4 females or as near as possible. The surviving offspring was euthanised after weaning. Also, the primary effect on parental animals was poor physical condition, resulting in premature mortality, growth reduction and reduced food consumption in both sexes at 750 mg/kg bw/day. These signs were seen with reduced severity at 200 mg/kg bw/day in males only. Thus, the NOAEL for systemic toxcity in parental animals was 50 mg/kg bw/day. Parental rats of the 750 mg/kg group showed a slight decrease in the fertility and conception indices. The poor physical condition of animals in the 750 mg/kg dose group might have been responsible for this effect; and as no histopathological evidence of toxicity to reproduction or infertility was noted the NOAEL for reproductive toxicity at least 750 mg/kg bw/day. In the offspring, increased postnatal loss and reduced viability were noted during PND 0-4 at 200 and 750 mg/kg bw/day, and with lower incidence at 50 mg/kg bw/day. Based on the results of the 28-day and 90-day study with FeNa-EDDHMA, it cannot be excluded that anaemia and/or impaired renal function may have been present that finally caused the observed litter losses. On the other hand, the finding in the low dose group consisted of only one female with total litter loss (15 pups) and 6 other females with one dead pup/litter which was within normal limits and might have occurred by chance. Therefore, with special regard to the low incidence and unusual distribution pattern of findings noted at 50 mg/kg bw/day, the NOAEL for developmental toxicity was considered 50 mg/kg bw/day under the conditions of this study. Effects at higher levels were considered closely related to (subclinical) maternal toxicity. 

Data Waiving for extended one-generation study:

The performance of an extended one-generation reproductive toxicity study is scientifically unjustified: no evidence of reproduction toxicology is seen in any tests available. A one-generation study with the structurally related substance FeNa-EDDHMA and a recently performed extended OECD 422 study with FeNa-EDDHA (with an additional high dose group with FeNa-EDDHMA for bridging purposes), administered via oral application, did not show evidence of reproductive toxicity, and developmental toxicity in these studies was only noted at doses clearly showing parental/maternal toxicity. In the developmental toxicity study no adverse effects on development were observed at all up to an exposure level of 500 mg/kg bw/day (highest tested dose).

In two repeated dose toxicity studies with oral administration (28-day and 90-day exposure) and in one 28-day repeated dose dermal toxicity study, no adverse effects were seen on the reproduction organs (no organ weight changes and no adverse histopathological findings noted on reproductive organs).

In summary, it is very unlikely that FeNa-EDDHA will cause adverse reproductive and/or developmental toxicity effects at doses without maternal systemic toxicity in an EOGRTS and conducting such a study is thus scientifically not justified. The available one-generation study with FeNa-EDDHMA (read across), the recently performed extended OECD 422 study with FeNa-EDDHA and FeNa-EDDHMA, and the developmental toxicity study with FeNa-EDDHA are sufficient for hazard assessment and risk characterisation. 

Short description of key information:
For the assessment of toxicity to reproduction/fertility, the read-across approach was applied using data from a one generation reproduction (OECD 415) toxicity study conducted with the test item FeNa-EDDHMA (EC number 405-420-1), which is a structural analogue of FeNa-EDDHA. This approach is considered justified since, in addition to the very similar physico-chemical properties of both substances, there are marked similarities with respect to their toxicological profiles as evidenced by: no acute toxicity, no skin or eye irritating properties, no mutagenic effects and comparable NOELs for repeated dose toxicity. For justification of read acoss see also section 13.

Effects on developmental toxicity

Description of key information

In a key developmental oral toxicity study (CIBA-GEIGY Limited, 1995) with FeNa-EDDHA in rats, the NOAEL/NOEL for developmental effects was established at the high dose level of 500 mg/kg bw/day based on the absence of embryo-/foetotoxic or teratogenic effects.

In addition, an extended OECD 422 study (with a 10-wk premating period) with FeNa-EDDHA has been performed. Regarding the results available for the reproductive and developmental toxicity of FeNa-EDDHA, at the high dose of 300 mg/kg bw/day (which was gradually reduced during the course of the study due to too high toxicity) there were notable effects on the ability of the females to give birth when they had strongly adverse reduced food consumption and body weights, and changes in haematology and clinical chemistry parameters (see above at reproduction). Most of these effects were predicted on maternal condition alone and were considered secondary to maternal performance. There were slight reductions in litter/pup weight in the mid dose FeNa-EDDHA group, but this was seen in the presence of clear maternal toxicity. There were no other findings which were indicative of effects on fertility, gestation, development of the conceptus or survival in lactation following administration of the test material for 10 weeks prior to and during gestation.

For MnNa2-EDTA, in an OECD414 study in rabbits (Raaf de - Beekhuijzen, 2016), slight developmental toxicity, which consisted of a non-statistically significant increase of caudal shift of pelvic girdle, was observed in presence of maternal toxicity at 100 mg/kg bw (with and without extra zinc) using the a structural analogue substance, MnNa2-EDTA. The mean litter incidences of this variation were 16.7%, 19.6%, 15.6%, 30.7% and 26.2% per litter at 0, 10, 30, 100 mg/kg bw and 100 mg/kg bw (extra zinc), respectively. However, the number of litters in which this finding was seen, was not different, viz. 15, 15, 11, 16 and 15 litters, respectively. These values were within the historical control range (max 38.5%). In addition, ‘caudal shift’ is often seen together with an increase in the number of ‘13th full ribs’ which was not the case in this study. In addition, at 100 mg/kg without extra zinc, the fetuses showed signs of retarded skeletal ossification. This was evidenced by higher (but non-statistically significant) incidences of unossified metacarpals and reduced ossification of the skull. Mean litter incidences of these variations were for unossified metacarpals 4.2%, 6.0%, 8.2%, 13.1%, 6.5% and for reduced ossification of the skull 0.8%, 1.8%, 0.0%, 2.4% and 1.6% per litter at 0, 10, 30, 100 mg/kg bw and 100 mg/kg bw (extra zinc), respectively. The two signs of retarded skeletal ossification in the 100 mg/kg bw group (without extra zinc) were seen in the presence of lower fetal body weights (means of 34.6 grams versus control value 38.1 grams) and lower maternal body weights. At 100 mg/kg with extra zinc, no delay in skeletal ossification was observed despite a slightly lower fetal body weight (mean of 36.1 grams versus control value 38.1 grams) and a slightly lower maternal body weight gain. These results showed that zinc supplementation amended the slight developmental delay noted in the 100 mg/kg bw group without extra zinc. The maternal NOAEL was considered to be 30 mg/kg bw as at 100 mg/kg bw a sustained reduced food intake was seen as well as reduced body weight gain which did not fully recover in animals that received 100 mg/kg without extra zinc. In all treated groups, there was a high incidence of gray discoloration of the urine. In addition, diarrhea and feces containing mucus were noted in a dose related manner at all dose levels. Increases in creatine kinase levels were seen in several animals of all test groups; the increases in creatine kinase levels were not statistically significant compared to the controls as a high variation within the groups was observed.

It should, however, be noted that rabbits will have had extra exposure to MnNa2-EDTA due to coprophagy. Rabbits (herbivores) do not have a complex ruminant digestive system. They extract extra nutrition from grass by giving their food a second pass through the gut. Rabbits produce cecotropes which are called 'soft feces’ or ‘night feces’. The cecotropes are the material resulting from the fermentation of food in a part of the digestive system, the cecum. Rabbits also excrete another kind of feces which is their typical hard fecal pellet, but they do not normally consume that. Cecotropes are nutrient-rich and are passed out of the body, like feces, but are re-ingested by the rabbit so that more nutrients can be absorbed. Cecotropes have twice the protein and half the fiber of their typical hard fecal pellets. They also contain high levels of vitamin K and B vitamins (Vitamin B 12 in particular). After ingestion, on the second pass through, the extra nutrients are absorbed by the small intestine. Without this process, many of the nutrients in the food would be lost and passed through the colon, and out as typical feces. In addition, rabbits have a more delicate gut microflora (diarrhea and faeces containing mucus were seen in the study) and it is well known that bacteriostatic substances such as chelating agents and biocidal substances disturb the balance of the rabbit intestinal/caecal microflora which in turn may lead to malnutrition and subsequent maternal toxicity (ECHA, 2016). These may be the reasons why rabbits are only used for examination of developmental toxicity and not used as the preferred species in repeated dose toxicity studies meant to establish health effects in humans. Finally, rabbits generally reduce their food intake within the last phase of their pregnancy by about 50% due to nestbuilding behavior and stress, which was also seen in the study with MnNa2-EDTA. This means that the zinc intake via the food is reduced by 50% whereas the administration of the chelate did not change. Altogether this may clearly explain a lower NOAEL in rabbits versus rats. Overall, rabbits are considered to be not the preferred species to investigate developmental effects induced by chelating substances which are absorbed to a low extent and might induce a malnutritional state which, however, is particularly induced via a substance-independent nutritional habit of rabbits and the potential metal-ion sequestration of chelates. Therefore and due to the fact that a rather low NOAEL of 10 mg/kg bw/day for systemic toxicity was identified in repeated dose studies with FeNa-EDDHA, and 25 mg/kg bw/day in the extended OECD 422 study, it is considered that no additional PNDT study in rabbits is required for FeNa-EDDHA.

 

Additional information

In a developmental toxicity study FeNa-EDDHA, it was administered once daily to groups of 24 mated female Sprague-Dawley derived rats by oral gavage at 5, 100 or 500 mg/kg bw/day from day 6 through day 15 of gestation. Control group females received the vehicle only. All dams were sacrificed on day 21 of the gestation period and foetuses removed for examination. In dams, there were no treatment-related clinical signs or incidences of mortality. The body weight gain was reduced at 500 mg/kg bw/day for the period from days 6 -16 of gestation, and reduced food consumption was also noted at this dose level. No adverse effects on pregnancy and no embryo-/foetotoxic effects were observed. There was no indication of teratogenic potential. On the basis of these results, the NOEL was 100 mg/kg bw/day for maternal toxicity and at least 500 mg/kg bw/day for develomental toxicity and teratogenicity.

Justification for classification or non-classification

The test item caused no developmental toxicity and no teratogenicity in the rat in a key developmental toxicity study according to test guideline OECD 414, and in a key developmental study in rabbits (OECD 414; read across). In an extended OECD 422 study, there were slight reductions in litter/pup weights, but there were no other findings which were indicative of effects on development of the conceptus or survival in lactation following administration of the test material for 10 weeks prior to and during gestation.

Effects on reproductive performance/fertility were assessed by means of a read-across approach using a one generation toxicity study according to test guideline OECD 415 conducted with a structural analogue FeNaEDDHMA (see also read across document in section 13). Only a slight change in reproductive performance was noted in this study at 750 mg/kg bw/day. In the absence of concomitant histopathological findings, this slight change in reproductive performance was considered to be secondary to the poor clinical condition of the animals at this dose level. Similarly, developmental effects were seen in this study at dose levels at pronounced maternal toxicity only. In the extended OECD 422 study with FeNa-EDDHA, at the high dose of 300 mg/kg bw/day (which was gradually reduced to 175 mg/kg bw/day) there were notable effects on the ability of the females to give birth when they had strongly adverse reduced food consumption and body weights, and changes in haematology and clinical chemistry parameters. Parental toxicity was high especially in females of the high FeNa-EDDHA group and it was finally decided to sacrifice the remainder of these females around gestation day 22. Most of these effects were predicted on maternal condition alone and were considered secondary to maternal performance. There were no other findings which were indicative of effects on fertility or gestation following administration of the test material for 10 weeks prior to and during gestation.

Accordingly, the substance is not subject to classification for toxicity to reproduction according to Regulation (EC) No 1272/2008 as amended for the ninth time in Regulation (EC) No 2016/1179.