Reaction mass of disodium [2,2'-(imino-κN)dibutanedioato-κ2O1,O4(4-)]cuprum(2-) and sodium sulphate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

218 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

High quality (there is sufficient data for hazard assessment).

Additional information

No reproductive toxicity studies are available for the target substance Cu(2Na)IDHA. Therefore, the data on free IDHA chelating agent and Cu(2Na)EDTA have been used to assess toxicity potential of Cu (2Na)IDHA to reproduction (please refer to read-across statement). Due to the fact that Cu(2Na)IDHA can probably be absorbed more extensively than Cu(2Na)EDTA, the toxicity results of studies conducted with inorganic copper compounds were also taken into account.

One-generation study conducted with IDHA, sodium salt

The purpose of this one-generation study was to evaluate possible effects of IDHA sodium salt on the entire reproduction process in Wistar rats (OECD 415; Eiben and Rinke, 2002, Project No. PH-32294). The substance was administered to groups of 25 male and 25 female rats each at concentrations of 0 (control), 1000, 4000 and 16000 ppm in demineralized drinking water (correspond to 0, 105.3 -140.0, 411.0 - 480.1 and 2081.4 - 2270.8 mg/kg bw in males and females, respectively). Parental F0 animals were pretreated over a period of about 10 weeks and allowed to mate over a period of up to three weeks. F1 offspring were nursed up to an age of four weeks. Clinical signs, body weights, food and water intake, mating performance, fertility, duration of pregnancy, estrus cycling and sperm parameters were examined in F0 rats. Litter size, percentage of males born and pup weight at birth as well as viability, rearing, lactation and body weight gain were studied in F1 offspring. Necropsies were done in all rats. Implantation sites were recorded in F0 females. Selected organs were weighed (F0 and F1) and histopathological evaluations were performed on some organs of F0 rats.

Mortality and clinical appearance in F0 animals were unchanged at levels of up to 4000 ppm. At 16000 ppm diarrhoea was noted in both sexes and 2/25 females exhibiting histopathologically a strong kidney damage were killed because of complications at their parturition. At 16000 ppm retarded body weights were evident in parental rats. The food intake was unchanged up to 16000 ppm. The uptake of drinking fluid was higher in treated F0 rats than in controls. At 1000 and 4000 ppm the increase in water intake is most likely a result of the enhanced salt content of the drinking fluid and not considered as adverse. At 16000 ppm, however, a correlation to diarrhoea and several morphological changes in the urinary tract described below and increase in kidney weights found in this group cannot be excluded. The reproduction parameters insemination index, mating performance, fertility index, gestation index, duration of pregnancy, life birth index, birth weights, percentages of males born, total number of pups born, prenatal loss, mean litter size at birth as well as viability, rearing and lactation index were not affected at levels of up to 16000 ppm.

The body weight development of F1 pups was retarded at 16000 ppm with the consequence that female pups exhibited reduced spleen weights. At 16000 ppm more pups were pale than in the other groups.

No test substance-related gross pathological findings were observed in F1 offspring up to 16000 ppm. The skeletal development of the offspring was unaffected. No adverse effect was seen in sperm parameters at 16000 ppm. F0 females exhibited no treatment effects on estrus cycling up to 16000 ppm.

At 16000 ppm degenerative and reparative alterations in the kidneys such as (macro-and microscopical) dilations and hyperplasias in the renal pelvis and increased amounts of renal mineralization, a reduction of male-specific hyaline droplet accumulation and basophilic tubuli occurred more frequently in male and/or female F0 rats. Slight hyperplasia of the transitional cell epithelium occurred in the urinary bladder. Furthermore, ureters of high dose females were dilated macroscopically and degeneratively changed. These changes are most likely due to the marked overload with sodium of the drinking fluid and indicate severe parental toxicity. In the pituitary of 16000 ppm females hypertrophic cells within the pars distalis and vacuolation of the pars nervosa were noted more frequently. In 16000 ppm females, pituitaries were macroscopically swollen and increased in their weights as a correlate to this. The toxicological relevance of these findings are debatable and most probably not compound specific, but due to general toxicity of this dose.

An increase in inflammatory cells of the cecum in 16000 ppm females is considered as a correlate to cecum dilations seen macroscopically and diarrhoea. Gross pathology and histopathology in remaining organs revealed no treatment-related lesions.

Thus, the concentration of 4000 ppm in the drinking fluid is established as the overall no observed adverse effect level (NOAEL) for the parent animals and reproduction parameters. The NOAEL for the fertility is 16000 ppm.

Combined oral repeated dose toxicity study with reproduction /developmental toxicity screening test with Cu(2Na)EDTA in rats

In this study, the possible effects of EDTA-CuNa2 on reproductive performance and development, and its sub-chronic toxicity were examined in groups of 12 male and 12 female Wistar rats. EDTA-CuNa2 was administered daily by gavage during a premating period of 10 weeks and during mating, gestation and lactation until postnatal day 4 (OECD 422; Lina, 2013; Report No. V20114). The dose levels were 0 (tap water only), 150, 500 and 1500 mg/kg bw/day. Due to mortality, the high-dose level was reduced to 1050 mg/kg bw/day from day 9 of the study.

All male and female rats of the high-dose group were found dead or killed in moribund condition before the start of the mating period. Three males of the mid-dose group were killed in moribund condition during or at the end of the mating period. Clinical signs observed in rats of the high-dose group and, to a lesser extent, in the mid-dose group included thin appearance, hunched posture, piloerection, blepharospasm, swollen abdomen, soft faeces and green watery discharge around perineum. Neurobehavioural observations and motor activity assessment did not indicate specific neurotoxic effects of the test substance. Ophthalmoscopic examination did not reveal any treatment-related changes.

Body weights were decreased in males of the high-dose group, and, from the end of the premating period, in males of the mid-dose group. During lactation, female body weights were increased in the remaining treatment groups. Feed intake was reduced in males of the high-dose group.

In the surviving rats (control, low- and mid-dose group) which were killed on day 90 (males) or on day 4 of lactation (females) the following effects have been observed in the mid-dose group: reduced body weight in males, increased weight of kidneys, spleen and testis and decreased weight of ovaries. Haematology and clinical chemistry findings are described in the section for repeated dose toxicity. The main gross finding in animals of the mid-dose group (and in intercurrently killed animals of the high-dose group) were enlarged intestines with green/watery contents, a pale and/or green appearance of the liver and kidneys, small epididymides and seminal vesicles, enlarged dark spleen, small thymus and a variety of changes in the stomach (see section: repeated dose toxicity for details). The decedent high-dose animals were subjected to microscopical examination only on the basis of macroscopic observations. The microscopic observations in this group confirmed the above histopathological changes.

There were no effects of the test substance on male fertility parameters (epididymal sperm motility, sperm count and sperm morphology, and testicular sperm count and daily sperm production). No treatment-related effects were seen in any of the reproductive or developmental indices.

Based on the effects in liver and kidneys in the low-dose group, the no-observed-effect level (NOEL) for parental toxicity was lower than 150 mg/kg bw/day. Because fertility parameters, reproductive performance and development were not affected by the test substance, the NOEL for reproduction and developmental effects was placed at ≥ 500 mg/kg bw/day.

Toxicity results of studies conducted with copper inorganic compounds

No effects on reproduction were seen in a two-generation study with rats at doses up to 23.6–43.8 mg Cu/kg bw and day as copper sulphate pentahydrate (SCOEL, 2013). NOAEL of 140 mg Cu/kg bw for reproductive effects was established for rats and mice in a 13 -week study (SCOEL, 2013). Inorganic copper compounds at dose levels at 12 mg Cu/kg bw /day did not induced reproductive adverse effects in minks (ATSDR, 2004). NOAEL of 66 and 68 mg Cu/kg bw /day was established for reproductive effects in male and female rats, respectively. For mice, NOAEL was 398 and 536 mg Cu/kg/day for males and females, respectively.

Estimation of an equivalent realistic NOAEL for Cu(2Na)IDHA

The same as in case of repeated dose toxicity, an estimated dose level for Cu(2Na)IDHA is considered more appropriate than the read-across from NOEL established for reproductive toxicity for Cu(2Na)EDTA or IDHA chelating agent because of possible underestimation of reproductive toxicity of copper originating from Cu(2Na)IDHA. For comparison, the toxicity of copper is higher (NOAEL of 23.6 mg Cu/kg bw in rats in 2-gen. study) than the toxicity of the chelating agent IDHA (NOAEL for fertility is 16000 ppm (corresponds to 2081 and 2270 mg/kg bw in male and females, respectively) and overall NOAEL and for reproduction is 4000 ppm (corresponds to 411 and 480 mg/kg bw in males and females, respectively)) or the read-across substance Cu(2Na)EDTA (NOEL of ≥ 150 mg/kg bw). Therefore, this lowest dose level of 23.6 mg Cu/kg bw without reproductive effects is taken for the estimation. It corresponds to 130.8 mg/kg bw of Cu(2Na)IDHA: ((MW of Cu(2Na)IDHA) is 354.7 g/mol/ MW of Cu 64 g/mol) x 23.6 g). Taking into account 60 % oral absorption established for Cu(2Na)IDHA, the estimated dose would result in 218 mg/kg bw: 130.8 x (100 %/60 %). This dose level is lower than 500 mg/kg bw, the NOEL for Cu(2Na)EDTA and lower than 411-480 mg/kg bw, NOAEL for IDHA, therefore it is considered sufficient to ensure no risk of reproductive effects in humans. Besides this, the dose of 218 mg/kg bw is based on an equivalent dose level for copper which would release in case of only full dissociation of copper complexes (worst-case). As basis for this calculation serves equation:

4H+ + CuIDHANa2 + H2O⇔H4IDHA + 2Na+ + Cu2+

Short description of key information:
- One-generation study with IDHA, sodium salt (OECD 415): 0, 1000, 4000 and 16000 ppm in drinking water, rats. NOAEL: 4000 ppm (overall toxicity and reproductive parameters); NOAEL of 16000 ppm for fertility;
- Combined study (OECD 422): gavage; 0, 150, 500 and 1500/1050 mg/kg bw; rats; NOAEL: 500 mg/kg bw;
- ATSDR (2004) and SCOEL (2013) data on reproductive toxicity of inorganic copper compounds: the lowest NOAELs are presented;
- Estimated dose levels: 218 mg/kg bw (reproductive toxicity); 0.12 mg/kg bw (developmental toxicity).

Justification for selection of Effect on fertility via oral route:
No study is selected since NOAEL is estimated for Cu(2Na)IDHA based on the lowest dose established for copper in a two-generation study. The reason is that the toxicity of Cu(2Na)IDHA is believed to be mediated by copper rather than by the chelate moiety. Additionally, IDHA (and its chelates) are expected to be absorbed more extensively due to the higher oral absorption of IDHA comparing to the absorption of EDTA and its salts.

Effects on developmental toxicity

Description of key information

- Developmental study  with IDHA, sodium salt (OECD 414): 0, 100, 300 and 1000 mg/kg bw; Wistar rats; NOAEL: 1000 mg/kg bw. 
- One-generation study with IDHA, sodium salt (OECD 415): 0, 1000, 4000 and 16000 ppm in drinking water, rats. NOAEL: 4000 ppm (overall toxicity, reproductive parameters and postnatal toxicity); NOAEL of 16000 ppm for teratogenicity;
- Combined study (OECD 422): gavage; 0, 150, 500 and 1500/1050 mg/kg bw; rats; NOAEL: 500 mg/kg bw;
- ATSDR (2004) and SCOEL (2013) data on developmental toxicity of inorganic copper compounds: the lowest NOAELs are presented. LOAEL 1.3– 1.6 mg Cu/kg bw /day in mice.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

0.12 mg/kg bw/day

Study duration:

chronic

Species:

other: human

Quality of whole database:

High quality (there is sufficient data for hazard assessment).

Additional information

No developmental toxicity studies are available for the target substance Cu(2Na)IDHA. The data on free IDHA chelating agent and Cu(2Na)EDTA have been used to assess developmental toxicity potential of Cu (2Na)IDHA (please refer to read-across statement). The toxicity results of studies conducted with inorganic copper compounds were also taken into account.

Developmental study conducted with IDHA, sodium salt

Groups of 25 inseminated female Wistar rats each were treated daily orally (by gavage) with IDHA, sodium salt (73.4 %) solved in demineralized water from day 6 to day 19 p.c. in doses of 0, 100, 300 and 1000 mg/kg body weight (bw)/day, respectively (OECD 414; Klaus, 2002, Report No. PH-32141). The fetuses were delivered by cesarean section on day 20 of gestation. Investigations were performed on general tolerance of the test compound by the females. Findings of maternal toxicity consisted of transiently marginally impaired feed consumption and body weight gain at the 1000 mg/kg dose level. Further on treatment relationship could not be excluded for marginally reduced carcass weight and corrected body weight gain. All other parameters evaluated, i.e. mortality and clinical signs, water intake and excreta, overall body weight development including final body weight and gross pathological observations were not affected by treatment at a dose level up to and including 1000 mg/kg bw/day.With respect to intrauterine development, gestation rate, postimplantation loss and accordingly the number of fetuses, fetal sex distribution, placental weight and appearance and fetal weight were not affected by treatment with IDS, Na-salt at a dose level up to and including 1000 mg/kg bw/day. External, skeletal and visceral evaluation of fetuses revealed neither toxicologically relevant treatment related effects at a dose level up to and including 1000 mg/kg bw/day. A teratogenic potential of IDHA, sodium salt was not evident at a dose level up to and including 1000 mg/kg bw/day.

Summarizing and evaluating all data investigated the following no-observed-effect levels (NOEL) were determined:

Maternal toxicity: 300 mg/kg bw/day

Developmental toxicity: 1000 mg/kg bw/day.

One-generation study conducted with IDHA, sodium salt

The purpose of this one-generation study was to evaluate possible effects of IDHA sodium salt on the entire reproduction process in Wistar rats (OECD 415; Eiben and Rinke, 2002, Project No. PH-32294). The substance was administered to groups of 25 male and 25 female rats each at concentrations of 0 (control), 1000, 4000 and 16000 ppm in demineralized drinking water (correspond to 0, 105.3 -140.0, 411.0 - 480.1 and 2081.4 - 2270.8 mg/kg bw in males and females, respectively). Parental F0 animals were pretreated over a period of about 10 weeks and allowed to mate over a period of up to three weeks. F1 offspring were nursed up to an age of four weeks.

The body weight development of F1 pups was retarded at 16000 ppm with the consequence that female pups exhibited reduced spleen weights. At 16000 ppm more pups were pale than in the other groups. No test substance-related gross pathological findings were observed in F1 offspring up to 16000 ppm. The skeletal development of the offspring was unaffected.

Combined oral repeated dose toxicity study with reproduction /developmental toxicity screening test with Cu(2Na)EDTA in rats

In this study, the possible effects of EDTA-CuNa2 on reproductive performance and development, and its sub-chronic toxicity were examined in groups of 12 male and 12 female Wistar rats. EDTA-CuNa2 was administered daily by gavage during a premating period of 10 weeks and during mating, gestation and lactation until postnatal day 4. The dose levels were 0 (tap water only), 150, 500 and 1500 mg/kg bw/day. Due to mortality, the high-dose level was reduced to 1050 mg/kg bw/day from day 9 of the study.

All male and female rats of the high-dose group were found dead or killed in moribund condition before the start of the mating period. Three males of the mid-dose group were killed in moribund condition during or at the end of the mating period. Clinical signs observed in rats of the high-dose group and, to a lesser extent, in the mid-dose group included thin appearance, hunched posture, piloerection, blepharospasm, swollen abdomen, soft faeces and green watery discharge around perineum. Neurobehavioural observations and motor activity assessment did not indicate specific neurotoxic effects of the test substance. Ophthalmoscopic examination did not reveal any treatment-related changes.

Body weights were decreased in males of the high-dose group, and, from the end of the premating period, in males of the mid-dose group. During lactation, female body weights were increased in the remaining treatment groups. Feed intake was reduced in males of the high-dose group.

In the surviving rats (control, low- and mid-dose group) which were killed on day 90 (males) or on day 4 of lactation (females) the following effects have been observed in the mid-dose group: reduced body weight in males, increased weight of kidneys, spleen and testis and decreased weight of ovaries. Haematology and clinical chemistry findings are described in the section for repeated dose toxicity. The main gross finding in animals of the mid-dose group (and in intercurrently killed animals of the high-dose group) were enlarged intestines with green/watery contents, a pale and/or green appearance of the liver and kidneys, small epididymides and seminal vesicles, enlarged dark spleen, small thymus and a variety of changes in the stomach (see section: repeated dose toxicity for details). The decedent high-dose animals were subjected to microscopical examination only on the basis of macroscopic observations. The microscopic observations in this group confirmed the above histopathological changes.

There were no effects of the test substance on male fertility parameters (epididymal sperm motility, sperm count and sperm morphology, and testicular sperm count and daily sperm production). No treatment-related effects were seen in any of the reproductive or developmental indices.

Based on the effects in liver and kidneys in the low-dose group, the no-observed-effect level (NOEL) for parental toxicity was lower than 150 mg/kg bw/day. Because fertility parameters, reproductive performance and development were not affected by the test substance, the NOEL for reproduction and developmental effects was placed at ≥ 500 mg/kg bw/day.

Toxicity results of studies conducted with copper inorganic compounds

Inorganic copper compounds at dose levels at 1 mg Cu/kg bw /day and higher induced a variety of developmental effects in animal studies (ATSDR, 2004). Among studies with different species and study durations, the lowest dose at which copper sulphate induced developmental effects was 1.3– 1.6 mg Cu/kg bw /day in mice. This dose corresponds to an air concentration of about 20 mg Cu/m³. At the recommended OEL of 0.01 mg Cu /m³, no developmental effects are expected to occur to humans (SCOEL, 2013). The recommended daily allowance (RDA) for copper is 0.013 mg/kg bw.

Estimation of an equivalent realistic NOAEL for developmental toxicity for Cu(2Na)IDHA

The same as in case of reproductive toxicity endpoint, the read-across from the NOAL of > 500 mg/kg bw established for the read-across substance Cu(2Na)EDTA for developmental effects (OECD 422) may underestimate the risk of developmental toxicity of copper originating from the exposure to Cu(2Na)IDHA. This is because of the higher oral absorption of IDHA (37 %) comparing to 5 % for EDTA or less than 1 -2 % for EDTA complexes. In this regard, developmental toxicity is expected to be mediated by elemental copper ions rather than that by free IDHA chelating agent. No developmental toxicity were observed in offspring from dams treated with the IDHA, sodium salt by the highest dose level (16000 ppm or 2270.8 mg/kg bw, OECD 415). In the developmental study with IDHA (OECD 414), sodium salt, NOAEL of 1000 mg/kg bw, the highest dose level, was established for developmental toxicity. Thus, an estimated dose level for Cu(2Na)IDHA is considered more appropriate than the read-across from NOAEL established for Cu(2Na)EDTA or IDHA chelating agent. No NOAEL exists for developmental effects for copper in animal studies; the lowest level known at which copper induced developmental effects was 1.3 -1.6 mg/kg bw in mice. Therefore, the recommended daily allowance (RDA) of 0.013 mg Cu/kg bw is considered to be a sufficiently safe level to estimate an equivalent dose for Cu(2Na)IDHA for developmental effects. It corresponds to 0.072 mg/kg bw for Cu(2Na)IDHA: ((MW of Cu(2Na)IDHA is 354.7 g/mol/ MW of Cu is 64 g/mol) x 0.013 g).

Taking into account 60 % oral absorption established for Cu(2Na)IDHA, the estimated NOAEL would be 0.12 mg/kg bw: 0.072 x (100 %/ 60 %).

This dose level is considered to be also an internal DNEL for systemic effects for Cu(2Na)IDHA (see DNEL derivation section).

Justification for selection of Effect on developmental toxicity: via oral route:
No study is selected since NOAEL for Cu(2Na)IDHA is estimated based on the recommended daily intake of 0.013 mg/kg bw established for copper by WHO. The reason is that the toxicity of Cu(2Na)IDHA is believed to be mediated by copper rather than by the chelate moiety. Additionally, IDHA (and its chelates) are expected to be absorbed more extensively due to the higher oral absorption of IDHA comparing to the absorption of EDTA and its salts.

Justification for classification or non-classification

Effects on reproduction and effects on fetal development have not been observed at levels up to 500 mg/kg bw in the combined repeated dose toxicity study with the read-across substance Cu(2Na)EDTA. At the next higher level of 1500/1050 mg/kg bw significant mortality occurred before mating took place. NOAEL of 411 -480 mg/kg bw for males and females, respectively, was established for the chelating agent IDHA for reproductive effects and 1000 mg/kg bw, the highest dose level tested, for developmental effects. However, NOAEL for reproductive and developmental effects for copper (element) are much lower: at 23.6 mg/kg bw there were no reproductive toxic effects in rats but developmental effects were still present at 1.3 mg Cu/kg bw in mice. Therefore, due to the possible higher absorption of Cu(2Na)IDHA complexes comparing to very low absorption of EDTA and its complexes, NOAEL of 218 mg/kg bw for fertility was estimated for Cu(2Na)IDHA based on NOAEL of 23.6 mg/kg bw for copper. Similarly, NOAEL of 0.072 mg/kg bw was estimated for developmental effects based on RDA for copper (0.013 mg/kg bw) established by WHO. Neither reproductive nor developmental effects can be expected at these dose levels.

Based on the results obtained in the available studies and taking into account the provisions laid down in Council Directive 67/548/EEC and CLP, classification with regard to toxicity to reproduction is not required.