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

Endpoint summary

Description of key information

- IDHA chelating agent: OECD 407; 28-day oral (gavage) study, rats, NOAEL: 200 and 1000 mg/kg bw in males and females, respectively;
- Cu(2Na)EDTA: OECD 422; rats, NOAEL: 150 mg/kg bw (males/females);
- Copper and its inorganic compounds (oral exposure): SCOEL, 2013: RDA of 0.09 mg/kg bw ;
- Copper and its inorganic compounds (inhalation exposure): SCOEL, 2013: OEL of 0.03 mg/m³ (inhalable fraction);
- Estimated dose levels for Cu(2Na)IDHA: NOAEL of 0.12 mg/kg bw for humans (oral); NOAEC of 0.28 mg/m³ for humans (inhalation).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

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).

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

0.28 mg/m³

Study duration:

chronic

Species:

other: human

Quality of whole database:

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

Additional information

No repeated dose 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 toxicity potential of Cu(2Na)IDHA at repeated exposures (please refer to read-across statement).

28 -Day oral (gavage) study with the chelating agent IDHA in rats

Iminodisuccinic acid, sodium salt was administered orally to Wistar rats (5 males and 5 females per dose) once a day, by gavage in target doses of 0 (vehicle control) - 40 -200 - 1000 mg/kg body weight over a period of 4 weeks. In addition, 5 male and 5 female rats per group were treated with the vehicle or 1000 mg/kg body weight and observed for reversibility, continuance or delayed occurrence of toxic effects during a recovery period of 14 days. Mortality was unaffected by treatment with the test substance. Appearance, clinical findings and general behaviour were not altered by treatment with the test substance up to and including 1000 mg/kg body weight. Growth and food consumption were not affected by the treatment.

Hematological investigations gave no indication of toxicologically relevant damage to blood, hematopoetic organs or coagulability up to and including 1000 mg/kg body weight. Clinical laboratory tests produced no evidence of treatment-related metabolic or organ damage.

Gross and histopathological investigations of various organs and tissues gave no indication of test-compound-related functional or morphological changes in both sexes up to and including 1000 mg/kg body weight. The organ weights were unaffected with the exception of a decrease of relative thymus weight in the recovery group (1000 mg/kg body weight). Taking into account the discrepancy of observations in the main groups and in the recovery groups and the fact that there was no evidence of treatment related effect in haematology examinations as well as in the histopathological examinations of thymus in the main group and the recovery group and of adrenals, spleen, draining and distant lymph nodes and bones in the main groups the decrease of relative thymus weight is considered to be Of no toxicological relevance.

The assessment of motor activity (horizontal activity) showed a wide variety of individual values. There was no effect on the motor activity up to 200 mg/kg body weight in both sexes. With 1000 mg/kg body weight the motor activity in the males in the main groups and in the recovery groups was below that of the respective control group at the end of the sampling period. Functional observations as another marker for neurotoxicity gave no evidence of a neurotoxic potential. In addition, the histopathological examination of brain, spinal cord and sciatic nerve gave no evidence of a neurotoxic action of the test material. In conclusion, taking into account all available data with regard to neurotoxicity, the toxicological relevance of the findings with regard to motor activity is questionable.

Under the conditions described the administration of Iminodisuccinic acid, sodium salt, to male and female rats was tolerated without treatment-related lesions up to and including 200 mg/kg body weight in the males and up to and including 1000 mg/kg body weight in the females. Therefore the no observed adverse effect level (NOEL) for the daily administration of Iminodisuccinic acid, sodium salt, is considered to be 200 mg/kg body weight in males and 1000 mg/kg body weight in females. The toxicological relevance of the isolated effect on motor activity in the males with 1000 mg/kg body weight is questionable.

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

In a study, the possible effects of Cu(2Na)EDTA on reproductive performance and development, and its sub-chronic toxicity were examined in groups of 12 male and 12 female Wistar rats (OECD 408, Lina, 2013; Report No. V2014). Cu(2Na)EDTA 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.

Haematology and clinical chemistry was conducted in females on day 65 (at the end of the premating period), and in males on day 85 (a few days prior to necropsy). At these time points, all males and most females of the high-dose group had died or had been killed.

The following changes in haematology were noted in the mid-dose group (now representing the highest dose level):

-       Prothrombin time was decreased in both sexes.

-       Red blood cell count was increased and MCV and MCH were decreased in males.

-       Total white blood cell counts and absolute neutrophils and monocytes counts were increased in males. 

The following changes in clinical chemistry were noted:

-       ALP, ASAT, ALAT and GGT activity were increased in males of the mid-dose group. In females ALP activity (mid-dose group) and ALAT activity (low- and mid-dose group) were decreased.

-       Bilirubin was increased in males of the mid-dose group

-       Creatinine was increased in the mid-dose group in both sexes

-       Urea, inorganic phosphate and calcium were increased in mid-dose males

-       Sodium was increased in males of the low- and mid-dose group. Potassium was increased in females of the mid-dose group.

The surviving rats (control, low- and mid-dose group) were killed on day 90 (males) or on day 4 of lactation (females).

-       Terminal body weights were decreased in males and increased in females of the mid-dose group.

-       The absolute and the relative weights of the heart were decreased in the mid-dose group in both sexes.

-       The relative weight of the kidneys was increased in males of the mid-dose group. In females of this group, the absolute kidney weight was increased.

-       The absolute and the relative weights of the spleen were increased in the mid-dose females.

-       The absolute and the relative weights of the ovaries were decreased in females of the mid-dose group.

-       The absolute weight of the testis was decreased in mid-dose males. 

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.

Microscopic examination revealed histopathological changes in the kidneys, the liver and the spleen.

-       The histopathological changes in the kidneys were characterised by tubular necrosis and degeneration, tubular epithelial cell karyomegaly and accumulation of brown pigment. These changes were mainly present in the mid-dose animals. However, tubular epithelial brown pigment was also noted in the kidneys of 6/10 low-dose males.

-       The changes in the liver were accumulation of periportal macrophages, especially in the mid-dose males, hepatocellular karyomegaly, brown pigment accumulation, bile duct hyperplasia and (multi)focal infiltration of mononuclear inflammatory cells. These changes were mainly present in the mid-dose animals. However, mononuclear cell infiltrate was also noted in the liver of 6/10 low-dose males and 3/10 low-dose females.

-       The changes in the spleen were accumulation of brown pigment and accumulation of macrophages in the white pulp in animals of the mid-dose group.

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.

Based on the histopathological effects in liver and kidneys noted in animals of the low-dose group, the no-observed-effect level (NOEL) for parental toxicity was lower than 150 mg/kg bw/day. However, because only limited effects were observed in the low-dose group, it was speculated that the NOEL was close to 150 mg/kg bw/day.

Toxicity results of studies conducted with copper inorganic compounds

Oral exposure

Copper is an essential nutrient that is incorporated into a number of biomolecules. The general population is exposed to copper through inhalation, consumption of food and water, and dermal contact with air, water, and soil that contains copper. The primary source of copper intake is the diet; however, the amount of copper in the diet usually does not exceed the average dietary requirements (RDAs) for copper. Drinking water is the primary source of excess copper (ATSDR, 2004).

In the United States, the median intake of copper from food is 0.93– 1.3 mg/day for adults (0.013–0.019 mg Cu/kg body weight/day using a 70-kg reference body weight). A recommended dietary allowance (RDA) of 0.9 mg/day (0.013 mg/kg/day) has been established.

Regarding human data, repeated oral exposure to copper by contaminated drinking water led to gastrointestinal effects similar to those observed after acute exposure (abdominal pain, nausea, vomiting, diarrhoea) (Greim, 2004 cited in SCOEL, 2013; ATSDR 2004). The most reliable case study in terms of exposure characterisation is that by Spitalny et al. (1984, cited in ATSDR, 2004 and SCOEL, 2013), which documents effect concentrations of 3.1–7.8 mg/L drinking water and a NOAEL of 1.58 mg/L. Controlled, well conducted studies with subacute to subchronic exposure of 60– 340 volunteers to copper (added to drinking water) revealed a LOAEL of 3–4 mg/L (0.073–0.092 mg Cu/kg × day) and a NOAEL of 1–2 mg/L (0.027–0.042 mg Cu/kg × day) for first gastrointestinal complaints (Araya et al 2003b, Pizarro et al 1999 cited in SCOEL, 2013).

Regarding animal data, mice were less susceptible to copper than rats. Kidney and liver toxicity, forestomach lesions as well as alterations in haematological and clinical chemistry parameters were common observations in the animal 14 -day drinking water study, 14 -day feeding study and in the combined repeated dose and reproductive/developmental toxicity study. NOAEL for animals: 26 mg/kg bw (ras; 14-day feeding study), 17 mg/kg bw (mice; 14-day feeding study); 2 mg Cu/kg bw /day in male rats and 0.83 mg Cu/kg bw /day in female rats (combined repeated dose and reproductive/ developmental study). LOAEL: 10 mg/kg bw (rats; 14-day drinking water study).

Inhalation exposure

A NOAEL of 0.36 mg/m³ has been estimated for acute sensory irritation in humans (SCOEL, 2013). It is not known, whether metal fume fever-like symptoms observed in employees exposed to copper dust at 0.12–0.36 mg/m³ is primarily dependent on concentration or on total dose (concentration × time product). Given all the uncertainties, a scientifically based STEL cannot be recommended. At the recommended OEL of 0.01 mg/m³, no developmental effects are expected to occur. Assuming an oral absorption rate of 30–40 %, which is typical for diets in developed societies (SFC 2003, cited in SCOEL, 2013), and an assumed 100 % absorption by inhalation, the daily difference of 0.8 mg/day would correspond to an inhalable air concentration of copper of 0.03–0.04 mg/m³ (5 days exposure/week with a breathing volume of 10 m³/8-hour day). To avoid systemic toxicity, the inhalable exposure to copper should be below this value. An OEL of 0.01 mg Cu/m³ for the respirable fraction is proposed. This OEL applies to copper and its inorganic compounds.

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

Cu(2Na)EDTA and Cu(2Na)IDHA are stable compounds but they can de-complex under acidic conditions of stomach, releasing copper ions which can become systemically available. Moreover, intestinal absorption of free IDHA chelating agent (37 %) is higher than oral absorption determined in experimental studies with free EDTA (5 %) or EDTA metal complexes (less than 1 -2 %). Therefore, the read-across from the LOAEL established for systemic effects for Cu(2Na)EDTA (150 mg/kg bw) or IDHA chelating agent (NOEL of 200 and 1000 mg/kg bw for males and females, respectively) can underestimate the risk of systemic toxicity of Cu(2Na)IDHA. Especially the risk of systemic toxicity of copper being systemic available after exposure to Cu(2Na)IDHA could be underestimated because of higher absorption percentage in case of IDHA.

Therefore, an estimated dose level based on the toxicity data available for copper is considered more appropriate. The lowest NOAEL of 2 mg Cu/kg bw /day in male rats and 0.83 mg Cu/kg bw /day in female rats were established in a combined repeated dose and reproductive/ developmental study (ATSDR, 2004) while NOAEL of 200 and 1000 mg/kg bw were established in a 28-day study for IDHA in males and females, respectively. According to equation:

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

0.83 mg Cu corresponds to 4.6 mg of Cu(2Na)IDHA ((MW of Cu(2Na)IDHA is 354.69 / MW of copper is 64) x 0.83). Taking into account 60 % oral absorption established for Cu(2Na)IDHA (please refer to toxicokinetic section), the estimated NOAEL would result in 7.8 mg/kg bw Cu(2Na)IDHA: 4.6 mg/kg bw x (100 %/ 60 %). 7.8 mg/kg bw is much lower than LOAEL of 150 mg/kg bw established for Cu(2Na)EDTA. There is also the recommended daily allowance (RDA) of 0.013 mg Cu/kg bw available. The value represents a sufficiently safe level to estimate an equivalent dose for Cu(2Na)IDHA for systemic effects for humans. It corresponds to 0.072 mg/kg bw for Cu(2Na)IDHA: ((MW of Cu(2Na)IDHA is 354.7 / MW of Cu is 64) x 0.013 mg/kg bw). Taking into account 60 % oral absorption established for Cu(2Na)IDHA, the estimated DNEL would be 0.12 mg/kg bw: 0.072 x (100 %/ 60 %).This dose level however may be overprotective but it can serve as an internal DNEL for systemic effects (no assessment factors are needed; see DNEL calculation section).

Similarly as for oral exposure route, an estimated NOAEC for humans for inhalation exposure route make sense to be derived. An OEL value of 0.01 mg/m³ exists for respirable fraction for copper and its inorganic compounds (SCOEL, 2013). Since no respirable fraction is expected for Cu(2Na)IDHA (particles of Cu(2Na)IDHA are > 100 µm), an OEL of 0.03-0.04 mg/m³ for inhalable fraction would be more appropriate. This limit would correspond to 0.17 mg/m³ of Cu(2Na)IDHA: ((MW of Cu(2Na)IDHA 354.7/ MW of copper 64) x 0.03 mg/m³). Taking into account 60 %absorption by inhalation (equal to oral absorption), the estimated NOAEC (=DNEL for inhalation, systemic effects) would result in 0.28 mg/m³: 0.17 x (100 %/60 %).

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
No study is selected since NOAEL is estimated for Cu(2Na)IDHA based on the RDA available for copper. 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 EDTA and its salts.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
No study is selected since NOAEL is estimated for Cu(2Na)IDHA based on the OEL available for inhalable fraction of coper. The reason is that the toxicity of Cu(2Na)IDHA is believed to be mediated by copper rather than by the chelate moiety. Cu(2Na)IDHA is expected to be swallowed after its deposition in the airways. Therefore, inhalation absorption, if occur, is confined to oral absorption. Additionally, IDHA (and its chelates) are expected to be absorbed more extensively due to the higher oral absorption of IDHA comparing to EDTA and its salts.

Justification for classification or non-classification

NOAEL of 200 and 1000 mg/kg bw were established for the chelating agent IDHA in the 28-day oral (gavage) study in male and female rats, respectively. No target organ toxicity was observed in this study. NOAEL of < 150 mg/kg bw was established in the combined study in rats with Cu(2Na)EDTA. Target organs were kidney and liver. For Cu(2Na)IDHA, an estimated systemic NOAEL (= DNEL) of 0.12 mg/kg bw was derived based on RDA of 0.013 mg/kg bw for copper established by WHO for humans. No target organ toxicity is expected at this dose level. Similarly, no systemic or target organ toxicity can be expected at the estimated concentration of 0.28 mg/m³ based on the existed OEL of 0.03 -0.04 mg/m³ for copper. Therefore, Cu(2Na)IDHA does not meet the criteria for classification and labelling for systemic target organ toxicity after repeated exposures (STOT-RE) in accordance with European regulation (EC) No. 1272/2008.