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Description of key information

Cu(2Na)IDHA is expected to be moderately absorbed after oral exposure, based on its high water solubility, negative logPow as well as absorption rates known for free IDHA and copper. The substance is poorly available for inhalation since it is in a micro granulated form with particles above 100 µm, has a low vapour pressure, and is highly hydrophilic. Cu(2Na)IDHA is also not expected to be absorbed following dermal exposure into the stratum corneum and into the epidermis, due to its high water solubility. Concerning its distribution in the body Cu(2Na)IDHA, if absorbed, is expected to be distributed mainly in the intravasal compartment, due to its high water solubility, and may be also transported to the liver. The substance does not indicate a significant potential for accumulation. Cu(2Na)IDHA is not expected to be significantly metabolised but to be eliminated unchanged via the bile and, to a lesser extent, via the urine. 

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential
Absorption rate - oral (%):
60
Absorption rate - dermal (%):
6
Absorption rate - inhalation (%):
60

Additional information

General

There are no ADME studies available for Cu(2Na)IDHA.The toxicokinetic profile of the test substance was not determined by actual absorption, distribution, metabolism or excretion measurements. Rather, the physical chemical properties of this substance were integrated with its toxicological data and the data on its structural analogues IDHA (iminodisuccinic acid) and Cu(2Na)EDTA to create a prediction of toxicokinetic behaviour. The results of toxicokinetic studies available for other aminopolycarboxylate chelate like Ethylenediaminetetraacetic acid calcium disodium salt (Ca(2Na)EDTA; CAS 62-33-9) and not chelated Ethylenediaminetetraacetic acid disodium salt (Na2EDTA; CAS 139-33-3) as well as copper metal ion (in form of salts) are also taken into account to assess the toxicokinetic behaviour of Cu(Na)IDHA.

There are acute (oral and dermal), irritation (skin and eye), and sensitization toxicity studies available for Cu(2Na)IDHA. The results of repeated dose toxicity studies and genetic toxicity studies conducted with the read-across substances chelating agent IDHA (iminodisuccinic acid) and Cu(2Na)EDTA have been used to cover the endpoints where the data are lacking for the target substance (please refer to read-across statement attached to the IUCLID file under section 13). The target substance and the read-across substances show very similar physical/chemical properties (high water solubility, negative logPow, no hydrolysis in water at environmentally pH values, low vapour pressure) and are thus believed to behave very similarly in aqueous solutions and in living organisms.The complexing metal ion copper, which is expected to be released under strong acidic conditions of stomach (pH < 3), is believed to contribute significantly to the toxicity observed in the long-term studies. Generally, toxicity of Cu(2Na)IDHA is expected to be mediated by excess exposure to elemental copper. Therefore, ADME data on copper have been considered to predict toxicokinetics behaviour of Cu(2Na)IDHA. Regarding toxicokinetic behaviour of Cu(2Na)IDHA in the complexed form, the substance is expected to exhibit similar biological activities with those of Cu(2Na)EDTA. In addition, similar patterns have been observed for the toxicological effects (e.g., available data showed similar level of acute oral and dermal toxicity, not irritating and not sensitisation properties and similar ecotoxicity potential). These common behaviours suggest a common mechanism and mode of action thereby providing evidence for the read-across between copper chelates of different aminopolycarboxylates (please refer to read-across statement).

Toxicological profile of Cu(2Na)IDHA

The target substance Cu(2Na)IDHA is moderately toxic by oral route of exposure (LD50 is between 300 – 2000 mg/kg bw; Gruszka, 2007, Report No. OS 46/06) and not toxic by dermal route of exposure in rats (LD50 is greater than 2000 mg/kg bw; Kropidło, 2010, Report No. DER -13/10). The substance is not a skin or eye irritant (Kropidło, 2010, Report No. DDR -15/10; Kropidło, 2010, Report No. ODR -13/10) and do not possess skin sensitisation potential (Kropidło, 2011, Report No. AI-3/10).

Effects in animal studies conducted with read-across substances which are relevant in the assessment of toxicokinetic behaviour of Cu(2Na)IDHA.

Long-term studies:

NOAEL of 1000 mg/kg bw, the highest dose tested, was established for females for the nearest analogue chelating agent IDHA in a sub-acute study in rats (Stropp and Popp, 1997; Report No. PH 26446). No toxicologically relevant clinical signs were noted in the animals treated with the substance. All blood chemistry parameters were unaffected and no toxicologically relevant findings were observed at necropsy. NOAEL of 200 mg/kg bw was established for males based on decreased motor activity.

The read-across substance Cu(2Na)EDTA produced adverse effects in animals treated with 150 mg/kg bw in the Combined oral repeated dose toxicity study with reproduction / developmental toxicity screening test (OECD 422; Lina, 2013; Report No. V20114). The target organs were liver and kidney.

The chelating agent IDHA and Cu(2Na)EDTA were no reproductive or developmental toxicants in animal studies. This was proven by an one-generation reproduction toxicity study (Eiben and Rinke, 2002, Report No. PH-32294), developmental study conducted with IDHA (Klaus, 2002, Report No. PH-32141) and the Combined oral repeated dose toxicity study with reproduction / developmental toxicity screening test conducted with Cu(2Na)EDTA (OECD 422; Lina, 2013; Report No. V20114).

Toxicokinetic study conducted with the chelating agent IDHA sodium salt:

In a toxicokinetic study, the pharmacokinetic behaviour (absorption, distribution, excretion) and metabolism of the complexing agent IDHA sodium salt for metal ions were investigated (OECD 417; Koester, 2007; Report No. M81819180). The test material was labelled with 14C and 13C in the 2 and 3 as well as 2' and 3' - positions of iminodisuccinic acid. The radiolabelled and unlabelled test material was administered to two groups of 5 male Wistar rats, respectively, at dose level of 1000 mg/kg bw. The rats received the test item by oral gavage as an aqueous solution. They were sacrificed 72 h after dosing. The total radioactivity that included the test item and possible metabolites was determined in plasma samples, the excreta (urine and faeces) as well as in organs and tissues. The metabolism was investigated by radio-HPLC and spectroscopic methods in selected urine samples and faeces extract. Further, osmolality in urine as well as pH-values and metals in urine and plasma samples collected from rats which received the unlabelled test item were investigated.
The kinetic and metabolic behaviour of IDHA sodium salt in male rats can be characterised by the following observations:
A first portion of the administered radioactivity was rapidly absorbed, widely distributed into organs and tissues and rapidly eliminated from the body via urine and faeces. For a second portion, the absorption and elimination periods were significant longer than for the first one which indicated a higher systemic exposure to the test item related radioactivity during this phase. An exact value for the absorption rate could not be determined from these observations. But taking the urinary excretion behaviour and the remaining radioactivity in the body at sacrifice into account it can be concluded that at least 37 % of the administered dose becomes systemically available.
The distribution of the radioactivity within the central compartments of the body (i.e. blood, liver, and kidney) showed a distinctive preference towards the liver and - to a lower extent - to the kidney which had the highest TRR-values at sacrifice.
IDHA sodium salt was metabolically stable as no metabolites were detected in the excreta in significant amounts.
The excretion of radioactivity via urine and faeces was fast and almost completed at 24 h after administration. The by far major part of the dose (68.7 %) was excreted with faeces and 34.7 % with urine. Relating to the administered dose, IDHA sodium salt amounted to 34.5 % in urine and 61.2 % in faeces. No single unknown metabolite was higher than 0.2 % in urine and 2 % in faeces. From the renal and faecal excretion rate as well as the low accumulation factor it was concluded that the residual radioactivity in the organs and tissues of the rat are subject to further elimination.
The administration of IDHA sodium salt led to transient changes of the urinary osmolality-and pH-values within the test period of 3 days. Based on a high variability of urine pH- and osmolality-values in animal species used in toxicological studies, these effects however were regarded as insignificant. Differences were obtained in the amount of metals in urine and plasma samples between treated and untreated rats.

Summary of toxicity effects of copper and its inorganic compounds:

Regarding copper toxicity, the most commonly reported adverse health effect in humans is gastrointestinal distress. Nausea, vomiting, and/or abdominal pain have been reported, usually occurring shortly after oral exposure (ATSDR, 2004; SCOEL, 2013). The observed effects are not usually persistent and gastrointestinal effects have not been linked with other health effects. Animal studies have also reported gastrointestinal effects (hyperplasia of forestomach mucosa) following ingestion of copper sulfate in the diet. Copper is also irritating to the respiratory tract. Coughing, sneezing, runny nose, pulmonary fibrosis, and increased vascularity of the nasal mucosa have been reported in workers exposed to copper dust. The liver is also a sensitive target of toxicity. Liver damage (necrosis, fibrosis, abnormal biomarkers of liver damage) have been reported in individuals ingesting lethal doses of copper sulfate. Recommended Daily Allowance (RDA) of 0.09 mg/kg bw was established by WHO (ATSDR, 2004).

In case of inhalation exposure to inorganic copper compounds, the critical effect is the local action on the respiratory tract, which includes an immunosuppression that is attributable to the disturbance of alveolar macrophage function (SCOEL, 2013).The effects (general feeling of discomfort, slight sensations of chills and warmth, stuffiness of the head) were reported by workers some weeks after the start of exposure. The OEL of 0.01 mg/m³ for respirable and 0.03-0.04 mg/m³ for inhalable fraction were established for copper and its inorganic compounds.

Toxicokinetic analysis of Cu(2Na)IDDHA

The substance Cu(2Na)IDHA is an odourless, dark blue solid in a microgranulated form (MW is 354.69 g/mol) at 20 °C. The substance is soluble in water (412 g/L at 20 °C; Lewandowska, 2011, Report No. BS-19/10-02) and has a negative partition coefficient (logPow = -3.09; Stegient-Nowicka, 2011, Report No. 2011/0119). It has a very low vapour pressure (3.02 x 10-7Pa; Petryka, 2013, Report No. BC-12/13) and it decomposes at 80 - 196 °C under atmospheric conditions (Lewandowska, 2011, Report No. BS-19/10-01. Hydrolysis as a function of pH does not apply as the substance forms very stable complexes (log of stability constant is12.88, Hyvönen et al., 2003). Therefore, chelate stability is more applicable instead. Cu(2Na)IDHA is stable over a wide pH range (3-12) but it is not stable under extremely acidic conditions (Lucena et al., 2003; Hyvönen et al., 2003). The following situation applies by pH below 3:

4H++ CuIDHANa2+ H2OH4IDHA + 2Na++ Cu2+

In the following, ADME parameters have been predicted based on data for copper, free IDHA, Cu(2Na)IDHA and Cu(2Na)EDTA.

 

Absorption

Oral absorption:

Oral absorption is favoured for small (with MW below 500 g/mol) water soluble molecules. Molecular weight of Cu(2Na)IDHA is 354.69 g/mol pointing to a possible absorption in the gastrointestinal (GI) tract while high water solubility (412 g/L) and the very low logPow value (-3.09) suggest that Cu(2Na)IDHA may be too hydrophilic to be readily absorbed via GI tract. The substance may be taken up by passive diffusion. As the substance’s molecular weight is lower than 500, it is likely to pass through aqueous pores or be carried through the gastrointestinal epithelial barrier by the bulk passage of water.However, the substance is instable under acidic condition (by pH under 3; Hyvönen et al., 2003). Therefore, the complex Cu(2Na)IDHA is expected to be in de-chelated form in the stomach: sodium ions, released Cu2+ions from the complex and free IDHA. In small intestines, free IDHA can chelate metal ions again because of increased pH. Thus, oral absorption of chelated Cu(2Na)IDHA will result from absorption of copper ions, sodium ions and free IDHA. Additionally, toxicity data on Cu(2Na)IDHA and Cu(2Na)EDTA can elucidate the pattern and rate of absorption. 

With regard to the toxicity data on Cu(2Na)IDHA and Cu(2Na)EDTA:

The substance produced toxic effects and death at 2000 mg/kg bw in rats in an acute oral toxicity study (LD50 > 300 - < 2000 mg/kg bw; Gruszka, 2007; Report No.OS-46/06). Similarly, the read-across substance Cu(2Na)EDTA was acutely toxic by oral (gavage) route in rats (LD50 is 890 mg/kg bw; BASF, 1985; Report No. 84 / 263) and produced clinical signs in the Combined oral repeated dose toxicity study with reproduction / developmental toxicity screening test (NOAEL < 150 mg/kg bw; Lina, 2013; Report No. V20114). These are the facts confirming absorption via the gastrointestinal tract (GI) tract. The toxicity observed in the acute toxicity study conducted with Cu(2Na)IDHA is probably more related to the released copper metal ions than to the toxicity of free IDHA (see toxicity profile of free IDHA).

With regard to the absorption data on free EDTA and other EDTA metal complexes:

In the toxicokinetic studies with other EDTA derivatives: EDTA-CaNa2 and Na salts of EDTA are poorly absorbed from the gastrointestinal tract (2 -18 % in rats; less than 5 % in humans; RAR, 2004). Poor absorption was observed also for sodium iron EDTA administered orally to animals and humans: “Only a very small fraction of the NaFeEDTA complex (less than 1 %) is absorbed intact and this is completely excreted in the urine. An additional small fraction (less than 5 %) of the EDTA moiety is absorbed, presumably bound to other metals in the gastrointestinal tract, and is also completely eliminated in the urine” (IPCS, 2014). In absorption studies with sodium iron EDTA, only a very small fraction of the sodium iron EDTA complex (less than 1–2 %) is absorbed intact and is rapidly and completely excreted via the kidneys in the urine (WHO, 2008)

With regard to the absorption data on free metal cations released from EDTA or IDHA metal complexes:

Regarding absorption of released copper from both IDHA and EDTA, the rates of their absorption are assumed to be dependent on the pH optimum at which chelated agents form efficiently complexes with metal cations. For instance, in absorption studies with sodium iron EDTA the fate of different complex species is investigated (WHO, 2008; IPCS, 2014). Administered orally, “iron (primarily Fe3+) remains complexed with EDTA under the acidic conditions prevailing in the stomach...". "The chelate holds the iron in solution as the pH rises in the upper small intestine, but the strength of the complex is progressively reduced allowing at least partial exchange with other metals and the release of some of the iron for absorption. Further results indicate that iron dissociates from the chelate and is released into the common non-haem iron pool before absorption” (IPCS, 2014).

With regard to the toxicity and ADME data on free IDHA chelating agent:

In the acute oral and sub-acute oral studies, conducted with IDHA chelating agent (in form of its sodium salt), only minimal effects were noted in treated animals. LD50 of greater than 2000 mg/kg bw in the acute study and NOAEL of 1000 mg/kg bw and 200 mg/kg bw were established for females and males in the sub-acute study, respectively (Stropp, 1996, Report No. T4060981; Stropp and Popp, 1997; Report No. PH 26446). The effects in males are demonstrating certain absorption of IDHA but it also shows its relatively low intrinsic toxicity. In the toxicokinetic study, the chelating agent IDHA was extensively absorbed and at least 37 % of the administered dose became systemically available in rats (Koester, 2007; Report No. M81819180).

With regard to copper (based on mostly inorganic copper compounds):

Copper is an essential element, which is incorporated in various proteins. It is a constituent of more than 20 enzymes (SCOEL, 2013). Physiologically normal levels of copper in the body are held constant by alterations in the rate and amount of copper absorption, compartmental distribution, and excretion (ATSDR, 2004). The site of maximal copper absorption is not known for humans, but it is assumed to be the stomach and duodenum; the average absorption efficiencies ranged from 24 to 60 % in presumably healthy adults. Copper is absorbed from the gastrointestinal tract as ionic copper or bound to amino acids or binding proteins (metallothionein). Numerous factors may affect copper absorption. These factors include: the amount of copper in the diet, competition with other metals, including zinc, iron, and cadmium and age (ATSDR, 2004). The absorption of copper appears to be inversely related to the amount of copper in the gastrointestinal tract (ATSDR, 2004).

Based on these data, it can be concluded that a moderate rate of absorption across the gastrointestinal tract epithelium will occur when Cu(2Na)IDHA applied orally. The complex Cu(2Na)IDHA is expected to dissociate at high acidic conditions of stomach. However in upper intestines, where pH raises, copper cation will tend to be complexed again with free ligand IDHA. Therefore, the absorbed fraction will result from intact Cu(2Na)IDHA and its released components: Cu2+ions and free IDHA. Intestinal uptake of released Cu2+ions is moderate (60 %), while intestinal uptake of sodium IDHA is at least 37 % comparing to 5 % of sodium EDTA. Therefore, oral absorption of intact copper IDHA chelates is expected to be somewhat higher than oral absorption of copper EDTA complexes or free EDTA.

In conclusion, taking into account gastrointestinal absorption percentages of 37 and 60 % for the chelating agent IDHA and free copper, respectively, as well as low absorption potential based on physico-chemical properties of Cu(2Na)IDHA, 60 % oral absorption (equal to the upper level known for copper ions) is considered appropriate in case of hazard assessment (DNEL derivation).

Absorption by inhalation:

Based on the low vapour pressure of Cu(2Na)IDHA, inhalation exposure is not likely. There are no particles with aerodynamic diameter less than 100 µm (Stefaniak, 2011; Report No. 2011/0118). Moreover, the final product has a micro granulated form. Thus, it is very unlikely, that big amounts of the substance reach the lung. In case of dust formation, it is expected that 100 % of the inhaled substance will be deposited in the upper respiratory tract, where the particles may be moved by mucociliary transport to the throat and where they are swallowed and enter the stomach. The particles are not expected to reach alveolar region. Nevertheless, if the substance reaches the lung, it is not very likely that the substance is taken up rapidly (based on physico-chemical properties). The substance is expected to be predominantly in chelated form since pH of healthy lungs is between 7.38 and 7.43 (Effros and Chinard, 1969). In case of a negligible fraction of released copper ions, no quantitative data exist for the rate of absorption by the inhalation route in humans (the absorption rate of fine particles is generally in the order of 50 %; SCOEL, 2013).
In an acute inhalation study with the read-across substance Cu(2Na)EDTA, it was absorbed by lungs of rats as confirmed by clinical signs and findings at necropsy (Jonker and van Triel, 2012). The substance was inhaled in form of aerosol (particles of 4.85 – 5.35 µm). The 4-h LC50 value exceeded 5.3 g/m³, therefore it does not trigger C&L. Thus, this indicates low systemic availability after inhalation and if bioavailable, low toxicity effects via this route of administration.

Based on this information, the absorption by inhalation is expected to confine to the amount of Cu(2Na)IDHA deposited in upper airways which can be swallowed. Therefore, as worst case, 60 % absorption (equal to the upper level known for copper ions) by inhalation is considered appropriate for the purposes of hazard assessment (DNEL derivation). This absorption rate covers also absorption of free IDHA chelating agent which is 37 %.

Dermal absorption:

Similarly, based on physical – chemical properties of Cu(2Na)IDHA, the substance is not likely to penetrate skin to a large extent due to its negative logPow: -3.09 and a very high water solubility: 421 g/L. Water solubility above 10.000 mg/L combined with a logPow value below 0 indicate that the substance may be too hydrophilic to cross the lipid rich environment of the stratum corneum. Dermal uptake for these substances will be low. The molecular weight of 354.69 g/mol indicates that a certain potential to penetrate the skin (< 500) exists. However, in case of such a hydrophilic substance dermal penetration is rather unlikely. This is supported by the findings of acute dermal toxicity studies of the target substance Cu(2Na)IDHA as well as free IDHA and Cu(2Na)EDTA where no systemic toxicity after exposure via the skin was noted (LD50 > 2000 mg/kg bw; Kropidło, 2010, Report No. DER -13/10; Stropp, 1997, Report No. T3061600; Beerens, 2010, Report No. 494023). Moreover, an acute dermal irritation / corrosion study in the rabbit (according to OECD 404) for Cu(2Na)IDHA did not demonstrate any irritation after 14 days (Kropidło, 2010, Report No. DDR-15/10). This information indicates that Cu(2Na)IDHA is unlikely to penetrate the skin. In a human study, EDTA-CaNa2 did not penetrate the skin, only 0.001 % was absorbed within 24 hours of administration (RAR, 2004). In case of dissociated complexes, copper ions uptake across intact skin would be expected to be extremely limited (ATSDR, 2004). The available in vivo data do not provide information on the rate and extent of absorption through intact skin following dermal exposure of humans or animals to copper (ATSDR, 2004; SCOEL, 2013). In vitro studies suggest that copper is poorly absorbed through intact skin. Less than 6 % of copper deposited on ex vivo human skin samples were absorbed (ATSDR, 2004). Low absorption potential through the skin would also apply to free IDHA chelating agent due to its high hydrophilicity (water solubility of 564 g/L; data for Baypure CX 100 (Bayer)).

Based on very low logPow values, high water solubility and absence of toxicity effects in animal studies conducted with different aminopolycarboxylate chelates: free chelating agent IDHA, Cu(2Na)IDHA, Cu(2Na)EDTA and Ca(2Na)EDTA, a similar behaviour regarding absorption through the skin is expected. Dermal absorption is considered to be negligible and equal to 6 % established for copper in an ex vivo study with human skin. This is the highest value known which will be used for hazard assessment: DNEL derivation.

Distribution and accumulative potential

When reaching the body, moderate amounts of Cu(2Na)IDHA resulted from the absorption are expected to be available for distribution. The amount absorbed into the body, will most likely exist only in the intravascular compartment (due to its low LogPow and high water solubility) and will not be distributed into the cells, as the cell membranes require a substance to be soluble also in lipids to be taken up. In a human study with Ca (2Na) EDTA, intravenously injected EDTA was excreted within 24 hours in the urine, 50 % of the substance in the first hour and 90 % within 7 hours (RAR, 2004). No test substance was detected in blood. On the other hand, Ca(2Na)EDTA was also excreted in the expired air in treated animals. This indicates a wide distribution potential and no accumulative potential.

With regard to copper ions from the decomplexed fraction of Cu(2Na)IDHA, no studies are known concerning the rate and extent of distribution of copper following dermal or inhalation exposure of humans or animals. Following ingestion of copper, copper levels in the blood rapidly rise. The copper predominantly bound to albumin which is transported to the liver and kidney (ATSDR, 2004). Further, copper can re-emerge into the plasma bound to ceruloplasmin and be transported to other tissues (ATSDR, 2004).

With regard to free chelating agent IDHA, the substance distributed predominantly towards the liver and - to a lower extent - to the kidney(Koester, 2007; Report No. M81819180).

In summary, based on these data and taking into account the fact that “substances with LogPow values of 3 or less would be unlikely to accumulate with the repeated intermittent exposure patterns normally encountered in the workplace” (TGD, Part 1; ECHA guidance R.7C, 2012), no enhanced risk for accumulation will be associated with Cu(2Na)IDHA.

Metabolism and excretion

No studies are available for Cu(2Na)IDHA and Cu(2Na)EDTA in which metabolites would be detected and studied in details. Due to the chelated nature of the molecule, metabolism in the human body will unlikely to occur. It is confirmed in the metabolism studies in animals and in humans with Ca(2Na) EDTA where the substance was rapidly excreted predominantly in faeces (more than 90 %). No metabolites were reported (RAR, 2004). In various in vitro and in in vivo genetic toxicity studies Cu(2Na)EDTA showed no effects with and without metabolising system. Metabolic activation leading to more toxic metabolites is thus not very likely. Further, due to the high stability constant of the copper chelate IDHA complex (K = 10-13) (Hyvönen et al., 2003), it is clear that it exerts a low reactivity in the organism. Therefore, it is assumed that most of this very water soluble copper fraction will be excreted unchanged in the chelated form mainly in the faeces in analogy with Ca(2Na)EDTA or other salts of EDTA. Small amount of absorbed fraction will be also excreted unchanged via the urine.

In case of dissociation of Cu(2Na)IDHA complexes in stomach under very acidic conditions, metabolism of free IDHA is expected to be similar to that described in the toxicokinetic study in rats (Koester, 2007; Report No. M81819180). The substance was metabolically stable as no metabolites were detected in significant amounts in the excreta. Thus, biotransformation of iminodisuccinate takes only place to a minor extent. With the aid of skin metabolism simulator*, a tool of the OECD QSAR Toolbox (v3.2, 2013), several metabolites were predicted for IDHA. They are all intermediates in mammals: oxaloacetate, aspartic acid, ß-alanine and pyruvate. The excretion pattern of IDHA were similar to those observed for Ca(2Na)EDTA (RAR, 2004). The excretion via urine and faeces was fast and almost completed at 24 h after administration. The major part of the administered dose (61.2 %) was excreted with faeces and 34.5 % with urine (Koester, 2007).

Regarding copper ions, the metabolism of copper consists mainly of its transfer to and from various organic ligands, most notably sulfhydryl and imidazole groups on amino acids and proteins (ATSDR, 2004). Several specific binding proteins for copper have been identified that are important in the uptake, storage, and release of copper from tissues. In the liver and other tissues, copper is stored bound to metallothionein and amino acids and in association with copper-dependent enzymes. Several studies have shown that copper exposure induces metallothionein synthesis (ATSDR, 2004). Ceruloplasmin is synthesized in the liver. Copper is incorporated into the molecule, and it is released from the liver. Copper exposure has also been shown to induce ceruloplasmin biosynthesis (ATSDR, 2004). The predominant elimination pathway of copper is the bile (SCOEL, 2013). After the oral administration of radioactive copper as copper acetate in healthy humans, 72 % was excreted in the faeces (ATSDR, 2004). A considerable fraction of faecal copper is of endogenous biliary origin. The remainder of the faecal copper is derived from unabsorbed copper and copper from desquamated mucosal cells. Copper in bile is associated with low molecular weight copper binding components as well as macromolecular binding species (ATSDR, 2004). Reabsorption of biliary copper is negligible (ATSDR, 2004). Small amounts are excreted via the urine (0.5–3.0 %; ATSDR, 2004; SCOEL, 2013).

*SMILES of IDHA used in the software Toolbox: OC(=O)CC(C(O)=O)NC(CC(O)=O)C(O)=O