Ferrate(1-), [[N,N'-1,2-ethanediylbis[N-[[2-(hydroxy-.kappa.O)phenyl]methyl]glycinato-.kappa.N,.kappa.O]](4-)]-, sodium (1:1), (OC-6-13)-

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics

Type of information:

other: expert statement

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: An extensive assessment of the toxicological behaviour of Fe(Na)HBED was performed, taking into account the chemical structure, the available physico-chemical-data and the available (eco-)toxicological data.

Data source

Reference

Reference Type:

other: Expert Statement

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Objective of study:

absorption

distribution

excretion

metabolism

toxicokinetics

Test guideline

Qualifier:

no guideline required

Principles of method if other than guideline:

An extensive assessment of the toxicological behaviour of Fe(Na)HBED was performed, taking into account the chemical structure, the available physico-chemical-data and the available (eco-)toxicological data.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

iron(3+) sodium 2-({2-[(carboxylatomethyl)[(2-oxidophenyl)methyl]amino]ethyl}[(2-oxidophenyl)methyl]amino)acetate

EC Number:

700-327-5

Cas Number:

1061328-86-6

Molecular formula:

C20H20FeN2NaO6

IUPAC Name:

iron(3+) sodium 2-({2-[(carboxylatomethyl)[(2-oxidophenyl)methyl]amino]ethyl}[(2-oxidophenyl)methyl]amino)acetate

Constituent 2

Reference substance name:

sodium [N-(2-{[(hydroxy-kO)acetyl][2-(hydroxy-kO)benzyl]amino-kN}ethyl)-N-[2-(hydroxy-kO)benzyl]glycinato(4-)-k2N,O]ferrate(1-)

IUPAC Name:

sodium [N-(2-{[(hydroxy-kO)acetyl][2-(hydroxy-kO)benzyl]amino-kN}ethyl)-N-[2-(hydroxy-kO)benzyl]glycinato(4-)-k2N,O]ferrate(1-)

Details on test material:

- Name of test material (as cited in study report): sodium [N-(2-{[(hydroxy-k O)acetyl][2-(hydroxy- k O)benzyl]amino-k N}ethyl)-N-[2-(hydroxy-k O)benzyl]glycinato(4-)- k 2 N,O]ferrate(1-)
- Substance type: chelate
- Physical state: solid

Radiolabelling:

other: not applicable in this expert statement

Test animals

Species:

other: not applicable

Strain:

other: not applicable

Details on test animals or test system and environmental conditions:

Not applicable

Administration / exposure

Route of administration:

other: all routes of administration are discussed in the expert statement

Vehicle:

other: not applicable

Details on exposure:

all routes of administration are discussed in the expert statement

Duration and frequency of treatment / exposure:

Not applicable

Doses / concentrations

Remarks:

Doses / Concentrations:
Not applicable

No. of animals per sex per dose / concentration:

Not applicable

Control animals:

other: not applicable

Positive control reference chemical:

Not applicable

Details on study design:

Not applicable

Details on dosing and sampling:

Not applicable

Statistics:

Not applicable

Results and discussion

Main ADME results

Main ADME results 1

Type:

absorption

Results:

oral, dermal and inhalatory absorption is most likely low / minimal

Main ADME results 2

Type:

distribution

Results:

Fe(Na)HBED is expected to be available only in the intravascular compartment

Main ADME results 3

Type:

metabolism

Results:

It is most likely that the substance of interest will not undergo extensive metabolism but be mainly excreted unchanged via the bile and to a smaller extent via the urine. However, it is possibly subject to glucuronidation.

Main ADME results 4

Type:

excretion

Results:

FeHBED is likely to be excreted unchanged. Its high water solubility leads normally to urinary excretion; however, in conjunction with the rather high MW & its chemical structure mainly biliary excretion is expected & possibly to a small extent via urine.

Toxicokinetic / pharmacokinetic studies

Details on absorption:

ABSORPTION OF Fe(Na)HBED
Taking into consideration the physicochemical properties of FeHBED, such as a rather high molecular mass, its steric structure, a very high stability constant, a non-existing hydrolysis, a ready water solubility and a low LogPow, it can be stated that Fe(Na)HBED is unlikely to be absorbed from GI tract.
Because of its very low vapour pressure, particle size distribution, a low LogPow and a high molecular mass and its steric structure, it can be stated that FeHBED is unlikely to be absorbed to a significant extent after exposure by the inhalation route.
As its molecular weight is close to 500, which indicates already a low potential to penetrate the skin. Additionally, the very low vapour pressure can be judged slightly advantageous for dermal uptake. However, the high water solubility is expected to hinder considerably the absorption following dermal exposure into the stratum corneum. Moreover, its low LogPow is not advantageous for absorption after dermal contact. This information indicates that FeHBED is unlikely to penetrate the skin.

Details on distribution in tissues:

In order to evaluate the distribution of Fe(Na)HBED, the physico-chemical properties: microgranulated, dust free particles distribution, high molecular mass, its spatial structure, low LogPow, readily solubility in water and low vapour pressure have been taken into account and indicate that Fe(Na)HBED is unlikely to permeate or penetrate GI tract. Therefore only a limited amount of Fe(Na)HBED is expected to be available for distribution. However, the minor amount absorbed into the body, will most likely exist only in the intravascular compartment (due to its high molecular weight and the low LogPow) 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. Because of its higher lipophilicity, HBED may be considered as better distributed into the tissues than Fe(Na)HBED.

Details on excretion:

Taking into consideration the high stability constant of K = 10-39 for FeHBED, it is clear that it exerts a low reactivity in the organism. The behaviour predicted for its metabolic fate supports this and the parent substance will be most likely excreted unchanged. Its high water solubility would lead normally to urinary excretion; however, in conjunction with the rather high molecular weight and its chemical structure mainly biliary excretion is expected and possibly to a small extent via the urine (may be also as conjugates with glucuronic acid). This finding is supported with the findings for HBED/NaHBED carried out in animals and humans, which have proven that the substance is easily excreted mainly biliary from the body.

Metabolite characterisation studies

Metabolites identified:

no

Details on metabolites:

It is most likely that the substance of interest will not undergo extensive metabolism but be mainly excreted unchanged via the bile and to a smaller extent via the urine. However, it is possibly subject to glucuronidation. In this case the possibility of entero-hepatic recycling, and the risk or a re-enter into system, does not bear any risk for the organism. Concerning the possibility of protein binding, this can not be ruled out, without adequate experimental data.

Any other information on results incl. tables

This assessment is intended to evaluate the toxicokinetic behaviour of sodium [N-(2-{[(hydroxy-k O)acetyl][2-(hydroxy- k O)benzyl]amino-k N}ethyl)-N-[2-(hydroxy-k O)benzyl]glycinato(4-)- k 2 N,O]ferrate(1-). This chemical substance belongs to the class of chelates (CAS-no. 1061328-86-6) and will be abbreviated in the following with FeHBED.

FeHBED is produced by reaction of iron ions with the chelating agent HBED (mostly in form of a sodium salt, NaHBED).

SUBSTANCE DESCRIPTION AND AVAILABLE DATA

IUPAC Name:

sodium [N-(2-{[(hydroxy-kO)acetyl][2-(hydroxy- kO)benzyl]amino-kN}ethyl)-N-[2-(hydroxy-kO)benzyl]glycinato(4-)- k2N,O]ferrate(1-)

Abbreviation:

FeHBED

CAS-no:

1061328-86-6

Molecular weight:

463.26 g/mol

Characteristics:

The substance FeHBED is at 20 °C a brown solid powder with a sweetish odour. The substance is soluble in water (34.55 g/L at 20°C) and has a LogPow of -1.96. It has a very low vapour pressure (5.75 E-04 Pa at 20°C). As already known from visual observations of the heating process, the melting and the decomposition of the test item start at ca. 250 °C). Hydrolysis as a function of pH does not apply as the substance forms extremely stable complexes. However, above pH 8, the stability is considerably reduced, despite that; hydrolysis is not expected, due to the lack of functional groups. The substance is, when administered orally or dermally to rats not toxic (LD50 > 2000 mg/kg bw). It is not an eye or skin irritant, but it has been shown to be skin sensitising when tested in the guinea pig maximisation test. Additionally, the substance was shown to be not mutagenic in a study according to OECD471. 

The activity of the iron ion in organisms

The role of iron in all living organisms - plants, animals and humans - is very well known and it is an important essential microelement for nearly all organisms, forming several oxidation states which differ by one election and it is required for the biological activity of many proteins mediating electron transfer and redox reactions. The importance of these effects can be seen by the redox potential of iron containing enzymes, which covers a range of nearly 1.000 mV. Moreover iron is required for metabolic functions which are important in oxygen transport and storage, cell respiration, DNA synthesis, photosynthesis and nitrogen fixation.

Therefore the intracellular concentration of iron requires tight control and is regulated both at the stage of uptake and in storage. Any perturbation of this homeostasis has severe pleiotropic effects on the physiology and development of living organisms.

Role of iron in humans

The essentiality of iron in humans was demonstrated already over two centuries ago and 60 to 70% of the iron in human body is found in the blood. Recommendations of US Food and Drug Administration are available, published as the so called RDA (Recommended Daily Allowances) for all micronutrients including iron, as iron is amongst others a major component of haemoglobin, myoglobine and cytochrome, all enzymes playing a critical role i.e. in the respiration processes. The recommended daily intake by humans ranges from 8 mg/day for males to 18 mg/ day for premenopausal females.

Even though iron is so important for metabolism, the bioavailability of iron is compromised by its low solubility. Therefore the chelates contribute considerably to an enhancement of the availability of iron in the biocircle, ensuring iron supply directly to the plant or solubilisation of native iron from soils.

The most common symptoms of iron deficiency are weakness, microcytic hypochromic anaemia, reduced immunity to infection, thalassemia. Iron deficiency anaemia is one of the ten most common human deficiencies and the effects of iron supplementation are significantly influenced by many predisposing biochemical factors.

The most important iron carrier in humans is transferrin, which also plays a major role in metallic ions movement in the body. Ferritin is the main factor in iron storage, located in marrow and affects iron metabolism.

The activity of the HBED anion in organisms

Interestingly the production of FeHBED follows the same mechanism as found in the body after dosing with NaHBED, it reacts with the iron-ion (Fe+3 ) and forms NaFeHBED.]

The chelating agent HBED has a very high affinity to iron and lacks acute toxicity. This is the reason for its use as a remedy for a number of diseases, such as iron overload, thalassemia, malaria, chronic haemolytic anaemia and acute iron poisoning. Medical tests, carried out in mammals and humans with β-thalassemia proved its safety up to 80 mg/kg bw. But experimental data in mice clearly demonstrate that orally administrated HBED - if not encapsulated in liposomes – was not effective in increasing iron elimination from the body. However, even in the most conservative approach any amount of chelating agent HBED present in tissues is very unlikely to cause any adverse side effects and this is supported by the fact, that HBED, used in much higher concentrations in rodents, dogs, primates and humans, didn’t cause any pathologic changes in tissues.

ABSORPTION OF FeHBED

In general, absorption of a chemical is possible, if the substance crosses biological membranes. This process requires a substance to be soluble, both in lipid and in water, and is also dependent on its molecular weight (substances with molecular weights below 500 are favourable for absorption). Generally, the absorption of chemicals which are surfactants or irritants may be enhanced, because of damage to cell membranes.

FeHBED would not be favourable for absorption, when taking its molecular weight (463.26 g/mol) into account. However, the substance is readily soluble in water (34.55 g/L), so it is apparent, that its absorption is hindered. The value of the LogPow (-1.96) shows the substance to be better soluble in water than in octanol (positive LogPow for lipophilic substances, negative LogPow for hydrophilic substances). Considering the above mentioned physico-chemical properties, the absorption into the body will not be favoured (LogPow between 0 and 4 are favourable for absorption).

In addition, as FeHBED is not irritating to the skin or to the eyes, the above mentioned enhancement of absorption for irritants, does not apply.

ORAL ABSORPTION

Regarding oral absorption, in the stomach, a hydrolysable substance will most likely undergo hydrolysis, as this is a favoured reaction in the acidic environment of the stomach. In the small intestine absorption occurs mainly via passive diffusion or lipophilic compounds may form micelles and be taken into the lymphatic system. Additionally, metabolism may occur by gut microflora or by enzymes in the gastrointestinal mucosa. However, the absorption of highly lipophilic substances (log P of 4 or above) may be limited by the inability of such substances to dissolve into gastrointestinal fluids and hence make contact with the mucosal surface. The absorption of such substances will be enhanced if they undergo micellular solubilisation by bile salts. Substances absorbed as micelles enter the circulation via the lymphatic system, bypassing the liver.

In order to evaluate the ability of FeHBED to be absorbed following oral administration, several physicochemical properties have been taken into account:

Molecular weight and spatial structure

The steric structure of FeHBED and its high molecular mass M = 463.26 g/mol rather complicate its ability to permeate through aqueous pores or to be carried through the epithelial barrier by the bulk passage of water.

The thesis, that its spatial structure hinders absorption and infiltration through cell membranes, is supported by a number of medical tests on oral absorption of HBED (or NaHBED, respectively. These studies have also shown a limited absorption from GI tract for HBED, which was due amongst others to the high molecular mass and the spatial structure.

Stability constant

When NaHBED reacts with Fe⁺³, this results in Na [FeHBED]. This is a dissociable compound, and the iron chelate FeHBED has a very high stability constant of K = 10^-39

                                                               [NaHBED]*[Fe]

where K =

                                                               Na[FeHBED]

NaHBED is so far recognized as strongest chelating agent used in fertigation to be applied in very high pH soils above 7,5 and calcareous soils

This value is the highest value known for iron chelates used in fertigation and is comparable with the one of haemoglobin, the strongest iron chelate ever.

For comparison, the stability constants of other iron chelates are given:

FeHBED                              K = 10^-39

FeEDDHA^*                         K = 10^-35

FeDTPA                             K = 10^-28

FeEDTA                             K = 10^-25

FeIDHA                              K = 10^-19

This high stability constant makes FeHBED a very effective substance to supply plants with iron in a.m. conditions. In fact, FeHBED is very stable (very high affinity of iron to HBED) and in the human or animal body no mechanisms exist, which are able to cleave the covalent bonds between the hexadentate molecule of the chelating agent HBED and the iron ion.

Hydrolysis

For FeHBED no neutral hydrolysis rate is evident. Therefore, in accordance with the above mentioned principles, it is very unlikely for FeHBED to be hydrolysed in the stomach.

Solubility in water

FeHBED is readily soluble in water 34.55 g/L at 20 °C, which equals to 0.31% Fe/L. Considering the concentrations applied in fertigation systems (approx. 0.0005% Fe) FeHBED has to be regarded as a hydrophilic substance.

The high water solubility in conjunction with the high molecular weight and the low LogPow results (see below) in negligible oral absorption rates.

Partition coefficient - LogPow

As LogPow values between 0 and 4 are favourable for absorption, the low LogPow of the registered substance (-1.96) allows the prediction, that absorption of FeHBED from GI via passive diffusion is unlikely because of the rate at which the substance partitions out of the GI fluid.

Summary of oral absorption

Taking into consideration the physicochemical properties of FeHBED, such as a rather high molecular mass, its steric structure, a very high stability constant, a non-existing hydrolysis, a ready water solubility and a low LogPow, it can be stated that FeHBED is unlikely to be absorbed from GI tract.

This thesis is supported by experimental data in monkeys obtained with HBED, which clearly demonstrate that oral administrated HBED (if not incorporated into liposomes) was not effective in increasing iron elimination from the body³. In contrast parenteral administered HBED increased iron elimination³.

In addition, oral toxicity tests with FeHBED in rats (acc. to OECD 420) gave an LD50 > 2000 mg/kg bw. This evidence endorses the thesis that absorption of FeHBED from the GI tract is limited.

ABSORPTION BY INHALATION

Concerning absorption in the respiratory tract, any gas or vapour has to be sufficiently lipophilic to cross the alveolar and capillary membranes (moderate LogPow values between 0-4 favourable for absorption). The rate of systemic uptake of very hydrophilic gases or vapours may be limited by the rate at which they partition out of the aqueous fluids (mucus) lining the respiratory tract and into the blood. Such substances may be transported out of the lungs with the mucus and swallowed or pass across the respiratory epithelium via aqueous membrane pores. Lipophilic substances (LogPow >0) have the potential to be absorbed directly across the respiratory tract epithelium. Very hydrophilic substances can be absorbed through aqueous pores (for substances with molecular weights below and around 200) or be retained in the mucus.

In order to evaluate the ability of FeHBED to be absorbed following inhalation administration, the following physicochemical properties have been taken into account:

Vapour pressure

The vapour pressure of FeHBED is very low (5.75×10-7 kPa at 20 °C), therefore exposure via inhalation is very unlikely to occur, as the substance is only minimally available in the atmosphere.

Particle size distribution

The distribution of the particle size, as a result of the improved drying process and the segregation operations during production, is rather high. The particle size distribution was determined and 17.2 % were above 500 µm, 42 % were 125 – 500 µm, 15 % were between 63 and 125 µm, 12.5 %were between 45 and 63 µM and only 13.3 % were below 45 µm. As the method used to determine (dry sieve) the particle size distribution is not very specific, when it comes to small diameters, it is likely that an even smaller fraction is really respirable / inhalable (< 10 / < 5 µm). Thus the final product has a microgranulated form. Therefore FeHBED is dust free and provides only a very limited risk for the inhalation of particles.

Partition coefficient - LogPow

The low LogPow of -1.96 does not support absorption through alveolar and capillary membranes.

However, the hydrophilicity of FeHBED could lead to a limited absorption into the aqueous fluids lining the respiratory track, followed by partition into the blood, but this way of absorption is not of significance, since for this process lower molecular weights are necessary.

Molecular weight and spatial structure

Absorption through aqueous pores is not considered to play a significant role as the molecular mass of FeHBED is too high for absorption through aqueous pores, as this is the only absorption possible, based on the high water solubility of the substance.

Summary of inhalatory absorption

Taking into consideration the physicochemical properties of FeHBED, such as its very low vapour pressure, particle size distribution, a low LogPow and a high molecular mass and its steric structure, it can be stated that FeHBED is unlikely to be absorbed to a significant extent after exposure by the inhalation route.

DERMAL ABSORPTION

In order to cross the skin, a compound must first penetrate into the stratum corneum and may subsequently reach the epidermis, the dermis and the vascular network. The stratum corneum provides its greatest barrier function against hydrophilic compounds, whereas the viable epidermis is most resistant to penetration by highly lipophilic compounds. Substances with a molecular weight below 100 are favourable for penetration of the skin and substances above 500 are normally not able to penetrate. The substance must be sufficiently soluble in water to partition from the stratum corneum into the epidermis. Therefore if the water solubility is below 1 mg/l, dermal uptake is likely to be low. Additionally LogPow values between 1 and 4 favour dermal absorption (values between 2 and 3 are optimal). Above 4, the rate of penetration may be limited by the rate of transfer between the stratum corneum and the epidermis, but uptake into the stratum corneum will be high. Above 6, the rate of transfer between the stratum corneum and the epidermis will be slow and will limit absorption across the skin. Uptake into the stratum corneum itself may be slow. Moreover vapours of substances with vapour pressures below 100 Pa are likely to be well absorbed and the amount absorbed dermally may be more than 10% of the amount that would be absorbed by inhalation. If the substance is a skin irritant or corrosive, damage to the skin surface may enhance penetration. During the whole absorption process into the skin, the compound may be subject to biotransformation.

In order to evaluate the ability of FeHBED to be absorbed following dermal absorption, several physico-chemical properties have been taken into account:

Molecular weight and spatial structure

As stated above a “molecular weight less than 100 favours dermal uptake“, therefore it is clear that the dermal absorption of FeHBED is not favoured only taking its molecular weight into account. However, its spatial structure is also hindering absorption as the molecule will be too broad to allow significant absorption. Therefore FeHBED with a molecular mass of M = 463.26 g/mol is very unlikely to penetrate the skin.

Solubility in water

It is already known “if water solubility is above 10.000 mg/l and the LogPow value below 0 the substance may be too hydrophilic to cross the lipid rich environment of the stratum corneum. The stratum corneum provides a great barrier against hydrophilic compounds. So dermal uptake for such substances will be low. FeHBED has a high water solubility of 34.550 mg/l and therefore a low lipid solubility and this, in conjunction with a very low LogPow, renders it unlikely to be absorbed through the skin.

Partition coefficient - LogPow

It has to be taken into account that “for substances with log P values <0, poor lipophilicity will limit penetration into the stratum corneum and hence dermal absorption. Values <–1 suggest that a substance is not likely to be sufficiently lipophilic to cross the stratum corneum, therefore dermal absorption is likely to be low”. As FeHBED has a LogPow of -1.96 is expected not to be absorbed through the skin.

Summary of dermal absorption

In case of FeHBED the molecular weight is 463.26, which indicates already a low potential to penetrate the skin. Additionally, the very low vapour pressure can be judged slightly advantageous for dermal uptake. However, the high water solubility is expected to hinder considerably the absorption following dermal exposure into the stratum corneum. Moreover, its low LogPow is not advantageous for absorption after dermal contact. This is supported by the findings of the low systemic toxicity of FeHBED after exposure via the skin (acute dermal toxicity study, no mortality after dermal application of 2000 mg/kg bw in rats). Moreover an acute dermal irritation / corrosion study in the rabbit (acc. to OECD 404) for FeHBED did not demonstrate any irritation after 14 days. This information indicates that FeHBED is unlikely to penetrate the skin.

DISTRIBUTION OF FeHBED

In general, the following principle applies: the smaller the molecule, the wider the distribution. A lipophilic molecule (LogPow >0) is likely to distribute into cells and the intracellular concentration may be higher than extracellular concentration particularly in fatty tissues. It’s not possible to foresee protein binding, which can limit the amount of a substance available for distribution. Furthermore, if a substance undergoes extensive first-pass metabolism, predictions made on the basis of the physico-chemical characteristics of the parent substance may not be applicable.

In order to evaluate the distribution of FeHBED, the physico-chemical properties: microgranulated, dust free particles distribution, high molecular mass, its spatial structure, low LogPow, readily solubility in water and low vapour pressure, indicate that FeHBED is unlikely to permeate or penetrate GI tract. Therefore only a limited amount of FeHBED is expected to be available for distribution. However, the minor amount absorbed into the body, will most likely exist only in the intravascular compartment (due to its high molecular weight and the low LogPow) 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.

ACCUMULATIVE POTENTIAL OF FeHBED

The potential for a substance to accumulate or to be retained within the body is a very important point. Lipophilic substances (LogPow >4) have the potential to accumulate within the body (mainly in the adipose tissue), if the dosing interval is shorter than 4 times the whole body half-life. Although there is no direct correlation between the lipophilicity of a substance and its biological half-life, substances with high LogPow values tend to have longer half-lives. Highly lipophilic substances (LogPow between 4 and 6) that come into contact with the skin can readily penetrate the lipid rich stratum corneum but are not well absorbed systemically. Although they may persist in the stratum corneum, they will eventually be cleared as the stratum corneum is sloughed off. A turnover time of 12 days has been quoted for skin epithelial cells

The accumulative potential of FeHBED is influenced by its availability in the organisms. Even though the substance will not be absorbed in higher extents as lain out above, FeHBED is not considered to concentrate in the human body (i.e. in adipose tissue), due to the fact that it is not a lipophilic substance. As it is known 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”, no enhanced risk for accumulation will be associated with the substance, due to its low LogPow of -1.96.

METABOLISM OF FeHBED

Route specific toxicity may result from several phenomena, such as hydrolysis within the gastrointestinal or respiratory tracts, also metabolism by gastrointestinal flora or within the gastrointestinal tract epithelia (mainly in the small intestine), respiratory tract epithelia (sites include the nasal cavity, tracheo-bronchial mucosa [Clara cells] and alveoli [type 2 cells]) and skin.

Due to the high stability constant of the iron chelate complex it is assumed that most of this iron fraction is excreted unchanged in the chelated form. Thus the already very water soluble native substance will be eliminated mainly in the faeces and to a lower extent in the urine. This thesis is supported by findings that another chelate iron dextran, investigated in monkeys for its induction of additional iron excretion¹, caused additional iron excretion mainly in the stool (92%) and little elimination via the urine (8%).

Even though phase I reaction are not expected to play a significant role, the molecular structure was investigated, to identify all possible sites for phase-1 reactions. The pre-existing carboxyl-groups are not expected to be subject to further phase I reactions. However, one possibility is that the nitrogen might be subject to oxidative desalkylation, yielding instable intermediates, which decompose to an secondary amine and an aldehyde. In addition, it is theoretically possible that the hydroxyl groups are subject to dehydration by alcohol dehydrogenase, yielding aldehydes / ketones. Moreover, the carbon double-bond might be cleaved and two hydrogens would be added. Additionally theoretically a cleavage of the cycle is possible, leading to the formation of either 2 methyl-groups or 1 methyl-group and CHOH-group. However, all these reactions are not likely to occur, also becausehydrolysis is judged not to apply for FeHBED.

The above mentioned functional groups (starting points for further metabolism) can react in phase 2 of the biotransformation with different molecules, leading to the formation of conjugations.As glucuronidation is quantitatively the most important phase II pathway for drugs and endogenous compounds, it is most probable to occur also for FeHBED. The enzyme required for glucuronidation is the UDP-glucuronosyltransferase (UGT), which metabolizes a broad range of structurally diverse endogenous and exogenous compounds. N-glucuronidation takes place mainly with aromatic amines and O-glucoronidation via the ester linkages pathway with carboxylic groups and both reactions increase significantly the water solubility, decrease the toxicity and lead often to subsequent excretion via the bile.

In addition FeHBED might be conjugated to activated sulphate or activated methionine.

However, concerning any possible metabolism, it has to be considered that the metabolites are not expected to exert relevant systemic but to be readily excreted. This is supported by many available tests for HBED in animals, included as supporting studies, which indicate no toxic effects on organs and tissues of exposed dogs, rats, mice and monkeys. Moreover, it can be expected that after HBED administrationin vivoFeHBED formation also occurred. No adverse effects detected in tissues and organs after HBED administration (and induced FeHBED formation) suggest that the chelating agent HBED as well as the registered substance FeHBED have very limited influence on the organism. Due to the lack of changes and no toxic effect observed, one may expect that metabolites of HBED and FeHBED are non toxic and/or easily excreted.

In conclusion, it is most likely that the substance of interest will not undergo extensive metabolism but be mainly excreted unchanged via the bile and to a smaller extent via the urine. However, it is possibly subject to glucuronidation. In this case the possibility of entero-hepatic recycling, and the risk or a re-enter into system, does not bear any risk for the organism. Concerning the possibility of protein binding, this can not be ruled out, without adequate experimental data.

EXCRETION OF FeHBED

The major routes of excretion for substances from the systemic circulation are in the urine and/or the faeces (via bile and directly from the gastrointestinal mucosa). For non-polar volatile substances and metabolites exhaled air is an important route of excretion. Substances that are excreted favourable in the urine tend to be water-soluble and of low molecular weight (below 300 in the rat) and be ionized at the pH of urine. Most will have been filtered out of the blood by the kidneys though a small amount may enter the urine directly by passive diffusion and there is the potential for reabsorption into the systemic circulation across the tubular epithelium. Substances that are excreted in the bile tend to be amphipathic (containing both polar and nonpolar regions), hydrophobic/strongly polar and have higher molecular weights and pass through the intestines before they are excreted in the faeces and as a result may undergo entero-hepatic recycling which will prolong their biological half-life. This is particularly a problem for conjugated molecules that are hydrolysed by gastrointestinal bacteria to form smaller more lipid soluble molecules that can then be reabsorbed from the GI tract Those substances less likely to recirculate are substances having strong polarity and high molecular weight of their own accord. Other substances excreted in the faeces are those that have diffused out of the systemic circulation into the GIT directly, substances which have been removed from the gastrointestinal mucosa by efflux mechanisms and non-absorbed substances that have been ingested or inhaled and subsequently swallowed. Non-ionized and lipid soluble molecules may be excreted in the saliva, where they may be swallowed again, or in the sweat. Highly lipophilic substances that have penetrated the stratum corneum but not penetrated the viable epidermis may be sloughed off with skin cells.

For FeHBED no data is available concerning its elimination. Taking into consideration the high stability constant of K = 10^-39 for FeHBED, it is clear that it exerts a low reactivity in the organism. The above mentioned behaviour predicted for its metabolic fate supports this thesis and the parent substance will be most likely excreted unchanged. Its high water solubility would lead normally to urinary excretion; however, in conjunction with the rather high molecular weight and its chemical structure mainly biliary excretion is expected and possibly to a small extent via the urine (may be also as conjugates with glucuronic acid). This thesis is supported with the findings for HBED/NaHBED in experiments carried out in animals and humans, which have proven that the substance is easily excreted mainly biliary from the body. In order to estimate the FeHBED excretion (Table 1) the data available for HBED obtained in the above mentioned experiments were used to perform the calculations.

Table 1. Calculated FeHBED excretion in response to HBED administration

Route	HBED administered (mg/kg bw)	Efficiency (%): Net iron excretion/total iron binding capacity of chelate administered	Fe balance (µg/kg bw.)	FeHBED excreted (mg/kg bw.):
SC bolus	32.4	14.2	-524	4.60
SC bolus	64.9	13.6	-899	8.83
20 min. i.v. inf.	64.9	7.7	-408	5.0
20 min. i.v. inf.	97.3	6.3	-570	6.13

 

SUMMARY OF THE TOXICOKINETICS OF FeHBED

In order to assess the toxicological behaviour of FeHBED(Sodium [N-(2-{[(hydroxy-kO)acetyl][2-(hydroxy-kO)benzyl]amino-kN}ethyl)-N-[2-(hydroxy-kO)benzyl]glycinato(4-)- k2 N,O] ferrate(1-)), the available and predicted physico-chemical and toxicological data have been evaluated. The substance is expected to be unlikely readily absorbed after oral exposure, based on its rather high molecular weight, its high water solubility and it’s LogPow of -1.96. This agrees with the high LD50 > 2000 mg/kg bw, determined in rats after oral exposure to FeHBED Concerning the absorption after exposure via inhalation, as the chemical has really low vapour pressure, it is clear, that the substance has a low availability for inhalation. FeHBED is not expected to be absorbed significantly following dermal exposure into the stratum corneum, due to its molecular weight, its LogPow and its high water solubility. Accordingly, its systemic toxicity via the skin has been proven to be low (no mortality after dermal application of 2000 mg/kg bw in rats). The substance is not expected to bear accumulative potential. FeHBED is not expected to be extensively metabolised but to be eliminated mainly via the bile (in cases as glucuronic acid conjugates) or due to its high water-solubility to a smaller extent oxidised or unchanged via the urine.

From the results of the toxicokinetics assessment it can be concluded, that uptake and resulting effects in the human body through all routes of exposure are negligible. Therefore, no further information is required to conclude in the toxicokinetic behaviour of FeHBED.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): other: no bioaccumulation potential based on assessment
An extensive Assessment of the toxicological behaviour of Fe(Na)HBED was performed (expert statement), taking into account the chemical structure, the available physico-chemical-data and the available (eco-)toxicological data.

Executive summary:

In order to assess the toxicological behaviour of Fe(Na)HBED(Sodium [N-(2-{[(hydroxy-kO)acetyl][2-(hydroxy-kO)benzyl]amino-kN}ethyl)-N-[2-(hydroxy-kO)benzyl]glycinato(4-)- k2 N,O] ferrate(1-)), the available and predicted physico-chemical and toxicological data have been evaluated. The substance is expected to be unlikely readily absorbed after oral exposure, based on its rather high molecular weight, its high water solubility and it’s LogPow of -1.96. This agrees with the high LD50 > 2000 mg/kg bw, determined in rats after oral exposure to Fe(Na)HBED Concerning the absorption after exposure via inhalation, as the chemical has really low calculated vapour pressure, it is clear, that the substance has a low availability for inhalation. Fe(Na)HBED is not expected to be absorbed significantly following dermal exposure into the stratum corneum, due to its molecular weight, its LogPow and its high water solubility. Accordingly, its systemic toxicity via the skin has been proven to be low (no mortality after dermal application of 2000 mg/kg bw in rats). The substance is not expected to bear accumulative potential. Fe(Na)HBED is not expected to be extensively metabolised but to be eliminated mainly via the bile (in cases as glucuronic acid conjugates) or due to its high water-solubility to a smaller extent oxidised or unchanged via the urine.

From the results of the toxicokinetics assessment it can be concluded, that uptake and resulting effects in the human body through all routes of exposure are negligible. Therefore, no further information is required to conclude in the toxicokinetic behaviour of Fe(Na)HBED.