Choline hydroxide

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics

Type of information:

other: Expert statement

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

No study covering all the relevant information was available, hence, an extensive assessment of the toxicokinetic behaviour of choline hydroxide was performed, taking into account the chemical structure, the available physico-chemical and toxicological data.

Data source

Reference

Reference Type:

other: Expert statement

Title:

Unnamed

Year:

2013

Report date:

2013

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 toxicokinetic behaviour of choline hydroxide was performed, taking into account the chemical structure, the available physico-chemical and toxicological data.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Choline hydroxide

EC Number:

204-625-1

EC Name:

Choline hydroxide

Cas Number:

123-41-1

Molecular formula:

C5H14NO.HO

IUPAC Name:

choline hydroxide

Test material form:

other: not applicable

Details on test material:

not applicable

Radiolabelling:

other: not applicable

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 relevant routes of administration are discussed in the expert statement

Vehicle:

other: not applicable

Details on exposure:

not applicable

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

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Absorption

In this chapter, the physico-chemical properties of the substance are used to draw general conclusions for its behaviour and how these properties will influence its oral, inhalatory and dermal absorption. Furthermore, these conclusions will be supported by the available literature data and studies.
In general, absorption of a chemical is possible, if the substance crosses biological membranes. In case where no transport mechanisms are involved, 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. This is the case for choline hydroxide, because it is a strong base (pKb = 5.06 for 0.0064-0.0403 M solutions (Seidel A, 2004)), the only distributed form of choline base, a 45 % solution has a pH value of 14.9, 45 % solution (Struyvelt, 2012) and it is classified as corrosive to the skin and eye. So, these effects must be additionally regarded when assessing the toxicokinetic behavior of choline base. In general, the absorption of the choline cation is well examined, and will be outlined in the following subchapters.

Absorption from the gastrointestinal tract

Under normal circumstances, choline is mainly present in the diet in the form of lecithin (PC), with less than 10 % present as either free base or sphingomyelin. Choline is released from lecithin and sphingomyelin by digestive enzymes of the gastrointestinal tract, although 50 % of the ingested lecithin enters the thoracic duct intact (McDowell LR, 2000). However, in the present statement the absorption of the unbound choline is relevant.
Since choline is a rather small, ionic and therefore hydrophilic molecule, passive diffusion through the lipid double layer is very unlikely. It can be rather assumed that the cation diffuses through pores, in this case likely charged ion channels, or will be transported actively by transporters.
In fact, choline is absorbed from the jejunum and ileum mainly by an energy-dependent carrier mechanism in the brush-border membrane. The unidirectional influx of choline appears to proceed primarily by a saturable, carrier-mediated process at low mucosal choline concentrations, as derived from jejunum tissue sample experiments. Here the influx rate is indicative as it is not a linear function. At high concentrations (> 4 mM) the influx rate is approximately linearly related to the mucosal choline concentration, suggesting that absorption by passive diffusion predominates. In this experiment, Km was determined as 0.6 mM and V as 132 nmol/cm² per h. Since the influx could be markedly reduced by either elimination of Na+ or addition of metabolic inhibitors, the conclusion could be drawn that the carrier-mediated mechanism is similar to the one of fructose: It demonstrates saturation kinetics but is independent of mucosal sodium, whereas usually the active transport of sugars and amino acids is based on a sodium-dependent mechanism (Kuczler FJ, 1977).
The choline carrier mechanism is moderately sensitive to hemicholinium-3 ((2S,2'S)-2,2'-biphenyl-4,4'-diylbis(2-hydroxy-4,4-dimethylmorpholin-4-ium), presynaptic choline re-uptake inhibiting drug) inhibition (McDowell LR, 2000; Garrow, TA, 2007). As shown in guinea pigs, choline is taken up by ileal cells about three times faster than by jejunal cells, which is due to the fact that choline is usually released from lecithin of the diet in the proximal and middle small intestine (Garrow TA, 2007). At low lumenal choline concentrations, in case choline was not already absorbed or metabolized in the upper GI tract, influx in the colonic mucosa is slower than in jejunum and appears to be solely attributed to simple diffusion (Kuczler FJ, 1977).
Later findings describe more precisely that choline, as a positively charged quaternary amine, requires protein-mediated transfer for its entrance into cells, as stated above. Biochemically, choline transport has been classified into three general phenomena based on kinetic studies with radiolabelled choline:
a) low-affinity (Km > 30-100 µM) facilitated diffusion that is insensitive to hemicholinium-3
b) intermediate-affinity sodium-independent transport that is moderately sensitive to hemicholinium-3 inhibition
c) high-affinity (Km < 10 µM) sodium-dependent transport that is very sensitive to hemicholinium-3 inhibition (Michel V, 2006; Garrow TA, 2007).
While the latter is unique to cholinergic neurons, the low- and intermediate affinity systems are used to transport through the plasma membrane or as intestinal transporters, but also for some other unique choline transport needs, such as blood-brain barrier and mitochondrial transporters (Garrow TA, 2007).
However, only one-third of the ingested choline appears to be absorbed intact. The major part is either oxidized to betaine or metabolized by the intestinal microflora to trimethylamine (De La Huerga J, 1953), whereas the latter is greatest when large amounts of choline are ingested; trimethylamine is excreted in the urine between 6 and 12 hours after consumption. Zeisel SH et al. showed, that 3 h after oral administration in humans, 64-65 % of the dose was absorbed; however, the form of choline was not stated (Zeisel SH, 1989).

Regarding the oral uptake, the hydroxide anion is only of minor importance. This is due to the fact that, if ingested orally, the contact time of the basic solution to the oesophagus is rather short for causing severe chemical burns which will be necessary to enlarge the oral uptake. Once reaching the stomach, the basic pH of the choline base solution will be immediately neutralized by the gastric acid. Only when ingesting large amounts of choline base, the neutralization capacity of the stomach acid will be used up. However, this scenario is unlikely due to expected pain in the oral cavity and pharynx caused by hydroxide. Hence, only effects of the choline cation have to be regarded as described above, and an absorption of choline, based on the data outlined above, can be estimated to be 50 %.

Absorption from the respiratory tract

Concerning absorption in the respiratory tract, any gas, vapour or other substances inhaled as respirable dust (i.e. particle size ≤ 15 µm) has to be sufficiently lipophilic to cross the alveolar and capillary membranes (moderate LogPow values between 0-4 are 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 on the other hand, such as choline can possibly be absorbed through aqueous pores via passive diffusion (for substances with molecular weights below and around 200) or be retained in the mucus. Data from the gastrointestinal absorption for high concentrations (Kuczler FJ, 1977) and the molecular weight of the cation (104.2 g/mol) suggest this possibility of absorption via passive diffusion. However, the high water solubility and negative logPow in general point to a very poor absorption and rather to a retention in the mucus.
Choline hydroxide, as the pure substance, has a really low calculated vapour pressure of 1.43x10-7 Pa (US EPA, 2000) and is likely to decompose at approx. 300 °C (BASF, 1983), which indicates a low availability for inhalation. Additionally, the most prevalent form is an aqueous solution, where no inhalable dusts are expected at all. Here, of course, the vapour pressure and boiling point are close to the ones of water and only relevant for the solvent.
Based on this data, it can be concluded that choline is not likely to be available for inhalation, due to its very low vapour pressure and its distribution as an aqueous solution.
Regarding the hydroxide anion, the absorption via the mucous membrane of the lungs can be generally considered as enhanced, in case choline hydroxide reaches the respiratory tract, because no neutralizing agents such as gastric acid compared to the stomach are available. However, since choline base has low availability for inhalation, the amount of hydroxide ions reaching the respiratory tract, and so the lung epithel, is only minor, and since the corrosivity due to hydroxide anions is a dose-dependent phenomenon, the enhancement of absorption due to corrosivity can be neglected, because the minor amount of OH- reaching the epithel would not be sufficient to cause any irritating and so absorption-enhancing effects.
In conclusion, it can be reasonably assumed, since choline is not very favourable for inhalatory absorption and the relevant transporting mechanisms for gastrointestinal absorption are not or at least not distinctively present in the lungs, that a maximum absorption of approx. 25 % of the applied amount of choline is reasonable for further risk assessment.

Absorption following dermal exposure

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 epidermis is most resistant to penetration by highly lipophilic compounds. Substances with a molecular weight below 100 are favourable for penetration through 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.
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 is most likely more than 10 % and less than 100 % 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 can be subject to biotransformation.

In case of choline hydroxide, the dissociated and hence charged organic cation will be predominantly present either due to the humidity of the skin, the humidity of the air, because choline base is hygroscopic, or the water present in the formulation in which it is distributed. The molecular weight of the cation is with 104,2 g/mol rather low, which in general indicates a certain potential to penetrate the skin. However, due to the negative logPow of -2.25, the high water solubility of > 486 g/L and hence hydrophilicity, it is nearly impossible for the choline cation only to penetrate the stratum corneum and to be absorbed via the intact skin. Furthermore, according to ECHAs Guidance document R.7C, quaternary ammonium salts in general can possibly bind to skin components, in this case the dead cells of the squamous epithelium. This possibility will furthermore contribute to the diminished absorption. Since the skin lacks a pronounced microflora compared to the intestines and the metabolic capacity compared to the liver, the minor amount of the choline cation which will nevertheless reach the blood stream, can be assumed to be absorbed mainly unmetabolized.
Although choline hydroxide has a very low vapour pressure, and the vapour pressure of the aqueous solution is mainly correlated to the one of water, this factor can be neglected because the effects resulting in non-absorption as described above are considered to be the more relevant ones.
Taking into account all these facts, it would normally be reasonable to assume a maximum dermal penetration of 10 % of the choline cation only as a worst case, which is compliant with ECHAs Guidance documents and scientifically reasonable when e.g. performing route-to-route extrapolations during risk assessment.

However, in general, if a substance is a skin irritant or corrosive, damage to the skin surface may enhance penetration (ECHA, 2008). This factor has to be regarded especially in the case of choline hydroxide, which exhibits due to the presence of hydroxide anions distinctive alkaline and hence corrosive properties. In case a corrosive chemical is applied to the skin, first, cell membranes in general can be damaged. Hence, the carrier mechanisms, which are normally required for choline transport in the cells of various tissues, such as organic cation transporters OTC1, OTC2 and OTC3, facilitating the low-affinity transport, and the carriers encoded in the CTL1-5 genes (choline-specific transporter-like proteins), facilitating the intermediate-affinity sodium-independent transport, are not required anymore for the uptake of the choline cation into the cell (Garrow TA, 2007). So, an increased influx in the damaged cell can be expected compared to cell with intact cell membranes. However, considering the fact that (1) the predominant effect for this increased influx is passive diffusion, which enables the choline cation to discharge from the cell in the same manner, and the fact that (2) in a chronic feed study in rats (Shivapurkar N, 1986, data from choline chloride (CC)) with an NOAEL > 1200 mg/kg bw/day CC or NOAEL > 1040 mg/kg bw/day choline base (recalculated), respectively, no local effects were seen in tissues capable for enhanced absorption, i.e. intestinal brush-border membrane, and the (3) low intrinsic toxicity of choline (LD50 ≥ 3500 mg/kg bw/day CC, BASF, 1963, data from choline chloride, corresponding to 3040 mg/kg bw/day choline base), it can be concluded that no local effects due to the cation are to be expected. In case any local effects, such as necrosis, are detected, they will be most likely due to the damage of cell compartments by the hydroxide.
Additionally, when applying a corrosive chemical to the skin and not instantaneous removing it, not only single cell membranes will be damaged, but also several layers of the skin. The stratum corneum consists of several layers of dead, cornified keratinocytes (i.e. corneocytes), which are linked by structural proteins forming an effective barrier. A prolonged exposure to hydroxides leads i.a. to the hydrolysis of these proteins, resulting in the disintegration of this barrier and finally severe chemical burns. Once that the stratum corneum is removed or at least made permeable, the primary barrier for hydrophilic compounds is not existent anymore and the free choline cation can easily reach the epidermis and from there be easily distributed mainly unmetabolized via the blood stream. Hence, the poor dermal absorption for the choline cation is not applicable anymore and the estimated absorption rate of 10 % must be adjusted upwards. An estimated absorption of 50 % choline hydroxide is considered reasonable, because the absorption is enhanced, but not equal to the bioavailability after an intravenous injection, because it can be expected that not the complete dermal dells will be damaged, and a certain barrier function emanates also from the epidermal cells.
After absorption, choline hydroxide, or the choline cation, may easily be distributed throughout the body via lymphatic or portal circulation.
When absorbed in the intestinal mucosa, choline is phosphorylated to phosphocholine, which is the primarily transported form of choline (bound to chylomicra) (Combs GF, 2008). When absorbed by the damaged skin, choline will circulate mostly unbound / unmetabolized through the vascular or lymphatic system. However, it is nearly irrelevant in which form the choline cation is distributed, because only the unbound cation can be absorbed by the target tissues. When applied intravenous, which is the most similar application route to the absorption of unmetabolized choline through the damaged skin, choline is rapidly eliminated from the plasma vie tissue uptake; however, with an increased plasma level of choline, urinary excretion is markedly increased, too (Buchman AL, 1994).

Details on distribution in tissues:

Distribution

After absorption, choline hydroxide as a quaternary amine salt may easily be distributed throughout the body via lymphatic or portal circulation due to its high water solubility and small molecular weight, because in general it can be stated that the smaller the molecule, the wider the distribution. Only based on the logPow (-2.25) of choline hydroxide, it could be assumed that the extracellular distribution is higher than the intracellular one, as the passage through the cell membrane, is hindered. But as stated above, for choline there are several protein-mediated mechanisms to effectively pass the membrane lipid barrier available. Also, choline is primarily transported in the form of phosphatidylcholine bound to chylomicra, which are subject to clearance to the lipoproteins that circulate to the peripheral tissues. Thus, choline is transported to the tissues predominantly as phospholipids associated with the plasma lipoproteins (Combs GF, 2008). In the tissues, choline is released in the free form by the actions of phospholipase C, which cleaves phosphatidylcholine to yield a diglyceride and phosphorylcholine and the latter is converted to free choline by alkaline phosphatase. However, peripheral tissues also contain phospholipase B activity and can therefore utilize the circulating form of choline producing glycerylphosphorylcholine. The latter can then be cleaved by glycerylphosphorylcholine diesterase to yield free choline. The brain also contains phospholipase D, which cleaves free choline directly from the circulating form (Combs GF, 2008).
Choline transport into the cell can be performed in three different ways:
The three organic cation transporters OTC1, OTC2 and OTC3, facilitating the low-affinity transport, are members of the solute carrier superfamily and are expressed in many tissues. This also applies to the intermediate-affinity sodium-independent transporters; many tissues express one or more of the CTL1-5 genes (choline-specific transporter-like proteins). The high-affinity transport on the other hand is unique to the cholinergic neurons of the brain, brain stem, and spinal cord. The required transporter CHT1 is also the limiting factor in the synthesis of the neurotransmitter acetylcholine (Garrow TA, 2007).

Many organs, such as lung, brain, placenta, kidney, and liver have acquired one or a combination of several choline transport systems, which allow them to fulfil specific functions. The central nervous system requires choline for the synthesis of membrane phospholipids and for acetylcholine. Here, a functioning choline transport is essential as neuronal cells are incompetent for choline de novo synthesis. The placenta possesses a choline transport mechanism, which is essential because a growing fetus has to derive all its metabolic choline requirements from its mother via the placenta (Michel V, 2006).
Special attention should be drawn to the choline uptake in the liver and kidney:
All ingested choline enters the hepatic circulation, making the liver, where there are very active biochemical pathways for choline metabolism, a significant ‘‘sink’’ for choline. The liver contains a transport system resembling the ‘‘low-affinity’’ facilitated diffusion, but multiple systems are also detectable under different conditions.
In vivo, systemically injected choline is accumulated by the liver, and in isolated perfused rat liver there is a significant net uptake of free choline. A saturable mechanism [Ka = 0.17 mM, Vmax = 0.84 µmol/g (dry weight) per min] and a nonsaturable mechanism (through which uptake is proportional to choline concentration presented to the liver) contribute to hepatic choline uptake. Free choline concentration within the hepatocytes is 50-100 nmol/g (wet weight). The remainder of the choline accumulated by the hepatocytes is metabolized to form betaine, phosphorylcholine, and lecithin (see 3.4). The rate at which liver takes up choline is sufficient to explain the extremely rapid disappearance of choline injected systemically (Zeisel SH, 1981, Michel V, 2006).
In the kidneys, within 15 min after a dose of radioactive choline is administered systemically a significant amount of label is found. Renal tubular transport of choline is of importance because it maintains the plasma choline concentration within relatively narrow limits by employing both net secretion and reabsorption. When choline is presented to the kidneys in excess of a species-specific threshold concentration, it will be excreted by the kidneys into the urine. If the plasma concentration is below this level, choline will be reabsorbed into the kidney and not excreted (Zeisel SH, 1981, Michel V, 2006).
Additionally, choline is extensively metabolized by the body and gastrointestinal microflora or the mucosa of the small intestine, so it is not applicable to focus only on the parent compound when outlining its distribution. In the following section, the distribution / accumulation of the several metabolites is described.

Accumulation

There are no known mechanisms to store choline in tissues. All of the molecules / metabolites that contain choline are either intermediates in pathways, signalling molecules, or reside in lipid bilayers providing structural and functional properties to membranes. So, it could also be stated that choline is stored in a certain mode anyway. Although pathway intermediates, particularly phosphocholine, can fluctuate because of recent intake or, which is toxicologically relevant, are accidently ingested in high amounts, there is no evidence indicating that these metabolite changes are nothing other than a transient mass effect. Rather, excess choline is catabolized via the choline oxidation pathway (Garrow TA, 2007). However, the findings of Buchman et al. suggest an at least partly storage of choline, presumably in liver or kidneys. After intravenous application of choline on four human volunteers, the plasma free choline levels failed after 24 h to return to baseline levels, although the concentration dropped rather fast after the end of the 12 h infusion period (Buchman AL, 1994).
In general, most of the choline in the body is found in the form of the phospholipids phosphatidylcholine (lecithine), lysophosphatidylcholine, choline plasmalogens, and sphingomyelin-essential components of all membranes. For example, lecithin is the predominant phospholipid in most mammalian cell membranes, and in rodent liver phosphatidylcholine accounts for 90 % of the total choline. Lysophosphatidylcholine and phosphocholine generally account for additional 7 - 9 %, whereas free choline is only 0.5 - 1 % of the total tissue choline. Liver and brain contain about 25 µmol total choline/g tissue, whereas muscle, heart and kidneys contain about 25 - 30 % of that found in liver and brain. Each organ has a similar distribution of choline metabolites except the kidneys, which contain significantly more glycerophosphocholine, presumably for osmotic control (Garrow TA, 2007, McDowell LR, 2000).

Details on excretion:

Excretion

In general, the major routes of excretion for substances from the systemic circulation are 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. These general assumptions are widely applicable for choline; however, it has to be kept in mind that the compound is intensely metabolized, transformed into essential molecules for normal body functions and therefore certain amounts of the compound remain in the body due to natural metabolic pathways.
After the ingestion of relatively large amounts of choline (15 mmol choline / kg bw. in rats), appreciable amounts (237 µmol (16 % of the dose)) reach the caecum and colon and are metabolized to form TMA by intestinal bacteria (Zeisel SH, 1989). When such an elevated amount of choline is ingested by normal persons (4 g (460 mg Nitrogen) choline base resp. 5.4 g (540 mg Nitrogen) choline chloride, in the form of choline bicarbonate), about 60 per cent appears in the urine as total trimethylamines (TTMA, including trimethylamine and trimethylamine oxide), mostly within 24 hours (De La Huerga J, 1953).
When choline is administered repeatedly by intravenous infusion over 12 h in human subjects, and hence bypassing intestinal and hepatic metabolism, plasma and urine level measurements revealed the following: The plasma choline levels increased to supranormal levels, as well as the urine levels did, during the choline infusion. When the infusion was terminated for 12 h, choline levels immediately dropped but did not reach the prior baseline. The elimination of choline is saturable at least at significant infusion rates, and a two-compartment model, in which elimination from the central compartment is saturable, gives the best fit. With an increased plasma level of choline, urinary excretion is markedly increased, too. Although many organs, predominantly the liver, contribute to the elimination of choline from the plasma, the Buchman et al. concluded that it is possible that the kidney could be more important in choline elimination than is readily apparent because no urinary choline metabolites were measured. Betaine and other choline metabolites, if found in the urine, can result not only from renal choline metabolism, but from choline uptake and metabolism by other organs as well.
In guinea pigs, in which choline and its metabolites are excreted in the urine, the major route of plasma choline clearance from the plasma is tissue uptake, predominantly by the liver and kidneys. Approximately 50 % of the administered choline are eliminated here by tissue uptake within three minutes. The kidneys extract approximately 30 % of the choline entering the renal artery in human subjects (Buchman AL, 1994).
In summary, when choline is accidently ingested at high doses, i.e. higher than required by the body, choline and its metabolites are readily excreted via the kidneys, presumably within 12 hours. Since choline is a required methyl donor, choline will be incorporated in normal cell compartments or pathways, and no excretion occurs, when applied in rather low levels.

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

Metabolism

Choline is extensively metabolized when entering the body. The intracellular routing of choline to its various metabolic pathways, phosphorylation, oxidation, and acetylation is cell and tissue specific. However, the attached picture provides an overview on the most relevant metabolites. Under normal circumstances, the amount of free choline is only 0.5 - 1 % per cent of the total tissue choline (Garrow TA, 2007).
When ingested, choline is degraded by the intestinal microflora to trimethylamine (TMA), partly dimethylamine. These compounds are also precursors of dimethylnitrosamine, which is classified as a carcinogen. At larger amounts (15 mmol choline / kg bw.), the authors found a disproportional large increase in TMA in rats, which supported the hypothesis that only when intestinal transport systems for the absorption of choline are overloaded, appreciable amounts of this nutrient reach the large bowel and are metabolized to form TMA by intestinal bacteria (Zeisel SH, 1989). Hence, the formation of TMA is only considerable when large amounts of choline are applied. Nevertheless, the chronic feeding study in rats (Shivarpurkar N, 1986) did not reveal a significant increase in carcinomas or mortality compared to control when applying choline chloride over 72 weeks at doses of 1200 mg/kg bw/day, corresponding to 1040 mg/kg bw/day choline hydroxide. Hence, the possible carcinogenic effects arising from TMA can be neglected. This conclusion is in congruence with the findings by De La Huerga et al.: The authors found that most of the metabolites are excreted as total trimethylamines, i.e. TMA or TMA oxide, but not further reacted, in the urine of humans (De La Huerga J, 1953).
After absorption in the intestinal mucosa, choline is phosphorylated to phosphocholine, which is the primarily transported form of choline (bound to chylomicra, see 3.2). Phosphocholine, or phosphorylcholine, will be furthermore transformed to structural membrane phospholipids, thereunder lecithine is the predominant (> 50 %) form, or phosphatidylcholine and sphingomyelin, which will be integrated in cell membranes. Also, choline can be transformed to signaling phospholipids, such as sphingosylphosphorylcholine or platelet-activating factor (acetyl-glyceryl-ether-phosphorylcholine), which is a mediator of many leukocyte functions, including platelet aggregation and degranulation, inflammation, and anaphylaxis.
Only in the central nervous system, choline is required for the synthesis of acetylcholine, a neurotransmitter, but also membrane phospholipids are synthesized as in other cells.
In the kidney and nervous system, choline is oxidized during the choline oxidation pathway by the choline dehydrogenase involving FAD to betaine aldehyde and finally transformed by betain-aldehyde dehydrogenase to betaine by utilization of NAD+. Betaine itself can undergo further transformation / degradation steps: It is transformed by betaine-homocysteine S-methyltransferase involving homocysteine to dimethylglycine, is further dehydrogenated under utilization of FAD twice, once by dimethylglycine dehydrogenase to sarcosine and once by sarcosine dehydrogenase to glycine. As the last step, glycine will be degraded by the glycine cleavage system to ammonium (NH4+) and carbon dioxide (CO2). Especially excess choline is catabolized via the choline oxidation pathway. This process requires enzymes found in the cytosol and mitochondria. Many tissues express some of the enzymes of choline oxidation, but the liver is the primary organ where the whole complement of enzymes is abundantly expressed. The oxidation of choline is an energy-yielding process that is anapleurotic to the one-carbon pool. Since the majority of one-carbon metabolism, on a whole body basis, occurs in the liver, the localization of choline oxidation in this organ is consistent with choline being a major one-carbon donor (Garrow TA, 2007).
Although methionine not being a derivative of choline, it is included in this scheme because the labelled methyl group of choline could be donated to homocystine to produce methionine, and hence the labelling in the choline could appear in methionine (Flower RJ, 1972).
Due to its methyl-donating properties, choline is part of intracellular, partly overlapping or intersecting metabolism pathways.
The pathways of choline and one-carbon metabolism intersect at the formation of methionine from homocysteine, as already mentioned above, predominantly present in the liver. Methionine is regenerated from homocysteine in a reaction catalysed by betaine-homocysteine methyltransferase, in which betaine, serves as a methyl donor. Hence, to be a source of methyl groups, choline must be converted to betaine, which has been shown to perform methylation functions as well as choline in some cases (McDowell LR, 2000).
Also, choline can be synthesized by the body itself, except in neuronal cells. Nevertheless, under normal circumstances, free choline is mainly gained from phosphatidylcholine, which is the predominant source of dietary choline and the main storage form in the body (Garrow TA, 2007). It can be transformed to glycerol phosphoryl choline by phospholipase B, which can be cleaved to choline under elimination of glycerol phosphate in peripheral tissues by glycerylphosphorylcholine diesterase, as already stated above. In tissues in general, phospholipase C cleaves phosphatidylcholine to yield a diglyceride and phosphorylcholine and the latter is converted to free choline by alkaline phosphatase. The brain also contains phospholipase D, which cleaves free choline directly from the circulating form, i.e. phosphatidylcholine bound to chylomicra (Combs GF, 2008).
In summary, it can be stated that choline is not only extensively absorbed and metabolized to essential molecules such as neurotransmitters or cell membrane compounds when applied, it is also synthesized by the body itself. It is essential to maintain normal body functions and a choline deficiency is even attributed to a large number of adverse effects, such as spontaneous carcinoma of the liver and generally increased sensitivity to carcinogens, or a ‘‘nonalcoholic fatty liver,’’ associated with accumulation of hepatic lipids and an increased sensitivity to inflammation (Michel V, 2006).

Any other information on results incl. tables

see attached expert statement

Applicant's summary and conclusion

Conclusions:

Choline hydroxide (unmetabolised) has no potential for bioaccumulation, but is incorporated in the body in form of essential molecules / metabolites, such as neurotransmitters (acetylcholine) or structural membrane phospholipids.
The present expert statement covers all relevant toxicokinetic parameters to assess the behaviour of choline hydroxide in the body, the available information is sufficient to enable one to perform a proper risk assessment. Hence, no further information needs to be gathered and further studies can be omitted due to animal welfare. In conclusion, choline has no potential for bioaccumulation in its non-metabolized form, and the incorporation of its metabolites does not bear any potential for adverse effects but is also required to maintain the normal functionality of the body. In case of an accidental high exposure, effective metabolism and clearance mechanisms take hold.

Executive summary:

Choline hydroxide was evaluated regarding its toxicokinetic behaviour, taking into account the available literature, toxicological and physico-chemical data.

Choline hydroxide is an important nutrient as a source of labile methyl groups, a precursor for several essential molecules for the body such as cell membrane components or neurotransmitter, and already present in diet and feed. Relevant here is the organic cation, choline.

The choline cation is poorly absorbed via the skin, however, the corrosive properties of the substance due to the hydroxide cation enhances dermal penetration, and a similar resorption compared to the oral route, i.e. estimated 50 %, can be assumed. Absorption via the respiratory epithelium, i.e. inhalative exposure is not relevant due to the limited generation of inhalable forms.

Choline is well absorbed after oral application from the jejunum and ileum mainly by a saturable, energy-dependent carrier mechanism in the brush-border membrane, to a lesser extent via passive diffusion. A similar mechanism applies for the membranes in many other tissues and enables choline to cross also the blood-brain barrier.

After absorption, choline salts can be easily distributed throughout the body via the lymphatic or portal circulation in the form of phosphatidylcholine bound to chylomicra. This form will be cleaved on-site by various, tissue-related enzymes, and choline can be absorbed. In many tissues, transport is mediated by low-affinity or intermediate-affinity sodium-independent transporters, whereas a high-affinity transport on the other hand is unique to the cholinergic neurons. All ingested choline enters the hepatic circulation, turning the liver into a significant first point of contact for the excessive metabolism for choline. Second, renal tubular transport of choline maintains the plasma choline concentration within relatively narrow limits by employing both net secretion and reabsorption. When choline is presented to the kidney in excess of a species-specific threshold concentration, it will be excreted via the kidneys into the urine. If the plasma concentration is below this level, choline will be reabsorbed by the kidneys and not excreted. In general, most of the choline in the body is found in the form of the phospholipids phosphatidylcholine (lecithine), lysophosphatidylcholine, choline plasmalogens, and sphingomyelin-essential components of all membranes.

This is due to an extensive metabolism of choline: Under normal circumstances, the amount of free choline is only 0.5 - 1 % per cent of the total tissue choline; excess choline is catabolized via the choline oxidation pathway. Other metabolites are phosphocholine, which will be furthermore transformed to structural membrane phospholipids, signalling phospholipids, such as sphingosylphosphorylcholine or platelet-activating factor, betaine, which can be transformed finally to NH4+ and CO2. Choline is part of intracellular, partly overlapping or intersecting metabolism pathways due to its methyl-donating properties and can be furthermore synthesized by the body itself or gained from phosphatidylcholine present in the diet.

So, choline is not only extensively absorbed after oral exposure and metabolized to essential molecules such as neurotransmitters or cell membrane compounds when applied, it is also synthesized by the body itself, and it is essential to maintain normal body functions.

Hence, when exposed to choline in the required ranges, nearly no choline will be excreted, When it is ingested, e.g. accidentally, in higher amounts, choline will be excreted mainly via the kidneys within approx. 12 hours, either as TMA after prior metabolism by the intestinal microflora, or as choline itself, or as betaine and other metabolites.

So in summary, choline has no potential for bioaccumulation in its non-metabolized form, and the incorporation of its metabolites does not bear a potential for adverse effects. Choline is also required to maintain the normal functionality of the body. In case of an accidental high exposure, effective metabolism and clearance mechanisms take hold.