Trimethylamine, N-oxide

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Passage across rat and human skin has been investigated employing excised skin circles in an in vitro diffusion cell apparatus

GLP compliance:

not specified

Specific details on test material used for the study:

CAS 62637-93-8 is the hydrated form of the notified substance Trimethylamine N-oxide (CAS 1184-78-7).
Radio labelled [U-14C] trimethylamine N-oxide dihydrate was prepared by the oxidation of [U-14C]-trimethylamine with hydrogen peroxide in aqueous alkali.
sp. act. 11.6 mCi/mol, radiochemical purity 99%

Radiolabelling:

yes

Species:

human

Sex:

not specified

Details on test animals or test system and environmental conditions:

Normal human abdominal skin sections obtained following surgical procedures

Type of coverage:

occlusive

Vehicle:

water

Doses:

concentration of the [U-14C]-compound in the receptor fluid was 1 mg/ml (as anhydrous trimethylamine N-oxide)

Details on in vitro test system (if applicable):

Normal human abdominal skin sections obtained following surgical procedures were provided by TNO Pharma and stored frozen flat at -20 °C until required.

Skin Preparation
Prior to experimentation, a small section of frozen skin was removed and allowed to defrost with the dermal side in contact with a saline solution (0.9% w/v). The skin was then placed epidermal side down on a plastic dissecting board where excess subcutaneous material and the thickness of the remaining skin samples measured using a micrometer gauge (human 0.76 ± 0.09 mm). Full thickness skin circles (1.7 cm diameter) were punched out using a circular steel cutter and placed in the flow through diffusion cells for 30 min to equilibrate. The integrity of the skin was assessed qualitatively by visual inspection and the use of a hand lens. Skin viability was demonstrated by the detection of peroxidase activity (6–150 lunits/mg protein, relative to lactoperoxidase) in homogenised skin samples (phosphate buffer 0.1 M pH7.0, containing protease inhibitors) employing hydrogen peroxide as substrate and 1,2-benzenediamine as the reaction indicator. However, the presence of peroxidase activity does not imply that enzymes that may metabolise trimethylamine are functioning.

In vitro flow-through diffusion cell system
The apparatus consisted of seven Teflon flow-through diffusion cells contained within a heated jacket held at 32 °C by a thermostatically controlled water circulator. Receptor fluid was pumped through the cells using a peristaltic multichannel cassette pump and known timed aliquots collected in a fraction collector. The receptor volume of each diffusion cell was 130 µl. Skin sections were placed into the donor chamber of the diffusion cells and secured using a threaded nut leaving an exposed area of skin measuring 0.32 cm². For all experiments occlusion of the skin was achieved by placing Teflon caps onto the threaded retaining nut positioned 2.9 cm above the skin surface (the air volume between skin and cap was 2.8 cm³).

Investigation of the transfer of trimethylamine and trimethylamine N-oxide from the dermal to epidermal surface
Aqueous solutions of [U-14C] trimethylamine N-oxide dihydrate were prepared and aliquots (20 µl) were applied via their addition to the receptor fluid thereby exposing the dermal side of the tissue. The receptor fluid flowing beneath the skin samples was a HEPES-buffered Hanks’ balanced salt solution (HEPES 25 mM, Hanks’ balanced salts 9.8 g/l, sodium bicarbonate 4 mM, gentamicin 50 mg/l) adjusted to pH 7.3. The initial concentration of the [U-14C]-compound in the receptor fluid was 1 mg/ml (as trimethylamine free base or anhydrous trimethylamine N-oxide). This radioactive receptor fluid (50 ml) was recirculated beneath the skin through the receptor chamber at a flow rate of 4 ml/h during the entire time-course of the experiment.

Any radioactivity that may have traversed the skin was removed from the epidermal surface by wiping with cotton wool swabs (three humidified with ethanol and one dry). The radioactivity in these swabs was quantified. In between these seven collection time-points (30 min, 1–6 h) the skin layer was occluded in an attempt to reduce any potential volatilisation of material.

Measurement of radioactivity
Triplicate aliquots (0.5 ml) of the various receptor fluids were added directly to scintillation cocktail, vortex mixed, and counted. All cotton wool swabs were left to extract directly into scintillation fluid (5 ml) for 48 h before counting. The skin circle itself was removed and digested overnight in ethanolic potassium hydroxide (1 ml; 1.5 M KOH in ethanol/water, 4:1 v/v) with triplicate aliquots (200 µl) of this digest being neutralised with glacial acetic acid, scintillation fluid added (15 ml), and the contents vortex mixed before counting. The threaded nut and cap (donor chamber) were removed and washed with ethanol (2 ml) and the skin cell (receptor chamber) was also washed in ethanol (5 ml).
Radioactivity within samples (receptor fluid, ethanol washes, digests, extracts) was quantified in vials containing scintillation cocktail by liquid scintillation spectrometry. Standard curves using skin digests and various quantities of receptor fluid, ethanol and scintillation fluid were employed to determine the degree of quench correction, with external standards being used for reference.

Chromatography and mass spectrometry
In some parallel studies during the first 6 h, certain acidified receptor fluid collections reduced in volume by lyophilization and also cotton wool swabs from the epidermal surface extracted into minimum quantities of ethanol (≈ 0.5–2 ml), were examined by chromatography. Thin-layer chromatography was performed on cellulose (0.1 mm thick) plates (20 x 20 cm, aluminium- backed) and developed in butan-1-ol, formic acid (90% v/v aq.), water (77;10;13 by vol.). The plates were then scanned for radioactivity. The Rf values obtained in this system were dimethylamine 0.38, trimethylamine, 0.42, trimethylamine N -oxide 0.51. Any radioactive areas of interest were eluted with ethanol and examined by mass spectrometry. High pressure liquid chromatography was achieved using a modified partition column connected to a Waters system and detected using a Waters 490 multiwavelength detector (214 nm) and a Ramona Raytest radioactive flow monitor incorporating a solid scintillation radio-detector. The mobile phase was 0.01 M aqueous sodium hydroxide with a flow rate of 1 ml/min. The limits of UV detection of dimethylamine, trimethylamine and trimethylamine N -oxide in this system were 0.25, 0.05 and 4.5 µg, respectively. Use of the radioactivity detector improved this sensitivity by an order of magnitude reaching down to 4 ng for amines derived from [14C]- trimethylamine (sp. act. 1.7Ci/mol) and to 750 ng for materials derived from [14C]-trimethylamine N -oxide (sp. act. 11.6 mCi/mol). Again, any peaks of interest were collected, acidified with HCl, reduced in volume and extracted with minimum quantities of ethanol for mass spectral examination. Electron impact mass spectrometry was undertaken on a Kratos MS80 Instrument with a source temperature of 200 °C using a direct insertion probe running at 70 eV.

Statistics and derived parameters
All data were presented as mean ± s.d. Where appropriate the data were compared using Student’s t-test (Gossett) for unpaired data. Differences between groups were considered statistically significant when p < 0:05. The flux constant, which was a measure of the steepest gradient on the cumulative excretion curves (maximum rate), was calculated from the data via linear regression and the permeability constant (Kp) was derived from the flux constant and the applied concentration.

Transfer  of  trimethylamine  and  trimethylamine N-oxide from the dermal to epidermal surface

Radioactivity from trimethylamine and its N-oxide were detected on the epidermal surface indicating that these compounds had passed through the skin from the underlying circulating medium to which they had been added. The N-oxide traversed the skin more readily than trimethylamine, with more of the N-oxide accumulating over the 6 h period (30.8 ± 12.1 µg/cm²/h) compared to that for the trimethylamine (1.2 ± 0.6 µg/cm²/h) experiments.

 

The actual shapes of the cumulative absorption graphs obtained were also interesting, with that for trimethylamine remaining linear throughout whereas that for the N-oxide showing an initial rapid transfer of material within the first hour or two and thereafter decreasing hour by hour until it approximated a linear trend (similar to trimethylamine) from 3 h onwards.

 

When the skin circles were examined at the end of the experiment, similar amounts of both materials were found within human tissues, with similar levels of trimethylamine-related radioactivity being found for the rat. However, considerably more radioactivity (≈100- fold) was detected following exposure to [14C]-trimethylamine N-oxide when compared to [14C]-trimethylamine (p < 0:001, Student’s t-test) suggesting a greater capacity within rat skin for the N-oxide.

 

Metabolism

Following the presentation of [14C]-trimethylamine to the dermal surface of both rat and human skin no evidence for demethylation to dimethylamine was obtained but a few percent (≈2%) of the radioactivity was identified as the N-oxide. This was confirmed by mass spectral examination. No N-oxide was found in the receptor fluid suggesting no chemical oxidation of the trimethylamine. Contrastingly, chromatography showed that the entire radioactivity measured following the [14C]-trimethyl- amine N-oxide experiments remained in the chemical form of the N-oxide with no reduction to trimethylamine or conversion (via formaldehyde loss) to dimethylamine.

 

Slight, but detectable, oxidation of trimethylamine to its N-oxide was observed during its passage across the skin. Flavin-monooxygenase activity, the enzyme that has been shown to undertake this N-oxidation reaction, has been detected in animal skin and mRNA coding for these enzymes has been measured in human skin and keratinocytes. The nature of the enzymes that are able to produce dimethylamine by demethylation of trimethylamine or via the removal of formaldehyde from trimethylamine N-oxide are more speculative, but may involve the cytochromes P450. Metabolism studies employing previously frozen skin samples are suspect owing to potential damage to the skin in the freezing and thawing process. Such damage could explain the relative lack of metabolism after epidermal application and the small amount of metabolites formed following exposure of the dermis. However, the major purpose of this part of the study was not to measure skin metabolism but to identify the chemical nature of the radioactive material that had passed across the skin. Any quantitative skin metabolism studies should be undertaken using fresh tissues.

Reference 2

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Passage across rat and human skin has been investigated employing excised skin circles in an in vitro diffusion cell apparatus

GLP compliance:

not specified

Specific details on test material used for the study:

CAS 62637-93-8 is the hydrated form of the notified substance Trimethylamine N-oxide (CAS 1184-78-7).
Radio labelled [U-14C] trimethylamine N-oxide dihydrate was prepared by the oxidation of [U-14C]-trimethylamine with hydrogen peroxide in aqueous alkali.
sp. act. 11.6 mCi/mol, radiochemical purity 99%

Radiolabelling:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

Male Sprague–Dawley rats (Harlan, Oxford Laboratory Animal Centre, Bicester, Oxfordshire, UK), weighing approximately 250 g

Type of coverage:

occlusive

Vehicle:

water

Doses:

concentration of the [U-14C]-compound in the receptor fluid was 1 mg/ml (as anhydrous trimethylamine N-oxide)

Details on in vitro test system (if applicable):

Male Sprague–Dawley rats weighing approximately 250 g, were sacrificed by carbon dioxide inhalation, the dorsal region was shaved with animal clippers and the skin dissected and frozen flat at -20 °C until required.

Skin Preparation
Prior to experimentation, a small section of frozen skin was removed and allowed to defrost with the dermal side in contact with a saline solution (0.9% w/v). The skin was then placed epidermal side down on a plastic dissecting board where excess subcutaneous material and the thickness of the remaining skin samples measured using a micrometer gauge (rat skin 0.90 ± 0.15 mm). Full thickness skin circles (1.7 cm diameter) were punched out using a circular steel cutter and placed in the flow through diffusion cells for 30 min to equilibrate. The integrity of the skin was assessed qualitatively by visual inspection and the use of a hand lens. Skin viability was demonstrated by the detection of peroxidase activity (6–150 lunits/mg protein, relative to lactoperoxidase) in homogenised skin samples (phosphate buffer 0.1 M pH7.0, containing protease inhibitors) employing hydrogen peroxide as substrate and 1,2-benzenediamine as the reaction indicator. However, the presence of peroxidase activity does not imply that enzymes that may metabolise trimethylamine are functioning.

In vitro flow-through diffusion cell system
The apparatus consisted of seven Teflon flow-through diffusion cells contained within a heated jacket held at 32 °C by a thermostatically controlled water circulator. Receptor fluid was pumped through the cells using a peristaltic multichannel cassette pump and known timed aliquots collected in a fraction collector. The receptor volume of each diffusion cell was 130 µl. Skin sections were placed into the donor chamber of the diffusion cells and secured using a threaded nut leaving an exposed area of skin measuring 0.32 cm². For all experiments occlusion of the skin was achieved by placing Teflon caps onto the threaded retaining nut positioned 2.9 cm above the skin surface (the air volume between skin and cap was 2.8 cm³).

Investigation of the transfer of trimethylamine and trimethylamine N-oxide from the dermal to epidermal surface
Aqueous solutions of [U-14C] trimethylamine N-oxide dihydrate were prepared and aliquots (20 µl) were applied via their addition to the receptor fluid thereby exposing the dermal side of the tissue. The receptor fluid flowing beneath the skin samples was a HEPES-buffered Hanks’ balanced salt solution (HEPES 25 mM, Hanks’ balanced salts 9.8 g/l, sodium bicarbonate 4 mM, gentamicin 50 mg/l) adjusted to pH 7.3. The initial concentration of the [U-14C]-compound in the receptor fluid was 1 mg/ml (as trimethylamine free base or anhydrous trimethylamine N-oxide). This radioactive receptor fluid (50 ml) was recirculated beneath the skin through the receptor chamber at a flow rate of 4 ml/h during the entire time-course of the experiment.

Any radioactivity that may have traversed the skin was removed from the epidermal surface by wiping with cotton wool swabs (three humidified with ethanol and one dry). The radioactivity in these swabs was quantified. In between these seven collection time-points (30 min, 1–6 h) the skin layer was occluded in an attempt to reduce any potential volatilisation of material.

Measurement of radioactivity
Triplicate aliquots (0.5 ml) of the various receptor fluids were added directly to scintillation cocktail, vortex mixed, and counted. All cotton wool swabs were left to extract directly into scintillation fluid (5 ml) for 48 h before counting. The skin circle itself was removed and digested overnight in ethanolic potassium hydroxide (1 ml; 1.5 M KOH in ethanol/water, 4:1 v/v) with triplicate aliquots (200 µl) of this digest being neutralised with glacial acetic acid, scintillation fluid added (15 ml), and the contents vortex mixed before counting. The threaded nut and cap (donor chamber) were removed and washed with ethanol (2 ml) and the skin cell (receptor chamber) was also washed in ethanol (5 ml).
Radioactivity within samples (receptor fluid, ethanol washes, digests, extracts) was quantified in vials containing scintillation cocktail by liquid scintillation spectrometry. Standard curves using skin digests and various quantities of receptor fluid, ethanol and scintillation fluid were employed to determine the degree of quench correction, with external standards being used for reference.

Chromatography and mass spectrometry
In some parallel studies during the first 6 h, certain acidified receptor fluid collections reduced in volume by lyophilization and also cotton wool swabs from the epidermal surface extracted into minimum quantities of ethanol (≈ 0.5–2 ml), were examined by chromatography. Thin-layer chromatography was performed on cellulose (0.1 mm thick) plates (20 x 20 cm, aluminium- backed) and developed in butan-1-ol, formic acid (90% v/v aq.), water (77;10;13 by vol.). The plates were then scanned for radioactivity. The Rf values obtained in this system were dimethylamine 0.38, trimethylamine, 0.42, trimethylamine N -oxide 0.51. Any radioactive areas of interest were eluted with ethanol and examined by mass spectrometry. High pressure liquid chromatography was achieved using a modified partition column connected to a Waters system and detected using a Waters 490 multiwavelength detector (214 nm) and a Ramona Raytest radioactive flow monitor incorporating a solid scintillation radio-detector. The mobile phase was 0.01 M aqueous sodium hydroxide with a flow rate of 1 ml/min. The limits of UV detection of dimethylamine, trimethylamine and trimethylamine N -oxide in this system were 0.25, 0.05 and 4.5 µg, respectively. Use of the radioactivity detector improved this sensitivity by an order of magnitude reaching down to 4 ng for amines derived from [14C]- trimethylamine (sp. act. 1.7Ci/mol) and to 750 ng for materials derived from [14C]-trimethylamine N -oxide (sp. act. 11.6 mCi/mol). Again, any peaks of interest were collected, acidified with HCl, reduced in volume and extracted with minimum quantities of ethanol for mass spectral examination. Electron impact mass spectrometry was undertaken on a Kratos MS80 Instrument with a source temperature of 200 °C using a direct insertion probe running at 70 eV.

Statistics and derived parameters
All data were presented as mean ± s.d. Where appropriate the data were compared using Student’s t-test (Gossett) for unpaired data. Differences between groups were considered statistically significant when p < 0:05. The flux constant, which was a measure of the steepest gradient on the cumulative excretion curves (maximum rate), was calculated from the data via linear regression and the permeability constant (Kp) was derived from the flux constant and the applied concentration.

Transfer  of  trimethylamine  and  trimethylamine N-oxide from the dermal to epidermal surface

Radioactivity from trimethylamine and its N-oxide were detected on the epidermal surface indicating that these compounds had passed through the skin from the underlying circulating medium to which they had been added. The N-oxide traversed the skin more readily than trimethylamine, with more of the N-oxide accumulating over the 6 h period (12.4 ± 10.4 µg/cm²/h) compared to that for the trimethylamine (2.3 ± 0.9 µg/cm²/h) experiments.

 

The actual shapes of the cumulative absorption graphs obtained were also interesting, with that for trimethylamine remaining linear throughout whereas that for the N-oxide showing an initial rapid transfer of material within the first hour or two and thereafter decreasing hour by hour until it approximated a linear trend (similar to trimethylamine) from 3 h onwards.

 

When the skin circles were examined at the end of the experiment, similar amounts of both materials were found within human tissues, with similar levels of trimethylamine-related radioactivity being found for the rat. However, considerably more radioactivity (≈100- fold) was detected following exposure to [14C]-trimethylamine N-oxide when compared to [14C]-trimethylamine (p < 0:001, Student’s t-test) suggesting a greater capacity within rat skin for the N-oxide.

 

Metabolism

Following the presentation of [14C]-trimethylamine to the dermal surface of both rat and human skin no evidence for demethylation to dimethylamine was obtained but a few percent (≈2%) of the radioactivity was identified as the N-oxide. This was confirmed by mass spectral examination. No N-oxide was found in the receptor fluid suggesting no chemical oxidation of the trimethylamine. Contrastingly, chromatography showed that the entire radioactivity measured following the [14C]-trimethyl- amine N-oxide experiments remained in the chemical form of the N-oxide with no reduction to trimethylamine or conversion (via formaldehyde loss) to dimethylamine.

 

Slight, but detectable, oxidation of trimethylamine to its N-oxide was observed during its passage across the skin. Flavin-monooxygenase activity, the enzyme that has been shown to undertake this N-oxidation reaction, has been detected in animal skin and mRNA coding for these enzymes has been measured in human skin and keratinocytes. The nature of the enzymes that are able to produce dimethylamine by demethylation of trimethylamine or via the removal of formaldehyde from trimethylamine N-oxide are more speculative, but may involve the cytochromes P450. Metabolism studies employing previously frozen skin samples are suspect owing to potential damage to the skin in the freezing and thawing process. Such damage could explain the relative lack of metabolism after epidermal application and the small amount of metabolites formed following exposure of the dermis. However, the major purpose of this part of the study was not to measure skin metabolism but to identify the chemical nature of the radioactive material that had passed across the skin. Any quantitative skin metabolism studies should be undertaken using fresh tissues.

Description of key information

Based on considerations of the physicochemical properties and the pathological findings following acute exposure, TMAO would be expected to be absorbed following oral, and dermal. Dermal absorption is further supported by the in vitro work of Kenyon et al (2004). Conservative absorption rates for this substance are set at 100% for oral and dermal routes of exposure.

TMAO is a low volatile (23.5 Pa) non-inhalable powder (D10 161.713 µm), therefore the possibility of exposure to aerosols, particles or droplets of inhalable size is unlikely. Due to the limited information available a worst case approach is applied, and absorption via inhalation is assumed to be as equivalent to the oral route.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

100

Absorption rate - dermal (%):

100

Absorption rate - inhalation (%):

100

Additional information

In accordance with Annex VIII, Column 1, 8.8.1, of Regulation (EC) 1907/2006 and with ‘Guidance on information requirements and chemical safety assessment Chapter R.7c: Endpoint specific guidance’ (ECHA, 2017), an assessment of the toxicokinetic behaviour of the target substance TMAO (Trimethylamine N-oxide; CAS #62637-93-8) was conducted to the extent that can be derived from the relevant available information. This comprises a qualitative assessment of the available substance specific data on physico-chemical and toxicological properties according to the Chapter R.7c Guidance document (ECHA, 2017) and taking into account further available information from source substances. The substance TMAO is a monoconstituent.

 

Physico-chemical properties

TMAO has a molecular weight 75.11 g/mol. It is a solid at 20 °C with a melting point at 100°C, a boiling point at 158°C and a water solubility of 793 g/L at 20 °C. The log Pow was measured to be -2.79 and the vapour pressure 23.5 Pa at 20 °C.

 

Absorption and distribution

Absorption is a function of the potential for a substance to diffuse across biological membranes. The most useful parameters providing information on this potential are the molecular weight, the octanol/water partition coefficient (log Pow) value and the water solubility. The log Pow value provides information on the relative solubility of the substance in water and lipids (ECHA, 2017).

 

Oral

In general, molecular weights below 500 and log Pow values between -1 and 4 are favourable for absorption via the gastrointestinal (GI) tract, provided that the substance is sufficiently water soluble (> 1 mg/L). Lipophilic compounds can be taken up by micellar solubilisation by bile salts, but this mechanism may be of particular importance for highly lipophilic compounds (log Pow > 4), in particular for those that are poorly soluble in water (≤ 1 mg/L) as these would otherwise be poorly absorbed. Solids must be dissolved before absorption; the degree depends on the water solubility (ECHA, 2017).

 

Some of the physico-chemical characteristics (molecular weight and water solubility) of the substance are in a range that indicate a good absorption from the gastrointestinal (GI-) tract following oral ingestion, while the log Pow may limit the uptake. TMAO is a water-soluble substance and will readily dissolve into the gastrointestinal fluids. As a very hydrophilic substance, absorption by passive diffusion may be limited by the rate at which the substance partitions out of the gastrointestinal fluid. However, as the molecular weight is low (less than 200) the substance may pass through aqueous pores or be carried through the epithelial barrier by the bulk passage of water.

 

The OECD 422 study reports treatment-related effects following oral exposure. Administration of TMAO dihydrate by once daily oral gavage in Han Wistar rats was associated with transient and recoverable effects on body weight and/or food consumption in females only at 150 and 300 mg/kg bw/day. Based on a consideration of the substance’s physicochemical properties and the available oral toxicity studies, a conservative default value for oral absorption of the substance is set as 100%.

 

Dermal

The in vitro assessment of dermal adsorption with [U-14C] trimethylamine N-oxide dihydrate (Kenyon et al., 2004) demonstrated that while TMAO dihydrate it potentially interacts by hydrogen-bonding where appropriate and appear to travel readily amidst both liquid and aqueous environments. As a result, skin seems to present little restriction to its movement and a conservative default value for dermal absorption of the substance is set as 100%.

 

Inhalation

TMAO is a solid with low vapour pressure (23.5 Pa at 20 °C) and high boiling point (above 150°C), and therefore a low volatility. Therefore, under normal use and handling conditions, inhalation exposure and availability for respiratory absorption of the substance in the form of particles will depend on the aerodynamic particle size (ECHA, 2017). The substance may also be available for inhalation absorption after inhalation of aerosols, if the substance is sprayed (e.g. as a formulated product). In humans, particles with aerodynamic diameters below 100 μm have the potential to be inhaled. Particles with aerodynamic diameters below 50 μm may reach the thoracic region and those below 15 μm the alveolar region of the respiratory tract. Particles deposited in the nasopharyngeal/thoracic region will mainly be cleared from the airways by the mucocilliary mechanism and swallowed. According to the data available on TMAO, the MMAD is at 414 µm, with D10 = 161 µm, D50 = 404 µm and D90 = 859 µm, indicating the TMAO as dry solid are mainly not inhalable/respirable particles.

 

The Log Pow is not particularly favorable to respiratory absorption (value < -1). However as TMAO is very hydrophilic, vapours may be retained within the mucus.

 

Due to the limited information available a worst case approach is applied, and absorption via inhalation is assumed to be as equivalent to the oral route; 100%.

 

Distribution and Accumulation

Distribution of a compound within the body depends on the physico-chemical properties of the substance; particularly the molecular weight, the lipophilic character and the water solubility. In general, the smaller the molecule, the wider the distribution. If the molecule is lipophilic, it is likely to distribute into cells and the intracellular concentration may be higher than extracellular concentration, particularly in fatty tissues (ECHA, 2017).

 

TMAO is a small water-soluble molecule and ions will diffuse through aqueous channels and pores. However it is a very hydrophilic molecule and its rate of diffusion across membranes could limit its distribution. No specific organ toxicity was observed in the OECD 422 animal studies with TMAO dihydrate.

 

No accumulation is expected due to the high water solubility and low Log Pow.

 

Metabolism/Excretion

Limited studies are available on radiolabelled TMAO.  The work of Al-Waiz, et al (1987a) shows that 25% of the radioactivity after administration of 14C-TMAO appears in the urine unmetabolized within three hours, too short a time for the TMAO to have reached the microbial contents of the large bowel for bacterial reduction to TMA to occur. Some retroverted metabolism to unoxidized TMA does occur. From studies with normal, healthy volunteers, results have shown that single oral doses of TMAO are readily absorbed and excreted in the urine in an apparently unchanged form (Al-Waiz, et al., 1987b).  After oral TMAO administration in four healthy subjects, >94% of the urinary TMA was in the form of TMAO and only <4% as the free base.

 

According to the data available TMAO is mainly excreted as such, with a potential for some retroverted metabolism to TMA by gut bacteria.

 

 

References:

Kenyon S, Carmichael PL, Khalaque S, Panchal S, Waring R, Harris R, et al. (2004). The passage of trimethylamine across rat and human skin. Food Chem Toxicol 42(10):1619-1628.

 

Al-Waiz, M., Mitchell, S.C., Idle, J.R. and Smith, R.L., 1987a. The metabolism of 14C-labelled trimethylamine and its N-oxide in man. Xenobiotica, 17(5), pp.551-558.

 

Al‐Waiz, M., Ayesh, R., Mitchell, S.C., Idle, J.R. and Smith, R.L., 1987b. Disclosure of the metabolic retroversion of trimethylamine N‐oxide in humans: A pharmacogenetic approach. Clinical Pharmacology & Therapeutics, 42(6), pp.608-612.