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Toxicological information

Dermal absorption

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Administrative data

dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference Type:
The passage of trimethylamine across rat and human skin
S. Kenyon, P.L. Carmichael, S. Khalaque, S. Panchal, R. Waring, R. Harris, R.L. Smith, S.C. Mitchell
Bibliographic source:
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.

Materials and methods

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

Test material

Reference substance name:
Reference substance 001
Cas Number:
Molecular formula:
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%

Test animals

not specified
Details on test animals or test system and environmental conditions:
Normal human abdominal skin sections obtained following surgical procedures

Administration / exposure

Type of coverage:
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.

Results and discussion

Any other information on results incl. tables

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.



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.

Applicant's summary and conclusion