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Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: acceptable, well-documented publication, which meets basic scientific principles
Objective of study:
distribution
excretion
metabolism
Principles of method if other than guideline:
Determination of tissue distribution, urinary excretion, possible other excretion path ways after intravenous or intraperitoneal administration of TMA to rats.
GLP compliance:
not specified
Radiolabelling:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
no data
Route of administration:
other: ip or iv
Vehicle:
other: solved in physiological saline, passing it through a 0.22 µm syringe filter, infusate volumen = 1 mL
Duration and frequency of treatment / exposure:
one injection to every rat in a group
Dose / conc.:
1 000 other: µmol bolus i.v. or i.p.
Remarks:
1000 µmol bolus i.v. or i.p.
Dose / conc.:
100 other: µmol bolus i.v. or i.p.
Remarks:
100 µmol bolus i.v. or i.p.
Dose / conc.:
5 other: µmol bolus i.v. or i.p.
Remarks:
< 5 µmol bolus i.v. or i.p.
No. of animals per sex per dose / concentration:
5
Control animals:
yes
Positive control reference chemical:
no data
Details on study design:
Routes of elimination: 5 male rats
Endogenous and bacterial synthesis: 5 male rats
Pharmacokinetics: 350-g male Sprague-Dawley rats (5 ?)
Details on dosing and sampling:
Blood samples of 150 to 350 µL were taken at 15 min, 30 min, and 1, 2,4, and 8 hr following the dose.
The blood concentrations obtained for iv or ip doses were indistinguishable, and the results therefore have been pooled.
Urine samples (24 h collections)
faeces samples
Monitoring of the exhalted air
To determine partitioning between blood and tissues, samples of blood, lung, heart, liver, kidney, intestine, and abdominal muscle were taken for analysis
Statistics:
using one-compartment model with first-order removal
mass balance equation for the one-compartment model
a plot of I^CB/JD) vs f has an intercept equal to ln( ) and a slope of -k/Vjy, allowing VD and k to be computed from linear regression.
slopes and intercepts for such plots determined by weighted least squares (weighting factor = inverse of the variance of each concentration).
Joint confidence intervals for VD and k (boundary of the constant F distribution).
Preliminary studies:
no data
Details on absorption:
When bolus doses of [14C]TMA (applied as the hydrochloride) were given ip, recovery of radioactivity in the urine was essentially complete, and respiratory excretion, faecal excretion, and accumulation in tissues of these amines or their metabolites were negligible. Varying amounts of TMA were oxidized to TMAO, the fraction oxidized decreasing at higher doses of TMA (Table 1).

TMA is absorbed from the diet (probably almost entirely in the small intestine) and synthesized by bacteria in the lower gut.
Details on distribution in tissues:
Blood concentrations of TMA were measured in rats for 8 hr following 5, 100, or 1000 µmol bolus i.v. or ip doses of radioisotopes or stable isotopes. At any given dose of TMA, the decay in blood concentration was approximately monoexponential. Values of VD greatly exceeded the size of the animals, suggesting that TMA is highly concentrated at one or more locations in the body. This was confirmed by measurements in tissue homogenates sampled 1 hr after a dose.

The tissue concentrations tended to exceed those in blood, sometimes by large factors. The tissue-to-blood ratios for DMA were typically ~2, with a value of 8 in kidney. The ratios for TMA tended to be lower than those for DMA, although still usually exceeding unity.
Details on excretion:
For TMA the entire dose as recovered in the 24 hr urine. For both DMA and TMA, — 1% of the dose was recovered in exhaled air as 14CO2, while a negligible amount was exhaled as the amine. These results demonstrate that both fecal and respiratory excretion of methylamines in normal rats are negligible. Retention of either the original methylamines or any carbon-based metabolites in tissues is also negligible.
Recovery of the DMA or TMA radiolabel was essentially complete at both dose levels.
Thus the fraction recovered as TMA increased with the TMA dose, as would be expected if TMA oxidation were saturable.

The results suggest that for a 100-µmol dose containing [14C]TMA (applied as the hydrochloride), ~20 % appears in rat urine as TMA, ~40 % as TMAO, and ~40 % as other (unidentified) compounds.

The finding of no difference in DMA excretion between oral and ip doses of TMA (Asatoor and Simenhoff, 1965) may be attributed to absorption of TMA in the upper G.I. tract, prior to its exposure to gut bacteria.
Metabolites identified:
yes
Details on metabolites:
Metabolites: 20 % appear in urine as TMA, mainly as (40 %) TMAO and 40 % as other metabolites. No apparent bacterial synthesis of TMA, actual bacterial synthesis might be masked by oxidation to TMAO and/or demethylation to DMA.

The simplest explanation for a large value of VD is the presence of high concentrations of the given substance in one or more tissues, relative to blood. High tissue concentrations may result from factors such as pH differences,1 solubility differences, or binding to proteins or other tissue constituents. If exchange of the substance between blood and tissues is so rapid that the tissue-to-blood concentration ratios remain very near their thermodynamic equilibrium values, then one-compartment kinetics will be strictly obeyed.

Because TMA followed one-compartment kinetics even at the earliest time point examined, there is no evidence of any need for a correction in VD for TMA.

The large volumes of distribution calculated for DMA and TMA, together with the direct evidence presented for their accumulation in selected tissues, strongly suggests that there are various regions outside the gastrointestinal contents which contain high concentrations of these methylamines.

An alternative explanation for the dose dependence of VD is active transport of DMA or TMA from extracellular to intracellular fluid.

Endogenous and Bacterial Synthesis: In both normal and germ-free rats, there was much more TMA ingested than excreted in the urine.

There was no apparent bacterial synthesis of TMA. Actual bacterial synthesis of TMA could have been masked by subsequent oxidation to TMAO and/or demethylation to DMA.

The concentration of endogenous TMA (25 ± 2 nmol/mL, n = 4) was higher than the peak concentration achieved following the 4 µmol dose, but still much lower than the peak value for the 100 -µmol dose. The time decay of the concentrations of labeled TMA in blood was qualitatively similar to that for DMA, except that there was no systematic deviation of the 15-min point from the best-fit straight line. Once again, a good approximation to one-compartment, linear behavior was exhibited over the relatively narrow range of concentrations achieved with any given dose, consistent with constancy of VD and k (see table 2)

TABLE 1 Radioisotope Balances for DMA and TMA in Normal Ratsa

 

DMA

TMA

Faeces

0.6 ± 0.1 (5)

0.8 ± 0.03 (5)

Blood

0.5 ± 0.2 (5)

0.04 ± 0.005 (5)

Lung

0.3 ± 0.04 (5)

< 0.001 (5)

Liver

2.3 ± 1.2 (5)

0.02 ± 0.004 (5)

Kidney

0.5 ± 0.05 (5)

0.003 ± 0.001 (5)

Urine

96 ± 2 (2)

96 ± 3 (2)

MA trapb

0.03 ± 0.01 (2)

0.05 ± 0.01 (2)

CO2

1 ± 0.2 (2)

0.8 ± 0.06 (2)

Totalc

101 ± 2

98 ± 3

aValues are percentages of dose recovered, expressed as means ± SE with the number of measurements (number of rats) in parentheses.
bMA, methylamine.
cError estimates for the totals were computed from the standard errors (SE) of the other entries using

TABLE 2 One-Compartment Pharmacokinetic Parameters for Dimethylamine and Trimethylaminea

Dose (µmol)

VD(mL)

k(ml/min)

 

Dimethylamine

 

2

2258 (1957-2440)

10.1 (9.2-10.7)

100

1041 (957-1112)

9.3 (8.8-9.6)

1000

375 (293-461)

4.1 (3.4-4.8)

 

Trimethylamineb

 

4 (TMA)

725 (463-1133)

4.0 (3.0-5.3)

100 (TMA)

344 (301-394)

3.5 (3.3-3.8)

1000 (TMA)

869 (791-955)

5.2 (4.8-5.5)

100 (TMAO)

172 (140-213)

2.0 (1.6-2.2)

aThe best-fit values are shown together with the 90% confidence intervals (in parentheses).
bThe doses were in the form of TMA or TMAO, as indicated.

Conclusions:
Interpretation of results: no bioaccumulation potential based on study results
TMA is mainly excreted as TMAO via the urinary tract and bears the potential to bioaccumulate in tissues.
Executive summary:

Preliminary it needs to be stated, that there are three sources of trimethylamine (TMA): diet, bacterial synthesis, and endogenous synthesis.

 

In 1994 Smith and Coworkers investigated the metabolism and excretion of methylamines in rats. They undertook these investigations to determine in detail the tissue distribution, urinary excretion, and possible other excretion path ways after administration of TMA-HCl to rats via injection (intravenous or intraperitoneal). The radiolabeled carbon in the trimethylamine-molecule permitted the detection of the mother compound and in addition the metabolites derived from TMA-HCl.

Blood samples were taken at 15 min, 30 min, and 1, 2,4, and 8 hr following the dose. And in conclusion the blood concentrations obtained for iv or ip doses were indistinguishable. Additionally urine samples (24 h collections), faeces samples and the exhaled air were investigated.

To determine partitioning between blood and tissues, samples of blood, lung, heart, liver, kidney, intestine, and abdominal muscle were also taken for analysis. When trimethylamine hydrochloride (TMA-HCl) is administered in vivo intravenously or intraperitoeally it is readily absorbed and uniformly distributed in the body, interestingly with the volume of distribution exceeding the animal size, suggesting that it is highly concentrated at one or more locations in the body (Smith, 1994).

The metabolism of TMA occurs mainly via oxidation to TMAO. About 40 % of the dose, administered to rats was excreted in the urine as TMAO, about 20 % were excreted as TMA and the remaining 40 % were other metabolites (Smith, 1994). The results indicate also a saturation of the metabolism of TMA (Smith, 1994), because with a raising dose of TMA the TMA fraction recovered in the urine raised too. Its elimination is primarily conducted via urine (96 % of the dose could be recovered in the urine after 24 hours) and only negligible excretion occurs via exhalation or via faeces (Smith, 1994). For both DMA and TMA, — 1% of the dose was recovered in exhaled air as 14CO2, while a negligible amount was exhaled as the amine. These results demonstrate that both faecal and respiratory excretion of methylamines in normal rats are negligible. Retention of either the original methylamines or any carbon-based metabolites in tissues is also negligible.

These findings indicate that the only important pathway for elimination of these methylamines (and their metabolites) from the body is urinary excretion.

Additionally there was no apparent bacterial synthesis of TMA, but actual bacterial synthesis might have been masked by oxidation to TMAO and/or demethylation to DMA.

As TMA can also undergo reaction to NDMA (N-nitrosodimethylamine), which is a potent carcinogen, this topic needs to be discussed here as well. The results from the study do not show that this pathway is a major one, so the risk is rather small. But because it cannot be stated that this does not occur at all, this possibility needs to be taken into account when judging the carcinogenic potential of TMA.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: acceptable well documented publication, which meets basic scientific principles
Objective of study:
metabolism
Principles of method if other than guideline:
Administration of TMA-HCL to rats (orally or intraperitoneally) and analytical verification of DMA in Urine, to test whether TMA-HCl can be transformed in DMA and excreted that way.
GLP compliance:
no
Radiolabelling:
no
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals or test system and environmental conditions:
Male albino rats (Wistar, 200-250 g), fed on Oxoid rat diet (Oxoid Ltd., Great Britain). The rats were placed in metabolic cages for 24 hours and basal urine was collected. After an interval of 2 days, the particular substance to be investigated was adiministered sand 24 h urine was collected again.
Route of administration:
intraperitoneal
Vehicle:
physiological saline
Duration and frequency of treatment / exposure:
50 mg trimethylamine hydrochloride (intraperitoneal)
100 mg trimethylamine hydrochloride (oral)
Dose / conc.:
50 other: mg (intraperitoneal injection)
Dose / conc.:
100 other: mg (oral administration)
No. of animals per sex per dose / concentration:
4 male Wistar rats
Control animals:
yes
Details on study design:
Trimethylamine as intermediate in the production of urinary dimethylamine Since trimethylamine is a normal constituent of urine and is derived from choline by the action of intestinal bacteria, it seemed likely to be an intermediate in the metabolism of choline to dimethylamine. To verify this, the effect of oral and intraperitoneal administration of trimethylamine hydrochloride was studied in two groups of rats. There was a marked excretion of dimethylamine in both groups, indicating that the secondary amine is produced by demethylation of trimethylamine.
Details on dosing and sampling:
The rats were placed in metabolic cages for 24 hours and basal urine was collected. After an interval of 2 days, the particular substance to be investigated was adiministered sand 24 h urine was collected again. A few dorps of 0.1 N HCl were placed in the containers used for urine collections. Urine specimens were stored in a deep-freeze.
Details on excretion:
Results of experiments with trimethylamine hydrochloride suggest that trimethylamine is an intermediate in the conversion of choline to dimethylamine.
Metabolites identified:
yes
Details on metabolites:
TMA is in part transformed to DMA and excreted via urine.
Conclusions:
Interpretation of results: no bioaccumulation potential based on study results
Results of experiments with trimethylamine suggest that trimethylamine is an intermediate in the conversion of choline to dimethylamine.
Executive summary:

The study performed by Asatoor et al, in 1965, already gives important data concerning DMA. They investigated whether DMA is generated in the mammalian body by feeding rats (in cases also administered via injection) with either choline chloride, lecithin, [14C]methylamine, or [Me14C]choline chloride (orally) or egg lecithin or trimethylamine hydrochloride (orally) or trimethylamine hydrochloride (intraperitoneal) and then analytical verification of DMA in Urine, to test whether these substances can be transformed in DMA and excreted that way. They could prove that DMA is a product of demethylation of trimethylamine. Thus, the conversion of choline to dimethylamine takes place in two stages; first, breakdown of choline to trimethylamine by intestinal bacteria and secondly, demethylation of the latter to dimethylamine. Furthermore TMA is in part metabolised to DMA, which is then excreted mainly via urine. Additionally MMA is partly methylated to form DMA. So these results are in accordance with the results obtained by Ischiwata et al. 1984.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: acceptable well-documented publication, which meets basic scientific principles
Objective of study:
metabolism
Principles of method if other than guideline:
This study was undertaken to investigate the contribution of demethylation to trimethylamine (applied as the hydrochloride) metabolism in man and its quantitative importance when compared to N-oxidation. The metabolism of orally administered trimethylamine (applied as the hydrochloride) has been studied in 4 male volunteers at 2 dose levels.
GLP compliance:
no
Radiolabelling:
no
Species:
human
Route of administration:
oral: capsule
Vehicle:
unchanged (no vehicle)
Duration and frequency of treatment / exposure:
one dose of 300 mg free base, then 1 week later to the same persons 600 mg free base
Dose / conc.:
300 other: mg free base
Remarks:
Doses / Concentrations: 300 mg free base
Dose / conc.:
600 other: mg free base
Remarks:
Doses / Concentrations: 600 mg free base
Control animals:
other: In all cases, control urine samples were collected from the volunteers during the day preceding the experiment.
Details on dosing and sampling:
urine collected the following 0--8 h into bottles containing hydrochloric acid (2 mL, 4 M).
Dimethylamine and trimethylamine were measured in the urine by headspace gas chromatography as previously described.
Trimethylamine N-oxide was measured as an increase in trimethylamine after reduction with acidified titanous sulphate.
Details on excretion:
Analysis of the control urines collected before the study revealed the presence of relatively small amounts of trimethylamine (10.4 +/- 2.3 µmol; mean +/- 1 S.D.), trimethylamine N-oxide (311.3 +/- 130.6 µmol) and dimethylamine (97.5 +/- 32.4 µmol), presumably arising from normal dietary sources and intermediary metabolism. After the oral dose of 300 mg trimethylamine there was a subsequent marked increase in unchanged compound (11.0-fold: P< 0.025; Student t-test) and trimethylamine N-oxide (10.2-fold; P < 0.001) content of the urine whereas dimethylamine levels increased insignificantly (1.3-fold; P < 0.25). Following the 600 mg dose there was again a large rise in the urinary output of trimethylamine (38.7-fold; P <: 0.001) and its N-oxide (23.9-fold; P < 0.001} whilst the value for the dimethylamine was only slightly raised (1.8-fold; P <: 0.1).
Metabolites identified:
yes
Details on metabolites:
N-Oxidation was the major route of metabolism whilst N-demethylation was negligible and only significant at the higher dose level.
Conclusions:
Interpretation of results: no bioaccumulation potential based on study results N-oxidation as the major route of metabolism in man
Executive summary:

In 1987, Al-Waiz et al. investigated the excretion of trimethylamine hydrochloride by 4 male human volunteers after oral ingestion of 2 dose levels (300 or 600 mg free base). At both dose levels over 95% of the administered trimethylamine was excreted in the N-oxide form, confirming N-oxidation as the major route of metabolism in man. In absolute terms, the amount of free trimethylamine detected after giving the larger dose increased nearly 40-fold from control values to give 404.2 + 61.6 µmol in the 0--8 h urine sample and imparted a fish-like odour to the urine. If demethylation was an important pathway for the degradation of trimethylamine in man then it would be expected that as the dose increased and more free trimethylamine becomes available, more dimethylamine would be produced. This clearly is not the case. Probably the majority of urinary dimethylamine is under normal circumstances of endogenous origin, presumably arising from the methylation of methylamine produced via intermediary metabolism. So the small part of ingested trimethylamine that is transformed to dimethylamine could be metabolized to nitrosamines. However, even small amounts of dimethylamine, if nitrosated, maybe sufficient to elicit a potentially carcinogenic response.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: acceptable well-documented publication, which meets basic scientific principles
Objective of study:
metabolism
Principles of method if other than guideline:
Oral administration of equimolar amounts (1 mmol/kg body weight) of potential amine precursors to male Wistar rats and investigation for their potential role as precursors of dimethylamine, using a previously validated rat model (Zhang et al., 1994b).
GLP compliance:
no
Radiolabelling:
no
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals or test system and environmental conditions:
Male Wistar rats (200 ± 250 g body weight) permitted to acclimatize in a controlled environment (24 °C, 12-hr light/dark cycle, 50 % humidity) with food (Labsure CRM pellets; K&K Greef Ltd, Croydon, UK) and water freely available throughout the experiment.
housed separately in metal metabolism cages (Radleys Ltd, Saffron Walden, UK)
Route of administration:
other: orogastric intubation
Vehicle:
water
Dose / conc.:
1 other: mmol/kg bw
No. of animals per sex per dose / concentration:
4 animals per group
Control animals:
other: a control 0 ± 24-hr urine collected into flasks containing hydrochloric acid (1 mL, 6 M) which was added to prevent microbial growth and maintain excreted dimethylamine as its water soluble hydrochloride salt.
Details on dosing and sampling:
Collection of the following 0 ± 24-hr urine.
All urine volumes were recorded and aliquots frozen (20 °C) until anlaysis.
Statistics:
Urinary excretion (0 ± 24-hr) of dimethylamine before and after administration of the compounds under investigation was compared statistically using Student's (Gossett) t-test.
Preliminary studies:
It has been accepted generally that dimethylamine arises from ingested choline via a bacterial degradation product and the intermediate trimethylamine with subsequent demethylation (Asatoor and Simenhof, 1965; Lowis et al., 1985; Zeisel et al., 1983).
Details on excretion:
Those compounds containing the monomethylamino function (methylamine, N-methylglycine) did not give rise to any significant increases in urinary dimethylamine output, suggesting that methylation of monomethylamines is not a major source of dimethylamine.
Of those compounds containing a trimethylamino function, the largest increase (+355 %) of dimethylamine was measured following trimethylamine N-oxide administration (12.9321.13 % dose).
Trimethylamine and choline only elicited slight increases in dimethylamine output (+51 %, 1.60 +/-0.80 % dose; +11 %, 0.60+/-0.36 % dose, respectively).
Rats excreted 1.4 mg diethylamine/kg/day in the urine. Administration of 1 mmol/kg bm (60 mg) increased the dimethylamine excretion to 2.1 mg/kg/day, that is 1.6 % of the trimethylamine dose. (for comparison: trimethylamine-N-oxide resulted in the excretion of 7.5 mg diethylamine /kg/day).
Metabolites identified:
yes
Details on metabolites:
MMA is not precursor of DMA. TMA is transformed in rather large quantities into DMA. Choline is a precursor of DMA.

The daily excretion of dimethylamine from rats with free access to food pellets was relatively constant.

Conclusions:
Interpretation of results: no bioaccumulation potential based on study results
Oral administration of 1 mmol/kg bw of potential amine precursors to male Wistar rats produced only small increases in urinary dimethylamine after choline (+11 %; 0.60 +/-0.36 % dose), dimethylaminopropanol (+32 %; 1.49 +/-0.30 % dose), dimethylaminoethyl chloride (+110 % 5.38 +/- 1.72 % dose) and trimethylamine (+51 %; 1.6 +/-0.80 % dose) input, whereas significantly larger increases were found following trimethylamine N-oxide ingestion (+355 %; 12.93 +/-1.13 % dose; t-test, P <0.001). These data suggest that trimethylamine N-oxide is a major dietary source of dimethylamine, by direct conversion and not by sequential reduction (to trimethylamine) and demethylation, and that in this respect it is of greater importance, on a molar basis, than choline.
Executive summary:

In 1998, Zhang et al. investigated the excretion of DMA after administration of various related amine precursors (among other trimethylamine applied as the hydrochloride). They found trimethylamine N-oxide (at a dose rate of 1 mmol/kg body weight) to be 20 times more effcient at providing dimethylamine than an equivalent dose of choline. Additionally less than 1 % of the choline dose was converted to dimethylamine. Slightly more of an equivalent trimethylamine dose (1.6 %) was converted to dimethylamine.

So trimethylamine N-oxide is undoubtedly a significant dietary source of dimethylamine and its carries the potential of beeing converted to carcinogenic nitrosodimethylamine. However, other dietary sources of dimethylamine probably lie undetected amidst the myriad of anutrient chemicals within foodstuffs, and the continued excretion of small amounts of dimethylamine from antibiotic treated and germfree rats (Asatoor et al., 1967; Zeisel et al., 1985) implies that dietary components, although probably the major source, are not the only originator of dimethylamine.

Endpoint:
basic toxicokinetics 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
Objective of study:
metabolism
Qualifier:
no guideline followed
Principles of method if other than guideline:
Enzyme assay: TMA (applied as the hydrochloride) was oxidized in vitro in rat liver microsomes from male Sprague-Dawley rats to TMA N-oxide and N-demethylated to dimethylamine (DMA)
GLP compliance:
no
Specific details on test material used for the study:
1 or 1.5 mg/mL and TMA.HCl in 0.01 Μ HCl at 250 μΜ concentration
Radiolabelling:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
no details given
Route of administration:
other: no administration, ex vivo assay
Vehicle:
unchanged (no vehicle)
Duration and frequency of treatment / exposure:
no exposure (ex vivo assay)
Dose / conc.:
1.5 other: mg/mL
Type:
metabolism
Results:
P-450-catalysis of TMA (applied as the hydrochloride) oxidation is negligible or non-existent.
Metabolites identified:
yes
Remarks:
TMA N-oxide (TMAO) and N-demethylated to dimethylamine (DMA)
Details on metabolites:
The FAD-containing monooxygenase-catalyzed N-oxidation and demethylation of trimethylamine (applied as the hydrochloride) in rat liver microsomes.

In order to select a suitable TMA concentration (applied as the hydrochloride) for Vmax, TMA metabolism was followed by substrate disappearance (measuring residual substrate) and TMAO formation. Both assays gave the Km value for TMA of 15 μΜ, using Lineweaver-Burk analysis (double reciprocal plot). It is known that in the absence of NADP or NADPH, the FADcontaining monooxygenases (FMO) are irreversibly inactivated at 37 °C during 30 minutes. In order to inactivate FMO without affecting cytochrome P-450 monooxygenases, the microsomes were preincubated without the NADPH-generating system (NADPH-GS) at 37 °C for only 10 min. A possible minor decrease in P-450 activity during incubation of another series of samples at 37 °C with the NADPH-GS was checked in a third series of samples preincubated at 37 °C with NADPH-GS for 2 min. It became obvious that at the saturating TMA concentration of 250 μΜ (applied as the hydrochloride), an appreciable P-450 catalysis could be ruled out, since there was no difference between the 2 min and 10 min preincubation with NADPH-GS. FMO catalysis was apparent in the almost complete inactivation of the microsomal metabolism of TMA to TMAO and DMA: there was between 1 and 3% of residual activity. The lack of catalysis by cytochrome P-450 monooxygenases was also checked in looking for the effect of carbon monoxide on TMA oxidation. The incubation in 100% oxygen did not influence TMA demethylation in most experiments, but doubled the N-oxygenation and even 50% oxygen/50% CO mixture significantly stimulated the rate of N-oxygenation in control microsomes. In the 80% CO/20% O2 gas mixture in the incubation, the rate of demethylation and N-oxygenation were similar to that in air. These results confirmed that P-450-catalysis of TMA oxidation is negligible or non-existent.

Conclusions:
TMA (applied as the hydrochloride) was oxidized in vitro in rat liver microsomes from male Sprague-Dawley rats to TMA N-oxide and N-demethylated to dimethylamine (DMA).
Executive summary:

Trimethylaminuria (TMAuria), the excessive urinary excretion of the odorous trimethylamine (TMA), accompanies elimination of TMA in sweat and corresponding "fish-odor" syndrome. TMA (applied as the hydrochloride) was oxidized in vitro in rat liver microsomes from male Sprague-Dawley rats to TMA N-oxide and N-demethylated to dimethylamine (DMA). Both reactions were inhibited to 1-3% of normal activity by preincubation of microsomes without NADPH-generating system at 37 °C for 10 minutes indicating the FAD-containing monooxygenase-catalyzed reactions. On the other hand, the reactions were not inhibited by gas phase containing up to 80% carbon monoxide/20% oxygen mixture. The results are compatible with the hypothesis that in rat liver microsomes the N-oxygenation and N-demethylation of TMA are catalyzed only or predominantly by FAD-containing monooxygenases, and the cytochrome P-450 monooxygenases play a negligible, if any, role.

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1992-1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable well documented publication, which meets basic scientific principles.
Objective of study:
metabolism
Principles of method if other than guideline:
The in vitro oxidation of trimethylamine (TMA; applied as the hydrochloride) to TMA N-oxide (TMAO) and dimethylamine (DMA) was studied in rat liver microsomes.
(role of flavin-containing monooxygenase and cytochrome P450 in the N-oxidation and N-demethylation of TMA (applied as the hydrochloride) in rat liver microsomes).
GLP compliance:
no
Radiolabelling:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
Male Sprague-Dawley rats, 180-200g, were fed laboratory rat chow and water ad lib. for 1 week after receipt from the Charles River Co.
To induce different forms of cytochrome P450, the rats were subjected to the following treatments:
(a) ethanol, administered as a 15% water solution for 3 days, rats killed on day 4;
(b) sodium phenobarbital, i.p. in saline, 75 mg kg/day for 3 days, rats killed on day 4;
(c) 3- methylcholanthrene, i.p. in corn oil, 25 mg/kg/day for 3 days, rats killed on day 4;
(d) pregnenolone lblu-carbonitrile, i.g. in corn oil at five doses, 25 mg/kg each, twice daily, killed 12 hr after dose 5; and
(e) control rats were untreated or given a single i.g./ i.p. dose of corn oil, 0.5 mL/rat 24 hr before being killed.

Then thirty-three per cent liver homogenates (in 154 mM KCl/50 mM Tris, pH 7.4, buffer) were prepared in a Potter-Elvehjem glass/ teflon homogenizer and centrifuged at 9,000g for 20min at 4”. The supernatant fractions were then centrifuged at 105,000 g for 60 min washed once with a solution containing 154 mM KCl and 10 mM EDTA, resuspended in 0.25 M sucrose (15-20 mg protein/mL), and stored at -70”.
Route of administration:
other: addition to the enzyme assay preparation
Vehicle:
other: in 0.01 M HCl
Duration and frequency of treatment / exposure:
Incubations were for 10 min at 37°.
Remarks:
Doses / Concentrations:
appropriate concentrations of TMA HCl in 0.01 M HCI
The initial TMA concentration (applied as the hydrochloride) used in TMA disappearance studies was 40 or 80 PM, and a 250 µM TMA concentration (applied as the hydrochloride) was used for studies on the formation of HCHO, DMA or TMAO to provide near-maximum velocity. Even at 1250 µM, TMA (applied as the hydrochloride) was not inhibitive.

The apparent K, for the formation of DMA and TMAO was estimated using an initial TMA (applied as the hydrochloride) concentration of 5, 10, 20, 50, 100, 250 and 500 µM
No. of animals per sex per dose / concentration:
not applicable
Control animals:
other: not applicable
Preliminary studies:
In humans, TMA is oxidized to TMAO (major metabolite) and dimethylamine (DMA, minor metabolite), which are excreted in the urine along with small amounts of unmetabolized TMA [ Al-Waiz et al.]. In these studies, about 95% of an administered dose of TMA is oxidized and excreted in the urine as TMAO, whereas less than 1% of a dose of TMA is demethylated and excreted in the urine as DMA.
Details on absorption:
not applicable
Details on distribution in tissues:
not applicable
Details on metabolites:
Trimethylamine N-oxygenation and N-demethylation in rat liver microsomes: production of DMA and TMAO

The apparent km-values for the metabolism of TMA and DMA and TMAO were determined. The Km value for DMA formation in five independent experiments with control microsomes was 92 * 16 PM (mean * SD) and the Km, for TMAO formation was 18.2 + 5.7m. solution.

At pH 7.4, TMA-HCI changes immediately to TMA which is volatile, and during the fifth minute of incubation only 7% of the TMA was in the 2-mL water (microsomal) phase, and 93% was in the 20 mL gas phase of the incubation vials (partition coefficient was calculated to be 0.86).

Since the partition coefficient is independent of concentration, the Km values could be recalculated to give a Km of 7.33 + 1.33 pM for DMA formation and 1.44 +:0.45 PM for TMAO formation.

In rats pretreated with phenobarbital, 3-methylcholanthrene, or ethanol for 3 days, there was little or no effect on the N-demethylation or N-oxygenation of TMA. In some studies where pregnenolone 16cu-carbonitrile was administered once every 12 hr for five doses and the animals killed 12 hr after the fifth dose, there was a small increase in the N-demethylation of TMA but not in the N-oxygenation reaction, whereas in other experiments there was no effect on either the demethylation or N-oxygenation. The overall conclusion of our studies was that pretreatment of rats with phenobarbital, 3-methylcholanthrene, ethanol or pregnenolone 16a-carbonitrile had little or no effect on the N-oxygenation or demethylation of TMA, and there was no evidence that inducible cytochrome P450 catalyze these reactions.

Studies were initiated to investigate the effects of carbon monoxide (CO) on the metabolism of TMA by rat liver microsomes. It was found that a CO/O2 ratio of 1 (50 % CO/SO% O2) or 4 (80 % CO/20 % O2) did inhibit TMAO or DMA formation by microsomes from control or pregnenolone l&xcarbonitrile-pretreated rats. A CO/O, ratio of 9 (90 % CO/10 % O2) had little or no inhibitory effect on TMA metabolism by control microsomes (the demethylation and N-oxygenation combined was unchanged) and a small inhibitory effect (30 %) on TMA metabolism by microsomes from pregnenolone 16rr-carbonitrile-treated rats. The small inhibitory effect on TMA metabolism that was observed may have resulted from a decrease in O2 tension since the N-oxygenation of TMA was more rapid in 100 % oxygen and even 50 % oxygen (despite the presence of 50 % CO) than in air. The overall results indicate that CO had little or no inhibitory effect on the demethylation and N-oxygenation of TMA. The data also indicate that changing the atmosphere in the incubation vial from 20 % oxygen/ 80 % nitrogen (air) to 100 % oxygen and even 50 % oxygen/50 % CO had a selective stimulatory effect on the N-oxygenation of TMA and did not affect TMA demethylation. The importance of the microsomal flavincontaining monooxygenase for the metabolism of TMA was evaluated with methimazole (a substrate and potent inhibitor of this enzyme). The results indicate that methimazole caused a concentration-dependent inhibition in the metabolism of TMA to DMA, suggesting that TMA demethylation is catalyzed by flavin-containing monooxygenase.

Effect of “thermal inactivation” of flavin monooxygenase. Since flavin monooxygenase is irreversibly inactivated by incubation at 37 °C for 30 min in the absence of NADPH, whereas cytochrome P450 retains catalytic activity under these conditions, we investigated the effect of preincubating liver microsomes without or with the NADPH-generating system at 37°C on the subsequent metabolism of TMA in the presence of NADPH. They selected a 10-min preincubation time to decrease the possibility of cytochrome P450 inactivation. The results demonstrated that preincubation of liver microsomes from control or pregnenolone lk-carbonitrilepretreated rats for 10min at 37 °C in the absence of the NADPH-generating system resulted in more than 95 % inhibition of TMA N-oxygenation and N-demethylation. The addition of an NADPH-generating system during the 10 min/37°C preincubation (prevention of thermal inactivation of flavin-containing monooxygenase) protected the TMA-oxidizing activity of liver microsomes. In additional studies, it was observed that even a 2-min preincubation of liver microsomes at 37° in the absence of NADPH resulted in a 60l-80 % decrease in the rate of TMA metabolism as measured by TMA disappearance and DMA formation. These results indicate the importance of flavin monooxygenase for TMA N-oxygenation and N-demethylation by rat liver microsomes.

Conclusions:
Interpretation of results: no bioaccumulation potential based on study results
The overall conclusion of the studies was that pretreatment of rats with phenobarbital, 3-methylcholanthrene, ethanol or pregnenolone 16a-carbonitrile had little or no effect on the N-oxygenation or demethylation of TMA (applied as the hydrochloride), and there was no evidence that inducible cytochrome P450 catalyze these reactions.
It was found that a CO/O2 ratio of 1 (50 % CO/5O % O2) or 4 (80 % CO/20 % O2) did inhibit TMAO or DMA formation by microsomes from control or pregnenolone 16alpha-carbonitrile-pretreated rats.
The overall results indicate that CO had little or no inhibitory effect on the demethylation and N-oxygenation of TMA (applied as the hydrochloride). The data also indicate that changing the atmosphere in the incubation vial from 20 % oxygen/ 80 % nitrogen (air) to 100 % oxygen and even 50 % oxygen/50 % CO had a selective stimulatory effect on the N-oxygenation of TMA and did not affect TMA demethylation.
The results indicate that methimazole caused a concentration-dependent inhibition in the metabolism of TMA to DMA, suggesting that TMA demethylation is catalyzed by flavin-containing monooxygenase.
The results demonstrated that preincubation of liver microsomes from control or pregnenolone lk-carbonitrile pretreated rats for 10 min at 37 °C in the absence of the NADPH-generating system resulted in more than 95 % inhibition of TMA N-oxygenation and N-demethylation.
The results indicate the importance of flavin monooxygenase for TMA N-oxygenation and Ndemethylation by rat liver microsomes
Executive summary:

In 1993 Gut et al., investigated the in vitro oxidation of trimethylamine (TMA, appled as the hydrochloride) to TMA N-oxide (TMAO) and dimethylamine (DMA) in rat liver microsomes. In details the role of flavin-containing monooxygenase and cytochrome P450 in the N-oxidation and N-demethylation of TMA was studied. Pretreatment of rats with phenobarbital, 3-methylcholanthrene, ethanol or pregnenolone 16 alpha-carbonitrile had little or no effect on the liver microsomal metabolism of TMA to TMAO or DMA. Changing the atmosphere in the incubation vessel from 20 % oxygen/80 % nitrogen (air) to 100 % oxygen had a selective stimulatory effect on the N-oxygenation of TMA but did not affect TMA N-demethylation. In addition, the Km for TMA N-demethylation was 5-fold higher than for the N-oxygenation reaction. The results of these studies suggest that the enzyme systems responsible for N-demethylation and N-oxygenation are different and that they are under different regulatory control. Carbon monoxide (CO/O2 = 80/20) had little or no inhibitory effect on either the N-demethylation or N-oxygenation of TMA by liver microsomes from control or pregnenolone 16 alpha- carbonitrile-treated rats. Additional studies indicated that methimazole, an inhibitor of FAD-containing monooxygenase (FMO), was a potent inhibitor of TMA oxidation. Evidence was presented that FMOs are the major enzymes responsible for N-demethylation and N-oxygenation of TMA in rat liver microsomes and that cytochrome P450 enzymes do not play a major role in the metabolism of TMA to TMAO or DMA. But since N-demethylation of tertiary amines is an unusual reaction for FMO, a possibility of another thermolabile enzyme catalyzing N-demethylation of TMA cannot be excluded entirely.

Endpoint:
basic toxicokinetics in vivo
Remarks:
Urinary Excretion of TMA and TMAO after TMA Administration
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
Objective of study:
excretion
metabolism
Qualifier:
no guideline followed
Principles of method if other than guideline:
A comparison of the capacity to N-oxygenate TMA between the rat and the suncus (Family: Soricidae, Order: Insectivora) was made for TMA metabolism.
GLP compliance:
not specified
Specific details on test material used for the study:
no details given
Radiolabelling:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
and Suncus murinus
Details on species / strain selection:
Suncus murinus and Sprague-Dawley rat
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Suncus marinus were obtained from Clea Japan (Tokyo, Japan). Sprague–Dawley rats were obtained from Japan SLC (Shizuoka, Japan).
- Age at study initiation: Suncus marinus (6 weeks old), Sprague-Dawley rats (7 weeks old)
- Housing: metabolic cages
- Diet (e.g. ad libitum): commercial chow (Clea Japan Inc.) ad libitum
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +- 1 °C
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
intraperitoneal
Vehicle:
physiological saline
Duration and frequency of treatment / exposure:
one injection
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
Doses / Concentrations: 10 mg/kg
No. of animals per sex per dose / concentration:
4
Control animals:
no
Positive control reference chemical:
no positive control
Details on study design:
no details given
Details on dosing and sampling:
TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine
- Time and frequency of sampling: 24 h

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine
- Time and frequency of sampling: 24 h
- From how many animals: (samples pooled or not) : 4 (pooled)
- Method type(s) for identification: GC-4CM and GC-17A
Type:
excretion
Results:
Suncus marinus: 40 +/- 4 % of the dose was recovered as parental TMA and 28 +/- 3 % as TMAO in urine
Type:
excretion
Results:
rat: 6.3 +/- 0.3 of the dose was recovered as parental TMA and 60 +/- 4 % as TMAO in urine
Type:
metabolism
Results:
Suncus marinus: The metabolic ratio of TMA was calculated to be 1.42 +/- 0.05
Type:
metabolism
Results:
rat: The metabolic ratio of TMA was calculated to be 0.11 +/- 0.01
Details on excretion:
The cumulative excretion of TMA and TMAO in the urine for 24 h after intraperitoneal administration of TMA to suncus and rats was determined. In the suncus, 40 +/- 4 % of the dose was recovered as parental TMA and 28 +/- 3 % as TMAO in urine, while the recoveries were 6.3 +/- 0.3 and 60 +/- 4 % as TMA and TMAO, respectively, in the rat. The metabolic ratio of TMA was calculated to be 1.42 +/- 0.05 and 0.11 +/- 0.01 in suncus and rats, respectively.
Metabolites identified:
yes
Remarks:
TMAO
Details on metabolites:
FMO-mediated TMA N-oxygenase activity was low in suncus liver microsomes. After i.p. administration of 10 mg/kg bw TMA, the suncus secreted 39.6 % of the dose as TMA in the urine after 24 hours, whereas the rat only excreted 6.3 % as TMA.

The results of the in vitro enzyme activity assay are presented in the following:

Kinetic Analysis of TMA N-Oxygenase

The capacity of microsomes from the suncus liver, lung, and kidney to oxidize TMA to yield TMA N-oxide was determined. Among the organs examined, the liver showed the highest activity of TMA N- oxygenase. The activity of the N-oxygenase in hepatic microsomes was 11 and 23 times higher than those of lung and kidney microsomes, respectively (data not shown), while the activity of microsomes from the suncus liver was much lower than that from the rat liver. Eadie–Hofstee plots of the TMA N-oxygenation by suncus liver microsomes showed a biphasic pattern, suggesting that more than two enzymes were involved in this reaction. The low Km component in the suncus showed a twofold higher Km (55 vs. 31 M) and a fourfold lower Vmax (0.61 vs 2.5 nmol/min/mg protein) values than those obtained using rat liver microsomes, resulting in a sevenfold lower Vmax/Km (11 vs 82 L/min/mg protein) value.

Requirement of Cofactors and the Effects of Inhibitors on TMA N-Oxygenation

To investigate the properties of enzymes involved in TMA N-oxygenation, the requirement of cofactors and the effects of inhibitors of FMO and cytochrome P450 (CYP) on TMA N-oxygenation were examined using suncus liver microsomes at TMA concentrations of 50 mM and 1 mM. The results suggest that the N-oxygenation of TMA is catalyzed mainly by FMO, not by CYP, even in the suncus, which showed very low FMO activity compared to the rat.

Table 1: Michaelis–Menten Kinetic Parameters for the TMA N-Oxygenation in Liver Microsomes from Suncus and Rats

      Enzyme 1           Enzyme 2
   Km 1 (µM)  Vmax 1 (nmol/min/mg protein)  Vmax/Km 1 (µL/min/mg protein)   Km 2 (µM)   Vmax 2 (nmol/min/mg protein)   Vmax/Km 2 (µL/min/mg protein)
 Suncus marinus  55 +/- 3  0.61 +/- 0.08  11 +/- 1  3600 +/- 800  3.7 +/- 0.6  1.1 +/- 0.2
 rat  31 +/- 3  2.5 +/- 0.1  82 +/- 6  450 +/- 36  1.3 +/- 0.0  3.0 +/- 0.2
Conclusions:
The present study revealed that FMO-mediated TMA N-oxygenase activity was low in suncus liver microsomes. Furthermore, the suncus showed higher cumulative urinary excretion of unchanged TMA than the rat. These results are in accordance with the observation that TMA N-oxygenase is present at low levels in extrahepatic tissues. Thus, the results shown here support the possibility that low hepatic FMO activity contributes to a low total body clearance of TMA in patients.
Executive summary:

In vitro and in vivo N-oxygenation of trimethylamine (TMA, applied as the hydrochloride) in the suncus (Suncus murinus) was investigated. The N-oxygenation of TMA has been thought to be catalyzed by flavin-containing monooxygenase (FMO). In a previous study, it was found that the levels of mRNAs for FMOs were extremely low in the suncus. Thus, the intention was to evaluate the capacity of the suncus to N-oxygenate TMA compared to the rat. Eadie–Hofstee plots of the TMA N-oxygenation by suncus liver microsomes showed a biphasic pattern, suggesting that more than two enzymes were involved in this reaction. The low Km component in the suncus showed a twofold higher Km (55 vs. 31 mM) and a fourfold lower Vmax (0.61 vs 2.5 nmol/min/mg protein) values than those obtained using rat liver microsomes, resulting in a sevenfold lower Vmax/Km (11 vs 82 ml/min/mg protein) value. After an intraperitoneal administration of TMA (10 mg/kg body wt), the suncus excreted 39.6 % of the dose in 24-h urine as TMA, whereas the rats excreted 6.3 %. Metabolic ratio in the TMA N-oxygenation was 1.42 and 0.11 in the suncus and the rat, respectively. These results indicate that the suncus can be an animal model for a poor metabolizer phenotype in TMA metabolism.

Description of key information

Bioaccumulation potential result:
Smith, Wishnok, Deen, 1994, toxicology and applied pharmacology 125, 296-308, administration of TMA-HCl intraperitoneally or intravenously to rats and investigation of the distribution, metabolism and excretion.
Asatoor et al., 1965, Biochimica et biophysica acta, 111 (1965) 384-392, administration of choline chloride (intraperitoneal) or [Me14C]choline chloride (orally) or egg lecithin or trimethylamine hydrochloride (orally) or trimethylamine hydrochloride (intraperitoneal) [14C]methylamine (intraperitoneal) or L[Me-14C]Methionine (intraperitoneal) or 14C-MMA to rats (orally or intraperitoneally) and analytical verification of DMA in Urine, to test whether these substances can be transformed in DMA and excreted that way.
Al-Waiz M. et al., 1987, Toxicology 43, 117-121, (1987), investigation of the contribution of demethylation to trimethylamine metabolism in man and its quantitative importance when compared to N-oxidation. The metabolism of orally administered trimethylamine (applied as the hydrochloride salt) has been studied in 4 male volunteers at 2 dose levels.
Zhang et al, 1998, Food and Chemical Toxicology, 36, 923-27, 1998, oral administration of equimolar amounts (1 mmol/kg body weight) of potential amine precursors to male Wistar rats and investigation for their potential role as precursors of dimethylamine.
Gut I. and Conney A.H., Drug Metabo. Drug Interact, (9),|201-208, (1991), study of the in vitro oxidation of trimethylammonium hydrochloride by rat liver microsomes from male Spragque-Dawley rats

Gut I. and Conney A.H., 1993, Biochem. Pharmacol., 46(2), 239-244, (1993), study of the in vitro oxidation of trimethylamine (TMA) to TMA N-oxide (TMAO) and dimethylamine (DMA) in rat liver microsomes.

Mushiroda, T. et al., 2000, Tox. Appl. Pharmacol., 162, 44-48, 2000, investigation of the in vitro and in vivo N-oxygenation of Trymethylamine (TMA, appliedas the hydrochloride) in the suncus (Suncus murinus).

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

The metabolism, disposition and toxicokinetics of trimethylammonium hydrochloride (TMA-HCl) have been characterized. First of all, it is important to take into account, that trimethylamine (TMA) has three different sources: the diet (for example in fish), bacterial synthesis in the intestinal tract and endogenous synthesis. In particular, TMA can be build in the body during bacterial degradation of choline inside the intestinal tract but variations in choline intake that might occur as part of consumption of normal foods are not likely to significantly increase TMA and DMA production, and therefore change our exposure to precursors of nitrosamines only somewhat. The possibility of bacterial synthesis of TMA, could not be proven in a study conducted by Smith et al. in 1994, even though in these experiments the process might have been masked by the oxidation of TMA to TMAO. Normally the dietary intake of preformed DMA and TMA and endogenous production of these amines are likely to be far more significant sources. In total the knowledge of the endogenous occurrence influences mainly the categorization and classification of trimethylamine.

In rats, trimethylamine was rapidly absorbed, uniformly distributed (interestingly the VD exceeds the animal size, so it has to be stated, that TMA must accumulate in some tissues more than in others) and metabolized with the majority of radioactivity eliminated as trimethylamine-oxide (about 40 %) via urine in both sexes of rats. As metabolite mainly trimethylamine-oxide is build, via flavin-dependent monooxigenases (also responsible for the demethylation to DMA), about 20 % of the compound is excreted unchanged as trimethylamine and the remaining 40 % are eliminated as different other metabolites, including dimethylamine. TMAO and TMA don't exert any carcinogenic potential. But there exists also the possibility of TMA being primarily metabolized to DMA and then to N-Nitrosodimethylamine, which is a potent carcinogen. Nevertheless in a study conducted with trimethylamine hydrochloride in human volunteers, the amount of trimethylamine eliminated as TMAO was found to be over 95 % of the administered dose, additionally the demethylation of TMA to DMA was found not to be an important pathway; so the risk of considerable amounts of DMA, which is then in part metabolized to N-Nitrosodimethylamine is rather low. It is not possible to exclude the possibility of nitrosamine formation, but it seems rather unlikely that a significant amount of nitrosamine can be built, since less than 5 % of the dose are available as DMA for the transformation to N-Nitrosodimethylamine. The excretion of trimethylamine is conducted mainly via the urine, and only negligible amount are excreted via faeces or exhaled.

Based on the results obtained in these studies, trimethylamine (and trimethylammonium hydrochloride) may be considered to be a rapidly absorbed and as trimethylamine-oxide well eliminated substance in rats and humans. It bears the possibility to be metabolized to dimethylamine, which can in part be transformed to the carcinogenic substance N-Nitrosodimethylamine.

 

Discussion on bioaccumulation potential result:

Preliminary it needs to be stated, that there are three sources of trimethylamine (TMA): diet, bacterial synthesis, and endogenous synthesis.

In 1994 Smith and Coworkers investigated the metabolism and excretion of methylamines in rats. They undertook these investigations to determine in detail the tissue distribution, urinary excretion, and possible other excretion path ways after administration of TMA-HCl to rats via injection (intravenous or intraperitoneal). The radiolabeled carbon in the trimethylamine-molecule permitted the detection of the mother compound and in addition the metabolites derived from TMA-HCl.

Blood samples were taken at 15 min, 30 min, and 1, 2, 4, and 8 hr following the dose. And in conclusion the blood concentrations obtained for iv or ip doses were indistinguishable. Additionally urine samples (24 h collections), faeces samples and the exhaled air were investigated. To determine partitioning between blood and tissues, samples of blood, lung, heart, liver, kidney, intestine, and abdominal muscle were also taken for analysis. When trimethylamine hydrochloride (TMA-HCl) is administered in vivo intravenously or intraperitoneally it is readily absorbed and uniformly distributed in the body, interestingly with the volume of distribution exceeding the animal size, suggesting that it is highly concentrated at one or more locations in the body (Smith, 1994).

The metabolism of TMA occurs mainly via oxidation to TMAO. About 40% of the dose, administered to rats was excreted in the urine as TMAO, about 20 % were excreted as TMA and the remaining 40 % where other metabolites (Smith, 1994). The results indicate also a saturation of the metabolism of TMA (Smith, 1994), because with a raising dose of TMA the TMA fraction recovered in the urine raised too. Its elimination is primarily conducted via urine (96 % of the dose could be recovered in the urine after 24 hours) and only negligible excretion occurs via exhalation or via feces (Smith, 1994). For both DMA and TMA, — 1% of the dose was recovered in exhaled air as 14CO2, while a negligible amount was exhaled as the amine. These results demonstrate that both faecal and respiratory excretion of methylamines in normal rats are negligible. Retention of either the original methylamines or any carbon-based metabolites in tissues is also negligible. These findings indicate that the only important pathway for elimination of these methylamines (and their metabolites) from the body is urinary excretion.

Additionally there was no apparent bacterial synthesis of TMA, but actual bacterial synthesis might have been masked by oxidation to TMAO and/or demethylation to DMA.As TMA can also undergo reaction to NDMA (N-nitrosodimethylamine), which is a potent carcinogen, this topic needs to be discussed here as well. The results from the study do not show that this pathway is a major one, so the risk it rather small. But because it cannot be stated that this does not occur at all, this possibility needs to be taken into account when judging the carcinogenic potential of TMA.

 

The study performed by Asatoor et al, in 1965, already gives important data concerning DMA production. They investigated whether DMA is generated in the mammalian body by feeding rats (in cases also administered via injection) with either choline chloride, lecithin, [14C]methylamine, or [Me14C]choline chloride (orally) or egg lecithin or trimethylamine hydrochloride (orally) or trimethylamine hydrochloride (intraperitoneal) and then analytical verification of DMA in Urine, to test whether these substances can be transformed in DMA and excreted that way. They could prove that DMA is a product of demethylation of trimethylamine. Thus, the conversion of choline to dimethylamine takes place in two stages; first, breakdown of choline to trimethylamine by intestinal bacteria and secondly, demethylation of the latter to dimethylamine. Furthermore TMA is in part metabolized to DMA, which is then excreted mainly via urine. Additionally MMA is partly methylated to form DMA. So these results are in accordance with the results obtained by Ischiwata et al. 1984.

 

In 1987 Al-Waiz et al. investigated the excretion of trimethylamine hydrochloride by 4 male human volunteers after oral ingestion of 2 dose levels (300 or 600 mg free base). At both dose levels over 95% of the administered trimethylamine was excreted in the N-oxide form, confirming N-oxidation as the major route of metabolism in man. In absolute terms, the amount of free trimethylamine detected after giving the larger dose increased nearly 40-fold from control values to give 404.2 +/- 61.6 µmol in the 0--8 h urine sample and imparted a fish-like odour to the urine. If demethylation was an important pathway for the degradation of trimethylamine in man then it would be expected that as the dose increased and more free trimethylamine becomes available, more dimethylamine would be produced. This clearly is not the case. Probably the majority of urinary dimethylamine is under normal circumstances of endogenous origin, presumably arising from the methylation of methylamine produced via intermediary metabolism. So the small part of ingested trimethylamine that is transformed to dimethylamine could be metabolized to nitrosamines. However, even small amounts of dimethylamine, if nitrosated, may be sufficient to elicit a potentially carcinogenic response.

 

In 1998, Zhang et al. investigated the excretion of DMA after administration of various related amine precursors. They found trimethylamine N-oxide (at a dose rate of 1 mmol/kg body weight) to be 20 times more efficient at providing dimethylamine than an equivalent dose of choline. Additionally less than 1 % of the choline dose was converted to dimethylamine. Slightly more of an equivalent trimethylamine dose (1.6%) was converted to dimethylamine.

So trimethylamine N-oxide is undoubtedly a significant dietary source of dimethylamine and its carries the potential of being converted to carcinogenic nitrosodimethylamine. However, other dietary sources of dimethylamine probably lie undetected amidst the myriad of anutrient chemicals within foodstuffs, and the continued excretion of small amounts of dimethylamine from antibiotic treated and germfree rats (Asatoor et al., 1967; Zeisel et al., 1985) implies that dietary components, although probably the major source, are not the only originator of dimethylamine.

 

In 1991, Gut et al., investigated the oxidation of Trimethylammonium hydrochloride by rat liver microsomes. They found that TMA (applied as the hydrochloride) was oxidized in vitro in rat liver microsomes from male Sprague-Dawley rats to TMA N-oxide and N-demethylated to dimethylamine (DMA). Both reactions were inhibited to 1-3% of normal activity by preincubation of microsomes without NADPH-generating system at 37 °C for 10 minutes indicating the FAD-containing monooxygenase-catalyzed reactions. On the other hand, the reactions were not inhibited by gas phase containing up to 80% carbon monoxide/20% oxygen mixture. The results are compatible with the hypothesis that in rat liver microsomes the N-oxygenation and N-demethylation of TMA are catalyzed only or predominantly by FAD-containing monooxygenases, and the cytochrome P-450 monooxygenases play a negligible, if any, role.

 

In 1993, Gut et al., further investigated the in vitro oxidation of trimethylamine (TMA) to TMA N-oxide (TMAO) and dimethylamine (DMA) in rat liver microsomes. In details the role of flavin-containing monooxygenase and cytochrome P450 in the N-oxidation and N-demethylation of TMA was studied. Pretreatment of rats with phenobarbital, 3-methylcholanthrene, ethanol or pregnenolone 16 alpha-carbonitrile had little or no effect on the liver microsomal metabolism of TMA to TMAO or DMA. Changing the atmosphere in the incubation vessel from 20% oxygen/80% nitrogen (air) to 100% oxygen had a selective stimulatory effect on the N-oxygenation of TMA but did not affect TMA N-demethylation. In addition, the Km for TMA N-demethylation was 5-fold higher than for the N-oxygenation reaction. The results of these studies suggest that the enzyme systems responsible for N-demethylation and N-oxygenation are different and that they are under different regulatory control. Carbon monoxide (CO/O2 = 80/20) had little or no inhibitory effect on either the N-demethylation or N-oxygenation of TMA by liver microsomes from control or pregnenolone 16 alpha-carbonitrile-treated rats. Additional studies indicated that methimazole, an inhibitor of FAD-containing monooxygenase (FMO), was a potent inhibitor of TMA oxidation. Evidence was presented that FMOs are the major enzymes responsible for N-demethylation and N-oxygenation of TMA in rat liver microsomes and that cytochrome P450 enzymes do not play a major role in the metabolism of TMA to TMAO or DMA. But since N-demethylation of tertiary amines is an unusual reaction for FMO, a possibility of another thermolabile enzyme catalyzing N-demethylation of TMA cannot be excluded entirely.

 

In 2000, Mushiroda et al. investigated the in vitro and in vivo N-oxygenation of trimethylamine (TMA, applied as the hydrochloride) in the suncus (Suncus murinus). The N-oxygenation of TMA has been thought to be catalyzed by flavin-containing monooxygenase (FMO). In a previous study, it was found that the levels of mRNAs for FMOs were extremely low in the suncus. Thus, the intention was to evaluate the capacity of the suncus to N-oxygenate TMA compared to the rat. Eadie–Hofstee plots of the TMA N-oxygenation by suncus liver microsomes showed a biphasic pattern, suggesting that more than two enzymes were involved in this reaction. The low Km component in the suncus showed a twofold higher Km (55 vs. 31 mM) and a fourfold lower Vmax (0.61 vs 2.5 nmol/min/mg protein) values than those obtained using rat liver microsomes, resulting in a sevenfold lower Vmax/Km (11 vs 82 ml/min/mg protein) value. After an intraperitoneal administration of TMA (10 mg/kg body wt), the suncus excreted 39.6 % of the dose in 24-h urine as TMA, whereas the rats excreted 6.3 %. Metabolic ratio in the TMA N-oxygenation was 1.42 and 0.11 in the suncus and the rat, respectively. These results indicate that the suncus can be an animal model for a poor metabolizer phenotype in TMA metabolism.