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Basic toxicokinetics

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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

Data source

Reference
Reference Type:
publication
Title:
Metabolism and excretion of methylamines in rats.
Author:
Smith, J L; Wishnok, J S; Deen, W M
Year:
1994
Bibliographic source:
Tox. Appl. Pharmacol., 125, 296-308, (1994)

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): trimethylamine hydrochloride
- Physical state: solid

[14C]trimethylamine hydrochloride
trimethylamine-d6 hydrochloride [(CD3)3NH • HCl]
trimethylamine-d9 hydrochloride [(CD2H)3NH • HCl]
Radiolabelling:
yes

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
no data

Administration / exposure

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
Doses / concentrationsopen allclose all
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:
5
Control animals:
yes
Positive control:
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).

Results and discussion

Preliminary studies:
no data

Toxicokinetic / pharmacokinetic studies

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.

Metabolite characterisation studies

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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.