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Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993
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:
Toxicology and applied pharmacology 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 DMA-HCl to rats.
GLP compliance:
no

Test material

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

[14C]Dimethylamine hydrochloride
dimethylamine-d6 hydrochloride [(CD3)2NH • HCl]
dimethylamine [13C2] hydrochloride
Radiolabelling:
yes
Remarks:
C14

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding labs, WIlmington, MA
- Weight at study initiation: 300 g
- Housing: stainless steel rack metabolism cages, later Nalgene acrylic metabolism cages
- Individual metabolism cages: yes
- Diet (e.g. ad libitum): ProLab 3000, Old Mother Hubard, Lowell, MA ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 days

Administration / exposure

Route of administration:
other: intravenous or intraperitoneal
Vehicle:
other: solved in physiological saline, passing it through a 0.22 µm syringe filter, infusate volumen = 1 mL
Details on exposure:
bolus doses of [14C]DMA-HCl were given ip
Duration and frequency of treatment / exposure:
one injection to every rat in a group
Doses / concentrationsopen allclose all
Dose / conc.:
5 other: µmol bolus i.v. or i.p.
Remarks:
Doses / Concentrations: < 5
Dose / conc.:
100 other: µmol bolus i.v. or i.p.
Dose / conc.:
1 000 other: 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 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)
feces samples
Monitoring of the exhalted air
Determination of partitioning between blood and tissues,. analysis of samples of blood, lung, heart, liver, kidney, intestine, and abdominal muscle
Statistics:
one-compartment model with first-order removal
mass balance equation for the one-compartment model
plot of In(CB/D) vs t has an intercept equal to ln(Vd -1) and a slope of -k/Vd, 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
Main ADME resultsopen allclose all
Type:
excretion
Results:
GFR = 2.5 mg/min
Type:
excretion
Results:
renal plasma flow rate (RPF) = 10 mL/min
Type:
excretion
Results:
renal blood flow rate (RBF) = 20 mL/min
Type:
excretion
Results:
apparent toal clearance = 9-10 ml/min

Toxicokinetic / pharmacokinetic studies

Details on absorption:
no data because administered via injection
Details on distribution in tissues:
The mean blood peak concentration of labeled DMA achieved following the 2-µmol dose was comparable to the concentration of endogenous DMA, which was found to be 2.5 ± 0.2 nmol/mL (n = 5). Except for the earliest (15 min) time point, the data for any given dose fell on a straight line, as predicted by the one-compartment, linear model. The lines shown are the least-squares fits to the data for / > 30 min. However, contrary to expectations for one-compartment, linear behavior, the intercepts and slopes of these lines depended on D. Thus, the apparent volume of distribution and clearance were dose dependent. As the dose of DMA injected was increased, there were decreases in the calculated values of both VD and k. The magnitude of these decreases was such that, in general, there was no overlap of the 90% confidence intervals for these parameters for the different doses.

The tissue-to-blood concentration ratios measured 1 hr following low doses of [14C]DMA or [14C]TMA 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 volumes of distribution for DMA and TMA in similar-size rats are 2260 and 725 mL, respectively.
From the percentage weight gain over the 5-day period, changes in VD of 40 mL/24 hr for DMA and 7 mL/24 hr for TMA or TMAO were estimated . This implies increases in total body content of DMA, TMA, and TMAO of 0.3, 1.1, and 4.5 µmol/ kg body wt/24 hr, respectively.
Details on excretion:
For DMA, < 1% of the dose was recovered in each of feces, blood, lung, and kidney, while —2% of the radioactivity remained in the liver. The largest portion of the dose, 96%, was recovered in the urine in the first 24 hr following the dose. (Urinary recovery between 24 and 72 hr was found to be negligible.) The total recovery was complete (101%) (Table1)
For both DMA and TMA, — 1% of the dose was recovered in exhaled air as 14C02, 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. The urinary recoveries shown for the stable isotopes include not only the administered amine, but also the other two amines assayed by GC/MS. Despite inclusion of all three methylamines, the stable isotope recoveries were substantially lower than those of the radioisotopes. Comparing the recoveries of 100 µmol doses measured with radioisotopes or stable isotopes, the lower values with the latter suggest that significant amounts of the urinary radioactivity were in the form of nonmethylamine metabolites (-20% for DMA and -40% for TMA).

When the DMA stable isotope was administered, there were no detectable amounts of TMA or TMAO stable isotopes in the urine.
Toxicokinetic parametersopen allclose all
Test no.:
#1
Toxicokinetic parameters:
other: VD 2258 mL (at 2 µmol), 1041 mlL(at 100 µmol), 375 mL (at 1000 µmol)
Test no.:
#2
Toxicokinetic parameters:
other: k = 10.1 mL/min ( at 2 µmol), 9.3 mL/min (at 100 µmol), 4.1 mL/min (at 1000 µmol)

Metabolite characterisation studies

Metabolites identified:
not measured
Details on metabolites:
The concentration of endogenous DMA = 2.5 ± 0.2 nmol/mL.
DMA is excreted mainly via urine. The applied method did not allow identifying metabolites.
Thesis: at the intermediate dose level ~20% of the DMA appeared in urine as metabolites

Any other information on results incl. tables

Endogenous and Bacterial Synthesis: much less DMA was excreted by the germ-free rats than by normal rats. Since dietary intakes of DMA were comparable in the two groups, this implies that there was a great deal of DMA synthesis by gut bacteria in the normal rats. Our calculations show that there may have been a small amount of net endogenous synthesis of DMA, but net bacterial synthesis was about four times greater.

TABLE 1 Radioisotope Balances for DMA and TMA in Normal Ratsa

 

DMA

TMA

Feces

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

Applicant's summary and conclusion

Conclusions:
Interpretation of results: no bioaccumulation potential based on study results
DMA is mainly excreted via the urinary tract and bears the some potential to circulate between tissues and the gastro intestinal tract.
Executive summary:

The study performed by Smith et al. in 1994, dealt with the metabolic fate of DMA or TMA after injection to rats. There are three sources of dimethylamine (DMA): diet, bacterial synthesis, and endogenous synthesis. Fish contains significant quantities of DMA and TMA, and is probably a major dietary source of methylamines. As TMAO is an end-product of nitrogen metabolism in fish, it can be metabolized to DMA and TMA by bacteria once fish have been killed. The endogenous concentration of DMA, was found to be 2.5 +/- 0.2 nmol/mL. So DMA enters the body from intestinal absorption of both dietary DMA and DMA formed by bacterial action in the lower gut or after endogenous synthesis after conversion of choline to DMA (see also Asatoor et al. 1965) and leaves by urinary excretion. The much greater urinary excretion of DMA by normal rats than by germ-free rats is clear evidence for significant synthesis of DMA by gut bacteria. Urine analysis following doses of stable isotopes showed also that DMA was not converted to TMA or TMAO. There is also some endogenous synthesis of DMA, possibly from monomethylamine (MMA). So the results of these metabolic balance studies indicate that there is net synthesis of DMA by gut bacteria and net consumption of TMAO by endogenous processes.

When dimethylamine hydrochloride (DMA-HCl) is administered in vivo intravenously or intraperitoneally it is readily absorbed (Smith, 1994). After the uptake it is 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 tissue-to-blood concentration ratio of 8 found for DMA in kidney greatly exceeds values for other body compartments, including gastric fluid (Smith et al., 1994). The apparent volume of distribution and clearance were dose-dependent. About 20 % of the administered DMA appear in the urine as non-methylamine metabolites.The experiments revealed, that the only important pathway for elimination of the three methylamines (and their metabolites) tested is urinary excretion. Fecal excretion, exhalation in breath, and retention in tissues are all negligible in normal rats. The experiments conducted showed that DMA is avidly secreted by the renal tubules, or there might exist as an alternative explanation for the dose dependence of the volume of distribution an active transport of DMA or TMA from extracellular to intracellular fluid. The results strongly suggest that there are various regions outside the gastrointestinal contents which contain high concentrations relative to the blood of these methylamines.