Registration Dossier

Toxicological information

Basic toxicokinetics

Currently viewing:

Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Older study on the test item itself similar to recent guidelines without GLP. There are some details lacking, but the given data indicate that the study was well and scientifically reasonably performed.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1979
Report Date:
1979

Materials and methods

Objective of study:
metabolism
Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Deviations:
not applicable
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): N-Methyl-2thion-thiazolidin
- Substance type: pure substance
- Other: substance is poorly soluble in water, slightly soluble in ethanol, soluble in ethyl acetate
Radiolabelling:
no

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Breeder Winkelmann, Kirchborchen, Germany
- Weight at study initiation: 180-210 g
- Housing: i.a. in metabolism cages

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
not specified
Duration and frequency of treatment / exposure:
Single oral application, exposure either 3h (exhalation) or 0.5, 1, 2, 4, 6, 24 h (blood examination)
Doses / concentrations
Remarks:
Doses / Concentrations:
500 mg/kg
No. of animals per sex per dose:
Total: at least 19 males per single dose of 500 mg/kg
Control animals:
no
Positive control:
none
Details on dosing and sampling:
PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, blood, exhalated air
- Time and frequency of sampling: after 1&3h (exhalation) resp. 0.5, 1, 2, 4, 6, 24 h (blood sampling)

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, blood, exhalated air
- Time and frequency of sampling: after 1&3h (exhalation) resp. 0.5, 1, 2, 4, 6, 24 h (blood sampling)
- Method type(s) for identification: GC-ECD, headspace-technique (CS2), GC, electrophoresis (metabolite-Identification)

Results and discussion

Main ADME results
Type:
metabolism
Results:
The substance is metabolized to N-Methyl-2on-thiazolidin and further biotransformation products which were detected in the urine, e.g. secondary amines

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
N-Methyl-2on-thiazolidin
secondary amines

Any other information on results incl. tables

Methodology and results

 

Determination of CS2 via GC-ECD-measurement (Headspace technique):

A concentration series of CS2 in n-hexane was made (1-50 ppm)

Each 100 µl of these solutions were placed in a glass vial (V = 20 ml) onto a small paper filter and closed with a gas-tight cap.

100 µl pure n-hexane served as control, vials were allowed to stand for 15 min at room temperature.

With a gas-tight syringe each 500 µl of the gas phase were removed through rubber membranes and separated on a column with 10% SE 30, detection was done with EDC. Retention time was 0.8 min, a linear correlation resulted from concentration and signal.

 

Examination of exhaled air of the rat for CS2:

500 mg/kg N-Methyl-2thion-thiazolidin were orally applied to one rat. Afterwards the animal was placed into an apparatus for isolation of constituents of exhaled air.

Via a water jet pump the exhaled air was sucked into a CO2/acetone cooling trap (-60°C). In an U-pipe first moisture was frozen and the dried air was lead through a n-hexane-filled washing flask which was replaced after 1h. Test ended after 3h.

The collected water was extracted with hexane. Both n-hexane washing fluids and extract was examined for CS” via GC-ECD-measurement (Headspace technique).

In no case CS2 was detected.

 

Examination of blood of the rat for CS2:

After oral gavage of 500 mg/kg N-Methyl-2thion-thiazolidin blood was sampled after 0.5, 1, 2, 4, and 6h. Each 200 µl whole blood were pipetted in glass vials onto filter paper and similarly as above analysed via headspace-technique for CS2.

In none of the samples CS2 was detected.

 

Determination of N-Methyl-2on-thiazolidin (=M1) in plasma:

1ml rat plasma from the same trial (500 mg/kg oral) were extracted 3x with 2 ml acetic acid ethyl ether. Extract was condensed to 1 ml and examined via gas chromatograph.

GC-data:

Column: 5% Carbowax 20M; WAW DMCS 60 - 80 mesh

Temperature program: 150°C for 2 min over 32 min to 220°C for 4 min

Detector: PND

With the given temperature program, besides the already known N-Methyl-2thion-thiazolidin, a second substance (M1) could be detected which was present in a much higher concentration than N-Methyl-2thion-thiazolidin. Similar retention times were observed for M1 and N-Methyl-2on-thiazolidin. M1 could be also isolated from the urine, mass spectrometry confirmed the proposed structure.

 

Time-dependent concentration of N-Methyl-2on-thiazolidin in plasma:

18 rats were dosed orally with 500 mg/kg N-Methyl-2thion-thiazolidin. After 0.5, 1, 2, 4, 6, and 24h blood of each three animals was taken and the concentration of M1 was determined in plasma, see attached file for details. The concentration of M1 is approx. 5-10 times higher than the one of N-Methyl-2thion-thiazolidin in plasma.

 

Recovery rate of N-Methyl-2on-thiazolidin in rat plasma:

Recovery rate was determined for 50 and 200 µmol/L of N-Methyl-2on-thiazolidin in plasma. Due to the relatively high volatility of the substance during condensation of the extracts, only a value of 70% was measured.

 

Isolation of N-Methyl-2on-thiazolidin (M1) from rat urine:

50 ml of the combined urine of rats orally dosed with 500 mg/kg N-Methyl-2thion-thiazolidin were extracted with 3x 60ml acetic acid ethyl ether. The extract was condensed to 1 ml and separated preparatively via thin layer chromatography (solvent: chloroform/acetic ester 1:9)

N-Methyl-2thion-thiazolidin shows on plates with fluorescence indicator a clear fluorescence extinction at 254 nm, M1 a weak extinction:

N-Methyl-2thion-thiazolidin: Rf = 0.54

M1: Rf = 0.31

N-Methyl-2on-thiazolidin: Rf = 0.31

The respective silica layers were isolated from the plate and the substances extracted with acetic ester. The as M1 isolated substance from the urine exhibited also in the GC the same retention time as N-Methyl-2on-thiazolidin (Rt = 1.5 min)

For structural confirmation a mass spectrum of each M1 and N-Methyl-2on-thiazolidin was prepared.

 

Incubation of N-Methyl-2thion-thiazolidin with liver microsomes:

Preparation of liver microsomes:

Males rats were pretreated with sodium phenobarbital (1% in drinking water over 1 week). 24h after stopping phenobarbital administration, livers were isolated and rinsed with ice-cooled 0.9% NaCl solution. Each 2g liver were homogenized with 8ml ice-cooled CaCl2 buffer (pH = 7.5) and centrifuged for 15 min at 4°C and 10,000g. Supernatant was decanted and after further centrifugation and washing steps the microsome preparation was obtained. From 2 g liver one may obtain ca. 10 ml homogenate, 1 ml homogenateca. 4 mg protein. Activity of microsomes was tested via hydroxylation of aniline to p-aminophenol.

 

Incubation:

1 mg N-Methyl-2thion-thiazolidin was solved in 100 µl isopropanol and incubated with 10 ml microsome homogenate in shaking water bath at 37°C.

1 mg N-Methyl-2thion-thiazolidin

10 ml microsome homogenate (2g liver)

1 ml MgCl2*6H2O (0.05m in 0.1m KCl)

1 ml NADP-Na (0.01m in KCl/Tris buffer)

1 ml Glucose-6-phosphate (0.1m in HCl Tris buffer)

50 µl glucose-6-phosphate dehydrogenase

2 ml KCl/Tris buffer

Total: 15.15 ml

Directly, after 30, 60 and 180 min 3ml of the incubation mixture were removed and 3x with 3ml acetic ester isolated, centrifuged and the organic phase was condensed to 1 ml. In the extract, N-Methyl-2thion-thiazolidin was determined chromatographically. As control, N-Methyl-2thion-thiazolidin was incubated with microsomes but without NADPH regenerating system, see attached file. After 180 min incubation without NADPH regenerating system, 82% of the original amount was recovered. At the incubation with NADPH regenerating system, a degradation of the original amount to 20% was found.

Similarly, the gas chromatogram showed a time-depending increasing peak with the retention time of N-Methyl-2on-thiazolidin.

 

Determination of further metabolites in the urine:

As N-Methyl-2thion-thiazolidin was excreted only to ca. 0.2% via urine and faeces, the major part is expected to be metabolised. As only the parent compound showed a distinctive extinction of fluorescence at 254 nm, it was searched, in order to detect the metabolites, for chemical colour reactions after chromatographical separation separation of urine and urine extracts.

 

Electrophoresis of urine samples:

As tests involving separation of urine and urine extract via thin layer chromatography on silica plates revealed that the formed metabolites must be very polar, electrophoretic separation was tried.

200 µl urine were mixed with 200 µl methanol and after centrifugation 50 µl of the supernatant were placed on silica plates (20x20 cm). The plate was sprayed with acetate buffer (pH = 4.5) and an electrophoretic separation at 400 V over 1 h was performed.

By spraying of sodium nitroprusside / acetaldehyde (for detection of secondary amines) some of the substances could be made visible as pink stains, see attachment. Compared to control urine, a distinctive increase of the stains was visible up to 30h after application. The successive decrease down to control level was reached after 76h. It was aimed to isolate the most intensively coloured substance via preparative electrophoresis and to determine its structure. The isolated substance showed in the gas chromatogram a similar retention time to peak no 8, see attachment. Mass spectrum did not allow structure determination of the isolated compound due to insufficient purity. For further spectroscopical examinations the isolated amount did not suffice.

 

Thin layer chromatography of urine samples:

20 µl of centrifuged urines were separate via thin layer chromatography in the system CH2Cl2 / CH3OH 8/2. After spraying of K2CrO4 / AgNO3 (for sulphur-containing substances) also several stains could be made visible, see attachment. An attribution to the still unknown peaks of the gas chromatogram was not possible.

 

Gas chromatography of urine extracts:

a) 1 ml urine was extracted 3 times with each 2 ml acetic acid ethyl ester and the extract was concentrated to 1 ml.

b) 1 ml urine was dropped on Na2SO4 and the solid was extracted 3 times with each 10ml CH2Cl2. The extract was concentrated to 1 ml.

 

Gas chromatographic parameters:

Column: 10% carbowax 20 M

Temperature program: 150°C for 2 min, increase 32°C per min to 220°C for 4 min.

Detector: PND

Attenuation: 1:256

Paper feed: 1 cm/min

Isolated peaks i.a.: M1, V-MTT, sec. amine

Detection was done via thermionic N-Detector. A detection with FID was not possible which hindered the successful utilisation of a GC-MS coupling for structure determination of further unknown metabolites.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study results
The study was assessed with Klimisch 2, the performed method was considered scientifically reasonable to assess the metabolism of N-Methyl-2thion-thiazolidin in the rat. Hence, the results can be considered as reliable. The given data indicate a metabolism of N-Methyl-2thion-thiazolidin to N-Methyl-2on-thiazolidin and a rather fast clearance within mostly 24h from the plasma via urine. Given data support the result that renal excretion of the unchanged substance was very low. Also further, very hydrophilic metabolites were found. Based on the available data, it can be concluded that N-Methyl-2thion-thiazolidin has no potential for bioaccumulation due to its excessive metabolism.
Executive summary:

A metabolism study was performed with N-Methyl-2thion-thiazolidin in rats.

After oral application of 500 mg/kg N-Methyl-2thion-thiazolidin to the rat, constituents of exhaled air were isolated via condensation over 3 hours. The condensate was tested for CS2 via gas chromatography (headspace technique). No CS2 could be detected. At similar dosage, CS2 could neither be found in the blood.

Via temperature-programmed gas chromatographic separation a second substance besides N-Methyl-2thion-thiazolidin could be detected in blood plasma. It was N-Methyl-2on-thiazolidin. This metabolite was present in a concentration 5-10 times higher than N-Methyl-2thion-thiazolidin. Kinetics of this substance in plasma was determined.

The metabolite N-Methyl-2on-thiazolidin was isolated from the urine. Its percentage is 2% of the initially applied amount N-Methyl-2thion-thiazolidin. By comparison with a reference substance and mass spectrometry its structure was confirmed.

By further separation techniques such as electrophoresis, thin layer chromatography and temperature-programmed gas chromatography several other biotransformation products were detected in the urine, e.g. secondary amines. Excretion occurred mainly 5h – 30h after application. The structures of these compounds could not be detected yet.

Based on the results, a bioaccumulation potential can be neglected.