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EC number: 217-614-1 | CAS number: 1908-87-8
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.
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
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 homogenate≙ca. 4 mg protein. Activity of microsomes was tested via hydroxylation of aniline to p-aminophenol.
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.
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.
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.
Calibration curve was perfectly linear.
For the determination of the recovery rate, preliminary tests with different organic solvents revealed that, with extraction with acetic acid ethyl ether of the test item out of an aqueous solution, a recovery rate of 100% was determined.
Plasma: A solution of the test item in aceton was added to each 1 ml plasma to obtain a solution of 10 and 100 µmol/L. Those were extracted with acetic acid ethyl ether. Double determination revealed an average recovery of 89%.
Urine: An analogues procedure revealed a recovery of 94%.
Faeces: 5g rat faeces were suspended in 10 ml water and homogenized with an Ultra-Turrax. The homogenate was mixed with 100 µl of a 10 mmol/L solution of the test item and successively extracted. Recovery was 40%.
Excretion of the test item in urine
The excretion of the test item in the urine of five rats receiving each 500 mg/kg via oral gavage was determined. Up to 48 h after gavage minor amounts of the test item could be detected.
Table 1: Total excretion of the test item in the urine
Test item in urine (0-76 h)
≙0.04% of the dose
≙0.29% of the dose
≙0.15% of the dose
≙0.11% of the dose
0.15% of the dose
As the table shows, the total excretion of the unchanged test item is very low. After enzymatic cleavage of the urine residues no further metabolites could be determined in the extracts via gas chromatography.
Excretion of the test item in the faeces
From the same experiment with 500 mg/kg the test item was determined in the faeces of three rats.
Table 2: Total excretion of the test item in the faeces
≙0.03% of the dose
≙0.10% of the dose
≙0.06% of the dose
0.06% of the dose
The total excretion of the unchanged test item is very low here, too.
The time-dependent excretion of the test item in the faeces is shown in table 3:
Table 3: Excretion of the test item in the faeces
Average of total excretion (3 rats)
Average of excretion per hour
0 – 5.5 h
5.5 – 23 h
23 – 47 h
47 – 76 h
N-Methyl-2thion-thiazolidin is completely absorbed after oral gavage even at high doses such as 500 mg/kg. Via faeces, an average of only 0.06% of unchanged substance were excreted.
As N-Methyl-2thion-thiazolidin is poorly soluble in water, renal elimination of unchanged substance was with an average of 0.15% also only minor.
Time-dependent concentration curve in plasma already shows the highest peak after 0.5h, both after gavage of 100 and 500 mg/kg. After a decrease of plasma concentration over 1-2 hours, due to after-resorption, a second maximum occurs after 4h (100 mg/kg) resp. 6h (500 mg/kg).
At the dose of 100 mg/kg there was 24h after absorption no N-Methyl-2thion-thiazolidin detectable in the plasma. At the higher dose, only minor concentrations were detected. The maxima after 0.5h and 4h resp. 6h are about equally high and reflect the dose relation very well.
100mg/kg / 500mg/kg = 1/5
15µmol/L / 90µmol/L = 1/6
In a study similar to OECD guideline 417, the absorption and excretion of N-Methyl-2thion-thiazolidin was assessed in the rat.
An analytical determination method for N-Methyl-2thion-thiazolidin was established. After extraction with acetic acid ethyl ether the substance could be detected in the extracts of plasma, urine and faeces.
After an acute oral gavage of 500 mg/kg to male Wistar rats up to 76h only an average of 0.06% of the original dose of unchanged N-Methyl-2thion-thiazolidin could be found. Resorption was complete also at the high dose.
Via the urine, also only 0.15% of the applied dose of the unchanged substance were excreted.
Excretion kinetics via faeces and urine were determined.
After acute oral gavage of 100 or 500 mg/kg the time-dependent concentration of N-Methyl-2thion-thiazolidin in plasma up to 24h after application was determined.
Concentration maxima of N-Methyl-2thion-thiazolidin are with 15 resp. 90 µmol/L not very high, reflect dose relation however very well.
Toxicokinetics, Absorption and Excretion: oral: gavage, male Wistar rats, 100 or 500 mg/kg, similar to OECD 417, determination in blood/plasma, urine, faeces: For plasma concentration, major peak were determined after 0.5 h as approx. 15 resp. 92 µmol/L and 4h as 15 µmol/L (100 mg/kg) resp. 6h as 85 µmol/L (500 mg/kg), 0.15% of original dose were found as total excretion (0-76 h)
Toxicokinetics, Metabolism: In vitro (rat microsomes) and in vivo (oral: gavage, male Wistar rats, 500 mg/kg), similar to OECD 417: Metabolism to N-Methyl-2on-thiazolidin, secondary amines and other unidentified, very soluble substances.
There are two Klimisch 2 studies on the registered substance itself, N-Methyl-2thion-thiazolidin, available, one focusing on absorption and excretion, one on metabolism.
Within the first study it was found that unchanged N-Methyl-2thion-thiazolidin was only excreted with an average of 0.06% of the original dose via faeces. Via the urine, also only 0.15% of the applied dose of the unchanged substance were excreted. The given data indicate a rather fast absorption based on plasma levels and a rather fast clearance within mostly 24h from the plasma. Concentration maxima of N-Methyl-2thion-thiazolidin are with 15 resp. 90 µmol/L upon gavage of100 resp. 500 mg/kgnot very high, reflect dose relation however very well.
Based on the available data, it cannot be stated definitively whether the substance will be excreted metabolized or accumulates in the body unmetabolized, as e.g. a metabolism by gut bacteria cannot be excluded or a re-distribution into fatty tissues. However, it was determined clearly that the absorption of the substance via the oral route was rather complete, hence, the oral absorption rate is set as 100%.
The information on its fate in the body could be obtained however from metabolism studies. The given data indicate a metabolism of N-Methyl-2thion-thiazolidin to N-Methyl-2on-thiazolidin and other biotransformation products and a rather fast clearance within mostly 24h resp. 30h from the plasma via urine. This information supports 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.
Data on the absorption via the dermal or inhalative route is however not given. So an assessment of the available information, e.g. physico-chemical data, will be performed in order to estimate the lacking absorption rates, as foreseen in ECHA’s guidance document R.7c. The following physico-chemical parameters of N-Methyl-2thion-thiazolidine will be taken into account when estimating its absorption into the body:
Molecular weight = 133.235 g/mol
Water solubility = 3.65 g/L @ 30°C, pH 4.7
Partition Coefficient: log Pow = 0.59 @ 20.3°C
Vapour pressure = 1.1 x 10E-04 hPa at 20°C, 2.2 x 10E-04 hPa at 25°C and 4.0 x 10E-03 hPa at 50°C
Melting point: 70.3°C
Boiling point: 341-344°C
Particle size: Substance is a solid which is produced and used as pellets only; hence, no inhalable particles need to be regarded
Skin / eye irritation: The substance is neither classified as skin nor eye irritant.
Taking into account the handling of the substance, its distribution form, the boiling point of 341-344°C and consequently a rather low vapour pressure of 2.2 x 10E-04 hPa at 25°C, exposure to the substance is negligible. Inhalation as a gas can be completely excluded. However, as a worst case scenario, e.g. improper handling of the solid and accidental exposure, although highly unlikely, the theoretical absorption via inhalation will be estimated.
With a rather low molecular weight of 133.235 g/mol, i.e. way below 500 g/mol, the substance is generally favourable for absorption. In general, assuming accidental exposure to the solid substance, either a prolonged exposure due to deposition and subsequent absorption or immediate absorption by micellular solubilisation has to be assumed. The latter mechanism may be of particular importance for highly lipophilic compounds (LogPow >4), particularly those that are poorly soluble in water (1 mg/l or less) and is hence not relevant here. To be readily soluble in blood, a gas, vapour or dust must be soluble in water and increasing water solubility would increase the amount absorbed per breath. However, the gas, vapour or dust must also be sufficiently lipophilic to cross the alveolar and capillary membranes. Therefore, a moderate log P value (between -1 and 4) would be favourable for absorption. Generally, liquids, solids in solution and water-soluble dusts would readily diffuse/dissolve into the mucus lining the respiratory tract. Hence, with a 0.59 @ 20.3°C and a water solubility of 3.65 g/L @ 30°C, the absorption of this fraction which may not be subjected to ciliary clearance can be considered as rather high.
In summary, taking into account the fraction of N-Methyl-2thion-thiazolidine being able to reach the lower respiratory tract, its potential to be absorbed and a certain precaution due to the lack of toxicokinetic test data, the inhalative absorption can be estimated to be 100% as a worst case assumption.
In order to cross the skin, a compound must first penetrate into the stratum corneum and may subsequently reach the epidermis, the dermis and the vascular network. The stratum corneum provides its greatest barrier function against hydrophilic compounds, whereas the epidermis is most resistant to penetration by highly lipophilic compounds. Substances with a molecular weight below 100 are favourable for penetration through the skin and substances above 500 g/mol are normally not able to penetrate. The substance must be sufficiently soluble in water to partition from the stratum corneum into the epidermis. Therefore, if the water solubility is below 1 mg/L, dermal uptake is likely to be low. Additionally, logPow values between 1 and 4 favour dermal absorption.
Above 4, the rate of penetration may be limited by the rate of transfer between the stratum corneum and the epidermis, but uptake into the stratum corneum will be high. If the substance is a skin irritant or corrosive, damage to the skin surface may enhance penetration. During the whole absorption process into the skin, the compound can be subject to biotransformation.
In case of N-Methyl-2thion-thiazolidine, an evaporation after skin contact does not need to be regarded due to the boiling point and low vapour pressure, and hence it can be assumed that the substance will remain on the skin until mechanical removal. Furthermore, since the substance is neither corrosive nor a skin irritant, additional absorption-enhancing effects can be disregarded, too.
The molecular weight is with 133.235 g/mol rather low, which in general indicates a certain potential to penetrate the skin. The logPow is only slightly below the guidance value of 1, i.e. 0.59 @ 20.3°C, and the water solubility of 3.65 g/L @ 30°C and pH 4.7 is definitively present, but not too excessive. So N-Methyl-2thion-thiazolidine is expected to be able to penetrate not only the epidermis, but also the stratum corneum to a non-negligible extent and hence to be absorbed via the skin.
Therefore, a distinct dermal absorption rate can be assumed, most reasonably a resulting maximum dermal penetration of N-Methyl-2thion-thiazolidine of 100% as a worst case, although it may be practically not be reached, which is compliant with i.a. ECHA’s Guidance documents and scientifically reasonable when e.g. performing route-to-route extrapolations during risk assessment.
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