Ethyl methyl sulphide

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

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:

other: Study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

metabolism

Principles of method if other than guideline:

Study of the in vitro metabolism of EMS and EMSO by rat microsomal fractions, and the involvement of cytochrome P450 and/or FMO in the S-oxidation of EMS and EMSO and delineates the role of particular cytochrome P450 isozymes in the S-oxidation of EMSO.

GLP compliance:

no

Radiolabelling:

no

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Bantin and Kinman Ltd (Aldbrough, UK)
- Age at study initiation: no data
- Weight at study initiation: 250± 30 g
- Fasting period before study:
- Housing: no data
- Individual metabolism cages: yes/no
- Diet: standard laboratory diet (RMEl) from SDS Ltd (Witham, UK) ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20
- Humidity (%): 50
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Details on study design:

Animal treatment and preparation of hepatic microsomes:
Animals were divided into groups of six rats each and treated with the following compounds once each day for 3 consecutive days: ß-naphthoflavone
(100 mg/kg), 3-methylcholanthrene (25 mg/kg) in corn oil, phenobarbital (80 mg/kg) in saline, dexamethasone (100 mg/kg) in distilled water containing 2% Tween 80 and clofibrate (200 mg/kg) in saline. The control groups were treated with the corresponding vehicle alone. The animais were starved ovemight before tissue preparation. All animals were sacrificed by cervical dislocation and the livers immediately excised for liver microsomes preparion by a standard ultracentrifugation method. The microsomal protein content was estimated by the colorimetrie method of Lowry et al. ( 1951 ). The difference between the absorbance at 450 and 490 nm of the sodium dithionite (5 mg) reduced microsomal sample and the reduced carbon monoxide complexed sample was measured and used to estimate the microsomal cytochrome P450 content.

Incubation procedures:
Incubations were carried out in duplicate with liver microsomes from the untreated and pretreated rat. Metabolism was initiated by the addition of
microsomal tissue and terminated after 0, 10, 20, 30 min or 1 h by adding NaOH (0.5 ml, 0.1 M) to the incubation mixture. Incubations were also carried out with microsomes from untreated animais in the presence of various metabolic inhibitors and activators using EMS or EMSO as substrate.

EMS and the internai standard (dimethyl sulphide) were extracted by a headspace procedure and separated satisfactorily using a column packed with 4% Carbowax 20 M/0.8% KOH on Carbopack B. EMSO, EMS02 and the internai standard n-propyl sul phone were separated satisfactorily using a 2-m column packed with 10% Carbowax 20 M on Chromosorb.

Gas chromatographie (GC) methods for the analysis of ethyl methyl sulphide (EMS) and its corresponding sulphoxide (EMSO) and sulphone (EMS02) in rat microsomes were developped.

Under the optimum conditions (incubation of 10 min and microsomal protein content of ca. 4 mg/ml), 10% of the initial EMS concentration (2.5 mM) was converted to the corresponding sulphoxide in rat liver microsomal incubations. However, < 0.1% of the sul phone was detected when rat liver microsomes were incubated with EMS. Similarly, 2.5% of the initial EMSO concentration (2.5 mM) was converted to the corresponding sulphone by rat liver microsomes (ca. 4 mg/ml protein) during an incubation of 30 min. However, no EMS was detected after incubation with EMSO under these conditions.

The estimated apparent Vmax and Km for the sulphoxidation of EMS were 3.8 ± 0.02 nmol/mg protein/min and 1.9±0.10 mM respectively. Vmax1, Vmax2 and Km1 and Km2 for the S-oxidation of EMSO were 0.5±0.01 and 0.2±0.01 nmol/mg protein/min and

0.7 ± 0.02 and 0.1 ± 0.00 mM respectively.

Studies with selective inducers and inhibitors of microsomal monooxygenases indicated that the sulphoxidation of EMS is mediated rnainly by FMO, whereas both FMO and cytochrome P450 are involved in the S-oxidation of EMSO.

Executive summary:

The in vitro metabolism of ethyl methyl sulphide (EMS) and ethyl methyl sulphoxide (EMSO) was evaluated in rat microsomal fractions. Under the optimum conditions (incubation of 10 min and microsomal protein content of ca. 4 mg/ml), 10% of the initial concentration (2.5 mM) was converted to the corresponding sulphoxide in rat liver microsomal incubations. However, < 0.1% of the ethyl methyl sulphone was detected when rat liver microsomes were incubated with. Similarly, 2.5% of the initial EMSO concentration (2.5 mM) was converted to the corresponding sulphone by rat liver microsomes (ca. 4 mg/ml protein) during an incubation of 30 min. However, no EMS was detected after incubation with EMSO under these conditions. The estimated apparent Vmax and Km for the sulphoxidation of were 3.8 ± 0.02 nmol/mg protein/min and 1.9±0.10 mM respectively. V_max1, V_max2 and K_m1 and K_m2 for the S-oxidation of EMSO were 0.5±0.01 and 0.2±0.01 nmol/mg protein/min and 0. 7 ± 0.02 and 0.1 ± 0.00 mM respectively. Studies with selective inducers and inhibitors of microsomal monooxygenases indicated that the sulphoxidation ofis mediated mainly by FMO, whereas both FMO and cytochrome P450 are involved in the S-oxidation of EMSO.

Reference 2

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: Study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

metabolism

Principles of method if other than guideline:

- characterization of the pharmacokinetics of EMS at different doses following intravenous and oral administration,
- evaluation of modifications of the pharmacokinetics of EMS generated by placing male wistar rats on chemically defined diets?
- evaluation of metabolite pharmacoklnetics in control rats and rats maintained on the synthetic diet after administration of EMS or the S-oxygenated metabolites metabolites.

GLP compliance:

no

Radiolabelling:

no

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Bantin and Kingman Limited (Aldbrough, Hull, U.K.)
- Age at study initiation: no data
- Weight at study initiation: 230-280g
- Fasting period before study: no data
- Housing: no data
- Individual metabolism cages: yes/no
- Diet: normal rat chow or synthetic diet [47% dextrln (type 2), 23% sucrose, 10% corn oil, 14% amino acids and supplemented with vitamins and minerals], ad libitum
- Water (e.g. ad libitum): no data
- Acclimation period: at least 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20
- Humidity (%): 50
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

other: oral and iv.

Vehicle:

not specified

Duration and frequency of treatment / exposure:

Single administration

Remarks:

Doses / Concentrations:
EMS: 40 mg/kg (orally); 10, 20 and 40 mg/kg (intravenously)
EMSO: 5 mg/kg (intravenously)
EMSO2: 15 mg/kg (intravenously)

No. of animals per sex per dose / concentration:

6

Control animals:

no

Details on study design:

The rats (n = 6) were surgically prepared under hypnorm/hypnovel anaesthesia by implanting indwelling cannula into the jugular veln and the carotid
artery, 24 h prior to drug administration and blood sampllng. EMS, EMSO or EMS02 were admlnistered as a single bolus dose through the jugular veln or orally by gavage.

Details on dosing and sampling:

Arterial blood samples (250 ml ??) were collected in heparinized tubes pre-dose and post-dose at 5, 15, 30, 45 mins, and at 1,2, 3,4,5, 6, 7, 8 and24 h.

Blood Jevels of EMS were analysed by a sensitive headspace gas chromatographic assay. The collected blood (250 ml) was placed in 5 ml serum bottles. The internal standard, dimethyl sulphide (25 ml of 2S mg/ml), was added and this was followed by addition of potassium carbonate (0.1 g). The stoppered vials were then vortex mixed and heated at 80°C for 20- 30 mins in a water bath. An aliquot (5 ml) of the headspace generated was injected directly onto the GLC column (2 m x 0.4 cm, 4% Carbowax 20M/0.8% w/w KOH on Carbopack B).
Blood levels of the sulphoxide and sulphone were monitored by a separate gas chromatography assay. To blood samples already analysed by headspace GLC, Sodium hydroxide (50 µl, 0.1 M), the internai standard, n-propyl sul phone (10 µl,4 mg/ml), and sodium chloride (0.1 g) were added. Samples were then extracted with dichloromethane (2 x S ml). The pooled extracts were evaporated to dryness and reconstituted in 20 µl of methanol. Aliqutos (5µl) of the reconstituted extracts were injected onto the GLC column (10% Carbowax 20 M on Chromosorb W (HP), 2 m x 0.4 cm).

Statistics:

Model independent pharmacokinetic parameters were calculated with the computer program Toplit veniion 1.0 using the standard non-compartmental equations.

One way analysis of variance ( ANOV A) was used to obtain probabllity values (p) and compare different treatment groups. A p value of less than 0.05 was considered statistically significant.

Details on absorption:

EMS (20 mg/kg, iv) was rapidly eliminated from rat blood with a terminal half-life of 0.14 h and was not detectable 1 h after administration. EMSO was detected in rat blood between 0.083 and 5 h. The blood levels of EMSO peaked at about 1 h and then declined with a slope that was shallower than that of the parent EMS. The appearance of detectable blood levels of EMSO2 was delayed to about 0.5 h after intravenous administration of EMS. The blood levels of EMSO2 then increased reaching a peak between 6 and 7 hours after administration of EMS and persisted in the systemic circulation 24 h post-dose.
The pharmacokinetics of EMS was linear over the dose range investigated.
The absorption of EMS after oral administration was rapid and the oral bioavailability was extensive (75%). After oral administration of EMS (40 mg/kg) the maximum blood concentration (Cmax.) was attained within the first 0.083 h of blood sampling and then dec1ined monoexponentially.

Metabolites identified:

yes

Details on metabolites:

Ethylmethylsulphoxide (EMSO)
Ethylmethylsulphone (EMSO2)

Executive summary:

Ethyl methyl sulphide (EMS), a simple dialkyl sulphide, is oxidized to the corresponding sulphoxide (EMSO) and sulphone (EMS02) derivatives both in vitro and in vivo. Two distinct enzymatic pathways appear to be involved in this sulphoxidation process; the flavin-containing monooxygenasen (FMO) is largely responsib1e for the S-oxidation of EMS to its sulphoxide while both cytochrome P-450 and FMO are involved in the further oxidation of the sulphoxide to the sulphone. The pharmacokinetics of EMS and its sulphoxide and sulphone metabolites were examined in male Wistar rats placed on normal rat chow and those placed on a synthetic diet. Blood levels of EMS were analysed by a sensitive headspace gas chromatography assay. A separate gas chromatography assay was developed to monitor the blood levels of EMSO and EMSO2.. The pharmacokinetics of EMS in control rats were linear from 10 to 40 mg/kg dose range. The blood concentration-time profile of EMS declined monoexponentially, EMS was rapidly eliminated from rat blood with a terminal half-life of 0.14h and was not detectable 1 h after administration. Following intravenous administration of EMSO (5 mg/kg), the blood concentration-time profile of EMSO declined with a terminal half -live (t1/2) of 1.46 h, about ten times longer than that of the parent sulphide. After administration of EMSO2 (15 mg/kg), the sulphone was metabolically stable and was eliminated very slowly from the blood. The in vivo disposition of EMS and EMSO were clearly altered in rats maintained on a synthetic diet following administration of EMS or EMSO. The pharmacokinetic data were consistent with a diminished drug oxidising capacity in rats placed on the synthetic diet and could serve as a useful probe for monitoring the regulation of FMO in animals.

Reference 3

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: Study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

excretion

metabolism

Principles of method if other than guideline:

The formation and excretion of the sulphoxides and sulphones of EMS in urine following administration of EMS in rats were examined, and the effects of the CYP inducers (phenobarbitone and ß-naphthoflavone) and FMO modulator (methimazole) on urinary excretion of the sulphoxides and sulphones were also evaluated in rats.
marker sulphides were also evaluated in rats.

GLP compliance:

no

Radiolabelling:

no

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories (Wilmington, MA, USA).
- Age at study initiation: no data
- Weight at study initiation: 230-280 g
- Fasting period before study: no data
- Housing: no data
- Individual metabolism cages: yes/no
- Diet : ad libitum
- Water: ad libitum
- Acclimation period: at least 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20
- Humidity (%): 50
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Duration and frequency of treatment / exposure:

Single administration

Remarks:

Doses / Concentrations:
50 mg/kg bw

No. of animals per sex per dose / concentration:

4

Control animals:

yes, concurrent vehicle

Details on study design:

Male Wistar rats in groups of four were pretreated with phenobarbitone (80 mg/kg/day in saline) intraperitoneally (i.p.), ß-naphthoflavone (100 mg/kg/day in corn oil, i.p.) or methimazole (50 mg/kg/day in saline) for three consecutive days. Rats in the control groups received an equivalent amount of the vehicles alone (1 mL/kg). On the fourth day, EMS (50 mg/kg in corn oil, 500 µL) was administered orally to the appropriate group of
rats pretreated with PB, ßNF, methimazole, saline or corn oil.

Details on dosing and sampling:

The animals were placed individually in glass metabolic cages and urine samples, free of faeces, collected at 24 h intervals (0-24, 24-48, 48-72 and 72-96 h} into tubes supported in Dewar flasks containing liquid nitrogen to prevent nonenzymic degradation of metabolic products. The urine samples were stored at -20°C until analysis.

Measurement of EMS and metabolites levels (GC):
The levels of EMS and its sulphoxide and sulphone in the urine from treated and untreated rats were monitored by a modified gas chromatography (GC) assay method. Urine levels of EMS were analysed by a sensitive headspace GC assay.

Identification of EMS metabolites (GCMS):
GC-MS was used to identify the sulphoxides and sulphones of EMS and CPMS in rat urine.

Assay validation:
To determine the precision and accuracy of the assays, known concentrations of EMS (0.5, 2 and 10) and its metabolites (2, 10 µg/mL and 40µg/mL) were spiked into blank rat urine and taken through the analytical procedure. The study was repeated on five separate occasions and the coefficient of variation (CV), a measure of precision and the mean percentage difference, MD(%), a measure of accuracy, were calculated. In order to determine the stability of EMS and its sulphoxides and sulphones in rat urine before extraction, aliquots (10 µg/mL) of the analytes were spiked into extraction tubes containing blank. rat urine (2 mL). The tubes were stored at 4 or -20°C before analysis. The tubes were analysed at various time intervals (up to 12 weeks) to determine the stability of the analytes. Prior to analysis, the tubes were spiked with the appropriate internai standard and taken through the analytical procedure.

Details on excretion:

The elimination of EMSO and EMSO2 was slow in male rats treated with EMS (50 mg/kg). After administration of EMS in rats, only 16% of the dose was accounted for in the urine samples collected over 96 h. The parent EMS was not detected in the urine and only a trace of EMSO ( <0.5%) was detected, mainly in the 0-24 h urine sample. EMSO2 was the major S-oxidation product in the urine of rats treated with EMS. Pretreatment of rats with PB or ßNF before oral administration of EMS did not significantly change the levels of the S-oxygenated metabolites excreted in rat urine (Table 2).
Pretreatment of rats with methimazole significantly decreased the S-oxidation of EMS in vivo. EMSO was not detected in the urine of rats pretreated with methimazole before administration of EMS while the amount of EMSO2 recovered in the urine of rats pretreated with methimazole was reduced to about one-third compared with the control group .

Metabolites identified:

yes

Details on metabolites:

Ethylmethylsulphoxide (EMSO) and ethylmethylsulphone (EMSO2)

The limit of quantification was 0.01 µg/mL for EMS in rat urine. The limit of quantification was 0.1 µg/mL for EMSO and EMSO2 in rat urine. EMSO and EMSO2 were stable for at least 3 months in rat urine stored at -20°C. In contrast, significant loss of EMS in rat urine was observed in samples stored for longer than 7 days at -20°C, probably due to the volatile nature of this compound.

Conclusions:

The results of this investigation indicate that EMS is oxidized to its corresponding sulphones, via a sulphoxide in rats. The low levels of the sulphoxides detected in urine suggest that the sulphoxides are short-lived in rats.

Executive summary:

In this investigation, the measurement and identification of the S-oxidation products of ethyl methyl sulphide (EMS) (and 4-chlorophenyl methyl sulphide (CPMS) and diphenyl sulphide (DPS), data not reported) in rat urine were carried out and a study of the effects of phenobarbitone (PB), ß-naphthoflavone ( ßNF) and methimazole on the urinary levels of their metabolites was conducted. Male Wistar rats (n = 4) were pretreated with PB (80 mg/kg/day in saline, i.p.), ßNF (100 mg/kg/day in corn oil, i.p.), methimazole (50 mg/kg/day in saline, i.p.) or the vehicles alone (1 mL/kg) for three consecutive days. After pretreatment, EMS (50 mg/kg in corn oil, 500 µL) was administered orally to the appropriate groups of rats. The animals were placed in metabolic cages and urine samples collected at 24 h intervals over 96 h. Chromatographie and spectroscopie techniques were used for the measurement and identification of the sulphoxides and sulphones of EMS in rat urine. Although only a trace of ethyl methyl sulphoxide (EMSO) was present in rat urine after administration of EMS, ethyl methyl sulphone (EMSO2) accounted for about 16% of the administered dose in the urine of male rats given EMS. In addition, pretreatment of rats with methimazole significantly decreased the S-oxidation of EMS. These results indicate that microsomal monooxygenases mediate the S-oxidation of EMS to its corresponding sulphone via a transient sulphoxide in rats.

Description of key information

Based on the data available, it is assumed that methylethylsulphide (MES) is extensively absorbed by the oral and inhalation routes. Due to its low boiling point (64°C), dermal absorption is assumed to be limited due to a rapid volatilization. MES is metabolised in the rat via S-oxidation to the sulphoxide (MESO) and to the sulphone (MESO2). The sulphoxide and sulphone are physiologically stable, and for the most part excreted unchanged.

Absorption

The assessment of the absorption profile of MES is based on the available toxicological data and the physicochemical properties as suggested by the REACH Guidance Chapter R.7c:

Molecular weight: 76.16 g/mole 

Vapeur pressure: 21.77 kPa @ 20°C

Water solubility: 6680 mg/L at 20°C 

Partition coefficient log Kow = 1.54

Oral

The low molecular weight, high water solubility and moderate log Kow are in favor of a significant absorption of MES by the oral route. Using a model to predict either high or low fraction absorbed for an orally administered, passively transported substance, the rates of absorption of DMS were 90% for a dose of 1 and 1000 mg (Danish QSAR). The absorption of 40 mg/kg of MES after oral administration was rapid and the oral bioavailability was extensive (75%) (Nnane et al., 2001).

Inhalation

The low molecular weight, high vapor pressure, high water solubility, moderate log Kow and mortality in the mortality in the acute inhalation toxicity study (Tansi, 1981) are in favor of a significant absorption of DMS by inhalation exposure. Therefore, according to the REACH Guidance, a default value of 100% inhalation absorption will be used.

Dermal

EMS is a highly volatile liquid, therefore absorption across the skin is limited by the rate at which the liquid evaporates off the skin surface.The rate of absorption of EMS was estimated using the IH SkinPerm model using a Kp derived from the EPI Dermwin model. For an instantaneous deposition of 1000 mg over 1000 cm² of skin or a deposition over time of 1 mg/cm²/h, MES is virtually not absorbed after 8 hours. Therefore, according to the REACH Guidance, a default value of 10% skin absorption will be used.

Metabolism

An extensive review of the metabolism of alkyl sulphides, sulphoxides/sulphones and sulphonates was performed by EFSA (2012). Once alkyl sulphides enter systemic circulation, they are rapidly oxidised to sulphoxides, and, depending on the structure of the sulphide, may be further oxidised to the sulphone (Figure 1). The products of S-oxidation reactions may react spontaneously with glutathione, and it is likely that they also exhibit reactivity towards nucleophilic sites in cellular macromolecules. The S-reaction is favoured by the presence of a lone reactive pair of electrons on divalent sulphur in monosulphides as shown by the excretion in the urine of dimethyl sulphoxide (DMSO) and dimethyl sulphone (DMSO₂) and to the expiration of a malodorous material (presumably DMS) after DMS subcutaneous administration to rabbits (Williams et al., 1966).
Figure 1. Biotransformation of sulphides substances.

SEE ATTACHED DOCUMENT

Although S-oxidation generally yields mixtures of sulphone and sulphoxide metabolites, the relative amounts of excretion products are dependent upon the polarity of the sulphide. In rats, polar aliphatic sulphides give rise to higher proportion of the sulphoxide metabolites (Damani, 1987). This is probably due to the water-solubility of the sulphoxides, which presumably limits their partitioning into the catalytic sites on the microsomal monooxygenase systems (P450 and FMO), involved in the S-oxidation reaction (Damani, 1987). The first oxidation from sulphide to sulphoxide is reversible, whereas the sulphone group is stable and is not reduced back to the sulphoxide; this latter irreversibility seems to be related to the substrate specificity of the reductase (Renwick, 1989).

EMS is oxidized to the corresponding sulphoxide (EMSO) and sulphone (EMSO₂) derivatives both in vitro and in vivo. Two distinct enzymatic pathways appear to be involved in this sulphoxidation process; the flavin-containing monooxygenase (FMO) is largely responsible for the S-oxidation of EMS to its sulphoxide while both cytochrome P-450 and FMO are involved in the further oxidation of the sulphoxide to the sulphone.

The in vitro metabolism of ethyl methyl sulphide (EMS) and ethyl methyl sulphoxide (EMSO) was evaluated in rat microsomal fractions (Nnane and Damani, 1999). Under the optimum conditions (incubation of 10 min and microsomal protein content of ca. 4 mg/ml), 10% of the initial EMS concentration (2.5 mM) was converted to the corresponding sulphoxide in rat liver microsomal incubations. However, < 0.1% of the EMSO₂was detected when rat liver microsomes were incubated with EMS. Similarly, 2.5% of the initial EMSO concentration (2.5 mM) was converted to the corresponding sulphone by rat liver microsomes (ca. 4 mg/ml protein) during an incubation of 30 min. However, no EMS was detected after incubation with EMSO under these conditions. The estimated apparent Vmax andKmfor the sulphoxidation of EMS were 3.8 ± 0.02 nmol/mg protein/min and 1.9±0.10 mM respectively. V_max1, V_max2and K_m1and K_m2 for the S-oxidation of EMSO were 0.5±0.01 and 0.2±0.01 nmol/mg protein/min and 0. 7 ± 0.02 and 0.1 ± 0.00 mM respectively. Studies with selective inducers and inhibitors of microsomal monooxygenases indicated that the sulphoxidation of EMS is mediated mainly by FMO, whereas both FMO and cytochrome P450 are involved in the S-oxidation of EMSO.

The measurement and identification of the S-oxidation products of ethyl methyl sulphide (EMS) in rat urine were carried out and a study of the effects of phenobarbitone (PB), ß-naphthoflavone (ßNF) and methimazole on the urinary levels of their metabolites was conducted (Nnane et al., 2005). Male Wistar rats (n = 4) were pretreated with PB (80 mg/kg/day in saline, i.p.),ßNF (100 mg/kg/day in corn oil, i.p.), methimazole (50 mg/kg/day in saline, i.p.) or the vehicles alone (1 mL/kg) for three consecutive days. After pretreatment, EMS (50 mg/kg in corn oil, 500 µL) was administered orally to the appropriate groups of rats. The animals were placed in metabolic cages and urine samples collected at 24 h intervals over 96 h. Chromatographic and spectroscopic techniques were used for the measurement and identification of the sulphoxides and sulphones of EMS in rat urine. Although only a trace of EMSO was present in rat urine after administration of EMS, EMSO₂ accounted for about 16% of the administered dose in the urine of male rats given EMS. In addition, pretreatment of rats with methimazole significantly decreased the S-oxidation of EMS. These results indicate that microsomal monooxygenases mediate the S-oxidation of EMS to its corresponding sulphone via a transient sulphoxide in rats.

Sulphides are sufficiently lipophilic to be efficiently absorbed from the gastrointestinal (GI) tract. Ethylmethyl sulphoxide (EMSO) and ethylmethyl sulphone (EMSO₂) are excreted in the urine as metabolites of EMS administered orally to rats (Nnane et al., 2005).

Toxicokinetics

The pharmacokinetics of EMS and its sulphoxide (EMSO) and sulphone (EMSO₂) metabolites were examined in male Wistar rats (Nnane et al., 2001). Blood levels of EMS were analysed by a sensitive headspace gas chromatography assay. A separate gas chromatography assay was developed to monitor the blood levels of EMSO and EMSO₂. EMS was administered orally by gavage as a single bolus dose of 40 mg/kg bw. Arterial blood samples were collected in heparinized tubes pre-dose and post-dose at 5, 15, 30, 45 mins, and at 1,2, 3,4,5, 6, 7, 8 and 24 h. The typical mean blood concentrations-time profiles of EMS, EMSO and EMSO₂ after administration of EMS are shown in figure 2.
Figure 2. Mean blood concentration or EMS, EMSO and EMSO₂ after oral administration or EMS (40 mg/kg) to male Wistar rats. Values represent the means (n = 6).

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The absorption of EMS after oral administration was rapid and the oral bioavailability was extensive (75%). The maximum blood concentration (Cmax) of EMS was attained within the first 5 mins of blood sampling and then decined monoexponentially (Figure 2). EMS was rapidly eliminated from rat blood with a terminal half-life of 0.14 h and was not detectable 1 h after administration. EMSO was detected in rat blood between 5 mins and 5 h. The blood levels of EMSO peaked at about 1 h and then declined with a slope that was shallower than that of the parent EMS. The appearance of detectable blood levels of EMSO₂ was delayed to about 0.5 h. The blood levels of EMSO₂ then increased reaching a peak between 6 and 7 hours after administration of EMS and persisted in the systemic circulation 24 h post dose.

Intravenous administration of EMSO (5 mg/kg) and EMSO₂ (15 mg/kg) to normal male rats was also carried out to determine their pharmacokinetic parameters. The blood concentration-time profile of EMSO declined exponentially with a terminal half-life (t_1/2) of 1.46 h, about ten times longer than that of the parent sulphide. EMSO was largely oxidised to the sulphone but no reduction to the sulphide (EMS) was observed. EMSO₂ accumulated and then persisted in the circulation for up to 24 h after administration of EMSO. After administration of EMSO₂ (15 mg/k.g), the sulphone was metabolically stable and was eliminated very slowly from the blood. Its total clearance was extremely low compared to that of its precursors.

The pharmacokinetic parameters of EMS, EMSO and EMSO₂ in male Wistar rats following intravenous administration of EMS (20 mg/kg) are presented in the following Table.

Table: Pharmacokinetic parameters of EMS, EMSO and EMSO₂ in male Wistar rats after intravenous dosing of EMS (20 mg/kg).

Parameters	EMS	EMSO	EMSO2
Cmax(mg.ml^-1)	-	6.85 ±0.48	12.13±0.55
Tmax(h)	-	0.82 ±0.12	6.17±0.75
AUC (µg.ml^-1.h)	0.93 ±0.30	26.66±5.76	711.8± 121.4
kel(h^-1)	5.96±2.31	0.26±0.093	0.0165 ± 0.002
t1/2(h)	0.14 ±0.04	2.59±0.62	42.74±5.74
Vd(L kg^-1)	4.06 ±0.69	-	-
Cl (ml.min^-1kg^-1)	398.8±150	-	-

Values represent mean± standard deviation (n = 6).
C_max: maximum plasma. Concentration
T_max: time to reach peak plasma concemration
AUC: extrapolated area under the plasma concentration versus time curve
K_el: elimination rate constant
t_1/2: elimination half-life
V_d: volume of distribution
Cl: clearance

The blood concentration-time profile of EMS declined with a terminal half-live (t_1/2) of 0.14 h, compared to 2.59 and 42.74 hours for EMSO and EMSO₂, respectively. The internal exposure to EMS, as reflected by an AUC of 0.93 µg.ml^-1.h, is insignificant when compared to the AUC of 26 and 711 µg.ml^-1.h for EMSO and EMSO₂, respectively.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

75

Absorption rate - dermal (%):

10

Absorption rate - inhalation (%):

100