Dimethyl disulphide

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Objective of study:

metabolism

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

To identify the number and proportions of its metabolites, dimethyl disulphide was incubated with fresh rat hepatocytes. Samples were removed after 0, 1, 2 and 4 hours and quenched in liquid nitrogen. All samples were analysed by GC-MS.

GLP compliance:

no

Remarks:

Although a claim of GLP compliance has not been made for this study, the laboratory procedures were conducted in accordance with the current GLP requirements of the UK MHRA and OECD.

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl disulphide

EC Number:

210-871-0

EC Name:

Dimethyl disulphide

Cas Number:

624-92-0

Molecular formula:

C2H6S2

IUPAC Name:

(methyldisulfanyl)methane

Test material form:

other: liquid

Details on test material:

Identity: Dimethyl disulphide
Chemical name: 2,3-Dithiabutane
Synonym: DMDS
Chemical Structure: CH3-S-S-CH3
Molecular formula: C2H6S2
Molecular weight: 94.20 g/mol
Batch: D58107
Purity: 99.8%.

Radiolabelling:

no

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Administration / exposure

Route of administration:

application in vitro

Vehicle:

other: incubation medium

Duration and frequency of treatment / exposure:

up to 4 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

588 other: nmol/L

Doses / concentrations 2

Dose / conc.:

177 other: nmol/L

Details on study design:

Dissolution of Test Substance
Stock solutions of Dimethyl disulphidewere prepared in incubation medium. The actual concentration of the formulation was determined after the incubation.
Reagents
General purpose reagents and solvents were of Analar grade (or a suitable alternative) and were obtained principally from VWR International Ltd, Rathburn Chemicals Ltd, Aldrich Chemical Company Ltd and Vickers Laboratories Ltd. Suitable liquid scintillants were obtained from Perkin Elmer Life Sciences Ltd.
Equipment
Radio-HPLC was performed on an Agilent 1100 chromatographic system with a Packard FSA 525TR radio-detector. HPLC data were captured on line with Laura software (version 3.4.7.52 SP8).
Test System
Source
Liver samples were obtained from one male and one female Sprague-Dawley rat. Hepatocytes were prepared by a two-step collagenase perfusion method.
The source of all tissue was recorded in the raw data and documented in the final report. The test system was identified by means of a label containing information such as species, lot number etc.
Cell Viability and [14C]7-Ethoxycoumamarin Metabolism
Cell viability of the hepatocyte suspensions was estimated by trypan blue exclusion. Metabolic capacity of the suspension cultures was determined by quantification of [14C]7-ethoxycoumarin metabolism.
Experimental Procedures
Cell Culture Medium
Leibovitz L-15 medium, pre-warmed toca.37°C, was used for all incubations.
Control matrix analysis
A sample of the control rat hepatocyte matrix (1 x 106cells/mL) was supplied to the Department of Environmental Sciences for analysis along with the incubation samples.
Dimethyl disulphide Incubations
All incubations and GC analysis was carried out in Headspace vials.
Dimethyl disulphide was incubated at a nominal concentration of 10 µmol/L with freshly prepared rat hepatocytes in suspension in Leibovitz L-15 medium (1 x 106cells/mL). Incubations were performed atca.37°C in a shaking water bath and terminated at 0, 1, 2 and 4 hours. The incubations were terminated by samples being frozen in liquid nitrogen. A blank incubation was carried out with either Leibovitz L-15 medium in the place of hepatocytes or in the place of DMDS.
Eight replicates at each time point were generated and supplied to the Department of Environmental Sciences and analysed by GC-MS.
[14C]7-Ethoxycoumarin Incubations
[14C]7-Ethoxycoumarin, at a nominal concentration of 50 µmol/L, was incubated in a suspension of freshly prepared rat hepatocytes at a nominal cell density of 1 x 106viable cells/mL in cell culture medium. Incubations were performed atca37°C in a shaking water bath and terminated after 0 and 4 hours. All incubations were carried out in duplicate.
The formation of 7-hydroxycoumarin and the corresponding glucuronide and sulphate conjugates was quantified by radio-HPLC.

Details on dosing and sampling:

sample analysis
Determination of 7-Ethoxycoumarin Metabolism
The formation of 7-hydroxycoumarin and the corresponding glucuronide and sulphate conjugates in the hepatocyte incubations was estimated by radio-HPLC.
Chemical Analysis
The concentration of Dimethyl disulphide and potential metabolites (dimethyl sulphide, dimethyl sulfone, dimethyl sulfoxide and methyl mercaptan) in the hepatocyte samples was measured using GC-MS.

Results and discussion

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

Dimethyl disulphide was metabolised extensively in fresh rat hepatocytes with the parent molecule concentrations of 66.0 and 92.3 nmol/L after 4 hours for replicate 3 and 4, respectively. As the amount of dimethyl disulphide decreased, the levels of metabolites were observed to increase at all time points
Two metabolites, dimethyl sulphide (DMS) and methyl mercaptan, were observed at measurable levels, greater than background levels in fresh rat hepatocytes after incubation for 4 hours. The major metabolite was attributed to methyl mercaptan with concentrations of 472 and 738 nmol/L at 4 hours in replicate 3 and 4, respectively.
Notably, the formation of the oxidation products of DMS, dimethylsulfoxide (DMSO) and dimethylsulfone (DMSO2)was not observed during the incubation with fresh rat hepatocytes.

Any other information on results incl. tables

Hepatocyte Viability and Metabolic Capacity

The cell viabilities of the fresh hepatocytes as measured by trypan blue exclusion were 71% and 67% in male and female rat, respectively. The cells were considered acceptable for use and were pooled prior to use.

Phase I metabolic capacity was assessed by the quantitative appearance of 7-hydroxycoumarin following incubation of [¹⁴C]7-ethoxycoumarin in suspensions of hepatocytes from rats (male and female pooled). Phase II activity was assessed by the quantitative appearance of the corresponding glucuronide and sulphate conjugates. The metabolic capacity assessment is shown in Table1. All hepatocyte preparations were found to have acceptable Phase I and Phase II metabolic capacity when compared to historical data.

Concentration of Dimethyl Disulphide in Hepatocyte incubations

The concentration of dimethyl disulphide was 588 and 177 nmol/L in the incubations with rat hepatocytes from replicate 3 and 4, respectively. This was lower than the anticipated concentration due to the volatile nature of handling the test compound. However, the concentration achieved still allowed the evaluation of the formation of the metabolites of dimethyl disulphide.

Incubation of Rat Hepatocytes in the Absence of Dimethyl Disulphide

Control incubations containing buffer instead of dimethyl disulphide were performed (Table2).

Following incubation of rat hepatocytes in the absence of dimethyl disulphide three peaks were observed at retention times corresponding to dimethyl disulphide, dimethyl sulphide and methyl mercaptan. Methyl mercaptan was the most abundant substance, whereas the 2 other metabolites were present at low levels. Therefore the concentrations in the control incubations were subtracted from the corresponding concentrations of parent and metabolites in the incubations with dimethyl disulphide and hepatocytes.

Incubation of Dimethyl Disulphide in the Absence of Hepatocytes

Control incubations containing buffer instead of hepatocytes were performed (Table3).

Following incubation of dimethyl disulphide in the absence of hepatocytes for 4 hours only dimethyl disulphide and methyl mercaptan were detected in the incubate samples.

As dimethyl disulphide is hydrolytically stable it is likely that methyl mercaptan was formed during the analysis, however, this could not be confirmed from data.

Incubation of Dimethyl Disulphide with Rat Hepatocytes

Dimethyl disulphide was metabolised extensively in fresh rat hepatocytes with the parent molecule concentrations of 66.0 and 92.3 nmol/L after 4 hours for replicate 3 and 4, respectively (Table4). As the amount of dimethyl disulphide decreased, the levels of metabolites were observed to increase at all time points

Two metabolites, dimethyl sulphide (DMS) and methyl mercaptan, were observed at measurable levels, greater than background levels in fresh rat hepatocytes after incubation for 4 hours. The major metabolite was attributed to methyl mercaptan with concentrations of 472 and 738 nmol/L at 4 hours in replicate 3 and 4, respectively (Table4).

Notably, the formation of the oxidation products of DMS, dimethylsulfoxide (DMSO) and dimethylsulfone (DMSO₂₎was not observed during the incubation with fresh rat hepatocytes.

Applicant's summary and conclusion

Conclusions:

Dimethyl disulphide, dimethyl sulphide and methyl mercaptan are present in the control hepatocyte solutions. Dimethyl disulphide was metabolised rapidly after incubation with fresh rat hepatocytes for up to 4 hours. Formation of both dimethyl sulphide and methyl mercaptan was observed over the 4 hour incubation. No formation of DMSO or DMSO2 was observed during the incubation with fresh rat hepatocytes.

Executive summary:

The objective of this study was to identify the number and proportions of the metabolites generated following incubation of dimethyl disulphide with fresh rat hepatocytes using a GC-MS method. Dimethyl disulphide was incubated with fresh rat hepatocytes. Samples were removed after 0, 1, 2 and 4 hours and quenched in liquid nitrogen. All samples were analysed by GC-MS. [¹⁴C]-7-ethoxycoumarin was also incubated with rat hepatocytes suspended in Leibovitz L-15 medium. Samples were terminated after 0 and 4 hours and all samples quantified by radio-HPLC for the formation of 7-hydroxycoumarin and glucuronide and sulphate conjugates. Dimethyl disulphide was metabolised rapidly after incubation with fresh rat hepatocytes for up to 4 hours. Methyl mercaptan was the main metabolite observed over the 4 hour incubation, dimethyl sulphide (DMS) was found at a low level. No formation of the oxidation products of DMS, dimethylsulfoxide (DMSO) or dimethylsulfone (DMSO₂) was observed during the incubation with fresh rat hepatocytes.