Carbon disulphide

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February-September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Objective of study:

toxicokinetics

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Determination of the whole blood toxicokinetics of carbon disulfide (CS2) and the plasma toxicokinetics of the CS2 metabolite 2-thiothiazolidine- 4-carboxylic acid (TTCA) in male Wistar Han rats following a single oral (gavage) or inhalation administration of CS2 and to determine the routes of elimination and excretion of CS2 and TTCA in male Wistar Han rats following a single oral (gavage) or inhalation administration of CS2.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Carbon disulphide

EC Number:

200-843-6

EC Name:

Carbon disulphide

Cas Number:

75-15-0

Molecular formula:

CS2

IUPAC Name:

methanedithione

Test material form:

liquid: volatile

Details on test material:

Batch 4680300
Complies to product sepcification: >99.5%

Specific details on test material used for the study:

Date of receipt: 16 January 2017
Storage: flammable cabinet at room T

Radiolabelling:

no

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

Fifty-three male Wistar Han rats were received at Charles River Ashland on 21 Feb 2017 from Charles River Raleigh. Each rat was inspected by a qualified technician upon receipt, judged to be in good health, and immediately placed in acclimation for at least 7 days. Animals were weighed and uniquely identified by a metal ear tag displaying a permanent animal number.
During the acclimation period and the Biological Phase, each rat was observed twice daily for changes in general appearance or behavior. All animals were group-housed (2 to 4/cage) following receipt in clean, solid-bottom cages with bedding material (Bed-O-Cobs®) or other suitable material in an environmentally controlled room. The cages were cleaned and changed routinely at a frequency in accordance with the
Guide for the Care and Use of Laboratory Animals (National Research Council, 2011).
Following dosing, animals used for the Excretion Phase were placed individually into glass metabolism cages for up to 72 hours. Individual cage cards were affixed to each cage displaying the animal number, group number, and study number. Enrichment devices were provided to
each animal for environmental enrichment and to aid in maintaining the animals’ oral health. Enrichment devices were not used in glass metabolism cages as the debris interferes with the analysis of the samples. The facilities at Charles River are fully accredited by the Association
for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International).
Animals were approximately 9 weeks of age and weighed between 213 g and 270 g at the time of dosing.

Administration / exposure

Route of administration:

other: oral or inhalation

Vehicle:

other: corn oil with 5% acetone

Remarks:

only for gavage dosing

Details on exposure:

Oral exposure
The test substance formulation was administered by a single oral (gavage) administration. Oral administrations were calculated on a mg/kg body weight basis using a dosage volume of 5 mL/kg. Duplicate aliquots of each oral dose formulation were transferred to the Analytical Chemistry
Department and analyzed to assess test substance concentration using a gas chromatography (GC) method with electron capture detection.
Inhalation exposure
The test substance was administered by a single whole-body inhalation exposure. The 3-h inhalation exposure was conducted using a 500-L glass and stainless steel whole-body exposure chamber. Vapors of the test substance were generated using a bubbler-type vaporization system (250 mL gas washing bottle, Ace Glass, Inc.; Vineland, NJ) filled with an appropriate amount of liquid test substance. Compressed nitrogen was metered into the inlet stem of the gas washing bottle and bubbled through a fritted disc and the liquid test substance produced concentrated vapors of the test substance. Nitrogen to the gas washing bottle was controlled using a regulator (Model No. 8802K, Coilhose Pneumatics Inc.; East Brunswick, NJ) and metered using a needle valve and Gilmont flowmeter (No. 11, Barnant Co./Gilmont Instruments; Barrington, IL) at a flow of 55.8 cc/min. The concentrated vapors of the test substance were delivered to the exposure chamber inlet and diluted to the desired atmosphere concentration by mixing with the chamber supply air prior to entering the chamber. Nominal exposure concentration was calculated for the test substance chamber from the total amount of test substance consumed during the exposure (as weighed prior to and at the termination of the generation) and the total volume of air that passed through the chamber during exposure. Total air volume was calculated by multiplying the mean ventilation rate by the exposure duration. Analyzed exposure concentrations were determined approximately every 15 minutes using a GC. Samples were collected from the approximate animal-breathing zone of the exposure chamber via 1/8-inch Teflon® tubing. The GC was calibrated using gas-phase standards prepared to contain a known vapor concentration of the test substance in 25-L Tedlar® gas bags (Supelco Analytical; Bellefonte, PA). The GC was calibrated with standards prepared and analyzed in triplicate. A calibration curve was prepared using standards prepared at 3 vapor concentrations spanning an appropriate range relative to the target exposure concentration.

Following dosing/end of exposure, excretion phase animals were placed in glass metabolism cages for separate collection of urine and feces.

Duration and frequency of treatment / exposure:

Single oral gavage dose or 3-h whole-body inhalation exposure.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

75 mg/kg bw (total dose)

Remarks:

oral

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw (total dose)

Remarks:

oral

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw (total dose)

Remarks:

oral

Doses / concentrations 4

Dose / conc.:

150 ppm (analytical)

Remarks:

inhalation

No. of animals per sex per dose / concentration:

9 male rats per group for toxicokinetics
3 male rats per group for determinations in urine, feces and expired air (carbon trap)

Control animals:

other: No, control animals are not needed for toxicokinetics as these are not exposed to the test substance

Positive control reference chemical:

No

Details on study design:

- Dose selection rationale: based on ECHA requirement to test 3, 30 and 300 ppm in an EOGRTS study
- Rationale for animal assignment (if not random): body weight

Details on dosing and sampling:

For the Toxicokinetic Phase, blood samples were collected from 3 animals/oral group/time point at approximately 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours postdose. Blood samples were collected from 3 animals/inhalation group/time point at approximately 0.083, 0.25, 0.5, 1, 4, 6, 8, 12, and 24 hours post-end of 3-h exposure. Samples of approximately 0.75 mL were collected via the jugular vein into tubes containing K2EDTA as the anticoagulant.
For the Excretion Phase, urine was collected from 3 animals/group on wet ice at approximately 0 to 6, 6 to 12, 12 to 24, 24 to 48, and 48 to 72 hours postdose/end of exposure. Feces were collected from 3 animals/group on wet ice at approximately 0 to 12, 12 to 24, 24 to 48, and 48 to 72 hours postdose/end of exposure. The expired air from each metabolism unit was drawn through a trap containing approximately 120 mL water and through an activated carbon trap for volatile organic compounds. The activated carbon trap consisted of 2 in-line cartridges each consisting of approximately 5 g of activated carbon capped on either end by glass wool. The activated carbon trap was collected at 0 to 6, 6 to 12, 12 to 24, 24 to 48, and 48 to 72 hours postdose/end of exposure. Urine, feces, and carbon trap samples were stored frozen at approximately -70°C.

Statistics:

Not applicable

Results and discussion

Main ADME results

Type:

metabolism

Results:

Approximately 1% of the dose was recovered as the metabolite TTCA in the urine (oral and inhalation). In feces TTCA concentrations measured accounted for 0.01% of the dose or less.

Toxicokinetic / pharmacokinetic studies

Details on absorption:

After a single oral (gavage) dose of CS2 administered to rats at 75 mg/kg, the whole blood Cmax was 15,500 ng/mL, and AUClast was 188,000 h*ng/mL. The terminal elimination phase T1/2 was 11.1 hours.
After a single oral (gavage) dose of CS2 administered to rats at 150 mg/kg, the whole blood Cmax was 29,500 ng/mL, and AUClast was 459,000 h*ng/mL. The terminal elimination phase T1/2 was 16.1 hours.
After a single oral (gavage) dose of CS2 administered to rats at 300 mg/kg, the whole blood Cmax was 53,400 ng/mL, and AUClast was 766,000 h*ng/mL. The terminal elimination phase T1/2 was 14.7 hours.
After a single inhalation exposure of CS2 to rats at 150 ppm over 3 hours, the whole blood Cmax was 11,300 ng/mL, and AUClast was 110,000 h*ng/mL. The terminal elimination phase T1/2 was 22.6 hours.
Dose-normalized AUClast ranged from 2510 (h*ng/mL)/(mg/kg) to 3060 (h*ng/mL)/(mg/kg) for the oral dose groups. The narrow range of values and the absence of any trend relative to dose level indicates that the whole blood exposure to CS2 was dose-proportional. Summing the whole
blood exposure of 110,000 h*ng/mL measured after the completion of a 3-hour inhalation exposure plus the calculated exposure during the inhalation phase (Cmax 11,300 ng/mL * 3 h) results in overall CS2 exposure (AUC) of 143,900 h*ng/mL following a 150 ppm inhalation dose. Extrapolating the inhalation exposure on the plot of oral dose versus exposure indicated that the inhalation dose described is roughly equivalent to an oral dose of 30 mg/kg.
The 2-thiothiazolidine-4-carboxylic acid (TTCA) concentrations in plasma were much lower than CS2 concentrations in whole blood, with Cmax ranging from 242 ng/mL to 421 ng/mL across all groups and dose-normalized AUClast in the range of 20.6 to 27.8 (h*ng/mL)/(mg/kg) following an oral dose. Like CS2, TTCA exposure was also dose-proportional among oral doses. The terminal elimination phase T1/2 ranged from 3.86 to 6.73 hours in the oral dose groups and was 1.58 hours in the inhalation group.

Details on excretion:

Based on the excretion data, approximately 11% to 19% of the dose was recovered as CS2 in the urine and 17% to 32% of the dose was recovered as CS2 in the expired air following an oral dose administered at 75 mg/kg, 150 mg/kg, or 300 mg/kg to male rats. The amount of CS2 recovered in the air and urine increased with increasing oral dose level, suggesting saturation of binding sites and/or metabolic clearance processes at higher doses. However, there was no notable change in the fraction eliminated in either matrix (urine or expired air) at the 2 lowest doses, and a minor shift to greater pulmonary excretion at the highest dose. The CS2 in expired air was primarily excreted in the first 12 hours, while CS2 continued to be excreted in urine across the time course. Using a calculated inhalation dose of 30 mg/kg extrapolated as above, but only using the AUC after the completion of the inhalation exposure, approximately 44% of the dose was recovered as CS2 in urine and expired air, and the distribution between the 2 matrices reflected a similar pattern as observed after the oral dose.
Approximately 1% of the dose was recovered as TTCA in the urine following an oral dose, reflecting the lower circulating concentration of TTCA versus CS2 in the whole blood and plasma. Similar recovery was observed after inhalation dosing. With the exception of 1 time point, all feces samples were below the quantitation limit (BQL) for CS2. In addition, the TTCA concentrations measured accounted for 0.01% of the dose or less
across the entire 72-hour collection period, indicating that biliary excretion is not a route of CS2 or TTCA excretion in rats.
Overall recovery of CS2 and TTCA represented approximately 31% to 52% of the administered dose among all dose levels and routes of administration. Because notable concentrations of CS2 were still being eliminated in the urine at 72 hours postdose, it is likely that CS2 and metabolites remained in the rat at the termination of the study. In addition, while quantitative recovery is desired, CS2 is known to undergo multiple metabolic transformations in vivo, and these metabolites were not measured as part of this study. Potential changes in biliary excretion of CYP-related metabolites would not be detected in this experiment, but, even with apparent variations in the degree of total excretion in urine and expired air between dose groups, the relative amount excreted in each matrix was consistent and there was no indication that biliary excretion was a significant contributor to clearance of CS2 or TTCA.

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

TTCA was the only metabolite measured

Applicant's summary and conclusion

Conclusions:

The study demonstrates that systemic exposure to CS2, as evaluated by whole blood AUClast, is dose-proportional and that the distribution of CS2 and TTCA in the urine and expired air does not differ by dose or by dose route. In addition, based on the data collected for TTCA and CS2, single and repeated oral doses can be scaled to match the desired equivalent inhalation exposure.

Executive summary:

The objectives of this study were to determine the whole blood toxicokinetics of carbon disulfide and the plasma toxicokinetics of the carbon disulfide metabolite 2-thiothiazolidine-4-carboxylic acid in male Wistar Han rats following a single oral (gavage) or inhalation administration of carbon disulfide and to determine the routes of elimination and excretion of carbon disulfide and 2 -thiothiazolidine-

4-carboxylic acid in male Wistar Han rats following a single oral (gavage) or inhalation administration of carbon disulfide.

For the Toxicokinetics Phase, 3 dose groups received a single oral (gavage) dose of carbon disulfide at 75, 150, or 300 mg/kg and 1 dose group (Group 4) received a 3-hour inhalation exposure to carbon disulfide at 150 ppm. All groups consisted of 9 male Wistar Han rats. Following dosing, blood samples were collected from 3 animals/group/time point in Group 1, Group 2, and Group 3 at approximately 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours. Following the end of exposure for Group 4, blood samples were collected from 3 animals/time point at approximately 0.083, 0.25, 0.5, 1, 4, 6, 8, 12, and 24 hours.

For the Excretion Phase, 3 dose groups received a single oral (gavage) dose of carbon disulfide at 75, 150, or 300 mg/kg and 1 dose group received a 3-hour inhalation exposure to carbon disulfide at 150 ppm. All groups consisted of 3 male Wistar Han rats. Following dosing/end of exposure, animals were placed into glass metabolism cages for separate collection of urine, feces, and activated carbon trap through 72 hours.

After a single oral (gavage) dose of carbon disulfide administered to rats at 75 mg/kg, the whole blood Cmax was 15,500 ng/mL, and AUClast was 188,000 h*ng/mL. The terminal elimination phase T1/2 was 11.1 hours. After a single oral (gavage) dose of carbon disulfide administered to rats at 150 mg/kg, the whole blood Cmax was 29,500 ng/mL, and AUClast was 459,000 h*ng/mL. The terminal elimination phase T1/2 was 16.1 hours. After a single oral (gavage) dose of carbon disulfide administered to rats at 300 mg/kg, the whole blood Cmax was 53,400 ng/mL, and AUClast was 766,000 h*ng/mL. The terminal elimination phase T1/2 was 14.7 hours.

After a single inhalation exposure to carbon disulfide administered to rats at 150 ppm over 3 hours, the whole blood Cmax was 11,300 ng/mL, and AUClast was 110,000 h*ng/mL. The terminal elimination phase T1/2 was 22.6 hours.

Dose-normalized AUClast ranged from 2510 (h*ng/mL)/(mg/kg) to 3060 (h*ng/mL)/(mg/kg) for the oral dose groups. The narrow range of values and the absence of any trend relative to dose level indicates that the whole blood exposure to carbon disulfide was dose-proportional. Summing the whole blood exposure of 110,000 h*ng/mL measured after the completion of a 3-hour inhalation exposure plus the calculated exposure during the Inhalation Phase (Cmax 11300 ng/mL * 3 h) results in overall carbon disulfide exposure (AUC) of 143,900 h*ng/mL following a 150 ppm inhalation dose. Extrapolating the inhalation exposure on the plot of oral dose versus exposure indicated that a 110,000 h*ng/mL inhalation dose is roughly equivalent to an oral dose of 30 mg/kg and an inhalation dose of 143,900 h*ng/mL is rougly equivalent to an oral dose of 44 mg/kg.

The 2-thiothiazolidine-4-carboxylic acid concentrations in plasma were much lower than carbon disulfide concentrations in whole blood, with Cmax ranging from 242 ng/mL to 421 ng/mL across all groups and dose-normalized AUClast in the range of 20.6 to 27.8 (h*ng/mL)/(mg/kg) following an oral dose. Like carbon disulfide, 2-thiothiazolidine-4-carboxylic acid exposure was also dose-proportional among oral doses. The terminal elimination phase T1/2 ranged from 3.86 to 6.73 hours in the oral dose groups and was 1.58 hours in the inhalation group. Based on the excretion data, approximately 11% to 19% of the dose was recovered as carbon

disulfide in the urine and 17% to 32% of the dose was recovered as carbon disulfide in the expired air following an oral dose. The amount of carbon disulfide recovered in the air and urine increased with increasing oral dose level, suggesting saturation of binding sites and/or metabolic clearance processes at higher doses. However, there was no notable change in the fraction eliminated in either matrix (urine or expired air) at the 2 lowest doses, and a minor shift to greater pulmonary excretion at the highest dose. Carbon disulfide in expired air was primarily excreted in the first 12 hours, while carbon disulfide in urine continued to be excreted across the time course. Using a calculated inhalation dose of 30 mg/kg extrapolated as above, but only using the AUC after the completion of the inhalation exposure, approximately 44% of the dose was recovered as carbon disulfide in urine and expired air, and the distribution between the 2 matrices reflected a similar pattern as observed after the oral dose.

Approximately 1% of the dose was recovered as 2-thiothiazolidine-4-carboxylic acid in the urine following an oral dose, reflecting the lower circulating concentration of 2-thiothiazolidine-4-carboxylic acid versus carbon disulfide in the whole blood and plasma. Similar recovery was observed after inhalation dosing.

With the exception of 1 time point, all feces samples were below the quantitation limit for carbon disulfide. In addition, the 2-thiothiazolidine-4-carboxylic acid concentrations measured accounted for 0.01% of the dose or less across the entire 72-hour collection period, indicating that biliary excretion is not a route of carbon disulfide or 2-thiothiazolidine-4-carboxylic acid excretion in rats.

Overall recovery of carbon disulfide and 2-thiothiazolidine-4-carboxylic acid represented approximately 31% to 52% of the administered dose among all dose levels and routes of administration. Because notable concentrations of carbon disulfide were still being eliminated in the urine at 72 hours postdose, it is likely that carbon disulfide and metabolites remained in the rat at the termination of the study. In addition, while quantitative recovery is desired, carbon disulfide is known to undergo multiple metabolic transformations in vivo, and these metabolites were not measured as part of this study. Potential changes in biliary excretion of CYP-related metabolites would not be detected in this experiment, but even with apparent variations in the degree of total excretion in urine and expired air between dose groups, the relative amount excreted in each matrix was consistent and there was no indication that biliary excretion was a significant contributor to clearance of carbon disulfide or 2-thiothiazolidine-4-carboxylic acid.

The study demonstrates that systemic exposure to carbon disulfide, as evaluated by whole blood AUClast, is dose-proportional and that the distribution of carbon disulfide and 2-thiothiazolidine-4-carboxylic acid in the urine and expired air does not differ by dose or by dose route. In addition, single and, based on the data collected for 2 -thiothiazolidine-4-carboxylic acid and carbon disulfide, repeated oral doses can be scaled to match the desired equivalent inhalation exposure.