Sodium O-isobutyl dithiocarbonate

Specific investigations: other studies

Administrative data

Endpoint:

mechanistic studies

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Well-documented publication and acceptable for assessment. Carbon disulphide is both a reagent in the manufacture, as well as a decomposition product of xanthates. Sodium isobutyl xanthate readily decomposes to carbon disulphide, especially in the presence of moisture/water. Therefore, the health effects of carbon disulphide (CS2) need to be considered in the assessment of sodium isobutyl xanthate.

Data source

Reference

Reference Type:

publication

Title:

Covalent cross-linking of erythrocyte spectrin by carbon disulfide in vivo

Author:

Valentine LM, Graham DG, and Anthony DC

Year:

1993

Bibliographic source:

Toxicology and Applied Pharmacology 121:71-77

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study addresses the mechanism of neurotoxicity of CS2 in rats, dosed by intraperitoneal injection. Particularly, its ability to covalently cross-link to a cytoskeletal protein of the erythrocytes, spectrin , is examined.The authors assess whether cross-linking of spectrin after exposure to CS2, can be used as a biomarker for neurotoxicity.

GLP compliance:

not specified

Type of method:

in vivo

Endpoint addressed:

neurotoxicity

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:

dithioxomethane

Test material form:

solid: compact

Details on test material:

Carbon disulphide is both a reagent in the manufacture, as well as a decomposition product of xanthates. Sodium isobutyl xanthate readily decomposes to carbon disulphide, especially in the presence of moisture/water. Therefore, the health effects of carbon disulphide (CS2) need to be considered in the assessment of sodium isobutyl xanthate.
- Name of test material (as cited in study report): carbon disulfide (obtained from Fisher Chemicals, Fair Lawn, NJ)
- Analytical purity: not mentioned, but probably >99.9%; an internet search on the company provider was performed

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Diet: ad libitum
- Water: ad libitum


ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

corn oil

Details on exposure:

Rats were dosed once daily by intraperitoneal injection with corn oil or CS2 in corn oil at 2 or 5 mmol/kg (total volume 0.15-0.4 ml) for periods of 21 and 42 days.

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

no data

Duration of treatment / exposure:

21 or 42 d

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 2, 5 nmol/kg
Basis:
nominal conc.

Control animals:

yes, concurrent vehicle

Examinations

Examinations:

The ability of the rats to return their hind limbs under their body after forced extension to the rear was examined. Body weights of the animals were recorded each week. Within 24 hr of the last treatment rats were exsanguinated under chloral hydrate anesthesia and sections of the muscular branch of the posterior tibial nerve were fixed using 4% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2. Nerve sections were postfixed in 2% osmium tetroxide, dehydrated in graded alcohols and propylene oxide, and embedded in Epon. Thick sections were stained using toluidine blue.

Results and discussion

Details on results:

No evidence of hind limb paralysis was observed. No axonal swellings were observed in any exposed group.

Any other information on results incl. tables

The SDS-PAGE showed two major bands in the region of 200 -250 kDa in all groups, that correspond to alpha- and beta-spectrin, and represented the proteins with the highest molecular weight in the controls. In the CS2 exposed groups, additional eluting proteins appeared in the 410 kDa region, as two distinct bands, with a more diffuse, less intensely staining region between the major bands. Based on the following peptide mapping, these two extra bands represent the spectrin dimer that failed to separate due to cross-linking. The selective base hydrolysis, suggested the presence of both thiourea and dithiocarbamate ester cross-linking structures in spectrin dimers. No spectrin dimer was detected in any of the control samples.

Applicant's summary and conclusion

Conclusions:

The study reveals that CS2 exposure results in covalent cross-linking of erythrocyte spectrin in vivo, after intraperitoneal injection of rats.
Carbon disulphide is both a reagent in the manufacture, as well as a decomposition product of xanthates. Sodium isobutyl xanthate readily decomposes to carbon disulphide, especially in the presence of moisture/water. Therefore, the health effects of carbon disulphide (CS2) need to be considered in the assessment of sodium isobutyl xanthate.

Executive summary:

What follows is the original abstract of the publication with slight modifications

Covalent cross-linking of proteins by CS2 has been demonstrated in vitro and represents a potential mechanism for the toxicity of this compound. In the present investigation the ability of CS2 to cross-link proteins covalently in vivo is demonstrated using denaturing polyacrylamide gel electrophoresis. Intraperitoneal injection of CS2 in rats at 2 or 5 mmoL/kg for 21 or 42 days produced several high-molecular-weight (~410 kDa) proteins eluted from erythrocyte membranes which were not present in control animals. Limited proteolysis of the high-molecular-weight protein bands, monomeric alpha-spectrin, and monomeric beta-spectrin using endoproteinase glu-C, followed by peptide mapping on denaturing polyacrylamide gels, showed the high-molecular-weight proteins to be alpha, beta heterodimers. The production of multiple heterodimers exhibiting different distances of migration was consistent with the existence of several preferred sites for cross-linking. Evidence for the presence of dithiocarbamate ester and thiourea cross-linking structures in spectrin dimers was obtained using selective base hydrolysis. No spectrin dimer was detected in control animals, and dimer formation demonstrated a cumulative dose response in CS2-treated rats. The longevity of red blood cells, the cumulative dose response, and the stability of the cross-linking structures endows spectrin cross-linking with the potential to serve as a biomarker of chronic low-level exposures to CS2 and may provide a means to correlate pathological changes with existing methods of CS2 exposure monitoring. The ability of CS2 to covalently cross-link erythrocyte spectrin suggests that CS2 may also cross-link other proteins in vivo and supports covalent cross-linking of proteins as a possible molecular mechanism through which CS2 manifests toxicity. If so, then spectrin cross-linking may parallel cross-linking reactions in the axon and provide a sensitive, pre-neurotoxic biomarker of this molecular event.