Carbon disulphide

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Similar or equivalent to OECD guidline but non-GLP conform. Well-documented publication with experimental details. Sufficient for assesment.

Data source

Reference

Reference Type:

publication

Title:

Activation of lysosomal degradative pathway in spinal cord tissues of carbon disulfide-treated rats

Author:

Yuan Gao, Shasha Wang, Anji Yi, Ruirui Kou, Keqin Xie, Fuyong Song

Year:

2014

Bibliographic source:

Chemico-Biological Interactions 219: 76–82

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

GLP compliance:

no

Limit test:

no

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 animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Experimental Animal Center of Shandong University
- Weight at study initiation: 250–280 g
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 50
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

Carbon disulfide was dissolved in corn oil and administered to rats at 3 mL/kg

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

6 weeks

Frequency of treatment:

6 times per week

Doses / concentrations

Remarks:

Doses / Concentrations:
200, 400, 600 mg/kg bw/d
Basis:

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

OTHER: Transmission electron microscopy analysis were performed using the ultra-thin sections of the lumbar spinal cord to investigate the autophagy-lysosomal activity in rat spinal cord. The expression of autophagy-lysosome associatd proteins (LC3, Ambra1, Atg1 and UVRAG) were measured by Western blotting analysis. The protein level of LAMP-1 and cathepsin B in spinal cord were also measured by Western blot. Proteolytic activities of cathepsins B in spinal cord were determined by using Inno-Zyme cathepsins B Activity Kit.

Statistics:

Statistical analysis was performed with one-way analysis of variance (ANOVA), followed by Dunnett post hoc test, which was provided by SPSS 13.0 statistical software. The differences were significant at P < 0.05.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY: Progressive gait abnormality was observed. At the beginning of the experiment, rats exhibited a normal, unaffected gait. On week 3, the rats exposed to 600 mg/kg bw/d CS2 showed slightly gait abnormality, i.e. uncoordinated placement, overcompensated movements, or splaying slightly of hindlimbs, and walking on tiptoes. At the end of the experiment, most of the animals showed moderately abnormal gait (obvious movement abnormalities characterized by markedly splaying hindlimbs, ataxia, swaying, stumbling); and about 30% of animals showed severely abnormal gait (crawling, unable to support weight completely). Furthermore, the rats exposed to 400 mg/kg bw/d CS2 began to show slight to moderate gait abnormality until week 5 after exposure, and lasted to the end of exposure. By contrast, no clinical signs were observed in the control and 200 mg/kg bw/d CS2 groups throughout the experiment. High-dose CS2 also resulted in the occurrence of hypokinesia, muscular rigidity and resting tremor in rats. For example, visible tremors primarily occurred in rat’s body part while rats holding their posture at rest. Moreover, it can be occasionally seen in the rear or front limbs of rats. A greater proportion of tremors in rats occurred when exposed to a higher dose of CS2.

BODY WEIGHT AND WEIGHT GAIN: During the first week of exposure, the rats kept the normal growth of body weight. As the intoxication continued, the mean weight gain of animal began to slow down gradually.

OTHER FINDINGS:
- Transmission electron microscopy analysis: Although no significant change in autophagosome number was observed in CS2-treated animal, the number of lysosomes in neuron soma of CS2-treated animal was markedly increased.
- Protein expression of LC3: When compared with the control, the level of LC3-II in rats treated with 200, 400, and 600 mg/kg bw/d CS2, increased by 33.6%, 52.9%, and 108.1% (P<0.05), respectively. The ratio of LC3-II to LC3-I was also significantly elevated in CS2-treated groups.
- Protein levels of Atg1, UVRAG, and Ambra1: When compared with the control, Atg1 increased by 62.3%, 46.8%, and 29.2% (P < 0.05), UVRAG increased by 30.9%, 31.3%, and 39.1% (P < 0.05) in 200, 400, and 600 mg/kg bw/d CS2-treated groups, respectively. The level of Ambra1 remained unaffected.
- Protein levels of LAMP-1 and cathepsin: When compared with the control, the levels of LAMP-1 in rats treated with 200, 400, and 600 mg/kg CS2 were elevated by 30.2%, 47.6%, and 48.1% (P < 0.05), respectively. Accordingly, the levels of cathepsin B were elevated by 22.1%, 39.6%, and 42.5% (P < 0.05), respectively.
- Cathepsins B activity: Cathepsins B activity in rats treated with 200, 400, 600 mg/kg bw/d CS2 increased by 34.7%, 45.1%, and 57.8% (P < 0.05), respectively, compared to the control.

Effect levels

Dose descriptor:

NOAEL

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In conclusion, no clinical signs were observed in the 200 mg/kg bw/d CS2 group. CS2 intoxication was associated with the activation of lysosomal degradative machinery.

Executive summary:

In the present study, male Wistar rats were randomly divided into three experimental groups and one control group. The rats in experimental groups were treated with CS2 by gavage at dosages of 200, 400 and 600 mg/ kg bw/d respectively, six times per week for 6 weeks. During the first week of exposure, rats kept the normal growth of body weight. Afterwards, the mean weight gain of animal began to slow down gradually. On week 3, the rats exposed to 600 mg/kg bw/d CS2 showed slightly gait abnormality. At the end of the experiment, most of the animals showed moderately abnormal gait and about 30% of animals showed severely abnormal gait. Rats exposed to 400 mg/kg bw/d CS2 showed slight to moderate gait abnormality until 5 week after exposure, and lasted to the end of exposure. By contrast, no clinical signs were observed in the control and 200 mg/kg bw/d CS2 groups throughout the experiment. The formation of autophagosomes and lysosomes in motor neurons of rat spinal cord was observed by transmission electron microscopy, the level of autophagy-related proteins, lysosome-associated membrane protein 1 (LAMP-1), and cathepsin B in spinal cord tissues was determined by Western blot analysis, and the activity of cathepsin B was measured by fluorescence assay. The results demonstrated that the number of lysosomes in motor neurons was markedly increased in CS2-treated rats. In the meantime, the administration of CS2 significantly increased the level of microtubule-associated protein light chain 3-II (LC3-II), Atg1, UVRAG and LAMP-1 in rat spinal cord. Furthermore, the content and activity of cathepsin B in rat spinal cord also showed a significant elevation. Taken together, this study suggested that CS2 intoxication was associated with the activation of lysosomal degradative machinery, which might play a protective role against CS2-induced neuronal damage.