Carbon disulphide

Endpoint summary

Description of key information

see discussion below

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 April 2010 to 22 June 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

no

GLP compliance:

yes

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

Animals: Rat, RccHan: WIST(SPF)

Rationale: Recognized by international guidelines as a recommended test system

Breeder: Harlan Laboratories B.V., Kreuzelweg 53, 5961 Horst / The Netherlands

Number of Animals per Group: 3 females
Total Number of Animals: 6 females
Age (when treated): 10 weeks
Body Weight Range (when treated): 174.8 g – 189.0 g

Identification: Unique cage number and corresponding color-coded spots on the tail. The animals were marked at acclimatization start.
Randomization: Selected by hand at time of delivery. No computer generated randomization program.

Acclimatization: Under laboratory conditions, after health examination. Only animals without any visible signs of illness were used for the study.

Environmental Conditions: Standard Laboratory Conditions. Air-conditioned with 10-15 air changes per hour, and continuously monitored environment with a room temperature of 22 ± 3 °C and a relative humidity between 30-70%, automatically controlled light cycle of 12 hours light and 12 hours dark and music played during the daytime light period.

Accommodation: In groups of three in Makrolon type-4 cages with wire mesh tops and standard softwood bedding (‘Lignocel’ J. Rettenmaier&Söhne GmbH&CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland).

Diet: Pelleted standard Provimi Kliba 3433 rat/mouse maintenance diet, batch no. 83/09 (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) ad libitum (except for the overnight fasting period prior to treatment and approximately 3-4 hours post dose). Results of analyses for contaminants are archived at Harlan Laboratories Ltd.

Water: Community tap water from Füllinsdorf ad libitum. Results of bacteriological, chemical and contaminant analyses are archived at Harlan Laboratories Ltd.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

VEHICLE:
The vehicle was chosen after a non-GLP solubility trial which was performed before the study initiation date. Since the Sponsor indicated that the test item was barely soluble in water and potentially could be chemically modified by polyethylene glycol, corn oil was used as appropriate vehicle for the oral applications. A preparation of 20% (w/w) in corn oil resulted in a white to yellow emulsion, which was considered orally applicable.

Batch Number: 049103168
Source: Carl Roth GmbH & Co., 76185 Karlsruhe / Germany
Stability of the Vehicle: Stable under storage conditions
Expiry Date: 31-Jan-2014
Storage Conditions: At room temperature (range of 20 ± 5 °C), light protected.
Safety Precautions: Routine hygienic procedures were used to ensure the health and safety of the personnel.

DOSE FORMULATION:
The substance was present at levels of 200 mg/ml in the vehicle.
Test material was made into a solution with the vehicle at a dose level of 2000 mg/kg.

The dose formulations were prepared shortly before each dosing occasion using a magnetic stirrer as homogenizer. The test item was weighed into a tared glass beaker on a suitable precision balance and the vehicle added (weight:volume). Homogeneity of the test item in the vehicle was maintained during administration using a magnetic stirrer.

TEST ITEM ADMINISTRATION:
The animals received a single dose of the test item by oral gavage administration at 2000 mg/kg body weight after being fasted for approximately 18 hours (access to water was permitted). Food was provided again approximately 3 hours after dosing. The dosing volume was 10 mL/kg body weight.

Doses:

2000 mg/kg body weight

No. of animals per sex per dose:

3 females per dose

Control animals:

no

Details on study design:

OBSERVATIONS:
Viability / Mortality: Daily during acclimatization. Once before treatment and within the first 30 minutes and at approximately 1, 2, 3 and 5 hours after treatment on test day 1 (in common with the clinical signs). Twice daily during days 2 – 15.

Clinical Signs: Daily during acclimatization and treatment. Additionally, within the first 30 minutes and at approximately 1, 2, 3 and 5 hours after administration on test day 1.

Body Weights: On test days 1 (prior to administration), 8 and 15.

Necropsy: All animals were killed at the end of the observation period by carbon dioxide asphyxiation and discarded after macroscopic examinations were performed. An external examination and opening of the abdominal and thoracic cavities for examinations of major organs was performed. The appearance of any macroscopic abnormalities was recorded. No organs or tissues were retained.

Statistics:

No statistical analysis was performed.

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

Individual mortality data are given in Attachment 1 - Table 1. No intercurrent deaths occurred during the course of the study.

Clinical signs:

other: Individual clinical observations are given in Attachment 2 - Table 2. Slightly ruffled fur was observed in all animals within the first day (from the first 30 minutes or 1 hour to 1, 2, 3 or 5 hours observation) and persisted up to 2 days in one animal af

Gross pathology:

Individual necropsy findings are given in Attachment 4 - Table 4. No macroscopic findings were recorded at necropsy.

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The median lethal dose of carbon disulfide after single oral administration to female rats, observed over a period of 14 days, is: LD50 (female rat): greater than 2000 mg/kg body weight.
Based upon the referred classification criteria (Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008), Carbon disulfide is not classified with respect to acute oral toxicity in the rat.

Executive summary:

Two groups, each consisting of three female RccHan:WIST (SPF) rats, were treated with Carbon disulfide by single oral gavage administration at a dosage of 2000 mg/kg body weight. The test item was formulated in corn oil at a concentration of 0.2 g/mL and administered at a dosing volume of 10 mL/kg. The animals were examined daily during the acclimatization period and mortality, viability and clinical signs were recorded. All animals were examined for clinical signs immediately before treatment, within the first 30 minutes and approximately 1, 2, 3 and 5 hours after treatment on day 1 and once daily during test days 2-15. Mortality/viability was recorded immediately before treatment, within the first 30 minutes and approximately 1, 2, 3 and 5 hours after administration on test day 1 (with the clinical signs) and twice daily during days 2-15. Body weights were recorded on day 1 (prior to administration) and on days 8 and 15. All animals were necropsied and examined macroscopically. No intercurrent deaths occurred during the course of the study. Slightly ruffled fur was observed in all animals on test day 1 and in one animal also on test day 2. The body weight of the animals was within the range commonly recorded for this strain and age.

No macroscopic findings were recorded at necropsy. The median lethal dose of Carbon disulfide after single oral administration to female rats, observed over a period of 14 days, is: LD50 (female rat): greater than 2000 mg/kg body weight.

Based upon the referred classification criteria (Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008), carbon disulfide is not classified with respect to acute oral toxicity in the rat.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

Well performed GLP study with an LD50 > 2000 mg/kg bw supported by results from published studies.

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 15-Jun-2010, Experimental Completion Date: 29-Jun-2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study conducted in compliance with OECD403 except that it was not indicated why particle size distribution was not measured as the test atmosphere was generated as an aerosol

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.2 (Acute Toxicity (Inhalation))

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1300 (Acute inhalation toxicity)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

traditional method

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., Kreuzelweg 53, 5961 NM Horst, Netherlands
- Age at study initiation: 9 weeks
- Weight at study initiation: males: 254.6 to 274.2 g, females: 169.9 to 183.7 g
- Fasting period before study: none
- Housing: Animals were housed in groups of 5 of the same sex in Makrolon® type-IV cages with wire mesh tops and standard softwood bedding ("Lignocel" J. Rettenmaier & Söhne GmbH & Co KG, 73494 Rosenberg / Germany, imported by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) including paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey, UK)
- Diet (e.g. ad libitum): Animals were housed in groups of 5 of the same sex in Makrolon® type-IV cages with wire mesh tops and standard softwood bedding ("Lignocel" J. Rettenmaier & Söhne GmbH & Co KG, 73494 Rosenberg / Germany, imported by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) including paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey, UK)
- Water (e.g. ad libitum): Community tap water from Füllinsdorf ad libitum in water bottles, except during the period when they were restrained in exposure tubes


ENVIRONMENTAL CONDITIONS
- Temperature (°C):
- Humidity (%): 30-70 %
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

This study was performed in an AAALAC-accredited laboratory in accordance with the Swiss Animal Protection Law under license no. 49.

IN-LIFE DATES: From: To: 15-Jun-2010 to 29--Jun-2010

Route of administration:

inhalation: vapour

Type of inhalation exposure:

nose only

Vehicle:

clean air

Remark on MMAD/GSD:

The test atmosphere was generated as a liquid aerosol. In view of the high vapour pressure it can be assumed that droplets will have evaporated on their way to the test animals and that the test animals have been exposed to a vapour test atmosphere. However, in the test report it was not indicated why particle size distribution was not measured.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The test atmosphere was generated using a Hudson nebulizer connected to a step dose pump. The entire polyethylene injector inside the nebulizer was replaced by a stainless steel injector. The concentration of the test item in the inhalation chamber was controlled by regulating the flow of the test item to the inhalation tower and by the addition of dilution air
- Exposure chamber volume: not applicable
- Method of holding animals in test chamber: The animals were confined separately in restraint tubes which were positioned radially around the exposure chamber
- Source and rate of air: compressed air was supplied by means of an oil free compressor and passed respiratory quality filters before it was introduced to the exposure system
- Method of conditioning air: respiratory quality filters
- System of generating particulates/aerosols: The test atmosphere was generated using a Hudson nebulizer connected to a step dose pump. The entire polyethylene injector inside the nebulizer was replaced by a stainless steel injector. The concentration of the test item in the inhalation chamber was controlled by regulating the flow of the test item to the inhalation tower and by the addition of dilution air
- Method of particle size determination: test item was generated as liquid aerosol which evaporated so that animals were inhaling vapour
- Treatment of exhaust air: filtered
- Temperature, humidity, pressure in air chamber: 23.5 °C, 2.4 % relative humididty, 20.0 % oxygen

TEST ATMOSPHERE
- Brief description of analytical method used: The concentration was measured at least 4 times per hour of exposure per on-line gas chromatography. The analyses were performed according to the conditions listed below.

Column: DB-624 (30m x 0.320mm x 1.80µm)
Injector: 225°C
Oven: 100 °C for 0.1min; then 50°C/min to 250°C for 0 min.
Detector: µECD, 260°C

Calibration:
A calibration curve ranging between concentrations of approximately 2.5 mg/L to approximately 14 mg/L was constructed from the test item in gas bags as part of the technical trials. The calibration gas bags were prepared at each concentration.

Acceptance Criteria:
The coefficient of variation was < 10% for all calibration gas bag samples at each concentration. The correlation coefficient of the used regression was 0.995 and therefore within the acceptance criteria.

Standards constructed from the test item in gas bags were sampled prior to initiation of each exposure at the chamber-line and used to check the integrity of the sampling line and check the GC calibration. Plots of the peak area used for the calibration were used to assess trends regarding system stability. The acceptance criterion for standard samples was an accuracy of 90 - 110% of the theoretical value.

- Samples taken from breathing zone: yes

VEHICLE
- Composition of vehicle (if applicable): none

TEST ATMOSPHERE (if not tabulated)
- Particle size distribution: not measured (vapour)

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

Actual concentration: 10.35 mg/L air
Nominal concentration: 12.23 mg/L air

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Observations for viability were recorded once before exposure on the day of exposure (test day 1), three times during exposure, immediately and 1 h after exposure on test day 1 and twice daily during the observation period. Each animal was examined three times during exposure, immediately and 1 h after exposure on test day 1 and once daily during the observation period. Observations were detailed and carefully recorded using explicitly defined scales as appropriate. Only grossly abnormal signs were detectable during exposure as the animals were restrained in the exposure tubes. The body weight of each animal was recorded on test days 1 (before exposure), 2, 4, 8 and 15 (before necropsy).

- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight

Statistics:

not needed

Sex:

male/female

Dose descriptor:

LC50

Effect level:

10.35 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Mortality:

three males and two females

Clinical signs:

other: Tachypnea was recorded in all animals during and immediately after exposure. Tachypnea persisted in all surviving animals until test day 2. Hunched or prostrate posture and/or decreased activity were observed in most of the animals one hour after end of e

Body weight:

From test day 1 to test day 2, slight to moderate body weight loss was noted in all surviving animals. Thereafter normal body weight development was recorded in these animals.

Gross pathology:

There were no macroscopic findings that were considered to be related to treatment with the test item. Red discoloration of the lung was recorded in animals that died. This finding was considered to be due to delayed necropsy.

Other findings:

none

Test Atmosphere Conditions

Temperature, relative humidity and oxygen concentration during exposure were considered to be satisfactory for this type of study. Relative humidity values were quite low as dry air was used for atmosphere generation.

 

Data on temperature, relative humidity and oxygen concentration are presented in the following table.

 

Recording Time [hours:min]	O2Concentration [Vol %]	Temperature [°C]	Relative Humidity [% RH]
08:00	20.2	23.9	3.6
08:30	20.1	23.4	2.5
09:00	20.1	23.6	2.4
09:30	20.0	23.9	2.3
10:00	19.9	23.4	2.3
10:30	19.9	23.4	2.3
11:00	19.8	23.3	2.2
11:30	19.8	23.3	2.2
12:00	19.8	23.4	2.2
Mean	20.0	23.5	2.4
St. Dev.	0.2	0.2	0.5
N	9	9	9

 

Determination of Nominal Atmosphere Concentration

The nominal concentration was 12.23 mg/L air.

 

Actual (Chemical) Determination of Atmosphere Concentrations

The mean actual concentration determined was 10.35 mg/L air as targeted. Details on chemically determined atmosphere concentrations are presented in the following tables:

 

Measurement	Chemical Atmosphere Concentration mg/L air
1	14.01
2	12.03
3	14.13
4	13.88
5	11.52
6	12.42
7	11.54
8	6.36
9	10.51
10	9.65
11	10.67
12	10.57
13	9.39
14	12.08
15	11.41
16	12.30
17	6.86
18	11.24
19	9.98
20	10.74
21	11.16
22	9.17
23	10.26
24	12.22
25	6.98
26	11.22
27	6.10
28	12.67
29	11.22
30	5.34
31	11.29
32	10.97
33	6.62
34	10.47
35	5.13
36	10.96
37	8.40
38	8.50
39	12.75
40	13.41
41	8.79
42	9.42
43	12.02
44	11.14
45	8.10
46	9.38
47	13.00
48	7.92
Mean	10.35
SD	2.3
n	48

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

Based on the results of this study, the 4-h LC50 of carbon disulfide obtained in this study was 10.35 mg/L air (actual concentration as vapour). There was no indication of relevant sex-related differences in toxicity of the test item.

Executive summary:

A group of five male and five female albino rats [RccHan^TM:WIST(SPF)] was exposed by nose-only, flow-past inhalation for four hours to the test item at a chemically determined mean concentration of 10.35 mg/L air. All animals were observed for clinical signs and mortality during the inhalation exposure and the subsequent 14-day observation period. Body weights were recorded prior to exposure on test day 1, and during the observation period on test days 2, 4, 8 and 15 before necropsy. On test day 15 all animals were sacrificed and necropsied. The ranges of aerosol concentration, temperature, relative humidity, oxygen content and airflow rate measured during the exposure were considered to be satisfactory for a study of this type. In addition, the test item was considered to be respirable to rats. Five animals died during the first 24 hours after exposure. All other animals survived the scheduled observation period. Tachypnea was recorded in all animals during exposure and persisted until test day 2 in the surviving ones. Hunched or prostrate posture and / or decreased activity were recorded in most of the animals after exposure up to day 2. A transient effect on body weight was observed. There were no macroscopic findings that were considered to be related to treatment with the test item.

 

Based on the results of this study, the 4 -h LC₅₀ of Carbon Disulfide obtained in this study was 10.35 mg/L air (actual concentration).There was no indication of relevant sex-related differences in toxicity of the test item.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LC50

Value:

10 350 mg/m³ air

Quality of whole database:

Well performed GLP study with a 4-h LC50 value of 10.35 mg/L.

Additional information

Two standard GLP studies were performed so as to assess the acute oral and inhalation toxicity of CS2, and were summarized as key studies under the relevant endpoint, respectively:

In the oral study, six female rats received a single dose of carbon disulfide of 2000 mg/kg bw by gavage. The substance was formulated in corn oil. No animal deaths occured during the conduction of the study and the following 14 -day period. No alterations were recorded in body weights. After sacrifice, the gross pathological and histopathological examinations showed no treatment-related lesions. The LD50 for CS2 is greater than 2000 mg/kg bw.

In the inhalation study, five male and five female rats were exposed nose-only for 4 h, to an analytically confirmed concentration of 10.35 mg CS2/L air. Five animals died during the first 24 h, while all the others survived the subsequent 14 - day period. Tachypnea, as well as effects on the posture and/or decreased activity were observed, up to day 2. A slight decline was measured in the body weights. No macroscopic treatment-related effects were seen in the examination after necropsy. The study yielded a 4 -h LC50 of 10.35 mg/L.

Except for the two key studies, no other standard acute oral or inhalation toxicity studies were located in the literature. A large number of non-standard studies was identified based on the most recent and elaborate reviews. Due to the huge amount of data, since CS2 toxicity has been thoroughly investigated for many years, it was not possible to include all of them as separate endpoint study records. Parts of the reviews are reproduced as such, concerning each exposure route, respectively, being a separate endpoint study record. Besides this, a selection of some important publications was made based on the reviews, in order to provide a complete and up-to-date picture of the acute toxic effects of the substance. Robust summaries were prepared for some of the publications, while others were summarized based on information collected from reviews and on the published abstract. The selected studies are summarized under section 7.9 specific investigations, since they are not focused on the endpoints that are typically investigated in an acute toxicity study, but they are addressing either the effects of CS2 on particular systems and/or organs or its mechanistic action.

Oral

All acute oral toxicity studies gave LD50 values above the levels for classification, except for the study of Kanada et al. 1994. However, the study does not provide sufficient information on how this value is derived (not specified); the validity of this study is unclear.

With regard tp sublethal effects, the cardiovascular system and the liver seem to be the main targets of acute oral exposure. Subacute (4 w) carbon disulfide oral administration of 126 mg/kg bw resulted in direct cardiodepressive effects in rats, which might be mediated by disruption of the energy supply in the heart (Klapperstück et al., 1991). The acute toxicity in the liver is manifested by enzymatic disruptions. The findings of Masuda et al. (1986) support that CS2 impairs the liver drug-metabolizing system in mice after acute exposure of 3 -30 mg/kg bw; this effect is obvious prior to pathological signs in the liver. Adverse effects including hind-limb paralysis (400 mg/kg bw for 14 d; Jones-Price et al. 1984b), and decreased norepinephrine levels (300 mg/kg bw once; Kanada et al. 1994) have also been reported.

Inhalation

These acute studies have been done primarily in rodents. The LC50 values recorded were variable, depending on the species tested. The lowest LC50, 220 ppm, was reported by Gibson & Roberts (1971) who exposed mice to CS2 for 60 min. Nevertheless, the study is inadequately reported as regards the chemical purity of the test material, the analytical techniques used and the results regarding mortality rates; as such the study cannot be used for hazard assessment. Izmerov et al. (1982) reported an LC50 of 25 mg/l for rats (2 h) and of 10 mg/l for mice (2 h); however, no further information is presented for these studies. Methodological deficiencies do not allow a correct interpretation.

Acute effects on the nervous system including behavorial alterations, alterations of catecholamine levels, effects on the liver and on the cardiovascular system have also been studied. Exposure to CS2 (4 h/d) for 3 d at 1000 ppm resulted in neurobehavorial disturbances (Goldberg et al., 1964). An investigation on catecholamine metabolism (McKenna & DiStefano, 1977), showed declined levels of brain, adrenal and heart noradrenaline, as well as of adrenal dopamine, after exposure to 640 ppm for 4 or 8 h. Brain noradrenaline levels were decreased at 64 ppm. CS2 did not produce any histological lesions on the myocardium of male Wistar rats after exposure at 1280 ppm, for 1 or 2 d (Chandra et al., 1972). After acute CS2 exposure (803 ppm, 18 h), disturbances in the oxidative phosphorylation of brain mitochondria were detected, accompanied by decreased respiratory and cardiac rate (Tarkowski & Sobczak, 1971). In rats, exposure to 20 ppm for 8 h, the lowest concentration tested, also inhibited biotransformation of drugs and solvents and caused a decrease of the glycogen content of the liver. All effects were rapidly reversible within about 24 h, and no increase of liver enzymes in serum was observed (Freundt & Dreher 1969; Freundt & Kuttner 1969). A 2 -h exposure to 20 ppm and above resulted in adverse effects on the hepatic energy supply of rats (Freundt & Kurzinger, 1975). However, it is difiicult to assess all these findings as reporting was limited.

  

Dermal

No studies were identified on the lethal or non-lethal effects in animals after acute dermal exposure to carbon disulfide.

Justification for classification or non-classification

Based upon the classification criteria Regulation (EC) No 1272/2008, carbon disulfide needs to be classified for acute inhalation toxicity (vapour). The key GLP study resulted in a 4 -h LC50 value of 10.35 mg/L which corresponds to CLP acute inhalation toxicity cat. 4. CS2 does not need to be classified for acute oral toxicity.