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Toxicological information

Neurotoxicity

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Description of key information

There are conclusive but not suffcient data for the classification of substance S-allyl O-pentyl dithiocarbonate with regard to Neurotoxicity.

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Link to relevant study records

Referenceopen allclose all

Endpoint:
neurotoxicity: sub-chronic oral
Type of information:
other: published data
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Carbon disulphide (CAS number 75–15–0) is both reagents used in the manufacture of S-allyl O-pentyl dithiocarbonate. Therefore, Carbon disulphide (CAS number 75–15–0) need to be considered in the assessment of S-allyl O-pentyl dithiocarbonate.
Qualifier:
no guideline followed
Principles of method if other than guideline:
CS2 was administered by gavage to male rats for 12 w. In order to examine the mechanism of action of its neurotoxicity, cerebrum samples were analyzed for their content of six cytoskeletal proteins: NF-L, NF-M, NF-H (NF: neurofilaments), a-tubulin, b-tubulin, and b-actin by immunoblotting. Moreover, neurological tests were performed.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Experimental Animal Center of Shandong University
- Weight at study initiation: 180-200 g
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 50
- Photoperiod (hrs dark / hrs light): 12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
VEHICLE
- Justification for use and choice of vehicle (if other than water): no
- Concentration in vehicle: 3 ml/kg
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
12 w
Frequency of treatment:
daily, 5 d/w
Remarks:
Doses / Concentrations:
0, 300, and 500 mg/kg bw
Basis:
other: treatment by gavage
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle
Observations and clinical examinations performed and frequency:
BODY WEIGHT: Yes
- Time schedule for examinations: twice per week

Specific biochemical examinations:
Nerve homogenates (cerebrum) were centrifuged at 100,000 g for 1 h to yield a high-speed pellet (P) and a high-speed supernatant (S). According to previous experiments, the P fraction consists of polymerized triplet proteins and represents the triton-insoluble filamentous cytoskeletal network . The high-speed supernatant (S) likely contains tritonsoluble NF (neurofilament) proteins in the form of intermediate heterotetramers and monomers that represent the mobile population of exchangeable NF (neurofilament) proteins . Protein concentration of two fractions was measured with the use of a Protein assay Kit.

In order to investigate alterations of NF subunits monoclonal antibodies for NF-H, NF-M, and NF-L were used by immunoblotting.
Neurobehavioural examinations performed and frequency:
NEUROLOGICAL TESTING: performed twice per week; involving gait scores and rotarod performance. To measure gait abnormalities, rats were placed in an open field, and were observed for 3 min. Post-observation, a gait score was given from 1 to 4 corresponding to 1=a normal, unaffected gait; 2=a slightly abnormal gait (hindlimbs show uncoordinated placement, exaggerated or overcompensated movements, or are splayed slightly, walks on tiptoes); 3=moderately abnormal gait (obvious movement abnormalities characterized by markedly splaying hindlimbs, ataxia, swaying, rocking, lurching, stumbling); 4=severely abnormal gait (flat foot walk, hindlegs flat on surface, crawling, or unable to support weight). In the rotarod performance, rats were placed on an accelerating rotarod, and the retention time was recorded.
Statistics:
Mann-Whitney U-test, Kruskal-Wallis H-test, one-way analysis of variance, LSD's post hoc tests (P<0.05)
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 specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Clinical biochemistry findings:
effects observed, treatment-related
Behaviour (functional findings):
effects observed, treatment-related
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Details on results:
Exposure to CS2 resulted in decrease of body weight gain, progressive gait abnormalities and reductions in retention time on the accelarating rotarod. At the end of the exposure, most of the animals showed moderately abnormal gait. At the end of the week 12, rats treated with 300 mg/kg CS2 showed much less retention times than the control animals, while rats treated with 500 mg/kg CS2 hardly kept any retention on the ratorod.

Biochemical examinations
pellet(P): significant decrease in NF-H, NF-M and NF-L levels at both exposure doses, compared to the controls. Protein levels of alpha-tubulin remained at control levels, while these of beta-tubulin decreased significantly. The protein levels of beta-actin remained unchanged.
supernatant (S): significant decrease in NF-H, NF-M and NF-L levels at both exposure doses, compared to the controls. Both alpha- and beta-tubulin were increased significantly, indicating a possible translocation of proteins from one subcellular fraction to the other. A significant increase was observed in the levels of beta-actin only in the rats treated with 500 mg/kg bw.
Dose descriptor:
LOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: decreased body weight, progressive gait abnormalities, neurofilamentous accumulations in the cerebral cortex
Remarks on result:
other:
Conclusions:
Carbon disulfide exposure by gavage in rats (300 and 500 mg/kg bw) resulted in neurological effects, i.e. abnormal gait and reductions in retention time on the accelarating rotarod, as well as in significant reductions of neurofilaments (NF) subunits and increase of microtubules and microfilaments subunits, in cerebral cortex homogenates. The authors associate the changes in the NFs cerebrum proteins with the CS2 treatment, suggesting this as a possible mechanism inducing neuropathy.
Carbon disulphide (CAS number 75–15–0) is both reagents used in the manufacture of S-allyl O-pentyl dithiocarbonate. Therefore, Carbon disulphide (CAS number 75–15–0) need to be considered in the assessment of S-allyl O-pentyl dithiocarbonate.
Executive summary:

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

To investigate the mechanism of carbon disulfide-induced neuropathy, male wistar rats were administrated by gavage at dosage of 300 or 500 mg/kg carbon disulfide, five times per week for 12 weeks. By the end of the exposure, the animals produced a slight or moderate level of neurological deficits, respectively. Cerebrums of carbon disulfide-intoxicated rats and their age-matched controls were Triton-extracted and centrifuged at a high speed (100,000 X g) to yield a pellet fraction of neurofilament (NF) polymer and a corresponding supernatant fraction, which presumably contained mobile monomer. Then, the contents of six cytoskeletal protein (NF-L, NF-M, NF-H, alpha-tubulin, beta-tubulin, and beta-actin) in both fractions were determined by immunoblotting. Results showed that the contents of the three neurofilament subunits in the pellet and the supernatant fraction decreased significantly regardless of dose levels (P<0.01). As for microtubule proteins, in the pellet fraction of cerebrum, the levels of alpha-tubulin and beta-tubulin demonstrated some inconsistent changes. However, in the supernatant fractions, the content of alpha-tubulin and beta-tubulin increased significantly in both two dose groups (P<0.01). In comparison to neurofilament and tubulin proteins, the content of beta-actin changed less markedly, only the supernatant fraction of the high dose group displayed significant increase (P < 0.01), but the others remained unaffected. These findings suggested that the changes of cytoskeleton protein contents in rat cerebrum were associated with the intoxication of carbon disulfide, which might be involved in the development of carbon disulfide neurotoxicity.

Endpoint:
neurotoxicity: sub-chronic oral
Type of information:
other: published data
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Carbon disulphide (CAS number 75–15–0) is both reagents used in the manufacture of S-allyl O-pentyl dithiocarbonate. Therefore, Carbon disulphide (CAS number 75–15–0) need to be considered in the assessment of S-allyl O-pentyl dithiocarbonate.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Rats were exposed subchronically to CS2 by gavage, in order to investigate the alterations of neurofilament (NF) proteins in the spinal cord and the possible mechanism of action of the substance.In addition neurological testing was performed.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Experimental Animal Center of Shandong University
- Weight at study initiation: 180-200 g
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 50
- Photoperiod (hrs dark / hrs light): 12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
VEHICLE
- Justification for use and choice of vehicle (if other than water): no
- Concentration in vehicle: 3 ml/kg
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
12 w
Frequency of treatment:
daily, 5 d/w
Remarks:
Doses / Concentrations:
0, 300, and 500 mg/kg bw
Basis:
other: treatment by gavage
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle
Observations and clinical examinations performed and frequency:
BODY WEIGHT: Yes
- Time schedule for examinations: twice per week
Specific biochemical examinations:
Spinal cord homogenates were centrifuged at 100,000 g for 1 h to yield a high-speed pellet (P) and a high-speed supernatant (S). According to previous investigations, the P fraction consists of polymerized triplet proteins and represents the triton-insoluble filamentous cytoskeletal network . The high-speed supernatant (S) likely contains tritonsoluble NF (neurofilament) proteins in the form of intermediate heterotetramers and monomers that represent the mobile population of exchangeable NF (neurofilament) proteins . Protein concentration of two fractions was assayed with the use of a Protein assay Kit.

Protein fractions were subjected to SDS-PAGE and thereafter, alterations of NF subunits monoclonal antibodies for NF-H, NF-M, and NF-L were used by immunoblotting. In order to examine the contribution of calpains to the changes of NFs, the protein levels of µ-calpain and m-calpain in the S and P fraction were also tested with the use of immunoblotting. Calpain is a neutral proteinase in the Central Nervous System that can degrade the NF subunits.

To determine whether the decrement in NF proteins was due to corresponding reductions in NF gene expression, the effects of CS2 on NF-L,
NF-M, and NF-H mRNA were investigated with the use of RT-PCR. GADPH gene was used as an internal standard.
Neurobehavioural examinations performed and frequency:
NEUROLOGICAL TESTING: the onset and development of neurotoxicity were determined by gait scoring (animals were observed for 3 min in an open field, twice per week). A gait score was assigned from 1 to 4 where 1=a normal, unaffected gait; 2=a slightly abnormal gait (hindlimbs show uncoordinated placement, exaggerated or overcompensated movements, or are splayed slightly, walks on tiptoes); 3=moderately abnormal gait (obvious movement abnormalities characterized by markedly splaying hindlimbs, ataxia, swaying, rocking, lurching, stumbling); 4=severely abnormal gait (flat foot walk, hindlegs flat on surface, crawling, or unable to support weight).
Sacrifice and (histo)pathology:
- Time point of sacrifice: at the end of the 12 weeks
- Number of animals sacrificed: all animals, by decapitation
Statistics:
one-way analysis of variance, LSD's post hoc tests (P<0.05)
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 specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Clinical biochemistry findings:
effects observed, treatment-related
Behaviour (functional findings):
effects observed, treatment-related
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Details on results:
Exposure to CS2 resulted in decrease of body weight gain and progressive gait abnormalities. At the end of the experiment most of the animals exposed to 500 mg/kg bw showed moderately abnormal gait, while slight gait abnormality was seen in anmals treated with 300 mg/kg bw.

BIOCHEMICAL EXAMINATIONS
The results on the NFs protein content of the two fractions (supernatant, pellet):
pellet(P): significant decrease in NF-H, NF-M and NF-L at both exposure doses, except for NF-L isolated from animals treated with the low dose.
supernatant (S): significant decrease in NF-H, NF-M and NF-L levels only at the dose of 500 mg/kg bw, compared to the controls.

Independent of the dose levels the spinal cord (both fractions) content in µ-calpain was increased significantly. For m-calpain a decrease was observed in the supernatant fractions, while the pellets remained unaffected.

The results regarding alterations in NF expressions, revealed significant increases in the mRNA production of all three NF proteins.
Dose descriptor:
LOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: decreased body weight gain, slight gait abnormalities
Remarks on result:
other:
Conclusions:
Carbon disulfide exposure by gavege in rats (300 and 500 mg/kg bw) resulted in neurological deficits, i.e. abnormal gait, that might be related to the observed significant reductions of NF subunits spinal cord homogenates. The results indicate that NF alterations might be related to the developments of CS2 neurotoxicity.
Carbon disulphide (CAS number 75–15–0) is both reagents used in the manufacture of S-allyl O-pentyl dithiocarbonate. Therefore, Carbon disulphide (CAS number 75–15–0) need to be considered in the assessment of S-allyl O-pentyl dithiocarbonate.
Executive summary:

What follows is the original abstract of the publication

To investigate the mechanism of carbon disulfide-induced neuropathy, male Wistar rats were randomly divided into two experimental groups and one control group. The rats in two experimental groups were treated with carbon disulfide by gavage at dosages of 300 and 500 mg/kg/day, respectively, five times per week for 12 weeks. Spinal cords of carbon disulfide-intoxicated rats and their age-matched controls were Triton-extracted and ultracentrifuged to yield a pellet fraction of neurofilament (NF) polymer and a corresponding supernatant fraction. Then, the contents of NF triplet proteins (NF-H, NF-M, NF-L) and two calpain isoforms (m-calpain and µ-calpain) in both fractions were determined by immunoblotting. In the meantime, the mRNA levels of NF-H, NF-M, and NF-L in spinal cords were quantified using reverse transcriptase-polymerase chain reaction. Results showed that in the pellet fraction, the contents of three NF subunits in both treated groups decreased significantly except NF-L in low dose group. In the supernatant fraction, the pattern of NFs alteration varied according to dose-levels. Compared to controls, three neurofilmant subunits in the high dose group displayed significant reduction consistently. However, in the low dose group, they remained unaffected. As for calpains, the contents of µ-calpain in both fractions increased significantly regardless of carbon disulfide dose-levels. Meanwhile, m-calpain demonstrated a significant decline in the supernatant fraction, and remained unchangeable in the pellet fraction compared to the control group. Furthermore, the levels of mRNA expression of NF-H, NF-M, and NF-L genes were elevated consistently in CS2-treated groups. These findings suggested that carbon disulfide intoxication was associated with obvious alterations of NFs content in rat spinal cord, which might be involved in the development of carbon disulfide neurotoxicity.

Endpoint:
neurotoxicity: acute oral
Type of information:
other: published data
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Pentan-1-ol/Amyl alcohol is both reagents used in the manufacture of S-allyl O-pentyl dithiocarbonate. Therefore, pentan-1-ol/Amyl alcohol need to be considered in the assessment of S-allyl O-pentyl dithiocarbonate.
Qualifier:
no guideline available
Principles of method if other than guideline:
Evaluation of motor activity (rotarod), body temperature and blood levels of test subtance after acute oral treatment.
GLP compliance:
no
Limit test:
no
Species:
mouse
Strain:
other: Swiss-Cox
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Laboratory Supply Company, Indianapolis
- Age at study initiation: adult animals
- Weight at study initiation: 20 - 25 g
- Housing: 10 per cage in plastic cages (24 x 34 x 15 cm) with Bed-O-Cobs bedding
- Diet (e.g. ad libitum): Wayne Lab-Blox
- Water (e.g. ad libitum): tap water
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 25
- Photoperiod (hrs dark / hrs light): 10:14 (light: 06:00 - 20:00)
Route of administration:
oral: gavage
Vehicle:
other: Tween 80
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Since 1-pentanol was not completely miscible with water at the proportions used, the suspension was stabilized by the addition of a few drops of Tween 80
- Concentration in vehicle: 25, 50 and 100%
- Amount of vehicle (if gavage): 0.2 ml/10 g
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
acute treatment
Frequency of treatment:
once
Remarks:
Doses / Concentrations:
500, 1000 and 2000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
20 - 25
Control animals:
other: yes, concurrent vehicle; The actual testing procedure permitted each mouse to serve as its own control.
Details on study design:
- Dose selection rationale: the dose ranges to be used were selected by determination of the LD50 values for the test substances, then extrapolation to an LD10 and use of a "high" dose level that was 75% of this value. No lethality was seen in any animals.
- The actual testing procedure permitted each mouse to serve as its own control. On test day 1, the rectal temperature was measured, after which each mouse was tested on the rotarod, then given the appropriate dose of alcohol. At the predesignated time (10-120 min), the body temperature was determined, rotarod testing performed, and a blood sample obtained. This entire procedure (for all 75 mice in a group) was repeated, for each dose of each alcohol, on four additional occasions at intervals of 72, 48, 24 and 24 hours; these test days are referred to as II, III, IV and V, respectively.
- The experimental design was developed to permit the determination of body temperature, rotarod performance and levels of alcohol in blood at five time points (10, 20, 40, 80 and 120 min) after administration of one of the three different doses of each alcohol. This design was repeated on five different days (on the same animals); 72, 48 and 24 hours, respectively, after the initial dose. Thus, on the first test dose for each alcohol, 75 mice were weighed and and randomly divided into three groups of 25 mice each; further subdivision led to five groups of 5 mice each, corresponding to the five time points for testing. All testing was done in rooms maintained at 22-23°C.
Observations and clinical examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: No data

OPHTHALMOSCOPIC EXAMINATION: No data
Specific biochemical examinations:
NEUROPATHY TARGET ESTERASE (NTE) ACTIVITY: No

CHOLINESTERASE ACTIVITY: No
Neurobehavioural examinations performed and frequency:
FUNCTIONAL OBSERVATIONAL BATTERY: No

LOCOMOTOR ACTIVITY: Yes
Motor performance was determined with rotarod systems consisting of an 8 cm diameter screen-floor drum rotating at a constant speed of 10 rpm. Each animal was placed on the drum, and allowed to remain for 120 sec. If impairment was observed, the time at which the animal fell from the rotating drum was recorded.
Other examinations:
Body temperature was measured with a YSI Model 47 Scanning Tele-Thermometer equipped with a No. 401 thermistor rectal probe. Each animal was placed in a plastic restraining cage; the rectal probe was lubricated with peanut oil and gently inserted 3.0 cm into the rectum. Body temperature was recorded 30 sec after the time of insertion.
Statistics:
Body temperature, rotarod performance, and levels of alcohol in blood were analyzed by one-way analysis of variances (ANOVA) with repeated measures for each dose of each alcohol; significant differences within each ANOVA were further analyzed by post-hoc Newman-Keuls tests. Data for each alcohol were analyzed by two-way ANOVA with repeated measures to test for dose effects.
Details on results:
CLINICAL SIGNS AND MORTALITY
No lethality was seen in any treatment group.

NEUROPATHOLOGY
ROTAROD MOTOR ACTIVITY:
Analysis of the effects of all four alcohols on rotarod performance indicated that no significant differences occurred for the different test days; consequently test days 1-5 were combined. For each alcohol, a dose-related impairment of performance was observed. This impairment was time-related and was positively correlated, in magnitude, with the levels of blood alcohol; i.e. the greater the blood level of each alcohol, the greater was the degree of impairment of rotarod performance. The smallest dose of each alcohol was ineffective; with the exception of the largest dose of ethanol, all animals returned to normal activity by 120 min after the administration of alcohol.
OTHER FINDINGS
BLOOD LEVELS OF TEST SUBSTANCE: (Table 1)
The maximum blood levels of test substance were seen at the 10 min point (the same observation was made for the 4 additional alcohols tested, with the exception of ethanol, where the maximum blood level was seen at the 40 min point) after administration. Since no significant day-to-day differences were observed, all of these data were pooled and are presented in Table 1. Because of the limited number of time points available, precise pharmacokinetic comparisons of the alcohols could not be made; however, the fraction of dose absorbed appeared to be related inversely to chain length (ethanol > 1-propanol > 1-butanol > 1-pentanol). Half-life values were in the order: 1-butanol (92 min), ethanol (71 min), 1-pentanol (60 min), 1-propanol (57 min).

BODY TEMPERATURE: (Table 2)
Analysis of the individual data points over the 5 days indicated that no significant differences occurred between test days 1-5; therefore, these (data were combined). Although the magnitude of the hypothermic response to each of the alcohols was dose-related, the examination of the data shows that there was no consistent relationship between the time of maximal hypothermic effect and the time course of blood levels of the test substance (as well as of l-propanol and 1-butanol). In general, the maximum hypothermic response was observed at either 10 or 20 min after the administration of alcohol.
For ethanol, the time-course of effects, on test day 1 (first administration) significantly differed from the time courses seen with subsequent doses (test days 2, 3, 4, 5). In contrast, test days 2-5 did not differ from each other; consequently, these data are combined. On test day 1, the greatest hypothermic response was seen at 20 min after the 6.0 and 3.0 g/kg doses of ethanol. In contrast, on test days 2-5, the greatest response was seen at 40 min. In addition, the magnitudes of effects seen on test day 2 were greater than the corresponding values for test days 1-5. No correlation in time was observed between the hypothermic effects and the blood levels of ethanol on test day 1.
A comparison of the effect of all four alcohols on body temperature at the 20 min time point after administration show that the lines for 1-propanol, 1-butanol, 1-pentanol, and test days 1-5 of ethanol are parallel; the molar potency ranking for the hypothermic effect is 1-pentanol = 1-butanol > 1-propanol > ethanol; the latter two were significant. In contrast, the dose-effect line for test day 1 of ethanol is non-parallel, making comparison of direct potency impossible.
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw (total dose)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: other: motor activity (rotarod) reversible impairment (within 80 min) observed at higher dose levels
Remarks on result:
other:

Table 1: Time course of the concentration of test substance in blood

Dose (mg/kg bw)

Time after administration (min) [number of mice examined]

10

20

40

80

120

2000

16 ± 0 [20]

14 ± 2 [20]

11 ± 8 [9]

6 ± 2 [11]

ND

1000

11 ± 5 [22]

11 ± 7 [20]

7 ± 1 [9]

ND

ND

500

7.1 ± 1 [21]

5 ± 1 [17]

ND

ND

ND

Values in mg/dl; ND: concentration of alcohol in blood was below the limit of sensitivity for the method

Table 2: Effects of test substance on body temperature

Dose (mg/kg bw)

Time after administration (min) [number of mice examined]

10

20

40

80

120

2000

-2.5 ± 0.2 [20]

-3.3 ± 0.3 [20]

-3.0 ± 0.5 [19]

-1.0 ± 0.4 [11]

-0.5 ± 0.6 [12]

1000

-2.4 ± 0.2 [22]

-2.5 ± 0.5 [20]

-1.4 ± 0.3 [20]

-0.1±0.3 [20]

-0.1 ± 0.1 [17]

500

-1.4 ± 0.1 [21]

-0.7 ± 0.4 [17]

-0.2 ± 0.2  [17]

0.0  [17]

-0.4 ± 0.2 [20]

Values in °C

 

Conclusions:
Evaluation of motor activity (rotarod), body temperature and blood levels of test subtance after acute oral treatment were observed. NOAEL=500 mg/kg bw (total dose).
Pentan-1-ol/Amyl alcohol is both reagents used in the manufacture of S-allyl O-pentyl dithiocarbonate. Therefore, pentan-1-ol/Amyl alcohol need to be considered in the assessment of S-allyl O-pentyl dithiocarbonate.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LOAEL
300 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Effect on neurotoxicity: via inhalation route

Link to relevant study records

Referenceopen allclose all

Endpoint:
neurotoxicity: sub-chronic inhalation
Type of information:
other: published data
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Carbon disulphide (CAS number 75–15–0) is both reagents used in the manufacture of S-allyl O-pentyl dithiocarbonate. Therefore, Carbon disulphide (CAS number 75–15–0) need to be considered in the assessment of S-allyl O-pentyl dithiocarbonate.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Male Wistar rats were exposed to a high concentration of CS2 for 12 weeks via inhalation.A neurological testing was performed to investigate effects ion the behaviour of the animals. Thereafter, the experiments were made in order to elucidate the mechanism of action of CS2 on the nervous system.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male
Route of administration:
inhalation: vapour
Vehicle:
unchanged (no vehicle)
Details on exposure:
no data
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
no data
Duration of treatment / exposure:
2 h/d for 12w
Frequency of treatment:
daily, 5 d/w
Remarks:
Doses / Concentrations:
1250 mg/m3 (396 ppm)
Basis:
nominal conc.
No. of animals per sex per dose:
40 (50 control animals)
Control animals:
yes, sham-exposed
Positive control:
no
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
Clinical biochemistry findings:
effects observed, treatment-related
Behaviour (functional findings):
effects observed, treatment-related
Details on results:
CS2-induced decreases in weight gain that were time-related. The neurological testing showed slight abnormalities in gait after 6 w of treatment : uncoordinated placement, compensated movements, or splaying slightly of hind limbs. The mean gait score was 2.08±0.21. After 9 w the scoring showed moderate gait abnormality, which became almost severe at the end of the exposure (3.96±0.12), and was manifested as markedly splaying hindlimbs, ataxia, swaying, and stumbling.
The indexes measured showed alterations in animals treated with CS2, when compared to controls. ROS and MDA levels were obviously increased in the cerebral cortex, hippocampus, spinal cord and serum. SOD activities were slightly increased, as well as the GSH contents and GSH-Px, CAT, T-AOC activities in cerebral cortex, hippocampus, spinal cord and serum after 2, 4, 8 or 12 w of CS2 poisoning.
Dose descriptor:
LOAEC
Effect level:
396 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: marked gait abnormalities; changes in lipid peroxidation in the nerve tissue and in serum
Remarks on result:
other:
Dose descriptor:
LOAEC
Effect level:
1 250 mg/m³ air
Based on:
test mat.
Sex:
male
Basis for effect level:
other: marked gait abnormalities; changes in lipid peroxidation in the nerve tissue and in serum
Remarks on result:
other:

CS2-induced decreases in weight gain that were time-related. The neurological testing showed slight abnormalities in gait after 6 w of treatment : uncoordinated placement, compensated movements, or splaying slightly of hind limbs. The mean gait score was 2.08±0.21. After 9 w the scoring showed moderate gait abnormality, which became almost severe at the end of the exposure (3.96±0.12), and was manifested as markedly splaying hindlimbs, ataxia, swaying, and stumbling. The indexes measured showed alterations in animals treated with CS2, when compared to controls. ROS and MDA levels were obviously increased in the cerebral cortex, hippocampus, spinal cord and serum. SOD activities were slightly increased, as well as the GSH contents and GSH-Px, CAT, T-AOC activities in cerebral cortex, hippocampus, spinal cord and serum after 2, 4, 8 or 12 w of CS2 poisoning.

Conclusions:
CS2-inhalation poisoning at 396 ppm of rats for 12 weeks resulted in remarkable gait abnormalities, which were correlated with changes in the lipid peroxidation and the oxidant status in the nerve tissue and serum. It is suggested that such changes are associated with the CS2-induced neuropathy.
Carbon disulphide (CAS number 75–15–0) is both reagents used in the manufacture of S-allyl O-pentyl dithiocarbonate. Therefore, Carbon disulphide (CAS number 75–15–0) need to be considered in the assessment of S-allyl O-pentyl dithiocarbonate.
Executive summary:

What follows is the original abstract of the publication

To further elucidate the mechanism and determine the biomarker of neuropathy induced by carbon disulfide (CS2), we performed a longitudinal observational study of reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), glutathione peroxidase (GSH-Px), catalase (CAT), superoxide dismutase (SOD) and total antioxidative capacity (T-AOC) in rat cerebral cortex, hippocampus, spinal cord and serum after 0, 2, 4, 8 and 12 weeks of CS2 administration. CS2 exposure was found to markedly increase ROS and MDA levels in cerebral cortex, hippocampus, spinal cord and serum of rats in both time and symptom-dependent manners. Although SOD activities slightly increased, there was a decrease in the GSH contents and GSH-Px, CAT activities in cerebral cortex, hippocampus, spinal cord and serum after 2, 4, 8 or 12 weeks CS2 intoxication and at gait score of 2, 3, or 4. The activities of T-AOC also decreased in all three nerve tissues and serum as time went on and symptom developed. Furthermore, significant correlations between LPO and gait abnormality were observed as symptom developed. Oxidation stress also resulted in Ca2+ concentrations and calmodulin (CaM) levels increases in cerebral cortex, hippocampus and spinal cord. Thus, CS2 intoxication was associated with elevation of lipid peroxidation (LPO) and reduction of antioxidant status, and the time and symptom-dependent changes of these indexes in rats’ nerve tissues and serum suggested that ROS and concomitant LPO, at least in part, were involved in CS2-induced neuropathy.

Endpoint:
neurotoxicity: sub-chronic inhalation
Type of information:
other: published data
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Carbon disulphide (CAS number 75–15–0) is both reagents used in the manufacture of S-allyl O-pentyl dithiocarbonate. Therefore, Carbon disulphide (CAS number 75–15–0) need to be considered in the assessment of S-allyl O-pentyl dithiocarbonate.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The aim of the study was to examine the morphological progression and dose response of CS2 distal axonopathy in the muscular branch of the posterior tibial nerve (MBPTN) and spinal cord. Rats were exposed to various concentrations of CS2 by inhalation for various time points. This publication is only one part of a whole study that aimed at investigating CS2 neurotoxicity in a coordinated way (several endpoints included); see below for details.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Raleigh, NC)
- Age at study initiation: 8-9 weeks
- Housing: individually in wire-mesh cages within the exposure chambers
- Diet: ad libitum, only during non-exposure times
- Water: ad libitum
- Acclimatation period: 10-14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 49.1
- Photoperiod (hrs dark / hrs light): 12
Route of administration:
inhalation: vapour
Vehicle:
unchanged (no vehicle)
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Data taken from Sills et al. (1998a, Carbon disulfide neurotoxicity in rats: I. Introduction and study design. NeuroToxicology 19 (1): 83-88)

CS2 was vaporized mixed with conditioned air and delivered to the chambers.
- Air flow rate: 4 l/min
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Monitored every 15 min with infrared spectrophotometers; maintained at the 3% of the targeted concentrations throughout the study.
Duration of treatment / exposure:
6 h/d for 2, 4, 8 or 13 w
Frequency of treatment:
daily, 5 d/w
Remarks:
Doses / Concentrations:
0, 50, 500, 800 ppm (0, 156, 1558, 2493 mg/m3)
Basis:
nominal conc.
No. of animals per sex per dose:
9 (in the 13 week study an additional 9 animals per dose/sex were included)
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: according to Sill et al. (1998a; Carbon disulfide neurotoxicity in rats: I. Introduction and study design. NeuroToxicology 19 (1): 83-88) the exposure concentrations were selected based on previous studies (Gottfried et al., 1985) demonstrating that metabolic saturation occurs at approximately 600 ppm CS2. Concentrations above (800 ppm) and below (500 ppm) metabolic saturation were selected for use, as well as, a concentration close to the current TLV of 10 ppm (50 ppm).
Sacrifice and (histo)pathology:
- Number of animals sacrificed: all
- Procedures for perfusion: rats were ansthetized with sodium pentobarbital containing heparin, and perfused through the heart with 4% phosphate buffered paraformaldehyde followed by 4% phosphate buffered glutaraldehyde pH 7.4
- Number of animals perfused: all
Gross examinations were performed
- Tissues evaluated: sciatic nerve, muscular branch of the posterior tibial nerve and caudal tail nerve, multiple levels of the spinal cord including C1 and C2, and L1 and L2 (light & electron microscopy), whole brain (cerebrum, cerebellum, midbrain), heart, aorta, lung, trachea, liver, kidneys, nasal cavity, testes, epididymis, ovaries, oviducts, uterus and vagina

For sciatic nerve, muscular branch of the posterior tibial nerve and caudal tail nerve, multiple levels of the spinal cord including C1 and C2, and L1 and L2
- Type of staining: 1% toluidinine blue (light microscopy), lead citrate and uranyl acetate (electron microscopy)
- Thickness: 1 micron
- Embedding media: Epon resin

All the other organs except for testes/epididymes, were trimmed, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E). The testes and epididymes were embedded in glycol methacrylate and stained with periodic acid shift and H&E.
Other examinations:
All the other examinations can be seen in the relevant seperate study entry (see below for details, section 'any other information on materials and methods incl. tables').
Statistics:
Fischer's exact test
Clinical signs:
not specified
Mortality:
not specified
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Clinical biochemistry findings:
not specified
Behaviour (functional findings):
not specified
Gross pathological findings:
effects observed, treatment-related
Neuropathological findings:
effects observed, treatment-related
Details on results:
NEUROPATHOLOGY
PNS: The muscular branch of the posterior tibial nerve (MBPTN) was the most sensitive to CS2 toxicity, compared to the sciatic and caudal tail nerve. Animals in the exposure group of 800 ppm had focal to multifocal giant axonal swelling in the MBPTN starting from the 8th w, that evolved from minimal to moderate from week 8 to 13. Degeneration and regeneration were observed at 13 w. Axonal swelling or other morphological changes were not detected at weeks 2 and 4 or at lower concentrations. Ultrastractural examinations revealed accumulation of 10 nm neurofilaments in swollen axons. The swollen axons were usually accompanied by myelin sheath thinning (reduced number of myelin lamellae).

CNS: Similarly to the MBPTN, axonal swelling was prominent at 8 w in the spinal cord segments, but at both 500 and 800 ppm exposure levels. By 13 w it progressed to moderate. Lumbar spinal cord swelling was more pronounced, compared to the cervical cord segments. The swollen axons of the lumbar cord had accumulated 10 nm neurofilaments. Axonal swelling was not observed at weeks 2 and 4 or at 50 ppm.
Dose descriptor:
NOAEC
Effect level:
50 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no axonal swelling in the muscular branch of the porterior tibial nerve and spinal cord
Remarks on result:
other:
Dose descriptor:
NOAEC
Effect level:
156 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no axonal swelling in the muscular branch of the porterior tibial nerve and spinal cord
Remarks on result:
other:

OTHER FINDINGS Lesions related to CS2 exposure were not detected in the heart, brain, aorta, lung, trachea, liver, kidneys, nasal cavity, testes, epididymis, ovaries, oviducts, uterus and vagina.

Conclusions:
Lesions of axonal swelling were observed in the MBPTN after 8 w of exposure to 800 ppm, and in the spinal cord after 8 w of exposure to 500 and 800 ppm. No lesions related to CS2 exposure were detected in the heart, brain, aorta, lung, trachea, liver, kidneys, nasal cavity, testes, epididymis, ovaries, oviducts, uterus and vagina at 50 ppm.
Carbon disulphide (CAS number 75–15–0) is both reagents used in the manufacture of S-allyl O-pentyl dithiocarbonate. Therefore, Carbon disulphide (CAS number 75–15–0) need to be considered in the assessment of S-allyl O-pentyl dithiocarbonate.
Executive summary:

The aim of the study was to examine the morphological progression and dose response of CS2 distal axonopathy in the muscular branch of the posterior tibial nerve (MBPTN) and spinal cord. For this reason, male and female Fischer 344 rats were exposed to 0, 50, 500, 800 ppm (0, 156, 1558, 2493 mg/m3) of CS2, 6 h/d, 5 days per week, for 2, 4, 8 or 13 w, via inhalation. At 8 w axonal swelling was observed in the MBPTN of animals exposed at 800 ppm, that progressed to more severe at 13 w. Similarly, axonal swelling was seen in the cervical and lumpar spinal cord, after 8 w, but at both high exposure concentrations. Swelling was accompanied by thinning of the myelin sheath and accumulation of 10 nm neurofilaments. No lesions related to CS2 exposure were detected in the heart, brain, aorta, lung, trachea, liver, kidneys, nasal cavity, testes, epididymis, ovaries, oviducts, uterus and vagina.

Endpoint:
neurotoxicity: sub-chronic inhalation
Type of information:
other: published data
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Carbon disulphide (CAS number 75–15–0) is both reagents used in the manufacture of S-allyl O-pentyl dithiocarbonate. Therefore, Carbon disulphide (CAS number 75–15–0) need to be considered in the assessment of S-allyl O-pentyl dithiocarbonate.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The aim of the study is to validate the use of erythrocyte spectrin cross-linking as a toxicity biomarker of CS2. Rats were exposed to 50, 500 and 800 ppm via inhalation, for 2,4, 8 and 13 weeks. The study examines the ability of CS2 to cross-link neurofilaments proteins, as a mechanism of toxic action.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Raleigh, NC
- Age at study initiation: 6-7 weeks
- Housing: individually in Hazleton 1000 inhalation chambers
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 49.1%
Route of administration:
inhalation: vapour
Vehicle:
air
Details on exposure:
Carbon disulfide was vaporized, mixed with conditioned air (HEPA-filtered, charcoal-scrubbed, temperature and humidity controlled), and delivered to the inhalation chambers.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations in the inhalation chambers were continuously monitored by Fourier transform infrared spectophotometers and were within 3% of the target levels.
Duration of treatment / exposure:
6 h/d for 2, 4, 8, or 13 w
Frequency of treatment:
5 d/w
Remarks:
Doses / Concentrations:
0, 50, 500, or 800 ppm (0, 156, 1558, 2493 mg/m3)
Basis:
nominal conc.
No. of animals per sex per dose:
4 for controls and exposure groups at the 2, 4 and 8 week time points, and 8 for the 13 week exposure groups
Control animals:
yes, sham-exposed
Observations and clinical examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes
- Time schedule: twice a day

BODY WEIGHT: Yes
- Time schedule for examinations: no data

MORPHOLOGY
Rats were whole-body perfused with phosphate-buffered 4% paraformaldehyde followed by phosphate-buffered 4% glutaraldehyde, pH 7.4, at room temperature. A complete necropsy was performed. Sections of cervical and lumbar spinal cord were postfixed in 2% osmium tetroxide, dehydrated in graded alcohols and propylene oxide, and embedded in epoxy resin. For light microscopy, 1-µm-thick sections were stained with toluidine blue. For electron microscopy, 60-nm ultra-thin sections were stained with lead citrate and uranyl acetate and viewed on a Zeiss 10-A transmission electron microscope. Four males and four females were examined at each exposure level, including controls, at the 2-, 4-, and 8-week time points and eight animals of each sex were used for the 13-week exposure groups.
Specific biochemical examinations:
Isolation of erythrocyte spectrin was perfomed as described previously (Genter St Clair et al., 1988) from erythrocytes of 5 randomly selected male rats taken from each exposure group except for the 13-week groups for which 10 male rats were used.

Neurofilament proteins were isolated from rat spinal cord according to a published method (Liem and Hutchison, 1982). Three male rats were examined at each exposure level and duration.

Covalent cross-linking of erythrocyte spectrin was evaluated by electrophoresis on SDS-containing 5% polyacrylamide gels and visualized with silver stain. Spectrin preparations (~10 µg total protein) were boiled for 5 min in sample buffer containing 1% SDS and 2% dithiothreitol before electrophoresis. Monomer and dimer spectrin were quantified by densitometry using an ISCO gel scanner, Model 1312, in combination with an ISCO UA-5 absorbance/fluorescence detector and analyzed using HP Chem Station software.

Immunoblotting of spectrin and neurofilament preparations: Erythrocyte membrane proteins were separated using 5% homogeneous polyacrylamide gels containing SDS. Transfer to PVDF membranes was performed using the wet transfer method at 60 V for 1 hr. Immunoblotting of membranes was initiated by briefly washing the membrane in Tris-buffered saline (20 mM Tris, 0.5 M NaCl, pH 7.5) with 0.05% Tween-20 (TBST). The membranes were blocked with 3% bovine serum albumin or 3% nonfat dry milk in TBST for 1 hr at room temperature. After incubating with primary antibody (rabbit polyclonal anti-human erythrocyte spectrin at a dilution of 1:400) for 1 hr at room temperature the membranes were washed three times for 5 min in TBST, incubated with goat anti-rabbit horseradish peroxidase-conjugated antibody (1:2000 dilution) in TBST at room temperature, washed three times for 5 min in TBST, and visualized using chemilu-minescence.
Rat neurofilament preparations were separated using 7% homogeneous polyacrylamide gels containing SDS and transferred similarly. Mouse monoclonal antibodies to NFL, NFM, and NFH were used at 1:2000 dilution and secondary antiserum was horseradish peroxidase-conjugated goat anti-mouse IgG (1:8000). Horseradish peroxidase-conjugated antibodies were visualized using chemiluminescence. Slow-migrating protein immunoreactive to anti-NFL was quantified by scanning films using a Bio-Rad Model GS-700 Densitometer and Molecular Analyst Software.
Statistics:
Fischer's exact test
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 specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Clinical biochemistry findings:
not examined
Behaviour (functional findings):
not examined
Gross pathological findings:
not examined
Neuropathological findings:
effects observed, treatment-related
Details on results:
BODY WEIGHT AND WEIGHT GAIN
Dose related decreases were measured in body weight gain in both sexes, but they were not significant for the 50 ppm-exposure group. The decline was more pronounced in males.
Dose descriptor:
LOAEC
Effect level:
50 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: subneurotoxic effect: neurofilament cross-linking
Remarks on result:
other:
Dose descriptor:
LOAEC
Effect level:
156 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: subneurotoxic effect: neurofilament cross-linking
Remarks on result:
other:

Morphology: no axonal swellings were observed in animals exposed to the lowest concentration level. Eight- week exposure to 500 ppm resulted in mild axonal swelling, and 13-week exposure in diffuse giant axonal swellings in the cervical and lumbar regions of the spinal cord. All rats exposed to 800 ppm for 8 and 13 weeks had axonal swellings of the spinal cord, that increased in size and distribution, with exposure time. Electron microscopy examinations of swollen axons revealed a decline in the number of microtubulus and increased microfilamentous density, with loss of their normal longitudinal orientation.

SDS-PAGE/Immunoblotting of spectrin: two major bands occured in the range of 200 -250 kDa in all animals, representing the alpha- and beta-spectrin monomers. In rats exposed to CS2, in addition several protein bands were detected in the area of 410 kDa, corresponding probably to spectrin dimers. Regarding the amount of spectrin dimer expressed as a percentage of the total spectrin detected, in relation to time, a plateu was reached at the two high concentration levels at 4 or 8 weeks, while at 50 ppm a more gradual increase, that reaches a maximum at 13 weeks, is observed.

Immunoblotting of neurofilaments: treated rats samples gave higher molecular weight protein bands, immunoreactive for all 3 NF antibodies, that were not visible in the controls. Such findings were detected after exposure to all concentrations, even at 50 ppm. These results indicate that neurofilament cross-linking can be detected prior to the development of axonal swellings.

Conclusions:
The findings support that covalent cross-linking of neurofilament subunits (that contributes to the development of axonal swellings in the neurons), may be the neurotoxic mechanism of action of CS2. Neurofilament cross-linking was measured at all concentrations tested, and was time- and dose-dependent. Moreover, it was evident prior to neuron axonal swelling.
Carbon disulphide (CAS number 75–15–0) is both reagents used in the manufacture of S-allyl O-pentyl dithiocarbonate. Therefore, Carbon disulphide (CAS number 75–15–0) need to be considered in the assessment of S-allyl O-pentyl dithiocarbonate.
Executive summary:

What follows is the original abstract of the publication

Using model proteins, a mechanism for CS2-mediated covalent cross-linking of proteins has been demonstrated previously. The biologic importance of CS2 -promoted protein cross-linking is apparent as a possible dosimeter of CS2 exposure and as a potential mechanism to account for the identical neuropathies produced by 2,5-hexanedione and CS2 . The present investigation examines the utility of erythrocyte spectrin cross-linking as a biomarker of effect for inhalation exposure to CS2 and examines the ability of CS2 to cross-link neurofilament proteins, a potential neurotoxic target. Rats were exposed to CS2 via inhalation at control, 50-, 500-, and 800-ppm levels for 2, 4, 8, and 13 weeks and spectrin dimer formation was quantified using denaturing gel electropho resis and densitometry. Neurofilament preparations were also obtained from spinal cords and examined for cross-linking using Western blotting methods. The results obtained for protein cross- linking were compared to morphologic changes in the cervical and lumbar spinal cord using light and electron microscopy. The spectrin dimer exhibited a cumulative dose response and was detectable at both the 50-ppm level employed that did not produce axonal swellings and prior to the development of axonal swellings for the 500- and 800-ppm levels used. Neurofilament protein cross-linking involved all three subunits and the temporal relationship of cross-linking was consistent with a contributing role in the development of axonal swellings. These results establish the sensitivity of spectrin cross-linking for evaluating inhalation exposures and extend the similarities observed for 2,5-hexanedione and CS2 in both clinical settings and in vitro models to their effects exerted on neurofilaments in the axon.

Endpoint:
neurotoxicity: sub-chronic inhalation
Type of information:
other: published data
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Carbon disulphide (CAS number 75–15–0) is both reagents used in the manufacture of S-allyl O-pentyl dithiocarbonate. Therefore, Carbon disulphide (CAS number 75–15–0) need to be considered in the assessment of S-allyl O-pentyl dithiocarbonate.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The expression of the low affinity nerve growth factor receptor (NGF-R) mRNA was investigated, as an early and sensitive indicator of insult or damage to either the Schwann cell and its myelin or to the underlying axon, after CS2 exposure. Its expression was examined in the sciatic nerve of the animals. According to the authors, NGF-R and its mRNA are expressed in precursors of Schwan cells, and it is very low in mature PNS. However, pathological situations may lead to upregulation.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Raleigh, NC)
- Age at study initiation: 8-9 weeks
- Housing: individually in wire-mesh cages within the exposure chambers
- Diet: ad libitum, only during non-exposure times
- Water: ad libitum
- Acclimatation period: 10-14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 49.1
Route of administration:
inhalation: vapour
Vehicle:
unchanged (no vehicle)
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Data taken from Sills et al. (1998a, Carbon disulfide neurotoxicity in rats: I. Introduction and study design. NeuroToxicology 19 (1): 83-88)

CS2 was vaporized mixed with conditioned air and delivered to the chambers.
- Air flow rate: 4 l/min
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Monitored every 15 min with infrared spectrophotometers; maintained at the 3% of the targeted concentrations throughout the study.
Duration of treatment / exposure:
6 h/d for 2, 4, 8 or 13 w
Frequency of treatment:
daily, 5 d/w
Remarks:
Doses / Concentrations:
0, 50, 500, 800 ppm (0, 156, 1558, 2493 mg/m3)
Basis:
nominal conc.
No. of animals per sex per dose:
9, (in the 13 w study additional 9 animals were used)
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: according to Sills et al. (1998a; Carbon disulfide neurotoxicity in rats: I. Introduction and study design. NeuroToxicology 19 (1): 83-88) the exposure concentrations were selected based on previous studies (Gottfried et al., 1985) demonstrating that metabolic saturation occurs at approximately 600 ppm CS2. Concentrations above (800 ppm) and below (500 ppm) metabolic saturation were selected for use, as well as, a concentration close to the current TLV of 10 ppm (50 ppm).
Observations and clinical examinations performed and frequency:
Animals from each group were sacrificed and sciatic nerves, from the region of the dorsal root ganglia to the popliteal fossa, were excised and stored frozen. RNA was isolated and purified. Northern blot analysis was used.
Sacrifice and (histo)pathology:
Morhological examinations of the sciatic nerve and muscular branch of the posterior tibial nerve (MBPTN) were performed at the 800 ppm , in order to correlate any upregulation of NGF-R mRNA with morphological alterations.MBPTN was chosen since it is comprised of the distal portions of a subset of axons that have passed through the sciatic nerve.Rats were perfused with 4% phosphate buffered paraformaldehyde followed by 4% phosphate buffered glutaraldehyde pH 7.4. Sections of the sciatic nerve and of the muscular branch of the posterior tibial nerve were post-fixed in 2% osmium tetroxide, dehydrated in graded alcohols and propylene oxide and embedded in Epoxy resin. For light microscopy, 1 µm thick sections were stained with toluidine blue. For electron microscopy, 60 nm ultra thin sections were stained with lead citrate and uranyl acetate.
Clinical signs:
not specified
Mortality:
not specified
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Clinical biochemistry findings:
not specified
Behaviour (functional findings):
not specified
Gross pathological findings:
not specified
Neuropathological findings:
effects observed, treatment-related
Details on results:
Exposure to CS2 caused an upregulation of mRNA expression for NGF-R in the sciatic nerve that was dose- and time-dependent, at least for the two high concentrations. At 50 ppm mRNA levels were comparable to the controls. Despite this, no morphological changes were detected in the sciatic nerve of the rats. In the MBPTN axonal swelling, as well as changes in Schwann cells, were detected (at 800 ppm), but not prior to 8 w of exposure. The alterations consisted of abundant cytoplasm with increased organelles, including Golgi apparatus, rough endoplasmic reticulum, ribosomes, and mitochondria.
Dose descriptor:
NOAEC
Effect level:
50 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: mRNA expression of NGF-R
Remarks on result:
other:
Dose descriptor:
NOAEC
Effect level:
156 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: mRNA expression of NGF-R
Remarks on result:
other:

Exposure to CS2 caused an upregulation of mRNA expression for NGF-R in the sciatic nerve that was dose- and time-dependent, at least for the two high concentrations. At 50 ppm mRNA levels were comparable to the controls. Despite this, no morphological changes were detected in the sciatic nerve of the rats. In the MBPTN axonal swelling, as well as changes in Schwann cells, were detected (at 800 ppm), but not prior to 8 w of exposure. The alterations consisted of abundant cytoplasm with increased organelles, including Golgi apparatus, rough endoplasmic reticulum, ribosomes, and mitochondria.

Conclusions:
NGF-R mRNA upregulation was observed in the sciatic nerve at the 2 high concentrations, that were time and dose-associated. These increases were not accompanied by morphological changes in the nerve, suggesting that quantification of NGF-R mRNA can be a sensitive early indicator of CS2 neurotoxicity.
Carbon disulphide (CAS number 75–15–0) is both reagents used in the manufacture of S-allyl O-pentyl dithiocarbonate. Therefore, Carbon disulphide (CAS number 75–15–0) need to be considered in the assessment of S-allyl O-pentyl dithiocarbonate.
Executive summary:

What follows is the abstract of the original publication

Expression of the low-affinity nerve growth factor receptor (NGF-R) in the peripheral nervous system is regulated by Schwann cell-axonal contact. Steady-state mRNA levels for NGF-R are very low in the mature peripheral nervous system, but are markedly upregulated in sciatic nerve during both primary demyelination (tellurium exposure) and secondary demyelination (Wallerian degeneration). Upregulation also occurs in various subdegenerative axonopathy models where there is axonal atrophy, suggesting its usefulness as a marker for subtle perturbations in normal axon-Schwann cell interactions (Roberson et al., Mol Brain Res 1995; 28:231-238). To further test this hypothesis, we examined NGF-R mRNA expression in sciatic nerves of rats exposed to carbon disulfide (CS2), a toxicant known to cause a distal axonopathy. Adult rats were exposed to CS2 gas (50, 500, or 800 ppm, 6 hr/day, 5 days/wk) for 2-13 weeks. RNA was isolated from sciatic nerves and levels of mRNA for NGF-R determined by Northern blot analysis. NGF-R mRNA expression increased in a dose- and time-dependent manner. Message levels were already increased after 2 wks of exposure to 800 ppm CS2, and increased further with continued exposure. Morphological alterations were not apparent in the sciatic nerve, even at the highest dosage levels with the longest exposure times. Upregulation of NGF-R mRNA is thus an indicator of subtle alterations in the normal axon-Schwann cell relationship and provides a sensitive measure of CS2 neurotoxicity. Assay of this marker may also be useful as a rapid and very sensitive general screen for other compounds which are potentially toxic to the peripheral nervous system.

Endpoint:
neurotoxicity: chronic inhalation
Type of information:
other: published data
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Carbon disulphide (CAS number 75–15–0) is both reagents used in the manufacture of S-allyl O-pentyl dithiocarbonate. Therefore, Carbon disulphide (CAS number 75–15–0) need to be considered in the assessment of S-allyl O-pentyl dithiocarbonate.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Female mice were exposed to 0 or 800 ppm of CS2 via inhalation for 20 weeks. The neurologic function of the animals was examined with the use of functional observational battery (FOB).
GLP compliance:
not specified
Limit test:
no
Species:
mouse
Strain:
other: C57BL6
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan, Indiapolis, IN
- Age at study initiation: 9- to 10-weeks old
- Weight at study initiation: 20.7 ±14 g
- Fasting period before study: 3 days
- Housing: individually in Hazleton 1000 inhalation chambers
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 10-14 days
Route of administration:
inhalation: vapour
Vehicle:
unchanged (no vehicle)
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
CS2 was vaporized, mixed with conditioned air, and thereafter, brought into the exposure chambers.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The CS2 concentrations in the chambers were controlled with the use of Fourier transform infrared spectrophotometer, with a 20-m gas cell. CS2 levels were recorded every 1.5 min in the exposure group an every 15 min in the control chamber. Actual CS2 concentration was within 3% of the target concentration.
Duration of treatment / exposure:
6 h/d for 20 w
Frequency of treatment:
daily, 5 d/w
Remarks:
Doses / Concentrations:
0, 800 ppm
Basis:
other: target concentrations
No. of animals per sex per dose:
12 (800 ppm) and 11 (control)
Control animals:
yes, sham-exposed
Observations and clinical examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes
- Time schedule: 2 times daily
BODY WEIGHT: Yes
- Time schedule for examinations: weekly
Neurobehavioural examinations performed and frequency:
FUNCTIONAL OBSERVATIONAL BATTERY: Yes, the protocol used was according to Mc Daniel & Moser (1993)
- Parameters checked:
Open field-activity level (horizontal activity and rearing), arousal, posture, gait, and occurence of involuntary motor movements (tremors, convulsions)
- Duration of observation period for open field observations: 3 min

Reactivity to specific stimuli-the animals were put on a wire-mesh screen that was fast inverted. The time the animal needed to climb up to the top was measured. Animals hanging or dropping off were also recorded.

Autonomic function- the number of fecal boluses and urine pools in the open field was recorded, as well as excessive lacrimation and salivation. Other parameters examined: reactivity to tail-pinch and to sound, forelimb placing response

- Scoring criteria for excitability: mouse alertness in the open field and reactivity when removed from home cage
Sacrifice and (histo)pathology:
Morphology: the animals were prepared fro cardiac perfusions after FOB examinations. Gross necropsies were performed after the heart perfusion. Tissue samples were collected from the peripheral nervous system (sciatic nerve, tibial nerve, and muscular branch of the posterior tibial nerve), as well as from the central nervous system (spinal cord). The samples were dissected for light microscopy and electron microscopy. In addition 3 coronal brain samples were examined.
Other examinations:
Spectrin cross-linking: at 8 weeks of exposure, blood samples were collected fron all animals; spectrin was purified from the erythrocytes, lypophilized. The analysis was conducted with polyacylamide gel electrophoresis, according to the method of Laemmli (1970). In order to observe the proteininc bands silver staining was used. Monomeric and dimeric spectrin levels were measured with a BIO-RAD GS-700 imaging spectrophotometer and molecular analysis software, and the quantification of the dimer was expressed as a percentage of monomer plus dimer.
Clinical signs:
not specified
Mortality:
not specified
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Clinical biochemistry findings:
not specified
Behaviour (functional findings):
not specified
Gross pathological findings:
not specified
Neuropathological findings:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY: piloerection, ptosis, hunched posture

BODY WEIGHT AND WEIGHT GAIN: significant decreased in mean body weights after 20 w

NEUROBEHAVIOUR the results are presented in Table 1 (section any other information on results incl. tables): affected gait (uncoordinated placement of the hind limbs and ataxia), weakness, all animal fell in the inverted screen test, decreased rearing and locomotor movement

NEUROPATHOLOGY peripheral NS: the most evident effect was observed in the muscular branch of the posterior tibial nerve; 12/12 mice exhibited axonal swell swelling (distention of axonal diameter, thinning of myelin sheaths). Axonal swelling was not detected in section of the tibial and sciatic nerve. Central NS: 5/12 animals exhibited axonal swelling in lumbar spinal cord segments, but not in cervial spinal cord and in the brain.
Ultrastructurally, the swellings were compsed of neuroflament accumulation.

Spectrin cross-linking: a significant increase in alpha- and beta-heterodimers of spectrin were detected in the gel electrophoresis.
Dose descriptor:
NOAEL
Effect level:
800 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: see 'Remark'
Remarks on result:
not determinable
Remarks:
no NOAEL identified

Table 1: Summary of FOB results

Measure

Effects of CS2

Activity

Open field rearing

Decreased

Home cage activity

Decreased

Open field activity

Decreased

Neuromuscular & vestibular

Gait score

Changed

Ataxia score

Changed

Inverted screen test

Disrupted

Righting reflex

Not affected

Excitability

Alertness

Not affected

Handling reactivity

Increased

Autonomic

Lacrimation

Some

Defecation, urination

Not affected

Salivation

Not affected

Convulsive

Tremors

Some

Clonus

None

Sensorimotor

Tail pinch, click response

Not affected

Forelimb placing response

Not affected

General measures

Body weight

Decreased

Piloerection

Present

Body posture

Some hunched

Ptosis

Present

Conclusions:
Exposure of female mice to 800 ppm of CS2 via inhalation resulted in neurobehavorial effects, such as alterations in gait and disturbed function on an inverted screen test, as well as in decreased rearing and locomotor movement. Morphological examinations of peripheral and central neurons, revealed axonal swelling, characterized by massive neurofilamentous accumulation. The findings suggest that covalent cross-linking of neurofilaments might be the toxic mechanism of CS2.
Carbon disulphide (CAS number 75–15–0) is both reagents used in the manufacture of S-allyl O-pentyl dithiocarbonate. Therefore, Carbon disulphide (CAS number 75–15–0) need to be considered in the assessment of S-allyl O-pentyl dithiocarbonate.
Executive summary:

What follows is the original abstract of the publication

Female C57BL6 mice were exposed to 0 or 800 ppm carbon disulfide (CS2), 6 h/d, 5 d/wk for 20 weeks. The neurologic function of all mice was assessed once at the end of exposures using a functional observational battery. General health effects included a decrease in body weight gain, piloerection, hunched body posture, and ptosis. Treatment-related effects included altered gait (uncoordinated placement of hind limbs and ataxia) and impaired function on an inverted screen test. In addition, rearing and locomotor movement were decreased in treated mice. Focal to multifocal axonal swelling was seen predominantly in the muscular branch of the posterior tibial nerve, and occasionally giant axonal swelling was detected in the lumbar segment of the spinal cord. Electron microscopic

examination revealed swollen axons with massive accumulation of neurofilament proteins within the axoplasm. Covalent crosslinking of erythrocyte spectrin (surrogate protein to neurofilament protein) was demonstrated in mice exposed to CS2 but not in mice receiving filtered air. These data provide supportive evidence that covalent cross-linking of neurofilament proteins is a significant feature of the axonal swellings in mice produced by inhalation exposure to C22.

Endpoint:
neurotoxicity: sub-chronic inhalation
Type of information:
other: published data
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Carbon disulphide (CAS number 75–15–0) is both reagents used in the manufacture of S-allyl O-pentyl dithiocarbonate. Therefore, Carbon disulphide (CAS number 75–15–0) need to be considered in the assessment of S-allyl O-pentyl dithiocarbonate.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Rats were exposed to various concentrations of CS2, for several time points. HPLC together with UV detection was used to isolate and quantify alpha and beta chains of globin in blood of the CS2 -exposed animals. The degree of globin modification was compared to light microscopic and ultrastructral changes in the central and peripheral nervous system. The modified globin was also characterized by liquid chromatography interfaced with electrospray mass spectrometry.This publication is only one part of a whole study that aimed at investigating CS2 neurotoxicity in a coordinated way (several endpoints included); see below for details.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Raleigh, NC)
- Age at study initiation: 8-9 weeks
- Housing: individually in wire-mesh cages within the exposure chambers
- Diet: ad libitum, only during non-exposure times
- Water: ad libitum
- Acclimatation period: 10-14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 49.1
- Photoperiod (hrs dark / hrs light): 12
Route of administration:
inhalation: vapour
Vehicle:
unchanged (no vehicle)
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Data taken from Sills et al. (1998a, Carbon disulfide neurotoxicity in rats: I. Introduction and study design. NeuroToxicology 19 (1): 83-88)

CS2 was vaporized mixed with conditioned air and delivered to the chambers.
- Air flow rate: 4 l/min
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Monitored every 15 min with infrared spectrophotometers; maintained at the 3% of the targeted concentrations throughout the study.
Duration of treatment / exposure:
6 h/d for 2, 4, 8 or 13 w
Frequency of treatment:
daily, 5 d/w
Remarks:
Doses / Concentrations:
0, 50, 500, 800 ppm (0, 156, 1558, 2493 mg/m3)
Basis:
nominal conc.
No. of animals per sex per dose:
9 (in the 13 w experiment 9 more animals were used, per dose/sex)
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: according to Sills et al. (1998a; Carbon disulfide neurotoxicity in rats: I. Introduction and study design. NeuroToxicology 19 (1): 83-88) the exposure concentrations were selected based on previous studies (Gottfried et al., 1985) demonstrating that metabolic saturation occurs at approximately 600 ppm CS2. Concentrations above (800 ppm) and below (500 ppm) metabolic saturation were selected for use, as well as, a concentration close to the then current TLV of 10 ppm (50 ppm).
Observations and clinical examinations performed and frequency:
MORPHOLOGY
Rats were whole body perfused with 4% phosphate buffered paraformaldehyde followed by 4% phosphate buffered glutaraldehyde pH 7.4. A complete necropsy was performed. Sections of cervical and lumbar spinal cord were post-fixed in 2% osmium tetroxide, dehydrated in graded alcohols and propylene oxide and embedded in Epoxy resin. For light microscopy, 1 µm thick sections were stained with toluidine blue. For electron microscopy, 60 nm ultra thin sections were stained with lead citrate and uranyl acetate. Four males and four females were examined per group (except for the 13 w, were 8 animals/dose/sex were examined).
Other examinations:
All the other examinations can be seen in the relevant seperate study entry (see below for details, section 'any other information on materials and methods incl. tables').
Positive control:
no
Statistics:
no statistic applied
Clinical signs:
not examined
Mortality:
not examined
Body weight and weight changes:
not examined
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
Clinical biochemistry findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Details on results:
Morphology: the results are analyzed in the summary of Sills et al. (1998).
Analysis of globin samples by RP-HPLC showed peaks corresponding to a combined alpha and a combined beta peak. An additional peak that was not seen in the samples from control animals, eluting between the two peaks of alpha and beta globin chains was also detected. All concentrations gave significant quantities of modified globin after 2 weeks of exposure, which were time-dependent. The authors compared these results with the results of Valentine et al. (1997) in order to elucidate any correlation between globin modification and erythrocyte cross-linking. A linear relationship was observed.
Dose descriptor:
NOAEL
Effect level:
156 - 2 493 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
not determinable
Remarks:
no NOAEL identified

Morphology: the results are analyzed in the summary of Sills et al. (1998).

Analysis of globin samples by RP-HPLC showed peaks corresponding to a combined alpha and a combined beta peak. An additional peak that was not seen in the samples from control animals, eluting between the two peaks of alpha and beta globin chains was also detected. All concentrations gave significant quantities of modified globin after 2 weeks of exposure, which were time-dependent. The authors compared these results with the results of Valentine et al. (1997) in order to elucidate any correlation between globin modification and erythrocyte cross-linking. A linear relationship was observed.

Conclusions:
It has been demonstrated that CS2 exposure of rats results in covalent modification of globin in erythrocytes; this is paralled by spectrin crosslinking and neurofilaments cross-linking (Valentine et al. 1997).The findings suggest that hemoglobin might be a sensitive preneurotoxic biomarker of effects for CS2.
Carbon disulphide (CAS number 75–15–0) is both reagents used in the manufacture of S-allyl O-pentyl dithiocarbonate. Therefore, Carbon disulphide (CAS number 75–15–0) need to be considered in the assessment of S-allyl O-pentyl dithiocarbonate.
Executive summary:

What follows is the original abstract of the publication

Although the neurotoxicity of CS2 has been recognized for over a century, presently there is no accepted biomarker of effect for CS2 exposure. Previous investigations have supported covalent cross-linking of erythrocyte spectrin as a potential preneurotoxic marker reflective of the biochemical changes occuring within the axon. In the present investigation, the potential of using CS2 promoted modification of hemoglobin as a dosimeter for quantifying exposure to CS2 was evaluated. Liquid chromatography was used to isolate and measure α and β chains of globin duration. The degree of globin modification was compared to light microscopic and ultrastructural changes in the central and peripheral nervous systems to determine the temporal relationship of globin modification to the structural changes in the axon. Samples obtained from rats exposed to CS2 contained a globin chain not present in control samples. Analysis of the peak corresponding to the new chain using electrospray mass spectometry was consistent with the generation of a single dithicarbamate ester or thiourea intramolecular cross-link in the α 1 major chain. This altered globin chain was detectable both at the subneurotoxic level of exposure and prior to axonal structural changes at the neurotoxic levels of exposure used. The extent of modification was positively correlated to the exposure level and duration for all conditions examined. These findings support hemoglobin as a potential preneurotoxic biomarker of effect for CS2 possessing several practical advantages relative to the use of CS2 -mediated spectrin cross-linking.

Endpoint:
neurotoxicity: inhalation
Remarks:
other: subacute and subchronic
Type of information:
other:
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Carbon disulphide (CAS number 75–15–0) is both reagents used in the manufacture of S-allyl O-pentyl dithiocarbonate. Therefore, Carbon disulphide (CAS number 75–15–0) need to be considered in the assessment of S-allyl O-pentyl dithiocarbonate.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The aim of this investigation was to study the effects of CS2 on the neurobehaviour of rats, with the use of a functional observation battery (FOB). Rats were exposed via inhalation to several concentrations of CS2 and thereafter, FOB was used to measure several neurobehavorial parameters. This publication is only one part of a whole study that aimed at investigating CS2 neurotoxicity in a coordinated way (several endpoints included); see below for details.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
Some data were taken from Sills et al. (1998a, Carbon disulfide neurotoxicity in rats: I. Introduction and study design. NeuroToxicology 19 (1): 83-88)

TEST ANIMALS
- Source: Charles River Laboratories (Raleigh, NC)
- Age at study initiation: 8-9 weeks
- Housing: individually in wire-mesh cages within the exposure chambers
- Diet: ad libitum, only during non-exposure times
- Water: ad libitum
- Acclimatation period: 10-14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 49.1
- Photoperiod (hrs dark / hrs light): 12
Route of administration:
inhalation: vapour
Vehicle:
unchanged (no vehicle)
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Data taken from Sills et al. (1998a, Carbon disulfide neurotoxicity in rats: I. Introduction and study design. NeuroToxicology 19 (1): 83-88)

CS2 was vaporized mixed with conditioned air and delivered to the chambers.
- Air flow rate: 4 l/min
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Monitored every 15 min with infrared spectrophotometers; maintained within 3% of the targeted concentrations throughout the study.
Duration of treatment / exposure:
6 h/d for 2, 4, 8 or 13 w
Frequency of treatment:
daily, 5 d/w
Remarks:
Doses / Concentrations:
0, 50, 500, 800 ppm (0, 156, 1558, 2493 mg/m3)
Basis:
nominal conc.
No. of animals per sex per dose:
9 (groups exposed for 2, 4 or 8 w), 18 (group exposed for 13 w)
Control animals:
yes, sham-exposed
Observations and clinical examinations performed and frequency:
Clinical observations: emaciation, anatomy, posture, hypothermia, appendage fracture, swelling, tissue mass, fur appearance & discoloration, wound, abscess, ulcer, ptosis, exopthalmia, lacrimal discharge, nasal discharge, salivation, respirations, urination, defecation, activity, comatose, ataxia, circling, tremors, convulsions, paralysis.
Neurobehavioural examinations performed and frequency:
FUNCTIONAL OBSERVATIONAL BATTERY: Yes
- Parameters checked in table 1 were examined (see below).
- Same technicians used throughout testing: No data
- Technicians were blind to treatment status of animals: No data
- Site of testing: isolated room with restricted access
- Scoring criteria (if any): presented in McDaniel & Moser (1993)
- Duration of observation period for open field observations: 3 min
Sacrifice and (histo)pathology:
All rats were tested at the beggining of the study and the morning after the last exposure (approx 18 h after). The FOB testing was performed according to the protocol of McDaniel & Moser (1993). Following FOB testing, the animals were tested for nerve conduction velocity alterations (summary of Herr et al., 1998). The postexposure in vivo tests were then followed by perfusion for neuropathological studies (see summary of Sills et al., 1998) or for protein cross-linking (see summary of Valentine et al., 1998) and nerve growth factor-receptor gene expression studies in peripheral nerve (Towes et al., 1998
Positive control:
no
Statistics:
ANOVA with the use of a grouping factor of treatment (concentration) and repeated measures of test time (pre- and post-exposure), categorical modeling procedure (CATMOD), post-hoc analyses with Dunett's t-test
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 specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Clinical biochemistry findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY lethality not treatment related; postural abnormalites (hunched posture were seen mostly at the 800 ppm at all exposure durations); no other abnormalities were noted

BODY WEIGHT AND WEIGHT GAIN weight gain was adversely affected, significant changes from 500 ppm and above ;more prominent in males

NEUROBEHAVIOUR according to the results of the FOB analysis, the major effects were exerted on neuromotor function, typically affecting the hindlimbs. A clear dose- and time- response motive was observed regarding the neuromuscular changes. Hindlimb and forelimb grip strength values were decreased , in the 500 and 800 ppm groups, with the hindlimb strenght being the most affected. Gait changes were recorded uncoordinated pacement, evolving by the 13th week to disturbed hindlimb control; such changes were significant at 500 & 800 pmm. Mild tremors, as well as decreased responsiveness to visual stimulus, were seen in the 13-week study, only at the high dose level. No essential changes were seen in activity. Few effects were observed on the sensorimotor reactivity. In response to handling it was generally increased, and excitability in the open field was decreased, in rats tested after the shorter exposures (two and four weeks).
Dose descriptor:
NOAEC
Effect level:
50 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Remarks on result:
other:
Dose descriptor:
NOAEC
Effect level:
156 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Remarks on result:
other:
Conclusions:
CS2 exposure produced neuromotor dysfunction, mainly manifesting in the hindlimbs, evident as early as 2 weeks of exposure, that showed a dose- and time-associated trend. These effects were significant in groups exposed to 500 or 800 ppm, and not to 50 ppm. The 50 ppm is considered as a NOAEC of the present investigation.
Carbon disulphide (CAS number 75–15–0) is both reagents used in the manufacture of S-allyl O-pentyl dithiocarbonate. Therefore, Carbon disulphide (CAS number 75–15–0) need to be considered in the assessment of S-allyl O-pentyl dithiocarbonate.
Executive summary:

Young adult male and female Fischer 344 rats were exposed to CS2 at the following concentrations: 0, 50, 500, 800 ppm, 6 h/d, 5 d/w, for 2, 4, 8 or 13 weeks via inhalation. The neurobehavorial alterations were investigated with a functional obsrvation battery (FOB). All rats were also tested prior to exposure so as to get baseline values. Neuromuscular deficits which were more pronounced in the hindlimbs, were observed in both sexes. Alterations showed a dose- and time-dependent trend. Other effects, mostly observed after 13 weeks of exposure, included decreased responsiveness to a visula stimulus and mild tremors. Reactivity in response to handling was generally increased, and excitability in the open field was decreased, in rats tested after the shorter exposures (two and four weeks).

Endpoint:
neurotoxicity: acute inhalation
Type of information:
other: published data
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Pentan-1-ol/Amyl alcohol is both reagents used in the manufacture of S-allyl O-pentyl dithiocarbonate. Therefore, pentan-1-ol/Amyl alcohol need to be considered in the assessment of S-allyl O-pentyl dithiocarbonate.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Relative acute neurotoxicity of solvents: Isoeffective air concentrations of 48 compounds evaluated in mice after 4 h inhalation.
GLP compliance:
no
Limit test:
no
Species:
mouse
Strain:
other: H strain
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 2 - 4 months
- Weight at study initiation: 350 g
- Housing: 16 mice were housed in one plastic cage in climatized boxes
- Diet (e.g. ad libitum): peletted diet DOS2b; ad libitum



ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
inhalation
Vehicle:
not specified
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: in 80-liter glass chambers operated under dynamic conditions (Frantík and Kratochvíle, 1976).
- Method of holding animals in test chamber: small plastic boxes ventilated through diffusion tubes permitting almost instant stabilization of the air concentration in the respiration zone.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
using gas chromatography
Duration of treatment / exposure:
2 h
Remarks:
Doses / Concentrations:
3 concentrations; 25 - 75% of the maximum effect
Basis:
no data
No. of animals per sex per dose:
8
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: All animals were given 3 control tests at weekly intervals before the 1st exposure. Most animals went through 3 - 4 exposures to each concentration, and the interval between exposures was at least 3 weeks.
Observations and clinical examinations performed and frequency:
Additional testing required for neurotoxicity studies:
All animals were given three control tests at weekly intervals before the first exposure.
Measuring of the biological effect always started less than 1 min after removal from the exposure box.
A short electrical impulse (0.2 sec, 50 Hz, 90 V in mice) was applied through ear electrodes. Of the six time characteristics recorded, the velocity of tonic extension (i.e. reciprocal of the latency) in mice was the most sensitive and reproducible response measure. The median of individual control values was subtracted from the values observed after exposure. Group means of differences were corrected for the difference in the simultaneously tested control group and converted to relative values, i.e. to percentage of the arbitrary maximum values, which for mice were 1/0.5 sec. (These values are exceeded in control tests only exceptionally.)
Dose descriptor:
conc. level: Isoeffective concentration evoking 30% depression in recorded activity
Effect level:
2 600 ppm
Based on:
test mat.
Sex:
female
Remarks on result:
other:
Dose descriptor:
conc. level: Isoeffective concentration evoking 30% depression in recorded activity
Effect level:
9.55 mg/L air (nominal)
Based on:
test mat.
Sex:
female
Remarks on result:
other:

Isoeffective conc. of solvent in inhaled air evoking a 30% depression in recorded activity:

conc = 2600 ppm

one-sided 90% confidence interval: 980 ppm

slope of the regression: 0.011 %ppm

Conclusions:
Isoeffective conc. of solvent in inhaled air evoking a 30% depression in recorded activity: conc = 2600 ppm.
Pentan-1-ol/Amyl alcohol is both reagents used in the manufacture of S-allyl O-pentyl dithiocarbonate. Therefore, pentan-1-ol/Amyl alcohol need to be considered in the assessment of S-allyl O-pentyl dithiocarbonate.
Endpoint:
neurotoxicity: acute inhalation
Type of information:
other: published data
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Pentan-1-ol/Amyl alcohol is both reagents used in the manufacture of S-allyl O-pentyl dithiocarbonate. Therefore, pentan-1-ol/Amyl alcohol need to be considered in the assessment of S-allyl O-pentyl dithiocarbonate.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Relative acute neurotoxicity of solvents: Isoeffective air concentrations of 48 compounds evaluated in rats after 4 h inhalation.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 0.5 - 1 year
- Weight at study initiation: 350 g
- Housing: four rats were housed in one plastic cage in climatized boxes
- Diet (e.g. ad libitum): peletted diet DOS2b with a supraoptimal content of vitamins, in a limited amount of 12 g per day per animal


ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
inhalation
Vehicle:
not specified
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: in 80-liter glass chambers operated under dynamic conditions (Frantík and Kratochvíle, 1976).
- Method of holding animals in test chamber: small plastic boxes ventilated through diffusion tubes permitting almost instant stabilization of the air concentration in the respiration zone.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
using gas chromatography
Duration of treatment / exposure:
4 h
Remarks:
Doses / Concentrations:
3 concentrations; 25 - 75% of the maximum effect
Basis:
no data
No. of animals per sex per dose:
4
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: All animals were given 3 control tests at weekly intervals before the 1st exposure. Most animals went through 3 - 4 exposures to each concentration, and the interval between exposures was at least 3 weeks.
Neurobehavioural examinations performed and frequency:
Additional testing required for neurotoxicity studies:

All animals were given three control tests at weekly intervals before the first exposure.
Measuring of the biological effect always started less than 1 min after removal from the exposure box.
A short electrical impulse (0.2 sec, 50 Hz, 180 V in rats) was applied through ear electrodes. Of the six time characteristics recorded, the duration of tonic extension of hindlimbs in rats was the most sensitive and reproducible response measure. The median of individual control values was subtracted from the values observed after exposure. Group means of differences were corrected for the difference in the simultaneously tested control group and converted to relative values, i.e. to percentage of the arbitrary maximum values, which for rats were 8 sec. (These values are exceeded in control tests only exceptionally.)
Dose descriptor:
conc. level: Isoeffective concentration 30% depression in recorded activity
Effect level:
1 600 ppm
Based on:
test mat.
Sex:
male
Remarks on result:
other:
Dose descriptor:
conc. level: Isoeffective concentration 30% depression in recorded activity
Effect level:
5.85 mg/L air (nominal)
Based on:
test mat.
Sex:
male
Remarks on result:
other:

Isoeffective conc. of solvent in inhaled air evoking a 30% depression in recorded activity: conc = 1600 ppm one-sided 90% confidence interval: 245 ppm slope of the regression: 0.022 %ppm

Conclusions:
Isoeffective conc. of solvent in inhaled air evoking a 30% depression in recorded activity:conc = 1600 ppm.
Pentan-1-ol/Amyl alcohol is both reagents used in the manufacture of S-allyl O-pentyl dithiocarbonate. Therefore, pentan-1-ol/Amyl alcohol need to be considered in the assessment of S-allyl O-pentyl dithiocarbonate.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
156 mg/m³
Study duration:
subchronic
Species:
rat

Effect on neurotoxicity: via dermal route

Link to relevant study records
Reference
Endpoint:
neurotoxicity: sub-chronic oral
Type of information:
other: published data
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Carbon disulphide (CAS number 75–15–0) is both reagents used in the manufacture of S-allyl O-pentyl dithiocarbonate. Therefore, Carbon disulphide (CAS number 75–15–0) need to be considered in the assessment of S-allyl O-pentyl dithiocarbonate.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Rats were exposed subchronically to CS2 by gavage, in order to investigate the alterations of neurofilament (NF) proteins in the spinal cord and the possible mechanism of action of the substance.In addition neurological testing was performed.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Experimental Animal Center of Shandong University
- Weight at study initiation: 180-200 g
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 50
- Photoperiod (hrs dark / hrs light): 12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
VEHICLE
- Justification for use and choice of vehicle (if other than water): no
- Concentration in vehicle: 3 ml/kg
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
12 w
Frequency of treatment:
daily, 5 d/w
Remarks:
Doses / Concentrations:
0, 300, and 500 mg/kg bw
Basis:
other: treatment by gavage
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle
Observations and clinical examinations performed and frequency:
BODY WEIGHT: Yes
- Time schedule for examinations: twice per week
Specific biochemical examinations:
Spinal cord homogenates were centrifuged at 100,000 g for 1 h to yield a high-speed pellet (P) and a high-speed supernatant (S). According to previous investigations, the P fraction consists of polymerized triplet proteins and represents the triton-insoluble filamentous cytoskeletal network . The high-speed supernatant (S) likely contains tritonsoluble NF (neurofilament) proteins in the form of intermediate heterotetramers and monomers that represent the mobile population of exchangeable NF (neurofilament) proteins . Protein concentration of two fractions was assayed with the use of a Protein assay Kit.

Protein fractions were subjected to SDS-PAGE and thereafter, alterations of NF subunits monoclonal antibodies for NF-H, NF-M, and NF-L were used by immunoblotting. In order to examine the contribution of calpains to the changes of NFs, the protein levels of µ-calpain and m-calpain in the S and P fraction were also tested with the use of immunoblotting. Calpain is a neutral proteinase in the Central Nervous System that can degrade the NF subunits.

To determine whether the decrement in NF proteins was due to corresponding reductions in NF gene expression, the effects of CS2 on NF-L,
NF-M, and NF-H mRNA were investigated with the use of RT-PCR. GADPH gene was used as an internal standard.
Neurobehavioural examinations performed and frequency:
NEUROLOGICAL TESTING: the onset and development of neurotoxicity were determined by gait scoring (animals were observed for 3 min in an open field, twice per week). A gait score was assigned from 1 to 4 where 1=a normal, unaffected gait; 2=a slightly abnormal gait (hindlimbs show uncoordinated placement, exaggerated or overcompensated movements, or are splayed slightly, walks on tiptoes); 3=moderately abnormal gait (obvious movement abnormalities characterized by markedly splaying hindlimbs, ataxia, swaying, rocking, lurching, stumbling); 4=severely abnormal gait (flat foot walk, hindlegs flat on surface, crawling, or unable to support weight).
Sacrifice and (histo)pathology:
- Time point of sacrifice: at the end of the 12 weeks
- Number of animals sacrificed: all animals, by decapitation
Statistics:
one-way analysis of variance, LSD's post hoc tests (P<0.05)
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 specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Clinical biochemistry findings:
effects observed, treatment-related
Behaviour (functional findings):
effects observed, treatment-related
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Details on results:
Exposure to CS2 resulted in decrease of body weight gain and progressive gait abnormalities. At the end of the experiment most of the animals exposed to 500 mg/kg bw showed moderately abnormal gait, while slight gait abnormality was seen in anmals treated with 300 mg/kg bw.

BIOCHEMICAL EXAMINATIONS
The results on the NFs protein content of the two fractions (supernatant, pellet):
pellet(P): significant decrease in NF-H, NF-M and NF-L at both exposure doses, except for NF-L isolated from animals treated with the low dose.
supernatant (S): significant decrease in NF-H, NF-M and NF-L levels only at the dose of 500 mg/kg bw, compared to the controls.

Independent of the dose levels the spinal cord (both fractions) content in µ-calpain was increased significantly. For m-calpain a decrease was observed in the supernatant fractions, while the pellets remained unaffected.

The results regarding alterations in NF expressions, revealed significant increases in the mRNA production of all three NF proteins.
Dose descriptor:
LOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: decreased body weight gain, slight gait abnormalities
Remarks on result:
other:
Conclusions:
Carbon disulfide exposure by gavege in rats (300 and 500 mg/kg bw) resulted in neurological deficits, i.e. abnormal gait, that might be related to the observed significant reductions of NF subunits spinal cord homogenates. The results indicate that NF alterations might be related to the developments of CS2 neurotoxicity.
Carbon disulphide (CAS number 75–15–0) is both reagents used in the manufacture of S-allyl O-pentyl dithiocarbonate. Therefore, Carbon disulphide (CAS number 75–15–0) need to be considered in the assessment of S-allyl O-pentyl dithiocarbonate.
Executive summary:

What follows is the original abstract of the publication

To investigate the mechanism of carbon disulfide-induced neuropathy, male Wistar rats were randomly divided into two experimental groups and one control group. The rats in two experimental groups were treated with carbon disulfide by gavage at dosages of 300 and 500 mg/kg/day, respectively, five times per week for 12 weeks. Spinal cords of carbon disulfide-intoxicated rats and their age-matched controls were Triton-extracted and ultracentrifuged to yield a pellet fraction of neurofilament (NF) polymer and a corresponding supernatant fraction. Then, the contents of NF triplet proteins (NF-H, NF-M, NF-L) and two calpain isoforms (m-calpain and µ-calpain) in both fractions were determined by immunoblotting. In the meantime, the mRNA levels of NF-H, NF-M, and NF-L in spinal cords were quantified using reverse transcriptase-polymerase chain reaction. Results showed that in the pellet fraction, the contents of three NF subunits in both treated groups decreased significantly except NF-L in low dose group. In the supernatant fraction, the pattern of NFs alteration varied according to dose-levels. Compared to controls, three neurofilmant subunits in the high dose group displayed significant reduction consistently. However, in the low dose group, they remained unaffected. As for calpains, the contents of µ-calpain in both fractions increased significantly regardless of carbon disulfide dose-levels. Meanwhile, m-calpain demonstrated a significant decline in the supernatant fraction, and remained unchangeable in the pellet fraction compared to the control group. Furthermore, the levels of mRNA expression of NF-H, NF-M, and NF-L genes were elevated consistently in CS2-treated groups. These findings suggested that carbon disulfide intoxication was associated with obvious alterations of NFs content in rat spinal cord, which might be involved in the development of carbon disulfide neurotoxicity.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LOAEL
7.5 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
For dermal exposure we taken that:
-the average weight of rat is 250 g (200 -300 g),
-the dose is applied over an area which is approximately 10% of the total body surface=0.008 kg

corrected dermal LOAEL= oral LOAEL
300 mg/kg bw/dx 0.025 kg =
LOAELrat 7.5 mg/kg bw/day

Additional information

oral exposure

Carbon disulfide exposure by gavage in rats (300 and 500 mg/kg bw) resulted in neurological effects, i.e. abnormal gait and reductions in retention time on the accelarating rotarod, as well as in significant reductions of neurofilaments (NF) subunits and increase of microtubules and microfilaments subunits, in cerebral cortex homogenates. The authors associate the changes in the NFs cerebrum proteins with the CS2 treatment, suggesting this as a possible mechanism inducing neuropathy.

Carbon disulphide (CAS number 75–15–0) is both reagents used in the manufacture of S-allyl O-pentyl dithiocarbonate. Therefore, Carbon disulphide (CAS number 75–15–0) need to be considered in the assessment of S-allyl O-pentyl dithiocarbonate.

LOAEL=300 mg/kg bw day

 

dermal exposure

For dermal exposure we taken that:

-the average weight of rat is 250 g (200 -300 g),

-the dose is applied over an area which is approximately 10% of the total body surface=0.008 kg

 

corrected dermal LOAEL=    oral LOAEL

                                   300 mg/kg bw/dx 0.025 kg =                   

 LOAELrat     7.5 mg/kg bw/day

 

Inhalation exposure:

 

The aim of the study of Sills RC, et al.1998 was to examine the morphological progression and dose response of CS2 distal axonopathy in the muscular branch of the posterior tibial nerve (MBPTN) and spinal cord. For this reason, male and female Fischer 344 rats were exposed to 0, 50, 500, 800 ppm (0, 156, 1558, 2493 mg/m3) of CS2, 6 h/d, 5 days per week, for 2, 4, 8 or 13 w, via inhalation. At 8 w axonal swelling was observed in the MBPTN of animals exposed at 800 ppm, that progressed to more severe at 13 w. Similarly, axonal swelling was seen in the cervical and lumpar spinal cord, after 8 w, but at both high exposure concentrations. Swelling was accompanied by thinning of the myelin sheath and accumulation of 10 nm neurofilaments. No lesions related to CS2 exposure were detected in the heart, brain, aorta, lung, trachea, liver, kidneys, nasal cavity, testes, epididymis, ovaries, oviducts, uterus and vagina.

 

The NOAEC was 156 mg/m3 based on the no axonal swelling in the muscular branch of the porterior tibial nerve and spinal cord.

Carbon disulphide (CAS number 75–15–0) is both reagents used in the manufacture of S-allyl O-pentyl dithiocarbonate. Therefore, Carbon disulphide (CAS number 75–15–0) need to be considered in the assessment ofS-allyl O-pentyl dithiocarbonate.

Justification for classification or non-classification

There are conclusive but not suffcient data for the classification of substance S-allyl O-pentyl dithiocarbonate with regard to Neurotoxicity.