Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Repeated dose toxicity of 2-methylpropane-2-thiol has been investigated in a 13-week inhalation toxicity study (Ulrich, 1984) and an oral combined repeated dose/reproductive/developmental toxicity study (MHLW, 2006) in rats. In the 3-month inhalation study, the NOAEC for systemic toxicity was 196 ppm (721 mg/m3). In the oral repeated-dose toxicity study (OECD TG 422), based on decreased body weight reduction in females at 200 mg/kg/day, the NOAEL was considered to be 50 mg/kg/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: no data
- Age at study initiation: no data
- Weight at study initiation: 272.2-325.1 g for males, 188.1-235-9 g for females
- Housing:no data
- Diet (e.g. ad libitum): no data
- Water (e.g. ad libitum):no data
- Acclimation period:no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-27
- Humidity (%): 35-75
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12-12
Route of administration:
oral: gavage
Vehicle:
corn oil
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
no details available
Duration of treatment / exposure:
42-53 days
Frequency of treatment:
Once daily
Remarks:
Doses / Concentrations:
10, 50, 200 mg/kg bw/day
Basis:

No. of animals per sex per dose:
12 males and 17 females per group at 0 and 200 mg/kg bw/day and 12 males and 12 females per group at 10 and 50 mg/kg bw/day; 12 males and 12 females per group were used for mating. To investigate reversibility, a recovery period of 14 days was established for 5 males and 5 non-mated females at 0 and 200 mg/kg bw/day
Control animals:
yes, concurrent vehicle
Details on study design:
Post-exposure period: 14 days
Positive control:
not relevant
Observations and examinations performed and frequency:
Clinical observations and frequency:
General condition was observed 2 or 3 times a day throughout the administration period and once a day throughout the recovery period.

Body weight:
Body weight was measured on days 1 (before dosing), 4, 8, 11, 15, 18, 22, 25, 30, 32, 36, 39, and 42 of the administration period in males. In females, body weight was measured on days 1 (before dosing), 4, 8, 11, 15, 18, 22, 25, 30, 32, 36, 39, and 42 of the administration period. Body weight of pregnant females was measured on days 0, 7, 14, and 20 of gestation and days 0 and 4 of lactation. Body weight was measured at necropsy in both sexes.

Food consumption:
Food consumption was measured on days 1 (before dosing), 4, 8, 11, 15, 30, 32, 36, 39, and 42 of the administration period in males. In females, food consumption was measured on days 1 (before dosing), 4, 8, 11, 15, 30, 32, 36, 39, and 42 of the administration period, and on days 18, 22, and 25 in non-mated females. Food consumption of pregnant females was measured on days 1, 7, 14, and 20 of gestation and days 1 and 4 of lactation.

During the recovery period, body weight and food consumption were measured on days 1, 4, 8, 11, and 14 in both sexes.

Urinalysis:
Urinalysis was carried out for 5 males per group at 6 weeks of the administration period.

Functional observation battery:
Detailed clinical observation was carried out once a week in all animals throughout the administration period. In pregnant females, it was carried out on days 7, 14, and 20 of gestation and on day 4 of lactation. Sensory reaction test, grip strength, and motor activity were examined at 6 weeks of administration in males and on day 4 of lactation in pregnant females.

Hematology and blood biochemistry:
At necropsy, hematology and blood biochemistry were examined in 5 males and 5 females per group.
Sacrifice and pathology:
Necropsy was carried out at the day following the end of the administration and recovery periods.

Organ weights:
Organs were examined at necropsy. The brain, heart, liver, kidney, adrenal, thymus, thyroids, and spleen of 5 males and 5 females per group and the testis and epididymis of all males were weighed.

Microscopic examination:
The spleen, liver, and kidney in 5 males and 5 females of each group were microscopically examined at the end of the administration and recovery periods. The cerebrum, cerebellum, medulla oblongata, pituitary, thymus, thyroid, parathyroid, adrenal, heart, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, trachea, lung, urinary bladder, spinal cord, mesenteric lymph node, submaxillary lymph node, sciatic nerve, femur (included bone marrow), and sternum (included bone marrow) of 5 males and 5 females at 0 and 200 mg/kg bw/day were microscopically examined at the end of the administration period. The testis, epididymis, prostate, and seminal vesicles of 5 males at 0 and 200 mg/kg bw/day were microscopically examined at the end of the administration period. Further, the ovary, uterus, and vagina of 5 females at 0 and 200 mg/kg bw/day were microscopically examined at the end of the administration period. Additionally, kidney tissue was immunohistochemically examined by staining with anti-alpha2u-globulin.
Statistics:
Statistical methods: For numerical data, Dunnett's test (homogeneous) or Steel test (heterogeneous) was used. Steel Multiple comparison test was used for urinary of test paper method. Wilcoxon's rank sum test was used for detailed clinical observation data, sensory reaction test data. Fisher exact probability test for necropsy data and Mann-Whitney U-test method for histopathology data were used.
Clinical signs:
no effects observed
Description (incidence and severity):
No compound-related changes were observed in any animal.
Mortality:
no mortality observed
Description (incidence):
There was no death in any group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A low value was observed in both sexes at 200 mg/kg bw/day throughout the administration period. During the recovery period, a lower body weight was observed in females, however, their body weight gains throughout the recovery period were similar to those of the control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A low value or a tendency toward a low value was observed in males at 200 mg/kg bw/day on days 4 and 15 of administration and in females at 200 mg/kg bw/day throughout the administration period. During the recovery period, females exhibited lower food consumption on day 1 of the recovery period, but food consumption after day 4 of the recovery period was similar to the control group. A decrease in food consumption was observed in females at 10mg/kg on day 15 of the administration period. However, it was not observed at 50mg/kg and not considered to be a dose-related effect.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Males:
Administration period (Table 1): Decreases in erythrocyte count, hemoglobin, hematocrit, and MCHC, an increase in platelet count, and prolonged PT and APTT were observed at 200 mg/kg bw/day. A decrease in MCHC was observed at 50 mg/kg bw/day. A decrease in reticulocyte at 10 mg/kg was not considered to be a dose related effect because it was not observed at 50 mg/kg bw/day and higher.
Recovery period (Table 2): Decreases in hemoglobin and MCHC and an increase in reticulocyte ratio were observed at 200 mg/kg bw/day.

Females:
Administration period Table 3): A decrease in erythrocyte count and an increase in reticulocyte ratio were observed at 200 mg/kg bw/day. An increase in reticulocyte at 50 mg/kg bw/day was not considered to be a dose related effect. A shortening of the APTT was observed at 50 mg/kg bw/day and higher.
Recovery period (Table 4): Increases in MCV and MCH and a decrease in MCHC were observed at 200 mg/kg bw/day. An increase in basophile at 200 mg/kg was not considered to be a dose related effect because it was not observed at the end of administration period.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Males:
Administration period (Table 5): Decreases in alpha1-globulin, glucose and chlorine and increases in alpha2-globulin, albumin, gamma-GTP, total cholesterol, and phospholipids were observed at 200 mg/kg bw/day. Increases in total cholesterol and phospholipids at 50 mg/kg bw/day were observed.
Females:
Administration period (Table 6): Decreases in alpha1-globulin and glucose and increases in total protein, A/G ratio, albumin, total cholesterol, and phospholipids were observed at 200 mg/kg bw/day. An increase in total cholesterol was observed at 50 mg/kg bw/day. A decrease in ALP at 50 mg/kg bw/day was not considered to be a dose related effect because it was not observed at 100 mg/kg bw/day.
Recovery period: A decrease in creatinine was observed at 200mg/kg bw/day. However, it was not considered to be a dose related effect because this effect was not observed at the end of administration period.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Administration period (Tables 7 and 8): Increases in absolute and relative weights of the liver were observed in males at 50 mg/kg bw/day and above and in females at 200 mg/kg bw/day. In the kidney of males, absolute weight at 50 mg/kg bw/day and above, and relative weight at 10 mg/kg bw/day and above were significantly increased. A decrease in absolute thymus weight was observed in males at 200 mg/kg bw/day. There were increases in relative weights of the heart, thyroids, testes and epididymides in males at 200 mg/kg and of the heart in females at 200mg/kg. However, absolute weights of these organs were not changed, and no histopathological changes were observed in these organs. These changes were considered to be due to decreases in body weights. Increases in absolute and relative weights of the epididymides in males and of the thymus in females were observed at 50mg/kg, but these changes were not considered to be dose related effects because these effects were not observed at 200 mg/kg bw/day.
Recovery period (Tables 9 and 10): Increase in relative liver weight was observed in both sexes at 200 mg/kg bw/day. Furthermore, increases in absolute and relative weights of the kidneys were observed in males at 200 mg/kg bw/day. There were increases in absolute weight of the adrenal in males at 200mg/kg and relative weight of the kidney in females at 200 mg/kg bw/day. However, these effects were not considered to be dose related effects because these changes were not observed at the end of administration period.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Administration period: Enlargement and discoloration of the kidneys were observed in 1, 3, and 4 males at 10, 50, and 200 mg/kg bw/day, respectively. Liver enlargement was observed in 2 males at 200 mg/kg bw/day.
Recovery period: Kidney enlargement was observed in 1 male at 200 mg/kg bw/day.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Administration period (Tables 11 and 12): Histopathological changes were observed in the liver and spleen of both sexes and in the kidneys of males. The histopathological findings were as follows: centrilobular hypertrophy of hepatocytes in the liver in males at 50 mg/kg bw/day and above and in females at 200 mg/kg bw/day, hemosiderin deposits in the red pulp in the spleen of both sexes at 200 mg/kg bw/day, periportal fatty degeneration of hepatocytes in the liver in males at 50 mg/kg bw/day and above, basophilic renal tubules in the kidneys in males at 10 mg/kg bw/day and above and hyaline deposits in proximal tubular epithelial cells in the kidneys in males at 10 mg/kg bw/day and above, which indicates alpha2u-globulin nephropathy.
Recovery period (Tables 13 and 14): Hemosiderin deposits were observed in the red pulp of the spleen of both sexes, and basophilic renal tubules were observed in the kidneys in males at 200 mg/kg bw/day. Periportal fatty degeneration of hepatocytes in the liver was observed in 1 male and 1 female at 200 mg/kg bw/day.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Based on body weight reduction observed in female rats dosed at 200 mg/kg/day.
Critical effects observed:
not specified
Table 1: Haematological parameters in males (end of treatment)
Dose (mg/kg bw/day)
0
10
50
200
Erythrocyte count (10^4/uL)
815(29)
820(21)
768(39)
743(28)**
Hemoglobin(g/dL)
14.9(0.5)
14.9(0.6)
14.2(0.3)
13.7(0.5)**
Hematocrit (%)
42.2(1.2)
42.9(1.6)
40.9(0.9)
 39.8(1.8)*
MCHC (g/dL)
35.3(0.2)
34.9(0.2)
34.7(0.5)*
34.5(0.3)**
Reticulocyte (%)
2.3(0.2)
1.8(0.3)*
 2.2(0.2)
3.5(0.9)
Platelet count (10^4/uL)
95.1(5.9)
96.1(5.9)
106.6(9.1)
121.7(19.2)**
PT (sec)
14.1(1.1)
14.8(1.8)
16.5(4.2)
21.9(8.9)*
APTT (sec)
26.1(1.9)
27.9(4.3)
27.0(3.8)
38.1(7.5)**
*p<0.05, ** p<0.01; n=5
Mean (Standard Deviation)


    
Table 2: Haematological parameters in males (end of recovery)
Dose (mg/kg bw/day)
0
200
Hemoglobin (g/dL)
15.5(0.5)
14.5(0.6)*
MCHC (g/dL)
35.8(0.8)
34.7(0.6)*
Reticulocyte (%)
2.3(0.2)
2.8(0.3)*
Note: *, p<0.05; n=5
Mean (Standard Deviation)

Table 3: Haematological parameters in females (end of treatment)
Dose (mg/kg bw/day)
0
10
50
200
Erythrocyte count (10^4/uL)
656(36)
634(12)
640(21)
607(37)*
Reticulocyte (%)
4.4(0.7)
7.0(1.8)*
5.7(2.1)
6.4(0.5)*
APTT (sec)
19.6 (1.6)
17.8(1.0)
17.1(0.8)*
16.8(1.4)**


    
Table 4: Haematological parameters in females (end of recovery)
Dose (mg/kg bw/day)
0
200
MCV (fL)
52.3(0.9)
54.8(1.2)**
MCH (pg)
18.9(0.4)
19.5(0.3 )*
MCHC (g/dL)
36.2(0.4)
35.6(0.3)*
Basophile (%)
0.1(0.0)
0.2(0.1)*
Note: *, p<0.05, **; p<0.01; n=5


    
Table 5: Blood biochemistry parameters in males (end of treatment)
Dose (mg/kg bw/day)
0
10
50
200
alpha1-globulin (%)
17.9(2.6)
17.8(1.6)
16.8(2.3)
13.7(1.4)*
alpha2-globulin (%)
7.6(0.7)
8.0(0.4)
8.2(0.6)
8.8(0.8)*
Albumin (g/dL)
2.92(0.22)
3.04(0.18)*
2.93(0.16)
3.43(0.30)*
gamma-GTP (IU/L)
0.3(0.2)
0.5(0.4)
0.2(0.3)
0.8(0.4)*
Total cholesterol (mg/dL)
60(10)
73(17)
85(8)*
96(15)**
Phospholipid (mg/dL)
106(10)
126(20)
141(8)*
171(26)**
Glucose (mg/dL)
124(9)
117(16)
111(23)
79(9)**
Chlorine (mEq/L)
108.4(2.3)
107.6(1.6)
107.0(1.4)
105.1(2.1)*

Note: *, p<0.05 **; p<0.01; n=5
Mean (Standard Deviation)



    
Table 6: Blood biochemistry parameters in females (end of treatment)
Dose (mg/kg bw/day)
0
10
50
200
Total protein (g/dL)
5.2(0.3)
5.4(0.2)
5.4(0.5)
6.2(0.4)**
A/G Ratio
1.19(0.07)
1.12(0.12)
1.20(0.09)
1.36(0.11)*
alpha1-globulin (%)
18.4(1.2)
17.6(1.8)
17.9(0.7)
15.5(2.1)*
Albumin (g/dL)
2.85(0.18)
2.83(0.21)
2.92(0.25)
3.57(0.29)**
ALP (IU/L)
241(37)
223(75)
132(30)*
177(90)
Total cholesterol (mg/dL)
74(3)
80(7)
97(8)*
120(22)*
Phospholipid (mg/dL)
144(8)
145(10)
173(19)
223(32)**
Glucose (mg/dL)
121(7)
112(12)
107(14)
93(8)**
Note: *, p<0.05 **; p<0.01; n = 5
Mean (Standard Deviation)


Conclusions:
Given that the hematological effects observed in the study were slight in nature with no indication of any associated adverse clinical or histopathogical effects and that the centrilobular hepatocellular hypertrophy is likely an adaptive response, the NOAEL for this study is 50 mg/kg/day.

Executive summary:

In an OECD TG 422 study, Sprague-Dawley rats were dosed by oral gavage with 0, 10, 50, or 200 mg/kg 2-methylpropane-2-thiol for 42 to 53 days. A 14-day recovery period was included for the control and the 200 mg/kg dose groups. There was no mortality, effect on urinalysis, clinical signs of toxicity, changes in functional observational battery (FOB) measurements, or motor activity.

Decreased body weight was observed in the 200 mg/kg animals during the treatment period, with no difference between treated and control animals during the 14-day recovery period. At 200 mg/kg, there was a slight, but statistically significant decrease in erythrocyte count in males and females (-9% and -8%, respectively); and slightly decreased hemoglobin, hematocrit, and MCHC (-8%, -6%, and -2%, respectively) in males. Prothrombin time and activated partial thromboplastin time were significantly increased in the 200 mg/kg males, however, activated partial thromboplastin time was shortened in the 50 and 200 mg/kg females. Following the recovery period, hemoglobin levels and MCHC were slightly, but significantly decreased (both sexes), and reticulocyte count was increased in males. Serum chemistry changes in the 200 mg/kg animals included: decreased α1-globulin and glucose; and increased albumin, cholesterol, phospholipids, α2-globulin (males only), and γ-GTP (males only). There were no significant changes in the serum chemistry between the treated and control animals following the 14-day recovery period. Relative kidney weights were increased in males at all dose levels; relative liver weights were increased in the 50 and 200 mg/kg males and in the 200 mg/kg females; and relative heart weights were increased in the 200 mg/kg animals (both sexes). Histopathological changes were seen in the kidneys of all dosed male rats. These changes included basophilic tubules and hyaline droplets in proximal tubular cells, which are indicative of alpha2u-globulin nephropathy, an effect not considered relevant to humans. Centrilobular hepatocyte hypertrophy was noted in the 50 and 200 mg/kg males and in the 200 mg/kg females, and slight hemosiderin deposition was noted in the spleen of the 200 mg/kg males and females. Following the 14-day recovery period, hemosiderin was still present in the spleens of the 200 mg/kg animals, but there was no centrilobular hepatocyte hypertrophy in the females and the incidence and severity was reduced in males. Given that the hematological effects were slight in nature with no indication of any associated adverse clinical or histopathological effects and that the centrilobular hepatocellular hypertrophy is likely an adaptive response, the NOAEL for this study is 50 mg/kg/day (based on body weight reduction observed in female rats dosed at 200 mg/kg/day).

This study received a Klimisch Score of 2 and was classified as reliable with restriction because although it is a GLP guideline study, a study report in English was not available.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Reliable with restrictions (Klimisch score = 2)
System:
other: decreased body weight in females at 200 mg/kg bw/day

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
when compared to the 1981 guideline: no opthalmological examination, no coagulation parameters and blood electrolytes
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, Michigan
- Age at study initiation: not reported
- Weight at study initiation: 205-215 g for males, 161-164g for females
- Housing: individually (animals were maintained within the exposure chambers)
- Diet (e.g. ad libitum): Purina® Certified Pelleted Rodent Chow® #5002
- Water (e.g. ad libitum): tap water
- Acclimation period: 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 72-84
- Humidity (%): 32-68
- Air changes (per hr): animals were maintained within the exposure chambers
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
clean air
Details on inhalation exposure:
Animal Exposure Methods:
Exposures were conducted in 1 m3 glass and stainless steel exposure chambers. Air for chamber ventilation was supplied by an HVAC system separate from the general laboratory systems. This air was particulate filtered and controlled for temperature and humidity. Chamber airflow varied (between 130 and 300 liters/minute) depending on desired exposure concentrations. Exposure temperature and humidity were recorded each day after 3 and 6 hours of exposure, except on the one day each week when the exposure was shortened to 4.5 hours for animal observations. On these days the temperature and humidity were recorded at 3 and 4.5 hours (see Table A, below).

Exposure Atmosphere Generation Methods:
Vapor atmospheres of 2-methylpropane-2-thiol were generated utilizing a counter-current vaporization system. Due to the large range of liquid flow rates required, two types of fluid metering devices were used: a FMI pump and two Sage Syringe Drives.
The system operates as follows: a FMI pump or a Sage Syringe Drive delivers the test material at a known constant rate to the top of the glass bead column. Air is passed up the bead column in a counter-current manner relative to the liquid. Vaporization occurs within the bead column. The concentrated vapors are piped to the exposure chamber air inlet where dilution with chamber ventilation air reduces the concentration to the desired level. Prior to the initiation of each day's exposure, the generation system was brought up to its operational conditions. Table B (see below) summarizes the operational characteristics of the various generation systems used for 2-methylpropane-2-thiol.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Actual exposure concentrations were measured by non-dispersive IR utilizing Wilks MIRANT 1-A analyzers. These analyzers were calibrated by volumetric dilution of the test material in a closed-loop calibration system as recommended by the instrument's manufacturer. The calibration of the IR analyzer was checked prior to each day's exposure. Utilizing an automated sampling system, exposure concentrations in each chamber were monitored at approximately hourly intervals.
Table C (see below) summarizes the exposure concentration data by presenting the mean of the 13 weekly mean nominal and actual exposure concentrations.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6-hours per day, 5-days per week
Remarks:
Doses / Concentrations:
10, 100 and 200 ppm
Basis:
other: target concentration
Remarks:
Doses / Concentrations:
9, 97 and 196 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
15
Control animals:
yes, sham-exposed
Details on study design:
Post-exposure period: None
Observations and examinations performed and frequency:
GENERAL OBSERVATIONS
1. Appearance and Behavior
All animals were observed in detail individually for pharmacotoxic signs once each week.

2. Mortality
Twice each exposure day, prior to exposure and again after exposure, all animals were observed for mortality and overt toxicity. On non-exposure days observations were conducted once in the morning and once in the afternoon.

3. Body Weights
Body weights for all animals were recorded once each week on the day that detailed observations for pharmacotoxic signs were made. On the day that body weights and detailed observations were made exposures were shortened to approximately 4.5 hours.

CLINICAL LABORATORY TESTS
Whenever possible, hematological and serum biochemical evaluations were performed on the same 5-male and 5-female rats from each group prior to exposure and after approximately 6 and 12 weeks of exposure.
Blood was obtained via puncture of the orbital sinus plexus from rats fasted overnight (approximately 16 hours).
1. Hematology
Hematological determinations included: hemoglobin, hematocrit, erythrocyte countl, total leucocyte count, platelet count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), differential leucocyte count and sulfhemoglobin.
2. Biochemistry
Biochemical determinations included: blood urea nitrogen (BUN), alkaline phosphatase, total bilirubin, aspartate aminotransferase (formerly SGOT), alanine aminotransferase (formerly SGPT), total protein, albumin, A/G ratio (calculated), glucose and whole blood cholinesterase.

Sacrifice and pathology:
SACRIFICE AND MACROSCOPIC EXAMINATIONS
After approximately 13 weeks of inhalation exposure to the test material, all surviving rats were sacrificed by intraperitoneal sodium pentobarbital administration followed by exsanguination from the abdominal aorta. These and ail rats which died spontaneously or were sacrificed in extremis were subjected to complete postmortem examinations under the direct supervision of a pathologist.
The postmortem examination consisted of an evaluation for external abnormalities including palpable masses and an inspection of orifices. The skin was then reflected from a ventral midline incision taking care not to enter the thoracic cavity. The trachea was exposed and clamped, the thoracic cavity opened and the lungs removed and examined while inflated. The heart was then removed and weighed. The lungs and trachea were deflated and weighed then reinflated via the trachea with 10% neutral buffered formalin. The abdominal cavity was examined for abnormalities and the organs removed, weighed when appropriate and placed in fixative. The urinary bladder was inflated through the wall with the fixative and left unopened for examination after fixation. The skull was opened, the brain examined and removed and the eyes removed. The pituitary was examined in situ and removed with surrounding sella turcica. The muscle was stripped off the vertebral column and spinal cord fixed intact after cutting into the vertebral column in at least two separate sites.

ORGAN WEIGHTS
The following organs from all terminally sacrificed animals were trimmed free of fat and connective tissue and weighed:
heart, kidneys, lung and trachea, testes or ovaries (after fixation), liver, brain, spleen, adrenal (after fixation)

HISTOPATHOLOGY
Microscopic examination of fixed hematoxylin-eosin stained paraffin sections was performed for all animals from the control and high level groups (also at low and mid dose for lungs and kidneys) , including those dying during the course of the study, sacrificed in extremis or from the terminal sacrifice. The following tissues were examined by an IRDC staff pathologist:
adrenals (both), aorta, brain (3 sections), cecum, colon, esophagus, eye and optic nerve, gonade, heart, kidney (both), liver, lungs (all 5 lobes), tumors
lymph nodes (bronchial, cervical, mesenteric) and any other tissue(s) with lesions, nasal turbinate, pancreas, pituitary, prostate/uterus, salivary gland (submaxillary), skin, small intestine (jejunum), spleen, sternum (bone marrow), stomach, trachea, thyroid/parathyroid (if present, in section), urinary bladder.
Statistics:
Generally, when the number of animals in any one group was equal-toor-less-than seven, non-parametric analyses were conducted utilizing the Kruskal-Wallis one-way analysis of variance followed, where appropriate, with the Mann-Whitney U. In those cases where the number of animals in all groups was greater than seven and the measurements were at least on an interval scale (continuous data) parametric analyses were conducted utilizing Bartlett's Chi-square test for homogeneity of variance, followed where appropriate, by an analysis of variance and then where appropriate by Dunnett's -t. In all cases the level of rejection was at the five percent level.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
In general, the exposed animals displayed no pharmacotoxic signs that were considered to be exposure-related. Alopecia and some instances of ocular discharge were observed.
Mortality:
no mortality observed
Description (incidence):
Only one animal died during the course of the study, a male in Group VI. The death may have been related to blood collection trauma, as it died shortly after blood collection for the 6-week interval.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no statistically significant exposure-related effects on body weight in males or females. It appears that there could be some dose-response effect on body weight in males, but this observation vas not as apparent in female rats. Therefore, these apparent dose-response trends are not considered to be of toxicological importance.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
After approximately 6 weeks of exposure, the erythrocyte count was slightly decreased (-7%) in female rats of Group VI when compared to the female control group. The values obtained are not considered to be outside a "normal" range and are not considered to be of biological significance.
After approximately 12 weeks of exposure, the erythrocyte count was slightly decreased in female rats of Groups VI (-5%) and VII (-4%) when compared to the female control group . The erythrocyte count for females falls within the range of historical normal values, with the control group being toward the high end of that range. Thus, the apparent dose-response reduction in erythrocyte count of females may be a random occurrence and of no toxicological significance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
After approximately 6 weeks of exposure, the only parameter for which a statistically significant difference was observed was for the slightly elevated BUN (+34%) of Group VI males. Due to the fact that this was a singular occurrence and that the value is still within the "normal" range, this difference is probably not biologically important.
After approximately 12 weeks of exposure, there were no statistically significant differences observed in any of the exposure groups with respect to any of the parameters when judged against the control group.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute and relative kidney weights were statistically significantly elevated in males of Groups VI (+24% and +19%) and VII (+23% and +28%). These observed differences may also be exposure-related, as the differences are observed in the two highest exposure groups for t-butyl mercaptan. Again, no such effect was observed in the females exposed to the same test material. Various other statistically significant differences observed with respect to organ weights are not considered to be biologically meaningful.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test article-related microscopic changes were observed in lungs of male and female rats and in the kidneys of the male rats.
There was a compound related increase in alveolar macrophages among males and females of the mid dose (97 ppm) and high dose (196 ppm) groups exposed to t-butyl mercaptan. At the mid dose level, 5 of 15 males and 3 of 15 females were affected. All lesions were trace in severity. At the high dose level 14 of 15 males and 12 of 15 females were affected. One male and one female were mild and all of the others were trace in severity. This lesion did not occur at the low dose level (9 ppm) and it was considered to be the no effect level.
Evaluation of the kidneys of male rats exposed to t-butyl mercaptan at 196 ppm showed trace to moderate chronic nephrosis in 14 of the 15 animals. The lesion consisted of varying degrees of multifocal degeneration of the proximal convoluted tubules, tubular regeneration and inflammatory cell infiltration of the interstitium. This lesion, nephrosis and/or regeneration occurred in the mid and low dose groups males, 7 of 15 at the low dose level and 13 of 15 at the mid dose level. It was considered to be compound related at all three dose levels.
Incidental non-treatment-related lesions were observed in adrenal, heart, liver, branchial and cervical lymph nodes, nasal turbinates, prostate, testes, trachea, eye, stomach and thymus.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Dose descriptor:
NOAEC
Remarks:
systemic toxicity
Effect level:
>= 196 ppm (analytical)
Sex:
male/female
Basis for effect level:
clinical biochemistry
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Remarks on result:
other: 196 ppm = 721 mg/m3
Critical effects observed:
not specified

Table 1 : male animals, mean body weight (g)

Week

Mean bw (sd)

Group I (control)

Group V (10 ppm)

Group VI (100 ppm)

Group VII (200 ppm)

ALLOC

205(7.2)

210(8.3)

215(8.5)

210 (9.5)

1

278(15.7)

289(17.4)

296(16.5)

270(25.4)

2

312(16.8)

325(21.1)

330(21.8)

308(20.9)

3

342(19.2)

359(26.4

360(24.5)

338(24.6

4

370(21.4)

383(31.2)

382(31.5)

359(28.2)

5

380(23.0)

399(30.1)

385(53.8)

376(36.7)

6

398(24.5)

415(33.4)

400(46.7)

392(37.5)

7

410(24.8)

427(37.9)

431(44.1)

402(39.0)

8

432(31.7)

448(40.3)

450(50.0)

421(38.0)

9

451(31.9)

468(42.6)

468(48.2)

442(41.4)

10

457(34.4)

473(46.4)

470(47.6)

448(44.1)

11

460(36.6)

482(47.9)

477(50.1)

452(42.4)

12

463(36.7)

479(48.8)

476(54.4)

442(55.9)

13

463(41.8)

481(51.3)

486(57.0)

449(62.0)

Table 2 : female animals, mean body weight (g)

Week

Mean bw (sd)

Group I (control)

Group V (10 ppm)

Group VI (100 ppm)

Group VII (200 ppm)

ALLOC

162(6.0)

161(6.4)

163(5.9)

164(4.4)

1

194(11.4)

193(13.1)

194(9.2)

195(10.2)

2

209(12.0)

209(14.0)

207(9.8)

212(12.5)

3

216 (13.7)

220(13.8)

218(11.9)

222(11.7)

4

229(15.3)

227(17.3)

228(10.3)

230(13.7)

5

233(17.5)

253(17.0)

233(12.7)

233(20.3)

6

239(18.5)

237(18.5)

236(14.7)

241(18.1)

7

244(20.2)

242(20.5)

241(14.2)

247(19.5)

8

249(21.6)

251(22.5)

248(13.8)

256(20.5)

9

256(22.2)

257(23.4)

257(14.2)

263(19.9)

10

258(21.6)

252(21.0)

255(25.6)

267(19.8)

11

263(22.7)

259(21.5)

254(18.7)

270(20.8)

12

262(25.7)

255(21.3)

254(21.8)

266(23.4)

13

267(24.5)

267(28.4)

254(30.2)

271(22.8)

Table 3: Mean(sd) hematological vales, week 6

Group

Sex

Leucocytes 103/cm3

RBC 106/cm3

Hb g/dl

Ht %

MCV µ3

MCH g

MCHC g/dl

Platelets 103/cm3

Neutro-philes seg/100WBC

Lympho-cytes /100WBC

SulHb g/dl

I

M

12.7(3.14)

8.10(0.462)

17.6(1.20)

47.1(3.02)

58(1.5)

21.8(0.38)

37.4(0.40)

647(53.9)

22(10.7)

74(9.7)

0(0)

V

M

11.7(3.01)

8.18(0.371)

18.1(0.71)

47.2(2.05)

58(0.8)

22.1(0.50)

38.2(0.60)

637(60.2)

21(11.1)

73(11.0)

0(0)

VI

M

20.8(19.8)

6.92(1.94)

15.3(4.10)

40.2(10.5)

58(1.8)

22.1(0.55)

31.9(0.51)

677(98.6)

18(6.2)

75(7.7)

0(0)

VII

M

14.6(3.55)

7.61(0.82)

16.7(0.96)

43.6(2.85)

57(3.0)

22.1(1.49)

38.4(0.54)

590(64.9)

23(5.5)

74(6.5)

0(0)

I

F

10.0(2.55)

7.66(0.23)

17.1(0.69)

45.2(2.05)

59(1.3)

22.4(0.46)

37.9(0.36)

629(50.7)

14(6.4)

83(6.1)

0(0)

V

F

8.2(2.93)

7.58(0.33)

16.7(0.49)

44.1(2.14)

59(1.2)

22.1(0.36)

37.5(0.90)

604(89.9)

17(10.9)

78(11.5)

0(0

VI

F

10.0(2.87)

7.18*(0.32)

16.4(1.21)

43.3(3.47)

60(2.6)

22.7(0.74)

37.8(0.72)

623(44.1)

19(6.6)

78(5.1)

0(0)

VII

F

9.7(1.62)

7.54(0.354)

16.8(0.62)

44.1(1.50)

59(1.0)

22.3(0.32)

38.1(0.46)

635(144.2)

19(3.7)

77(2.6)

0(0)

WBC -White blood cells Leucocytes

*Statistically different from the control group mean, p<0.05

Table 4: Mean(sd) hematological vales, week 12

Group

Sex

Leucocytes 103/cm3

RBC 106/cm3

Hb g/dl

Ht %

MCV µ3

MCH g

MCHC g/dl

Platelets 103/cm3

Neutro-philes seg/100WBC

Lympho-cytes /100WBC

SulHb g/dl

I

M

11.1(3.65)

8.37(0.217)

17.7(0.66)

50.1(2.50)

60(3.2)

21.1(0.26)

35.0(2.05)

766(51.9)

21(14.0)

77(15.0)

0(0)

V

M

10.2(1.31)

8.67(0.329)

18.1(0.75)

50.5(1.74)

58(2.6)

20.8(0.44)

35.8(0.90)

737(93.2)

19(3.6)

78(3.1)

0(0)

VI

M

9.8(2.60)

8.11(0.547)

17.6(0.86)

48.6(1.87)

58(2.1)

21.2(0.50)

36.3(1.23)

754(33.8)

20(7.4)

77(6.2)

0(0)

VII

M

12.5(3.66)

7.71(1.469)

16.1(2.91)

47.0(8.40)

61(7.1)

21.2(0.92)

34.8(2.48)

925(289.6)

21(12.6)

76(12.0)

0(0)

I

F

9.4(3.10)

7.91(0.130)

17.6(0.60)

49.8(3.23)

63(4.7)

22.2(0.76)

35.3(2.38)

762(140.0)

13(3.8)

83(4.1)

0(0)

V

F

7.2(2.66)

7.99(0.248)

17.8(0.75)

49.6(3.02)

62(2.1)

22.3(0.81)

36.1(1.43)

714(151.2)

14(1.8)

84(2.6)

0(0)

VI

F

9.4(2.89)

7.55*(0.203)

17.1(0.57)

46.8(1.02)

62(0.7)

22.6(0.35)

36.5(0.51)

762(81.0)

20(11.5)

74(10.0)

0(0)

VII

F

7.1(0.63)

7.63*(0.187)

17,1(0.86)

46.9(2.69)

62(2.1)

22.5(0.78)

36.5(0.75)

770(87.6)

14(2.7)

84(3.0)

0(0)

WBC - Whitebloodcells

* Significantly different from control group mean, p<0.05

** Significantly different from control group mean, p<0.01

Table 5: Mean (sd) biochemical Values, 6 Weeks

Group

Sex

BUN (mg/dl)

Alk. Phosp. (IU/L)

Total bilirubin (mg/dl)

AST (IU/l)

ALT (UI/l)

Total protein (g/dl)

Albunin (g/dl)

A/G ratio

Glucose (mg/dl)

Cholines-terase (µM/ml/min)

I

M

11.9(1.38)

97(14.8)

0.1(0.05)

92(26.6)

40(5.4)

7.2(0.34)

3.5(0.14)

1.0(0.05)

96(13.8)

4.2(0.39)

V

M

13.2(2.90)

85(20.7)

0.2(0.05)

90(16.5)

35(4.7)

6.8(0.26)

3.3(0.17)

1.0(0.05)

97(15.9)

4.3(0.25)

VI

M

16.0*(1.79)

75(16.4)

0.2(0.08)

100(42.2)

43(8.6)

6.9(0.89)

3.3(0.40)

0.9(0.04)

92(12.1)

3.8(0.91)

VII

M

14.3(1.66)

80(17.4)

0.2(0.05)

96(44.1)

38(4.5)

6.9(0.36)

3.4(0.12)

0.9(0.05)

92(10.3)

4.1(0.34)

I

F

15.2(1.04)

49(12.1)

0.2(0.05)

76(6.6)

32(4.8)

7.7(0.42)

3.8(0.29)

1.0(0.08)

97(9.4)

5.5(0.70)

V

F

14.6(1.88)

57(11.6)

0.2(0.08)

91(32.0)

33(8.8)

7.3(0.24)

3.7(0.19)

1.0(0.10)

101(14.0)

5.3(0.42)

VI

F

14.2(0.62)

52(11.5)

0.2(0.00)

101(36.1)

59(63.0)

7.5(0.36)

3.8(0.24)

1.0(0.05)

94(13.4)

5.7(0.71)

VII

F

16.0()2.76

59()15.0

0.2()0.05

93(6.8)

36(3.8)

7.7(0.49)

3.8(0.27)

1.0(0.08)

88(10.4)

5.4(0.62)

*Significanrly different from control group mean, p<0.05

Table 6: Mean (sd) biochemical Values, 12 Weeks

Group

Sex

BUN (mg/dl)

Alk. Phosp. (IU/L)

Total bilirubin (mg/dl)

AST (IU/l)

ALT (UI/l)

Total protein (g/dl)

Albunin (g/dl)

A/G ratio

Glucose (mg/dl)

Cholines-terase (µM/ml/min)

I

M

12.4(1.83)

71(15.8)

0.2(0.04)

89(19.2)

34((6.1)

7.1(0.42)

3.5(0.09)

1.0(0.10)

97(10.7)

4.1(0.17)

V

M

12.9(2.65)

63(13.6)

0.2(0.04)

99(23.0)

33(6.1)

7.1(0.29)

3.5(0.11)

1.0(0.07)

90(11.1)

4.4(0.22)

VI

M

14.9(1.17)

66(24.3)

0.2(0.04)

94(19.9)

36(7.5)

7.4(0.60)

3.6(0.30)

1.0(0.15)

90(13.3)

4.3(0.34)

VII

M

13.4(1.18)

59(11.0)

0.1(0.05)

106(20.3)

32(6.7)

6.7(0.38)

3.3(0.29)

1.0(0.08)

88(8.3)

4.2(0.41)

I

F

12.6(1.80)

39(11.9)

0.2(0.04)

80(3.5)

30(6.2)

7.6(0.58)

4.0(0.33)

1.1(0.08)

91(18.1)

5.3(0.76)

V

F

12.6(3.30)

46(4.5)

0.2(0.04)

80(10.3)

29(1.8)

7.7(0.63)

4.0(0.28)

1.1(0.08)

110(22.2)

5.7(0.70)

VI

F

15.2(3.09)

43(12.5)

0.2(0.04)

89(11.3)

25(2.6)

7.5(0.33)

3.9(0.19)

1.1(0.05)

85(16.1)

5.2(0.74)

VII

F

12.1(1.08)

41(16.5)

0.2(0.00)

89(10.4)

28(3.5)

7.5(0.47)

3.9(0.23)

1.1(0.07)

96(17.0)

5.5(0.48)

Conclusions:
2-methylpropane-2-thiol induced an increase of kidney weights in male rats exposed to 97 and 196 ppm and a chronic nephropathy in all males. It has been shown in the OECD 422 study that 2-methylpropane-2-thiol induced male rat-specific hyaline droplets nephropathy with no relevance for human risk assessment (Hard et al., 2009). The NOAEC for systemic toxicity is higher or equal to 196 ppm.

References:
Hard GC, Johnson KJ and Cohen SM (2009) A comparison of rat chronic progressive nephropathy with human renal disease-implications for human risk assessment. CRIT-REV-TOXICOL, 39, 332-346.
Takahashi K, Lindamood C and Maronpot RP (1993) Retrospective Study of Possible alpha-2µ-Globulin Nephropathy and Associated Cell Proliferation in Male Fischer 344 Rats Dosed with t-Butyl Alcohol. ENVIRON-HEALTH-PERSPECT, 101(SUPPL 5), 281-286.
Executive summary:

In a 13-week inhalation toxicity study, male and female Sprague-Dawley rats (15/sex/dose) were exposed (whole body) to 2 -methylpropane-2-thiol at measured concentrations 0, 9, 97, or 196 ppm (0, 33, 357 or 721 mg/m3, respectively) for six hours per day, five days per week. 

There were no deaths, clinical signs of toxicity or body weight changes observed. Blood urea nitrogen was statistically significantly different from the control group at the 6-week interval only for the 97 ppm exposure group; this change was not considered biologically relevant because it occurred at only one time interval. Statistically significant differences in erythrocyte count were only found in females at 6-week (97 ppm) and 12-week (97 and 196 ppm). The clinical pathology endpoints that differed from concurrent controls remained within the range of historical control values and were not considered to be biologically or toxicologically relevant. No compound-related macroscopic lesions were observed in any of the rats that were sacrificed at the termination of the study or those that died during the course of the study. There was a compound related increase in alveolar macrophages among males and females of the mid dose (97 ppm) and high dose (196 ppm) groups exposed to t-butyl mercaptan. At the mid dose level, 5 of 15 males and 3 of 15 females were affected. All lesions were trace in severity. At the high dose level 14 of 15 males and 12 of 15 females were affected. One male and one female were mild and all of the others were trace in severity. This lesion did not occur at the low dose level (9 ppm). Toxicologically significant increases in the mean weights of kidneys occurred in male rats exposed to 97 and 196 ppm. There was a compound and concentration-related increase in chronic nephrosis (varying degrees of multifocal degeneration of the proximal convoluted tubules, tubular regeneration, and inflammatory cell infiltration of the interstitium) in 14 of 15 animals at the high concentration (196 ppm). The lesion was also noted in 13 of 15 at the mid concentration (97 ppm) and 7 of 15 animals at the low concentration (9 ppm). However, findings in the kidneys of males were considered to be rat-specific with no relevance for human risk assessment.

The NOAEC for systemic toxicity was determined to be ≥ 196 ppm (721 mg/m3).

This study received a Klimisch score of 2 and is classified as reliable with restriction.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
721 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
Reliable with restrictions (Klimisch score = 2)

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not reported
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
yes
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Orient Bio Co. (Seoul, Republic of Korea)
- Age at study initiation: 6 weeks
- Housing: individually in wire-bottomed stainless-steel mesh cages that were placed in exposure chambers
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Details on inhalation exposure:
The test animals were either exposed to butane-2-thiol or fresh air for 6 h per day, 5 days a week for a period of 13 weeks. The inhalation
exposure was carried out from 10:00 to 16:00 in a stainless-steel chamber (1000 L). The experimental design was based on the usual working
schedule for workers, as well as the major exposure route for the test chemical.

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Gas Generator (Shibata Co. Japan) connected to a whole-body stainless steel exposure chamber (Shibata Co. Japan)
- Method of holding animals in test chamber: Animals were housed individually in wire-bottom stainless steel cages that were placed inside the exposure chambers.
- Source and rate of air: Not reported
- Method of conditioning air: Air mixed with sec-butanethiol vapors conditioned at 30°C by passage through a thermostatic condenser
- System of generating particulates/aerosols: Fresh air was bubbled through the liquid sec-butanethiol to generate sec-butanethiol vapor-air mixture.

TEST ATMOSPHERE
- Brief description of analytical method used: The apparatus and conditions used for detecting sec-butanethiol by gas chromatography (Shimadzu Co., Japan) were as follows: detector, flame ionization detector; column, 0.5 m silicon DC-200 15% chromosorb with a mesh of 80/100; detector temperature, 150°C; oven temperature, 130°C; oven temperature, 200°C; injector temperature, 200°C; and injection volume, 1 ml of sample gas.
- Samples taken from breathing zone: yes

VEHICLE (if applicable)
- Vehicle: Fresh air
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The mean concentration was measured by gas chromatography every 30 min for 6 h and was taken as the concentration value for each day. This was then averaged over the 13-week exposure period in order to obtain the mean and standard deviations.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hrs per day, 5 days per week
Remarks:
Doses / Concentrations:
25, 100 and 400 ppm
Basis:
other: target conc.
Remarks:
Doses / Concentrations:
25.1 ± 1.44, 99.6 ± 3.78, and 403.4 ± 32.75 ppm
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
92, 367, 1488 mg/m3
Basis:
analytical conc.
No. of animals per sex per dose:
10 per sex per dose
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: The experimental concentrations were selected based on the results of a preliminary dose-range finding study in which rats (5/sex/dose) were exposed to sec-butanethiol via whole-body inhalation at concentrations of 62.5, 125, 250, and 500 ppm for 2 weeks. At a concentration of 500 ppm, both males and females showed suppressed body weight gain and decreased food intake. A dose of 250 ppm produced only a slight decrease in body weight gain. On the contrary, there were no treatment-related effects of clinical signs, body weight, or food intake at ≤125 ppm. On the basis of these results, 400 ppm was used as the high dose, and the doses of 100 and 25 ppm were selected as medium and low doses, respectively, using a scaling factor of 4.
- Rationale for animal assignment (if not random): Random assignment
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily (before and after exposure)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily (before and after exposure)

BODY WEIGHT: Yes
- Time schedule for examinations: Prior to beginning of exposure and once/week during the experimental period.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes (The amounts of food were calculated before they were supplied to the cages, and the remnants were measured the next day in order to calculate the difference, which was regarded as daily food consumption (g/rat/day)).

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to exposure and during the last week of the experimental period.
- Dose groups that were examined: All animals in each dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to sacrifice
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: 10/sex/dose
- Parameters checked in table [No.1] in the results section were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to sacrifice
- Animals fasted: Yes
- How many animals: 10/sex/dose
- Parameters checked in table [No.2] in the results section were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: During last week of exposure
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters such as specific gravity, pH, protein, glucose, ketone body, occult blood, bilirubin, urobilinogen, nitrite, and leukocyte contents were examined.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

ORGAN WEIGHTS: Yes
400 ppm concentrations of sec-butanethiol and a vehicle control group exposed to
fresh air only. Each group consisted of 10 rats of each gender.
The absolute and relative (organ-to-body weight ratios) weights of the following organs were measured: brain, heart, thymus, lung, liver, spleen, kidney, adrenal, testis, epididymis, and/or ovary.

HISTOPATHOLOGY: Yes
The following tissues were obtained from all animals: skin, mammary gland, spleen, pancreas, jejunum, stomach, duodenum, ileum, cecum, colon, mesenteric lymph node, salivary gland, submandibular lymph node, ovaries, uterus, vagina, urinary bladder, epididymides, prostates, seminal vesicles, rectum, kidneys, adrenal glands, liver, sternum, thymus, heart, lung, trachea, esophagus, thyroids (including parathyroids), tongue, aorta, sciatic nerve, skeletal muscle, femur, thoracic spinal cord, Harderian glands, brain, pituitary gland, eyes, testes, nasal cavity, nasal turbinates, and Zymbal glands. Eyes and testes were preserved in Davidson’s fixative and Bouin’s fixative, respectively. Other tissues were fixed with a 10% neutral buffered formalin solution. The tissues were routinely processed, embedded in paraffin, and sectioned at 3–5 lm. The sections were stained with hematoxylin–eosin for microscopic examination. The nasal passages and nasal turbinates were decalcified prior to being embedded and sectioned. The nasal cavity was sectioned at levels posterior to the upper incisors, the incisive papilla, the second palatine ridge, and the first molar teeth (Young, 1981). All the organs and tissues removed from the animals in the vehicle control group and the treatment groups were examined microscopically. All gross lesions, as defined in the study of pathology, were also included in the examination.
Statistics:
The variance of numerical data was checked using Bartlett’s test (1937). For homogeneous variances, data was subjected to a one-way analysis of variance (ANOVA) and, for non-homogenous variances; data was analyzed by Kruskal-Wallis’ non-parametric ANOVA (1952). If either of the tests showed a significant difference among the groups, the data was analyzed by the multiple comparison procedure of the Dunnett’s post-hoc test (1964). Clinical signs, necropsy findings, and histopathological findings were represented as frequencies, and were subjected to Fisher’s exact probability test (1970) where necessary. All statistical analyses were conducted with Statistical Analysis Software (SAS Institute, Inc., 1997). The significant probability values P < 0.05 (*) or P < 0.01 (**) are represented with asterisks.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Although not statistically significant, the body weight of male rats was slightly lower in the 400 ppm group from Day 7 of the test to termination when compared to the control group. In contrast, the body weight of male rats was slightly higher in the 25 ppm group from Day 14 of the test to termination when compared with the control group. Statistically significant suppression of body weight gain was observed in female rats on days 28 and 42 to 90 of treatment in the 25 ppm group, and on days 14 through 90 of treatment in the 400 ppm group when compared to control animals.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In males, the amount of food consumed was significantly lower on Days 0 and 7 of treatment in the 400 ppm group (11.7 ± 3.9 and 20.7 ± 1.5 g) than in the control group (22.6 ± 2.4 and 24.3 ± 2.3 g). In females, food consumption of the 400 ppm group was also decreased significantly on Days 0 and 7 of treatment (9.6 ± 3.5 and 13.6 ± 1.8 g) than in the control group (18.2 ± 2.7 and 18.8 ± 3.3 g).
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In males, the serum value of ALT in the 400 ppm group decreased significantly, while BUN levels in the 25 ppm group increased significantly compared to the control group. In females, the serum level of ALT decreased significantly, while the total bilirubin increased significantly in the 400 ppm group compared to the control group.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In males, the relative weights of the liver and kidneys in the 400 ppm group increased significantly in a dose-dependent manner compared to those of the control group. In females, the relative weights of the kidneys, brain, lung, and heart in the 400 ppm group also significantly increased in a dose-dependent manner compared to those of the control group.
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathological changes were observed in the liver, kidneys, spleen, lung, heart, thymus, adrenals, and nasal turbinates in the treatment groups. Of the histopathological findings observed in this study, the incidences of tubular hyaline droplets, granular cast, pyelonephritis, and tubular degeneration/regeneration in the kidneys and hemosiderin pigment in the spleen were significantly higher in the males of the 400 ppm group than those in the control group, but not in the females of the group. The degree of the histopathological changes observed in the 400 ppm group was slight-to-moderate. The other histopathological findings observed in both genders of the treatment groups were also found in the control group or were determined to be spontaneous changes.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Dose descriptor:
NOAEC
Remarks:
systemic and respiratory tract effects
Effect level:
99.6 ppm (analytical)
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
food consumption and compound intake
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Remarks on result:
other: 99.6 ppm = 367 mg/m3
Dose descriptor:
LOAEC
Effect level:
403.4 ppm (analytical)
Sex:
male/female
Basis for effect level:
body weight and weight gain
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Remarks on result:
other: 403.4 ppm = 1488 mg/m3
Critical effects observed:
not specified

Table 1. Hematological results of male and female rats after inhalation of butane-2 -thiol for 13 weeks.a

Parameters

Dosage (ppm)

 

 

 

 

0

25

100

400

No. of rats

10

10

10

10

Male

RBC (x1012/L)

9.21 ± 0.55

9.15 ± 0.32

9.10 ± 0.33

8.46 ± 0.40**

HB (g/dl)

15.96 ± 0.54

15.82 ± 0.46

15.95 ± 0.49

15.11 ± 0.64**

HCT (%)

45.06 ± 2.55

44.06 ± 1.53

44.56 ± 1.61

42.22 ± 2.39*

MCV (fl)

48.95 ± 1.51

48.18 ± 1.42

48.97 ± 1.15

49.88 ± 1.40

MCH (pg)

17.36 ± 0.75

17.29 ± 0.42

17.55 ± 0.54

17.87 ± 0.81

MCHC (g/dl)

35.48 ± 1.13

35.94 ± 0.82

35.81 ± 0.70

35.81 ± 0.74

PLT (x109/L)

1167.80 ± 154.88

1085.90 ± 75.83

1063.60 ± 100.15

1132.80 ± 211.73

WBC (x109/L)

7.22 ± 1.43

6.91 ± 2.23

7.47 ± 1.49

6.60 ± 1.26

Female

RBC (x1012/L)

8.78 ± 0.36

8.80 ± 0.33

8.72 ± 0.21

7.58 ± 0.49**

HB (g/dl)

16.47 ± 0.57

16.23 ± 0.57

16.27 ± 0.47

14.33 ± 1.47**

HCT (%)

45.48 ± 1.41

45.38 ± 1.55

45.17 ± 1.76

40.54 ± 3.48**

MCV (fl)

51.84 ± 1.17

51.55 ± 0.94

51.77 ± 1.15

53.42 ± 1.59**

MCH (pg)

18.76 ± 0.34

18.45 ± 0.41

18.66 ± 0.34

18.88 ± 0.95

MCHC (g/dl)

36.18 ± 0.47

35.78 ± 0.59

36.04 ± 0.69

35.32 ± 0.86*

PLT (x109/L)

1263.80 ± 128.66

1230.60 ± 152.82

1084.50 ± 129.72

1239.20 ± 403.24

WBC (x109/L)

4.80 ± 1.19

5.69 ± 1.43

5.85 ± 2.34

6.59 ± 2.98

RBC, red blood cells; HB, hemoglobin; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin

concentration; PLT, platelet; and WBC, white blood cells.

a Values are presented as means ± SD.

* Indicates a significant difference at P < 0.05 compared with the control group.

** Indicates a significant difference at P < 0.01 compared with the control group.

 

Table 2. Serum biochemical findings of male and female rats after inhalation of butane-2 -thiol for 13 weeks.a

Parameters

Dosage (ppm)

 

 

 

 

0

25

100

400

No. of rats

10

10

10

10

Male

AST (IU/l)

75.6 ± 16.45

76.5 ± 23.14

69.1 ± 8.27

65.9 ± 6.44

ALT (IU/dl)

50.9 ± 19.89

51.6 ± 19.95

43.1 ± 7.77

34.2 ± 3.88*

ALP (mg/dl)

302.3 ± 76.52

313.6 ± 55.56

317.3 ± 53.31

306.8 ± 71.70

BUN (mg/dl)

16.99 ± 14.8

19.35 ± 3.23*

18.96 ± 2.00

19.04 ± 1.27

CRTN (mg/dl)

0.62 ± 0.04

0.64 ± 0.05

0.66 ± 0.05

0.65 ± 0.08

GLU (mg/dl)

225.2 ± 40.93

209.8 ± 31.48

212.1 ± 32.90

223.8 ± 40.27

LDH (IU/l)

371.2 ± 217.67

323.6 ± 142.94

242.4 ± 134.77

382.2 ± 229.16

T-CHO (mg/dl)

72.4 ± 8.92

84.2 ± 13.80

80.4 ± 11.86

71.2 ± 9.91

T-BIL182.4 ± 57.88(mg/dl)

0.03 ± 0.01

0.04 ± 0.01

0.04 ± 0.01

0.04 ± 0.01

TP (g/dl)

6.64 ± 0.31

6.82 ± 0.30

6.89 ± 0.36

6.93 ± 0.52

Female

AST (IU/l)

97.2 ± 30.69

93.9 ± 23.95

96.8 ± 49.70

85.8 ± 36.82

ALT (IU/dl)

65.3 ± 24.26

62.6 ± 16.80

52.9 ± 18.18

40.2 ± 11.03*

ALP (mg/dl)

189.7 ± 98.05

216.6 ± 81.05

203.9 ± 60.41

182.4 ± 57.88

BUN (mg/dl)

16.83 ± 2.04

17.51 ± 2.38

17.94 ± 2.28

17.06 ± 2.31

CRTN (mg/dl)

0.63 ± 0.05

0.61 ± 0.06

0.65 ± 0.05

0.60 ± 0.05

GLU (mg/dl)

173.3 ± 27.20

181.0 ± 30.22

209.8 ± 35.18

196.4 ± 40.45

LDH (IU/l)

359.6 ± 195.40

375.9 ± 208.35

320.9 ± 155.29

288.0 ± 162.82

T-CHO (mg/dl)

115.5 ± 11.70

104.5 ± 11.94

104.8 ± 16.39

104.4 ± 13.41

T-BIL (mg/dl)

0.03 ± 0.01

0.04 ± 0.00

0.04 ± 0.01

0.05 ± 0.02**

TP (g/dl)

7.54 ± 0.28

7.38 ± 0.30

7.25 ± 0.25

7.35 ± 0.47

AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; BUN, blood urea nitrogen; CRTN, creatinine; GLU, glucose; LDH, lactate dehydrogenase;

T-CHO, total cholesterol; T-BIL, total bilirubin; and TP, total protein.

aValues are presented as means ± SD.

* Indicates a significant difference at P < 0.05 compared with the control group.

** Indicates a significant difference at P < 0.01 compared with the control group.

Table 3. Relative organ weights of male and female rats after inhalation of butane-2-thiol for 13 weeksa.

 

Parameters

Dosage

 

0

25

100

400

No. of rats

10

10

10

10

Male Rats

Body weight at term

459.2 ± 56.9

 

496.4 ± 56.8

467.8 ± 31.0

443.1 ± 42.9

 

Liver

2.90 ± 0.13

3.04 ± 0.26

3.09 ± 0.21

3.45 ± 0.22**

Kidney: right

0.29 ± 0.02

0.31 ± 0.03

0.31 ± 0.03

0.35 ± 0.04**

Adrenal: right

0.007 ± 0.001

0.007 ± 0.001

 0.006 ± 0.001

0.008 ± 0.002

Spleen

0.16 ± 0.02

0.16 ± 0.02

0.17 ± 0.02

0.17 ± 0.02

Testes: right

0.39 ± 0.07

0.39 ± 0.04

0.38 ± 0.07

0.40 ± 0.12

Epididymis: Right

0.15 ± 0.02

0.14 ± 0.01

0.14 ± 0.03

0.15 ± 0.04

Brain

0.51 ± 0.05

0.48 ± 0.04

0.50 ± 0.03

0.53 ± 0.06

Lung

0.38 ± 0.03

0.36 ± 0.03

0.33 ± 0.07

0.39 ± 0.03

Heart

0.28 ± 0.03

0.28 ± 0.02

0.29 ± 0.02

0.30 ± 0.03

Thymus

0.09 ± 0.02

0.10 ± 0.01

0.10 ± 0.02

0.08 ± 0.03

Female Rats

Body weight at term

311.8 ± 16.80

283.7 ± 22.90

293.8 ± 25.0

270.9 ± 27.70

Liver

2.96 ± 0.20

3.04 ± 0.26

2.93 ± 0.23

3.01 ± 1.07

Kidney: right

0.31 ± 0.02

0.32 ± 0.02

0.33 ± 0.04

0.35 ± 0.02**

Adrenal: right

0.015 ± 0.003

0.014 ± 0.006

0.015 ± 0.003

0.015 ± 0.006

Spleen

0.19 ± 0.01

0.19 ± 0.02

0.19 ± 0.02

0.21 ± 0.08

Ovary: right

0.02 ± 0.01

0.02 ± 0.01

0.02 ± 0.01

0.03 ± 0.00

Brain

0.74 ± 0.04

0.78 ± 0.05

0.78 ± 0.06

0.83 ± 0.06**

Lung

0.46 ± 0.04

0.51 ± 0.03

0.53 ± 0.01

0.54 ± 0.06*

Heart

0.33 ± 0.03

0.34 ± 0.01

0.35 ± 0.03

0.36 ± 0.03*

Thymus

0.14 ± 0.02

0.13 ± 0.02

0.13 ± 0.02

0.14 ± 0.02

a Values are presented as means ± SD (%)

* Indicates a significant difference at P < 0.05 compared to control group

** Indicates a significant difference at P < 0.01 compared to control group

Table 4. Number of histopathological in male and female rats after inhalation of butane-2-thiol for 13 weeks.

 

Parameters

Dosage (ppm)

 

0

25

100

400

No. of rats

10

10

10

10

Male Rats

Liver

 

 

 

 

Centriobular hepatocyte hypertrophy

0

-

-

2

Focal inflammation

0

-

-

0

Kidneys

 

 

 

 

Tubular hyaline droplets

0

0

1

10*

Granular cast

0

0

2

10*

Protein cast

0

0

0

1

Pyelonephritis

0

1

1

10*

Tubular degeneration/regeneration

0

0

0

10*

Mineralization

0

0

0

0

Spleen

 

 

 

 

Extramedullary hepatopoeisis

2

0

2

4

Hemosiderin pigments

0

1

3

10*

Lung

 

 

 

 

Inflammation

1

-

-

1

Mineralization

1

-

-

0

Heart

 

 

 

 

Cardiomyopathy

7

-

-

6

Thymus

 

 

 

 

Atrophy

0

-

-

2

Adrenals

 

 

 

 

Cortical vacuolation

6

-

-

7

Nasal turbinates

 

 

 

 

Eosinophillic inclusions

0

0

2

6

Mineralization

0

0

0

3

Female Rats

Liver

 

 

 

 

Centriobular hepatocyte hypertrophy

0

-

-

0

Focal inflammation

0

-

-

1

Kidneys

 

 

 

 

Tubular hyaline droplets

0

0

0

0

Granular cast

0

0

0

2

Protein cast

0

0

0

1

Pyelonephritis

0

1

0

5

Tubular degeneration/regeneration

0

0

0

2

Mineralization

7

4

3

5

Spleen

 

 

 

 

Extramedullary hepatopoeisis

0

0

0

3

Hemosiderin pigments

5

4

5

9

Lung

 

 

 

 

Inflammation

2

-

-

3

Mineralization

0

-

-

0

Heart

 

 

 

 

Cardiomyopathy

1

-

-

2

Thymus

 

 

 

 

Atrophy

0

-

-

0

Adrenals

 

 

 

 

Cortical vacuolation

0

-

-

0

Nasal turbinates

 

 

 

 

Eosinophillic inclusions

1

1

0

2

Mineralization

0

0

0

2

* Indicates a significant difference at P < 0.05 compared to control group

Conclusions:
Based on the treatment-related effects observed (erythrocyte, kidneys, liver, and nasal turbinates), the LOAEC is 1.48 mg/L (403.4 ppm or 1488 mg/m3) and the NOAEC is 0.367 mg/L (99.6 ppm or 367 mg/m3).
Executive summary:

In a 90-day inhalation toxicity study, butane-2-thiol was administered to 10 Sprague-Dawley rats/sex/concentration by dynamic whole body exposure at concentrations of 0, 0.092, 0.367, or 1.488 mg/L (0, 25.1, 99.6, or 403.4 ppm) for 6 hours per day, 5 days/week for a total of 91 days. 

No treatment-related toxic symptoms or mortality were observed in any of the animals treated with butane-2 -thiol during the experimental period.  In males, the amount of food consumed was significantly lower on Days 0 and 7 of treatment in the 1.488 mg/L (403.4 ppm) group (11.7 ± 3.9 and 20.7 ± 1.5 g) than in the control group (22.6 ± 2.4 and 24.3 ± 2.3 g). In females, food consumption of the group exposed to 1.488 mg/L (403.4 ppm) was also decreased significantly on Days 0 and 7 of treatment (9.6 ± 3.5 and 13.6 ± 1.8 g) than in the control group (18.2 ± 2.7 and 18.8 ± 3.3 g). Significant decreases in body weight gain were observed in females in the high concentration group and decreases in RBC, haemoglobin and hematocrit levels were reported in both male and female animals in the 1.488 mg/L (403.4 ppm) group. In males, the relative weights of the liver and kidneys in the 1.488 mg/L (403.4 ppm) group were increased significantly in a dose-dependent manner compared to those of the control group. In females, the relative weights of the kidneys, brain, lung, and heart in the 1.488 mg/L (403.4 ppm) group were also significantly increased in a dose-dependent manner compared to those of the control group. No gross pathological changes were observed at necropsy, however histopathological alterations observed predominantly in the 1.488 mg/L (403.4 ppm) groups, included centrilobular hepatocyte hypertrophy in the liver of males (which corresponded with increased liver weights) and tubular hyaline droplets, granular cast, pyelonephritis, and tubular degeneration/regeneration in the kidneys (severe in male animals), extramedullary haematopoiesis and hemosiderin pigment in the spleen, and eosinophilic inclusions and mineralization in the nasal olfactory epithelium. Based on these findings the target organs of butane-2-thiol were determined to be the erythrocyte, kidneys, liver, and nasal turbinates in rats. The LOAEC is 1.488 mg/L (403.4 ppm or 1488 mg/m3) and the NOAEC is 0.367 mg/L (99.6 ppm or 367 mg/m3).

This study received a Klimisch score of 1 and is classified as reliable without restriction because it was well conducted and documented. The methods, timeframe and selection are the same as the OECD guidelines for subchronic inhalation, and it is compliant with GLP regulations.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
367 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
Reliable with restrictions (Klimisch score = 2)

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Oral Route

In an OECD TG 422 study, Sprague-Dawley rats were dosed by oral gavage with 0, 10, 50, or 200 mg/kg 2-methylpropane-2-thiol for 42 to 53 days. A 14-day recovery period was included for the control and the 200 mg/kg dose groups. There was no mortality, effect on urinalysis, clinical signs of toxicity, changes in functional observational battery (FOB) measurements, or motor activity (MHLW, 2006; Klimisch score = 2

 

Decreased body weight was observed in the 200 mg/kg animals during the treatment period, with no difference between treated and control animals during the 14-day recovery period. At 200 mg/kg, there was a slight, but statistically significant decrease in erythrocyte count in males and females (-9% and -8%, respectively); and slightly decreased hemoglobin, hematocrit, and MCHC (-8%, -6%, and -2%, respectively) in males. Prothrombin time and activated partial thromboplastin time were significantly increased in the 200 mg/kg males, nevertheless, activated partial thromboplastin time was shortened in the 50 and 200 mg/kg females. Following the recovery period, hemoglobin levels and MCHC were slightly, however, significantly decreased (both sexes), and reticulocyte count was increased in males. Serum chemistry changes in the 200 mg/kg animals included: decreased α1-globulin and glucose; and increased albumin, cholesterol, phospholipids, α2-globulin (males only), and γ-GTP (males only). There were no significant changes in the serum chemistry between the treated and control animals following the 14-day recovery period. Relative kidney weights were increased in males at all dose levels; relative liver weights were increased in the 50 and 200 mg/kg males and in the 200 mg/kg females; and relative heart weights were increased in the 200 mg/kg animals (both sexes). Histopathological changes were seen in the kidneys of all dosed male rats. These changes included basophilic tubules and hyaline droplets in proximal tubular cells, which are indicative of alpha2u-globulin nephropathy, an effect not considered relevant to humans. Centrilobular hepatocyte hypertrophy was noted in the 50 and 200 mg/kg males and in the 200 mg/kg females, and slight hemosiderin deposition was noted in the spleen of the 200 mg/kg males and females. Following the 14-day recovery period, hemosiderin was still present in the spleens of the 200 mg/kg animals, but there was no centrilobular hepatocyte hypertrophy in the females and the incidence and severity was reduced in males. Given that the hematological effects were slight in nature with no indication of any associated adverse clinical or histopathological effects and that the centrilobular hepatocellular hypertrophy is likely an adaptive response, the NOAEL for this study is 50 mg/kg/day (based on decreased body weight in females dosed at 200 mg/kg/day).

 

Inhalation Route

One key 90-day study is available for 2-methylpropane-2-thiol. Additionally, one subchronic inhalation toxicity study on a structural analogue (butane-2-thiol) can be used as read across for 2-methylpropane-2-thiol.

 

In a key 90-day inhalation toxicity study (Ulrich, 1984; Klimisch score = 2), male and female Sprague-Dawley rats (15/sex/dose) were exposed (whole body) to 2-methylpropane-2-thiol at measured concentrations 0, 9, 97, or 196 ppm (0, 33, 357 or 721 mg/m3, respectively) for six hours per day, five days per week. There were no deaths, clinical signs of toxicity or body weight changes observed. Blood urea nitrogen was statistically significantly different from the control group at the 6-week interval only for the 97 ppm exposure group; this change was not considered biologically relevant because it occurred at only one time interval. Statistically significant differences in erythrocyte count were only found in females at 6-week (97 ppm) and 12-week (97 and 196 ppm). The clinical pathology endpoints that differed from concurrent controls remained within the range of historical control values and were not considered to be biologically or toxicologically relevant. No compound-related macroscopic lesions were observed in any of the rats that were sacrificed at the termination of the study or those that died during the course of the study. There was a compound related increase in alveolar macrophages among males and females of the mid dose (97 ppm) and high dose (196 ppm) groups exposed to 2-methylpropane-2-thiol. At the mid dose level, 5 of 15 males and 3 of 15 females were affected. All lesions were trace in severity. At the high dose level 14 of 15 males and 12 of 15 females were affected. One male and one female were mild and all of the others were trace in severity. This lesion did not occur at the low dose level (9 ppm). Toxicologically significant increases in the mean weights of kidneys occurred in male rats exposed to 97 and 196 ppm. There was a compound and concentration-related increase in chronic nephrosis (varying degrees of multifocal degeneration of the proximal convoluted tubules, tubular regeneration, and inflammatory cell infiltration of the interstitium) in 14 of 15 animals at the high concentration (196 ppm). The lesion was also noted in 13 of 15 at the mid concentration (97 ppm) and 7 of 15 animals at the low concentration (9 ppm). However, findings in the kidneys of males were considered to be rat-specific with no relevance for human risk assessment. The NOAEC for systemic toxicity was determined to be ≥ 196 ppm (721 mg/m³).

To assess local effects on the respiratory tract a 90-day inhalation toxicity study (Kim et al., 2009; Klimisch score = 1) for the structural analogue, butane-2 -thiol, was read-across to the target substance, 2 -methylpropane-2-thiol. The substance was administered to 10 Sprague-Dawley rats/sex/concentration by whole body exposure at concentrations of 0, 0.092, 0.367, or 1.488 mg/L (0, 25.1, 99.6, or 403.4 ppm) for 6 hours per day, 5 days/week for a total of 91 days. No treatment-related toxic symptoms or mortality were observed in any of the animals treated with butane-2 -thiol during the experimental period.  In males, the amount of food consumed was significantly lower on Days 0 and 7 of treatment in the 1.488 mg/L (403.4 ppm) group (11.7 ± 3.9 and 20.7 ± 1.5 g) than in the control group (22.6 ± 2.4 and 24.3 ± 2.3 g). In females, food consumption of the group exposed to 1.488 mg/L (403.4 ppm) was also decreased significantly on Days 0 and 7 of treatment (9.6 ± 3.5 and 13.6 ± 1.8 g) than in the control group (18.2 ± 2.7 and 18.8 ± 3.3 g). Significant decreases in body weight gain were observed in females in the high concentration group and decreases in RBC, haemoglobin and hematocrit levels were reported in both male and female animals in the 1.488 mg/L (403.4 ppm) group. In males, the relative weights of the liver and kidneys in the 1.488 mg/L (403.4 ppm) group were increased significantly in a dose-dependent manner compared to those of the control group. In females, the relative weights of the kidneys, brain, lung, and heart in the 1.488 mg/L (403.4 ppm) group were also significantly increased in a dose-dependent manner compared to those of the control group. No gross pathological changes were observed at necropsy, however histopathological alterations observed predominantly in the 1.488 mg/L (403.4 ppm) groups, included centrilobular hepatocyte hypertrophy in the liver of males (which corresponded with increased liver weights) and tubular hyaline droplets, granular cast, pyelonephritis, and tubular degeneration/regeneration in the kidneys (severe in male animals), extramedullary haematopoiesis and hemosiderin pigment in the spleen, and eosinophilic inclusions and mineralization in the nasal olfactory epithelium. Based on these findings the target organs of butane-2-thiol were determined to be the erythrocyte, kidneys, liver, and nasal turbinates in rats. The LOAEC is 1.488 mg/L (403.4 ppm or 1488 mg/m³) and the NOAEC is 0.367 mg/L (99.6 ppm or 367 mg/m³).

 

 

Justification for classification or non-classification

According to REGULATION (EC) No 1272/2008 and the available data for the repeated dose toxicity:

 

- Specific target organ toxicity after repeated exposure: Hematological changes seen in the oral and inhalation studies on 2-methylpropane-2-thiol and butane-2 -thiol, respectively, appear to be the result of increased RBC destruction in the spleen, as indicated by the presence of hemosiderin pigment. These effects were slight in nature with no indication of any associated adverse clinical or histopathological effects in either study. It is believed that the centrilobular hepatocellular hypertrophy is an adaptive response which is reversible upon cessation of exposure. Therefore, 2-methylpropane-2-thiol is not classified for specific target organ toxicity after repeated exposure.