Registration Dossier

Administrative data

Description of key information

Kidney changes indicative of alpha2u-globulin nephropathy were observed in male rats in the OECD test guideline 422 oral gavage study on 2-methylpropane-2-thiol (MHLW, 2006) and in the 90-day inhalation study on butane-2-thiol (Kim et al., 2009).  Alpha2u-globulin nephropathy is a male rat-specific effect that is not relevant to humans. Hemosiderin accumulation in the spleen following oral and inhalation exposure to 2-methylpropane-2-thiol and butane-2-thiol, respectively, appears to be the result of increased destruction of RBC in the spleen. This is supported by the hematological changes of decreased red blood cells, hemoglobin, and hematocrit. However, this finding was slight in nature and is not considered adverse. Centrilobular hepatocellular hypertrophy was also observed in both male and female rats the oral study on 2-methylpropane-2-thiol. The nasal cavity effects observed in rats exposed by inhalation to butane-2-thiol are likely due to a localized chronic irritation.  There were also increased alveolar macrophages (described as trace severity) in the respiratory tract of rats exposed to butane-1-thiol. However, in the absence of any other histopathological changes in that study, it is questionable whether this finding is indicative of a localized irritation response.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study was classified as reliable with restriction because although it is a GLP guideline study, a study report in English was not available for data verification. However, this study is peer reviewed and considered sufficient for this endpoint.
Reason / purpose:
reference to other study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
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
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no death in any group.
No compound-related changes were observed in any animal.

BODY WEIGHT AND WEIGHT GAIN
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, but their body weight gains throughout the recovery period were similar to those of the control group.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption tended to be low compared with controls 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 bw/day on day 15 of the administration period. However, it was not observed at 50mg/kg bw/day and not considered to be a dose-related effect.

HAEMATOLOGY
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 bw/day 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 bw/day was not considered to be a dose related effect because it was not observed at the end of administration period.

CLINICAL CHEMISTRY
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 compared with the controls. However it was not considered to be an adverse effect because it was not observed at the end of administration period.

URINALYSIS
No test substance-related changes were observed.

NEUROBEHAVIOUR
No compound-related changes were observed in any animal in the functional observation battery.

ORGAN WEIGHTS
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 bw/day, 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 200 mg/kg bw/day and relative weight of the kidney in females at 200 mg/kg bw/day. However these effects were not considered to be adverse because they were not observed at the end of administration period.

GROSS PATHOLOGY
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.

HISTOPATHOLOGY: NON-NEOPLASTIC
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.
Dose descriptor:
NOAEL
Effect level:
>= 200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No findings considered adverse.
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:
In a study conducted to OECD test guideline 422 and in compliance with GLP, it was concluded that none of the effects observed were adverse and relevant to humans. The NOAEL for 2-methypropane-2-thiol in this study was therefore ≥200 mg/kg bw/day (MHLW, 2006).

Executive summary:

In an OECD TG 422 study, Sprague-Dawley rats were dosed by oral gavage with 0, 10, 50, or 200 mg/kg bw/day 2-methylpropane-2-thiol for 42 to 53 days. A 14-day recovery period was included for the control and the 200 mg/kg bw/day 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).

Decreased body weight was observed in the 200 mg/kg bw/day animals during the treatment period, with no difference between treated and control animals during the 14-day recovery period, so this finding was concluded to be non-adverse (by the EPSR author). At 200 mg/kg bw/day, 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 compared with the controls. Prothrombin time and activated partial thromboplastin time were significantly increased in the 200 mg/kg bw/day males, but activated partial thromboplastin time was shortened in the 50 and 200 mg/kg bw/day 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 bw/day 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 bw/day males and in the 200 mg/kg bw/day females; and relative heart weights were increased in the 200 mg/kg bw/day 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 bw/day males and in the 200 mg/kg bw/day females, and slight hemosiderin deposition was noted in the spleen of the 200 mg/kg bw day males and females. Following the 14-day recovery period, hemosiderin was still present in the spleens of the 200 mg/kg bw/day animals, but there was no centrilobular hepatocyte hypertophy 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 histopathogical effects and that the centrilobular hepatocellular hypertrophy is likely an adaptive response, the NOAEL for this study is 200 mg/kg bw/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:
no adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
1982-07-20
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study was classified as reliable without restriction because it was a well-documented study and followed OECD Guideline 413. Read-across is considered to be scientifically valid and reliability 2.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
no data reported on the age or fasting of the animals; calibration of exposure analyzers assumed chemical was 100% pure; lacked description of the control group and dose selection; and food consumption.
GLP compliance:
yes (incl. certificate)
Remarks:
International Research and Development corporation Quality Assurance Department
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
other: Charles River CD (albino)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, Michigan
- Weight at study initiation: 200 to 270 grams
- Housing: caged individually
- Diet (e.g. ad libitum): Purina Certified Pelleted Rodent Chow ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: four days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2 to 29.4°C
- Humidity (%): 28 to 68
- Photoperiod (hrs dark / hrs light): 12 hours


IN-LIFE DATES: From: 1981-03-23 To: 1981-06-19
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1 m3 glass and stainless steel chamber
- Source and rate of air: HVAC system, rate varied between 130 and 300 litres/minute depending on desired exposure concentrations
- Method of conditioning air: particulate filtered and controlled for temperature and humidity
- System of generating particulates/aerosols: pump or Sage Syringe Drive delivers test material to the top of the glass bead column, air is passed up the bead column in a counter-current manner relative to the liquid, vaporisation occurs within the bead column, vapours are piped to exposure chamber air outlet where dilution with chamber ventilation air reduces the concentration to the desired level.
- Temperature, humidity, pressure in air chamber:
- Air flow rate: rate varied between 130 and 300 litres/minute depending on desired exposure concentrations


TEST ATMOSPHERE
- Brief description of analytical method used: Nominal concentrations were calculated by recording the weight of test material contained in each syringe or reservoir prior to and after each exposure. The calculated difference was divided by the total volume of air passed through the chamber during each exposure to determine the nominal concentration.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Actual exposure concentrations were measured by non-dispersive IR utilising analysers. Analyzers were prime calibrated once before initiation of the study and then at monthly intervals. Calibrations were performed in triplicate, responses were averaged and plotted, and a linear least-squares fit was applied. Exposure was monitored at approximately hourly intervals and from this the mean and standard deviation was determined.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours per day, 5 days per week
Remarks:
Doses / Concentrations:
0.03, 0.26, 0.55 mg/L (33, 260, 550 mg/m3)
Basis:
nominal conc.
No. of animals per sex per dose:
15
Control animals:
yes, concurrent no treatment
Details on study design:
No data reported
Positive control:
No data reported
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once per week
- Cage side observations checked in table [No. 6 for week thirteen] were included.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to exposure, 6 weeks, and 12 weeks


BODY WEIGHT: Yes
- Time schedule for examinations: once per week


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to exposure, 6 weeks, and 12 weeks
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 10
- Parameters checked in Appendix C were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to exposure, 6 weeks, and 12 weeks
- Animals fasted: No data reported
- How many animals: 10
- Parameters checked in appendix D were examined.


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No


OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
No data reported
Statistics:
Generally, when the number of animals in any one group was equal-to-or-less-than seven, non-parametric analyses were conducted utilizing the Kruskal-Wallis one-way analysis of variance followed, where appropriate, with the Mann-Whitney. In those cases where the number of animals in all groups was greater than seven and the measurements were at least on an internal 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:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
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
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY Animals displayed no pharmacotoxic signs that were considered to be exposure-related. Two animals died during the course of the study, a male in Group IV and a female in Group III. The cause of death for the female is unknown. The death of the male may have been related to blood collection trauma, as it died shortly after blood collection.

BODY WEIGHT AND WEIGHT GAIN 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 with respect to test materials, but this observation was not as apparent in female rats. Therefore, the apparent dose-response trends are not considered to be of toxicological importance.

FOOD CONSUMPTION
No data reported

FOOD EFFICIENCY
No data reported

WATER CONSUMPTION
No data reported

OPHTHALMOSCOPIC EXAMINATION
No data reported

HAEMATOLOGY There were no significant differences observed between any of the groups for any of the hematology parameters at the pre-initiation interval. There is only one parameter for which a significant difference was observed for the individual 6-week hematology values. The erythrocyte count was decreased in female rats of Group IV 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. The individual 12-week hematology values had three parameters for which statistically significant differences for females of various exposure groups were found when evaluated against female control animals: Group IV had a high segmented neutrophil count, the erythrocyte counts for Groups III and IV were low and the lymphocyte count for Group IV was significantly lower. The observed statistical significant differences obtained for erythrocyte count, lymphocytes, and segmented neutrophils for Group IV are not considered to be of toxicologic importance.


CLINICAL CHEMISTRY There were no significant differences observed between any of the groups for any of the clinical chemistry parameters at the pre-initiation interval or the 12 week interval. The only parameter for which a statistically significant difference was observed was for the slightly elevated BUNN of Group IV 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.

URINALYSIS
No data reported

NEUROBEHAVIOUR
No data reported

ORGAN WEIGHTS Absolute lung and trachea weight for Group III males and relative lung and trachea weight for Groups III and IV males was statistically significantly different from the control group. No such effects were observed in the females exposed to the same test material. These effects may be related to the n-butyl mercaptan exposure because the lung is a logical target organ and the effect was observed at both of the higher exposure levels.

GROSS PATHOLOGY Test article-related microscopic changes were observed in lungs of male and female rats from the test material. The lung lesions consisted of interstitial fibrosis, which involved one or more of all lobes, a diffuse distribution was evident. In the areas of fibroplasia, a minimal number of chronic inflammatory cells, was also present. However, their distribution was sparse, so that the overall picture presented was one of interstitial fibroplasia. Interstitial inflammatory cell infiltrate, composed primarily of chronic inflammatory cells, occurred in the control and test material exposed rats. Young fibroblasts were also seen sparsely distributed in these area. The distribution and incidence of this lesion in control and the exposed rats were indicative of its spontaneous nature and no compound effect was evident with respect to this lesion. Incidental non-treatment-related lesions were observed in adrenal, heart, liver, bronchial and cervical lymph nodes, nasal turbinates, prostate, testes, trachea, eye, stomach, and thymus.

OTHER FINDINGS
MACROSCOPIC PATHOLOGY Two rats died during the study but neither of these animals or any of the animals terminally sacrificed showed any test article related to macroscopic changes.
Dose descriptor:
NOAEC
Remarks:
systemic
Effect level:
550 mg/m³ air (analytical)
Sex:
male/female
Basis for effect level:
other: No adverse systemic effects observed.
Critical effects observed:
not specified
Conclusions:
In a 90-day inhalation toxicity study, butane-1-thiol was administered to 15 Sprague-Dawley rats/sex/concentration by dynamic whole body exposure at concentrations of 0, 0.033, 0.26, or 0.55 mg/L (0, 9, 70, or 150 ppm) for 6 hours per day, 5 days/week for a total of 91 days. Based on this observation, the local NOAEC based on effects on the respiratory tract was 0.033 mg/l (33 mg/m3), but treatment-related systemic toxicity were not observed, so the NOAEC for systemic toxicity is >=0.55 mg/l (550 mg/m3). (Ulrich, 1982).
Executive summary:
In a 90-day inhalation toxicity study, butane-1-thiol was administered to 15 Sprague-Dawley rats/sex/concentration by dynamic whole body exposure at concentrations of 0, 0.033, 0.26, or 0.55 mg/L (0, 9, 70, or 150 ppm) for 6 hours per day, 5 days/week for a total of 91 days (Ulrich, 1982).

 

There were no statistically significant compound related effects in appearance and behaviour, mortality, body weight, hematology, clinical chemistry, or macroscopic pathology. Organ weights of the lung and trachea were statistically different form some groups compared to the control, microscopic pathology changes were observed in lungs of male and female rats. Male rats treated with the test material at all test concentrations exhibited exposure related nephrosis of the kidney but this effect was not seen in female rats.  An increased incidence of alveolar macrophages was observed in male and female rats treated with butane-1-thiol at concentrations of 0.26 and 0.55 mg/L. Based on this observation, the local NOAEC based on effects on the respiratory tract was 0.033 mg/l (33 mg/m3), but treatment-related systemic toxicity were not observed, so the NOAEC for systemic toxicity is >=0.55 mg/l (550 mg/m3).

 

This study received a Klimisch score of 2 and was classified as reliable without restriction because it was a well-documented study and followed OECD Guideline 413.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
550 mg/m³
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

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

Mode of Action Analysis / Human Relevance Framework

Additional information

Oral Repeat Dose Toxicity

 

No oral repeat dose toxicity data were identified for ethanethiol. However, there is an oral repeat dose toxicity study on another C2-C4 alkyl mercaptan, 2-methylpropane-2-thiol, which can be used as read-across to ethanethiol. 

 

In an OECD TG 422 study (MHLW, 2006; Klimisch score = 2), Sprague-Dawley rats were dosed by oral gavage with 0, 10, 50, or 200 mg/kg bw/day 2-methylpropane-2-thiol for 42 to 53 days. A 14-day recovery period was included for the control and the 200 mg/kg bw/day 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 bw/day animals during the treatment period, with no difference between treated and control animals during the 14-day recovery period, therefore this finding was not considered to be adverse (by the study reviewer). At 200 mg/kg bw/day, 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 bw/day males, but activated partial thromboplastin time was shortened in the 50 and 200 mg/kg bw/day 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 bw/day 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 bw/day males and in the 200 mg/kg bw/day females; and relative heart weights were increased in the 200 mg/kg bw/day 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 bw/day males and in the 200 mg/kg bw/day females, and slight hemosiderin deposition was noted in the spleen of the 200 mg/kg bw/day males and females. Following the 14-day recovery period, hemosiderin was still present in the spleens of the 200 mg/kg bw/day animals, but there was no centrilobular hepatocyte hypertophy 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 histopathogical effects and that the centrilobular hepatocellular hypertrophy is likely an adaptive response, the NOAEL for this study is 200 mg/kg bw/day.

Inhalation Repeat Dose Toxicity

No studies evaluating the subchronic toxicity of ethanethiol by the inhalation route were identified. Subchronic inhalation toxicity studies on other C2-C4 alkyl mercaptans (butane-1-thiol, butane-2-thiol, and 2-methylpropane-2-thiol) can be used as read across for ethanethiol.

Adverse systemic effects relevant to humans were not observed in any of the available read-across studies. The key study tested up to the highest concentration.

In the key 90-day inhalation toxicity study (Ulrich, 1982a; Klimisch score = 2), butane-1-thiol was administered to groups 15 Sprague-Dawley rats by whole body exposure at concentrations of 0, 0.033, 0.26, and 0.55 mg/L (0, 9, 70, and 150 ppm, respectively) for 6 hours per day, 5 days per week, for a total of 91 days. No statistically significant compound related effects in haematology, clinical chemistry, appearance, behavior, body weight, or mortality were observed. However, organ weights (lungs and trachea) of some test group animals were different from the control group. Microscopic pathology changes were observed in the lungs of both male and female rats. Male rats treated with the test material at all test concentrations exhibited exposure related nephrosis of the kidney but this effect was not seen in female rats. An increased incidence of alveolar macrophages was observed in male and female rats treated with butane-1 -thiol at concentrations of 0.26 and 0.55 mg/L.

In a supporting 90-day inhalation toxicity study (Kim et al., 2009; Klimisch score = 2), butane-2 -thiol 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. However, the effects on the liver and kidneys are not adverse effects relevant to humans, and the effects on the nasal cavity are local (NOAEC = 99.6 ppm, 367 mg/m3). The haematology findings were slight in nature and not considered adverse. Therefore the NOAEC for systemic effects is ≥1.48 mg/L (403.4 ppm or 1488 mg/m3).

In a supporting 90-day inhalation toxicity study (Ulrich, 1982a, 1983, 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 -methypropane-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).Based on this observation, the local NOAEC based on effects on the respiratory tract was 0.033 mg/l (33 mg/m3), but treatment-related systemic toxicity were not observed, so the NOAEC for systemic toxicity is ≥0.55 mg/l (550 mg/m3).

The study with butane-1-thiol (Ulrich, 1992) was selected as key study for derivation of DNELs because:

·        It gives the worst-case NOAEC value

·        The test substance is the closest available chemical structure to the registration substance, with both containing a primary thiol group.

.  

Dermal Repeat Dose Toxicity

There are no dermal repeated dose toxicity studies available to ethanethiol or other Category members.

Read-Across Justification

To reduce animal testing REACH recommends to make use of a read-across approach where appropriate based on the high accordance in properties relevant for the specific endpoint.

There are no data on the repeated dose toxicity for ethanethiol, therefore good quality studies on three other Category members,

butane-1-thiol, butane-2-thiol and 2-methylpropane-2-thiol

have been read-across to assess the potential for ethanethiol to cause repeated dose toxicity via the oral and inhalation routes.

The low molecular weight aliphatic alkylthiols are volatile organic liquids of moderate aqueous solubility and low n-octanol-water partition coefficient. The substances all contain a single thiol (-SH) functional group. The acid dissociation constant (pKa) of the thiol group is approximately 10 therefore the substances can be considered as weak acids which will not be significantly ionised at physiologically relevant pH. There are oral and inhalation data for repeated dose toxicity available for read-across substances butane-1-thiol, butane-2-thiol and 2-methylpropane-2-thiol. Overall there is a consistent pattern of adaptive liver hypertrophy and species-specific alpha2u-globulin nephropathy. There were also slight and inconsistent signs of respiratory irritation in the repeated dose inhalation studies. None of the findings in the repeated dose studies were considered adverse and the NOAEL/NOAECs were the highest doses tested. Based on the similar pattern of metabolism expected for these substances, once absorbed the distribution to tissues and excretion is expected to be similar. Since the structures of the read across substances and ethanethiol are very similar, it is acceptable to assume that the systemic effects of ethanethiol following repeated oral and inhalation will be the same as those for the read across substances.



Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The key study was conducted to OECD test guideline 422 and in compliance with GLP.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
The key study was conducted to OECD test guideline 413 and in compliance with GLP. Adverse systemic effects relevant to humans were not observed in any of the available read-across studies. The worst-case value was selected for DNEL derivation.

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, ethanethiol is not classified for specific target organ toxicity after repeated exposure.