Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
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.
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guideline
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

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
TS: 2-methyl-2-propanethiol
Purity 99.0-99.9%
Lot No. 200501-001

Test animals

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

Administration / exposure

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
Doses / concentrations
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

Examinations

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.

Results and discussion

Results of examinations

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 was 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)
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.

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 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 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. However it was not considered to be a dose related effect because this effect 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, 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 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.

Effect levels

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.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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)

Applicant's summary and conclusion

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, but 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 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 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.