Propane-1-thiol

Endpoint summary

Description of key information

There are no repeated dose toxicity data for n-propyl mercaptan, (1-propanethiol; CAS 107-03-9), therefore data were read-across from the structurally analogous substances tert-butyl mercaptan (2-methylpropane-2-thiol; CAS 75-66-1) and n-butyl mercaptan (1-butanethiol; CAS 109-79-5). 1-Propanethiol (NPM, target), 1-butanethiol (source) and 2-methylpropane-2-thiol (source) all contain a thiol (-SH) functional group with a branched or linear aliphatic carbon chain.

In a combined repeated dose toxicity study with the reproduction / developmental toxicity screening test) with 2-methylpropane-2-thiol, conducted according to OECD Test Guideline 422 and in compliance with GLP, the NOAEL was concluded to be 50 mg/kg bw/day based on body weight reduction observed in female rats dosed at 200 mg/kg bw/day (MHLW, 2006).  

In a 13-week inhalation toxicity study, conducted according to OECD Test Guideline 413 and in compliance with GLP, 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. The NOAEC for systemic toxicity is higher or equal to 196 ppm (equivalent to 721 mg/m3) (Ulrich, 1982a).

In a 13-week inhalation toxicity study, conducted according to OECD Test Guideline 413 and in compliance with GLP, the concluded NOAEC for 1-butanethiol was 150 ppm (550 mg/m3 analytical) based on no adverse systemic effects observed up to the highest concentration tested (Ulrich, 1982a).

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:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

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 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

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

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 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.

Key result

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, 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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to 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

Dose / conc.:

10 ppm

Remarks:

target concentration

Dose / conc.:

100 ppm

Remarks:

target concentration

Dose / conc.:

200 ppm

Remarks:

target concentration

Dose / conc.:

9 ppm (analytical)

Dose / conc.:

97 ppm (analytical)

Dose / conc.:

196 ppm (analytical)

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:

no effects observed

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:

no effects observed

Description (incidence and severity):

(Tables 1 and 2)
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):

(Tables 3 and 4)
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):

(Tables 5 and 6)
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

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

(Table 7)
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

Description (incidence and severity):

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.

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 wercaptan 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

Key result

Dose descriptor:

NOAEC

Remarks:

systemic toxicity

Effect level:

>= 196 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse systemic effects observed.

Remarks on result:

other: equivalent to 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 10^3/cm^3	RBC 10^6/cm^3	Hb g/dl	Ht %	MCV µ^3	MCH g	MCHC g/dl	Platelets 10^3/cm^3	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 10^3/cm^3	RBC 10^6/cm^3	Hb g/dl	Ht %	MCV µ^3	MCH g	MCHC g/dl	Platelets 10^3/cm^3	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.

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1982-07-20

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)

Deviations:

yes

Remarks:

when compared to the 1981 guideline: 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

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
- 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:

clean air

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: 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. The mean chamber temperature was around 77 °F). The relative humidity was between 46 and 51 %.
- 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. Actual exposure concentrations were measured by non-dispersive IR utilizing Wilks MIRAN 1-A analyzers.
- 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

Dose / conc.:

0 ppm

Remarks:

control

Dose / conc.:

10 ppm

Remarks:

desired concentration

Dose / conc.:

75 ppm

Remarks:

desired concentration

Dose / conc.:

150 ppm

Remarks:

desired concentration

Dose / conc.:

0 ppm (nominal)

Dose / conc.:

11 ppm (nominal)

Dose / conc.:

86 ppm (nominal)

Dose / conc.:

187 ppm (nominal)

Dose / conc.:

0 ppm (analytical)

Dose / conc.:

9 ppm (analytical)

Remarks:

equivalent to 33 mg/m³ air (analytical) (group II)

Dose / conc.:

70 ppm (analytical)

Remarks:

equivalent to 260 mg/m³ air (analytical) (group III)

Dose / conc.:

150 ppm (analytical)

Remarks:

equivalent to 550 mg/m³ air (analytical) (group IV)

No. of animals per sex per dose:

15

Control animals:

yes, concurrent no treatment

Details on study design:

Post-exposure period: None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once per week for appearance and behaviour; twice a day for mortality and overt toxicity

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: Yes, overnight
- How many animals: 5 males and 5 females
- 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.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to exposure, 6 weeks, and 12 weeks
- Animals fasted: Yes, overnight
- How many animals: 5 males and 5 females
- 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.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

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 a ll 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.
Additional microscopic examination was performed on formalin fixed hematoxylin - eosin stained paraffin sections of kidney (2) and lung (all 5 lobes) from all animals in Groups II and III. In addition, lung sections from all animals in Groups I and IV were re-examined and those diagnoses are also included.

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:

no effects observed

Description (incidence and severity):

Animals displayed no pharmacotoxic signs that were considered to be exposure-related. Alopecia and some instances of ocular discharge were observed.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female in Group III died during the course of the study. The cause of death for the female is unknown.

Body weight and weight changes:

no effects observed

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 with respect to test material, 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 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, non-treatment-related

Description (incidence and severity):

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 biochemistry findings:

no effects observed

Description (incidence and severity):

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.

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 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 pathological findings:

no effects observed

Description (incidence and severity):

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.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

At the first histological examination, test article-related microscopic changes were observed in lungs of male and female rats. 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.

At the second histological examination of lung and kidney of all groups, none of the rats exposed to n-butyl mercaptan showed any test article related microscopie lesions at mid and low dosage levels.
Upon re-evaluation of the high dose lung tissues from n-butyl mercaptan, it was determined that there was no compound related fibrosis in these animals. The change previously reported as fibrosis was an artifact resulting from compression of alveoli at the time of tissue trimming. There was a compound related increase in alveolar macrophages present among males and females of the high dose (150 ppm) n-butyl mercaptan group. Eleven of 15 males and 12 of 15 females were affected. The lesion was trace in severity for all but a single male which was mild. This lesion did not occur at the low or mid dose group. The mid dose group (70 ppm) was a no effect level for n-butyl mercaptan.
Lesions resembling spontaneous viral disease occurred in the lungs of some male and female rats from group 3 treated with n-butyl mercaptan. The lesions were similar to those of Sendai Virus infection but this could not be determined conclusively from microscopic examination of the lungs.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEC

Remarks:

systemic toxicity

Effect level:

>= 560 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

Critical effects observed:

not specified

See attachment for animal data tables.

Conclusions:

In a 13-week inhalation toxicity study, conducted according to OECD Test Guideline 413 and in compliance with GLP, the concluded NOAEC was 150 ppm (550 mg/m3 analytical) based on no adverse systemic effects observed up to the highest concentration tested.

Executive summary:

In a 13-week inhalation toxicity study, male and female Sprague-Dawley rats (15/sex/group) were exposed (whole body) to butane-1-thiol (n-butyl mercaptan)at target concentrations of 0, 10, 75 and 150 ppm (0, 33, 260 and 550 mg/m3 analytical, respectively) for six hours per day, five days per week (Ulrich, 1982a, 1983 and 1984).There were no deaths, clinical signs of toxicity, body weight changes, effects on urinalysis or any behavioral changes observed. One female died during week 3 of unknown cause and one male died of blood collection trauma after the 6 week blood collection. Female rats exhibited a statistically significant decrease in red blood cells when compared to controls at week 12 for the 75 ppm group and a similar decrease was noted for the 150 ppm group at weeks 6 and 12. A statistically significant elevation of neutrophils and a corresponding decrease of lymphocytes were noted for the 150 ppm group of females when compared to controls at week 12. None of these changes were considered to have shifted out of the normal range for rats and therefore were not considered to be biologically significant. No effects on hematology were observed in males. Lung weights were statistically elevated for males in the 75 and 150 ppm groups when compared to controls. The only microscopic finding attributable to the test material was an increase in alveolar macrophages of trace severity present among males and females of the high concentration (150 ppm) group only which was not considered as adverse. Eleven of 15 males and 12 of 15 females were affected. Based on the findings in this study, the NOAEC was 150 ppm (550 mg/m3 analytical).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

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

There are no repeated dose toxicity data for n-propyl mercaptan, (1-propanethiol; CAS 107-03-9), therefore data were read-across from the structurally analogous substances tert-butyl mercaptan (2-methylpropane-2-thiol; CAS 75-66-1) and n-butyl mercaptan (1-butanethiol; CAS 109-79-5). 1-Propanethiol (NPM, target), 1-butanethiol (source) and 2-methylpropane-2-thiol (source) all contain a thiol (-SH) functional group with a branched or linear aliphatic carbon chain. See attachment to Section 13 for justification of read-across.

In a combined repeated dose toxicity study with the reproduction / developmental toxicity screening test) with 2-methylpropane-2-thiol, conducted according to OECD Test Guideline 422 and in compliance with GLP (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. 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 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 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 50 mg/kg bw/day (based on decreased body weight in females dosed at 200 mg/kg bw/day).

In a 90-day inhalation toxicity study (Ulrich, 1982a), conducted according to OECD Test Guideline 413 and in compliance with GLP, 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).

In a 13-week inhalation toxicity study, conducted according to OECD Test Guideline 413 and in compliance with GLP, male and female Sprague-Dawley rats (15/sex/group) were exposed (whole body) to butane-1-thiol (n-butyl mercaptan)at target concentrations of 0, 10, 75 and 150 ppm (0, 33, 260 and 550 mg/m3 analytical, respectively) for six hours per day, five days per week (Ulrich, 1982a).There were no deaths, clinical signs of toxicity, body weight changes, effects on urinalysis or any behavioral changes observed. One female died during week 3 of unknown cause and one male died of blood collection trauma after the 6 week blood collection. Female rats exhibited a statistically significant decrease in red blood cells when compared to controls at week 12 for the 75 ppm group and a similar decrease was noted for the 150 ppm group at weeks 6 and 12. A statistically significant elevation of neutrophils and a corresponding decrease of lymphocytes were noted for the 150 ppm group of females when compared to controls at week 12. None of these changes were considered to have shifted out of the normal range for rats and therefore were not considered to be biologically significant. No effects on hematology were observed in males. Lung weights were statistically elevated for males in the 75 and 150 ppm groups when compared to controls. The only microscopic finding attributable to the test material was an increase in alveolar macrophages of trace severity present among males and females of the high concentration (150 ppm) group only which was not considered as adverse. Eleven of 15 males and 12 of 15 females were affected. Based on the findings in this study, the NOAEC was ≥150 ppm (550 mg/m3 analytical).

 

 

Justification for classification or non-classification

Based on the available read-across data for tert-butyl mercaptan (2-methylpropane-2-thiol; CAS 75-66-1) and and n-butyl mercaptan (1-butanethiol; CAS 109-79-5), no classification for target oral toxicity following repeated exposure is required for the registered substance, 1-propanethiol, according to Regulation (EC) No 1272/2008.