Benzimidazole-2-thiol

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Data source

Reference

Reference Type:

publication

Title:

Toxicity Study by Oral Administration To Rats.

Author:

Kawasaki et al.

Year:

1998

Bibliographic source:

The Journal of Toxicological Sciences

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

The present 28-day repeated dose oral toxicity study of test substance followed by 2-week recovery examination in Wistar rats was conducted to evaluate adverse effects and their reversibility

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Benzimidazole-2-thiol

EC Number:

209-502-6

EC Name:

Benzimidazole-2-thiol

Cas Number:

583-39-1

Molecular formula:

C7H6N2S

IUPAC Name:

1H-benzimidazole-2-thiol

Details on test material:

SOURCE OF TEST MATERIAL
- Test Identity: 2-MBI (benzimidazole-2-thiol)
- Source and lot/batch No.of test material: Ouchi Shinko Chemical Ind., Ltd.(Osaka, Japan)
- Purity: >95%
- Substance type: Organic
- Physical state: Solid
- Molecular formula: C7H6N2S
- Molecular weight: 150.204 g/mol

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:2-MBI was dissolved or suspended in corn oil and administered to rats by the i.g route using disposable gavage tube.

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

No data

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SLC Co. (Shizuoka, Japan)
- Age at study initiation: 5 weeks old
- Weight at study initiation: male 92 g, females 80g
- Fasting period before study: 16 hours
- Housing: Rats were housed in plastic cages (5 rats/cage) using chip bedding.
- Diet (e.g. ad libitum): The basal pellet diet (F-2); ad libitum
- Water (e.g. ad libitum): Tap water; ad libitum
- Acclimation period: One week prior to the initiation of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24±1°C
- Humidity (%):55±5%
- Photoperiod (hrs dark / hrs light): 12 hr light/dark cycle

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

No data

Vehicle:

corn oil

Remarks:

2%

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: test chemical was dissolved or suspended in corn oil and administered to rats by the i.g route using disposable gavage tube.

DIET PREPARATION
- Rate of preparation of diet (frequency): No data
- Mixing appropriate amounts with (Type of food): No data
- Storage temperature of food: No data

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil
- Concentration in vehicle: 0, 2.0, 10.0 or 50.0 mg/Kg
- Amount of vehicle (if gavage): No data
- Lot/batch no. (if required): No data
- Purity: No data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of test substance in corn oil was examined by HPLC after extracting the test compound with methanol. The test chemical was confirmed to be stable for atleast 1 week at room temperature

Duration of treatment / exposure:

28 days (14 days recovery period)

Frequency of treatment:

Daily for two consecutive weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 other: mg/kg

Remarks:

Treatment period

Doses / concentrations 2

Dose / conc.:

2 other: mg/kg

Remarks:

Treatment period

Doses / concentrations 3

Dose / conc.:

10 other: mg/kg

Remarks:

Treatment period

Doses / concentrations 4

Dose / conc.:

50 other: mg/kg

Remarks:

Treatment group

Doses / concentrations 5

Dose / conc.:

0 other: mg/kg

Remarks:

Recovery period

Doses / concentrations 6

Dose / conc.:

50 other: mg/kg

Remarks:

Recovery period

No. of animals per sex per dose:

Total: 80
0 mg/Kg: 10 males and 10 females
2.0 mg/Kg: 10 males and 10 females
10.0 mg/Kg: 10 males and 10 females
50.0 mg/Kg: 10 males and 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: For the dose-determining study, male and female rats (5 rats/group) were orally administered 80 mg, 40, 20, 10 and 5 mg/kg of 2-MBI for a consecutive 2 weeks. Taking into account the observed reduction in body weight gain in groups receiving more than 40 mg/kg and the 2 weeks longer treatment period for the 28-day repeated oral toxicity study, doses of 0 (control, corn oil alone), 2, 10 and 50 mg/kg of 2-MBI were administered by gavage to groups of 10 male and 10 female rats for 28 consecutive days.
.
- Rationale for animal assignment (if not random): No data
- Rationale for selecting satellite groups: Yes
- Post-exposure recovery period in satellite groups: No data
- Section schedule rationale (if not random): No data

Positive control:

No data

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: No data
- Cage side observations checked in table [No.?] were included. The animals were observed for mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:Clinical signs were monitored throughout the study.

BODY WEIGHT: Yes
- Time schedule for examinations: Three days prior to the initiation of the treatment. On the first day of treatment, and then twice weekly throughout the study.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes;once a week

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified
- Time schedule for examinations: No data

OPHTHALMOSCOPIC EXAMINATION: Not specified
- Time schedule for examinations: No data
- Dose groups that were examined: No data

HAEMATOLOGY: Yes
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 5 animals/sex
- Parameters checked : Red blood cells (REC), hemoglobin (Hb), hematocrict (HCT), mean corpuscular volume (MCV), mean hemo-globin concentration (MHC), mean corpuscular homoglobin concentration (MCHC), platelets (PLT) and white blood cells {WBC), blood clotting time

CLINICAL CHEMISTRY: Yes
- Animals fasted: No data
- How many animals: 5 animals/sex
- Parameters checked : Total protein (TP), albumin (ALB), blood urea nitrogen (BUN), creatinine (CRN), glucose (GLC),non esterified fatty acid (NEFA), phospholipid (PL),triglyceride (TG), total cholesterol (T-CHO), free cholesterol (F-CHO), alkaline phosphatase (ALP), amylase (AMY), cholinesterase (CHE). aspartate aminotransferase (AST), alanine aminotransferase (ALT). gamma -glutamyl transpeptidase ( y -GTP), leucine aminopeptidase CLAP ), lactate dehydrogenase (LDH). calcium (Ca),magnesium (Mg2+), inorganic posphorus (Pi), sodium (Na+), potassium (K+). and chlorine (CI-) were analyzed.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The weights of the brain, heart, lungs,liver, kidneys, spleen, adrenals, testes, ovaries, pituitary, thymus, submaxillary glands and thyroid glands of each animals were measured.

HISTOPATHOLOGY: Yes
The brain, heart, lungs, liver, kidneys, spleen, adrenals, testes, ovaries, pituitary, thymus, submaxillary glands and thyroid glands and the esophagus, stomach, small and large intestine, pancreas, ischiatic nerve, urinary bladder, seminal vesicles, uterus, prostate and, mysenteric lymph nodes as well as samples of spinal cord, skeletal muscle and bone marrow were fixed in 10% buffered formalin solution for routine histological processing. Paraffin sections were stained with hematoxylin and eosin for histopatbological examination.

Statistics:

All quantitative data, except for the histopathological findings were statistically analyzed by one-way analysis of variance (ANOVA) techniques with Dunnett's or Scheff ’s multiple comparison procedures. Significance was established at the p<0.05 level.

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 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:

effects observed, treatment-related

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

Clinical signs and mortality:
Mortlaity: No mortality was noted in male and female rats
Clinical signs: No signs of toxicity were observed in the tested animals

Body weight and weight gain: Decrease in body weight gain was observed. The rats of both sexes receiving 50 mg/kg showed emaciation and severe suppression in body weight gain one week after treatment started. No significant differences in body weight gain were observed in the groups that received 10 mg/kg 2-MBI or less.

Food consumption and compound intake: Decreased food consumption one week after treatment started was observed.

Food efficiency: No data

Water consumption and compound intake: No data

Opthalmoscopic examination: No data

Haematology: At the termination of the treatment, significant decreases in WBC in the 10 and 50 mg/kg female rats were observed. At the end of the recovery period, RBC, Hb and HCT were significantly decreased in both the male and female 50 mg/kg groups showing delayed onset of anemia. Increased active partial thromboplastin time was observed for both the males and females receiving 50 mg/kg.

Clinical chemistry: Serum levels of TP, BUN, PL, T-CHO, F-CHO,CHE and ɣ -GTP were significantly increased in both males and females given 50 mg/kg. ALP,K+ and Pi were decreased significantly in male rats receiving more than 10 mg/kg , and Na+ was increased significantly in the 50 mg/kg trated animals. CHO and PL levels remained significantly high 2 weeks after termination.

Urinanalysis: No data

Neurobehaviour: No data

Organ weights: Dose related inncreases in absolute and relative weights of thyroid, liver and kidney were observed. At 10 mg/kg, the mean absolute and relative thyroid weights in both sexes were approximately 3 times those of the control rats and in the 50mg/kg dose group the increase in relative thyroid weight was more than 10-fold. Dose related decreases in absolute and relative thymus weights in all the treatment groups of male and female rats and a derease in relative spleen weight in both males and females receiving 50 mg/kg were also observed. Significant increases in brain, lung, adrenal and pituitary gland weights and decreases in heart and sabmaxilIary gland and of relative organ weights were also observed with 50 mg/kg. Reduction in thymus weight was also observed in a dose-dependent manner without significant histopathological alteration.

Gross pathology: Marked enlargement of thyroid glands and thymus involution were evident. Diffuse hyperplasia of tall and columnar epithelial cells of follicles, and decrease in colloid and thickening of fibrous capsule appeared.

Histopathology:Hypertrophic cells in the anterior pituitary glands were found, Calcification of the collecting tubules in kidneay and fatty changes in adrenal cortex was observed

Effect levels

Dose descriptor:

NOEL

Effect level:

< 2 other: mg/kg/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on the significant decrease in thymus weight in the 2 mg/kg 2-MBI treatment group.

Remarks on result:

other: No toxic effects were observed

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table: Hematological findings for rats after 28 days and 2 week recovery period

Group	Treatment				Recovery
No. of animals	Control 5	2 mg/Kg 5	10 mg/Kg 5	50 mg/Kg 5	Control 5	50 mg/Kg 5
Male
RBC	9.00±0.10	9.07±0.37	8.60±0.19	8.66±0.25	9.86±0.19	7.49±0.13**
Hb	15.8±0.4	15.9±0.4	15.4±0.4	15.7±0.4	16.3±0.3	13.7±0.2**
HCT	47.1±0.4	46.7±1.6	45.8±1.6	44.0±1.5**	48.0±0.6	39.8±0.9**
PLT	0.81±0.08	0. 84±0.09	0.79±0.18	0.63±0.05**	0.84±0.13	1.06±0.08*
WBC	7.36±0.83	6.66±0.36	6.58±0.35	6.46±0.83	8.22±0.68	6.30±0.40
PT	14.6±0.6	14.3±0.8	14.8±0.8	14.9±0.9	16.3±0.5	15.2±0.3**
APTT	24.7±2.1	24.2±4.1	25.6±2.7	35.6 v0.8*	30.0±6.7	25.2±2.6
Female
RBC	9.01±0.14	9.02±0.12	8.77±0.58	9.00±0.51	8.58±0.20	6.64±0.08
Hb	16.2±0.2	16.2±0.3	15.9±1.0	16.3 v0.7	15.6±0.3	12.7 v0.5**
HCT	46.2±1.0	45.8±1.0	45.1±31	45.1±2.6	44.6±0.9	34.8± 0.6**
PLT	0.72±0.08	0.71±0.21	0.78±0.16	0.67±0.15	1.04±0.06	1.22±0.08**
WBC	8.78±1.05	7.62±1.33	5.98±1.06**	5.24±0.31**	5.84±0.65	6.24±1.2

*, **: Significantly different from the relevant control at p<0.05, p<0.01, respectively

Table no 2: Biochemical findings for male rats after 28 days of treatment with test substance & 2-week recovery Period

Groups	Treatment				Recovery
	control	2mg/Kg	10 mg/Kg	50 mg/Kg	Control 5	50 mg/Kg
TP	5.98±0.16	6.01 ±0.15	6.20 ±0.22	6.99 ±0.08	6.32 ±0.16	5.79 ±0.10
ALB	4.25 ±0.07	4.24 ±0.12	4.33± 0.10	4.86 ±0.06	4.38 ±0.05	3.94 ±0.09
BUN	8.89±1.26	8.09±0.89	6.65±0.31	12.77±0.49	12.1±1.40	11.1±0.4
CRN	0.28±0.03	0.27±0.04	0.24±0.02	0.28±0.02	0.34±0.07	0.24±0.02
GLC	118±4	120±13	124±6	124±6	130±7	108±8
PL	103±3	108± 9	107± 9	226± 16	121± 5	160± 12
TG	76± 15	82 ±14	74 ±23	65 ±10	157± 23	85± 12
T-CHO	52± 4	54± 6	65± 7	180 ±11	59± 2	105 ±11
F-CHO	7.6± 1.6	7.1± 2.3	10.0 ±1.7	46.4± 3.5	11.4 ±1.6	23.1 ±3.7
ALP	323± 29	312 ±9	206 ±24	187± 26	198± 11	262 ±28
AST	82± 8	81± 4	69± 8	54± 3	62± 16	64± 6
CHE	174 ±15	186± 21	536 ±53	2013± 289	173 ±32	480± 42
-GTP	1.29± 0.40	1.33 ±0.22	1.42 ±0.37	1.98 ±0.09	0.01± 0	0.01± 0
LAP	47± 2	45± 1	47± 2	65± 4	42± 2	48± 2
Ca	9.9± 0.1	9.9± 0.2	9.9 ±0.2	9.8± 0.3	10.5± 0.1	10.2± 0.2
Pi	7.9 ±0.3	7.7 ±0.3	7.1± 0.3	5.4± 0.2	7.3± 0.3	7.6± 0.3
Na	137± 1	137± 1	138 ±1	142 ±1	136± 1	136± 1
K	5.2± 0.2	4.9± 0.3	4.7± 0.2	3.5 ±0.1	4.3± 0.3	5.0± 0.2
Cl	100± 0	99± 1	98 ±2	98± 1	99± 1	103± 1

Table no : Biochemical findings for female rats after 28 days of treatment with test substance & 2-week recovery Period

Groups	Treatment				Recovery
	control	2mg/Kg	10 mg/Kg	50 mg/Kg	Control 5	50 mg/Kg
TP	5.92 0.16	5.81 0.15	5.88 0.06	6.70 0.23	6.29 0.23	6.31 1.05
ALP	4.30± 0.12	4.29± 0.10	4.29± 0.05	4.60± 0.14	4.44± 0.19	3.78± 0.08
A/G	2.67± 0.18	2.83± 0.11	2.71± 0.10	2.19± 0.13	2.41± 0.15	1.66± 0.5
CRN	0.29 ±0.04	0.29 ±0.04	0.27± 0.03	0.41 ±0.07	0.32± 0.01	0.30± 0.02
PL	154 ±8	139 ±9	123± 7	265 ±22	173± 14	187 ±14
TG	52± 2	49± 12	41± 4	65 ±12	55± 11	64 ±15
T-CHO	85± 7	74± 5	73± 6	208 ±22	95± 4	121± 13
F-CHO	19.0± 2.0	15.8± 1.4	15.2± 1.7	59.1± 4.0	23.3± 1.5	30.0± 3.1
ALP	200± 29	200± 28	138± 7	183± 51	131± 12	153± 18
AST	74± 7	75± 6	68± 4	62± 4	63± 4	61± 5
CHE	1230 ±235	1350± 146	1380± 152	2096± 41	1566± 293	1421± 147
-GTP	0.70 ±0.18	0.57± 0.21	0.69± 0.24	1.25± 0.31	0.03 ±0.02	0.10± 0.21
LAP	46± 3	44 1	45 1	68 3	43 3	48 3
Pi	6.4 0.3	6.1± 0.2	6.1± 0.3	5.5± 0.2	5.1± 0.3	6.8 ±0.3
Na	138± 0	138± 1	140± 1	143± 2	137± 0	135± 1
K	4.5± 0.2	4.4± 0.2	3.9± 0.1	3.1± 0.1	4.4± 0.2	4.6 ±0.1

 

 

Applicant's summary and conclusion

Conclusions:

The no-observed adverse effect level (NOAEL) of test substance in oral gavage study was considered to be less than 2 mg/kg/day based on the significant decrease in thymus weight in the 2 mg/kg test substance treatment group.

Executive summary:

In order to determine the oral toxicity of test substance, a 28-day repeated dose toxicity study in Wistar rats followed by observation over a 14-day recovery period was conducted at dose levels of 0, 2, 10 and 50 mg/kg administered by gavage. No toxic deaths occurred due to test substance treatment. Decreases of body weight gain and food consumption in the 50 mg/kg dose group were observed during the second half of the treatment period. In addition, hematological examination and serum biochemical tests revealed decreased white blood cells and hemoglobin and increased serum urea nitrogen, cholesterol, phospholipid, ɣ-glutamyl transpeptidase and the Na+/K+ ratio in the 50 mg/kg dose group. Marked thyroid enlargement (to 10 fold the control weight), histopathologically associated with diffuse hyperplasia of follicles with decreased colloid and thickening of the fibrous capsule, was found. Reduction in thymus weight was also observed in a dose-dependent manner without significant histopathological alteration. The no-observed adverse effect level (NOAEL) of test substance in oral gavage study was found to be less than 2 mg/kg/day based on the significant decrease in thymus weight in the 2 mg/kg test substance treatment group.