Benzimidazole-2-thiol

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Peer reviewed journal research article

Data source

Reference

Reference Type:

publication

Title:

Prechronic Inhalation Toxicity Studies of test chemical in F344/N Rats

Author:

CHARLES L. GAWORSKI,

Year:

1991

Bibliographic source:

FUNDAMENTAL AND APPLIED TOXICOLOGY ( 1991)

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

A 13 weeks subchronic inhalation study was conducted to investigate the toxicologic effects of test substance aerosols and to identify target organs, differences in sensitivity between sexes, and dose-response relationships with repeated exposures at various concentrations.

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:

- Name of test material : 2-Mercaptobenzimidazole (2-MBI)
- Substance type: Organic
- Physical state: Solid

Test animals

Species:

rat

Strain:

Fischer 344

Details on species / strain selection:

No data

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, NY).
- Age at study initiation: 6 to 7 weeks of age
- Housing: Animals were maintained in stainless steel wire-mesh cages in 2-m3 inhalation chambers.
- Diet (e.g. ad libitum): NIH-07 open formula diet; ad libitum except during exposures.
- Water (e.g. ad libitum): Filtered City of Chicago drinking water was supplied ad libitum via an automatic watering system.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 75 +/- 3°F
- Humidity (%):55 ± 15%
- Air changes (per hr): 15 ± 2 changes per hour.
- Photoperiod (hrs dark / hrs light): A 12-hr light/dark cycle (6 AM to 6 PM light)

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

< 3 µm

Remarks on MMAD:

MMAD / GSD: The mass median aerodynamic diameter (MMAD) was less than 3.0 µm and the geometric standard deviation (σg) was in the range of 1.9 to 3.0.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: A wet dispersion technique using a pneumatic spray nozzle was devised to generate aerosols of test chemical from its 10% aqueous suspension.
- Source and rate of air: 0.03-in.diameter orifice at a pressure of 250 psig.
- System of generating particulates/aerosols: aerosol spray was collected with a cyclone mist collector for use in the aerosol generators.
- Temperature, humidity, pressure in air chamber: 150°C
- Method of particle size determination: The cyclone was designed to remove any droplets or natomized liquid greater than 15 pm diameter.

TEST ATMOSPHERE
- Brief description of analytical method used: RAMS real-time aerosol monitors, Quartz Crystal Microbalance (QCM)-based cascade impactor
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

-6 hr/day, 5 day/week, for 13 weeks.
-A control group of 10 rats/sex was exposed to filtered air. At least two consecutive exposures were given immediately prior to the scheduled termination.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/m³ air

Doses / concentrations 2

Dose / conc.:

3.1 mg/m³ air

Doses / concentrations 3

Dose / conc.:

6.2 mg/m³ air

Doses / concentrations 4

Dose / conc.:

12.5 mg/m³ air

Doses / concentrations 5

Dose / conc.:

25 mg/m³ air

Doses / concentrations 6

Dose / conc.:

50 mg/m³ air

No. of animals per sex per dose:

Groups of 10 rats/sex

Control animals:

yes

Details on study design:

Nineteen additional male rats were included in each of the control and the 3.1, 12.5, and 50 mg/m’ groups (special study) for scheduled collection of serum samples for thyroid hormone radioimmunoassay (RIA).

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations performed weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were measured at exposure initiation, weekly thereafter, and at necropsy.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 14 days
- Anaesthetic used for blood collection: Yes (with 70% CO2)
- Animals fasted: No
- How many animals: 5
- Parameters checked: Erythrocyte count and indices; leucocyte count and differentials; hemoglobin, hematocrit,reticulocyte, and platelet counts; and prothrombin and activated partial thromboblastin times (Hematologic determinations were performed with a Baker 9000 hematology analyzer).

CLINICAL CHEMISTRY: Yes
- Animals fasted: No
- How many animals: 5
- Parameters checked: albumin, total protein, blood urea nitrogen,creatinine, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatatase, glucose, total cholesterol,serum cholinesterase, sorbitol dehydrogenase, lactic dehydrogenase, total bihrubin, and free fatty acids.(clinical chemistry tests were performed with a Baker Centrifichem 500 automated analyzer (Serono-Baker, Allentown, PA).)

OTHER:
MORTALITY: YES
Animals were observed twice each day for mortality/moribundity

Sacrifice and pathology:

Rats necropsied at 2, 4, or 8 weeks of exposure.
All animals (excluding special study groups in the subchronic study) received a complete necropsy and were examined for gross lesions.

GROSS PATHOLOGY: Yes
Liver, thymus, thyroid, right kidney, right testis, heart, brain, and lung weights were measured.

HISTOPATHOLOGY: Yes
The following tissues were collected for histopathologic examination: gross lesions and tissue masses, lymph nodes (bronchial, mediastinal, mandibular, and mesenteric),
mammary gland with adjacent skin, thigh muscle, salivary gland, femur including marrow, rib (costochondral junction), nasal cavity and turbinates, tongue, larynx, pharnyx
and trachea, lung and mainstem bronchi, heart and aorta,thymus, thyroids, parathyroids, esophagus, stomach, large and small intestines, liver, pancreas, spleen, kidneys, adrenals, urinary bladder, preputial or clitoral glands, prostate, testes, epididymides, seminal vesicles, scrotal sac, vagina, ovaries, uterus, brain and pituitary, spinal cord, sciatic nerve, eyes, and Zymbal’s glands. Tissues were fixed in 10% neutral buffered formalin, trimmed, embedded in paraffin, sectioned, and stained with hematoxylin and
eosin.

Statistics:

Organ weights and organ weight/body weight ratios were analyzed by one-way (by sex) analysis of variance (ANOVA) followed by a Dunnett’s test when a significant F ratio was obtained. Thyroid hormone data were analyzed by ANOVA followed by either a Dunnett’s test, a one sample t test, or a Mann-Whitney test (RS/Explore software, version 1.1. Serial No. V-658, BBN RS/Expert Limited Partnership, BBN Software Products Corp., Cambridge, MA). TSH results were analyzed following exclusion of outliers, as determined by the method of Dixon ( 1953). Clinical chemistry results were analyzed by ANOVA and a Dunnett’s test using LABCAT software (Innovative Programming Associates, Inc., Princeton, NJ).The level of significance was p ≤ 0.05.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The principle clinical signs of toxicity seen in rats exposed at 25 or 50 mg/m3 included hunched posture, emaciation, and hypoactivity.

Mortality:

mortality observed, treatment-related

Description (incidence):

Ten of the 19 male rats and all 10 females exposed to 50 mg/m3 died. One female control rat also died.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A dose-related decrease in body weight gain occurred in both sexes exposed to 25 mg/m3 or greater, with adverse changes generally apparent after 3-4 weeks of exposure. Comparison of initial and final body weight group means indicated essentially no weight gain during the exposure period in either sex exposed at the 50 mg/m3 level or in females exposed at 25 mg/m’. No significant adverse body weight effects were seen in the rats exposed at concentrations of 12.5 mg/m3, or less.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Concentration-dependent anemia with decreased RBC counts and hemoglobin and hematocrit values was seen at levels of 12.5 mg/m3 and above. In the male rats of the 50 mg/m3 group and the female rats of the 25 mg/m3 group, prothrombin and activated partial thromboplastin times were increased. The leukocyte counts were depressed for the males of all concentration groups.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The 25 mg/m3 males exhibited elevated levels of aspartate aminotransferase, alinine aminotransferase, alkaline phosphatase and sorbitol dehydrogenase activity, while both sexes of the 25 mg/m3 group had increased levels of blood urea nitrogen and cholesterol as well as decreased blood levels of free fatty acids.
With regard to the thyroid hormones, the male rats of the 12.5 g/m3 group showed a drop in T3 levels at the 2, 4, and 8 week ,which however, was reversible after 13 weeks. In the 50 mg/m3 goup, T3 level were depressed at 2 weeks, but found to have recovered to control levels by the end of the study.Test chemical also produced a marked dose-related reduction in T4 levels. At the highest test chemical exposure level no serum thyroxine was detected after 2 weeks of exposure, with levels remaining below the limit of detection for the remainder of the exposure. Rats exposed to 12.5 mg/m3 had decreased T4 at 2, 4,or 8 weeks, with recovery by termination of the study, while exposure at 3.1 mg/m3 did not significantly reduce T4 levels.

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

Description (incidence and severity):

Dose-related increases in absolute and relative thyroid weights occurred in both sexes of rats. At 6.2 mg/m3, the mean absolute and relative thyroid weights of either sex were approximately twice the values of the respective controls,with exposure at 25 or 50 mg/m3 resulting in increased relative weights of these organs of approximately 5 to 10 times the control weights. Increased relative liver weights were seen in males exposed at 6.2 mg/m3, or greater, and in females exposed at 3.1 mg/m3, or greater. Absolute and relative thymus weights were significantly reduced in both sexes exposed to test chemical.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Necropsy observations included dark/red adrenal glands, discolored skin of the toes, and enlarged thyroid glands in exposed rats.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological changes was seen in several organs associated with the most notable being organs of the endocrine system.
Histopathologically, both sexes of the groups receiving 3.13 g/m3 or higher doses exhibited thyroid follicular cell hyperplasia, the extent of which was concentration-dependent.
Adrenal cortical necrosis occurred in rats at the highest exposure level. This same lesion occurred at the 25 mg/m3 level, mixed with degeneration of the
zona reticularis of the adrenal cortex.
Thymic atrophy was noted with high incidence in rats exposed at 25 or 50 mg/m3. Hepatocyte hypertrophy occurred in the animals exposed at 50 mg/m3.
Histopathological lesions were noted in the kidneys (tubular atrophy and mineralisation from 25 mg/m3), the adrenal gands (necrosis and degeneration from 25 mg/m3), the pancreas (hyperplasia of islet cells from 25 mg/m3), the mysentric lymp nodes (hyperplasia from 25 mg/m3), the pituitary gland (cytoplasmic vacuolization from 12.5 mg/m3, proliferation of the thyrotrophic hormone-producing basophilic cells of the anterior adenohypophysis), the bone marrow (hypocellularity from 25 mg/m3) and the nasal cavity (cystic degeneration of the respiratory epithelium from 25 mg/3). Thyroid hyperplasia and decrease in thymus weight thus proved to be most sensitive parameters.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Effect levels

Dose descriptor:

LOAEC

Effect level:

3.1 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Subchronic inhalation study of test chemical : aerosol exposure conditions

 

Experimentally determined aerosol mass concentrations				Aerosol particle size distribution
Aerosol target concn (m/m3)	Mean (mg/m3)	%RSD	N	MMAD	σg
3.1	3.1	12.1	66	2.0	2.9
6.2	6.2	8.0	66	2.3	2.9
12.5	12.5	11.5	66	2.2	2.8
25.0	25.1	11.2	66	2.0	2.1
50.0	51.1	7.5	66	2.3	2.4

 

6 hr/day, 5 days/week

Determined from six daily gravimetric filter-collected aerosol samples over 66 exposure days.

The values are means of four determinations.

Relative standard deviation.

Mass median aerodynamic diameter and geometric standard deviation.

 

Applicant's summary and conclusion

Conclusions:

Thus, on the basis of the study the LOAEC (lowest observed adversed effect concentration) for test chemical in male and female rats was considered to be 3.13 mg/m3.

Executive summary:

The inhalation toxicity of test chemical was evaluated in male and female F344/N rats exposed 6 hr/day, 5 days/week to respirable aerosols generated by spray atomization of aqueous suspensions of the test chemicalpowder and subsequent drying of the resulting aerosols. Subchronic exposures were conducted at target concentrations of 0, 3.1, 6.2, 12.5, 25.0, and 50.0 mg/m3 of test chemical. Rats at ≥25 mg/m3 displayed hunched posture, hypoactivity, and reduced body weight gain, with compound related mortality at the highest exposure level. Anemia; increased SGPT, SGOT, alkaline phosphatase, sorbitol dehydrogenase, BUN, and cholesterol; and reduced free fatty acid were seen in rats at ≥25 mg/m3. Increased thyroid weight and thyroid follicular cell hyperplasia were noted in both sexes at ≥6.2 mg/m3, with reduced triicdothyronine and thyroxine levels in both sexes at ≥12.5 mg/m3. Thyroid follicular cell hyperplasia was also seen in rats at 3.1 mg/m3. Thymus weights were significantly reduced in both sexes at all exposure levels with liver weight increases at ≥6.2 mg/m3. Exposure-related histopathologic changes included pituitary cytoplasmic vacuolization, adrenal cortex necrosis, lymphoid depletion, thymic atrophy, liver cell hypertrophy, renal mineralization and tubular atrophy, and hypocellularity of the bone marrow. Effects observed even at lower concentration of 3.13 mg/m3 so that it was not possible to determine a no observable effet level in this study. Thus, on the basis of the study the LOAEC (lowest observed adversed effect concentration) was considered to be 3.1 mg/m3.