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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-11-12 to 2015-02-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
other: draft study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
adopted on 3rd October, 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): (Benzothiazol-2ylthio) Acetic Acid
- Physical state: light yellow white powder
- Analytical purity: 97.58 %
- Purity test date: 2014-05-05
- Lot/batch No.: 41273
- Expiration date of the lot/batch: 2016-01-23
- Storage condition of test material: Cool and dry (+2°C to + 8°C)

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% (w/v) in Milli-Q water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For homogeneity and active ingredient (a.i.) concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 and during week 4 of the treatment period and sent to Analytical R&D of Advinus Therapeutics Limited. For each set, duplicate samples were drawn from top, middle and bottom layers of each preparation. In case of control, duplicate samples were drawn only from middle layer.
The analysis was done as per the method described in Advinus Study No. G9831. One set of samples were analyzed for concentration analysis and the other set (second set) of samples were stored at ambient conditions in experimental room for reanalysis purpose as a backup.
Duration of treatment / exposure:
28 days followed by 14 day recovery
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
5 mg/kg bw/day (nominal)
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
40 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Main groups: 6 rats/sex/group
Recovery groups: 5 rats/sex/group
Control animals:
yes, concurrent vehicle
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, twice daily for morbidity and mortalities.

DETAILED CLINICAL OBSERVATIONS: Yes, twice daily during treatment and once daily during recovery period.

BODY WEIGHT: Yes
- Time schedule for examinations: For main groups, individual animal body weight was recorded on day 1, 8, 15, 22, 28 (prior to dosing). For recovery groups, individual animal body weight was recorded on day 1, 8, 15, 22, 29, 36 and 42.
Terminal fasting body weight was recorded only for main and recovery groups prior to sacrifice on day 29 and 43, respectively.

FOOD CONSUMPTION: Yes
For main groups, a known weight of food (food input) was provided on day 1. Food left over in the cage was recorded and replenished with known weight of food on day 8, 15 and 22 for main groups. Food consumption was calculated for day 1 to 8, 8 to 15, 15 to 22 and 22 to 28.
For recovery groups, a known weight of food (food input) was provided on day 1. Food left over in the cage was recorded and replenished with known weight of food on day 8, 15, 22, 29 and 36. Food consumption was calculated for day 1 to 8, 8 to 15, 15 to 22, 22 to 29, 29 to 36 and 36 to 42.
The food spillage was recorded during cage change with food left out measurement sessions.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before start of treatment period (during acclimatization day 5) in main and recovery groups, at the end of treatment period in main groups (week 4) and at the end of recovery period in recovery groups (week 6). Before examination, mydriasis was induced using 1% solution of Tropicamide

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 29 (main groups) and day 43 (recovery groups)
- Anaesthetic used for blood collection: Yes. The blood samples were collected from the abdominal aorta under isoflurane anesthesia.
- Animals fasted: Yes
- How many animals: all
Parameters checked: Red Blood Cells, Haemoglobin, Haematocrit, Mean Corpuscular Volume, Mean Corpuscular Haemoglobin, Mean Corpuscular Haemoglobin Concentration, Reticulocytes, White Blood Cells, Differential Leukocyte Counts, Platelets, Coagulation parameters.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 29 (main groups) and day 43 (recovery groups)
- Anaesthetic used for blood collection: Yes. The blood samples were collected from the abdominal aorta under isoflurane anesthesia.
- Animals fasted: Yes
- How many animals: all
- Parameters checked: Alanine Aminotransferase, Albumin, Alkaline Phosphatase, Albumin / Globulin ratio, Aspartate Amino Transferase, Gamma Glutamyl Transpeptidase, Blood Urea Nitrogen, Creatinine, Creatine Kinase, Calcium, Chloride, Globulin, Glucose, Inorganic phosphorous, Potassium
Sodium, Total cholesterol, Total Plasma Protein, Total Bilirubin, Triglycerides, Bile acids (Total)

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were collected from all rats at the end of the treatment period for main groups and at the end of the recovery period for recovery groups in urine collection tubes.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Specific gravity, Nitrite, pH, Proteins, Glucose, Ketone bodies, Urobilinogen, Bilirubin, Erythrocytes, Leukocytes, Appearance (colour and clarity), Volume

SPERM COUNT, MORPHOLOGYand MOTILITY
- Time schedule for examinations: Sperm evaluation was carried out for all male rats at terminal sacrifice at the end of treatment and recovery periods. At necropsy, right epididymis was collected and frozen (approximately at -20°C) for sperm count by manual method. Right vas deferens was collected for evaluation of sperm motility and sperm morphology. Sperm motility was evaluated using the semen samples collected from the right vas deferens, immediately after necropsy using Hamilton- Thorne TOX-IVOS sperm analyzer.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
following organs/tissues were collected and weighed:
Adrenal glands, Aorta, Bone marrow smear, Brain (cerebrum, cerebellum, medulla/pons), Cecum, Colon, Duodenum, Epididymides, Esophagus, Eyes (with optic nerve), Gross lesions, Femur bone with distal joint, Femoral muscle (Skeletal Muscle), Heart, Harderian glands, Ileum with Peyer’s Patches, Jejunum, Kidneys, Lacrimal glands, Larynx, Liver, Lungs, Mesenteric lymph node, Mandibular lymph node, Mammary gland, Nerves, sciatic, Ovaries, Pancreas, Pituitary, Prostate, Rectum, Salivary glands (submandibular, sub lingual and parotid), Seminal vesicles and coagulating glands, Skin, Spinal cord (cervical, thoracic and lumbar), Spleen, Sternum with marrow, Stomach, Testes, Thymus, Thyroid with Parathyroids, Tongue, Trachea, Urinary bladder, Uterus with cervix, Vagina.
Histopathological examination was carried out on the preserved organs and tissues from rats in the vehicle control (G1), high dose (G5) group. There were no test item related histopathological changes observed in any organ/tissue in high dose (G5) group and hence microscopic evaluation was not performed in lower dose groups (G2, G3 and G4) and recovery group (G1R, G4R and G5R) rats.
Statistics:
Data captured using ProvantisTM: Parameters such as body weight, food consumption, organ weights and derived data such as net body weight gains and organ weight ratios, clinical pathology data - hematology (Prothrombin Time, Activated Partial Thromboplastin Time data was entered retrospectively in Provantis) and clinical chemistry was analysed using ProvantisTM built-in statistical tests.
The statistical analysis of the sperm parameters was carried out using licensed copies of SYSTAT Statistical package version 12.0. The data was tested for homogeneity of variances (Levene’s test) within the group before performing One-way analysis of variance (ANOVA).
The data pertaining to males and female rats were evaluated separately.
All analyses and comparisons were evaluated at the 5% (P≤0.05) level. Statistically significant differences (P≤0.05), indicated by the aforementioned tests were designated by the superscripts throughout the report as stated below:
+/-: Significantly higher / lower than the respective vehicle control group (main groups).
a+/a-: Significantly higher / lower than the respective vehicle control recovery group.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
no clinical signs
Mortality:
no mortality observed
Description (incidence):
no motralities,
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Apparent decreased terminal body weight was observed in females at 100mg/kg bwt/day without any toxicological significant effect and the changes were not observed at the end of recovery period.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Significant increase in the kidney weights (absolute and/or relative) was observed in males at 100 mg/kg bwt/day and in females at 40 and 100 mg/kg bwt/day dose. The altered kidney weight was higher than vehicle control and in-house historical control data, but was not associated with any related microscopic changes. Therefore, the toxicological significance of increased reversible kidney weights remains unclear.
Increased relative liver weight in males and females (apparent change) of the 100mg/kg bwt/day group might be considered as an adaptive reaction and was not of any toxicological significance. Decreased absolute thymus weights in female 100 mg/kg bwt/day group were considered incidental and not of any toxicological significance. These findings were not associated with corresponding gross and/or microscopic changes. Alterations in liver and thymus weights were reversible at the end of recovery period and were comparable to in-house historical control data
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
40 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Any other information on results incl. tables

Clinical signs/Mortalities:

There were no test item-related clinical signs or mortalities observed in males and females in all the treated groups either during the treatment period or recovery period.

Ophthalmological examination:

Ophthalmological examination did not reveal any abnormalities in males and females in all the treated groups either during the treatment period or recovery period.

Body weights:

There were no test item-related variations observed in the mean body weights and net body weight gains in all tested dose groups either during the treatment period or recovery period in both sexes.

Food consumption:

The food consumption was unaffected by the treatment at all the tested dose groups during the treatment period and recovery period in males and females.

Haematology:

No toxicological significant alterations were noted in any of the tested groups of both the sexes at the end of main and recovery period.

Coagulation:

There were no test item related changes in PT and APTT among the dose groups tested of both the genders.

Clinical Chemistry:

The following significant differences were observed in males and females.

- Decreased total cholesterol levels in males at 100 mg/kg bwt/day were considered toxicologically insignificant as there was no corresponding decrease in food intake and terminal body weights. This change was reversible after 14 days recovery period.

- Increased triglycerides in females at 100mg/kg bwt/day were considered incidental/ toxicologically insignificant as it was not associated with any microscopic changes in the liver. This change was reversible at the end of recovery period.

- All other changes which attained statistical significances at termination of treatment and recovery groups were considered incidental/toxicologically insignificant as they fall within in-house historical control data.

Urine Analysis:

There were no test item related changes in any of the urine parameter analyzed in both sexes at the end of main and recovery groups.

Anatomic Pathology

Apparent decreased terminal body weight was observed in females at 100mg/kg bwt/day without any toxicological significant effect and the changes were not observed at the end of recovery period.

Significant increase in the kidney weights (absolute and/or relative) was observed in males at 100 mg/kg bwt/day and in females at 40 and 100 mg/kg bwt/day dose. The altered kidney weight was higher than vehicle control and in-house historical control data, but was not associated with any related microscopic changes. Therefore, the toxicological significance of increased reversible kidney weights remains unclear.

Increased relative liver weight in males and females (apparent change) of the 100mg/kg bwt/day group might be considered as an adaptive reaction and was not of any toxicological significance. Decreased absolute thymus weights in female 100 mg/kg bwt/day group were considered incidental and not of any toxicological significance. These findings were not associated with corresponding gross and/or microscopic changes. Alterations in liver and thymus weights were reversible at the end of recovery period and were comparable to in-house historical control data.

All sperm parameters evaluated were unaffected by (Benzothiazol-2-ylthio) acetic acid as high as 100 mg/kg bwt/day.

There were no gross lesions observed across the toxicity group rats attributed to test item administration.

No significant histologic alterations attributable to treatment were noted in any of the male and female rats.

Applicant's summary and conclusion

Conclusions:
Based on the findings, it is concluded that the No Observed Adverse Effect Level (NOAEL) for (Benzothiazol-2-ylthio) acetic acid is 100 mg/kg bwt/day (highest dose tested) when administered repeatedly for 28 days to Wistar rats by oral route.
Executive summary:

The purpose of this repeated dose oral toxicity study was to assess the toxicity potential of test item (Benzothiazol-2-ylthio) acetic acid in Wistar rats when administered orally by gavage for 28 consecutive days and also intended to assess the reversibility of any effects following 14 days recovery period. This study provided information on important toxic effects, target organs and an estimate of a No Observed Adverse Effect Level (NOAEL).

The main toxicity groups (G1 to G5) consisted of 6 rats/sex/group and recovery groups (G1R, G4R and G5R) consisted of 5 rats/sex/group. The test item (Benzothiazol-2-ylthio) acetic acid was suspended in vehicle [0.5% (w/v) Carboxymethyl cellulose sodium salt in Milli-Q water] and administered by oral gavage at the dose levels of 5 (G2), 15 (G3), 40 (G4/G4R) and 100 (G5/G5R) mg/kg bwt/day for 28 consecutive days. Animals in the vehicle control group (G1/G1R) were administered the vehicle alone. The dose volume administered was 10 mL/kg/day.

The stability and homogeneity of the test item in the vehicle was determined before the initiation of the treatment at 1 and 100 mg/mL concentration levels. The results indicated that the test item in the dosing formulation was homogeneous and stable for 8 days at room temperature and refrigeration condition. The dose formulations were analyzed for (Benzothiazol-2-ylthio) acetic acid concentration and homogeneity on Day 1 and week 4 of the treatment period. The results indicated that the analyzed concentrations were within the ±15% of variations from the nominal concentrations.

All rats were observed for clinical signs (twice daily), mortality (twice daily), ophthalmological examination (prior to initiation of treatment and at terminal sacrifice), changes in body weight (weekly) and food consumption (weekly). Clinical pathology investigations were performed at the end of the treatment (Day 29 - G1 to G5) and at the end of the recovery period (Day 43- G1R, G4R and G5R). Rats from main groups (G1 to G5) were sacrificed on day 29. The rats of recovery group (G1R and G4R) were sacrificed on day 43 (14-day recovery period).

All main and recovery group rats were subjected to detailed gross pathological examination at terminal sacrifice and specified organs were weighed. Sperm evaluation was carried out for all surviving male rats at terminal sacrifice at the end of treatment and recovery periods. Histological examination was performed for all the preserved tissues/organs from all animals in the vehicle control group (G1) and high dose (G5) group of (Benzothiazol-2-ylthio) acetic acid. In addition, all the gross lesions were examined from all the groups.

Under the experimental conditions employed, the following results were obtained:

(Benzothiazol-2-ylthio) acetic acid at the doses of 5, 15, 40 and 100 mg/kg Bwt/day, administered to Wistar rats for 28 consecutive days revealed no effects on clinical signs, mortality, body weights, net body weight gains and food consumption in both sexes.

There were no test item-related changes in haematology, coagulation, clinical chemistry and urinalysis parameters at all the tested dose levels.

There were no test item-related changes observed in terminal fasting body weights, organ weights and gross and microscopic changes at all the tested dose groups.

There were no test item-related changes in sperm parameters (vas deferens sperm motility, cauda epididymal sperm counts and sperm morphology).

Based on the above findings, it is concluded that the No Observed Adverse Effect Level (NOAEL) for (Benzothiazol-2-ylthio) acetic acid is 100 mg/kg/day (highest dose tested) when administered repeatedly for 28 days to Wistar rats by oral route.