Tetrabutylammonium bromide

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 2022 - Jan 2023

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tetrabutylammonium bromide

EC Number:

216-699-2

EC Name:

Tetrabutylammonium bromide

Cas Number:

1643-19-2

Molecular formula:

C16H36N.Br

IUPAC Name:

tetrabutylazanium bromide

Test material form:

solid: crystalline

Remarks:

White to off white crystalline powder

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals
Body weight ranges upon receipt: 126 - 173 g (males) and 98 - 141 g (females).
Age at the time of dosing: 7 - 8 weeks.

Housing
Prior to receipt of animals for the study, the study room, racks and cages were cleaned and disinfected. During the study, the floor of the experimental room and work tops were swept and mopped with disinfectant solution at least once every day and and more frequently when required. Cages were cleaned at regular intervals. Two to three rats were housed in each polycarbonate cage (length 37 cm X breadth 21 cm X height 20 cm). Rotation of the cage positions in each rack was carried once a week to nullify any possible effects arising from the cage placement. Sterilized corn-cob produced from pure corn, dried and free from dust, procured from approved supplier, was used as bedding material. It was renewed as often as was necessary to keep the animals dry and clean.
Bedding material of batch no. 222 (Krishna Corncob industries, Aurangabad) was used.

Environmental conditions
The room temperature was maintained between 21.0 and 22.5°C and the relative humidity achieved was in the range of 53.2 and 63.7%. Artificial light was set to give a cycle of 12 hours light and 12 hours dark. Adequately filtered air with at least 12 changes per hour was provided.

Diet
A conventional laboratory pellet diet (Batch No. 040522, 040722, 040822) from approved Supplier (Nutrivet Life Sciences, Pune) was available ad libitum.

Water
Aquaguard™ filtered drinking water was available ad libitum in bottles. Samples of the drinking water were periodically subjected to tests for bacteriological and chemical contaminants. Acceptance criteria as per IS 10500 (2012) were met for the samples that were periodically checked.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Distilled water was used as vehicle.

Details on oral exposure:

Each dose in the study was given by oral gavage at a fixed volume of 10 ml/kg bw, based on the most recently recorded body weight for the animal.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration and homogeneity assessment of the dose formulation of Tetrabutyl ammonium bromide (CAS 1643-19-2) was performed on Day 2, 43 and 85. The results of the analyzed formulation were 2.969, 11.942 and 35.203 mg/ml for Day 2; and 2.942, 11.540 and 34.828 mg/ml for Day 43; and 2.983, 11.746, 35.715 mg/ml for day 85 for low, mid and high dose respectively. Overall mean concentrations of does formulations were found within the acceptance limits of ± 20% (from 96.55% to 99.91%) of the nominal concentrations for all the formulations, and the relative standard deviations (RSD) of the top, middle and bottom layers were less than 10% (from 0.66% to 2.10%) in all analyses. Therefore, all dose formulations in this study were considered accurate, precise and homogenous.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

360 mg/kg bw/day (nominal)

Remarks:

G4

Doses / concentrations 2

Dose / conc.:

120 mg/kg bw/day (nominal)

Remarks:

G3

Doses / concentrations 3

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

G2

Doses / concentrations 4

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control group (G1)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

During the acclimatisation phase, male and female animals were separately randomized into four different experimental groups, based on most recently recorded body weights, using excel generated tables as per the internal SOP at the test facility. After randomisation, it was ensured that individual body weights were within ±20% of the respective group means. The mean body weights were analyzed by One-way ANOVA and it was found that the group means were statistically comparable to each other within each sex.

Dose levels were selected based on an OECD 422 study with Tetrabutyl ammonium bromide (CAS No. 1643-19-2) in rats (National Institute of Technology and Evaluation, Japan). In this study, 2 deaths were observed at 600 mg/kg (one male, one female). The male rat died already during the 2nd week of treatment and the female died during gestation. Before death, the male showed fracture of incisors, soft stool and a decrease in the amount of faeces and the female showed staining of lower abdominal fur and staggering gait. At 600 mg/kg/day, the following histopathological findings attributed to the test item were observed: diffuse hyperplasia in the mucosa in the cecum, hypertrophy of perilobular hepatocytes, cell infiltration in the mucosa in the cecum, colon and rectum, cell debris in crypt in the rectum, erosion in the duodenum, atrophy of lymphoid follicle in the spleen, erosion in the glandular stomach, atrophy and atrophy of the thymus. Target organs were identified to be the gastrointestinal tract and liver. NOEL was considered to be 60 mg/kg/day based on the histopathological findings in the cecum at 180 mg/kg/day whereas NOAEL was considered to be 180 mg/kg/day. Based on the above data and considering the significantly longer exposure duration in OECD 408, 360 mg/kg/day was selected as the highest dose level for the study.

Positive control:

None

Examinations

Observations and examinations performed and frequency:

Mortality/Morbidity
All animals were observed twice daily (once before and once after the day’s activities) for any morbidity and/or mortality, throughout the acclimatization and treatment period.

Clinical Signs
All animals were observed daily for clinical signs after dose administration. These observations were regularly performed approximately 1 hour after dose administration since onset of clinical signs after oral gavage can be anticipated in that time frame.

Detailed Clinical Examination
All animals were subject to a detailed clinical examination one day before treatment and weekly thereafter throughout the treatment periods. The animals were examined outside the home cages and the observations included, but were not limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions & excretions and autonomic activity (eg.piloerectio, pupil size, respiratory pattern). The gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (eg. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) were also checked for and recorded.The gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (eg. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) were also checked for and recorded.

Body Weight and Feed Consumption
All animals were weighed once on receipt, once during randomization, once at the start of treatment and weekly thereafter up to terminal sacrifice. These data were used to compare absolute weights and to calculate body weight gains throughout the experimental period. All animals fasted overnight prior to euthanasia and all animals were weighed before necropsy to compare terminal body weights and to calculate relative organ weights. The weight of feed provided and that leftover were recorded for each cage once every week starting from Day 1 of treatment. This data was used to calculate mean feed consumption by each animal throughout the experimental period.

Ophthalmological Examination
Ophthalmological examinationswere performed on all animals before treatment (during acclimatization period) and in all G1 and G4 animals during the final week of dosing. Since no abnormal findings were found in any of the animals in G4, the analysis was not extended to G2 and G3. Before ophthalmologic examination, mydriasis was induced using 1% Tropicamide (Batch No.TR-2108).

Estrus Cycle
The estrus cycle stage was determined in each female rat in G1, G2, G3 and G4 by microscopic examination of the vaginal smears. The examination was carried out on the last day before scheduled sacrifice.

Motor Activity and Behavioural Observation and Functional Observational Battery
Sensory reactivity to stimuli, assessment of grip strength, hind limb foot splay and motor activity assessment were conducted in G1, G2, G3 and G4 groups during the last week of treatment. Animals were subjected to examinations of various functional parameters which included: motor activity measurements using OPTO–VARIMEX 4 (Serial No.: 100180, manufactured by: Columbus Instruments, USA, supplied by: Gentech Marketing and Distribution, New Delhi, India), an automated animal activity measuring system; fore limb and hind limb grip strength, using grip strength meter (Serial No.: 0167-005L, manufactured by: Columbus Instruments, USA, supplied by: Gentech Marketing and Distribution, New Delhi, India); hind limb foot splay record and sensory reactivity measurements.

Sacrifice and pathology:

Blood samples for hematological and clinical biochemistry evaluations were collected on the day of sacrifice (prior to necropsy). The animals were fasted overnight and the blood samples were collected prior to euthanasia from retro-orbital plexuses. Samples were transferredinto vials without anticoagulant for clinical biochemistry endpoints, into 4% EDTA (potassium salt) for hematology endpoints, and into 3.8% sodium citrate for coagulation assessment i.e. Prothrombin (PT) and Activated Partial Prothrombin Time (aPTT). The hematological and clinical biochemistry evaluations were carried out shortly after collection.

Hematology
The following hematologic parameters were estimated using an auto-analyzer (Exigo Vet Hematology Analyzer, serial number 55061, Boule Medical AB, Sweden) and the Robonic-Eazy Clot Activated Clot Time Coagulation Machine Analyser (Serial number CG0231211RBK; Robonik India Pvt Ltd).
Parameters Unit
Activated Partial Thromboplastin time (aPTT) Sec
Hemoglobin (HGB) g/dl
Hematocrit (HCT) %
Mean Corpuscular Volume (MCV) fL
Mean Corpuscular Hemoglobin (MCH) pg/cell
Mean Corpuscular Hemoglobin Concentration (MCHC) g/dl
Platelet Count (PLT) 109/L
Prothombin Time (PT) Sec
Total Erythrocyte Count (RBC) 1012/L
Total Leukocyte count (WBC) 109/L
HDL mg/dl
LDL mg/dl
Blood smears were prepared from the collected blood, stained with Leishman’s stain and observed for differential leukocyte counts (%). Blood smears for reticulocytes (%) were also prepared and evaluated.

Clinical Biochemistry
The following parameters were estimated in serum by using an auto analyzer (VITALAB Selectra E, Bimedis Inc, USA) and the EnSpire Multimode Plate Reader (Serial Number 23001588; Perkin Elmer Inc, USA).
ParametersP Unit
Alanine amino transferase (ALT) U/l
Aspartate amino transferase (AST) U/l
Albumin (Alb) g/dl
Alkaline phosphatase (ALP) U/l
Alb/ Glb (A:G) – Calculated -
Blood urea nitrogen (BUN) – Calculated mg/dl
Bile acids µmol/l
Cholesterol (Chol) mg/dl
Calcium mg/dl
Creatinine (Crea) mg/dl
Glucose (Glu) mg/dl
Gamma-Glutamyl Transpeptidase (GGT) U/I
Globulin (Glob) – Calculated g/dl
Phosphorus mg/dl
Triglycerides (TRIG) mg/dl
Total Protein (TP) g/dl
Total Bilirubin (T.Bil) mg/dl
Urea mg/dl
T3 pg/ml
T4 ng/ml
TSH µIU/ml
The following electrolytes were estimated in serum using the 9180 Electrolyte Analyzer (Serial number 24656; Roche Holding AG, Switzerland).
Sodium (Na) mmol/l
Potassium (K) mmol/l
Chloride (Cl) mmol/l

Urinalysis
Urine samples for urinalysis were collected on the day of sacrifice (prior to necropsy). The animals were housed in metabolic cages overnight and the urine samples were collected in the morning hours prior to necropsy. Water for drinking was available ad libitum during the fasting hours. Colour, appearance and volume of the urine were recorded by visual examinations and. the following parameters below were analyzed using the Uri Scan Optima- Semi Auto Urine Analyser (Serial number RS232C; YD Diagnostics Corp, South Korea).
Parameters Unit
Blood / Blood Cell RBC/µl
Bilirubin mg/dl
Glucose mg/dl
Ketone mg/dl
Leucocytes WBC/µl
Microscopic Parameters* -
Nitrite Positive/Negative
Protein mg/dl
pH -
Specific Gravity -
Urobilinogen mg/dl
*Microscopic parameters included observation of urine sediments (after centrifugation) for presence of epithelial cells, red blood cells, pus cells (white blood cells), casts, crystals and other sediments.

Bone Marrow Smear Examination
Bone marrow smear (from femur) were prepared at the time of necropsy. Since no treatment-related effects on erythropoiesis were noted during the hematologic evaluations, the bone marrow smear samples were not examined.

Pathology
Gross Pathology
All animals were sacrificed on Day 91 (after overnight fasting) and subjected to detailed gross pathological examinations. Each animal was weighed, euthanized by CO2 asphyxiation, and examined externally. All orifices and the cranial, thoracic, and visceral cavities were opened and examined macroscopically.

Organ Weight
Organs marked with asterisk below were trimmed of adherent tissue/fat and weighed, prior to preservation in a fixative. Organs were weighed immediately after they were collected. Organs were kept in normal saline till they were weighed. For paired organs, the combined weight was recorded.

Histopathology
Except the eyes and testes, which were fixed using Modified Davidson’s fluid, the organs listed in the table below were preserved in 10 % neutral buffered formalin (NBF) for the subsequent histopathological examination.
Adrenals* Pancreas
Aorta Peyer's Patches
Bone (femur) with joint # Pituitary * (weighing after fixation)
Brain (cerebrum,cerebellum,mid brain)* Prostate and Seminal vesicle with coagulating glands as a whole *
Cecum Rectum
Colon Salivary glands
Duodenum Sciatic Nerve
Epididymides * Skeletal muscle
Eyes with optic nerve Skin
Gross lesion (if any) Spinal Cord (cervical, mid-thoracic and lumbar)
Heart * Spleen *
Ileum Sternum with marrow #
Jejunum Stomach
Kidneys * Testes *
Liver * Thymus*
Lung $ Thyroid Parathyroids * (weighing after fixation)
Mammary glands Trachea
Mesenteric and Mandibular lymph node Urinary Bladder *
Oesophagus Uterus with Vagina *
Ovaries * -

# Decalcified prior to sectioning; *Organ Weight; $Inflated with 10 % NBF
Full histopathological examinations were carried out on the preserved organs and tissues of all animals in G1 and G4. Histopathological examinations on the kidneys were extended to G2 and G3, as treatment-related effects in the kidneys were observed in G4. All gross lesions were examined histologically.

Statistics:

Raw data was analysed using statistical software Sigma Plot v14.0. All data was summarized in tabular form and the means and standard deviation were calculated using the software. All continuous data (body weight, percent change in body weight with respect to day 1, feed consumption, haematology, clinical chemistry, absolute and relative organ weights, etc. were checked first for their normality and homogeneity, and then analysed using one-way ANOVA (or unpaired t-test) for comparison of means. Heterogneous data was analysed using ANOVA on ranks Kruskal-Wallis test on ranks. For means found significantly different by ANOVA, Dunnett’s post-hoc test was applied to identify the group pairs with significant differences. P values less than or equal to 0.05 were accepted as being statistically significant.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of toxicity were observed at any dose level.

Mortality:

no mortality observed

Description (incidence):

All animals survived to scheduled sacrifice.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No significant changes in body weight or body weight gain were observed, apart from a significant increase in body weight in female rats in G3 on day 78 (mean, 262.4 gram) compared to G1 (mean, 242.1 gram) that was considered incidental and not toxicologically relevant.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

For male rats, no significant changes in food intake were observed. In female rats, significant increases in food intake were observed from day 8-15, 22-29, 29-36, and 36-43 in G2, and from day 1-8, 29-36, 39-43, and 50-57 in G3, as compared to G1. For the female rats in G4, a significant decrease in food intake was observed from day 15-22 (mean, 15.9 g/rat/day) as compared to G1 (mean, 17.2 g/rat/day). The observed changes in food intake in G2 and G3 lacked dose-dependency and for G2, G3, and G4, no associated effects on body weight or body weight gain were observed. The observed changes in food intake in the female aminals were therefore not considered to be toxicologically relevant

Food efficiency:

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No eye abnormalities were observed in the study.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Significant changes in hematology were limited to a significant increase mean corpuscular volume (MCV) in male rats in G4 (mean, 49.81 fL) compared to G1 (mean, 46.51 fL) and a significant increase in mean corpuscular hemoglobin (MCH) in male rats in G4 (mean, 17.64 pg/cell) compared to G1 (mean, 16.54 pg/cell). Kidneys are responsible for erythropoietin production and the observed changes in MCV and MCH in male rats in G4 could be corroborated with histopathological changes in the kidneys, most notably increased incidences of interstitial haemorrhages, as described below. Therefore, the observed changes in MCV and MCH in male rats in G4 were considered to be adverse effects of treatment. MCV tended to be higher in female rats in G4 (mean, 52.19 fL) compared to G1 (mean, 50.36 fL) and this plausible biologically significant effect could also be corroborated with a slight increase in the incidence of interstitial haemorrhages in the kidneys as described below.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

For male rats, noteworthy changes in clinical chemistry included a significant increase in creatinine levels in G4 males (mean, 0.56 mg/dl) compared to G1 (mean, 0.48 mg/dl); significant increases in sodium levels in G2 (mean, 144.7 mmol/l), G3 (mean, 147.9 mmol/l) and G4 (mean, 151.3 mmol/l) compared to G1 (mean, 142.3 mmol/l); and significant increases in chloride levels in G3 (mean, 122.9 mmol/l) and G4 (mean, 115.8 mmol/l) compared to G1 (mean, 85.1 mmol/l). For female rats, noteworthy changes in clinical chemistry included a significant decrease in urea levels in G4 (mean, 27.7 mg/dl) compared to G1 (mean, 37.1 mg/dl); a significant increase in LDL cholesterol in G4 (mean, 12.6 mg/dl) compared to G1 (mean, 9.5 mg/dl); a significant decrease in blood urea nitrogen (BUN) in G4 (mean, 12.9 mg/dl) compared to G1 (mean, 17.4 mg/dl); significant increases in sodium levels in G3 (mean, 146.7 mmol/l) and G4 (mean, 150.9 mmol/l) compared to G1 (mean,141.9 mmol/l); and significant increases in chloride levels in G2 (mean, 105.3 mmol/l), G3 (mean, 111.9 mmol/l) and G4 (mean, 110.0 mmol/l) compared to G1 (mean, 91.9 mmol/l).
Kidneys usually play an important role in the regulation of fluid and electrolyte balance in the body and kidney damage can be associated with an abrupt reduction in renal function causing imbalance in electrolytes associated with various pathological conditions (Perlman RL,Heung M and Ix JH (2013). The observed changes in creatinine levels, serum electrolyte levels, urea, BUN in G3/G4 in male/females could be corroborated with histopathological findings in the kidneys as described below and were therefore considered adverse effects of treatment. The significant increase in LDL cholesterol in G4 females may be attributed to the test item in since lipid profile can be adversely affected by uremic toxins following kidney damage. As such, the significant increase in LDL cholesterol in G4 females was also considered to be an adverse effect of treatment. The significant changes in serum electrolyte levels in G2 in male and females could not be corroborated with any histopathological changes in the kidneys. For G2 female rats, the significant changes in serum electrolyte levels could be corroborated with significant changes in motoractivity as described below As such the significant changes in serum electrolytes in G2 females were considered to be adverse effects of treatment.
No significant changes in serum triiodothyronine (T3), thyroxine (T4), or thyroid stimulating hormone (TSH) were observed in any of the groups, except from a significant increase in TSH in G3 males (mean 0.49 µlU/ml) compared to G1 males (mean, 0.42 µlU/ml) that was considered incidental and not toxicologically relevant.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

In male rats, statistically/biologically significant changes in FOB included a significant decrease in stereotypic time in G4 (mean, 107.7 sec) compared to G1 (mean, 140.3 sec), a significant decrease in burst of stereotypic movements in G4 (mean, 76.1 counts) compared to G1 (mean, 96.2 counts), and a significant decrease in horizontal counts in G4 (mean, 1421.20 counts) when compared to G1 (mean, 1873.80 counts). In female rats, statistically/biologically significant changes in FOB included significant increases in distance travelled in G2 (mean, 2052.8 cm), G3 (mean, 2197.4 cm) and G4 (mean, 2405.5 cm) compared to G1 (mean, 1372.3 cm); significant decreases in resting time in G2 (mean, 152.5 sec), G3 (mean,167.8 sec) and G4 (mean, 144.7 sec) compared to G1 (mean, 253.6 sec); significant increases in ambulatory time in G2 (mean, 308.1 sec), G3 (mean, 303.7 sec) and G4 (mean, 331.3 sec) compared to G1 (mean, 215.1 sec); significant increases in horizontal counts in G2 (mean, 1821.3 counts), G3 (mean, 1804.7 counts) and G4 (mean, 1886.7 counts) compared to G1 (mean, 1207.0 counts); significant increases in ambulatory counts in G2 (mean, 1206.7 counts), G3 (mean, 1243.6 counts) and G4 (mean, 1293.0 counts) compared to G1 (mean, 752.7 counts); significant increases in clockwise rotations in G2 (mean, 27.5 counts), G3 (mean, 26.1 counts) and G4 (mean, 27.8 counts compared to G1 (mean, 15.3 counts); and a significant increases in counter clockwise rotations in G2 (mean, 26.5 counts), G3 (mean, 29.8 counts) and G4 (mean, 32.3 counts) compared to G1 (mean, 17.2 counts). These observed changes in motor activity in G4 males and in G2, G3, and G4 females were considered to be adverse effects of treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Significant changes in organ weight were limited to a significant increase in absolute spleen weight in G2 males (mean, 0.8922 gram) compared to G1 (mean, 0.7115 gram); a significant increase in uterus weight in G2 females (mean, 0.8057 gram) compared to G1 (mean, 0.5679 gram); and a significant decrease in the relative weight of the prostate with seminal vesicle in G3 males (mean, 0.5975) compared to G1 (mean, 0.7241). These observed changes in organ weight lacked dose-dependency and were therefore considered to be incidental and not toxicologically relevant.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Gross findings were limited to one incidence of reduced testes (mild in severity) in one male rat in G2 and one incidence of haemorrhage in the thymus (minimal in severity) in one female rat in G3. None of these findings were attributed to the test item due to lack of dose dependency.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The following histopathological findings were observed during the microscopic examinations:
Kidneys: Interstitial haemorrhages, minimal in severity (G3 Males: 4/10, G3 Females: 3/10, and G4 Females: 3/10); interstitial haemorrhages, mild in severity (G4 Males: 6/10 and G4 Females: 2/10); interstitial MNC infiltration, minimal in severity (G4 Males: 3/10, G1 Females: 1/10, and G3 Females: 1/10); tubular cast, minimal in severity (G4 Females: 1/10); and tubular cast, mild in severity (G4 Females: 4/10). These observed lesions in G3 and G4 could be corroborated with changes in haematology and clinical biochemistry data and were considered to be adverse.
Liver: Microgranuloma, minimal in severity (G1 Males: 1/10, G4 Males: 3/10, G1 Females: 3/10, and G4 Females: 1/10); single cell necrosis, minimal in severity (G1 Male: 1/10); and MNC infiltration, minimal in severity (G4 Males: 1/10, G1 Females: 2/10 and G4 Females: 4/10). These observed lesions in G1 and G4 were considered incidental in nature and therefore not toxicologically relevant.
Testis: Bilateral tubular degeneration and atrophy, minimal in severity (G2 Males: 1/10). This observation lacked dose dependency and was therefore considered to be incidental in nature.
Thymus:Haemorrhage, minimal in severity (G3 Females: 1/10). This observation lacked dose dependency and was therefore considered to be incidental in nature.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

No significant changes in estrous cyclicity were observed.

Effect levels

1

Key result

Dose descriptor:

LOAEL

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

behaviour (functional findings)

serum/plasma biochemistry

2

Key result

Dose descriptor:

NOAEL

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable because of methodological limitations

Remarks:

NOAEL for female rats could not be established due to adverse effects observed at 30 mg/kg bw/day.

3

Key result

Dose descriptor:

LOAEL

Effect level:

120 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

serum/plasma biochemistry

4

Key result

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

ophthalmological examination

organ weights and organ / body weight ratios

serum/plasma biochemistry

serum/plasma hormone analyses

urinalysis

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Table 1 :Summary of Mortality and Morbidity

 Sex: Male

Group	Dose (mg/kg bw)	No. of Incidence / No. of Animals	Observation
G1	0	10/10	No mortality/ No morbidity
G2	30	10/10	No mortality/ No morbidity
G3	120	10/10	No mortality/ No morbidity
G4	360	10/10	No mortality/ No Morbidity

 

 Sex: Female

Group	Dose (mg/kg bw)	No. of Incidence / No. of Animals	Observation
G1	0	10/10	No mortality/ No morbidity
G2	30	10/10	No mortality/ No morbidity
G3	120	10/10	No mortality/ No morbidity
G4	360	10/10	No mortality/ No Morbidity

 

Table 2: Summary of General Clinical Signs Observation

 Sex: Male

Group	Dose (mg/kg bw)	Clinical Signs	No. of Incidence / No. of Animals
G1	0	Normal	10/10
G2	30	Normal	10/10
G3	120	Normal	10/10
G4	360	Normal	10/10

 

 Sex: Female

Group	Dose (mg/kg bw)	Clinical Signs	No. of Incidence / No. of Animals
G1	0	Normal	10/10
G2	30	Normal	10/10
G3	120	Normal	10/10
G4	360	Normal	10/10

 

Table 3: Detailed Clinical Examinations

Sex: Male
Group	Dose (mg/kg body weight)	Clinical Signs	No. of Incidence / No. of Animals
G1	0	No Abnormalities Detected	10/10
G2	30	No Abnormalities Detected	10/10
G3	120	No Abnormalities Detected	10/10
G4	360	No Abnormalities Detected	10/10

Key: NAD; No abnormalities detected.

 

Sex: Female
Group	Dose (mg/kg body weight)	Clinical Signs	No. of Incidence / No. of Animals
G1	0	No Abnormalities Detected	10/10
G2	30	No Abnormalities Detected	10/10
G3	120	No Abnormalities Detected	10/10
G4	360	No Abnormalities Detected	10/10

Key: NAD; No abnormalities detected.

 

Table 4: Summary of Ophthalmological Examination

 Week: Terminal                                                                                            

Group		G1	G4	G1	G4
Sex		Male		Female
Dose (mg/kg bw)		0	360	0	360
Eye	Observation	No. of Incidence / No. of Animals
Terminal
Right	NAD	10/10	10/10	10/10	10/10
Left	NAD	10/10	10/10	10/10	10/10

Key: NAD; No abnormalities detected

 

Table 5: Summary of Body Weight (g)

Sex: Male
Dose (mg/kg bw)		01	08	15	22	29	36	43	50	57	64	71	78	85	90	91
G1 & 0	Mean	250.60	278.40	298.40	316.10	336.10	340.50	357.30	372.00	381.90	398.60	403.40	410.40	420.10	412.40	391.50
	SD	20.27	21.52	27.14	26.43	25.86	27.06	27.09	30.27	31.18	34.52	35.10	35.06	37.84	40.34	38.54
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
G2 & 30	Mean	256.20	286.60	309.70	330.80	351.60	360.00	373.90	389.80	401.00	414.60	421.60	431.50	441.60	437.60	417.90
	SD	18.72	18.88	20.84	22.94	24.80	35.39	36.45	39.36	42.86	49.25	49.28	50.93	56.19	54.48	53.81
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
G3 & 120	Mean	255.40	281.60	306.90	331.30	347.30	362.40	375.40	385.10	396.50	411.00	415.70	426.70	435.40	427.10	407.30
	SD	17.67	15.68	18.37	22.53	35.76	40.44	40.99	51.09	51.17	50.03	47.35	44.92	47.49	48.45	46.70
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
G4 & 360	Mean	251.00	277.30	302.80	324.10	352.20	367.50	378.30	391.90	398.50	414.40	415.20	423.80	428.90	424.90	403.40
	SD	21.16	32.54	32.74	29.11	26.91	34.02	30.46	34.38	33.85	42.69	45.97	47.78	46.21	42.79	39.52
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10

Keys: bw= body weight, N = Number of animals, SD = Standard deviation.

                                                                                                                       

Table 5: Summary of Body Weight (g) (Continued)

Sex: Female
Dose (mg/kg bw)		01	08	15	22	29	36	43	50	57	64	71	78	85	90	91
G1 & 0	Mean	181.70	192.00	204.00	210.70	217.20	224.00	228.10	232.10	236.90	240.40	240.10	242.10	245.40	244.80	231.00
	SD	11.80	14.13	15.17	16.98	17.54	18.70	18.43	20.08	22.25	23.10	22.26	22.00	23.54	23.31	22.18
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
G2 & 30	Mean	184.90	192.80	210.30	219.20	226.80	233.40	239.10	245.00	246.50	253.70	253.00	259.00	261.60	256.30	242.00
	SD	12.93	11.17	13.04	13.13	12.19	14.04	14.94	16.59	18.20	18.54	18.78	18.58	18.91	17.94	16.32
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
G3 & 120	Mean	182.60	196.80	211.20	216.30	227.40	237.30	242.10	244.90	248.30	257.30	259.50	262.40↑	266.90	266.70	251.50
	SD	10.75	10.35	10.95	10.04	8.04	9.87	9.71	13.21	10.61	14.23	15.86	14.64	14.14	16.78	16.24
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
G4 & 360	Mean	180.90	190.00	208.80	216.40	223.80	228.70	234.40	236.20	237.90	244.40	242.80	245.90	249.80	245.20	233.50
	SD	11.20	13.02	8.84	10.30	11.08	13.65	13.91	14.47	16.09	16.20	18.47	17.41	19.19	19.72	18.94
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10

Keys: bw= body weight, N = Number of animals, SD = Standard deviation. ,↑: increased as compared to control (P<0.05)

 

Table 6: Summary of Percent Change in Body weight with Respect to Day 1

Sex: Male
Dose (mg/kg bw)		01	08	15	22	29	36	43	50	57	64	71	78	85	90	91
G1 & 0	Mean	11.14	19.03	26.18	34.24	36.09	42.83	48.70	52.63	59.35	61.27	64.10	68.00	64.83	56.53	11.14
	SD	2.77	3.92	4.72	5.52	8.24	8.46	9.90	9.74	11.98	12.31	12.53	14.06	14.21	14.25	2.77
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
G2 & 30	Mean	11.96	21.01	29.28	37.38	40.43	45.91	52.10	56.46	61.70	64.45	68.32	72.16	70.61	62.89	11.96
	SD	3.30	4.60	6.14	6.29	7.61	8.83	9.77	11.36	13.41	13.48	14.00	15.19	14.57	14.40	3.30
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
G3 & 120	Mean	10.46	20.43	29.96	36.05	41.93	47.14	51.04	55.53	61.28	63.01	67.31	70.64	67.47	59.74	10.46
	SD	5.39	7.13	8.02	11.23	12.89	14.42	19.23	19.40	19.31	17.16	16.44	16.38	17.68	17.33	5.39
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
G4 & 360	Mean	10.28	20.58	29.27	40.62	46.69	51.27	56.65	59.39	65.56	65.77	69.25	71.37	69.85	61.25	10.28
	SD	6.54	7.84	8.05	8.54	11.34	13.16	14.14	15.20	16.77	17.18	18.30	18.29	17.67	16.38	6.54
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10

Keys: bw= body weight, N = Number of animals, SD = Standard Deviation.

 

Table 6: Summary of Percent Change in Body weight with Respect to Day 1 (Continued)

Sex: Female
Dose (mg/kg bw)		01	08	15	22	29	36	43	50	57	64	71	78	85	90	91
G1 & 0	Mean	5.68	12.34	15.95	19.56	23.37	25.67	27.77	30.40	32.36	32.25	33.34	35.16	34.79	27.19	5.68
	SD	4.15	5.87	5.57	6.38	8.53	8.96	8.23	9.41	10.60	10.82	10.50	11.32	10.75	10.20	4.15
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
G2 & 30	Mean	4.37	13.91	18.85	23.04	26.68	29.86	33.05	33.86	37.74	37.48	40.70	42.07	39.15	31.40	4.37
	SD	2.15	5.31	7.66	8.57	10.50	12.04	12.67	13.34	13.35	14.72	14.67	14.02	13.10	12.34	2.15
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
G3 & 120	Mean	7.85	15.80	18.63	24.74	30.27	32.96	34.51	36.32	41.44	42.73	44.19	46.76	46.74	38.38	7.85
	SD	3.31	4.95	5.36	4.93	8.17	9.11	10.71	8.75	13.02	14.63	12.68	13.75	15.73	14.95	3.31
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
G4 & 360	Mean	5.05	15.60	19.80	24.00	26.67	29.86	30.80	31.71	35.43	34.57	36.30	38.40	35.85	29.40	5.05
	SD	3.75	4.41	5.10	7.76	8.27	9.02	8.35	8.73	10.73	12.16	11.80	12.07	12.28	12.17	3.75
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10

Keys: bw: body weight, N = Number of animals, SD = Standard Deviation.

 

Table 7: Summary of Feed Consumption (g/animal/day)                                                  

Sex: Male
Dose (mg/kg bw)		1-8	8-15	15-22	22-29	29-36	36-43	43-50	50-57	57-64	64-71	71-78	78-85	85-90
G1 & 0	Mean	25.5	25.9	25.3	23.8	23.3	23.8	24.5	24.6	24.8	25.2	27.6	25.6	16.2
	SD	4.0	5.2	3.5	2.3	2.2	2.4	2.1	2.0	2.0	2.0	4.7	2.2	1.3
	N	10	10	10	10	10	10	10	10	10	10	10	10	10
G2 & 30	Mean	25.4	25.7	25.8	24.4	22.3	22.6	23.8	24.4	24.1	26.2	27.3	25.3	16.5
	SD	3.6	3.0	2.9	3.1	4.8	2.7	3.0	3.5	4.0	3.6	3.7	3.9	2.5
	N	10	10	10	10	10	10	10	10	10	10	10	10	10
G3 & 120	Mean	24.2	25.4	25.6	23.4	23.7	23.0	22.6	23.3	23.4	24.5	25.1	24.8	15.9
	SD	2.2	1.3	1.5	2.7	2.2	1.8	2.5	1.9	1.4	1.8	2.0	2.8	1.3
	N	10	10	10	10	10	10	10	10	10	10	10	10	10
G4 & 360	Mean	23.6	23.4	24.7	24.6	23.9	23.1	23.3	23.4	22.9	23.8	25.1	24.2	15.1
	SD	4.4	2.9	1.1	1.3	0.9	0.8	0.3	1.0	1.7	1.2	2.5	1.8	0.6
	N	10	10	10	10	10	10	10	10	10	10	10	10	10

Keys: bw: body weight, N = Number of animals, SD = Standard Deviation

 

Table 7:Summary of Feed Consumption (g/animal/day) (Continued)    

Sex: Female
Dose (mg/kg bw)		1-8	8-15	15-22	22-29	29-36	36-43	43-50	50-57	57-64	64-71	71-78	78-85	85-90
G1 & 0	Mean	17.0	16.4	17.2	15.7	15.6	14.8	15.9	15.6	14.6	14.7	14.9	15.7	9.8
	SD	0.8	0.6	1.0	1.0	0.9	0.4	2.6	1.3	0.6	0.9	1.1	1.7	0.6
	N	10	10	10	10	10	10	10	10	10	10	10	10	10
G2 & 30	Mean	18.7	18.5↑	18.2	18.3↑	17.3↑	16.7↑	16.8	16.5	15.8	16.2	15.9	16.2	10.4
	SD	1.1	1.2	0.8	1.9	0.8	1.1	1.4	1.5	1.3	1.1	1.2	0.9	0.6
	N	10	10	10	10	10	10	10	10	10	10	10	10	10
G3 & 120	Mean	18.8↑	18.2	17.9	17.9	17.5↑	16.9↑↑	17.3	18.2↑	16.5	16.0	16.8	17.1	11.1
	SD	0.8	1.4	0.4	0.5	0.7	1.0	1.2	0.2	2.4	1.3	1.4	1.4	2.0
	N	10	10	10	10	10	10	10	10	10	10	10	10	10
G4 & 360	Mean	16.8	16.5	15.9↓	16.2	15.4	15.3	15.3	15.2	15.0	14.8	15.1	15.3	9.0
	SD	1.1	0.6	0.3	1.4	1.1	0.3	0.8	0.5	0.8	0.6	0.7	1.1	0.9
	N	10	10	10	10	10	10	10	10	10	10	10	10	10

Keys: bw: body weight, N = Number of animals, SD = Standard Deviation,↑: increased as compared to control (P<0.05) ,↑↑: increased as compared to control (P<0.01), ↓: decreased as compared to control (P<0.05),

 

Table 8: Summary of Functional Observation Battery/Neurobehavioral Observation Record

Group (Number of animals per group)		G1(10)	G2(10)	G3(10)	G4 (10)
Week : Terminal                                                                                                    Sex: Male
Dose (mg/kg body weight)		0	30	120	360
Parameter	Observation	Number of animals showing observations
Posture	Curled up, often asleep	10	10	10	10
	Sitting B	0	0	0	0
	Sitting C	0	0	0	0
	Sitting A	0	0	0	0
	Rearing	0	0	0	0
Convulsion	Absent	10	10	10	10
Ease of removming from the cage	Very easy	10	10	10	10
	Easy	0	0	0	0
	Moderately difficult	0	0	0	0
Handling reactivity	Easy	10	10	10	10
	Moderately easy	0	0	0	0
Palpebral closure	Eyelids wide open	10	10	10	10
Lacrimation	None	10	10	10	10
Eye Examination	Absent	10	10	10	10
Piloerection	Absent	10	10	10	10
Skin Examination	Absent	10	10	10	10
Salivation	None	10	10	10	10
Gait	Normal	10	10	10	10
Mobility	Normal	10	10	10	10
Arousal	Normal	10	10	10	10
Respiration	Normal	10	10	10	10
Tonic Movement	Absent	10	10	10	10
Clonic Movement	Absent	10	10	10	10
Stereotypy	Absent	10	10	10	10
Bizzare Behaviour	Absent	10	10	10	10
Number of Rears	Mean	11.90	11.10	9.20	8.50
	SD	2.23	1.37	1.93	1.78
Vocalization Count	Mean	0.00	0.00	0.00	0.00
	SD	0.00	0.00	0.00	0.00
No. of urine pools	Mean	1.50	1.60	1.50	0.80
	SD	0.97	0.84	0.71	0.63
No. of faecal bolus	Mean	1.30	0.60	1.80	0.70
	SD	0.67	0.70	1.69	0.67

Key: SD= standard deviation.

Table 8: Summary of Functional Observation Battery/Neurobehavioral Observation Record (Continued)

Group (Number of animals per group)		G1(10)	G2(10)	G3(10)	G4 (10)
Week : TerminalSex: Female
Dose (mg/kg body weight)		0	30	120	360
Parameter	Observation	Number of animals showing observations
Posture	Curled up, often asleep	10	10	10	10
	Sitting B	0	0	0	0
	Sitting C	0	0	0	0
	Sitting A	0	0	0	0
	Rearing	0	0	0	0
Convulsion	Absent	10	10	10	10
Ease of removming from the cage	Very easy	10	10	10	10
	Easy	0	0	0	0
	Moderately difficult	0	0	0	0
Handling reactivity	Easy	10	10	10	10
	Moderately easy	0	0	0	0
Palpebral closure	Eyelids wide open	10	10	10	10
Lacrimation	None	10	10	10	10
Eye Examination	Absent	10	10	10	10
Piloerection	Absent	10	10	10	10
Skin Examination	Absent	10	10	10	10
Salivation	None	10	10	10	10
Gait	Normal	10	10	10	10
Mobility	Normal	10	10	10	10
Arousal	Normal	10	10	10	10
Respiration	Normal	10	10	10	10
Tonic Movement	Absent	10	10	10	10
Clonic Movement	Absent	10	10	10	10
Stereotypy	Absent	10	10	10	10
Bizzare Behaviour	Absent	10	10	10	10
Number of Rears	Mean	12.90	11.60	8.60	8.40
	SD	2.81	2.37	2.27	1.71
Vocalization Count	Mean	0.00	0.00	0.00	0.00
	SD	0.00	0.00	0.00	0.00
No. of urine pools	Mean	2.10	2.30	1.80	1.60
	SD	1.66	1.83	1.87	0.97
No. of faecal bolus	Mean	1.20	1.20	1.30	1.20
	SD	1.14	1.03	0.67	0.79

Key: SD= standard deviation.

 

Table 9: Summary of Sensory Activity Measurement Records

Sex: Male
Group (Number of animals)		G1 (10)	G2 (10)	G3 (10)	G4(10)
Dose (mg/kg body weight)		0	30	120	360
Parameters	Observation	Number of Animals Showing Observations
Approach response	Fast	10	10	10	10
Touch response	Normal	10	10	10	10
Click response	Normal	10	10	10	10
Pupil response	Normal	10	10	10	10
Air righting reflex	Normal	10	10	10	10
Tail pinch response	Flinch	10	10	10	10

 

 

Table 9: Summary of Sensory Activity Measurement Records (Continued)

Sex: Female
Group (Number of animals)		G1 (10)	G2 (10)	G3 (10)	G4(10)
Dose (mg/kg body weight)		0	30	120	360
Parameters	Observation	Number of Animals Showing Observations
Approach response	Fast	10	10	10	10
Touch response	Normal	10	10	10	10
Click response	Normal	10	10	10	10
Pupil response	Normal	10	10	10	10
Air righting reflex	Normal	10	10	10	10
Tail pinch response	Flinch	10	10	10	10

 

Table 10: Mean Foot Splay Record Main Study

Sex: Male
Group (N)	G1 (10)		G2 (10)		G3 (10)		G4 (10)
Dose (mg/kg body weight)	0		30		120		360
Parameter	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Foot Splay (mm)	111.13	24.19	98.17	23.78	85.33	14.79	93.50	17.86
Sex: Female
Group (N)	G1 (10)		G2 (10)		G3 (10)		G4 (10)
Parameter	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Foot Splay (mm)	86.57	16.81	86.27	13.61	86.37	21.46	93.70	13.57

Keys: N = Number of animals in group, R= Recovery, SD = Standard deviation, mm= millimeter.

 

Table 11: Summary of Grip strength

Sex: Male
Group	G1(10)		G2(10)		G3(10)		G4(10)
Dose (mg/kg bw)	0		30		120		360
Parameter	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Forelimb (grams force)	1050	95.60	1011.37	168.22	904.27	60.24	844.53	155.38
Hindlimb (grams force)	531.37	38.52	522.60	38.49	504.70	41.43	516.63	42.53
Sex: Female
Group	G1(10)		G2(10)		G3(10)		G4(10)
Dose (mg/kg bw)	0		30		120		360
Parameter	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Forelimb (grams force)	726	50.43	703.57	75.66	790.17	74.58	814.40	63.12
Hindlimb (grams force)	338.23	31.68	335.47	58.65	367.70	55.31	322.63	35.34

Key: SD= standard deviation.

 

 

  Table 12: Mean Motor Activity Record Main Study

Dose (mg/kg body weight)	0		30		120		360
Sex: Male
Group (N)	G1 (10)		G2 (10)		G3 (10)		G4 (10)
Parameter	Mean	SD	Mean	SD	Mean	SD	Mean	SD
DT(cm)	2065.90	337.07	1581.20	556.19	1884.20	597.67	2025.10	971.27
RT(sec)	142.60	24.72	198.70	61.79	172.80	63.42	191.80	103.03
ST (sec)	140.30	19.01	136.80	22.95	119.50	9.23	107.70↓	23.79
AT(sec)	317.10	34.89	264.50	67.08	307.70	62.39	300.50	101.72
BSM	96.20	6.92	92.90	13.44	86.20	7.33	76.10↓↓	16.24
HC	1873.80	300.30	1441.00	427.19	1441.50	505.78	1421.20↓	636.95
AC	1244.50	246.28	893.90	323.26	1045.20	346.45	1141.00	553.19
HB	1.00	0.00	1.20	1.75	0.60	0.52	1.20	0.63
CR	34.50	8.44	21.60	9.16	26.00	11.93	24.90	13.88
CCR	26.30	6.11	23.90	12.64	26.60	11.06	26.40	13.33
Sex: Female
Group (N)	G1 (10)		G2 (10)		G3 (10)		G4 (10)
Parameter	Mean	SD	Mean	SD	Mean	SD	Mean	SD
DT(cm)	1372.30	610.33	2052.80	417.37	2197.40↑	732.81	2405.50↑↑	789.97
RT(sec)	253.60	90.56	152.50	35.11	167.80	56.42	144.70↓	76.18
ST (sec)	131.30	36.03	139.40	21.70	128.50	18.34	124.00	28.81
AT(sec)	215.10	75.46	308.10↑↑	37.43	303.70↑	63.44	331.30↑↑↑	77.94
BSM	85.80	17.54	89.00	9.15	91.50	9.95	88.40	16.71
HC	1207.00	580.22	1821.30↑	390.25	1804.70↑	572.57	1886.70↑	575.24
AC	752.70	419.97	1206.70↑	265.43	1243.60↑	453.04	1293.00	433.25
HB	0.70	1.57	0.00	0.00	0.10	0.32	0.40	0.52
CR	15.30	10.03	27.50	11.35	26.10	10.89	27.80↑	11.39
CCR	17.20	13.82	26.50	10.42	29.80	12.99	32.30↑	12.70

Keys: DT=Distance travelled (cm), RT=Resting time (sec), ST=Stereotypic time (sec), AT=Ambulatory time (sec), BSM= Burst of stereotypic movements, HC= Horizontal counts, AC=Ambulatory count, HB=Horizontal break, CR=Clockwise rotation and CCR=Counter Clockwise rotation and N = Number of animals in the group, SD= Standard Deviation, cm= Centimeter, sec= seconds, SD= standard deviation, N=No. of animals.

 

Table 13: Absolute Organ Weight (g)

Sex: Male

Group		G1		G2
Dose (mg/Kg)		0		30
Organ	No. of Animals	Mean	SD	Mean	SD
Body weight	10	391.5000	38.5379	417.9000	53.8092
Brain	10	2.0882	0.1281	2.1372	0.1427
Adrenals	10	0.0767	0.0164	0.0747	0.0140
Testes	10	3.5679	0.3882	3.3821	0.3621
Epididymis	10	1.5378	0.1356	1.5228	0.1829
Prostate and Seminal vesicle	10	2.8429	0.6506	2.7116	0.4172
Heart	10	1.2697	0.1102	1.3115	0.1528
Liver	10	11.7107	2.2078	13.5756	2.3159
Kidneys	10	2.9739	0.3727	3.1869	0.4982
Spleen	10	0.7115	0.1168	0.8922↑	0.1468
Thymus	10	0.3512	0.0626	0.3465	0.0726
Thyroid/Parathyroid	10	0.0214	0.0041	0.0205	0.0037
Pitutary	10	0.0130	0.0015	0.0125	0.0022
Group		G3		G4
Dose (mg/Kg)		120		360
Organ	No. of Animals	Mean	SD	Mean	SD
Body weight	10	407.3000	46.7001	403.4000	39.5171
Brain	10	2.0857	0.0803	2.1232	0.1253
Adrenals	10	0.0806	0.0137	0.0843	0.0168
Testes	10	3.3317	0.3805	3.2489	0.2375
Epididymis	10	1.5165	0.1356	1.4912	0.1524
Prostate and Seminal vesicle	10	2.4167	0.3886	2.4522	0.3900
Heart	10	1.2707	0.1252	1.2870	0.1101
Liver	10	13.1193	1.6177	12.2915	1.2275
Kidneys	10	2.9434	0.2905	2.9161	0.2700
Spleen	10	0.7984	0.1654	0.7876	0.1144
Thymus	10	0.4017	0.0912	0.3508	0.1163
Thyroid/Parathyroid	10	0.0217	0.0020	0.0234	0.0022
Pitutary	10	0.0128	0.0011	0.0128	0.0016

 

Table 13: Absolute Organ Weight (g) (Continued)

Sex: Female

Group		G1		G2
Dose (mg/Kg)		0		30
Organ	No. of Animals	Mean	SD	Mean	SD
Body weight	10	231.0000	22.1761	242.0000	16.3231
Brain	10	1.9132	0.1084	1.9524	0.1050
Adrenals	10	0.0843	0.0145	0.0811	0.0152
Uterus	10	0.5679	0.1807	0.8057↑	0.2359
Ovaries	10	0.1420	0.0256	0.1307	0.0125
Heart	10	0.7963	0.0855	0.8812	0.1278
Liver	10	7.1794	0.9708	8.0657	1.0710
Kidneys	10	1.7949	0.1986	1.9129	0.1409
Spleen	10	0.5625	0.1341	0.5736	0.1356
Thymus	10	0.3260	0.0600	0.3310	0.0536
Thyroid/Parathyroid	10	0.0189	0.0022	0.0189	0.0025
Pitutary	10	0.0156	0.0013	0.0165	0.0020
Group		G3		G4
Dose (mg/Kg)		120		360
Organ	No. of Animals	Mean	SD	Mean	SD
Body weight	10	251.50	16.2361	233.5000	18.9400
Brain	10	1.9440	0.1430	1.9657	0.0647
Adrenals	10	0.0856	0.0126	0.0936	0.0152
Uterus	10	0.6685	0.1457	0.6158	0.2409
Ovaries	10	0.1581	0.0363	0.1616	0.0251
Heart	10	0.9002	0.0981	0.8553	0.0620
Liver	10	8.0313	1.4899	7.4647	0.8717
Kidneys	10	1.8903	0.1614	1.9918	0.1619
Spleen	10	0.6030	0.1399	0.5464	0.0717
Thymus	10	0.3631	0.0616	0.3333	0.0657
Thyroid/Parathyroid	10	0.0186	0.0019	0.0174	0.0032
Pitutary	10	0.0177	0.0035	0.0143	0.0021

 

Key: SD= Standard deviation. ,↑: increased as compared to control (P<0.05)

 

Table 14: Organ Weight Relative to Body Weight (g %)

                                                                                                                                    Sex: Male

Group		G1		G2
Dose (mg/Kg)		0		30
Organ	No. of Animals	Mean	SD	Mean	SD
Body weight	10	391.5000	38.5379	417.9000	53.8092
Brain	10	0.5357	0.0289	0.5161	0.0475
Adrenals	10	0.0200	0.0058	0.0179	0.0024
Testes	10	0.9159	0.1034	0.8233	0.1491
Epididymis	10	0.3936	0.0200	0.3671	0.0428
Prostate and Seminal vesicle	10	0.7241	0.1411	0.6501	0.0683
Heart	10	0.3263	0.0330	0.3151	0.0227
Liver	10	2.9739	0.3388	3.2384	0.2156
Kidneys	10	0.7583	0.0411	0.7615	0.0524
Spleen	10	0.1824	0.0282	0.2152	0.0342
Thymus	10	0.0906	0.0187	0.0832	0.0156
Thyroid/Parathyroid	10	0.0055	0.0013	0.0049	0.0009
Pitutary	10	0.0033	0.0003	0.0030	0.0006
Group		G3		G4
Dose (mg/Kg)		120		360
Organ	No. of Animals	Mean	SD	Mean	SD
Body weight	10	407.3000	46.7001	403.4000	39.5171
Brain	10	0.5174	0.0547	0.5310	0.0616
Adrenals	10	0.0199	0.0034	0.0213	0.0055
Testes	10	0.8239	0.1014	0.8113	0.0891
Epididymis	10	0.3754	0.0423	0.3709	0.0325
Prostate and Seminal vesicle	10	0.5975↓	0.1004	0.6110	0.0990
Heart	10	0.3130	0.0171	0.3201	0.0216
Liver	10	3.2244	0.2040	3.0738	0.4486
Kidneys	10	0.7244	0.0346	0.7268	0.0765
Spleen	10	0.1966	0.0377	0.1970	0.0377
Thymus	10	0.0996	0.0241	0.0866	0.0260
Thyroid/Parathyroid	10	0.0054	0.0009	0.0059	0.0007
Pitutary	10	0.0032	0.0003	0.0032	0.0005

Keys: SD = Standard deviation, N = Number of animals in group ↓: Decreased as compared to control (P<0.05)

 

Table 14: Organ Weight Relative to Body Weight (g %)

Sex: Female

Group		G1		G2
Dose (mg/Kg)		0		30
Organ	No. of Animals	Mean	SD	Mean	SD
Body weight	10	231.0000	22.1761	242.0000	16.3231
Brain	10	0.8361	0.1002	0.8097	0.0654
Adrenals	10	0.0367	0.0070	0.0335	0.0055
Uterus	10	0.2457	0.0743	0.3350↑	0.1008
Ovaries	10	0.0615	0.0102	0.0542	0.0063
Heart	10	0.3450	0.0235	0.3635	0.0383
Liver	10	3.0998	0.1962	3.3299	0.3724
Kidneys	10	0.7765	0.0346	0.7928	0.0699
Spleen	10	0.2408	0.0378	0.2370	0.0541
Thymus	10	0.1416	0.0252	0.1368	0.0205
Thyroid/Parathyroid	10	0.0082	0.0007	0.0078	0.0011
Pitutary	10	0.0068	0.0008	0.0069	0.0012
Group		G3		G4
Dose (mg/Kg)		120		360
Organ	No. of Animals	Mean	SD	Mean	SD
Body weight	10	251.5000	16.2361	233.5000	18.9400
Brain	10	0.7735	0.0396	0.8463	0.0662
Adrenals	10	0.0340	0.0043	0.0401	0.0057
Uterus	10	0.2678	0.0663	0.2625	0.0946
Ovaries	10	0.0626	0.0120	0.0697	0.0125
Heart	10	0.3584	0.0361	0.3668	0.0145
Liver	10	3.1924	0.5331	3.1913	0.1807
Kidneys	10	0.7520	0.0510	0.8559	0.0722
Spleen	10	0.2389	0.0494	0.2344	0.0283
Thymus	10	0.1439	0.0186	0.1430	0.0263
Thyroid/Parathyroid	10	0.0074	0.0007	0.0075	0.0012
Pitutary	10	0.0071	0.0014	0.0062	0.0008

Keys: SD = Standard deviation, N = Number of animals in group ↑: increased as compared to control (P<0.05)

 

Table 15: Hematology

                                                                                                                               Sex: Male

Group (N)	G1 (10)		G2 (10)		G3 (10)		G4 (10)
Dose (mg/kg bw)	0		30		120		360
Parameter	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Total Erythrocyte Count (RBC)	8.86	0.32	8.61	0.36	8.66	0.40	8.43	0.23
Hematocrit (HCT)	41.17	0.81	40.66	1.60	40.72	1.59	41.97	1.19
Mean Corpuscular Volume (MCV)	46.51	1.45	47.20	1.32	46.99	1.20	49.81↑↑↑	1.47
Hemoglobin (HGB)	14.66	0.31	14.47	0.55	14.51	0.60	14.84	0.52
MCH	16.54	0.47	16.81	0.47	16.77	0.46	17.64↑↑↑	0.60
MCHC	35.63	0.22	35.65	0.29	35.68	0.23	35.56	0.25
Platelet Count (PLT)	653.70	64.77	616.70	87.52	615.60	101.28	589.20	81.79
Total Leukocyte count (WBC)	9.72	3.06	10.50	2.32	9.13	2.88	9.71	2.40
Neutrophil	27.50	2.37	27.70	1.89	27.50	2.27	27.80	2.62
Lymphocyte	72.00	2.49	71.80	1.93	71.80	1.81	71.70	2.36
Monocyte	0.30	0.48	0.30	0.48	0.40	0.52	0.30	0.48
Eosinophil	0.20	0.42	0.20	0.42	0.30	0.48	0.20	0.42
Basophil	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
Prothrombin Time (PT)	18.28	1.42	19.27	1.32	17.72	1.08	19.11	2.29
(aPTT)	29.01	1.73	28.35	1.43	27.69	1.79	28.32	1.84
Reticulocyte	1.18	0.11	1.18	0.08	1.19	0.07	1.19	0.06

Keys: SD = Standard deviation, N = Number of animals in group, MCH; Mean Corpuscular Hemoglobin, MCHC; Mean Corpuscular Hemoglobin Concentration, aPTT; Activated Partial thromboplastin Time.:  ↑↑↑ increased as compared to control (P<0.001),

 

Table 15:Hematology (Continued)

                                                                                                                               Sex: Female

Group (N)	G1 (10)		G2 (10)		G3 (10)		G4 (10)
Dose (mg/kg bw)	0		30		120		360
Parameter	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Total Erythrocyte Count (RBC)	7.93	0.41	8.14	0.32	7.82	0.31	7.96	0.34
Hematocrit (HCT)	39.97	2.63	41.55	0.96	40.51	1.66	41.56	1.29
Mean Corpuscular Volume (MCV)	50.36	2.22	51.06	1.73	51.84	2.03	52.19	1.19
Hemoglobin (HGB)	13.65	0.56	13.73	0.43	13.27	0.54	13.76	0.35
MCH	17.24	0.48	16.87	0.55	16.99	0.52	17.28	0.39
MCHC	34.30	1.31	33.09	0.57	32.79	0.53	33.13	0.37
Platelet Count (PLT)	583.00	65.23	640.30	99.76	602.40	63.52	608.80	74.95
Total Leukocyte count (WBC)	6.72	2.70	6.03	1.14	6.09	1.74	6.75	0.79
Neutrophil	27.30	2.41	27.30	2.45	27.60	2.41	27.70	3.13
Lymphocyte	72.20	2.57	72.20	2.15	72.00	2.21	71.80	3.26
Monocyte	0.30	0.48	0.30	0.48	0.20	0.42	0.30	0.48
Eosinophil	0.20	0.42	0.20	0.42	0.20	0.42	0.20	0.42
Basophil	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
Prothrombin Time (PT)	18.60	1.20	17.48	0.49	19.08	0.95	18.76	0.70
(aPTT)	28.25	0.68	28.52	1.46	27.47	1.36	28.95	1.32
Reticulocyte	1.16	0.12	1.15	0.07	1.18	0.08	1.17	0.07

Keys: SD = Standard deviation, N = Number of animals in group ↓: decreased as compared to control (P<0.05) MCH; Mean Corpuscular Hemoglobin, MCHC; Mean Corpuscular Hemoglobin Concentration, aPTT; Activated Partial thromboplastin Time

 

Table 16: Clinical Chemistry

Sex: Male

Group (N)	G1 (10)		G2 (10)
Dose (mg/kg bw)	0		30
Parameter	Mean	SD	Mean	SD
Glucose (mg/dl)	85.08	11.24	84.82	21.40
Cholesterol (mg/dl)	55.76	6.91	56.91	7.72
Triglyceride (mg/dl)	76.32	19.10	74.81	20.93
ALT (U/l)	90.28	11.21	79.67	29.14
AST (U/l)	125.98	18.40	136.12	28.19
GGT(U/I)	1.04	0.57	1.22	0.83
Creatinine (mg/dl)	0.48	0.07	0.53	0.03
Albumin (g/dl)	3.48	0.12	3.39	0.16
Total Protein (g/dl)	6.57	0.22	6.52	0.37
Total Bilirubin (mg/dl)	0.22	0.06	0.34	0.48
Urea (mg/dl)	34.22	3.49	33.59	4.04
Phosphorus(mg/dl)	4.92	0.45	5.29	0.37
Calcium (mg/dl)	10.40	0.42	10.76	1.43
ALP (U/l)	307.93	61.81	294.20	68.66
Bile Acid (µmol/l)	22.00	8.79	35.95	51.36
HDL Cholesterol (mg/dl)	24.74	3.90	26.39	4.42
LDL Cholesterol (mg/dl)	20.56	2.38	20.97	3.86
BUN (mg/dl)	16.07	1.80	16.00	2.60
A/G Ratio	1.14	0.11	1.09	0.08
Globulin (g/dl)	3.08	0.23	3.13	0.30
Na (mmol/l)	142.33	0.87	144.70↑	2.26
K (mmol/l)	5.02	0.27	5.12	0.34
Cl (mmol/l)	85.08	11.24	103.90	2.08

Keys: SD = Standard deviation, N = Number of animals in group.↑: increased as compared to control (P<0.05)

 

Table 16: Clinical Chemistry (Continued)

Sex: Male

Group (N)	G3 (10)		G4 (10)
Dose (mg/kg bw)	0		30
Parameter	Mean	SD	Mean	SD
Glucose (mg/dl)	90.08	14.16	77.97	17.90
Cholesterol (mg/dl)	64.26	9.35	54.89	6.81
Triglyceride (mg/dl)	71.13	26.32	67.66	25.04
ALT (U/l)	72.36	18.47	80.02	14.12
AST (U/l)	143.43	25.17	134.08	21.34
GGT(U/I)	0.93	0.43	1.32	0.42
Creatinine (mg/dl)	0.52	0.05	0.56↑↑↑	0.04
Albumin (g/dl)	3.48	0.14	3.49	0.11
Total Protein (g/dl)	6.60	0.26	6.64	0.26
Total Bilirubin (mg/dl)	0.18	0.04	0.22	0.04
Urea (mg/dl)	27.99↓↓	4.72	33.29	4.39
Phosphorus(mg/dl)	4.88	1.59	5.37	0.63
Calcium (mg/dl)	10.28	0.35	10.51	0.40
ALP (U/l)	290.46	74.14	289.10	75.17
Bile Acid (µmol/l)	19.57	4.08	26.01	5.99
HDL Cholesterol (mg/dl)	31.97↑↑	6.89	27.61	3.97
LDL Cholesterol (mg/dl)	22.17	3.78	20.50	2.23
BUN (mg/dl)	13.08↓↓	2.21	15.55	2.05
A/G Ratio	1.12	0.06	1.11	0.10
Globulin (g/dl)	3.12	0.17	3.16	0.24
Na (mmol/l)	147.90↑↑↑	2.13	151.30↑↑↑	1.64
K (mmol/l)	5.07	0.35	5.33	0.50
Cl (mmol/l)	122.90↑↑↑	17.35	115.80↑↑↑	1.69

Keys: SD = Standard deviation, N = Number of animals in group, bw= Body weight, ↑: increased as compared to control (P<0.05), .:  ↑↑↑ increased as compared to control (P<0.001),  ↓↓ decreased as compared to control (P<0.01),  ↑↑  increased  as compared to control (P<0.01).

 

Table 16: Clinical Chemistry (Continued)

Sex: Female

Group (N)	G1 (10)		G2 (10)
Dose (mg/kg bw)	0		30
Parameter	Mean	SD	Mean	SD
Glucose (mg/dl)	76.85	15.83	72.48	10.70
Cholesterol (mg/dl)	45.91	6.21	50.53	6.25
Triglyceride (mg/dl)	48.15	12.70	69.79	24.09
ALT (U/l)	78.58	11.26	90.45	23.94
AST (U/l)	128.53	23.81	145.36	31.66
GGT(U/I)	1.40	0.84	2.20	2.28
Creatinine (mg/dl)	0.60	0.04	0.55	0.05
Albumin (g/dl)	3.55	0.16	3.74	0.08
Total Protein (g/dl)	6.67	0.24	6.82	0.26
Total Bilirubin (mg/dl)	0.19	0.03	0.21	0.06
Urea (mg/dl)	37.14	5.69	33.22	4.13
Phosphorus(mg/dl)	4.27	0.99	4.09	0.63
Calcium (mg/dl)	10.32	0.32	10.59	0.37
ALP (U/l)	194.62	88.57	159.10	50.78
Bile Acid (µmol/l)	18.45	3.98	20.68	5.38
HDL Cholesterol (mg/dl)	28.95	4.78	32.37	5.00
LDL Cholesterol (mg/dl)	9.52	1.53	9.94	1.35
BUN (mg/dl)	17.38	2.68	15.52	1.93
A/G Ratio	1.14	0.11	1.23	0.10
Globulin (g/dl)	3.13	0.24	3.07	0.25
Na (mmol/l)	141.90	1.85	144.40	1.26
K (mmol/l)	4.68	0.47	4.66	0.46
Cl (mmol/l)	91.90	31.99	105.30↑↑↑	0.67

Keys: SD = Standard deviation, N = Number of animals in group,↑↑↑: significant increase as at P<0.001.

 

Table 16: Clinical Chemistry (Continued)

Sex: Female

Group (N)	G3 (10)		G4 (10)
Dose (mg/kg bw)	0		30
Parameter	Mean	SD	Mean	SD
Glucose (mg/dl)	77.48	15.37	86.68	11.91
Cholesterol (mg/dl)	55.38	9.25	51.64	7.02
Triglyceride (mg/dl)	54.47	18.26	50.72	15.64
ALT (U/l)	75.17	33.13	70.36	31.72
AST (U/l)	138.94	34.33	159.53	62.76
GGT(U/I)	1.10	0.42	1.65	0.94
Creatinine (mg/dl)	0.57	0.09	0.56	0.08
Albumin (g/dl)	3.88	0.26	3.78	0.15
Total Protein (g/dl)	7.01	0.31	6.93	0.37
Total Bilirubin (mg/dl)	2.20	6.32	0.16	0.05
Urea (mg/dl)	32.39	7.03	27.69↓↓↓	4.82
Phosphorus(mg/dl)	4.16	0.55	4.27	0.59
Calcium (mg/dl)	10.67	0.37	10.67	0.39
ALP (U/l)	154.54	48.64	195.27	90.77
Bile Acid (µmol/l)	18.36	4.71	15.53	2.85
HDL Cholesterol (mg/dl)	35.13	7.35	31.19	5.78
LDL Cholesterol (mg/dl)	11.20	2.25	12.57↑↑↑	1.90
BUN (mg/dl)	15.14	3.29	12.88↓↓↓	2.35
A/G Ratio	2.18	2.86	1.21	0.09
Globulin (g/dl)	3.13	0.29	3.15	0.28
Na (mmol/l)	146.70↑↑	2.36	150.90↑↑↑	2.77
K (mmol/l)	4.88	0.15	5.02↑	0.43
Cl (mmol/l)	111.90↑↑↑	2.13	110.00↑↑↑	2.05

Keys: SD = Standard deviation, N = Number of animals in group,↑: increased as compared to control (P<0.05). ,↑: increased as compared to control (P<0.05), .:  ↑↑↑ increased as compared to control (P<0.001), ↑↑ decreased as compared to control (P<0.01), ↓↓↓ increased as compared to control (P<0.001)

 

Table 17: Summary of Urine Analysis Physiochemical Parameters

Physiochemical parameters
Parameters		Unit	Observation	Group 1		Group 2		Group 3		Group 4
				Mean	SD	Mean	SD	Mean	SD	Mean		SD
pH		-	-	6.40	0.210	6.00	0.470	6.05	0.550	6.05		0.640
Sp. Gravity		-	-	1.020	0.003	1.020	0.002	1.020	0.003	1.020		0.003
Volume		ml	-	8.42	5.205	7.76	4.692	6.39	4.077	7.83		4.464
Male
Total No. of Observations
Color				Pos	Neg	Pos	Neg	Pos	Neg	Pos	Neg
	Pale Yellow	-	-	10	0	10	0	10	0	10	0
	Yellow	-	-	0	10	0	10	0	10	0	10
	Dark yellow	-	-	0	10	0	10	0	10	0	10
	Yellowish Brown	-	-	0	10	0	10	0	10	0	10
	Brown	-	-	0	10	0	10	0	10	0	10
Appearance	Clear	-	-	10	0	10	0	10	0	10	0
	Turbid	-	-	0	10	0	10	0	10	0	10
	Hazy	-	-	0	10	0	10	0	10	0	10
Blood / Blood Cell		RBC/µl	-	0	10	0	10	0	10	0	10
Bilirubin		mg/dl	-	0	10	0	10	0	10	0	10
Urobilinogen		mg/dl	-	0	10	0	10	0	10	0	10
Ketone		mg/dl	-	0	10	0	10	0	10	0	10
Protein		mg/dl	-	0	10	0	10	0	10	0	10
Nitrite		-	-	0	10	0	10	0	10	0	10
Glucose		mg/dl	-	0	10	0	10	0	10	0	10
Leucocytes		WBC/µl	-	0	10	0	10	0	10	0	10

 

Table 17: Summary of Urine Analysis Physiochemical Parameters (Continued)

Physiochemical parameters
Parameters		Unit	Observation	Group 1			Group 2			Group 3			Group 4
				Mean		SD	Mean		SD	Mean		SD	Mean		SD
pH		-	-	6.40		0.210	6.00		0.470	6.05		0.550	6.05		0.640
Sp. Gravity		-	-	1.020		0.003	1.020		0.002	1.020		0.003	1.020		0.003
Volume		ml	-	3.39		2.165	5.20		3.198	4.55		1.613	4.83		2.063
Female
Total No. of Observations
Colour				Pos	Neg		Pos	Neg		Pos	Neg		Pos	Neg
	Pale Yellow	-	-	10	0		10	0		10	0		10	0
	Yellow	-	-	0	10		0	10		0	10		0	10
	Dark yellow	-	-	0	10		0	10		0	10		0	10
	Yellowish Brown	-	-	0	10		0	10		0	10		0	10
	Brown	-	-	0	10		0	10		0	10		0	10
Appearance	Clear	-	-	10	0		10	0		10	0		10	0
	Turbid	-	-	0	10		0	10		0	10		0	10
	Hazy	-	-	0	10		0	10		0	10		0	10
Blood / Blood Cell		RBC/µl	-	0	10		0	10		0	10		0	10
Bilirubin		mg/dl	-	0	10		0	10		0	10		0	10
Urobilinogen		mg/dl	-	0	10		0	10		0	10		0	10
Ketone		mg/dl	-	0	10		0	10		0	10		0	10
Protein		mg/dl	-	0	10		0	10		0	10		0	10
Nitrite		-	-	0	10		0	10		0	10		0	10
Glucose		mg/dl	-	0	10		0	10		0	10		0	10
Leucocytes		WBC/µl	-	0	10		0	10		0	10		0	10

 

Table 17: Summary of Urine Analysis Microscopic Parameters

Parameters		Sex	Unit/	Group 1	Group 2	Group 3	Group 4
Epithelial cells	Absent	Male	/ h.p.f.	8	7	7	7
	1-2			2	3	3	3
	3-6			0	0	0	0
	6-12			0	0	0	0
	>12			0	0	0	0
	N			10	10	10	10
Pus cells	Absent		/ h.p.f.	4	5	5	8
	1-2			6	5	5	2
	3-6			0	0	0	0
	6-12			0	0	0	0
	>12			0	0	0	0
	N			10	10	10	10
RBCs	Absent		/ h.p.f.	10	9	10	10
	1-2			0	0	0	0
	3-6			0	0	0	0
	6-12			0	0	0	0
	>12			0	0	0	0
	N			10	9	10	10
Epithelial cast	Absent		/ h.p.f.	7	8	6	7
	1-2			3	2	4	3
	3-6			0	0	0	0
	>6			0	0	0	0
	N			10	10	10	10
White cell cast	Absent		/ h.p.f.	10	10	10	10
	1-2	Male		0	0	0	0
	3-6			0	0	0	0
	>6			0	0	0	0
	N			10	10	10	10
Red cell cast	Absent		/ h.p.f.	10	10	10	10
	1-2			0	0	0	0
	3-6			0	0	0	0
	>6			0	0	0	0
	N			10	10	10	10
Waxy cast	Absent		/ h.p.f.	10	10	10	10
	1-2			0	0	0	0
	3-6			0	0	0	0
	>6			0	0	0	0
	N			10	10	10	10
Granularcells	Absent		/ h.p.f.	7	8	6	6
	1-2			3	2	4	4
	3-6			0	0	0	0
	>6			0	0	0	0
	N			10	10	10	10
hyaline cast	Absent		/ h.p.f.	10	10	10	10
	1-2			0	0	0	0
	3-6			0	0	0	0
	>6			0	0	0	0
	N			10	10	10	10
Triple Phosphate	Absent		/ h.p.f.	6	5	2	10
	OCC			0	0	0	0
	Few	Male		0	0	0	0
	Many			0	0	0	0
	N			6	5	2	10
Calcium Oxalate	Absent		/ h.p.f.	7	5	8	5
	OCC			0	0	0	0
	Few			0	0	0	0
	Many			0	0	0	0
	N			7	5	8	5
Calcium Carbonate	Absent		/ h.p.f.	10	10	10	10
	OCC			0	0	0	0
	Few			0	0	0	0
	Many			0	0	0	0
	N			10	10	10	10
Sperm	Absent		/ h.p.f.	3	2	2	1
	OCC			0	0	0	0
	Few			0	0	0	0
	Many			0	0	0	0
	N			3	2	2	1
Bacteria	Absent		/ h.p.f.	10	10	10	10
	OCC			0	0	0	0
	Few			0	0	0	0
	Many			0	0	0	0
	N			10	10	10	10

                                                     

 

Table 17: Summary of Urine Analysis Microscopic Parameters (Continued)

Parameters		Sex	Unit	Group 1	Group 2	Group 3	Group 4
Epithelial cells	Absent	Female	/ h.p.f.	6	8	5	5
	1-2			4	2	5	5
	3-6			0	0	0	0
	6-12			0	0	0	0
	>12			0	0	0	0
	N			10	10	10	10
Pus cells	Absent		/ h.p.f.	6	8	4	4
	1-2			4	2	6	6
	3-6			0	0	0	0
	6-12			0	0	0	0
	>12			0	0	0	0
	N			10	10	10	10
RBCs	Absent		/ h.p.f.	10	10	10	10
	1-2			0	0	0	0
	3-6			0	0	0	0
	6-12			0	0	0	0
	>12			0	0	0	0
	N			10	10	10	10
Epithelial cast	Absent		/ h.p.f.	6	8	6	6
	1-2			4	2	4	4
	3-6			0	0	0	0
	>6			0	0	0	0
	N			10	10	10	10
White cell cast	Absent		/ h.p.f.	10	10	10	10
	1-2			0	0	0	0
	3-6			0	0	0	0
	>6	Female		0	0	0	0
	N			10	10	10	10
Red cell cast	Absent		/ h.p.f.	10	10	10	10
	1-2			0	0	0	0
	3-6			0	0	0	0
	>6			0	0	0	0
	N			10	10	10	10
Waxy cast	Absent		/ h.p.f.	10	10	10	10
	1-2			0	0	0	0
	3-6			0	0	0	0
	>6			0	0	0	0
	N			10	10	10	10
Granularcells	Absent		/ h.p.f.	7	9	6	8
	1-2			3	1	4	2
	3-6			0	0	0	0
	>6			0	0	0	0
	N			10	10	10	10
hyaline cast	Absent		/ h.p.f.	10	10	10	10
	1-2			0	0	0	0
	3-6			0	0	0	0
	>6			0	0	0	0
	N			10	10	10	10
Triple Phosphate	Absent		/ h.p.f.	3	4	4	4
	OCC			0	0	0	0
	Few			0	0	0	0
	Many	Female		0	0	0	0
	N			3	4	4	4
Calcium Oxalate	Absent		/ h.p.f.	5	7	6	8
	OCC			0	0	0	0
	Few			0	0	0	0
	Many			0	0	0	0
	N			5	7	6	8
Calcium Carbonate	Absent		/ h.p.f.	10	10	10	10
	OCC			0	0	0	0
	Few			0	0	0	0
	Many			0	0	0	0
	N			10	10	10	10
Sperm	Absent		/ h.p.f.	10	10	10	10
	OCC			0	0	0	0
	Few			0	0	0	0
	Many			0	0	0	0
	N			10	10	10	10
Bacteria	Absent		/ h.p.f.	10	10	10	10
	OCC			0	0	0	0
	Few			0	0	0	0
	Many			0	0	0	0
	N			10	10	10	10

 

 

Table 18: Mean Hormone levels (T3, T4 & TSH)

Male
Group	Dose (mg/kg)		T3 (pg/ml)	T4 (ng/ml)	TSH (µIU/ml)
G1	0	Mean	329.45	12.87	0.42
		SD	108.62	3.75	0.06
		N	10	10	10
G2	30	Mean	329.00	11.92	0.43
		SD	120.56	1.63	0.05
		N	10	10	10
G3	120	Mean	383.10	12.24	0.49↑
		SD	130.19	1.13	0.06
		N	10	10	10
G4	360	Mean	371.60	11.97	0.43
		SD	178.48	1.69	0.04
		N	10	10	10

Key: SD: Standard deviation; N: No. of animals, ↑=significant increase at p<0.05

 

 

 

Female
Group	Dose (mg/kg)		T3 (pg/ml)	T4 (ng/ml)	TSH (µIU/ml)
G1	0	Mean	418.70	12.54	0.46
		SD	174.62	1.46	0.06
		N	10	10	10
G2	30	Mean	374.20	11.45	0.53
		SD	60.18	1.70	0.17
		N	10	10	10
G3	120	Mean	416.60	12.13	0.47
		SD	151.02	1.13	0.05
		N	10	10	10
G4	360	Mean	379.05	12.55	0.46
		SD	186.92	1.12	0.08
		N	10	10	10

Key: SD: Standard deviation; N: No. of animals

 

 

Table 19 Summary of Gross Pathology Observation

(Male and female)

Group	G1	G2	G3	G4	G1	G2	G3	G4
Dose (mg/kg)	0	30	120	360	0	30	120	360
Sex	Male				Female
Total Number of Animals Observed	10	10	10	10	10	10	10	10
Organ & Lesion
Testes
Reduced size,minimal	0	1	0	0	./.	./.	./.	./.
Thymus
Haemorrhage, minimal	0	0	0	0	0	0	1	0

Note: All organs/tissues as per study plan were observed.

 

 

 

Table 20 Microscopic Pathology Observation

 

Group	G1	G2		G3		G4		G1	G2	G3		G4
Dose (mg/kg)	0	30		120		360		0	30	120		360
Sex	Male							Female
Total Number of Animals Observed	10		10		10		10	10	10		10	10
No abnormality detected	8		9		6		1	5	10		6	0
Liver
Microgranuloma, minimal	1	NA		NA		3		3	NA	NA		1
Single cell necrosis, minimal	1	NA		NA		0		0	NA	NA		0
MNC infiltration, minimal	0	NA		NA		1		2	NA	NA		4
Kidneys
Interstitial haemorrhages, minimal	0	0		4		0		0	0	3		3
Interstitial haemorrhages, mild	0	0		0		6		0	0	0		2
Interstitial MNC infiltration, minimal	0	0		0		3		1	0	1		0
Tubular cast, minimal	0	0		0		0		0	0	0		1
Tubular cast, mild	0	0		0		0		0	0	0		4
Gross Lesions
Testes
Tubular degeneration and atrophy, minimal	NA	1		NA		NA		NA	NA	NA		NA
Thymus
Haemorrhage, minimal	0	NA		NA		0		NA	NA	1		NA

 

Note: All of the preserved organs of groups G1 and G4 and gross observation of G2 and G3 were observed and the organs with abnormality are listed.

Applicant's summary and conclusion

Conclusions:

Based on the above data and under the experimental conditions used in this study, the following effect levels were derived:
• NOAEL for male rats was considered to be 30 mg/kg bw/day.
• LOAEL for male rats was considered to be 120 mg/kg bw/day based on significant changes in clinical biochemistry (electrolyte levels) and histopathological findings in the kidneys (interstitial haemorrhage of minimal in severity) at 120 mg/kg bw/day.
• NOAEL for female rats could not be established due to adverse effects observed at 30 mg/kg bw/day.
• LOAEL for female rats was considered to be 30 mg/kg bw/day based on biologically significant changes in FOB and clinical biochemistry (electrolyte levels) at 30 mg/kg bw/day.

Executive summary:

The objective of this study (performed according to OECD 408 and GLP) was to provide information on the effects of repeated oral administration of Tetrabutyl ammonium bromide (CAS No. 1643-19-2) once a day in Wistar rats for a minimum period of 90 days. A total of 80 Wistar rats (40 males and 40 females) were randomised into four experimental groups, with each group containing 10 animals per sex. The groups, viz., G1, G2, G3 and G4 received 0 (vehicle), 30, 120 and 360 mg/kg bw/day of the test item, respectively. Dose administration was through oral gavage and the vehicle used in this study was distilled water. All groups were treated with the test item for a period of 90 days, afterwhich all animals were euthanized. Each dose in the study was given at a fixed volume of 10 ml/kg bw, based on the most recently recorded body weight for the animal. To ensure accurate administration of the test item, the concentrations and homogeneity of the dose formulations were verified through formulation analysis on three occasions i.e. on Day 2, Day 43 and Day 85 of treatment. The results were found within acceptable limits on all occasions. Observations on the animals encompassed mortality/morbidity, clinical signs, detailed clinical examinations, body weight, body weight changes, feed consumption, ophthalmological examination, functional observatory battery (FOB), hematology, clinical biochemistry, urinalysis, organ weight, gross pathology, and histopathology.

No mortality and no clinical signs of toxicity were observed at any dose level. No significant changes in body weight, body weight gain, or food intake attributed to the test item were observed. No eye abnormalities were observed in the study. In male rats, statistically/ biologically significant changes in FOB included a significant decrease in stereotypic time in G4 (mean, 107.7 sec) compared to G1 (mean, 140.3 sec), a significant decrease in burst of stereotypic movements in G4 (mean, 76.1 counts) compared to G1 (mean, 96.2 counts), and a significant decrease in horizontal count in G4 (mean, 1421.20 counts) when compared to G1 (mean, 1873.80 counts). In female rats, statistically/biologically significant changes in FOB included significant increases in distance travelled in G2 (mean, 2052.8 cm), G3 (mean, 2197.4 cm) and G4 (mean, 2405.5 cm) compared to G1 (mean, 1372.3 cm); significant decreases in resting time in G2 (mean, 152.5 sec), G3 (mean, 167.8 sec) and G4 (mean, 144.7 sec) compared to G1 (mean, 253.6 sec); significant increases in ambulatory time in G2 (mean, 308.1 sec), G3 (mean, 303.7 sec) and G4 (mean, 331.3 sec) compared to G1 (mean, 215.1 sec); significant increases in horizontal counts in G2 (mean, 1821.3 counts), G3 (mean, 1804.7 counts) and G4 (mean, 1886.7 counts) compared to G1 (mean, 1207.0 counts); significant increases in ambulatory counts in G2 (mean, 1206.7 counts), G3 (mean, 1243.6 counts) and G4 (mean, 1293.0 counts) compared to G1 (mean, 752.7 counts); significant increases in clockwise rotations in G2 (mean, 27.5 counts), G3 (mean, 26.1 counts) and G4 (mean, 27.8 counts compared to G1 (mean, 15.3 counts); and a significant increases in counter clockwise rotations in G2 (mean, 26.5 counts), G3 (mean, 29.8 counts) and G4 (mean, 32.3 counts) compared to G1 (mean, 17.2 counts). These observed changes in motor activity in G4 males and in G2, G3, and G4 females were considered to be adverse effects of treatment.For male rats, noteworthy changes in clinical chemistry included a significant increase in creatinine levels in G4 males (mean, 0.56 mg/dl) compared to G1 (mean, 0.48 mg/dl); significant increases in sodium levels in G2 (mean, 144.7 mmol/l), G3 (mean, 147.9 mmol/l) and G4 (mean, 151.3 mmol/l) compared to G1 (mean, 142.3 mmol/l); and significant increases in chloride levels in G3 (mean, 122.9 mmol/l) and G4 (mean, 115.8 mmol/l) compared to G1 (mean, 85.1 mmol/l). For female rats, noteworthy changes in clinical chemistry included a significant decrease in urea levels in G4 (mean, 27.7 mg/dl) compared to G1 (mean, 37.1 mg/dl); a significant increase in LDL cholesterol in G4 (mean, 12.6 mg/dl) compared to G1 (mean, 9.5 mg/dl); a significant decrease in blood urea nitrogen (BUN) in G4 (mean,  12.9 mg/dl) compared to G1 (mean, 17.4 mg/dl); significant increases in sodium levels in G3 (mean, 146.7 mmol/l) and G4 (mean, 150.9 mmol/l) compared to G1 (mean, 141.9 mmol/l); and significant increases in chloride levels in G2 (mean, 105.3 mmol/l), G3 (mean, 111.9 mmol/l) and G4 (mean, 110.0 mmol/l) compared to G1 (mean, 91.9 mmol/l). The observed changes in creatinine levels, serum electrolyte levels, urea, BUN in G3 and/or G4 in male and/or females could be corroborated with histopathological findings in the kidneys and were therefore considered adverse effects of treatment. The significant increase in LDL cholesterol in G4 females may be attributed to the test item in since lipid profile can be adversely affected byuremic toxins following any damage to the kidneys. As such, the significant increase in LDL cholesterol in G4 females was also considered to be an adverse effect of treatment. The significant changes in serum electrolyte levels in G2 in male and females could not be corroborated with any histopathological changes in the kidneys. For G2 female rats, the significant changes in serum electrolyte levels could be corroborated with significant changes in motoractivity. As such the significant changes in serum electrolytes in G2 females were considered to be adverse effects of treatment. No significant changes in serum  triiodothyronine (T3), thyroxine (T4), or thyroid stimulating hormone (TSH) were observed in any of the groups, except from a significant increase in TSH in G3 males (mean 0.49 µlU/ml) compared to G1 males (mean, 0.42 µlU/ml) that was considered incidental and not toxicologically relevant. Significant changes in hematology were limited to a significant increase mean corpuscular volume (MCV) in male rats in G4 (mean, 49.81 fL) compared to G1 (mean, 46.51 fL) and a significant increase in mean corpuscular hemoglobin (MCH) in male rats in G4 (mean, 17.64 pg/cell) compared to G1 (mean, 16.54 pg/cell). Kidneys are responsible for erythropoietin production and the observed changes in MCV and MCH in male rats in G4 could be corroborated with histopathological changes in the kidneys, most notably increased incidences of interstitial haemorrhages. Therefore, the observed changes in MCV and MCH in male rats in G4 were considered to be adverse effects of treatment. MCV tended to be higher in female rats in G4 (mean, 52.19 fL) compared to G1 (mean, 50.36 fL) and this plausible biological effect could also be corroborated with a slight increase in the incidence of interstitial haemorrhages in the kidneys. No significant changes in absolute or relative organ weight attributed to the test item were observed. No gross findings attributed to the test item were observed. Histopathological findings attributed to the test item were observed exclusively in the kidneys in G3 and G4 and included the following: interstitial haemorrhages, minimal in severity (G3 Males: 4/10, G3 Females: 3/10, and G4 Females: 3/10); interstitial haemorrhages, mild in severity (G4 Males: 6/10 and G4 Females: 2/10); interstitial MNC infiltration, minimal in severity (G4 Males: 3/10, G1 Females: 1/10, and G3 Females: 1/10); tubular cast, minimal in severity (G4 Females: 1/10); and tubular cast, mild in severity (G4 Females: 4/10). These observed lesions in G3 and G4 could be corroborated with changes in haematology and clinical biochemistry data and were considered to be adverse.

Conclusion

Based on the above data and under the experimental conditions used in this study, the following effect levels were derived:

• NOAEL for male rats was considered to be 30 mg/kg bw/day.


• LOAEL for male rats was considered to be 120 mg/kg bw/day based on significant changes in clinical biochemistry (electrolyte levels) and histopathological findings in the kidneys (interstitial haemorrhage of minimal in severity) at 120 mg/kg bw/day.


• NOAEL for female rats could not be established due to adverse effects observed at 30 mg/kg bw/day.


• LOAEL for female rats was considered to be 30 mg/kg bw/day based on biologically significant changes in FOB and clinical biochemistry (electrolyte levels) at 30 mg/kg bw/day.