N-tert-butylacrylamide

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 2022 - Jan 2023

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Report date:

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:

N-tert.-butyl acrylamide

Cas Number:

107-58-4

Molecular formula:

C7H13NO

IUPAC Name:

N-tert.-butyl acrylamide

Test material form:

solid: particulate/powder

Remarks:

White powder

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CPCSEA approved animals (under license from Charles River Labs) from Hylasco Biotechnology (India) Pvt. Ltd.
Hyderabad, 500 078
- Females (if applicable) nulliparous and non-pregnant: nulliparous & non pregnant
- Age at study initiation: 6 - 7 weeks
- Weight at study initiation: 179.59 – 229.62 g (Males) and 132.12 – 164.82 g (Females)
- Housing: Animals of the same sex and group was housed in a maximum of three per cage in standard polycarbonate cages (size: 412mm x 290mm x 190mm). The cages were fitted with stainless steel mesh top grill with facilities for holding pelleted food and drinking water bottle.
- Diet (e.g. ad libitum): The pellet feed (Purina Lab Diet 5L79 RAT AND MOUSE 18% (Lot No.25MAR20223) manufactured by PMI Nutrition International) was available ad libitum throughout acclimatization and experimental period. Feed sample were analysed and determined to comply with the requirement of FSSC 22000.
- Water (e.g. ad libitum): Reverse osmosis water was available ad libitum throughout the study period. The test results of collected water samples met acceptance criteria as per IS 10500:2012 for drinking water.
- Acclimation period: The male animals were acclimatized for seven days and female animals were acclimatized for eight days to laboratory conditions and was observed for clinical signs once daily. Veterinary examination of all the animals was performed during acclimatization. Ophthalmological examination of all animals was performed during acclimatization.
- Bedding material: Autoclaved corncob (Lot No.13 and 14) was used as bedding material and was changed along with the cage at least once a week. The material was manufactured by Rowan Agro Nature Pvt Ltd. The corncob samples passed the requirement of the Food Safety Standard Regulation (2011) with respected to tested parameters.

ENVIRONMENTAL CONDITIONS: Animals was housed in an environmentally monitored air-conditioned room maintained at a temperature of 19.6 to 23.9°C, relative humidity of 46 to 71 % and with 12 hours light and 12 hours dark cycle. Temperature and humidity was monitored round the clock and recorded once daily.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% CMC in Milli-Q water.

Details on oral exposure:

The dose volume administered to each rat was at an equivolume of 10 mL/kg body weight. The volume of formulation administered was adjusted based on the recent body weight of the individual rat.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation analysis was performed by the Analytical chemistry department at Vipragen Biosciences Private Limited. The stability of the test item was analyzed in a separate study by HPLC (Study No.: VBPL-ANL-168-G-U-2022) before the start of treatment. The samples were collected in duplicates (5 mL each) from the top, middle and bottom layers from all dose concentrations, covered with aluminium foil and stored at room temperature (bench top) for 6 hours. Further, the prepared formulation samples were covered with aluminium foil and stored at 2 to 8 °C for 24 hours, 72 hours and 8 days. An aliquot was taken at post-storage for each dose concentration and analyzed for stability evaluation. Results of all dose concentrations indicated that the test item formulations in 0.5% CMC in Milli-Q water were stable for 6 hours under room temperature, and for 8 days at 2 to 8 °C. Sampling and analysis of formulations were performed during week 1, week 7 and week 13 of the treatment to determine the homogeneity and concentration using HPLC. The samples were collected in duplicates (5 mL each) from the top, middle and bottom layers from all dose concentrations, and in duplicates (5 mL each) from the middle layer from vehicle control. Results of all dose concentrations were found to be acceptable as the overall mean concentrations were within ±15% (from 95.13% to 99.29%) of the nominal concentrations, and the relative standard deviation (RSD) of top, middle and bottom layers were less than 10% (from 0.21% to 1.3%).

Duration of treatment / exposure:

90 days

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

450 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:

G1 (Vehicle control)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Healthy rats were weighed and grouped before the initiation of treatment and allocated to their respective treatment groups using a body weight based stratified randomization method using MS Excel spreadsheets. Mean body weights of each group before the start of the treatment did not exceed ±20% of the mean body weight in each sex and group. Body weights of the animals was analyzed statistically to rule out the statistically significant differences between groups of same sex.

Positive control:

None

Examinations

Observations and examinations performed and frequency:

Mortality and morbidity:
The animals were observed for morbidity and mortality twice a day. On the weekends and public holidays, the animals were observed at least once a day.

Clinical signs:
All animals were observed for clinical signs of toxicity during the treatment period. General clinical signs of toxicity were carried out once per day throughout the treatment period. Detailed clinical signs of toxicity were carried out before dosing (during acclimatization) and once a week thereafter. These observations were made outside the home cage, in a standard arena and at similar times on each occasion. Care was taken to ensure that variations during observations are minimal. Observation included, but not be limited to, changes in skin, fur, eyes, and mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g., lacrimation, piloerection, pupil size, and respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g., excessive grooming, repetitive circling) or bizarre behaviour (e.g., self-mutilation, walking backwards).

Body weight:
Body weights were recorded on day 1 of treatment, once a week thereafter, on the day before fasting, and on the day after fasting (before terminal sacrifice).

Food intake:
Leftover feed was recorded once a week (coinciding with body weight recordings) and feed input was weighed and recorded based on consumption requirements. Feed consumed was calculated and presented as feed consumption per rat per day (g/rat/day).

Functional parameters:
The animals were subjected to functional observatory battery (FOB) examinations during the last week of treatment. The FOB included the following:
a) Home Cage Observations
Each rat was observed in the home cage for activity level and scored.
b) Handheld Observations
Observations were made while holding the subject animal. Animals were observed for lacrimation and salivation, hair coat characteristics (color/staining, alopecia, and piloerection), degree of eyelid closure, ocular abnormalities and muscle tone or mass. Each observation was recorded using a scoring/ranking system.
c) Open Field Observation
Each rat was placed in an open arena, on a flat surface with a clean absorbent paper and observed for at least 2 minutes. Absorbent paper was replaced for each rat. During this observation period, each rat was evaluated as it moved freely/unperturbed, and the following parameters were recorded using a scoring/ranking system:
• Activity (locomotion).
• Rearings for a standard time.
• Observation of reactivity and arousal.
• Observation of gait and postural characteristics.
• Observation of involuntary/abnormal motor movements (tremors, twitches, clonic
convulsions, tonic convulsions, Stereotypic behavior and bizarre behavior).
d) Sensory Reactivity Measurements
While the rat was in the open field arena and after two minutes of observation, the following sensory reactivity measurements were performed and the observations were recorded using a scoring/ranking system:
• Approach response
• Touch response
• Click response
• Tail-pinch response
• Pupil response
• Aerial righting reflex
e) Landing Hind limbs Foot splay
The landing hind limbs foot splay was performed by dropping each rat on a horizontal surface (e.g., table surface) from a short height and measuring the distance between the hind feet upon landing. The hind feet of the rat were gently pressed to an ink pad just prior to testing. The rat was dropped from a height of approximately 30 cm on to a recording paper sheet, which had the details viz., Study No., animal No. and group. A clean recording paper sheet was used for each rat. A total of 3 readings was recorded for each rat and the average of three individual foot splay values were presented in the report.
f) Grip Strength Performance
Forelimbs and hind limbs grip strength performance was measured using a grip strength meter (Orchid Scientific & Innovative India Pvt. Ltd; Model: GSM 02RS). Three trials for both fore and hind limb will be conducted for each rat. The average value (forelimb and hind limb, respectively) was calculated and presented in the report.
g) Motor Activity
Locomotor activity was tested for 5 minutes using an activity measuring system (Orchid Scientific & Innovative India Pvt. Ltd; Model: ACT01). The displayed values for each rat were documented in the raw data and presented in the report.

Ophthalmic examinations:
Ophthalmic examination were performed in all animals of G1, G2, G3, and G4 before initiation of treatment and in all animals of G1 and G4 during the last week of treatment, prior to blood collection for clinical pathology. Ophthalmic examinations were carried using a 3.5v Coaxial Ophthalmoscope (Welch Allyn Inc., USA). Before examination, mydriasis was induced using a 1% solution of Tropicamide.

Serum hormone levels:
Total serum levels of T4, T3, and TSH, respectively, were measured on samples obtained from each animal at study termination on day 91. For this, separate blood samples were collected in a plain tube and serum was collected after centrifugation. Aliquots of serum samples were stored at -20˚C or lesser until measurement. Serum thyroid hormone levels were measured using a commercially available assay kit.

Urinalysis:
At the last week of treatment, all rats were housed in metabolic cages overnight (reverse osmosis water available ad libitum without feed) and urine samples were collected early in the morning before blood collection. Color, appearance and volume of the urine were recorded after visual observation. The other parameters were analyzed using a Cobas u411 Urine Analyzer, Roche, USA.

Table 1. Urine Parameters
Sr. No. Parameters Unit
1 Appearance -
2 Volume mL
3 Specific gravity -
4 pH -
5 Leucocytes (WBC) Cells/µL
6 Protein mg/dL
7 Glucose -
8 Erythrocytes (RBC) Cells/µL

Sacrifice and pathology:

CLINICAL LABORATORY INVESTIGATIONS:
At the end of the experiment period, blood and urine samples were collected from all surviving animals. All animals were placed individually in metabolic cages and fasted overnight for approximately 16 hours with access to water ad libitum. Urine samples were collected early in the morning. Blood samples were collected early in the day to reduce biological variation caused by circadian rhythms. Blood samples were collected from the retro-orbital plexus using micro glass capillary tube under isoflurane anaesthesia. Samples were collected for hematology, coagulation assessment and clinical chemistry. For hematology and coagulation assessment, blood samples were collected in tubes containing 10% dipotassium ethylene diamine tetra acetic acid (K2-EDTA) and sodium citrate, respectively. For clinical chemistry, blood samples were collected in a plain tube and serum was collected after centrifugation.

HEMATOLOGY
The following haematological parameters were analyzed at the end of the experimental period by using a SCIL Vet ABC hematology analyzer, Horiba Medical ABX S.A., USA.

Table 2. Hematology Parameters
Sr. No. Parameters Unit
1 Total Leukocyte Count (WBC) 103 cells/mm3
2 Erythrocyte Count (RBC) 106 cells/mm3
3 Hemoglobin (HGB) g/dL
4 Hematocrit (HCT) %
5 Platelet Count 103 cells/mm3
6 Mean corpuscular volume (MCV) µm3
7 Mean Corpuscular Hemoglobin (MCH) pg
8 Mean Corpuscular Hemoglobin Concentration (MCHC) g/dL
9 Differential Leucocytes Count (DLC) %
10 Reticulocytes Counts %

Reticulocytes and the differential leucocyte counts were carried out manually. Blood smear was prepared by using standard techniques and stained with Giemsa stain (For DLC) and Reticulocyte diluting fluid (for reticulocyte). The cell count will be expressed as %. A total of 100 cells will be counted per blood smear and the following cells count will be recorded: Lymphocytes (LYM), Monocytes (MOM), Neutrophils (NEU), Eosinophils (EOS), and Basophils (BAS).

COAGULATION PARAMETERS
Blood samples collected at the end of the experimental period from all animals were subjected to coagulation analysis. Blood samples were centrifuged for 10 minutes at 2500 rpm for the isolation of plasma by using Stago Max coagulation analyser (Diagnostica stago, 92600 Asnieres, France).

Table 3. Coagulation Parameters
Sr. No. Parameters Unit
1 Prothrombin Time (PT) Seconds
2 Activated Partial Thromboplastin Time (APTT) Seconds

CLINICAL CHEMISTRY
The following clinical chemistry parameters were measured at the end of the experiment period for all surviving animals by using a Randox Daytona Plus, London, UK. Electrolytes were analyzed by using a 9180 Electrolyte Analyzer, Roche, USA.

Table 4. Clinical Chemistry Parameters
Sr. No. Parameters Unit
1 Total Protein (T.Pro) g/L
2 Albumin (ALB) g/L
3 Alanine Aminotransferase (ALT) U/L
4 Aspartate Aminotransferase (AST) U/L
5 Alkaline Phosphatase (ALP) U/L
6 Total Bile acids (TBA) µmol/L
7 Globulin – Calculative (GLOB) g/L
8 Glucose (GLU) mg/dL
9 Gamma Glutamyl Transpeptidase (GGT) U/L
10 High Density Lipoprotein (HDL) mg/dL
11 Low Density Lipoprotein (LDL) mg/dL
12 Total Cholesterol (TCHO) mg/dL
13 Creatinine (CRE) mg/dL
14 Serum Urea (URE) mg/dL
15 Total bilirubin (TBIL) mg/dL
16 Triglycerides (TRIG) mg/dL
17 Blood Urea Nitrogen (BUN) – Calculative mg/dL
18 Calcium (Ca) mg/dL
19 Phosphorous (I.Phos) mg/dL
20 Chloride (Cl) mmol/L
21 Sodium (Na+) mmol/L
22 Potassium (K+) mmol/L

PATHOLOGY
All animals were subjected to detailed pathological examination at the end of the experiment period. Vaginal smears were examined from all females on the day of necropsy to determine the stage of estrous cycle and to correlate that data with the histopathological findings.

NECROPSY AND GROSS PATHOLOGY
At the end of the observation period, all the surviving animals were euthanized by exsanguination under CO2 asphyxiation. All animals in the study were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

ORGAN WEIGHTS
On the completion of the gross pathological examination, the tissues and organs noted in the following table were collected and weighed from all the rats. 10 % Neutral Buffered Formalin (NBF) was used for fixation.

Table 6. List of Organs for Weighing
Sr. No. Tissue
1 Liver
2 Kidneys
3 Adrenals
4 Spleen
5 Heart
6 Thymus
7 Brain
8 Testes/ Ovaries
9 Epididymides/Uterus
10 Prostate + seminal vesicles with coagulating glands
11 Thyroid gland (after fixation)
12 Pituitary gland (after fixation)

Note 1: Paired organs were weighed together.
Note 2: Tissues were trimmed off for any adherent tissue and their wet weight was taken as soon as possible after dissection to avoid drying.

TISSUE COLLECTION AND PRESERVATION
The following tissues were collected from all the surviving animals and preserved in 10% neutral buffered formalin.

Table 7. List of Organs for Histopathology
Sr. No. Tissue
1 Adrenal glands
2 Aorta
3 Axillary/neck lymph nodes
4 Bone marrow smear (femur)
5 Brain (cerebrum, cerebellum, medulla/pons)
6 Cecum
7 Colon
8 Duodenum
9 Epididymides
10 Oesophagus
11 Eyes (with optic nerve) a
12 Femur bone with bone marrow along the joint
13 Gross lesions
14 Heart
15 Ileum with Peyer’s Patch
16 Jejunum
17 Kidneys
18 Liver
19 Lungs b
20 Mammary gland (male and female)
21 Mesenteric lymph nodes
22 Nerve, sciatic
23 Ovaries
24 Pancreas
25 Pituitary gland
26 Prostate + seminal vesicles with coagulating glands
27 Rectum
28 Salivary gland
29 Skeletal muscle
30 Skin
31 Spinal cord (cervical, thoracic and lumbar)
32 Spleen
33 Stomach
34 Testes a
35 Thymus
36 Thyroid with Parathyroids
37 Trachea
38 Urinary bladder b
39 Uterus with cervix
40 Vagina
Key: a = were collected in modified Davidson’s fluid; b= infused with formalin at necropsy.

HISTOPATHOLOGY
The histopathological examination was carried out on the preserved organs/tissues of vehicle control (G1) and high dose group (G4) rats. In addition, all gross lesions were examined microscopically. As no test item related histopathological changes were observed in high dose groups (G4), histopathological examinations were not performed for lower dose groups (i.e. G2 and G3). The tissues were processed for routine paraffin embedding and tissue sections were stained with Mayer’s Haematoxylin and Eosin stain. In addition, testes were sectioned at 4 micron and stained with PAS (Periodic acid–Schiff) reagent and Haematoxylin to aid in qualitative assessment of spermatogenesis. Unused tissues were archived.

Statistics:

The following statistical methods were used to analyze the body weight, feed consumption, organ weights as well as clinical pathology data.
• Data was summarized in tabular form. Statistical analysis was performed using Graphpad Prism (Version 8.1.2).
• All the data was checked for normality and homogeneity before the comparisons.
• Data for each group of animals were subjected to parametric tests such as one way ANOVA (analysis of variance) along with multiple comparisons. Non normal/non homogenous data was subjected to non-parametric tests. Values were given as mean ± standard deviation (SD).
• All analyses and comparisons were evaluated at the 5% (P ≤ 0.05) level.
• All the statistically significant values were indicated with * asterisk symbol in the respective summary tables.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Lethargy was observed in male rats in G4 (range: 1-5 animals) on day 44 and each day onwards, up to terminal sacrifice. This lethargy was observed 10-15 minutes after dosing, persisted up to about 60 minutes after dosing, and was considered to be an adverse effect of treatment. No clinical signs of toxicity were observed in G2 or G3 males or in any of the female groups.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significant decreases in body weight were observed in G4 males on day 57 (mean, 480.98 gram), day 64 (mean, 499.31 gram), and after fasting (mean, 525.06 gram) compared to G1 (mean, 518.31 gram, 535.65 gram, and 569.15 gram on day 57, 64, and after fasting, respectively). Furthermore in G4 males, significant decreases in body weight gain were observed between day 15-22 (mean, G4: 45.01 gram; G1: 54.24 gram), 29-36 (mean, G4: 28.66 gram; G1: 34.82 gram), 50-57 (mean, G4: 17.84 gram; G1: 27.17 gram) and 85-90 (mean, G4: 2.08 gram; G1: 11.27 g). These observed changes body weight and body weight gain in G4 males were considered to be adverse effects of treatment, as body weight and body weight gain were generally lower in G4 males compared to G1 males throughout the study period. No other significant changes in body weight or body weight gain were observed, with the exception of a significant increase in body weight gain in G2 females between day 43-50 (mean 16.12 gram) compared to G1 (mean, 4.47 gram), a significant increases in body weight gain in G4 females between day 43-50 (mean, 14.73 gram) compared to G1 (mean, 4.47 gram), and a significant increase in body weight gain in G4 females between day 78-85 (mean, 9.93 gram) compared to G1 (mean, 6.07 gram). These significant increases in body weight gain in G2 and G4 females were considered to be incidental and not toxicologically significant.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In G4 males, a significant decrease in food intake was observed between day 85-90 (mean, 31.41 gram) compared to G1 (mean, 34.35 gram). In G4 females, a significant increase in food intake was observed between day 43-50 (mean, 22.87 gram) compared to G1 (mean, 20.40 gram). No other significant changes in food intake were observed. The decrease in food intake in G4 male rats was associated with a tendency of lower body weight in G4 males on day 90 (mean 548.53 gram) compared to G1 males (mean, 591.49 gram) and was therefore considered treatment-related. The significant increase in food intake in G4 females was not considered to be toxicologically relevant, as no adverse effects on body weight or body weight gain were observed in the group.

Food efficiency:

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ocular abnormalities were observed in the study.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant changes in hematology in male rats included a statistically significant decrease in WBC in G4 (mean, 6.85 x 103/mm3 cells) compared to G1 (mean, 8.74 x 103/mm3 cells) and a statistically significant increase in MCHC in G4 (mean, 34.39 g/dL) compared to G1 (mean, 33.79 g/dL).

Statistically significant changes in hematology in female rats included statistically significant decreases in RBC in G3 (mean, 7.98 x 106/mm3 cells) and G4 (mean, 7.73 106/mm3 cells) compared to G1 (mean, 8.43 106/mm3 cells); a statistically significant increase in platelet count in G4 (mean, 1040.80 x 103/mm3 cells) compared to G1 (mean, 875.70 x 103/mm3 cells); a statistically significant increase in MCV in G4 (mean, G4: 56.00 µm3) compared to G1 (mean, 54.00 µm3); a statistically significant increase in MCHC in G4 (mean, 36.63 g/dL) compared to G1 (mean, 34.47 g/dL); and statistically significant increases in MCH in G3 (mean, 20.14 pg) and G4 (mean, 23.55 pg) compared to G1 (mean, 18.61 pg).

No significant changes in coagulation parameters were observed.

The observed changes in haematology in G4 males and in G3 and G4 females were within historical control ranges of the test facility and could not be corroborated with any clinical symptoms, any changes in organ weight, or any gross or histopathological findings. Therefore, the observed changes in hematology in this study were not considered to be toxicologically relevant.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant changes in clinical chemistry in male rats included significant decreases in glucose in G2 (mean, 113.02 mg/dL), G3 (mean, 109.56 mg/dL) and G4 (mean, 108.85 mg/dL) compared to G1 (mean, 128.42 mg/dL); a significant increase in urea in G4 (mean, 35.54 mg/dL) compared to G1 (mean, 29.85 mg/dL); significant decreases in creatinine in G2 (mean, 0.49 mg/dL) and G4 (mean, 0.49 mg/dL) compared to G1 (mean, 0.54 mg/dL); a significant decrease in total cholesterol in G3 (mean, 57.81 mg/dL) compared to G1 (mean, 77.55 mg/dL); a significant decrease in AST in G4 (mean, 120.12 U/L) compared to G1 (mean, 163.50 U/L); a significant increase in Total Protein in G4 (mean, 71.34 g/L) compared to G1 (mean, 65.93 g/L); a significant increase in HDL in G2 (mean, 15.48 mg/dL) compared to G1 (mean, 12.35 mg/dL); a significant decrease in LDL in G4 (mean, 5.00 g/L) compared to G1 (mean, 7.68 g/L); significant increases in ALB in G3 (mean, 44.63 g/L) and G4 (mean, 47.17 g/L) compared to G1 (mean, 42.06 g/L); a significant decrease in calcium in G2 (mean, 10.20 mg/dL) compared to G1 (mean, 10.81 mg/dL); a significant increase in phosphorous in G4 (mean, 8.74 mg/dL) compared to G1 (mean, 7.99 mg/dL); a significant decrease in globulin in G2 (mean, 22.54 g/L) compared to G1 (mean, 23.97 g/L); a significant increase in TBA in G4 (mean, 42.02 µmol/L) compared to G1 (mean, 23.37 µmol/L); a significant increase in BUN in G4 (mean, 16.61 mg/dL) compared to G1 (mean, 13.95 mg/dL); significant increases in sodium in G3 (mean, 143.70 mmol/L) and G4 (mean, 143.00 mmol/L) compared to G1 (mean, 140.80 mmol/L); and a significant increase in chloride in G3 (mean, 103.70 mmol/L) compared to G1 (mean,101.40 mmol/L).
Statistically significant changes in clinical chemistry in female rats included a significant increase in urea in G4 (mean, 40.18 mg/dL) compared to G1 (mean, 32.63 mg/dL); a significant decrease in creatinine levels in G4 (mean, 0.51 mg/dL) compared to G1 (mean, 0.57 mg/dL); a significant increase in total protein in G4 (mean, 74.81 g/L) compared to G1 (mean, 68.34 g/L); significant increases in HDL in G3 (mean, 15.59 mg/dL) and G4 (mean, 19.45 mg/dL) compared to G1 (mean, 12.39 mg/dL); a significant decrease in LDL in G4 (mean, 3.92 mg/dL) compared to G1 (mean, 7.98 mg/dL); a significant increase in albumin in G4 (mean, 49.79 g/L) compared to G1 (mean, 44.93 g/L); a significant increase in calcium in G4 (mean, 10.50 mg/dL) compared to G1 (mean, 9.36 mg/dL); and a significant increase in BUN in G4 (mean, 19.13 mg/dL) compared to G1 (mean, 15.54 mg/dL).
The observed changes in clinical chemistry in G2, G3 and G4 males and in G3 and G4 females could not be correlated with any clinical symptoms or any gross or histopathological findings. The significant increases in urea and BUN in G4 females compared to G1 could be corroborated with a significant increase in absolute kidney weight in G4 females compared to G1 females. However, since no treatment-related gross or histopathological findings were made in the kidneys in G4 females, the observed changes in urea and BUN in G4 females were not considered to be adverse.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant changes in thyroid hormone levels in male rats included significant increases in T3 levels in G2 (mean, 1409.62 ng/mL), G3 (mean, 1434.39 ng/mL) and G4 (mean, 1419.42 ng/mL) compared to G1 (mean, 1231.01ng/mL). Statistically significant changes in thyroid hormone levels in female rats included a significant increase in T4 levels in G3 (mean, 47.92 pg/mL) compared to G1 (mean, 35.06 pg/mL) and significant increases in TSH levels in G3 (mean, 733.41 pg/mL) and G4 (mean, 755.83 pg/mL) compared to G1 (mean, 500.20 pg/mL). Since no significant changes in absolute or relative thyroid weight were observed and all thyroids examined in the study were normal upon the gross and histopathological examinations, the observed changes in T3, T4 and TSH levels in the male and female rats were not considered to be adverse.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No significant changes in urinalysis were observed.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No significant changes in the functional observatory battery were observed, with the exception of statistically significant decreases in landing hindlimb foot splay in G2 females (mean, 43.30 mm) and G4 females (mean, 46.97 mm) compared to G1 (mean, 60.93 mm).

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant changes in organ weight in male rats included a significant increase in absolute liver weight in G4 (mean, 21.40 grams) compared to G1 (mean, 18.62 grams); a significant increase in absolute kidneys weight in G4 (mean, 4.38 grams) compared to G1 (mean, 3.78 grams); significant increases in relative organ weight of liver (mean, G4: 4.09%; G1: 3.29 %), kidneys (mean, G4: 0.84%; G1: 0.67%), adrenals (mean, G4: 0.02%; G1: 0.01%), heart (mean, G4: 0.35 %; G1: 0.30 %) and prostate + seminal vesicles with coagulating glands (mean, G4: 0.72%; G1: 0.64%). Statistically significant changes in organ weight in female rats included a significant increase in absolute liver weight in G4 (mean, 11.57 grams) compared to G1 (mean, 10.04 grams), a significant increase in absolute kidneys weight in G4 (mean, 2.32 grams) compared to G1 (mean, 2.10 grams), a significant increase in absolute brain weight in G2 (mean, G2: 2.09 grams) compared to G1, 2.03 grams); and significant increases in relative organ weights of liver (mean, G4: 4.11 %; G1:3.55 %) and kidneys (mean, G4: 0.82%; G1: 0.74%). The significant changes in absolute and relative organ weight in G4 males and in G2 and G4 females could not be correlated with any gross or histopathological findings and were therefore considered to be incidental and not toxicologically significant.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No gross pathological lesions were observed, with the exception of small sized testes (bilateral) and small sized epididymides (bilateral) in one G1 male rat.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Histopathological findings included the following: aspermia (bilateral) in the epididymides in one G1 male rat; atrophy (moderate in severity) in the seminiferous tubules (bilateral, diffuse) in one G1 male rat; infiltration of mononucleated cells (minimal in severity) in the renal pelvis of the kidneys in one G1 female rat and one G4 female rat; and cystic dilatation (minimal in severity) in the uterus in one G1 female rat. No treatment related findings were observed during the qualitative assessment of spermatogenesis stages or during the morphological examination of interstitial testicular cell structures in high dose group (G4) male animals when compared to the vehicle control group (G1). Testes of one G1 male animal did not show any stages of spermatogenesis due to bilateral atrophy of the testes. The stage of estrous cycle in each female rat in G1 and G4 at the terminal sacrifice was in correlation with the histology of respective female reproductive organs. The observed microscopic findings in the G1 and G4 were considered incidental and not toxicologically relevant.

Histopathological findings: neoplastic:

no effects observed

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

>= 450 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

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

other endocrine activity endpoints

2

Key result

Dose descriptor:

NOAEL

Effect level:

>= 120 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

other endocrine activity endpoints

3

Key result

Dose descriptor:

LOAEL

Effect level:

<= 450 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

clinical signs

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

TABLE 1 - SUMMARY OF CLINICAL SIGNS AND MORTALITY – MALES

Clinical Signs  and  Mortality– Males

Groups

Dose (mg/kg bw/day)

Mortality/Morbidity  (From day 1 to 91)

Days

1 to 43

44

45

46

47

48

49

50

51

52

53

54

55

56

G1

0

0/10

Normal

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

G2

30

0/10

Normal

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

G3

120

0/10

Normal

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

G4

450

0/10

Normal

10/10

8/10

7/10

8/10

5/10

8/10

9/10

7/10

8/10

7/10

8/10

6/10

7/10

6/10

Lethargy

0/10

2/10

3/10

2/10

5/10

2/10

1/10

3/10

2/10

3/10

2/10

4/10

3/10

4/10

Key:  n=10, G1- Vehicle control, G2-Low dose, G3- Mid dose, G4-High dose.

 

TABLE 1 (CONTD.)  SUMMARY OF CLINICAL SIGNS AND MORTALITY – MALES

Clinical Signs  and  Mortality– Males

Groups

Dose (mg/kg bw/day)

Days

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

G1

0

Normal

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

G2

30

Normal

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

G3

120

Normal

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

G4

450

Normal

6/10

6/10

6/10

7/10

7/10

6/10

6/10

6/10

6/10

7/10

6/10

6/10

6/10

8/10

5/10

7/10

7/10

7/10

Lethargy

4/10

4/10

4/10

3/10

3/10

4/10

4/10

4/10

4/10

3/10

4/10

4/10

4/10

2/10

5/10

3/10

3/10

3/10

Key:  n=10, G1- Vehicle control, G2-Low dose, G3- Mid dose, G4-High dose.

 

 

TABLE 1 (CONTD.)  SUMMARY OF CLINICAL SIGNS AND MORTALITY – MALES

Clinical Signs  and  Mortality– Males

Groups

Dose (mg/kg bw/day)

Days

75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

G1

0

Normal

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

G2

30

Normal

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

G3

120

Normal

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

10/10

G4

450

Normal

7/10

7/10

7/10

9/10

7/10

7/10

7/10

7/10

8/10

7/10

8/10

7/10

8/10

5/10

5/10

7/10

10/10

Lethargy

3/10

3/10

3/10

1/10

3/10

3/10

3/10

3/10

2/10

3/10

2/10

3/10

2/10

5/10

5/10

3/10

0/10

Key:  n=10, G1- Vehicle control, G2-Low dose, G3- Mid dose, G4-High dose.

 

TABLE 2 - SUMMARY OF CLINICAL SIGNS AND MORTALITY – FEMALES                                                                                                                                                              

Clinical Signs  and  Mortality – Females
Groups	Dose (mg/kg bw/day)	Mortality/Morbidity  (From day 1 to 91)	Clinical Signs (from day 1 to 91)
			Normal
G1	0	0/10	10/10
G2	30	0/10	10/10
G3	120	0/10	10/10
G4	450	0/10	10/10

 Key: n=10, G1- Vehicle control, G2-Low dose, G3- Mid dose, G4- High dose.

 

TABLE 3 SUMMARY OF DETAILED CLINICAL EXAMINATION – MALES

Home cage observation	Days	Groups
		G1	G2	G3	G4
	Pre dose	3R,4S,3A	4R,3S,3A	2R,2S,6A	5R,2S,3A
Animal body position / Posture   R – Rearing S – Sitting / Standing Normally A – Asleep	8	4R,4S,2A	4R,4S,2A	4R,4S,2A	3R,4S,3A
	15	4R,4S,2A	3R,5S,2A	4R,4S,2A	3R,5S,2A
	22	1R,6S,3A	2R,5S,3A	2R,6S,2A	5R,3S,2A
	29	4R,4S,2A	4R,5S,0A	4R,5S,1A	3R,4S,2A
	36	3R,6S,1A	1R,7S,2A	3R,6S,1A	3R,5S,2A
	43	4R,5S,1A	4R,3S,3A	3R,5S,1A	3R,6S,1A
	50	5R,5S,0A	4R,4S,2A	4R,4S,2A	5R,3S,2A
	57	5R,5S,0A	6R,3S,1A	5R,4S,1A	4R,4S,2A
	64	3R,6S,1A	5R,4S,1A	4R,4S,2A	3R,4S,3A
	73	5R,4S,1A	4R,4S,2A	4R,4S,2A	5R,5S,0A
	78	4R,6S,0A	4R,6S,0A	3R,4S,3A	3R,4S,3A
	85	4R,6S,0A	4R,6S,0A	2R,5S,3A	3R,4S,3A
Respiration Normal	Pre dose,8,15,22,29,36, 43,50,57,64,73,78,85	10/10	10/10	10/10	10/10
Vocalization Normal		10/10	10/10	10/10	10/10
Hand-held observation
Ease of removal Easy/Normal	Pre dose,8,15,22,29,36, 43,50,57,64,73,78,85	10/10	10/10	10/10	10/10
Handling reactivity Easy to handle/ normal		10/10	10/10	10/10	10/10
Palpebral closure Open		10/10	10/10	10/10	10/10
Lacrimation No lacrimation		10/10	10/10	10/10	10/10
Nasal discharge No discharge		10/10	10/10	10/10	10/10
Salivation No salivation		10/10	10/10	10/10	10/10
Teeth Normal		10/10	10/10	10/10	10/10
Fur coat/ Skin Normal		10/10	10/10	10/10	10/10
Muscle tone/ Mass Normal		10/10	10/10	10/10	10/10
Perineum wetness None/normal		10/10	10/10	10/10	10/10
Tail Normal		10/10	10/10	10/10	10/10

                Key: n=10, G1- Vehicle control, G2-Low dose, G3-High dose, G4- High dose.

 

TABLE 3 (CONTD.)  SUMMARY OF DETAILED CLINICAL EXAMINATION - MALES

Open-field observation	Days	Groups
		G1	G2	G3	G4
Gait Head is horizontal, abdomen rises slightly above floor, and body moves up and down slightly during walking	Pre dose,8,15,22,29,36, 43,50,57,64,73,78,85	10/10	10/10	10/10	10/10
Arousal Normal		10/10	10/10	10/10	10/10
Clonic convulsion None/ normal		10/10	10/10	10/10	10/10
Tonic Convulsion None/ normal		10/10	10/10	10/10	10/10
Stereotype None		10/10	10/10	10/10	10/10
Bizarre /Abnormal behaviour(s)  None		10/10	10/10	10/10	10/10
Fecal consistency Formed/ normal		10/10	10/10	10/10	10/10

 Key: n=10, G1- Vehicle control, G2-Low dose, G3-High dose, G4- High dose.

 

TABLE 3 (CONTD.)  SUMMARY OF DETAILED CLINICAL EXAMINATION – MALES

Open- field observation (contd.)                                  Rearing count
		PD	8	15	22	29	36	43	50	57	64	73	78	85
G1	Mean	13.40	15.90	18.50	16.90	16.20	15.60	14.20	11.80	15.80	18.90	19.00	19.00	18.50
	SD	2.07	2.60	1.78	1.79	1.87	1.71	1.48	1.23	1.93	1.91	1.94	1.83	1.58
G2	Mean	13.30	14.90	18.40	15.90	15.90	16.30	13.70	10.50	14.40	18.80	18.80	17.80	18.40
	SD	2.11	2.33	2.01	1.79	1.73	2.16	1.64	1.43	1.43	1.75	1.81	2.74	1.71
G3	Mean	12.90	16.50	18.80	16.50	15.40	16.50	13.70	10.70	14.10	17.90	18.30	17.70	17.60
	SD	2.47	2.95	1.87	1.84	1.71	2.27	1.77	1.06	1.91	1.66	1.89	2.11	1.71
G4	Mean	13.70	16.90	18.20	16.40	15.40	15.90	13.80	11.00	14.90	18.20	19.10	17.70	16.70
	SD	2.36	2.28	1.93	1.78	1.35	1.66	1.69	1.25	2.28	1.81	1.60	2.21	2.00
Urine pools (count)
G1	Mean	4.70	4.40	4.60	4.10	4.60	3.50	2.20	3.00	2.10	3.50	3.30	3.10	2.90
	SD	1.42	1.96	0.97	0.99	1.51	1.08	0.63	0.82	0.74	1.08	0.95	0.74	1.10
G2	Mean	5.90	5.50	4.20	3.30	5.50	3.80	2.60	3.90	2.90	3.40	2.60	2.60	2.80
	SD	1.66	1.08	1.48	1.16	1.58	1.03	0.70	0.74	1.20	1.07	0.70	1.26	0.79
G3	Mean	5.40	4.30	3.50	4.20	5.00	3.30	3.30	3.40	3.00	3.30	2.80	2.90	3.00
	SD	1.43	1.89	1.27	0.92	1.25	1.16	1.16	0.97	1.05	0.95	0.92	0.74	0.82
G4	Mean	4.30	5.10	4.50	4.20	5.20	3.00	3.50	3.50	1.90	3.30	2.20	2.70	2.40
	SD	1.49	1.37	1.18	1.32	1.48	1.05	1.08	0.85	0.74	1.16	1.55	0.82	0.84
Faecal (count)
G1	Mean	2.10	1.90	1.70	0.70	2.20	1.70	1.40	1.30	1.90	1.50	1.60	1.60	1.70
	SD	1.20	1.10	1.42	0.67	0.63	0.95	0.52	0.48	0.74	1.08	1.07	0.70	0.82
G2	Mean	3.50	1.60	1.40	0.90	2.50	1.20	2.00	1.20	1.30	1.70	1.50	1.80	1.70
	SD	1.08	0.97	0.70	0.74	1.08	0.92	0.67	1.03	0.67	0.95	1.08	0.79	0.82
G3	Mean	3.00	1.70	1.50	1.20	1.90	1.40	1.50	1.00	1.70	1.40	1.50	1.80	1.70
	SD	1.25	0.95	0.53	0.79	0.99	0.70	0.85	0.82	0.95	0.97	1.08	0.79	0.82
G4	Mean	2.80	1.90	1.40	1.20	2.00	0.80	1.20	1.10	1.20	1.70	0.30	1.60	1.60
	SD	1.03	1.10	0.70	1.03	1.15	0.63	0.63	0.99	0.63	0.95	0.48	0.84	0.70

Key: n=10, G1- Vehicle control, G2-Low dose, G3-High dose, G4- High dose.

 

 

TABLE 4 SUMMARY OF DETAILED CLINICAL EXAMINATION – FEMALES

Home cage observation	Days	Groups
		G1	G2	G3	G4
	Pre dose	2R,5S,3A	2R,3S,5A	5R,2S,3A	2R,6S,2A
Animal body position / Posture   R – Rearing S – Sitting / Standing Normally A – Asleep	8	5R,3S,2A	4R,5S,1A	3R,4S,3A	3R,5S,2A
	15	3R,5S,2A	4R,4S,2A	3R,5S,2A	3R,4S,2A
	22	3R,5S,2A	3R,5S,2A	3R,4S,3A	3R,6S,1A
	29	3R,6S,1A	3R,5S,2A	3R,6S,1A	4R,5S,1A
	36	6R,4S,0A	4R,5S,1A	3R,5S,2A	2R,7S,1A
	43	5R,3S,2A	3R,4S,3A	4R,4S,2A	4R,5S,1A
	50	4R,4S,2A	3R,4S,3A	3R,4S,3A	3R,5S,2A
	57	3R,5S,2A	3R,5S,2A	3R,6S,1A	4R,4S,2A
	64	4R,5S,1A	3R,5S,2A	4R,5S,1A	3R,4S,3A
	73	6R,3S,1A	6R,3S,1A	5R,2S,3A	5R,3S,2A
	78	4R,6S,0A	3R,6S,1A	3R,5S,2A	3R,4S,3A
	85	4R,5S,1A	4R,5S,1A	3R,5S,2A	3R,4S,3A
Respiration Normal	Pre dose,8,15,22,29,36, 43,50,57,64,73,78,85	10/10	10/10	10/10	10/10
Vocalization Normal		10/10	10/10	10/10	10/10
Hand-held observation
Ease of removal Easy/Normal	Pre dose,8,15,22,29,36, 43,50,57,64,73,78,85	10/10	10/10	10/10	10/10
Handling reactivity Easy to handle/ normal		10/10	10/10	10/10	10/10
Palpebral closure Open		10/10	10/10	10/10	10/10
Lacrimation No lacrimation		10/10	10/10	10/10	10/10
Nasal discharge No discharge		10/10	10/10	10/10	10/10
Salivation No salivation		10/10	10/10	10/10	10/10
Teeth Normal		10/10	10/10	10/10	10/10
Fur coat/ Skin Normal		10/10	10/10	10/10	10/10
Muscle tone/ Mass Normal		10/10	10/10	10/10	10/10
Perineum wetness None/normal		10/10	10/10	10/10	10/10
Tail Normal		10/10	10/10	10/10	10/10

 Key: n=10, G1- Vehicle control, G2-Low dose, G3- Mid dose, G4-High dose.

 

 

TABLE 4 (CONTD.) - SUMMARY OF DETAILED CLINICAL EXAMINATION – FEMALES

Open-field observation	Days	Groups
		G1	G2	G3	G4
Gait Head is horizontal, abdomen rises slightly above floor, and body moves up and down slightly during walking	Pre dose,8,15,22,29,36, 43,50,57,64,73,78,85	10/10	10/10	10/10	10/10
Arousal Normal		10/10	10/10	10/10	10/10
Clonic convulsion None/ normal		10/10	10/10	10/10	10/10
Tonic Convulsion None/ normal		10/10	10/10	10/10	10/10
Stereotype None		10/10	10/10	10/10	10/10
Bizarre /Abnormal behaviour(s)  None		10/10	10/10	10/10	10/10
Fecal consistency Formed/ normal		10/10	10/10	10/10	10/10

Key: n=10, G1- Vehicle control, G2-Low dose, G3- Mid dose, G4-High dose.

 

 

 

TABLE 4 (CONTD.) - SUMMARY OF DETAILED CLINICAL EXAMINATION – FEMALES

Open- field observation (contd.)                                  Rearing count
		PD	8	15	22	29	36	43	50	57	64	73	78	85
G1	Mean	18.80	19.30	19.70	20.50	15.40	17.20	17.40	18.00	19.40	19.40	17.90	18.40	18.70
	SD	2.39	2.21	1.49	1.58	1.51	1.48	2.22	1.83	2.01	1.51	2.28	1.90	1.49
G2	Mean	19.00	18.50	19.80	20.30	15.60	17.40	17.10	18.20	18.30	18.80	16.60	18.30	16.90
	SD	2.26	1.78	1.87	2.00	1.78	1.26	1.66	2.04	1.89	1.32	2.22	1.49	2.13
G3	Mean	18.80	18.20	18.70	19.80	15.80	17.40	17.20	17.40	18.00	17.60	17.20	18.30	16.00
	SD	2.39	1.69	2.21	1.99	1.55	1.26	1.69	2.07	2.16	2.07	2.86	1.89	2.62
G4	Mean	20.00	18.50	19.30	20.00	16.50	17.00	15.70	17.90	18.50	20.60	16.80	17.70	15.80
	SD	2.45	2.22	1.77	2.45	1.58	1.15	2.06	2.18	2.42	1.96	2.74	2.21	2.53
Urine pools (count)
G1	Mean	5.60	3.60	3.50	3.50	4.70	5.10	4.80	3.30	3.40	3.40	1.90	2.80	2.70
	SD	1.51	1.43	1.27	1.27	1.25	0.99	1.14	1.16	1.17	1.07	1.37	0.79	0.95
G2	Mean	5.50	4.30	3.20	3.10	4.70	5.80	4.20	3.20	3.30	3.20	1.80	3.10	2.70
	SD	1.58	1.49	1.03	0.99	1.34	1.32	0.79	1.03	1.16	1.03	1.62	0.74	0.95
G3	Mean	5.40	4.60	3.70	3.10	5.20	5.50	3.70	3.30	3.10	3.40	2.00	2.90	2.50
	SD	1.26	1.26	1.16	0.99	1.32	1.08	0.95	0.95	1.20	0.97	1.33	0.88	0.97
G4	Mean	5.70	4.40	3.60	3.30	4.70	5.60	3.90	3.00	3.20	3.30	2.40	3.10	2.30
	SD	0.95	1.84	1.43	1.16	1.57	0.97	0.74	1.05	1.23	0.95	1.35	0.99	1.06
Faecal (count)
G1	Mean	2.00	1.60	1.30	1.20	2.20	1.70	2.20	1.80	1.60	1.30	0.50	1.70	1.70
	SD	1.05	1.26	0.95	0.79	1.14	1.16	0.63	1.03	1.07	1.16	0.53	0.82	0.82
G2	Mean	2.10	1.40	1.40	1.00	2.00	1.90	1.60	1.50	1.50	1.50	0.80	1.50	1.70
	SD	0.99	0.70	0.84	0.82	0.94	0.99	1.07	0.85	1.08	1.08	0.42	0.71	0.82
G3	Mean	1.80	1.80	1.80	1.30	2.40	2.00	1.50	1.20	1.50	1.50	0.40	1.50	1.70
	SD	0.92	1.23	1.32	0.95	0.97	0.94	1.35	1.03	1.08	0.97	0.52	0.71	0.82
G4	Mean	1.60	1.40	1.20	1.20	2.20	1.80	1.30	1.00	1.50	1.40	0.30	1.70	1.60
	SD	0.97	0.84	0.92	1.03	0.92	1.14	0.82	0.82	1.08	0.97	0.48	0.82	0.70

Key: n=10, G1- Vehicle control, G2-Low dose, G3-High dose, G4- High dose.

 

 

TABLE 5 - SUMMARY OF OPHTHALMOLOGICAL EXAMINATION – MALES

Ophthalmological Examination – Males
Groups	Dose (mg/kg bw/day)	Observations	Left Eye	Right Eye
G1	0	NAD	10/10	10/10
G4	450	NAD	10/10	10/10

Key: n= 10; G1 = Vehicle Control; G4 = High dose; NAD = No Abnormality Detected.

 

Note: No abnormalities were detected in both eyes, before treatment in any of the Males.

 

TABLE 6 - SUMMARY OF OPHTHALMOLOGICAL EXAMINATION – FEMALES

Ophthalmological Examination – Females
Groups	Dose (mg/kg bw/day)	Observations	Left Eye	Right Eye
G1	0	NAD	10/10	10/10
G4	450	NAD	10/10	10/10

Key: n= 10; G1 = Vehicle Control; G4 = High dose; NAD = No Abnormality Detected.

 

Note: No abnormalities were detected in both eyes, before treatment in any of the Females.

 

TABLE 7 - SUMMARY OF BODY WEIGHTS – MALES

	Body Weight (g) – Males
	Group / Dose (mg/kg bw/day)	Day	01	08	15	22	29	36	43	50	57	64	71	78	85	90
	G1	Mean	201.23	258.68	310.81	365.04	400.69	435.52	462.75	491.14	518.31	535.65	551.98	563.66	580.22	591.49
0		SD	13.11	12.96	19.21	19.24	23.36	27.16	30.39	35.67	36.31	41.92	42.52	44.86	45.22	48.70
		N	10	10	10	10	10	10	10	10	10	10	10	10	10	10
	G2	Mean	203.92	258.25	314.15	361.35	392.18	422.77	448.09	473.42	493.98	516.29	529.60	538.90	555.34	562.42
30		SD	16.63	18.24	20.36	21.23	22.79	27.38	29.21	31.32	35.16	39.55	42.46	44.07	46.99	45.03
		N	10	10	10	10	10	10	10	10	10	10	10	10	10	10
	G3	Mean	202.33	259.06	311.80	360.67	391.46	412.49	450.47	475.55	495.45	515.56	531.12	542.37	557.96	564.26
120		SD	16.15	16.16	15.60	14.49	14.86	14.88	13.37	14.19	19.30	19.87	19.77	21.42	23.27	23.18
		N	10	10	10	10	10	10	10	10	10	10	10	10	10	10
G4		Mean	200.83	252.16	307.69	352.70	385.34	414.00	437.23	463.14	480.98*	499.31*	517.26	530.72	546.45	548.53
450		SD	12.68	13.88	13.84	12.55	12.45	15.26	18.39	20.35	22.45	24.48	29.58	33.85	35.91	35.54
		N	10	10	10	10	10	10	10	10	10	10	10	10	10	10

        Key: n=10, G1- Vehicle control, G2-Low dose, G3-High dose, G4- High dose, *= Statistically significant from vehicle control (P ≤ 0.05).

 

 

TABLE 8 – SUMMARY OF BODY WEIGHTS – FEMALES                                                                                                                                                                               

Body Weight (g) – Females
Group / Dose (mg/kg bw/day)	Day	01	08	15	22	29	36	43	50	57	64	71	78	85	90
G1	Mean	151.38	176.02	203.34	224.05	236.25	247.75	258.18	262.65	270.05	279.98	285.13	287.59	293.66	296.10
	SD	9.13	7.88	7.89	11.49	13.33	15.37	16.19	18.43	16.50	17.48	18.06	17.80	17.50	16.25
0	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10
G2	Mean	151.96	175.96	204.78	223.09	240.55	249.72	259.19	275.31	279.26	284.63	290.14	292.68	300.11	302.38
	SD	8.89	8.60	8.90	13.63	14.42	14.85	16.09	17.28	17.52	17.16	17.93	19.79	20.77	21.85
30	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10
G3	Mean	153.50	176.22	204.20	223.60	241.12	250.10	260.61	270.37	275.44	283.68	287.84	291.54	298.53	300.89
	SD	8.54	16.87	20.75	23.78	25.61	28.38	23.73	22.25	24.84	27.76	26.52	26.98	25.98	25.74
120	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10
G4	Mean	152.86	173.02	201.69	217.44	230.99	246.44	256.28	271.01	276.97	287.43	290.83	294.04	303.97	305.99
	SD	8.10	14.13	11.88	9.88	12.56	13.50	13.30	13.49	16.37	19.02	20.34	21.05	20.37	22.18
450	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10

            Key: n=10, G1- Vehicle control, G2-Low dose, G3-High dose, G4- High dose.

 

   

TABLE 9 - SUMMARY OF NET BODY WEIGHT GAIN – MALES

Net Body Weight Gain (g) - Males
Group / Dose (mg/kg bw/day)	From: To	01-08	08-15	15-22	22-29	29-36	36-43	43-50	50-57	57-64	64-71	71-78	78-85	85-90	1-90	% Gain
G1	Mean	57.45	52.12	54.24	35.65	34.82	27.23	28.39	27.17	17.34	16.33	11.68	16.56	11.27	390.26	194.93
	SD	6.09	16.33	9.55	6.28	5.49	5.69	6.73	3.65	8.69	4.84	5.32	3.36	5.96	49.62	29.84
0	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
G2	Mean	54.33	55.90	47.20	30.84	30.59	25.32	25.33	20.56	22.32	13.30	9.31	16.44	7.08	358.50	177.19
	SD	5.69	7.09	6.77	6.69	6.88	7.56	6.65	5.27	16.76	6.50	4.44	4.22	4.13	45.32	29.26
30	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
G3	Mean	56.74	52.73	48.88	30.79	33.59	25.43	25.08	19.91	20.11	15.56	11.24	15.60	6.30	361.94	180.23
	SD	7.24	5.82	3.62	3.79	2.75	5.56	2.82	8.26	4.88	4.46	4.57	3.47	5.00	23.80	21.62
120	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
G4	Mean	51.33	55.54	45.01*	32.65	28.66*	23.23	25.91	17.84*	18.33	17.95	13.46	15.73	2.08*	347.70	174.54
	SD	5.19	16.06	7.78	6.12	4.56	6.64	4.09	14.79	6.78	7.47	5.26	5.86	3.93	42.49	29.60
450	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10

           Key: n=10, G1- Vehicle control, G2-Low dose, G3-High dose, G4- High dose, *= Statistically significant from vehicle control (P ≤ 0.05).

 

TABLE 10 - SUMMARY OF NET BODY WEIGHT GAIN – FEMALES

Net Body Weight Gain (g) - Females
Group / Dose (mg/kg bw/day)	From: To	01-08	08-15	15-22	22-29	29-36	36-43	43-50	50-57	57-64	64-71	71-78	78-85	85-90	1-90	% Gain
G1	Mean	24.64	27.32	20.71	12.19	11.50	10.43	4.47	7.40	9.92	5.16	2.46	6.07	2.45	144.73	96.11
	SD	8.64	7.17	5.39	4.46	6.92	5.51	5.96	6.38	4.75	4.18	4.10	3.76	3.90	16.60	13.96
0	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
G2	Mean	24.00	28.82	18.30	17.47	9.17	9.47	16.12*	3.94	5.37	5.51	2.54	7.42	2.27	150.42	99.18
	SD	6.85	7.88	10.42	6.81	4.41	8.71	2.44	5.16	5.75	7.31	5.21	3.24	4.45	18.52	12.63
30	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
G3	Mean	22.71	27.99	19.40	17.52	8.98	10.51	9.75	5.07	8.24	4.16	3.70	7.00	2.36	147.39	95.89
	SD	11.64	9.96	4.28	6.29	9.25	6.39	10.06	7.62	6.75	5.43	4.67	3.21	3.92	19.85	10.83
120	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
G4	Mean	20.16	28.67	15.75	13.56	15.45	9.84	14.73*	5.96	10.47	3.40	3.21	9.93*	2.02	153.13	100.36
	SD	9.31	6.10	7.12	7.51	7.75	6.63	10.33	5.54	10.74	8.79	5.06	3.61	5.88	19.48	13.06
450	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10

           Key: n=10, G1- Vehicle control, G2-Low dose, G3-High dose, G4- High dose, *= Statistically significant from vehicle control (P ≤ 0.05).

 

 

TABLE 11 - SUMMARY OF AVERAGE FOOD INTAKE – MALES

Average Feed Consumption (g/Rat/day) - Males
Groups Dose (mg/kg bw/day)	Days
	From: To	01-08	08-15	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	28.79	30.58	33.78	30.77	31.49	31.24	32.37	32.10	31.18	32.26	32.49	31.98	34.35
	SD	0.80	1.94	1.36	1.69	1.12	1.39	1.15	0.82	0.65	1.29	1.22	1.05	1.87
	N	10	10	10	10	10	10	10	10	10	10	10	10	10
G2 30	Mean	29.11	31.36	34.11	31.31	30.87	30.90	31.47	31.56	30.56	32.00	31.68	31.29	33.48
	SD	0.85	1.13	0.59	1.03	0.85	0.82	1.08	1.19	0.72	0.77	1.34	0.93	1.88
	N	10	10	10	10	10	10	10	10	10	10	10	10	10
G3 120	Mean	28.80	29.64	32.86	30.22	31.05	30.83	31.63	31.22	31.40	30.88	32.57	31.74	32.82
	SD	1.08	1.01	0.72	0.78	1.02	1.03	1.21	1.49	0.38	1.08	1.31	0.76	1.31
	N	10	10	10	10	10	10	10	10	10	10	10	10	10
G4 450	Mean	29.05	31.00	33.91	31.07	30.84	30.52	31.88	31.84	30.26	33.74	34.12	31.45	31.41*
	SD	0.70	1.18	1.06	1.11	1.41	1.47	1.35	1.55	0.43	1.32	1.22	1.05	0.73
	N	10	10	10	10	10	10	10	10	10	10	10	10	10

       Key: n=10, G1- Vehicle control, G2-Low dose, G3-High dose, G4- High dose, *= Statistically significant from vehicle control (P ≤ 0.05).

 

 

TABLE 12 - SUMMARY OF AVERAGE FOOD INTAKE – FEMALES                                                                                                 

Average Feed Consumption (g/Rat/day) - Females
Groups Dose (mg/kg bw/day)	Days
	From: To	01-08	08-15	15-22	22-29	29-36	36-43	43-50	50-57	57-64	64-71	71-78	78-85	85-90
G1	Mean	20.23	20.23	21.23	19.75	19.94	19.55	20.40	20.25	20.80	20.73	21.15	20.88	25.85
	SD	0.70	2.62	0.86	1.27	1.69	1.08	0.97	0.61	0.71	1.14	1.11	0.84	0.99
	N	10	10	10	10	10	10	10	10	10	10	10	10	10
G2	Mean	19.72	20.98	20.95	20.82	20.07	19.35	21.11	19.07	20.39	21.56	21.65	21.08	25.72
	SD	1.42	1.47	2.23	2.53	0.98	1.07	0.92	1.17	0.63	3.24	3.07	0.93	3.15
	N	10	10	10	10	10	10	10	10	10	10	10	10	10
G3	Mean	20.52	21.17	21.81	21.55	20.16	20.00	20.90	19.93	21.05	21.94	20.38	21.35	25.32
	SD	1.66	2.09	1.38	2.27	0.35	0.71	0.70	1.52	1.07	2.70	1.07	0.84	1.19
	N	10	10	10	10	10	10	10	10	10	10	10	10	10
G4	Mean	19.67	20.95	21.37	20.67	20.50	21.12	22.87*	21.66	21.03	22.14	21.30	20.86	24.00
	SD	1.28	0.84	0.44	1.36	0.58	0.97	1.13	0.78	0.79	0.73	0.38	0.55	1.93
	N	10	10	10	10	10	10	10	10	10	10	10	10	10

        Key: n=10, G1- Vehicle control, G2-Low dose, G3-High dose, G4- High dose. *= Statistically significant from vehicle control (P ≤ 0.05).

 

TABLE 13 - SUMMARY OF FUNCTIONAL OBSERVATION BATTERY (MALES)

Parameter		Groups
		G1	G2	G3	G4
Home cage observations
Posture -  (R-Rearing S-Sitting)		8R 2S	6R 4S	8R 2S	6R 4S
Respiration - Normal		10/10	10/10	10/10	10/10
Clonic involuntary movement - None/normal		10/10	10/10	10/10	10/10
Tonic involuntary movement - None/normal		10/10	10/10	10/10	10/10
Vocalization - None/normal		10/10	10/10	10/10	10/10
Convulsions - Absent - N		10/10	10/10	10/10	10/10
Hand-held observations
Reactivity - Easy/ normal (animal does not resist)		10/10	10/10	10/10	10/10
Handling - Squeaks or does not squeak but exhibit mild resistance; easy to handle/ normal		10/10	10/10	10/10	10/10
Palpebral closure - Open		10/10	10/10	10/10	10/10
Lacrimation - No lacrimation/ normal		10/10	10/10	10/10	10/10
Salivation - No salivation/ normal		10/10	10/10	10/10	10/10
Piloerection - None/ normal		10/10	10/10	10/10	10/10
Perineum Wetness - Absence of wetness		10/10	10/10	10/10	10/10
Body Temperature (ºC)	Mean	37.89	37.59	37.52	37.71
	SD	0.40	0.49	0.50	0.46

    Key: n=10, G1- Vehicle control, G2-Low dose, G3-High dose, G4- High dose.

 

TABLE 13 (CONDT.) - SUMMARY OF FUNCTIONAL OBSERVATION BATTERY (MALES)

Parameter		Groups
		G1	G2	G3	G4
Open field activity
Clonic involuntary movement - None/ Normal		10/10	10/10	10/10	10/10
Tonic involuntary movement - None/ Normal		10/10	10/10	10/10	10/10
Gait - Head is horizontal, abdomen rises slightly above floor, and body moves up and down slightly during walking		10/10	10/10	10/10	10/10
Movement - Normal		10/10	10/10	10/10	10/10
Arousal - Normal (keeps guard up and engages in exploratory activity)		10/10	10/10	10/10	10/10
Tremors - None		10/10	10/10	10/10	10/10
Occurrence of stereotype - None		10/10	10/10	10/10	10/10
Abnormal behavior - None		10/10	10/10	10/10	10/10
Number of defecation	Mean	1.20	1.80	1.60	1.50
	SD	0.98	0.98	0.92	0.81
Number of Urination	Mean	2.50	3.20	2.80	2.70
	SD	1.02	1.08	1.25	0.90
Number of Rearing	Mean	19.90	20.40	21.40	21.20
	SD	3.05	1.50	1.36	1.54
Sensory Reactivity Measurements
Approach Response - Slowly approaches, sniffs and pulls back/ normal		10/10	10/10	10/10	10/10
Touch Response - Slowly approaches, sniffs and pulls back/ normal		10/10	10/10	10/10	10/10
Eyelid reflex - Blink/ Normal		10/10	10/10	10/10	10/10
Pinna reflex - Auricle twitches/ normal		10/10	10/10	10/10	10/10

    Key: n=10, G1- Vehicle control, G2-Low dose, G3-High dose, G4- High dose.

TABLE 13 (CONDT.) - SUMMARY OF FUNCTIONAL OBSERVATION BATTERY (MALES)

Sensory Reactivity Measurements Condt.
Sound response - Mild reaction, hears sound/normal			10/10	10/10	10/10	10/10
Tail pinch response - Looks back, moves forward and lightly squeaks/normal			10/10	10/10	10/10	10/10
Pupillary reflex - Contracts			10/10	10/10	10/10	10/10
Aerial righting reflex - Lands on four limbs/ normal			10/10	10/10	10/10	10/10
Tail limb reflex - Present			10/10	10/10	10/10	10/10
Nervous and muscle measurement
Abdominal tone - Normal (proper hardness)			10/10	10/10	10/10	10/10
Limb tone - Normal (proper hardness)			10/10	10/10	10/10	10/10
Locomotor activity using actimeter	X	Mean	566.10	556.70	560.90	567.20
		SD	85.12	47.14	52.65	49.95
	Y	Mean	505.20	508.70	513.60	526.90
		SD	56.89	33.42	39.72	25.09
	Z	Mean	553.70	552.40	566.60	556.50
		SD	41.18	54.22	23.75	51.97
	Total	Mean	1625.00	1617.80	1641.10	1650.60
		SD	175.61	109.93	107.29	104.34
Grip Strength (gf)	Fore limb	Mean	759.00	773.27	781.77	774.97
		SD	57.51	49.84	30.76	20.39
	Hind limb	Mean	486.13	487.53	491.73	488.37
		SD	8.30	10.73	3.28	3.51
Landing hind limb foot splay (mm)		Mean	75.90	59.47	64.63	62.93
		SD	12.07	15.48	13.12	16.00

Key: n=10, G1- Vehicle control, G2-Low dose, G3-High dose, G4 - High dose.

TABLE  14 - SUMMARY OF FUNCTIONAL OBSERVATION BATTERY (FEMALES)

Parameter		Groups
		G1	G2	G3	G4
Home cage observations
Posture -  (R-Rearing S-Sitting)		8R 2S	6R 4S	8R 2S	6R 4S
Respiration - Normal		10/10	10/10	10/10	10/10
Clonic involuntary movement - None/normal		10/10	10/10	10/10	10/10
Tonic involuntary movement - None/normal		10/10	10/10	10/10	10/10
Vocalization - None/normal		10/10	10/10	10/10	10/10
Convulsions - Absent - N		10/10	10/10	10/10	10/10
Hand-held observations
Reactivity - Easy/ normal (animal does not resist)		10/10	10/10	10/10	10/10
Handling - Squeaks or does not squeak but exhibit mild resistance; easy to handle/ normal		10/10	10/10	10/10	10/10
Palpebral closure - Open		10/10	10/10	10/10	10/10
Lacrimation - No lacrimation/ normal		10/10	10/10	10/10	10/10
Salivation - No salivation/ normal		10/10	10/10	10/10	10/10
Piloerection - None/ normal		10/10	10/10	10/10	10/10
Perineum Wetness - Absence of wetness		10/10	10/10	10/10	10/10
Body Temperature (ºC)	Mean	37.87	38.00	37.97	38.14
	SD	0.30	0.51	0.50	0.42

Key: n=10, G1- Vehicle control, G2-Low dose, G3-High dose, G4- High dose.

 

TABLE 14 (CONDT.) - SUMMARY OF FUNCTIONAL OBSERVATION BATTERY (FEMALES)

Parameter		Groups
		G1	G2	G3	G4
Open field activity
Clonic involuntary movement - None/ Normal		10/10	10/10	10/10	10/10
Tonic involuntary movement - None/ Normal		10/10	10/10	10/10	10/10
Gait - Head is horizontal, abdomen rises slightly above floor, and body moves up and down slightly during walking		10/10	10/10	10/10	10/10
Movement - Normal		10/10	10/10	10/10	10/10
Arousal - Normal (keeps guard up and engages in exploratory activity)		10/10	10/10	10/10	10/10
Tremors - None		10/10	10/10	10/10	10/10
Occurrence of stereotype - None		10/10	10/10	10/10	10/10
Abnormal behavior - None		10/10	10/10	10/10	10/10
Number of defecation	Mean	1.60	1.80	1.40	3.00
	SD	0.92	0.75	1.02	0.77
Number of Urination	Mean	2.50	2.10	3.00	2.20
	SD	1.02	1.30	0.77	1.17
Number of Rearing	Mean	21.70	21.30	21.20	21.30
	SD	1.49	1.55	1.83	1.55
Sensory Reactivity Measurements
Approach Response - Slowly approaches, sniffs and pulls back/ normal		10/10	10/10	10/10	10/10
Touch Response - Slowly approaches, sniffs and pulls back/ normal		10/10	10/10	10/10	10/10
Eyelid reflex - Blink/ Normal		10/10	10/10	10/10	10/10
Pinna reflex - Auricle twitches/ normal		10/10	10/10	10/10	10/10

Key: n=10, G1- Vehicle control, G2-Low dose, G3-High dose, G4- High dose.

TABLE 14 (CONDT.) - SUMMARY OF FUNCTIONAL OBSERVATION BATTERY (FEMALES)

Sound response - Mild reaction, hears sound/normal			10/10	10/10	10/10	10/10
Tail pinch response - Looks back, moves forward and lightly squeaks/normal			10/10	10/10	10/10	10/10
Pupillary reflex - Contracts			10/10	10/10	10/10	10/10
Aerial righting reflex - Lands on four limbs/ normal			10/10	10/10	10/10	10/10
Tail limb reflex - Present			10/10	10/10	10/10	10/10
Nervous and muscle measurement
Abdominal tone - Normal (proper hardness)			10/10	10/10	10/10	10/10
Limb tone - Normal (proper hardness)			10/10	10/10	10/10	10/10
Locomotor activity using actimeter	X	Mean	596.50	602.00	587.80	592.50
		SD	44.36	39.32	16.66	16.60
	Y	Mean	562.70	561.10	560.20	566.10
		SD	30.50	26.18	25.91	10.88
	Z	Mean	582.50	582.90	584.20	587.50
		SD	33.25	28.69	13.88	19.22
	Total	Mean	1741.70	1746.00	1742.20	1746.10
		SD	105.81	88.54	49.13	44.33
Grip Strength (gf)	Fore limb	Mean	708.90	702.60	699.97	712.17
		SD	30.16	34.51	34.21	23.75
	Hind limb	Mean	372.67	382.80	375.77	378.30
		SD	10.78	10.74	11.67	12.78
Landing hind limb foot splay (mm)		Mean	60.93	43.30*	64.43	46.97*
		SD	11.61	9.07	6.47	11.40

Key: n=10, G1- Vehicle control, G2-Low dose, G3-High dose, G4- High dose, *= Statistically significant from vehicle control (P ≤ 0.05).

 

TABLE 15 - SUMMARY OF ESTRUS CYCLE

Stages of Oestrus Cycle
Oestrus Cycle  Stage	G1	G2	G3	G4
P	0	1	1	1
P/E	2	0	1	1
E	2	2	1	2
E/M	0	0	2	1
M	2	2	2	2
M/D	1	2	0	1
D	2	2	2	1
D/P	1	1	1	1

Key: P = Proestrus; E= Estrous; M = Metestrus; D = Diestrus; Transitional stages – P/E, E/M, M/D and D/P.

 G1 = Vehicle Control; G2 = Low dose; G3 = High dose, n=10.

 

Table 16 - Summary of THYROID HORMONE ANALYSIS – MALES                                                                                                                                                 

Sex: Male		Parameters
Groups Dose(mg/kg bw/day)		T3	T4	TSH
		ng/mL	pg/mL	pg/mL
G1 0	Mean	1231.01	24.83	551.49
	SD	100.57	7.73	170.97
	N	10	10	10
G2 30	Mean	1409.62 *	26.46	559.41
	SD	148.55	5.45	160.81
	N	10	10	10
G3 120	Mean	1434.39 *	26.23	489.45
	SD	202.50	5.19	198.36
	N	10	10	10
G4 450	Mean	1419.42 *	26.47	583.93
	SD	140.31	2.58	263.58
	N	10	10	10

Key: n=10, G1 = Vehicle Control; G2 = Low dose; G3= Mid dose, G4 = High dose; *= Statistically significant from vehicle control (P ≤ 0.05).

 

 

Table 17 - Summary of THYROID HORMONE ANALYSIS – FEMALES

Sex: Female		Parameters
Groups Dose(mg/kg bw/day)		T3	T4	TSH
		ng/mL	pg/mL	pg/mL
G1 0	Mean	1139.28	35.06	500.20
	SD	120.73	6.28	119.13
	N	10	10	10
G2 30	Mean	1067.56	42.17	616.84
	SD	154.46	4.55	161.29
	N	10	10	10
G3 120	Mean	1043.01	47.92 *	733.41 *
	SD	121.78	3.34	146.62
	N	10	10	10
G4 450	Mean	1162.13	40.06	755.83*
	SD	191.52	7.68	203.37
	N	10	10	10

Key: n=10, G1 = Vehicle Control; G2 = Low dose; G3= Mid dose, G4 = High dose; *= Statistically significant from vehicle control (P ≤ 0.05).

CLINICAL PATHOLOGY DATA:

TABLE 1 - SUMMARY OF HEMATOLOGICAL PARAMETERS– MALES         

Group Dose (mg/kg/day)		Hematological Parameters– Males
		WBC	RBC	Hb	HCT	PLT	MCV	MCH	MCHC	NEU	LYM	EOS	MON	BAS	Retic
		103 cells/ mm3	106/mm3	g/dl	%	103 cells/ mm3	µm3	pg	g/dl	%	%	%	%	%	%
G1 0	Mean	8.74	9.41	16.22	48.03	876.90	51.20	17.28	33.79	28.70	68.90	2.10	0.30	0.00	26.90
	SD	1.88	0.59	0.48	1.78	75.58	1.93	0.81	0.52	5.64	5.86	0.57	0.48	0.00	2.47
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10
G2 30	Mean	7.00	9.44	16.29	48.33	914.70	51.30	17.23	33.68	31.10	66.80	1.80	0.30	0.00	27.50
	SD	2.14	0.36	0.81	2.48	65.27	1.95	0.63	0.34	3.60	3.55	0.42	0.48	0.00	3.06
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10
G3 120	Mean	7.25	9.39	16.47	49.04	909.30	52.20	17.55	33.56	30.20	67.00	2.20	0.60	0.00	24.80
	SD	1.23	0.29	0.44	1.00	54.16	1.62	0.57	0.65	5.29	5.29	0.63	0.70	0.00	5.05
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10
G4 450	Mean	6.85*	9.15	16.45	47.84	959.00	52.20	18.00	34.39*	31.20	66.80	2.00	0.00	0.00	28.80
	SD	1.40	0.34	0.62	1.57	112.15	1.40	0.74	0.47	5.59	5.87	0.94	0.00	0.00	4.66
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10

  Key:  *= Statistically significant from vehicle control (P ≤ 0.05); G1=Vehicle control; G2=low dose; G3=mid dose; G4=high dose.

TABLE 2 - SUMMARY OF HEMATOLOGICAL PARAMETERS– FEMALES

Group Dose (mg/kg/day)		Hematological Parameters– Females
		WBC	RBC	Hb	HCT	PLT	MCV	MCH	MCHC	NEU	LYM	EOS	MON	BAS	Retic
		103 cells/ mm3	106/mm3	g/dl	%	103 cells/ mm3	µm3	pg	g/dl	%	%	%	%	%	%
G1 0	Mean	5.58	8.43	15.67	45.52	875.70	54.00	18.61	34.47	21.80	76.30	1.40	0.50	0.00	25.20
	SD	0.70	0.21	0.45	1.24	59.09	1.25	0.87	1.50	6.03	6.75	0.70	0.53	0.00	1.69
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10
G2 30	Mean	4.86	8.39	15.70	45.25	872.30	54.00	18.73	34.74	22.80	75.20	1.70	0.30	0.00	26.30
	SD	1.17	0.52	1.03	3.16	76.91	1.76	0.62	0.59	5.27	5.37	0.82	0.48	0.00	4.22
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10
G3 120	Mean	4.85	7.98*	16.05	44.69	939.40	55.90	20.14*	35.94	22.90	75.10	1.60	0.40	0.00	25.70
	SD	0.98	0.33	0.39	1.01	131.87	1.66	0.65	0.41	2.69	2.81	0.97	0.52	0.00	3.92
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10
G4 450	Mean	5.25	7.73*	15.90	43.39	1040.80*	56.00*	23.55*	36.63*	25.80	71.70	1.60	0.90	0.00	28.20
	SD	1.50	0.39	0.69	2.22	116.74	1.56	9.59	0.52	5.49	5.58	0.52	0.74	0.00	2.86
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10

Key:  *= Statistically significant from vehicle control (P ≤ 0.05); G1=Vehicle control; G2=low dose; G3=mid dose; G4=high dose.

 

TABLE 3 - SUMMARY OF COAGULATION PARAMTERS - MALES                                                                                                              

Groups Dose (mg/kg/day)		Coagulation Parameters- Males
		PT	APTT
		Seconds	Seconds
G1 0	Mean	13.22	19.51
	SD	2.38	1.99
	N	10	10
G2 30	Mean	13.12	19.75
	SD	2.00	5.03
	N	10	10
G3 120	Mean	13.02	19.54
	SD	1.18	3.24
	N	10	10
G4 450	Mean	12.25	18.75
	SD	0.89	2.43
	N	10	10

                    Key:  G1=Vehicle control; G2=low dose; G3=mid dose; G4=high dose.

 

TABLE 4 - SUMMARY OF COAGULATION PARAMTERS – FEMALES                                                                                 

Groups Dose (mg/kg/day)		Coagulation Parameters- Females
		PT	APTT
		Seconds	Seconds
G1 0	Mean	13.87	21.96
	SD	3.10	3.66
	N	10	10
G2 30	Mean	12.81	19.15
	SD	1.17	1.38
	N	10	10
G3 120	Mean	12.86	22.77
	SD	2.06	5.14
	N	10	10
G4 450	Mean	12.74	25.60
	SD	0.96	4.37
	N	10	10

                        Key:  G1=Vehicle control; G2=low dose; G3=mid dose; G4=high dose.

 

TABLE 5- SUMMARY OF CLINICAL CHEMISTRY PARAMETERS – MALES

Groups Dose (mg/kg/day)		Clinical Chemistry Parameters – Males
		GLU	UREA	CRE	TCHO	TRIG	AST	ALT	TP	HDL	LDL	ALB
		mg/dL	mg/dL	mg/dL	mg/dL	U/L	U/L	g/L	g/L	mg/dL	g/L	g/L
G1 0	Mean	128.42	29.85	0.54	77.55	107.37	163.50	80.81	65.93	12.35	7.68	42.06
	SD	8.91	3.47	0.05	17.87	26.34	54.72	41.76	2.01	1.95	3.08	1.04
	N	10	10	10	10	10	10	10	10	10	10	10
G2 30	Mean	113.02*	32.29	0.49*	58.55	85.77	146.56	60.60	71.98	15.48*	12.02	38.88
	SD	9.45	3.37	0.04	7.44	21.77	18.84	10.91	23.89	18.17	18.54	9.77
	N	10	10	10	10	10	10	10	10	10	10	10
G3 120	Mean	109.56*	33.39	0.51	57.81*	124.93	139.91	75.09	68.23	11.42	5.75	44.63*
	SD	8.07	4.83	0.04	20.20	36.08	45.67	31.28	2.85	0.95	1.83	1.59
	N	10	10	10	10	10	10	10	10	10	10	10
G4 450	Mean	108.85*	35.54*	0.49*	59.44	95.59	120.12*	79.57	71.34*	12.41	5.00*	47.17*
	SD	15.71	6.45	0.03	9.40	68.27	10.74	18.91	2.28	2.76	1.12	1.13
	N	10	10	10	10	10	10	10	10	10	10	10

   Key:  *= Statistically significant from vehicle control (P ≤ 0.05); G1=Vehicle control; G2=low dose; G3=mid dose; G4=high dose.

 

TABLE 5 (CONTD.) - SUMMARY OF CLINICAL CHEMISTRY PARAMETERS– MALES

Groups Dose (mg/kg/day)		Clinical Chemistry Parameters – Males
		T.BIL	Ca	PHO	GLOB	TBA	ALP	GGT	BUN	NA	K	Cl
		mg/dL	mg/dL	mg/dL	-	U/L	U/L	U/L	mmol/L	mmol/L	mmol/L	mmol/L
G1 0	Mean	0.11	10.81	7.99	23.97	23.37	86.04	0.00	13.95	140.80	4.68	101.40
	SD	0.05	0.36	0.49	1.30	12.84	15.97	0.00	1.62	1.40	0.30	1.43
	N	10	10	10	10	10	10	10	10	10	10	10
G2 30	Mean	0.07	10.20*	7.61	22.54*	23.45	75.29	0.00	15.09	141.70	4.74	102.00
	SD	0.07	0.45	0.38	0.94	11.66	29.62	0.00	1.57	1.25	0.30	2.11
	N	10	10	10	10	10	10	10	10	10	10	10
G3 120	Mean	0.07	10.91	8.31	23.75	21.96	77.38	0.00	15.60	143.70*	4.84	103.70*
	SD	0.03	0.20	0.57	1.25	13.34	17.75	0.00	2.26	1.16	0.28	1.34
	N	10	10	10	10	10	10	10	10	10	10	10
G4 450	Mean	0.10	10.96	8.74*	24.17	42.02*	95.29	0.00	16.61*	143.00*	4.52	102.20
	SD	0.07	0.27	0.56	1.36	23.26	21.79	0.00	3.01	1.56	0.28	0.79
	N	10	10	10	10	10	10	10	10	10	10	10

  Key:  *= Statistically significant from vehicle control (P ≤ 0.05); G1=Vehicle control; G2=low dose; G3=mid dose; G4=high dose.; - = Not Applicable.

 

TABLE 6- SUMMARY OF CLINICAL CHEMISTRY PARAMETERS – FEMALES

Groups Dose (mg/kg/day)		Clinical Chemistry Parameters – Females
		GLU	UREA	CRE	TCHO	TRIG	AST	ALT	TP	HDL	LDL	ALB
		mg/dL	mg/dL	mg/dL	mg/dL	U/L	U/L	g/L	g/L	mg/dL	g/L	g/L
G1 0	Mean	109.05	32.63	0.57	61.64	71.04	122.38	45.86	68.34	12.39	7.98	44.93
	SD	17.81	4.08	0.06	19.60	15.12	13.88	7.40	3.68	1.65	3.90	2.38
	N	10	10	10	10	10	10	10	10	10	10	10
G2 30	Mean	107.49	36.16	0.57	57.60	63.67	134.23	51.79	67.69	13.28	4.76	44.29
	SD	17.71	4.34	0.06	19.25	8.63	40.29	17.21	3.64	3.64	2.15	1.82
	N	10	10	10	10	10	10	10	10	10	10	10
G3 120	Mean	98.75	36.49	0.55	56.87	65.09	124.19	44.67	69.70	15.59*	4.56	46.15
	SD	33.26	3.20	0.04	10.25	10.50	12.60	8.72	3.51	2.59	1.73	2.40
	N	10	10	10	10	10	10	10	10	10	10	10
G4 450	Mean	112.15	40.18*	0.51*	51.98	75.37	126.92	58.36	74.81*	19.45*	3.92*	49.79*
	SD	14.42	6.87	0.06	9.86	13.69	27.58	15.38	4.33	5.58	0.91	2.63
	N	10	10	10	10	10	10	10	10	10	10	10

 Key:  *= Statistically significant from vehicle control (P ≤ 0.05); G1=Vehicle control; G2=low dose; G3=mid dose; G4=high dose.

 

TABLE 6 (CONTD.) - SUMMARY OF CLINICAL CHEMISTRY PARAMETERS– FEMALES

 

Groups Dose (mg/kg/day)		Clinical Chemistry Parameters – Females
		T.BIL	Ca	PHO	GLOB	TBA	ALP	GGT	BUN	NA	K	Cl
		mg/dL	mg/dL	mg/dL	-	U/L	U/L	U/L	mmol/L	mmol/L	mmol/L	mmol/L
G1 0	Mean	0.15	9.36	5.42	23.41	27.89	43.80	0.00	15.54	141.00	4.16	102.00
	SD	0.05	0.16	0.50	1.67	15.29	9.83	0.00	1.94	1.05	0.19	2.62
	N	10	10	10	10	10	10	10	10	10	10	10
G2 30	Mean	0.16	9.42	5.43	23.40	49.87	43.64	0.11	17.22	140.60	4.08	102.30
	SD	0.05	0.22	0.86	2.04	32.14	21.33	0.27	2.06	1.35	0.19	0.67
	N	10	10	10	10	10	10	10	10	10	10	10
G3 120	Mean	0.17	9.65	5.07	23.55	38.11	45.18	0.01	17.37	141.60	3.99	102.50
	SD	0.04	0.39	0.58	1.70	28.72	10.19	0.03	1.52	1.17	0.21	2.07
	N	10	10	10	10	10	10	10	10	10	10	10
G4 450	Mean	0.14	10.50*	5.70	25.02	43.50	49.54	0.00	19.13*	142.30	4.19	104.30
	SD	0.07	0.21	0.38	2.03	27.21	14.06	0.00	3.27	1.34	0.38	2.54
	N	10	10	10	10	10	10	10	10	10	10	10

Key:  *= Statistically significant from vehicle control (P ≤ 0.05); G1=Vehicle control; G2=low dose; G3=mid dose; G4=high dose.; - = Not Applicable.

                                 

TABLE 7 - SUMMARY OF URINALYSIS PARAMETERS – MALES

Urinalysis Parameters – Males     Groups Dose (mg/kg/day)
Parameters		G1 0	G2 30	G3     120	G4      450
Appearance	Clear	10	07	06	07
	Slightly Turbid	00	03	04	03
Volume (ml)	Mean	10.50	9.50	9.00	9.00
	SD	2.84	5.50	3.16	3.16
Specific Gravity	Mean	1.02	1.02	1.02	1.02
	SD	0.00	0.00	0.00	0.00
pH	Mean	7.60	7.60	7.90	7.90
	SD	0.52	0.52	0.32	0.32
Leucocytes WBC (Cells/µL)	Neg	07	05	03	04
	25	02	04	07	06
	100	01	01	00	00
	500	00	00	00	00
Protein (mg/dL)	Neg	09	07	06	07
	25	01	03	04	03
	75	00	00	00	00
Erythrocytes (RBC) (Cells/µL)	Pos	00	00	00	00
	Neg	09	10	10	10
	10	01	00	00	00

Key:  G1=Vehicle control; G2=low dose; G3=mid dose; G4=high dose

 

TABLE 8 - SUMMARY OF URINALYSIS PARAMETERS – FEMALES

Urinalysis Parameters – Females Groups Dose (mg/kg/day)
Parameters		G1 0	G2 30	G3 120	G4 450
Appearance	Clear	09	08	09	09
	Slightly Turbid	01	02	01	01
Volume (ml)	Mean	13.50	13.00	12.50	12.50
	SD	4.12	4.83	4.86	3.54
Specific Gravity	Mean	1.01	1.01	1.01	1.01
	SD	0.00	0.01	0.01	0.00
pH	Mean	7.80	7.60	7.10	7.20
	SD	1.03	1.43	1.45	1.55
Leucocytes WBC (Cells/µL)	Neg	03	03	03	03
	25	07	07	07	05
	100	00	00	00	02
	500	00	00	00	00
Protein (mg/dL)	Neg	06	06	04	06
	25	04	04	06	04
	75	00	00	00	00
Erythrocytes (RBC) (Cells/µL)	Pos	00	00	00	00
	Neg	03	05	05	05
	10	07	05	05	05

Key:  G1=Vehicle control; G2=low dose; G3=mid dose; G4=high dose

 

TABLE 9 - SUMMARY OF TERMINAL FASTING BODY WEIGHT AND ABSOLUTE ORGAN WEIGHTS (g) – MALES

Groups Dose (mg/kg/day)		Fasting Body Weight And Absolute Organ Weights (g) – Males
		FBW	Liver	Kidneys	Adrenals	Spleen	Heart	Thymus	Brain	Testes	Epididymis	Prostate+seminal vesicles with coagulating glands	Thyroid gland	Pituitary gland
		(g)	(g)	(g)	(g)	(g)	(g)	(g)	(g)	(g)	(g)	(g)	(g)	(g)
G1 0	Mean	569.15	18.62	3.78	0.08	1.08	1.72	0.61	2.33	4.01	1.56	3.64	0.13	0.02
	SD	46.85	1.46	0.43	0.02	0.16	0.25	0.15	0.18	1.28	0.28	0.40	0.02	0.01
	N	10	10	10	10	10	10	10	10	10	10	10	10	10
G2 30	Mean	540.34	18.11	3.93	0.09	1.01	1.76	0.60	2.25	4.14	1.69	3.58	0.13	0.02
	SD	44.76	0.75	0.19	0.01	0.06	0.15	0.07	0.09	0.38	0.09	0.24	0.02	0.00
	N	10	10	10	10	10	10	10	10	10	10	10	10	10
G3 120	Mean	541.54	19.31	4.02	0.09	1.10	1.70	0.60	2.20	3.87	1.64	3.67	0.13	0.02
	SD	25.48	0.66	0.19	0.01	0.13	0.09	0.09	0.12	0.19	0.16	0.22	0.02	0.01
	N	10	10	10	10	10	10	10	10	10	10	10	10	10
G4 450	Mean	525.06*	21.40*	4.38*	0.09	1.05	1.83	0.55	2.24	3.77	1.63	3.78	0.14	0.02
	SD	34.38	1.28	0.08	0.01	0.09	0.11	0.04	0.15	0.19	0.13	0.22	0.01	0.01
	N	10	10	10	10	10	10	10	10	10	10	10	10	10

Key:  *= Statistically significant from vehicle control (P ≤ 0.05); G1=Vehicle control; G2=low dose; G3=mid dose; G4=high dose

 

TABLE 10 - SUMMARY OF TERMINAL FASTING BODY WEIGHT AND ABSOLUTE ORGAN WEIGHTS – FEMALES

Groups Dose (mg/kg/day)		Fasting Body Weight And Absolute Organ Weights (g) – Females
		FBW	Liver	Kidneys	Adrenals	Spleen	Heart	Thymus	Brain	Ovaries	Uterus	Thyroid gland	Pituitary gland
		(g)	(g)	(g)	(g)	(g)	(g)	(g)	(g)	(g)	(g)	(g)	(g)
G1 0	Mean	283.17	10.04	2.10	0.11	0.66	1.08	0.49	2.03	0.26	0.85	0.12	0.02
	SD	17.26	0.96	0.11	0.02	0.09	0.03	0.03	0.05	0.02	0.17	0.01	0.01
	N	10	10	10	10	10	10	10	10	10	10	10	10
G2 30	Mean	286.54	10.33	2.14	0.12	0.69	1.10	0.49	2.09*	0.26	0.79	0.12	0.02
	SD	18.30	0.67	0.13	0.01	0.07	0.10	0.06	0.06	0.02	0.16	0.01	0.01
	N	10	10	10	10	10	10	10	10	10	10	10	10
G3 120	Mean	283.19	10.21	2.14	0.12	0.70	1.12	0.46	2.06	0.27	0.88	0.12	0.02
	SD	25.41	0.92	0.17	0.02	0.06	0.11	0.05	0.05	0.03	0.07	0.01	0.01
	N	10	10	10	10	10	10	10	10	10	10	10	10
G4 450	Mean	283.44	11.57*	2.32*	0.11	0.74	1.18	0.46	2.07	0.26	0.88	0.12	0.02
	SD	21.27	0.44	0.15	0.01	0.08	0.12	0.04	0.07	0.03	0.06	0.01	0.00
	N	10	10	10	10	10	10	10	10	10	10	10	10

Key:  *= Statistically significant from vehicle control (P ≤ 0.05); G1=Vehicle control; G2=low dose; G3=mid dose; G4=high dose

 

TABLE 11 - SUMMARY OF RELATIVE ORGAN WEIGHTS – MALES

Groups Dose (mg/kg/day)		Fasting Body Weight And Absolute Organ Weights (g) – Males
		Liver	Kidneys	Adrenals	Spleen	Heart	Thymus	Brain	Testes	Epididymis	Prostate+seminal vesicles with coagulating glands	Thyroid gland	Pituitary gland
		(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)
G1 0	Mean	3.29	0.67	0.01	0.19	0.30	0.11	0.41	0.72	0.28	0.64	0.02	0.00
	SD	0.40	0.11	0.00	0.02	0.05	0.02	0.03	0.25	0.05	0.09	0.00	0.00
	N	10	10	10	10	10	10	10	10	10	10	10	10
G2 30	Mean	3.37	0.73	0.02	0.19	0.33	0.11	0.42	0.77	0.32*	0.67	0.02	0.00
	SD	0.27	0.05	0.00	0.02	0.03	0.01	0.04	0.09	0.04	0.06	0.00	0.00
	N	10	10	10	10	10	10	10	10	10	10	10	10
G3 120	Mean	3.57	0.74	0.02	0.20	0.31	0.11	0.41	0.72	0.30	0.68	0.02	0.00
	SD	0.17	0.03	0.00	0.02	0.02	0.02	0.03	0.05	0.02	0.07	0.00	0.00
	N	10	10	10	10	10	10	10	10	10	10	10	10
G4 450	Mean	4.09*	0.84*	0.02*	0.20	0.35*	0.11	0.43	0.72	0.31	0.72*	0.03	0.00
	SD	0.40	0.06	0.00	0.01	0.04	0.01	0.05	0.03	0.02	0.07	0.00	0.00
	N	10	10	10	10	10	10	10	10	10	10	10	10

Key:  *= Statistically significant from vehicle control (P ≤ 0.05); G1=Vehicle control; G2=low dose; G3=mid dose; G4=high dose.

 

TABLE 12 - SUMMARY OF RELATIVE ORGAN WEIGHTS – FEMALES

Groups Dose (mg/kg/day)		Fasting Body Weight And Absolute Organ Weights (g) – Females
		Liver	Kidneys	Adrenals	Spleen	Heart	Thymus	Brain	Ovaries	Uterus	Thyroid gland	Pituitary gland
		(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)
G1 0	Mean	3.55	0.74	0.04	0.23	0.38	0.17	0.72	0.09	0.30	0.04	0.01
	SD	0.37	0.06	0.01	0.04	0.02	0.02	0.05	0.01	0.06	0.00	0.00
	N	10	10	10	10	10	10	10	10	10	10	10
G2 30	Mean	3.61	0.75	0.04	0.24	0.39	0.17	0.73	0.09	0.28	0.04	0.01
	SD	0.26	0.07	0.00	0.03	0.02	0.02	0.04	0.01	0.05	0.00	0.00
	N	10	10	10	10	10	10	10	10	10	10	10
G3 120	Mean	3.62	0.76	0.04	0.25	0.40	0.16	0.73	0.10	0.31	0.04	0.01
	SD	0.30	0.05	0.01	0.02	0.05	0.02	0.07	0.01	0.04	0.01	0.00
	N	10	10	10	10	10	10	10	10	10	10	10
G4 450	Mean	4.11*	0.82*	0.04	0.26	0.42	0.16	0.74	0.09	0.31	0.04	0.01
	SD	0.40	0.07	0.00	0.03	0.05	0.02	0.06	0.01	0.03	0.00	0.00
	N	10	10	10	10	10	10	10	10	10	10	10

Key:  *= Statistically significant from vehicle control (P ≤ 0.05); G1=Vehicle control; G2=low dose; G3=mid dose; G4=high dose.

 

TABLE 13 - SUMMARY OF EXTERNAL AND GROSS PATHOLOGICAL OBSERVATIONS – MALES

Group Dose (mg/kg bw/ day)	G1 0	G2 30	G3 120	G4 450
No. of Animals Observed	10/10	10/10	10/10	10/10
External Observations^
NAD	10/10	10/10	10/10	10/10
Internal Observations^
NAD	9/10	10/10	10/10	10/10
Testes (bilateral) – small sized	1/10	0/10	0/10	0/10
Epididymides (bilateral) – small sized	1/10	0/10	0/10	0/10

Key: NAD= No Abnormality Detected

 

 

TABLE 14 - SUMMARY OF EXTERNAL AND GROSS PATHOLOGICAL OBSERVATIONS – FEMALES

Group Dose (mg/kg bw/ day)	G1 0	G2 30	G3 120	G4 450
No. of Animals Observed	10/10	10/10	10/10	10/10
External Observations^
NAD	10/10	10/10	10/10	10/10
Internal Observations^
NAD	10/10	10/10	10/10	10/10

Key: NAD= No Abnormality Detected

 

TABLE 15 - SUMMARY OF HISTOPATHOLOGICAL OBSERVATIONS – MALES AND FEMALES

Tissue: Observations		Group Dose (mg/kg bw/ day)
		Male		Female
		G1 0	G4 450	G1 0	G4 450
Epididymides:
Aspermia, Bilateral	Present	1	-	-	-
Testes:
Atrophy, seminiferous tubules, bilateral, Diffuse	Moderate	1	-	-	-
Testes (PAS stain):
Stage I to XIV	Absent	1	-	-	-
Kidneys:
Infiltration, MNC, renal pelvis, Focal	Minimal	-	-	1	1
Uterus:
Dilation, cystic, glandular, Focal	Minimal	-	-	1	-

         Key: - MNC – Mono Nuclear Cells, Organs/Tissues with no abnormality detected (NAD) were not included in this table.

 

Applicant's summary and conclusion

Conclusions:

NOAEL in male rats was considered to be 120 mg/kg bw/day, since no adverse effects could be detected at this dose level. LOAEL in male rats was considered to be 450 mg/kg bw/day based on clinical signs of toxicity (lethargy) and adverse effects on body weight and body weight gain. NOAEL in female rats was considered to be 450 mg/kg bw/day since no adverse effects in female rats could be detected in the study.

Executive summary:

The aim of this study was to evaluate the toxicity of N-tert.-butyl acrylamide (CAS No. 107-58-4) following oral (gavage) administration to Wistar Rats for 90 consecutive days. The study was designed to provide information on systemic toxicity and target organ toxicity and to establish the sub-chronic NOAEL and LOAEL values for N-tert.-butyl acrylamide (CAS No. 107-58-4) in Wistar rats. One vehicle control (G1) and three graded dose levels of 30 (G2), 120 (G3) and 450 (G4) mg/kg bw/day, with each group consisting of 10 males and 10 female rats were used in the study. The test item, i.e. N-tert.-butyl acrylamide (TBAA) (CAS No.:107-58-4) in 0.5% CMC (in Milli-Q water), was administered by oral gavage to animals of both sex at the doses of 30 (G2), 120 (G3) and 450 (G4) mg/kg bw/day for 90 consecutive days. Similarly, the vehicle (i.e. 0.5% CMC in Milli-Q water) was administered to the male and female rats in the vehicle control group (G1) for 90 consecutive days. Oral administration was done at a fixed dose volume of 10 mL/kg bw. The stability of the test item formulation was determined prior to start of treatment and the results indicated that the formulations were stable for 7 days at 2 to 8 ºC. Dose formulations were prepared and were administered to rats allocated to the respective dose groups within the stability period. The formulation analyses were performed during the first week, the 7^th week, and the 13^th week of treatment. The results revealed that the formulations were considered acceptable as the overall mean concentrations were within ±15% (from 95.13% to 99.29%) of the nominal concentrations, and the relative standard deviation (RSD) of top, middle and bottom layers were less than 10% (from 0.21% to 1.3%). The animals were observed for mortality and morbidity twice daily during the study period. On the weekends and public holidays, the animals were observed at least once a day. General clinical signs were recorded once daily and detailed clinical signs were recorded before dosing (during acclimatization) and once a week thereafter. Ophthalmological examinations were performed in all animals of G1, G2, G3, and G4 before initiation of treatment and in all animals of G1 and G4 during the last week of treatment (i.e. 13^th week of treatment). The animals were subjected to functional observation examinations, which included homecage observation, openfield activity, sensory reactivity measurements, locomotor activity using actimeter, grip strength and landing hindlimb foot splay, during the last week of treatment. Body weights were recorded on day 1 of treatment, once a week thereafter, on the day before fasting, and after fasting before necropsy. Leftover feed was recorded once a week (coinciding with body weight recordings) and feed input was weighed and recorded based on consumption requirements. Feed consumed was calculate and presented as feed consumption per rat per day (g/rat/day). Triiodothyronine (T3), Thyroxine (T4) and Thyroid Stimulating Hormone (TSH) serum levels were recorded from blood samples collected before necropsy. Blood and urine samples were collected at the end of the treatment periods for clinical pathology investigations. All the animals were subjected to detailed gross necropsy and specified organs were collected and weighed. The histopathological examinations were carried out on the preserved organs of vehicle control (G1) and high dose group (G4) rats. No mortality or morbidity was observed at any dose level. Lethargy was observed in male rats in G4 (range: 1-5 animals) on day 44 and each day onwards, up to terminal sacrifice. This lethargy was observed 10-15 minutes after dosing, persisted up to about 60 minutes after dosing, and was considered to be an adverse effect of treatment. No clinical signs of toxicity were observed in G2 or G3 males or in any of the female groups. Significant decreases in body weight were observed in G4 males on day 57 (mean, 480.98 gram), day 64 (mean, 499.31 gram), and after fasting (mean, 525.06 gram) compared to G1 (mean, 518.31 gram, 535.65 gram, and 569.15 gram on day 57, 64, and after fasting, respectively). Furthermore in G4 males, significant decreases in body weight gain were observed between day 15-22 (mean, G4: 45.01 gram; G1: 54.24 gram), 29-36 (mean, G4: 28.66 gram; G1: 34.82 gram), 50-57 (mean, G4: 17.84 gram; G1: 27.17 gram) and 85-90 (mean, G4: 2.08 gram; G1: 11.27 g). These observed changes body weight and body weight gain in G4 males were considered to be adverse effects of treatment, as body weight and body weight gain were generally lower in G4 males compared to G1 males throughout the study period. No other toxicologically significant changes in body weight or body weight gain were observed. In G4 male rats, a significant decrease in food intake was observed between day 85-90 (mean, 31.41 gram) compared to G1 (mean, 34.35 gram). This decrease in food intake was associated with a tendency of lower body weight in G4 males on day 90 (mean 548.53 gram) compared to G1 males (mean, 591.49 gram) and was therefore considered toxicologically relevant. No toxicologically significant changes in the functional observatory battery were observed. No ocular abnormalities were observed in the study. Several changes in thyroid hormone levels and in hematology were observed in G2, G3 and G4, but none could be corroborated with any clinical symptoms, any significant changes in absolute or relative organ weight, or with any gross or histopathological findings. Therefore, the observed changes in thyroid hormone levels and in hematology were not considered to be toxicologically relevant. No significant changes in coagulation parameters were observed. Noteworthy changes in clinical chemistry included a significant increase in urea in G4 females (mean, 40.18 mg/dL) compared to G1 (mean, 32.63 mg/dL) and a significant increase in BUN in G4 females (mean 19.13 mg/dL) compared to G1 (mean, 15.54 mg/dL) that could be corroborated with a significant increase in absolute kidney weight in G4 females compared to G1. However, since no treatment-related gross or histopathological findings were made in the kidneys in G4 females, the observed changes in urea and BUN in G4 females were not considered to be adverse. No significant changes in urinalysis were observed. Some statistically significant changes in absolute and relative organ weight were observed in G2 and G4, but none could be correlated with any gross or histopathological findings. Therefore, the observed changes in organ weight in G2 and G4 were not considered to be adverse. No gross pathological lesions were observed, with the exception of small sized testes (bilateral) and small sized epididymides (bilateral) in one G1 male rat. Histopathological findings included the following: aspermia (bilateral) in the epididymides in one G1 male rat; atrophy (moderate in severity) in the seminiferous tubules (bilateral, diffuse) in one G1 male rat; infiltration of mononucleated cells (minimal in severity) in the renal pelvis of the kidneys in one G1 female rat and one G4 female rat; and cystic dilatation (minimal in severity) in the uterus in one G1 female rat. No treatment related findings were observed during the qualitative assessment of spermatogenesis stages or during the morphological examination of interstitial testicular cell structures in high dose group (G4) male animals when compared to the vehicle control group (G1). Testes of one G1 male animal did not show any stages of spermatogenesis due to bilateral atrophy of the testes. The stage of estrous cycle in each female rat in G1 and G4 at the terminal sacrifice was in correlation with the histology of respective female reproductive organs. The observed microscopic findings in the G1 and G4 were considered incidental and therefore not toxicologically relevant. Based on the results from this study, the following effect levels were concluded for N-tert.-butyl acrylamide (CAS No. 107-58-4):

• NOAEL in male rats was considered to be 120 mg/kg bw/day, since no adverse effects could be detected at this dose level.  


• LOAEL in male rats was considered to be 450 mg/kg bw/day based on clinical signs of toxicity (lethargy) and adverse effects on body weight and body weight gain.


• NOAEL in female rats was considered to be 450 mg/kg bw/day since no adverse effects in female rats could be detected in the study.