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Description of key information

Repeated dose toxicity


OECD 407:


NOAEL in male rats was considered at 500 mg/kg. The observed effects in female rats treated at 500 mg/kg in the main study included significant decreases in body weight gain and indications of minor kidney toxicity. These effects were considered to be reversible based on lack of similar effects in the recovery groups of female rats treated at 500 mg/kg. The observed effects in female rats treated at 500 mg/kg in the main study were considered adverse and only reversible upon cessation of treatment. NOAEL in female rats was therefore considered at 250 mg/kg.


OECD 408:


NOAEL in male rats was considered to be 120 mg/kg bw/day, based on clinical signs of toxicity (lethargy) and significant decreases in body weight and body weight gain at 450 mg/kg bw/day. NOAEL in female rats was considered to be 450 mg/kg bw/day, as no adverse effects were observed at this dose level in the female rats.


Repeated dose toxicity- Inhalation route


According to Annex IX of the REACH regulation, testing by the inhalation route is appropriate only if exposure of humans via inhalation is likely. Taking into account the particle size distribution of N-tert-butylacrylamide and that the exposure via the inhalation route is unlikely, this end point was considered for waiver.


 


Repeated dose toxicity- Dermal route


The acute toxicity value for N-tert-butylacrylamide (as provided in section 7.2.3) is >2000 mg/kg body weight. Also, given the use of the chemical; repeated exposure by the dermal route is unlikely since the use of gloves is common practice in industries. Hence this end point was considered for waiver.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

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)
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
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.
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
Dose / conc.:
450 mg/kg bw/day (nominal)
Remarks:
G4
Dose / conc.:
120 mg/kg bw/day (nominal)
Remarks:
G3
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
G2
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
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.
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
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
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
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
Key result
Critical effects observed:
no
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.

 
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 7th week, and the 13th 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. 13th 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.   

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 Sep 2014 - 12 Nov 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from a CRO study report
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
2008
Deviations:
no
Remarks:
ophthalmic examinations conducted prior to treatment (all animals), at the end of the dosing period (main study), and at the end of the recovery period (recovery groups).
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: In-House Bred Animal Facility
- Age at study initiation: 6 - 8 weeks
- Weight at study initiation: Male : 137-191 g, Female: 137- 165 g
- Fasting period before study: No data available
- Housing: Animals were housed in Polycarbonate cages. Cage rotation was carried out weekly and cleaned at regular intervals. Animals were bedded on sterilized corn cob produced from pure corn, dried and free from dust. Floor of the experimental room and work tops were swept and mopped with disinfectant solution every day or as on requirement.
- Diet (e.g. ad libitum): A conventional laboratory pelleted diet, ad libitum.
- Water (e.g. ad libitum): Aqua guard filtered drinking water in bottles, ad libitum.
- Acclimation period: Male: 6 days, Female: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.00 to 23.10 °C
- Humidity (%): 49.90 to 69.40%.
- Air changes (per hr): Adequately filtered air 12 times per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark artificial light.

IN-LIFE DATES: From: September 22, 2014
To: October 20, 2014
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Substance was weighed and dissolved in a vehicle (corn oil) to achieve desired concentration of test item. Dose formulation was freshly prepared daily. At the time of dosing, dose formulation was kept on the magnetic stirrer to maintain the homogeneity of test item.

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

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil was used as a vehicle based on the solubility testing.
- Concentration in vehicle: 0, 125, 250 and 500 mg/kg/day
- Amount of vehicle (if gavage): 1 ml/100g body weight
- Lot/batch no. (if required): MKBQ9948V and MKBG9426V
- Purity: No data available
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and concentration of the substance in dose formulation was analysed by a validated analytical method, at the start of treatment (on day 1) and on day 21.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Dose / conc.:
0 other: mg/kg/day
Dose / conc.:
125 other: mg/kg/day
Dose / conc.:
250 other: mg/kg/day
Dose / conc.:
500 other: mg/kg/day
No. of animals per sex per dose:
Total : 60
0 mg/kg/day: 5 male, 5 female
125 mg/kg/day: 5 male, 5 female
250 mg/kg/day: 5 male, 5 female
500 mg/kg/day: 5 male, 5 female

Recovery:
0 mg/kg/day: 5 male, 5 female
500 mg/kg/day: 5 male, 5 female
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the information provided by Sponsor.
- Rationale for animal assignment (if not random): No data available
- Rationale for selecting satellite groups: No data available
- Post-exposure recovery period in satellite groups: No data available
- Section schedule rationale (if not random): No data available
Positive control:
Not included.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily (morning and evening)
- Cage side observations checked in table [No.?] were included: Morbidity and mortality were observed.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first treatment and weekly thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: At start of treatment and thereafter weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data available
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data available

FOOD EFFICIENCY: No data available
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data available

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data available
- Time schedule for examinations: No data available

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to treatment (During Acclimatization Period) and at the end of the dosing (main groups) and recovery periods (recovery group) were examined.
- Dose groups that were examined: All 60 animals were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On termination day, just prior to necropsy.
- Anaesthetic used for blood collection: No data available
- Animals fasted: Yes, overnight (approximately 16-18 hr).
- How many animals: All 60 animals were examined.
- Parameters checked in table [No.?] were examined: Total Erythrocyte Count (RBC), Hematocrit (HCT), Mean Corpuscular Volume (MCV), Hemoglobin (HGB), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Platelet Count (PLT), Total Leukocyte count (WBC), Prothombin Time (PT) and Activated Partial Thromboplastin time (aPTT) were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On termination day, just prior to necropsy.
- Animals fasted: Yes, overnight (approximately 16-18 hr).
- How many animals: All 60 animals were examined.
- Parameters checked in table [No.?] were examined: Glucose (Glu), Cholesterol (Chol), Triglycerides (TRIG), Alanine amino transferase (ALT), Aspartate amino transferase (AST), Gamma-Glutamyl Transpeptidase (GGT), Calcium, Creatine Kinase (CK), Albumin, Total Protein (TP), Creatinine (Crea), Total Bilirubin (T.Bil), Phosphorus, Alkaline phosphatase (ALP), Urea, Lactate Dehydrogenase (LD), Sodium (Na), Potassium (K), Chloride (Cl), Blood urea nitrogen (BUN), Globulin (Glob), A/G and Bile acids were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: No data available
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data available
- Parameters checked in table [No.?] were examined: Colour, appearance, urine volume, Blood / Blood Cell, Bilirubin, Urobilinogen, Ketone, Protein, Nitrite, Glucose, pH, Specific Gravity, Leucocytes, Microscopical Parameters were examined.

NEUROBEHAVIOURAL EXAMINATION: No data available
- Time schedule for examinations: No data available
- Dose groups that were examined: No data available
- Battery of functions tested: sensory activity / grip strength / motor activity / other: Yes, Sensory reactivity to stimuli, grip strength, hind limb foot splay and motor activity were tested.

OTHER:
Organ weight: Absolute and relative organ weights were examined.

Organ examined: Liver, kidneys, adrenals, testes, epdidymides, Prostate and Seminal vesicle with coagulating glands, uterus with cervix, ovary with oviduct, thymus, spleen, brain and heart were examined.
Bone Marrow Smear were also examined
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were fasted overnight before necropsy. Animals were sacrificed by using over dose of CO2 and examined externally and macroscopically.

HISTOPATHOLOGY: Yes
All tissues were preserved in 10 % neutral buffered formalin (except eyes and testes; which were fixed using Modified Davidson fluid for 24 hr and then transferred to 10 % neutral buffered formalin (NBF) for preservation) for subsequent histopathological examintion.

Organ examined:
Adrenals, Bone (femur) with joint, Brain (cerebrum,cerebellum,mid brain), Cecum, Colon, Duodenum, Epididymides, Eyes with optic nerve, Gross lesion, Ileum, Jejunum, Kidneys, Liver, Lung, Mammary glands, Mesenteric and Mandibular lymph node, Oesophagus and Ovary with oviduct were examined.
Statistics:
Statistical analysis of raw data was performed by using statistical software Sigma Plot 11.0. The mean and standard deviation was calculated using the software and all data was summarized in tabular form. All continuous data (body weight, feed consumption, haematology, clinical chemistry, absolute and relative organ weights) were checked for their homogeneity using Bartlett’s test. All homogenous data was analysed using ANOVA and data showing significance in their variances was subjected to Dunnett’s t-test. All heterogeneous data was analysed using F test and Student’s t-test.
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 planned death.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Female rats treated at 500 mg/kg (main study) showed significant decreases in % body weight gain compared to the control group during all weeks of treatment. No other significant changes in body weight gain were observed. No significant changes in absolute body weight were observed, however female rats treated at 500 mg/kg tended to be of lower weight during all weeks of treatment compared to the control group (by 5.9% on week 1; by 6.5% on week 2; by 6.9% on week 3; and by 7.7% on week 4).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No significant changes in food intake were observed with the exception of few significant decreases in food intake in the recovery groups of female rats treated at 500 mg/kg as compared to the control group that were considered incidental and toxicologically non-significant.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No eye abnormalities were observed at any dose level.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Significance changes in haematology included a significant increase in neutrophils in female rats treated at 500 mg/kg (main study); significant decreases in MCV and WBC in recovery female rats treated at 500 mg/kg; a significant increase in platelets in recovery female rats treated at 500 mg/kg; and a significant decrease in activated partial thromboplastin time in male rats treated at 500 mg/kg. None of these changes in haematology were considered to be of toxicological significance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Significant changes in clinical chemistry included minimal changes (less than 4% vs controls) in sodium levels in male rats treated at 125, 250 and 500 mg/kg (main study); significant decrease in lactate dehydrogenase in male rats treated at 500 mg/kg (main study); significant decrease in creatinine kinase in male rats treated at 500 mg/kg (main study); significant increases in alkaline phosphatase in male rats treated at 250 and 500 mg/kg (main study); significant increase in alanine amino transferase in male rats treated at 500 mg/kg (main study); significant increases in urea nitrogen, urea, total bilirubin, and calcium in female rats treated at 500 mg/kg (main study); significant increases in urea nitrogen, urea, albumin, alkaline phosphatase, potassium, chloride, and bile acids in recovery male rats treated at 500 mg/kg; significant increase in sodium levels in recovery female rats treated at 500 mg/kg; and a significant decreases in cholesterol in recovery male rats treated at 500 mg/kg. The observed changes in enzyme levels in male rats treated at 250 and 500 mg/kg were attributed to the test item since they could be corroborated with increased liver weights and morphological changes, as explained in detail below. The observed changes in urea nitrogen and urea in female rats treated at 500 mg/kg were considered as possibly treatment related as they could be corroborated with histopathological changes in the kidneys, as explain in detail below. The other changes in clinical chemistry were considered to be of no toxicological significance.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
No significant changes in urinalysis were observed, with the exception of a significant increase in urine volume in recovery female rats treated at 500 mg/kg that was considered incidental.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No significant changes in sensory reactivity were observed at any dose level. No significant changes in functionality were observed, with the exception of significant increases in hind limb grip strength in male rats treated at 125 and 500 mg/kg (main study) and a significant decrease in hind limb grip strength in recovery male rats treated at 500 mg/kg. The observed changed in hind limb grip strength were inconsistent and could not be corroborated with any other observations in the study. Therefore, they were considered to be of no toxicological significance.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Noteworthy changes in organ weight included significant increases in liver weight in male rats treated at 125, 250 and 500 mg/kg (main study), a significant increase in relative liver weight in male rats at 250 and 500 mg/kg (main study) and significant increases in relative kidney weight in male rats at 250 and 500 mg/kg (main study).
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross pathological findings were observed in any of the animals.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Noteworthy changes in histopathology included increased incidence of hepatocellular hypertrophy (mild in severity) in 4 of 5 male rats treated at 500 mg/kg; bile duct proliferation in liver (minimal or mild in severity) in 2 of 5 male rats treated at 500 mg/kg; cystic degeneration (minimal in severity) in kidneys in 1 of 5 female rats treated at 500 mg/kg; and tubular degeneration (minimal or mild in severity) in 2 of 5 female rats treated at 500 mg/kg. Other histopathological findings occurred at comparable incidences at 500 and 0 mg/kg (main study) and no remarkable changes in histopathology were observed in the recovery groups at 500 mg/kg.
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
NOAEL
Effect level:
500 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
urinalysis
Dose descriptor:
NOAEL
Effect level:
250 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
urinalysis
Dose descriptor:
LOAEL
Effect level:
500 mg/kg bw/day (nominal)
Sex:
female
Basis for effect level:
body weight and weight gain
histopathology: non-neoplastic
Key result
Critical effects observed:
no

Mortality and Morbidity

Group

Treatment

Dose (mg/kg b.wt.)

No. of Animals/

Group

Day of Observations

Observation

G1

Control

0

5

1-29

NMM

G2

Low

125

5

1-29

NMM

G3

Mid

250

5

1-29

NMM

G4

High

500

5

1-29

NMM

G1-R

Control -Recovery

0

5

1-43

NMM

G4-R

High- Recovery

500

5

1-43

NMM

Group

Treatment

Dose (mg/kg b.wt.)

No. of Animals/

Group

Day of Observations

Observation

G1

Control

0

5

1-29

NMM

G2

Low

125

5

1-29

NMM

G3

Mid

250

5

1-29

NMM

G4

High

500

5

1-29

NMM

G1-R

Control -Recovery

0

5

1-43

NMM

G4-R

High- Recovery

500

5

1-43

NMM

Key:NMM = No mortality and morbidity observed.

Mean Absolute Organ Weight (g)

Sex:Male

Organs

 

Group 1

Group 2

Group 3

Group 4

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Body weight (g)

251.80

21.09

239.20

24.78

240.60

18.37

234.60

11.48

Brain

1.910

0.062

1.935

0.053

1.966

0.051

1.900

0.057

Adrenals

0.046

0.013

0.059

0.007

0.053

0.009

0.051

0.010

S.V. With Coagulating gland and prostrate

1.1831

0.22882

1.11382

0.167243

0.8658

0.1214

1.1246

0.12127

Testes

3.003

0.246

2.857

0.296

2.966

0.167

2.810

0.150

Epdidymides

0.902

0.164

1.004

0.135

0.958

0.106

0.954

0.045

Heart

1.044

0.088

1.007

0.078

0.998

0.069

0.911

0.043

Liver

10.450

0.919

11.804

1.553

13.062

1.580

13.492

1.428

Kidneys

2.294

0.219

2.271

0.261

2.575

0.202

2.427

0.192

Spleen

1.356

0.434

1.142

0.332

1.136

0.196

0.905

0.198

Thymus

0.301

0.071

0.311

0.044

0.299

0.071

0.241

0.056

Organs

 

Group 1-R

Group 4-R

Mean

SD

Mean

SD

Body weight (g)

273.40

20.21

280.80

15.39

Brain

1.935

0.120

1.927

0.067

Adrenals

0.056

0.009

0.064

0.008

S.V. With Coagulating gland and prostrate

1.5541

0.4754

1.4925

0.260007

Testes

3.210

0.166

3.168

0.254

Epdidymides

1.145

0.063

1.112

0.153

Heart

1.148

0.080

1.175

0.125

Liver

10.861

0.878

10.788

0.658

Kidneys

2.417

0.304

2.368

0.199

Spleen

1.002

0.106

1.048

0.285

Thymus

0.296

0.106

0.349

0.090

Sex:Female

Organs

Group 1

Group 2

Group 3

Group 4

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Body weight (g)

185.40

15.13

173.00

4.00

178.40

7.37

169.60

5.41

Brain

1.791

0.076

1.692

0.091

1.728

0.040

1.789

0.087

Adrenals

0.059

0.011

0.051

0.011

0.061

0.011

0.064

0.012

Ovary

0.092

0.012

0.106

0.032

0.111

0.024

0.091

0.015

Uterus

0.720

0.120

0.645

0.107

0.561

0.152

0.553

0.115

Heart

0.853

0.118

0.725

0.027

0.731

0.063

0.725

0.079

Liver

8.231

0.754

7.106

0.640

7.743

0.646

8.403

0.586

Kidneys

1.582

0.132

1.412

0.098

1.470

0.097

1.585

0.177

Spleen

0.719

0.279

0.502

0.122

0.485

0.120

0.516

0.225

Thymus

0.300

0.049

0.266

0.081

0.235

0.036

0.242

0.042

Organs

Group 1-R

Group 4-R

Mean

SD

Mean

SD

Body weight (g)

196.60

9.96

188.40

8.50

Brain

1.792

0.087

1.800

0.059

Adrenals

0.077

0.007

0.090

0.006

Ovary

0.164

0.015

0.170

0.018

Uterus

0.777

0.305

0.723

0.120

Heart

0.846

0.054

0.782

0.045

Liver

7.429

0.677

7.394

0.560

Kidneys

1.684

0.058

1.522

0.110

Spleen

0.776

0.126

0.441

0.054

Thymus

0.281

0.062

0.320

0.037

Conclusions:
NOAEL in male rats was considered at 500 mg/kg. The observed effects in female rats treated at 500 mg/kg in the main study included significant decreases in body weight gain and possible minor kidney toxicity. These effects were considered to be reversible based on lack of similar effects in the recovery groups of female rats treated at 500 mg/kg. The observed effects in female rats treated at 500 mg/kg in the main study were considered adverse and only reversible upon cessation of treatment. NOAEL in female rats was therefore considered at 250 mg/kg.
Executive summary:

The substance was given by oral gavage to 5 rats per sex per dose at 0 (vehicle; corn oil), 125, 250 and 500 mg/kg/day for a total of 28 days (main study). The substance was also given by oral gavage to additional 5 rats per sex per dose at 0 (corn oil) and 500 mg/kg/day that were allowed to recovery for 2 weeks after the final dose (recovery groups). The study was conducted according to GLP and OECD 407 (2008) with extra inclusion of ophthalmic examinations conducted prior to treatment (all animals), at the end of the dosing period (main study), and at the end of the recovery period (recovery groups). All animals survived to planned death and there were no clinical signs of toxicity at any dose level. No eye abnormalities were observed at any dose level. Female rats treated at 500 mg/kg (main study) showed significant decreases in % body weight gain compared to the control group during all weeks of treatment. No other significant changes in body weight gain were observed. No significant changes in absolute body weight were observed, however female rats treated at 500 mg/kg tended to be of lower weight during all weeks of treatment compared to the control group (by 5.9% on week 1; by 6.5% on week 2; by 6.9% on week 3; and by 7.7% on week 4). No significant changes in food intake were observed with the exception of few significant decreases in food intake in the recovery groups of female rats treated at 500 mg/kg as compared to the control group that were considered incidental and toxicologically non-significant. No significant changes in sensory reactivity were observed at any dose level. No significant changes in functionality were observed, with the exception of significant increases in hind limb grip strength in male rats treated at 125 and 500 mg/kg (main study) and a significant decrease in hind limb grip strength in recovery male rats treated at 500 mg/kg. The observed changed in hind limb grip strength were inconsistent and could not be corroborated with any other observations in the study. Therefore, they were considered to be of no toxicological significance. Significance changes in haematology included a significant increase in neutrophils in female rats treated at 500 mg/kg (main study); significant decreases in MCV and WBC in recovery female rats treated at 500 mg/kg; a significant increase in platelets in recovery female rats treated at 500 mg/kg; and a significant decrease in activated partial thromboplastin time in male rats treated at 500 mg/kg. None of these changes in haematology were considered to be of toxicological significance. Significant changes in clinical chemistry included minimal changes (less than 4% vs controls) in sodium levels in male rats treated at 125, 250 and 500 mg/kg (main study); significant decrease in lactate dehydrogenase in male rats treated at 500 mg/kg (main study); significant decrease in creatinine kinase in male rats treated at 500 mg/kg (main study); significant increases in alkaline phosphatase in male rats treated at 250 and 500 mg/kg (main study); significant increase in alanine amino transferase in male rats treated at 500 mg/kg (main study); significant increases in urea nitrogen, urea, total bilirubin, and calcium in female rats treated at 500 mg/kg (main study); significant increases in urea nitrogen, urea, albumin, alkaline phosphatase, potassium, chloride, and bile acids in recovery male rats treated at 500 mg/kg; significant increase in sodium levels in recovery female rats treated at 500 mg/kg; and a significant decreases in cholesterol in recovery male rats treated at 500 mg/kg. The observed changes in enzyme levels in male rats treated at 250 and 500 mg/kg were attributed to the test item since they could be corroborated with increased liver weights and morphological changes, as explained in detail below. The observed changes in urea nitrogen and urea in female rats treated at 500 mg/kg were considered as possibly treatment related as they could be corroborated with histopathological changes in the kidneys, as explain in detail below. The other changes in clinical chemistry were considered to be of no toxicological significance. No significant changes in urinalysis were observed, with the exception of a significant increase in urine volume in recovery female rats treated at 500 mg/kg that was considered incidental. Noteworthy changes in organ weight included significant increases in liver weight in male rats treated at 125, 250 and 500 mg/kg (main study), a significant increase in relative liver weight in male rats at 250 and 500 mg/kg (main study) and significant increases in relative kidney weight in male rats at 250 and 500 mg/kg (main study). No gross pathological findings were observed in any of the animals. Noteworthy changes in histopathology included increased incidence of hepatocellular hypertrophy (mild in severity) in 4 of 5 male rats treated at 500 mg/kg; bile duct proliferation in liver (minimal or mild in severity) in 2 of 5 male rats treated at 500 mg/kg; cystic degeneration (minimal in severity) in kidneys in 1 of 5 female rats treated at 500 mg/kg; and tubular degeneration (minimal or mild in severity) in 2 of 5 female rats treated at 500 mg/kg. Other histopathological findings occurred at comparable incidences at 500 and 0 mg/kg (main study) and no remarkable changes in histopathology were observed in the recovery groups at 500 mg/kg. In conclusion, the observed effects in male rats treated at 250 and 500 mg/kg in the main study were considered adaptive, non-adverse, and reversible based on lack of similar effects in the recovery group of male rats treated at 500 mg/kg. NOAEL in male rats was therefore considered at 500 mg/kg. The observed effects in female rats treated at 500 mg/kg in the main study included significant decreases in body weight gain and possible minor kidney toxicity. These effects were considered to be reversible based on lack of similar effects in the recovery groups of female rats treated at 500 mg/kg. The observed effects in female rats treated at 500 mg/kg in the main study were considered adverse and only reversible upon cessation of treatment. NOAEL in female rats was therefore considered at 250 mg/kg.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
120 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Klimisch 1

Additional information

Justification for classification or non-classification

In the OECD 407 study with the substance, adverse effects were limited to significant decreases in body weight gain and some indications of mild kidney toxicity in female rats treated at 500 mg/kg bw/day (highest dose tested). No such effects were observed in the recovery group of female rats treated at 500 mg/kg bw/day. Therefore, the adverse effects in female rats treated at 500 mg/kg bw/day in the OECD 407 were considered reversible and not taken as significant or severe signs of toxicity that would justify classification for STOT RE as per CLP regulation. In the OECD 408 study with the substance, adverse effects were limited to clinical signs of toxicity in male rats treated at 450 mg/kg bw/day (highest dose tested) and significant decreases in body weight and body weight gain in male rats treated at 450 mg/kg bw/day. The lethargy was transient during the treatment period and the significant decreases in body weight were slight/moderate compared to the control group (about 5-7% lower throughout the second half of the treatment period). In both the OECD 407 and the OECD 408, the adverse effects, which were not marked in severity, were observed at dose levels above the CLP guidance values that would justify a classification for STOT RE, i.e. above 300 mg/kg bw/day for a 28-days study (using Haber's rule) and above 100 mg/kg bw/day for a 90-days study (refer to Section 3.9.2.9.6 in Regulation EC No. 1272/2008). The substance is therefore considered to be classified as Not Classified for STOT RE as per regulation Regulation EC No. 1272/2008.