Registration Dossier
Registration Dossier
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EC number: 216-699-2 | CAS number: 1643-19-2
Endpoint summary
Administrative data
Description of key information
Repeated dose toxicity: oral
OECD 407 study: NOAEL in male and female rats was established at 1000 mg/kg bw/day (oral gavage).
OECD 408 study: NOAEL in male rats was established at 30 mg/kg bw/day (low-dose). LOAEL in male rats was established at 120 mg/kg bw/day (mid-dose) based on significant changes in clinical chemistry (electrolyte levels) and histopathological findings in the kidneys (i.e. interstitial haemorrhage of minimal severity in 4 out of 10 males) at 120 mg/kg bw/day. NOAEL in female rats could not be determined due to significant changes in clinical chemistry (electrolyte levels) and significant changes in the functional observatory battery (indicative of hyperactivity) at 30 mg/kg bw/day (low-dose).
Repeated dose toxicity: inhalation
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 the substance the majority of the particles were found to be in the size of 150 micrometer to 106 micrometer which is much larger size range compared to the inhalable particulate matter. Thus, exposure to inhalable dust, mist and vapour of the test chemical is highly unlikely. Therefore this study is considered for waiver.
Repeated dose toxicity: dermal
The acute toxicity value for test chemical (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. Thus, it is expected that the test chemical shall not exhibit 28 day repeated dose toxicity by the dermal route. In addition, there is no data available that suggests that the test chemical shall exhibit repeated dose toxicity by the dermal route. 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
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- June 2022 - Jan 2023
- Reliability:
- 1 (reliable without restriction)
- 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:
- Animals
Body weight ranges upon receipt: 126 - 173 g (males) and 98 - 141 g (females).
Age at the time of dosing: 7 - 8 weeks.
Housing
Prior to receipt of animals for the study, the study room, racks and cages were cleaned and disinfected. During the study, the floor of the experimental room and work tops were swept and mopped with disinfectant solution at least once every day and and more frequently when required. Cages were cleaned at regular intervals. Two to three rats were housed in each polycarbonate cage (length 37 cm X breadth 21 cm X height 20 cm). Rotation of the cage positions in each rack was carried once a week to nullify any possible effects arising from the cage placement. Sterilized corn-cob produced from pure corn, dried and free from dust, procured from approved supplier, was used as bedding material. It was renewed as often as was necessary to keep the animals dry and clean.
Bedding material of batch no. 222 (Krishna Corncob industries, Aurangabad) was used.
Environmental conditions
The room temperature was maintained between 21.0 and 22.5°C and the relative humidity achieved was in the range of 53.2 and 63.7%. Artificial light was set to give a cycle of 12 hours light and 12 hours dark. Adequately filtered air with at least 12 changes per hour was provided.
Diet
A conventional laboratory pellet diet (Batch No. 040522, 040722, 040822) from approved Supplier (Nutrivet Life Sciences, Pune) was available ad libitum.
Water
Aquaguard™ filtered drinking water was available ad libitum in bottles. Samples of the drinking water were periodically subjected to tests for bacteriological and chemical contaminants. Acceptance criteria as per IS 10500 (2012) were met for the samples that were periodically checked. - Route of administration:
- oral: gavage
- Vehicle:
- water
- Remarks:
- Distilled water was used as vehicle.
- Details on oral exposure:
- Each dose in the study was given by oral gavage at a fixed volume of 10 ml/kg bw, based on the most recently recorded body weight for the animal.
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The concentration and homogeneity assessment of the dose formulation of Tetrabutyl ammonium bromide (CAS 1643-19-2) was performed on Day 2, 43 and 85. The results of the analyzed formulation were 2.969, 11.942 and 35.203 mg/ml for Day 2; and 2.942, 11.540 and 34.828 mg/ml for Day 43; and 2.983, 11.746, 35.715 mg/ml for day 85 for low, mid and high dose respectively. Overall mean concentrations of does formulations were found within the acceptance limits of ± 20% (from 96.55% to 99.91%) of the nominal concentrations for all the formulations, and the relative standard deviations (RSD) of the top, middle and bottom layers were less than 10% (from 0.66% to 2.10%) in all analyses. Therefore, all dose formulations in this study were considered accurate, precise and homogenous.
- Duration of treatment / exposure:
- 90 days
- Frequency of treatment:
- Once daily
- Dose / conc.:
- 360 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:
- Vehicle control group (G1)
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- During the acclimatisation phase, male and female animals were separately randomized into four different experimental groups, based on most recently recorded body weights, using excel generated tables as per the internal SOP at the test facility. After randomisation, it was ensured that individual body weights were within ±20% of the respective group means. The mean body weights were analyzed by One-way ANOVA and it was found that the group means were statistically comparable to each other within each sex.
Dose levels were selected based on an OECD 422 study with Tetrabutyl ammonium bromide (CAS No. 1643-19-2) in rats (National Institute of Technology and Evaluation, Japan). In this study, 2 deaths were observed at 600 mg/kg (one male, one female). The male rat died already during the 2nd week of treatment and the female died during gestation. Before death, the male showed fracture of incisors, soft stool and a decrease in the amount of faeces and the female showed staining of lower abdominal fur and staggering gait. At 600 mg/kg/day, the following histopathological findings attributed to the test item were observed: diffuse hyperplasia in the mucosa in the cecum, hypertrophy of perilobular hepatocytes, cell infiltration in the mucosa in the cecum, colon and rectum, cell debris in crypt in the rectum, erosion in the duodenum, atrophy of lymphoid follicle in the spleen, erosion in the glandular stomach, atrophy and atrophy of the thymus. Target organs were identified to be the gastrointestinal tract and liver. NOEL was considered to be 60 mg/kg/day based on the histopathological findings in the cecum at 180 mg/kg/day whereas NOAEL was considered to be 180 mg/kg/day. Based on the above data and considering the significantly longer exposure duration in OECD 408, 360 mg/kg/day was selected as the highest dose level for the study. - Positive control:
- None
- Observations and examinations performed and frequency:
- Mortality/Morbidity
All animals were observed twice daily (once before and once after the day’s activities) for any morbidity and/or mortality, throughout the acclimatization and treatment period.
Clinical Signs
All animals were observed daily for clinical signs after dose administration. These observations were regularly performed approximately 1 hour after dose administration since onset of clinical signs after oral gavage can be anticipated in that time frame.
Detailed Clinical Examination
All animals were subject to a detailed clinical examination one day before treatment and weekly thereafter throughout the treatment periods. The animals were examined outside the home cages and the observations included, but were not limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions & excretions and autonomic activity (eg.piloerectio, pupil size, respiratory pattern). The gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (eg. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) were also checked for and recorded.The gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (eg. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) were also checked for and recorded.
Body Weight and Feed Consumption
All animals were weighed once on receipt, once during randomization, once at the start of treatment and weekly thereafter up to terminal sacrifice. These data were used to compare absolute weights and to calculate body weight gains throughout the experimental period. All animals fasted overnight prior to euthanasia and all animals were weighed before necropsy to compare terminal body weights and to calculate relative organ weights. The weight of feed provided and that leftover were recorded for each cage once every week starting from Day 1 of treatment. This data was used to calculate mean feed consumption by each animal throughout the experimental period.
Ophthalmological Examination
Ophthalmological examinationswere performed on all animals before treatment (during acclimatization period) and in all G1 and G4 animals during the final week of dosing. Since no abnormal findings were found in any of the animals in G4, the analysis was not extended to G2 and G3. Before ophthalmologic examination, mydriasis was induced using 1% Tropicamide (Batch No.TR-2108).
Estrus Cycle
The estrus cycle stage was determined in each female rat in G1, G2, G3 and G4 by microscopic examination of the vaginal smears. The examination was carried out on the last day before scheduled sacrifice.
Motor Activity and Behavioural Observation and Functional Observational Battery
Sensory reactivity to stimuli, assessment of grip strength, hind limb foot splay and motor activity assessment were conducted in G1, G2, G3 and G4 groups during the last week of treatment. Animals were subjected to examinations of various functional parameters which included: motor activity measurements using OPTO–VARIMEX 4 (Serial No.: 100180, manufactured by: Columbus Instruments, USA, supplied by: Gentech Marketing and Distribution, New Delhi, India), an automated animal activity measuring system; fore limb and hind limb grip strength, using grip strength meter (Serial No.: 0167-005L, manufactured by: Columbus Instruments, USA, supplied by: Gentech Marketing and Distribution, New Delhi, India); hind limb foot splay record and sensory reactivity measurements. - Sacrifice and pathology:
- Blood samples for hematological and clinical biochemistry evaluations were collected on the day of sacrifice (prior to necropsy). The animals were fasted overnight and the blood samples were collected prior to euthanasia from retro-orbital plexuses. Samples were transferredinto vials without anticoagulant for clinical biochemistry endpoints, into 4% EDTA (potassium salt) for hematology endpoints, and into 3.8% sodium citrate for coagulation assessment i.e. Prothrombin (PT) and Activated Partial Prothrombin Time (aPTT). The hematological and clinical biochemistry evaluations were carried out shortly after collection.
Hematology
The following hematologic parameters were estimated using an auto-analyzer (Exigo Vet Hematology Analyzer, serial number 55061, Boule Medical AB, Sweden) and the Robonic-Eazy Clot Activated Clot Time Coagulation Machine Analyser (Serial number CG0231211RBK; Robonik India Pvt Ltd).
Parameters Unit
Activated Partial Thromboplastin time (aPTT) Sec
Hemoglobin (HGB) g/dl
Hematocrit (HCT) %
Mean Corpuscular Volume (MCV) fL
Mean Corpuscular Hemoglobin (MCH) pg/cell
Mean Corpuscular Hemoglobin Concentration (MCHC) g/dl
Platelet Count (PLT) 109/L
Prothombin Time (PT) Sec
Total Erythrocyte Count (RBC) 1012/L
Total Leukocyte count (WBC) 109/L
HDL mg/dl
LDL mg/dl
Blood smears were prepared from the collected blood, stained with Leishman’s stain and observed for differential leukocyte counts (%). Blood smears for reticulocytes (%) were also prepared and evaluated.
Clinical Biochemistry
The following parameters were estimated in serum by using an auto analyzer (VITALAB Selectra E, Bimedis Inc, USA) and the EnSpire Multimode Plate Reader (Serial Number 23001588; Perkin Elmer Inc, USA).
ParametersP Unit
Alanine amino transferase (ALT) U/l
Aspartate amino transferase (AST) U/l
Albumin (Alb) g/dl
Alkaline phosphatase (ALP) U/l
Alb/ Glb (A:G) – Calculated -
Blood urea nitrogen (BUN) – Calculated mg/dl
Bile acids µmol/l
Cholesterol (Chol) mg/dl
Calcium mg/dl
Creatinine (Crea) mg/dl
Glucose (Glu) mg/dl
Gamma-Glutamyl Transpeptidase (GGT) U/I
Globulin (Glob) – Calculated g/dl
Phosphorus mg/dl
Triglycerides (TRIG) mg/dl
Total Protein (TP) g/dl
Total Bilirubin (T.Bil) mg/dl
Urea mg/dl
T3 pg/ml
T4 ng/ml
TSH µIU/ml
The following electrolytes were estimated in serum using the 9180 Electrolyte Analyzer (Serial number 24656; Roche Holding AG, Switzerland).
Sodium (Na) mmol/l
Potassium (K) mmol/l
Chloride (Cl) mmol/l
Urinalysis
Urine samples for urinalysis were collected on the day of sacrifice (prior to necropsy). The animals were housed in metabolic cages overnight and the urine samples were collected in the morning hours prior to necropsy. Water for drinking was available ad libitum during the fasting hours. Colour, appearance and volume of the urine were recorded by visual examinations and. the following parameters below were analyzed using the Uri Scan Optima- Semi Auto Urine Analyser (Serial number RS232C; YD Diagnostics Corp, South Korea).
Parameters Unit
Blood / Blood Cell RBC/µl
Bilirubin mg/dl
Glucose mg/dl
Ketone mg/dl
Leucocytes WBC/µl
Microscopic Parameters* -
Nitrite Positive/Negative
Protein mg/dl
pH -
Specific Gravity -
Urobilinogen mg/dl
*Microscopic parameters included observation of urine sediments (after centrifugation) for presence of epithelial cells, red blood cells, pus cells (white blood cells), casts, crystals and other sediments.
Bone Marrow Smear Examination
Bone marrow smear (from femur) were prepared at the time of necropsy. Since no treatment-related effects on erythropoiesis were noted during the hematologic evaluations, the bone marrow smear samples were not examined.
Pathology
Gross Pathology
All animals were sacrificed on Day 91 (after overnight fasting) and subjected to detailed gross pathological examinations. Each animal was weighed, euthanized by CO2 asphyxiation, and examined externally. All orifices and the cranial, thoracic, and visceral cavities were opened and examined macroscopically.
Organ Weight
Organs marked with asterisk below were trimmed of adherent tissue/fat and weighed, prior to preservation in a fixative. Organs were weighed immediately after they were collected. Organs were kept in normal saline till they were weighed. For paired organs, the combined weight was recorded.
Histopathology
Except the eyes and testes, which were fixed using Modified Davidson’s fluid, the organs listed in the table below were preserved in 10 % neutral buffered formalin (NBF) for the subsequent histopathological examination.
Adrenals* Pancreas
Aorta Peyer's Patches
Bone (femur) with joint # Pituitary * (weighing after fixation)
Brain (cerebrum,cerebellum,mid brain)* Prostate and Seminal vesicle with coagulating glands as a whole *
Cecum Rectum
Colon Salivary glands
Duodenum Sciatic Nerve
Epididymides * Skeletal muscle
Eyes with optic nerve Skin
Gross lesion (if any) Spinal Cord (cervical, mid-thoracic and lumbar)
Heart * Spleen *
Ileum Sternum with marrow #
Jejunum Stomach
Kidneys * Testes *
Liver * Thymus*
Lung $ Thyroid Parathyroids * (weighing after fixation)
Mammary glands Trachea
Mesenteric and Mandibular lymph node Urinary Bladder *
Oesophagus Uterus with Vagina *
Ovaries * -
# Decalcified prior to sectioning; *Organ Weight; $Inflated with 10 % NBF
Full histopathological examinations were carried out on the preserved organs and tissues of all animals in G1 and G4. Histopathological examinations on the kidneys were extended to G2 and G3, as treatment-related effects in the kidneys were observed in G4. All gross lesions were examined histologically. - Statistics:
- Raw data was analysed using statistical software Sigma Plot v14.0. All data was summarized in tabular form and the means and standard deviation were calculated using the software. All continuous data (body weight, percent change in body weight with respect to day 1, feed consumption, haematology, clinical chemistry, absolute and relative organ weights, etc. were checked first for their normality and homogeneity, and then analysed using one-way ANOVA (or unpaired t-test) for comparison of means. Heterogneous data was analysed using ANOVA on ranks Kruskal-Wallis test on ranks. For means found significantly different by ANOVA, Dunnett’s post-hoc test was applied to identify the group pairs with significant differences. P values less than or equal to 0.05 were accepted as being statistically significant.
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- No clinical signs of toxicity were observed at any dose level.
- Mortality:
- no mortality observed
- Description (incidence):
- All animals survived to scheduled sacrifice.
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No significant changes in body weight or body weight gain were observed, apart from a significant increase in body weight in female rats in G3 on day 78 (mean, 262.4 gram) compared to G1 (mean, 242.1 gram) that was considered incidental and not toxicologically relevant.
- Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- For male rats, no significant changes in food intake were observed. In female rats, significant increases in food intake were observed from day 8-15, 22-29, 29-36, and 36-43 in G2, and from day 1-8, 29-36, 39-43, and 50-57 in G3, as compared to G1. For the female rats in G4, a significant decrease in food intake was observed from day 15-22 (mean, 15.9 g/rat/day) as compared to G1 (mean, 17.2 g/rat/day). The observed changes in food intake in G2 and G3 lacked dose-dependency and for G2, G3, and G4, no associated effects on body weight or body weight gain were observed. The observed changes in food intake in the female aminals were therefore not considered to be toxicologically relevant
- Food efficiency:
- not examined
- Ophthalmological findings:
- no effects observed
- Description (incidence and severity):
- No eye abnormalities were observed in the study.
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Significant changes in hematology were limited to a significant increase mean corpuscular volume (MCV) in male rats in G4 (mean, 49.81 fL) compared to G1 (mean, 46.51 fL) and a significant increase in mean corpuscular hemoglobin (MCH) in male rats in G4 (mean, 17.64 pg/cell) compared to G1 (mean, 16.54 pg/cell). Kidneys are responsible for erythropoietin production and the observed changes in MCV and MCH in male rats in G4 could be corroborated with histopathological changes in the kidneys, most notably increased incidences of interstitial haemorrhages, as described below. Therefore, the observed changes in MCV and MCH in male rats in G4 were considered to be adverse effects of treatment. MCV tended to be higher in female rats in G4 (mean, 52.19 fL) compared to G1 (mean, 50.36 fL) and this plausible biologically significant effect could also be corroborated with a slight increase in the incidence of interstitial haemorrhages in the kidneys as described below.
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- For male rats, noteworthy changes in clinical chemistry included a significant increase in creatinine levels in G4 males (mean, 0.56 mg/dl) compared to G1 (mean, 0.48 mg/dl); significant increases in sodium levels in G2 (mean, 144.7 mmol/l), G3 (mean, 147.9 mmol/l) and G4 (mean, 151.3 mmol/l) compared to G1 (mean, 142.3 mmol/l); and significant increases in chloride levels in G3 (mean, 122.9 mmol/l) and G4 (mean, 115.8 mmol/l) compared to G1 (mean, 85.1 mmol/l). For female rats, noteworthy changes in clinical chemistry included a significant decrease in urea levels in G4 (mean, 27.7 mg/dl) compared to G1 (mean, 37.1 mg/dl); a significant increase in LDL cholesterol in G4 (mean, 12.6 mg/dl) compared to G1 (mean, 9.5 mg/dl); a significant decrease in blood urea nitrogen (BUN) in G4 (mean, 12.9 mg/dl) compared to G1 (mean, 17.4 mg/dl); significant increases in sodium levels in G3 (mean, 146.7 mmol/l) and G4 (mean, 150.9 mmol/l) compared to G1 (mean,141.9 mmol/l); and significant increases in chloride levels in G2 (mean, 105.3 mmol/l), G3 (mean, 111.9 mmol/l) and G4 (mean, 110.0 mmol/l) compared to G1 (mean, 91.9 mmol/l).
Kidneys usually play an important role in the regulation of fluid and electrolyte balance in the body and kidney damage can be associated with an abrupt reduction in renal function causing imbalance in electrolytes associated with various pathological conditions (Perlman RL,Heung M and Ix JH (2013). The observed changes in creatinine levels, serum electrolyte levels, urea, BUN in G3/G4 in male/females could be corroborated with histopathological findings in the kidneys as described below and were therefore considered adverse effects of treatment. The significant increase in LDL cholesterol in G4 females may be attributed to the test item in since lipid profile can be adversely affected by uremic toxins following kidney damage. As such, the significant increase in LDL cholesterol in G4 females was also considered to be an adverse effect of treatment. The significant changes in serum electrolyte levels in G2 in male and females could not be corroborated with any histopathological changes in the kidneys. For G2 female rats, the significant changes in serum electrolyte levels could be corroborated with significant changes in motoractivity as described below As such the significant changes in serum electrolytes in G2 females were considered to be adverse effects of treatment.
No significant changes in serum triiodothyronine (T3), thyroxine (T4), or thyroid stimulating hormone (TSH) were observed in any of the groups, except from a significant increase in TSH in G3 males (mean 0.49 µlU/ml) compared to G1 males (mean, 0.42 µlU/ml) that was considered incidental and not toxicologically relevant. - Behaviour (functional findings):
- effects observed, treatment-related
- Description (incidence and severity):
- In male rats, statistically/biologically significant changes in FOB included a significant decrease in stereotypic time in G4 (mean, 107.7 sec) compared to G1 (mean, 140.3 sec), a significant decrease in burst of stereotypic movements in G4 (mean, 76.1 counts) compared to G1 (mean, 96.2 counts), and a significant decrease in horizontal counts in G4 (mean, 1421.20 counts) when compared to G1 (mean, 1873.80 counts). In female rats, statistically/biologically significant changes in FOB included significant increases in distance travelled in G2 (mean, 2052.8 cm), G3 (mean, 2197.4 cm) and G4 (mean, 2405.5 cm) compared to G1 (mean, 1372.3 cm); significant decreases in resting time in G2 (mean, 152.5 sec), G3 (mean,167.8 sec) and G4 (mean, 144.7 sec) compared to G1 (mean, 253.6 sec); significant increases in ambulatory time in G2 (mean, 308.1 sec), G3 (mean, 303.7 sec) and G4 (mean, 331.3 sec) compared to G1 (mean, 215.1 sec); significant increases in horizontal counts in G2 (mean, 1821.3 counts), G3 (mean, 1804.7 counts) and G4 (mean, 1886.7 counts) compared to G1 (mean, 1207.0 counts); significant increases in ambulatory counts in G2 (mean, 1206.7 counts), G3 (mean, 1243.6 counts) and G4 (mean, 1293.0 counts) compared to G1 (mean, 752.7 counts); significant increases in clockwise rotations in G2 (mean, 27.5 counts), G3 (mean, 26.1 counts) and G4 (mean, 27.8 counts compared to G1 (mean, 15.3 counts); and a significant increases in counter clockwise rotations in G2 (mean, 26.5 counts), G3 (mean, 29.8 counts) and G4 (mean, 32.3 counts) compared to G1 (mean, 17.2 counts). These observed changes in motor activity in G4 males and in G2, G3, and G4 females were considered to be adverse effects of treatment.
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Significant changes in organ weight were limited to a significant increase in absolute spleen weight in G2 males (mean, 0.8922 gram) compared to G1 (mean, 0.7115 gram); a significant increase in uterus weight in G2 females (mean, 0.8057 gram) compared to G1 (mean, 0.5679 gram); and a significant decrease in the relative weight of the prostate with seminal vesicle in G3 males (mean, 0.5975) compared to G1 (mean, 0.7241). These observed changes in organ weight lacked dose-dependency and were therefore considered to be incidental and not toxicologically relevant.
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Gross findings were limited to one incidence of reduced testes (mild in severity) in one male rat in G2 and one incidence of haemorrhage in the thymus (minimal in severity) in one female rat in G3. None of these findings were attributed to the test item due to lack of dose dependency.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- The following histopathological findings were observed during the microscopic examinations:
Kidneys: Interstitial haemorrhages, minimal in severity (G3 Males: 4/10, G3 Females: 3/10, and G4 Females: 3/10); interstitial haemorrhages, mild in severity (G4 Males: 6/10 and G4 Females: 2/10); interstitial MNC infiltration, minimal in severity (G4 Males: 3/10, G1 Females: 1/10, and G3 Females: 1/10); tubular cast, minimal in severity (G4 Females: 1/10); and tubular cast, mild in severity (G4 Females: 4/10). These observed lesions in G3 and G4 could be corroborated with changes in haematology and clinical biochemistry data and were considered to be adverse.
Liver: Microgranuloma, minimal in severity (G1 Males: 1/10, G4 Males: 3/10, G1 Females: 3/10, and G4 Females: 1/10); single cell necrosis, minimal in severity (G1 Male: 1/10); and MNC infiltration, minimal in severity (G4 Males: 1/10, G1 Females: 2/10 and G4 Females: 4/10). These observed lesions in G1 and G4 were considered incidental in nature and therefore not toxicologically relevant.
Testis: Bilateral tubular degeneration and atrophy, minimal in severity (G2 Males: 1/10). This observation lacked dose dependency and was therefore considered to be incidental in nature.
Thymus:Haemorrhage, minimal in severity (G3 Females: 1/10). This observation lacked dose dependency and was therefore considered to be incidental in nature. - Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- no effects observed
- Description (incidence and severity):
- No significant changes in estrous cyclicity were observed.
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 30 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- behaviour (functional findings)
- serum/plasma biochemistry
- Key result
- Dose descriptor:
- NOAEL
- Based on:
- test mat.
- Sex:
- female
- Remarks on result:
- not determinable because of methodological limitations
- Remarks:
- NOAEL for female rats could not be established due to adverse effects observed at 30 mg/kg bw/day.
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 120 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- histopathology: non-neoplastic
- serum/plasma biochemistry
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 30 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- behaviour (functional findings)
- body weight and weight gain
- clinical biochemistry
- clinical signs
- food consumption and compound intake
- gross pathology
- haematology
- histopathology: neoplastic
- histopathology: non-neoplastic
- mortality
- ophthalmological examination
- organ weights and organ / body weight ratios
- serum/plasma biochemistry
- serum/plasma hormone analyses
- urinalysis
- Key result
- Critical effects observed:
- no
- Conclusions:
- Based on the above data and under the experimental conditions used in this study, the following effect levels were derived:
• NOAEL for male rats was considered to be 30 mg/kg bw/day.
• LOAEL for male rats was considered to be 120 mg/kg bw/day based on significant changes in clinical biochemistry (electrolyte levels) and histopathological findings in the kidneys (interstitial haemorrhage of minimal in severity) at 120 mg/kg bw/day.
• NOAEL for female rats could not be established due to adverse effects observed at 30 mg/kg bw/day.
• LOAEL for female rats was considered to be 30 mg/kg bw/day based on biologically significant changes in FOB and clinical biochemistry (electrolyte levels) at 30 mg/kg bw/day. - Executive summary:
The objective of this study (performed according to OECD 408 and GLP) was to provide information on the effects of repeated oral administration of Tetrabutyl ammonium bromide (CAS No. 1643-19-2) once a day in Wistar rats for a minimum period of 90 days. A total of 80 Wistar rats (40 males and 40 females) were randomised into four experimental groups, with each group containing 10 animals per sex. The groups, viz., G1, G2, G3 and G4 received 0 (vehicle), 30, 120 and 360 mg/kg bw/day of the test item, respectively. Dose administration was through oral gavage and the vehicle used in this study was distilled water. All groups were treated with the test item for a period of 90 days, afterwhich all animals were euthanized. Each dose in the study was given at a fixed volume of 10 ml/kg bw, based on the most recently recorded body weight for the animal. To ensure accurate administration of the test item, the concentrations and homogeneity of the dose formulations were verified through formulation analysis on three occasions i.e. on Day 2, Day 43 and Day 85 of treatment. The results were found within acceptable limits on all occasions. Observations on the animals encompassed mortality/morbidity, clinical signs, detailed clinical examinations, body weight, body weight changes, feed consumption, ophthalmological examination, functional observatory battery (FOB), hematology, clinical biochemistry, urinalysis, organ weight, gross pathology, and histopathology.
No mortality and no clinical signs of toxicity were observed at any dose level. No significant changes in body weight, body weight gain, or food intake attributed to the test item were observed. No eye abnormalities were observed in the study. In male rats, statistically/ biologically significant changes in FOB included a significant decrease in stereotypic time in G4 (mean, 107.7 sec) compared to G1 (mean, 140.3 sec), a significant decrease in burst of stereotypic movements in G4 (mean, 76.1 counts) compared to G1 (mean, 96.2 counts), and a significant decrease in horizontal count in G4 (mean, 1421.20 counts) when compared to G1 (mean, 1873.80 counts). In female rats, statistically/biologically significant changes in FOB included significant increases in distance travelled in G2 (mean, 2052.8 cm), G3 (mean, 2197.4 cm) and G4 (mean, 2405.5 cm) compared to G1 (mean, 1372.3 cm); significant decreases in resting time in G2 (mean, 152.5 sec), G3 (mean, 167.8 sec) and G4 (mean, 144.7 sec) compared to G1 (mean, 253.6 sec); significant increases in ambulatory time in G2 (mean, 308.1 sec), G3 (mean, 303.7 sec) and G4 (mean, 331.3 sec) compared to G1 (mean, 215.1 sec); significant increases in horizontal counts in G2 (mean, 1821.3 counts), G3 (mean, 1804.7 counts) and G4 (mean, 1886.7 counts) compared to G1 (mean, 1207.0 counts); significant increases in ambulatory counts in G2 (mean, 1206.7 counts), G3 (mean, 1243.6 counts) and G4 (mean, 1293.0 counts) compared to G1 (mean, 752.7 counts); significant increases in clockwise rotations in G2 (mean, 27.5 counts), G3 (mean, 26.1 counts) and G4 (mean, 27.8 counts compared to G1 (mean, 15.3 counts); and a significant increases in counter clockwise rotations in G2 (mean, 26.5 counts), G3 (mean, 29.8 counts) and G4 (mean, 32.3 counts) compared to G1 (mean, 17.2 counts). These observed changes in motor activity in G4 males and in G2, G3, and G4 females were considered to be adverse effects of treatment.For male rats, noteworthy changes in clinical chemistry included a significant increase in creatinine levels in G4 males (mean, 0.56 mg/dl) compared to G1 (mean, 0.48 mg/dl); significant increases in sodium levels in G2 (mean, 144.7 mmol/l), G3 (mean, 147.9 mmol/l) and G4 (mean, 151.3 mmol/l) compared to G1 (mean, 142.3 mmol/l); and significant increases in chloride levels in G3 (mean, 122.9 mmol/l) and G4 (mean, 115.8 mmol/l) compared to G1 (mean, 85.1 mmol/l). For female rats, noteworthy changes in clinical chemistry included a significant decrease in urea levels in G4 (mean, 27.7 mg/dl) compared to G1 (mean, 37.1 mg/dl); a significant increase in LDL cholesterol in G4 (mean, 12.6 mg/dl) compared to G1 (mean, 9.5 mg/dl); a significant decrease in blood urea nitrogen (BUN) in G4 (mean, 12.9 mg/dl) compared to G1 (mean, 17.4 mg/dl); significant increases in sodium levels in G3 (mean, 146.7 mmol/l) and G4 (mean, 150.9 mmol/l) compared to G1 (mean, 141.9 mmol/l); and significant increases in chloride levels in G2 (mean, 105.3 mmol/l), G3 (mean, 111.9 mmol/l) and G4 (mean, 110.0 mmol/l) compared to G1 (mean, 91.9 mmol/l). The observed changes in creatinine levels, serum electrolyte levels, urea, BUN in G3 and/or G4 in male and/or females could be corroborated with histopathological findings in the kidneys and were therefore considered adverse effects of treatment. The significant increase in LDL cholesterol in G4 females may be attributed to the test item in since lipid profile can be adversely affected byuremic toxins following any damage to the kidneys. As such, the significant increase in LDL cholesterol in G4 females was also considered to be an adverse effect of treatment. The significant changes in serum electrolyte levels in G2 in male and females could not be corroborated with any histopathological changes in the kidneys. For G2 female rats, the significant changes in serum electrolyte levels could be corroborated with significant changes in motoractivity. As such the significant changes in serum electrolytes in G2 females were considered to be adverse effects of treatment. No significant changes in serum triiodothyronine (T3), thyroxine (T4), or thyroid stimulating hormone (TSH) were observed in any of the groups, except from a significant increase in TSH in G3 males (mean 0.49 µlU/ml) compared to G1 males (mean, 0.42 µlU/ml) that was considered incidental and not toxicologically relevant. Significant changes in hematology were limited to a significant increase mean corpuscular volume (MCV) in male rats in G4 (mean, 49.81 fL) compared to G1 (mean, 46.51 fL) and a significant increase in mean corpuscular hemoglobin (MCH) in male rats in G4 (mean, 17.64 pg/cell) compared to G1 (mean, 16.54 pg/cell). Kidneys are responsible for erythropoietin production and the observed changes in MCV and MCH in male rats in G4 could be corroborated with histopathological changes in the kidneys, most notably increased incidences of interstitial haemorrhages. Therefore, the observed changes in MCV and MCH in male rats in G4 were considered to be adverse effects of treatment. MCV tended to be higher in female rats in G4 (mean, 52.19 fL) compared to G1 (mean, 50.36 fL) and this plausible biological effect could also be corroborated with a slight increase in the incidence of interstitial haemorrhages in the kidneys. No significant changes in absolute or relative organ weight attributed to the test item were observed. No gross findings attributed to the test item were observed. Histopathological findings attributed to the test item were observed exclusively in the kidneys in G3 and G4 and included the following: interstitial haemorrhages, minimal in severity (G3 Males: 4/10, G3 Females: 3/10, and G4 Females: 3/10); interstitial haemorrhages, mild in severity (G4 Males: 6/10 and G4 Females: 2/10); interstitial MNC infiltration, minimal in severity (G4 Males: 3/10, G1 Females: 1/10, and G3 Females: 1/10); tubular cast, minimal in severity (G4 Females: 1/10); and tubular cast, mild in severity (G4 Females: 4/10). These observed lesions in G3 and G4 could be corroborated with changes in haematology and clinical biochemistry data and were considered to be adverse.
Based on the above data and under the experimental conditions used in this study, the following effect levels were derived:
- NOAEL for male rats was considered to be 30 mg/kg bw/day.
- LOAEL for male rats was considered to be 120 mg/kg bw/day based on significant changes in clinical biochemistry (electrolyte levels) and histopathological findings in the kidneys (interstitial haemorrhage of minimal in severity) at 120 mg/kg bw/day.
- NOAEL for female rats could not be established due to adverse effects observed at 30 mg/kg bw/day.
- LOAEL for female rats was considered to be 30 mg/kg bw/day based on biologically significant changes in FOB and clinical biochemistry (electrolyte levels) at 30 mg/kg bw/day.
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- Data is from a study report
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
- Principles of method if other than guideline:
- According to OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
- GLP compliance:
- yes
- Limit test:
- yes
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- Details on test animal
TEST ANIMALS
- Source: National Institute of Biosciences, Pune.
- Age at study initiation: 6 to 8 weeks old
- Weight at study initiation:
Male 167.38 g
Female 148.02 g
- Fasting period before study: No data available
- Housing: Animal was housed in polycarbonate cages. Three rats of same sex were housed together in each cage of size 39 cm X 28 cm X 14 cm. Paddy husk was used as bedding material.
- Diet (e.g. ad libitum): Rodent feed supplied by the Nutrivet Life Sciences, Pune, was provided ad libitum from individual feeders on cage top.
- Water (e.g. ad libitum): Water was provided ad libitum from individual bottles attached to the cages. Water was from a local source and passed through the reverse osmosis membrane before use.
- Acclimation period: 5 days prior to dosing.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C (actual range: 19.5 °C to 22.4 °C)
- Humidity (%):30% to 70% (actual range: 52.9% to 58.3%).
- Air changes (per hr): ten air changes per hour of 100% fresh air that has been passed through the HEPA filters.
- Photoperiod (hrs dark / hrs light): An artificial light and dark cycle of 12 hours each were provided.
IN-LIFE DATES:
Male: From: 10-06-2014
To: 08-07-2014
Female : From: 11-06-2014
To: 09-07-2014 - Route of administration:
- oral: gavage
- Vehicle:
- water
- Remarks:
- distill water
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS: The test chemical was dissolved in distilled water for preparation of solution(s). The solution(s) of test chemical was made at volumes suitable for daily use for 28 days. The solution(s) were prepared at concentrations of 0, 25, 50 and 100 mg/ml such that dosage of 0 (vehicle), 250, 500 and 1000 mg/kg body weight respectively were administered. The concentration of the test chemical was varied so as to maintain the dose volume constant at or upto 10 ml/kg body weight.
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): Distilled water was used as a vehicle.
- Concentration in vehicle: The solution(s) were prepared at concentrations of 0, 25, 50 and 100 mg/ml such that dosage of 0 (vehicle), 250, 500 and 1000 mg/kg body weight.
- Amount of vehicle (if gavage): 10 ml/kg bw
- Lot/batch no. (if required): No data available
- Purity: No data available - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The test chemical formulation samples were prepared day 1 (pre-dosing) and analyzed by HPLC analysis.
- Duration of treatment / exposure:
- 28 days consecutively
- Frequency of treatment:
- Once daily
- Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Remarks:
- Control Group (G1)
- Dose / conc.:
- 250 mg/kg bw/day (actual dose received)
- Remarks:
- Low Dose Group (G2)
- Dose / conc.:
- 500 mg/kg bw/day (actual dose received)
- Remarks:
- Mid Dose Group (G3)
- Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- Remarks:
- High Dose Group (G4)
- No. of animals per sex per dose:
- 0 mg/kg bw/day: 6 male, 6 female
250 mg/kg bw/day: 6 male, 6 female
500 mg/kg bw/day: 6 male, 6 female
1000 mg/kg bw/day: 6 male, 6 female - Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Based on results from a preliminary 14-day study their were no chenge in the survivel, body weight, Daily clinical observations and Gross pathological examination of 1000 mg/kg/bw/day group. Based on these results, the 28 day study dose levels were finalized as 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg body weight.
- Rationale for animal assignment (if not random): Animals were randomized by body weight.
- 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:
- No Data Available
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: No data available
- Cage side observations checked in table [No.?] were included. : Rats were observed for Behavior, Alterations, Vocalizations, Respiration and Palpebral closure.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily, At least once a week thereafter until scheduled sacrifice.
BODY WEIGHT: Yes
- Time schedule for examinations: On the day of randomization, first day of dosing, weekly thereafter and a fasting body weight at scheduled sacrifice on day 29.
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:
- 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: No Data Available
- Time schedule for examinations: No Data Available
- Dose groups that were examined: No Data Available
HAEMATOLOGY: Yes, By using Beckman Coulter haematology analyzer.
- Time schedule for collection of blood: At termination.
- Anaesthetic used for blood collection: No data available
- Animals fasted: Yes, overnight fasted prior to sampling.
- How many animals: Blood collected from all rats of 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg bw/day group at termination.
- Parameters checked/examined. : Hemoglobin, Red Blood Corpuscles, Hematocrit, Mean Corpuscular Volume, Mean Corpuscular Hemoglobin, Mean Corpuscular Hemoglobin Concentration, Platelets, White Blood Corpuscles, Neutrophils, Lymphocytes, Eosinophils, Monocytes, Basophil and Prothrombin time were checked.
CLINICAL CHEMISTRY: Yes, By using Dimension XpandPlus and Acculyte 5P.
- Time schedule for collection of blood: At termination.
- Animals fasted: Yes, overnight fasted prior to sampling.
- How many animals: Blood collected from all rats of 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg bw/day group at termination.
- Parameters checked/examined. : Total Protein, Blood Urea Nitrogen, Urea Nitrogen, Alanine Aminotransferase, Aspartate Aminotransferase, Alkaline Phosphatase, Gamma Glutamyl Transferase, Glucose, Calcium, Phosphorous, Albumin, Total Bilirubin, Creatinine , Total Cholesterol, Triglycerides, Globulin Calculated
Sodium, Potassium, Chloride were checked.
URINALYSIS: No data available
- Time schedule for collection of urine: No data available
- Metabolism cages used for collection of urine: No data available
- Animals fasted: No data available
- Parameters checked in table [No.?] were examined. : No data available
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
OTHER: Viability, Behavior in Home cage, Vocalizations, Respiration, Palpebral closure, Handling Observations, Urination, Defecation, Prominence of Eye, Lacrimation, Salivation, Piloerection, Examination of Skin / Fur, Stereotype Behaviour, Rearing (Rears), Clonic and Tonic Movements and Severity of Gait were observed. - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
Gross necropsy was conducted. All the animals of 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg/bw/day group were sacrificed and gross lesions were noted.
HISTOPATHOLOGY: Yes
Control and treated at the highest dose level of 1000 mg/kg were subjected to sacrifice.
Organs examined: Adrenals, Aorta, Brain (cerebrum, cerebellum and pons), Caecum, Cervix, Colon, Duodenum, Epididymides, Eyes, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs, Mesenteric Lymphnodes, Oesophagus, Ovaries, Pancreas, Pituitary gland, Pharyngeal Lymphnodes, Prostate, Rectum, Skeletal Muscles, Skin with Mammary Gland, Spleen, Sternum with bone marrow, Sciatic Nerve, Spinal Cord (Cervical, mid thoracic and lumbar), Stomach, Seminal Vesicles with Coagulation Gland, Testes, Thymus, Thyroid, Trachea, Vagina, Urinary Bladder and Uterus of 1000 mg/kg/bw/day group. - Other examinations:
- No Data Available
- Statistics:
- Data were analyzed for reporting group means and standard deviations with significance between the controls and treated groups, using in-house developed and validated MS-Excel 2003 based statistical software. All the parameters characterized by continuous data such as body weight, per cent body weight change, feed consumption, organ weight, relative organ weight, haematological and clinical chemistry data were subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analyses of Variance (ANOVA) and Dunnett’s t-test. Where the data did not meet the homogeneity of variance, Student’s t-test was performed to calculate significance.
Significance was calculated at 1% as well as 5% level and indicated in the summary tables as follows:
* = Significant than control at 95% level of confidence (p≤ 0.05).
** = Significant than control at 99% level of confidence (p≤ 0.01). - Clinical signs:
- no effects observed
- Description (incidence and severity):
- Daily clinical observations did not reveal any signs of toxicity in male and female animals from of 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg bw/day dose groups
- Mortality:
- no mortality observed
- Description (incidence):
- All animals survived throughout the study duration of 28 days.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- All the rat of 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg/bw/day dose groups exhibited normal body weight gain at the end of the study period of 28 days.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- Food intake of animals for control and 250 mg/kg, 500 mg/kg and 1000 mg/kg /bw/day dose groups were found to be normale throughout the study period of 28 days.
- Food efficiency:
- not specified
- Water consumption and compound intake (if drinking water study):
- not specified
- Ophthalmological findings:
- not specified
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- In males, Hb, Total RBC, HCT and MCV values were increased in all the dose groups. Also, MCH values were also increased for animals from 1000 mg/kg dose group. The MCHC values were decreased for animals from 250 mg/kg, 500 mg/kg and 1000 mg/kg dose groups. In females, in creased MCV values were observed for animals from 250 mg/kg and 1000 mg/kg dose groups. Also, increased MCH values were obtained for animals from 250 mg/kg and 1000 mg/kg. Decreased MCHC values were obtained for animals from 1000 mg/kg dose group.
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Biochemical investigations conducted at the end of dosing period on day 29, revealed following significant changes in the values of different parameters studied when compared with that of respective controls.
Male :
Bilirubin: Elevated levels were observed in animals from 500 mg/kg dose group (p≤0.05),
Sodium: Elevated levels were observed in animals from 250 mg/kg (p≤0.01) and 1000 mg/kg (p≤0.05) dose groups,
Chloride: Elevated levels were observed in animals from 250 mg/kg and 1000 mg/kg dose groups (p≤0.01),
Alanine Aminotransferase: Decreased levels were observed in animals from 250 mg/kg, 500 mg/kg and 1000 mg/kg dose groups (p≤0.05) and
Cholesterol: Decreased levels were observed in animals from 250 mg/kg dose group (p≤0.01).
Female :
Aspartate Aminotransferase: Elevated levels were observed in animals from 250 mg/kg dose group (p≤0.05),
Calcium: Elevated levels were observed in animals from 250 mg/kg and 500 mg/kg dose groups (p≤0.05),
Phosphorous: Elevated levels were observed in animals from 250 mg/kg (p≤0.01), 500 mg/kg (p≤0.05) and 1000 mg/kg (p≤0.01) dose groups,
Globulin: Elevated levels were observed in animals from 500 mg/kg dose group (p≤0.05),
Chloride: Elevated levels were observed in animals from 250 mg/kg and 500 mg/kg dose groups (p≤0.01) and
Blood Urea Nitrogen and Urea Nitrogen: Decreased levels were observed in animals from 1000 mg/kg dose group (p≤0.05). - Urinalysis findings:
- not specified
- Behaviour (functional findings):
- effects observed, non-treatment-related
- Description (incidence and severity):
- Sensory Reactivity Observations: All animals from control and different dose groups showed normal arousal level, visual response, touch response, auditory response, tail pinch response and visual placing response. Normal air righting reflex was observed in all animals from control and different dose groups in week 4.
Grip Strength: Lower values were observed in male animals from 250 mg/kg dose group (p≤0.05). This change was not dose dependent and was considered to be of no toxicological importance. Grip strength values observed in female animals for control and different dose groups were comparable.
Motor Activity: Higher values were observed in male animals from 250 mg/kg and 1000 mg/kg dose groups for first interval (p≤0.05). Lower values were observed in female animals from 1000 mg/kg dose group for first interval (p≤0.01) and for second interval (p≤0.05). These changes were within laboratory range and were considered to be of no toxicological importance. - Immunological findings:
- not specified
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Description (incidence and severity):
- In males, in comparison with controls organ weight data of male animals sacrificed on day 29, revealed increased relative weight of adrenals of animals from 250 mg/kg dose group (p≤0.01). In females. In comparison with controls organ weight data of female animals sacrificed on day 29, revealed increased relative weight of lungs of animals from 500 mg/kg dose group (p≤0.05) and increased relative weight of uterus of animals from 250 mg/kg dose group (p≤0.05). Although significant change in organ weights were observed in animals from different dose groups, no related gross pathological or histological changes were seen and findings were not dose dependent and hence considered to be of no toxicological importance.
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- Gross pathological examination in male and female animals from control and different treatment groups did not reveal any abnormality.
- Neuropathological findings:
- not specified
- Histopathological findings: non-neoplastic:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Histopathological examination revealed minimal focal to multifocal periportal mononuclear cell infiltration in the liver; minimal interstitial haemorrhages and/or mononuclear cells infiltration in the kidneys; minimal alveolar haemorrhages in the lungs; minimal multifocal haemosiderosis and/or diffused congestion in spleen; minimal eosinophilic infiltration and/or luminal dilatation in uterus; minimal luminal seminal coagulum in urinary bladder; minimal dilatation of zona reticularis and/or presence of accessory adrenocortical tissue in adrenals; minimal focal to multifocal haemorrhages in thymus; presence of ultimobranchial cysts in thyroid; in male or female animals from control and high dose group. All the changes observed in the control and high dose treatment group animals were similar, incidental and mode of death related, physiological and are covered in the facility historical data of the histopathology findings.
- Histopathological findings: neoplastic:
- not specified
- Other effects:
- not specified
- Details on results:
- Clinical signs and mortality:
Clinical signs: Daily clinical observations did not reveal any signs of toxicity in male and female animals from of 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg bw/day dose groups.
Mortality: No mortality were observed in any of the traeted groups of 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg bw/day.
Body weight and weight gain: All the rat of 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg/bw/day dose groups exhibited normal body weight gain at the end of the study period of 28 days.
Food consumption and compound intake: Food intake of animals for control and 250 mg/kg, 500 mg/kg and 1000 mg/kg /bw/day dose groups were found to be normal throughout the study period of 28 days.
Haematology: Statistically significant increase in the values of Hb, Total RBC, HCT and MCV of male rats dosed at 250 mg/kg, 500 mg/kg and 1000 mg/kg/bw/day, increased values of MCH of male rats dosed at 1000 mg/kg were observed. Increased values of MCV and MCH of female rats dosed at 250 mg/kg and 1000 mg/kg. In addition statistically significant decrease was observed in the values of MCHC of male rats dosed at 250 mg/kg, 500 mg/kg and 1000 mg/kg and female rats dosed at 1000 mg/kg. The increase / decrease in the values of different parameters were marginal and within the normal limits .
Clinical chemistry:
Significant increase of Bilirubin levels in male rats dosed of Tetra butyl ammonium bromide were observed at 500 mg/kg/bw/day.
Significant increase of Sodium and Chloride levels in male rats dosed at 250 mg/kg and 1000 mg/kg of the test chemical.
Significant increase of Aspartate Aminotransferase levels in female rats dosed at 250 mg/kg of the test chemical was observed.
Increase of Calcium and Chloride levels in female rats dosed at 250 mg/kg and 500 mg/kg of the test chemical.
Significant increase of Phosphorous levels was found in female rats dosed at 250 mg/kg, 500 mg/kg and 1000 mg/kg of the test chemical.
Statistically significant increase of Globulin levels was found in female rats dosed at 500 mg/kg of the test chemical.
Decrease level of Alanine Aminotransferase was found in male rats dosed at 250 mg/kg, 500 mg/kg and 1000 mg/kg of the test chemical.
Decrease level of Cholesterol was found in male rats dosed at 250 mg/kg.Statistically significant decrease level of Blood Urea Nitrogen and Urea Nitrogen in female rats dosed at 1000 mg/kg of the test chemical. Although there was an increase/decrease in the values of various biochemical parameters as mentioned above, the deviations were marginal and within the range of normal limits.
Neurobehavioural parameters:
Sensory Reactivity Observations: All animals from control and different dose groups showed normal arousal level, visual response, touch response, auditory response, tail pinch response and visual placing response. Normal air righting reflex was observed in all animals from control and different dose groups in week 4.
Grip Strength: Lower values were observed in male animals from 250 mg/kg dose group (p≤0.05). This change was not dose dependent and was considered to be of no toxicological importance. Grip strength values observed in female animals for control and different dose groups were comparable.
Motor Activity: Higher values were observed in male animals from 250 mg/kg and 1000 mg/kg dose groups for first interval (p≤0.05). Lower values were observed in female animals from 1000 mg/kg dose group for first interval (p≤0.01) and for second interval (p≤0.05). These changes were within laboratory range and were considered to be of no toxicological importance.
Organ weights: Organ weight of female animals revealed increased relative weights of liver of animals from 250 mg/kg and 500 mg/kg dose groups, increased relative weights of kidneys of animals from 250 mg/kg dose group and decreased relative weights of spleen of animals from 1000 mg/kg dose group.
Although significant change in organ weights were observed in animals from different dose groups, but the effect was not due to the test chemical.
Male animals revealed increased relative weights of adrenals at 250 mg/kg dose group. Female animals revealed increased relative weights of lungs at 500 mg/kg dose group and increased relative weights of uterus at 250 mg/kg dose group of the test chemical. Although significant change in organ weights were observed in 250 mg/kg/bw/day and 500 mg/kg/bw/day dose groups, But, findings were not dose dependent and hence considered to be of no toxicological importance.
Gross pathology: In male and female animals from control and 250 mg/kg/bw/day, 500 mg/kg/bw/day and 1000 mg/kg/bw/day of the test chemical. All the treatment groups did not reveal any abnormality.
Histopathology: Focal to multifocal periportal mononuclear cell infiltration in the liver; minimal interstitial haemorrhages and/or tubular dilatation in the kidneys; minimal alveolar haemorrhages and/or alveolar histiocytosis in the lungs; minimal multifocal haemosiderosis and/or diffused congestion in spleen; minimal eosinophilic infiltration and/or luminal dilatation and/or endometrial gland dilatation in uterus; minimal dilatation of zona reticularis and/or presence of accessory adrenocortical tissue in adrenals; minimal multifocal haemorrhages in thymus; presence of ultimobranchial cysts in thyroid; in male or female animals from control and high dose group. All the changes observed in the control and 1000 mg/kg/bw/day dose group animals were similar and there Is no toxic effect of the test chemical was found. - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/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: non-neoplastic
- mortality
- organ weights and organ / body weight ratios
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Key result
- Critical effects observed:
- no
- Conclusions:
- Based on all the available data, it was concluded that the test chemical did not cause any systemic toxicity at the highest dose tested. Therefore, it was concluded that the No observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg bw/day when Sprague-Dawley rats exposed to the test chemical orally for 28 days.
- Executive summary:
A subacute study according to the OECD TG 407 was conducted under GLP conditions to evaluate the toxic effects of repeated administration of test chemical in male and female Sprague-Dawley rats by gavage. The test chemical was administered to 6 animals/sex/species in distilled water at doses of 0, 250, 500 and 1000 mg/kg/bw/day for 28 days. Daily clinical observations did not reveal any signs of toxicity in male and female animals from of 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg bw/day dose groups. All animals survived throughout the study duration of 28 days. All the rat of 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg/bw/day dose groups exhibited normal body weight gain at the end of the study period of 28 days. Food intake of animals for control and 250 mg/kg, 500 mg/kg and 1000 mg/kg /bw/day dose groups were found to be normal throughout the study period of 28 days. In males, Hb, Total RBC, HCT and MCV values were increased in all the dose groups. Also, MCH values were also increased for animals from 1000 mg/kg dose group. The MCHC values were decreased for animals from 250 mg/kg, 500 mg/kg and 1000 mg/kg dose groups. In females, increased MCV values were observed for animals from 250 mg/kg and 1000 mg/kg dose groups. Also, increased MCH values were obtained for animals from 250 mg/kg and 1000 mg/kg. Decreased MCHC values were obtained for animals from 1000 mg/kg dose group. Biochemical investigations conducted at the end of dosing period on day 29, revealed following significant changes in the values of different parameters studied when compared with that of respective controls. In males, Elevated bilirubin levels were observed in animals from 500 mg/kg dose group (p≤0.05), Elevated sodium levels were observed in animals from 250 mg/kg (p≤0.01) and 1000 mg/kg (p≤0.05) dose groups, Elevated chloride levels were observed in animals from 250 mg/kg and 1000 mg/kg dose groups (p≤0.01), Decreased Alanine Aminotransferase levels were observed in animals from 250 mg/kg, 500 mg/kg and 1000 mg/kg dose groups (p≤0.05) and Decreased Cholesterol levels were observed in animals from 250 mg/kg dose group. In females, Elevated Aspartate aminotransferase levels were observed in animals from 250 mg/kg dose group (p≤0.05), Elevated calcium levels were observed in animals from 250 mg/kg and 500 mg/kg dose groups (p≤0.05), Elevated phosphorous levels were observed in animals from 250 mg/kg (p≤0.01), 500 mg/kg (p≤0.05) and 1000 mg/kg (p≤0.01) dose groups, Elevated globulin levels were observed in animals from 500 mg/kg dose group, Elevated chloride levels were observed in animals from 250 mg/kg and 500 mg/kg dose groups (p≤0.01) and Decreased Blood Urea Nitrogen and Urea Nitrogen levels were observed in animals from 1000 mg/kg dose group (p≤0.05). All animals from control and different dose groups showed normal arousal level, visual response, touch response, auditory response, tail pinch response and visual placing response. Normal air righting reflex was observed in all animals from control and different dose groups in week 4. Lower values of grip strength were observed in male animals from 250 mg/kg dose group (p≤0.05). This change was not dose dependent and was considered to be of no toxicological importance. Grip strength values observed in female animals for control and different dose groups were comparable. Higher values of motor activity were observed in male animals from 250 mg/kg and 1000 mg/kg dose groups for first interval (p≤0.05). Lower values were observed in female animals from 1000 mg/kg dose group for first interval (p≤0.01) and for second interval (p≤0.05). These changes were within laboratory range and were considered to be of no toxicological importance. In males, in comparison with controls organ weight data of male animals sacrificed on day 29, revealed increased relative weight of adrenals of animals from 250 mg/kg dose group (p≤0.01). In females. In comparison with controls organ weight data of female animals sacrificed on day 29, revealed increased relative weight of lungs of animals from 500 mg/kg dose group (p≤0.05) and increased relative weight of uterus of animals from 250 mg/kg dose group (p≤0.05). Although significant change in organ weights were observed in animals from different dose groups, no related gross pathological or histological changes were seen and findings were not dose dependent and hence considered to be of no toxicological importance. Gross pathological examination in male and female animals from control and different treatment groups did not reveal any abnormality. Histopathological examination revealed minimal focal to multifocal periportal mononuclear cell infiltration in the liver; minimal interstitial hemorrhages and/or mononuclear cells infiltration in the kidneys; minimal alveolar hemorrhages in the lungs; minimal multifocal haemosiderosis and/or diffused congestion in spleen; minimal eosinophilic infiltration and/or luminal dilatation in uterus; minimal luminal seminal coagulum in urinary bladder; minimal dilatation of zona reticularis and/or presence of accessory adrenocortical tissue in adrenals; minimal focal to multifocal hemorrhages in thymus; presence of ultimo branchial cysts in thyroid; in male or female animals from control and high dose group. All the changes observed in the control and high dose treatment group animals were similar, incidental and mode of death related, physiological and are covered in the facility historical data of the histopathology findings. Based on all the available data, it was concluded that the test chemical did not cause any systemic toxicity at the highest dose tested. Therefore, it was concluded that the No observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg bw/day when Sprague-Dawley rats exposed to the test chemical orally for 28 days.
Referenceopen allclose all
Table 1 :Summary of Mortality and Morbidity
Sex: Male
Group | Dose (mg/kg bw) | No. of Incidence / No. of Animals | Observation |
G1 | 0 | 10/10 | No mortality/ No morbidity |
G2 | 30 | 10/10 | No mortality/ No morbidity |
G3 | 120 | 10/10 | No mortality/ No morbidity |
G4 | 360 | 10/10 | No mortality/ No Morbidity |
Sex: Female
Group | Dose (mg/kg bw) | No. of Incidence / No. of Animals | Observation |
G1 | 0 | 10/10 | No mortality/ No morbidity |
G2 | 30 | 10/10 | No mortality/ No morbidity |
G3 | 120 | 10/10 | No mortality/ No morbidity |
G4 | 360 | 10/10 | No mortality/ No Morbidity |
Table 2: Summary of General Clinical Signs Observation
Sex: Male
Group | Dose (mg/kg bw) | Clinical Signs | No. of Incidence / No. of Animals |
G1 | 0 | Normal | 10/10 |
G2 | 30 | Normal | 10/10 |
G3 | 120 | Normal | 10/10 |
G4 | 360 | Normal | 10/10 |
Sex: Female
Group | Dose (mg/kg bw) | Clinical Signs | No. of Incidence / No. of Animals |
G1 | 0 | Normal | 10/10 |
G2 | 30 | Normal | 10/10 |
G3 | 120 | Normal | 10/10 |
G4 | 360 | Normal | 10/10 |
Table 3: Detailed Clinical Examinations
Sex: Male | |||
Group | Dose (mg/kg body weight) | Clinical Signs | No. of Incidence / No. of Animals |
G1 | 0 | No Abnormalities Detected | 10/10 |
G2 | 30 | No Abnormalities Detected | 10/10 |
G3 | 120 | No Abnormalities Detected | 10/10 |
G4 | 360 | No Abnormalities Detected | 10/10 |
Key: NAD; No abnormalities detected.
Sex: Female | |||
Group | Dose (mg/kg body weight) | Clinical Signs | No. of Incidence / No. of Animals |
G1 | 0 | No Abnormalities Detected | 10/10 |
G2 | 30 | No Abnormalities Detected | 10/10 |
G3 | 120 | No Abnormalities Detected | 10/10 |
G4 | 360 | No Abnormalities Detected | 10/10 |
Key: NAD; No abnormalities detected.
Table 4: Summary of Ophthalmological Examination
Week: Terminal
Group | G1 | G4 | G1 | G4 | |
Sex | Male | Female | |||
Dose (mg/kg bw) | 0 | 360 | 0 | 360 | |
Eye | Observation | No. of Incidence / No. of Animals | |||
Terminal | |||||
Right | NAD | 10/10 | 10/10 | 10/10 | 10/10 |
Left | NAD | 10/10 | 10/10 | 10/10 | 10/10 |
Key: NAD; No abnormalities detected
Table 5: Summary of Body Weight (g)
Sex: Male | ||||||||||||||||
Dose (mg/kg bw) |
| 01 | 08 | 15 | 22 | 29 | 36 | 43 | 50 | 57 | 64 | 71 | 78 | 85 | 90 | 91 |
G1 & 0 | Mean | 250.60 | 278.40 | 298.40 | 316.10 | 336.10 | 340.50 | 357.30 | 372.00 | 381.90 | 398.60 | 403.40 | 410.40 | 420.10 | 412.40 | 391.50 |
SD | 20.27 | 21.52 | 27.14 | 26.43 | 25.86 | 27.06 | 27.09 | 30.27 | 31.18 | 34.52 | 35.10 | 35.06 | 37.84 | 40.34 | 38.54 | |
N | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G2 & 30 | Mean | 256.20 | 286.60 | 309.70 | 330.80 | 351.60 | 360.00 | 373.90 | 389.80 | 401.00 | 414.60 | 421.60 | 431.50 | 441.60 | 437.60 | 417.90 |
SD | 18.72 | 18.88 | 20.84 | 22.94 | 24.80 | 35.39 | 36.45 | 39.36 | 42.86 | 49.25 | 49.28 | 50.93 | 56.19 | 54.48 | 53.81 | |
N | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G3 & 120 | Mean | 255.40 | 281.60 | 306.90 | 331.30 | 347.30 | 362.40 | 375.40 | 385.10 | 396.50 | 411.00 | 415.70 | 426.70 | 435.40 | 427.10 | 407.30 |
SD | 17.67 | 15.68 | 18.37 | 22.53 | 35.76 | 40.44 | 40.99 | 51.09 | 51.17 | 50.03 | 47.35 | 44.92 | 47.49 | 48.45 | 46.70 | |
N | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G4 & 360 | Mean | 251.00 | 277.30 | 302.80 | 324.10 | 352.20 | 367.50 | 378.30 | 391.90 | 398.50 | 414.40 | 415.20 | 423.80 | 428.90 | 424.90 | 403.40 |
SD | 21.16 | 32.54 | 32.74 | 29.11 | 26.91 | 34.02 | 30.46 | 34.38 | 33.85 | 42.69 | 45.97 | 47.78 | 46.21 | 42.79 | 39.52 | |
N | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
Keys: bw= body weight, N = Number of animals, SD = Standard deviation.
Table 5: Summary of Body Weight (g) (Continued)
Sex: Female | ||||||||||||||||
Dose (mg/kg bw) |
| 01 | 08 | 15 | 22 | 29 | 36 | 43 | 50 | 57 | 64 | 71 | 78 | 85 | 90 | 91 |
G1 & 0
| Mean | 181.70 | 192.00 | 204.00 | 210.70 | 217.20 | 224.00 | 228.10 | 232.10 | 236.90 | 240.40 | 240.10 | 242.10 | 245.40 | 244.80 | 231.00 |
SD | 11.80 | 14.13 | 15.17 | 16.98 | 17.54 | 18.70 | 18.43 | 20.08 | 22.25 | 23.10 | 22.26 | 22.00 | 23.54 | 23.31 | 22.18 | |
N | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G2 & 30 | Mean | 184.90 | 192.80 | 210.30 | 219.20 | 226.80 | 233.40 | 239.10 | 245.00 | 246.50 | 253.70 | 253.00 | 259.00 | 261.60 | 256.30 | 242.00 |
SD | 12.93 | 11.17 | 13.04 | 13.13 | 12.19 | 14.04 | 14.94 | 16.59 | 18.20 | 18.54 | 18.78 | 18.58 | 18.91 | 17.94 | 16.32 | |
N | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G3 & 120 | Mean | 182.60 | 196.80 | 211.20 | 216.30 | 227.40 | 237.30 | 242.10 | 244.90 | 248.30 | 257.30 | 259.50 | 262.40↑ | 266.90 | 266.70 | 251.50 |
SD | 10.75 | 10.35 | 10.95 | 10.04 | 8.04 | 9.87 | 9.71 | 13.21 | 10.61 | 14.23 | 15.86 | 14.64 | 14.14 | 16.78 | 16.24 | |
N | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G4 & 360 | Mean | 180.90 | 190.00 | 208.80 | 216.40 | 223.80 | 228.70 | 234.40 | 236.20 | 237.90 | 244.40 | 242.80 | 245.90 | 249.80 | 245.20 | 233.50 |
SD | 11.20 | 13.02 | 8.84 | 10.30 | 11.08 | 13.65 | 13.91 | 14.47 | 16.09 | 16.20 | 18.47 | 17.41 | 19.19 | 19.72 | 18.94 | |
N | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
Keys: bw= body weight, N = Number of animals, SD = Standard deviation. ,↑: increased as compared to control (P<0.05)
Table 6: Summary of Percent Change in Body weight with Respect to Day 1
Sex: Male | ||||||||||||||||
Dose (mg/kg bw) |
| 01 | 08 | 15 | 22 | 29 | 36 | 43 | 50 | 57 | 64 | 71 | 78 | 85 | 90 | 91 |
G1 & 0 | Mean | 11.14 | 19.03 | 26.18 | 34.24 | 36.09 | 42.83 | 48.70 | 52.63 | 59.35 | 61.27 | 64.10 | 68.00 | 64.83 | 56.53 | 11.14 |
SD | 2.77 | 3.92 | 4.72 | 5.52 | 8.24 | 8.46 | 9.90 | 9.74 | 11.98 | 12.31 | 12.53 | 14.06 | 14.21 | 14.25 | 2.77 | |
N | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G2 & 30 | Mean | 11.96 | 21.01 | 29.28 | 37.38 | 40.43 | 45.91 | 52.10 | 56.46 | 61.70 | 64.45 | 68.32 | 72.16 | 70.61 | 62.89 | 11.96 |
SD | 3.30 | 4.60 | 6.14 | 6.29 | 7.61 | 8.83 | 9.77 | 11.36 | 13.41 | 13.48 | 14.00 | 15.19 | 14.57 | 14.40 | 3.30 | |
N | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G3 & 120 | Mean | 10.46 | 20.43 | 29.96 | 36.05 | 41.93 | 47.14 | 51.04 | 55.53 | 61.28 | 63.01 | 67.31 | 70.64 | 67.47 | 59.74 | 10.46 |
SD | 5.39 | 7.13 | 8.02 | 11.23 | 12.89 | 14.42 | 19.23 | 19.40 | 19.31 | 17.16 | 16.44 | 16.38 | 17.68 | 17.33 | 5.39 | |
N | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G4 & 360 | Mean | 10.28 | 20.58 | 29.27 | 40.62 | 46.69 | 51.27 | 56.65 | 59.39 | 65.56 | 65.77 | 69.25 | 71.37 | 69.85 | 61.25 | 10.28 |
SD | 6.54 | 7.84 | 8.05 | 8.54 | 11.34 | 13.16 | 14.14 | 15.20 | 16.77 | 17.18 | 18.30 | 18.29 | 17.67 | 16.38 | 6.54 | |
N | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
Keys: bw= body weight, N = Number of animals, SD = Standard Deviation.
Table 6: Summary of Percent Change in Body weight with Respect to Day 1 (Continued)
Sex: Female | ||||||||||||||||
Dose (mg/kg bw) |
| 01 | 08 | 15 | 22 | 29 | 36 | 43 | 50 | 57 | 64 | 71 | 78 | 85 | 90 | 91 |
G1 & 0 | Mean | 5.68 | 12.34 | 15.95 | 19.56 | 23.37 | 25.67 | 27.77 | 30.40 | 32.36 | 32.25 | 33.34 | 35.16 | 34.79 | 27.19 | 5.68 |
SD | 4.15 | 5.87 | 5.57 | 6.38 | 8.53 | 8.96 | 8.23 | 9.41 | 10.60 | 10.82 | 10.50 | 11.32 | 10.75 | 10.20 | 4.15 | |
N | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G2 & 30 | Mean | 4.37 | 13.91 | 18.85 | 23.04 | 26.68 | 29.86 | 33.05 | 33.86 | 37.74 | 37.48 | 40.70 | 42.07 | 39.15 | 31.40 | 4.37 |
SD | 2.15 | 5.31 | 7.66 | 8.57 | 10.50 | 12.04 | 12.67 | 13.34 | 13.35 | 14.72 | 14.67 | 14.02 | 13.10 | 12.34 | 2.15 | |
N | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G3 & 120 | Mean | 7.85 | 15.80 | 18.63 | 24.74 | 30.27 | 32.96 | 34.51 | 36.32 | 41.44 | 42.73 | 44.19 | 46.76 | 46.74 | 38.38 | 7.85 |
SD | 3.31 | 4.95 | 5.36 | 4.93 | 8.17 | 9.11 | 10.71 | 8.75 | 13.02 | 14.63 | 12.68 | 13.75 | 15.73 | 14.95 | 3.31 | |
N | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G4 & 360 | Mean | 5.05 | 15.60 | 19.80 | 24.00 | 26.67 | 29.86 | 30.80 | 31.71 | 35.43 | 34.57 | 36.30 | 38.40 | 35.85 | 29.40 | 5.05 |
SD | 3.75 | 4.41 | 5.10 | 7.76 | 8.27 | 9.02 | 8.35 | 8.73 | 10.73 | 12.16 | 11.80 | 12.07 | 12.28 | 12.17 | 3.75 | |
N | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
Keys: bw: body weight, N = Number of animals, SD = Standard Deviation.
Table 7: Summary of Feed Consumption (g/animal/day)
Sex: Male | ||||||||||||||
Dose (mg/kg bw) |
| 1-8 | 8-15 | 15-22 | 22-29 | 29-36 | 36-43 | 43-50 | 50-57 | 57-64 | 64-71 | 71-78 | 78-85 | 85-90 |
G1 & 0 | Mean | 25.5 | 25.9 | 25.3 | 23.8 | 23.3 | 23.8 | 24.5 | 24.6 | 24.8 | 25.2 | 27.6 | 25.6 | 16.2 |
SD | 4.0 | 5.2 | 3.5 | 2.3 | 2.2 | 2.4 | 2.1 | 2.0 | 2.0 | 2.0 | 4.7 | 2.2 | 1.3 | |
N | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G2 & 30 | Mean | 25.4 | 25.7 | 25.8 | 24.4 | 22.3 | 22.6 | 23.8 | 24.4 | 24.1 | 26.2 | 27.3 | 25.3 | 16.5 |
SD | 3.6 | 3.0 | 2.9 | 3.1 | 4.8 | 2.7 | 3.0 | 3.5 | 4.0 | 3.6 | 3.7 | 3.9 | 2.5 | |
N | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G3 & 120 | Mean | 24.2 | 25.4 | 25.6 | 23.4 | 23.7 | 23.0 | 22.6 | 23.3 | 23.4 | 24.5 | 25.1 | 24.8 | 15.9 |
SD | 2.2 | 1.3 | 1.5 | 2.7 | 2.2 | 1.8 | 2.5 | 1.9 | 1.4 | 1.8 | 2.0 | 2.8 | 1.3 | |
N | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G4 & 360 | Mean | 23.6 | 23.4 | 24.7 | 24.6 | 23.9 | 23.1 | 23.3 | 23.4 | 22.9 | 23.8 | 25.1 | 24.2 | 15.1 |
SD | 4.4 | 2.9 | 1.1 | 1.3 | 0.9 | 0.8 | 0.3 | 1.0 | 1.7 | 1.2 | 2.5 | 1.8 | 0.6 | |
N | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
Keys: bw: body weight, N = Number of animals, SD = Standard Deviation
Table 7:Summary of Feed Consumption (g/animal/day) (Continued)
Sex: Female | ||||||||||||||
Dose (mg/kg bw) |
| 1-8 | 8-15 | 15-22 | 22-29 | 29-36 | 36-43 | 43-50 | 50-57 | 57-64 | 64-71 | 71-78 | 78-85 | 85-90 |
G1 & 0 | Mean | 17.0 | 16.4 | 17.2 | 15.7 | 15.6 | 14.8 | 15.9 | 15.6 | 14.6 | 14.7 | 14.9 | 15.7 | 9.8 |
SD | 0.8 | 0.6 | 1.0 | 1.0 | 0.9 | 0.4 | 2.6 | 1.3 | 0.6 | 0.9 | 1.1 | 1.7 | 0.6 | |
N | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G2 & 30 | Mean | 18.7 | 18.5↑ | 18.2 | 18.3↑ | 17.3↑ | 16.7↑ | 16.8 | 16.5 | 15.8 | 16.2 | 15.9 | 16.2 | 10.4 |
SD | 1.1 | 1.2 | 0.8 | 1.9 | 0.8 | 1.1 | 1.4 | 1.5 | 1.3 | 1.1 | 1.2 | 0.9 | 0.6 | |
N | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G3 & 120 | Mean | 18.8↑ | 18.2 | 17.9 | 17.9 | 17.5↑ | 16.9↑↑ | 17.3 | 18.2↑ | 16.5 | 16.0 | 16.8 | 17.1 | 11.1 |
SD | 0.8 | 1.4 | 0.4 | 0.5 | 0.7 | 1.0 | 1.2 | 0.2 | 2.4 | 1.3 | 1.4 | 1.4 | 2.0 | |
N | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G4 & 360 | Mean | 16.8 | 16.5 | 15.9↓ | 16.2 | 15.4 | 15.3 | 15.3 | 15.2 | 15.0 | 14.8 | 15.1 | 15.3 | 9.0 |
SD | 1.1 | 0.6 | 0.3 | 1.4 | 1.1 | 0.3 | 0.8 | 0.5 | 0.8 | 0.6 | 0.7 | 1.1 | 0.9 | |
N | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
Keys: bw: body weight, N = Number of animals, SD = Standard Deviation,↑: increased as compared to control (P<0.05) ,↑↑: increased as compared to control (P<0.01), ↓: decreased as compared to control (P<0.05),
Table 8: Summary of Functional Observation Battery/Neurobehavioral Observation Record
Group (Number of animals per group) | G1(10) | G2(10) | G3(10) | G4 (10) | |
Week : Terminal Sex: Male | |||||
Dose (mg/kg body weight) | 0 | 30 | 120 | 360 | |
Parameter | Observation | Number of animals showing observations | |||
Posture | Curled up, often asleep | 10 | 10 | 10 | 10 |
Sitting B | 0 | 0 | 0 | 0 | |
Sitting C | 0 | 0 | 0 | 0 | |
Sitting A | 0 | 0 | 0 | 0 | |
Rearing | 0 | 0 | 0 | 0 | |
Convulsion | Absent | 10 | 10 | 10 | 10 |
Ease of removming from the cage | Very easy | 10 | 10 | 10 | 10 |
Easy | 0 | 0 | 0 | 0 | |
Moderately difficult | 0 | 0 | 0 | 0 | |
Handling reactivity | Easy | 10 | 10 | 10 | 10 |
Moderately easy | 0 | 0 | 0 | 0 | |
Palpebral closure | Eyelids wide open | 10 | 10 | 10 | 10 |
Lacrimation | None | 10 | 10 | 10 | 10 |
Eye Examination | Absent | 10 | 10 | 10 | 10 |
Piloerection | Absent | 10 | 10 | 10 | 10 |
Skin Examination | Absent | 10 | 10 | 10 | 10 |
Salivation | None | 10 | 10 | 10 | 10 |
Gait | Normal | 10 | 10 | 10 | 10 |
Mobility | Normal | 10 | 10 | 10 | 10 |
Arousal | Normal | 10 | 10 | 10 | 10 |
Respiration | Normal | 10 | 10 | 10 | 10 |
Tonic Movement | Absent | 10 | 10 | 10 | 10 |
Clonic Movement | Absent | 10 | 10 | 10 | 10 |
Stereotypy | Absent | 10 | 10 | 10 | 10 |
Bizzare Behaviour | Absent | 10 | 10 | 10 | 10 |
Number of Rears | Mean | 11.90 | 11.10 | 9.20 | 8.50 |
SD | 2.23 | 1.37 | 1.93 | 1.78 | |
Vocalization Count | Mean | 0.00 | 0.00 | 0.00 | 0.00 |
SD | 0.00 | 0.00 | 0.00 | 0.00 | |
No. of urine pools | Mean | 1.50 | 1.60 | 1.50 | 0.80 |
SD | 0.97 | 0.84 | 0.71 | 0.63 | |
No. of faecal bolus | Mean | 1.30 | 0.60 | 1.80 | 0.70 |
SD | 0.67 | 0.70 | 1.69 | 0.67 | |
Table 8: Summary of Functional Observation Battery/Neurobehavioral Observation Record (Continued)
Group (Number of animals per group) | G1(10) | G2(10) | G3(10) | G4 (10) | |
Week : TerminalSex: Female | |||||
Dose (mg/kg body weight) | 0 | 30 | 120 | 360 | |
Parameter | Observation | Number of animals showing observations | |||
Posture | Curled up, often asleep | 10 | 10 | 10 | 10 |
Sitting B | 0 | 0 | 0 | 0 | |
Sitting C | 0 | 0 | 0 | 0 | |
Sitting A | 0 | 0 | 0 | 0 | |
Rearing | 0 | 0 | 0 | 0 | |
Convulsion | Absent | 10 | 10 | 10 | 10 |
Ease of removming from the cage | Very easy | 10 | 10 | 10 | 10 |
Easy | 0 | 0 | 0 | 0 | |
Moderately difficult | 0 | 0 | 0 | 0 | |
Handling reactivity | Easy | 10 | 10 | 10 | 10 |
Moderately easy | 0 | 0 | 0 | 0 | |
Palpebral closure | Eyelids wide open | 10 | 10 | 10 | 10 |
Lacrimation | None | 10 | 10 | 10 | 10 |
Eye Examination | Absent | 10 | 10 | 10 | 10 |
Piloerection | Absent | 10 | 10 | 10 | 10 |
Skin Examination | Absent | 10 | 10 | 10 | 10 |
Salivation | None | 10 | 10 | 10 | 10 |
Gait | Normal | 10 | 10 | 10 | 10 |
Mobility | Normal | 10 | 10 | 10 | 10 |
Arousal | Normal | 10 | 10 | 10 | 10 |
Respiration | Normal | 10 | 10 | 10 | 10 |
Tonic Movement | Absent | 10 | 10 | 10 | 10 |
Clonic Movement | Absent | 10 | 10 | 10 | 10 |
Stereotypy | Absent | 10 | 10 | 10 | 10 |
Bizzare Behaviour | Absent | 10 | 10 | 10 | 10 |
Number of Rears | Mean | 12.90 | 11.60 | 8.60 | 8.40 |
SD | 2.81 | 2.37 | 2.27 | 1.71 | |
Vocalization Count | Mean | 0.00 | 0.00 | 0.00 | 0.00 |
SD | 0.00 | 0.00 | 0.00 | 0.00 | |
No. of urine pools | Mean | 2.10 | 2.30 | 1.80 | 1.60 |
SD | 1.66 | 1.83 | 1.87 | 0.97 | |
No. of faecal bolus | Mean | 1.20 | 1.20 | 1.30 | 1.20 |
SD | 1.14 | 1.03 | 0.67 | 0.79 | |
Table 9: Summary of Sensory Activity Measurement Records
Sex: Male | |||||
Group (Number of animals) | G1 (10) | G2 (10) | G3 (10) | G4(10) | |
Dose (mg/kg body weight) | 0 | 30 | 120 | 360 | |
Parameters | Observation | Number of Animals Showing Observations | |||
Approach response | Fast | 10 | 10 | 10 | 10 |
Touch response | Normal | 10 | 10 | 10 | 10 |
Click response | Normal | 10 | 10 | 10 | 10 |
Pupil response | Normal | 10 | 10 | 10 | 10 |
Air righting reflex | Normal | 10 | 10 | 10 | 10 |
Tail pinch response | Flinch | 10 | 10 | 10 | 10 |
Table 9: Summary of Sensory Activity Measurement Records (Continued)
Sex: Female | |||||
Group (Number of animals) | G1 (10) | G2 (10) | G3 (10) | G4(10) | |
Dose (mg/kg body weight) | 0 | 30 | 120 | 360 | |
Parameters | Observation | Number of Animals Showing Observations | |||
Approach response | Fast | 10 | 10 | 10 | 10 |
Touch response | Normal | 10 | 10 | 10 | 10 |
Click response | Normal | 10 | 10 | 10 | 10 |
Pupil response | Normal | 10 | 10 | 10 | 10 |
Air righting reflex | Normal | 10 | 10 | 10 | 10 |
Tail pinch response | Flinch | 10 | 10 | 10 | 10 |
Table 10: Mean Foot Splay Record Main Study
Sex: Male | ||||||||
Group (N) | G1 (10) | G2 (10) | G3 (10) | G4 (10) | ||||
Dose (mg/kg body weight) | 0 | 30 | 120 | 360 | ||||
Parameter | Mean | SD | Mean | SD | Mean | SD | Mean | SD |
Foot Splay (mm) | 111.13 | 24.19 | 98.17 | 23.78 | 85.33 | 14.79 | 93.50 | 17.86 |
Sex: Female | ||||||||
Group (N) | G1 (10) | G2 (10) | G3 (10) | G4 (10) | ||||
Parameter | Mean | SD | Mean | SD | Mean | SD | Mean | SD |
Foot Splay (mm) | 86.57 | 16.81 | 86.27 | 13.61 | 86.37 | 21.46 | 93.70 | 13.57 |
Keys: N = Number of animals in group, R= Recovery, SD = Standard deviation, mm= millimeter.
Table 11: Summary of Grip strength
Sex: Male | ||||||||
Group | G1(10) | G2(10) | G3(10) | G4(10) | ||||
Dose (mg/kg bw) | 0 | 30 | 120 | 360 | ||||
Parameter | Mean | SD | Mean | SD | Mean | SD | Mean | SD |
Forelimb (grams force) | 1050 | 95.60 | 1011.37 | 168.22 | 904.27 | 60.24 | 844.53 | 155.38 |
Hindlimb (grams force) | 531.37 | 38.52 | 522.60 | 38.49 | 504.70 | 41.43 | 516.63 | 42.53 |
Sex: Female | ||||||||
Group | G1(10) | G2(10) | G3(10) | G4(10) | ||||
Dose (mg/kg bw) | 0 | 30 | 120 | 360 | ||||
Parameter | Mean | SD | Mean | SD | Mean | SD | Mean | SD |
Forelimb (grams force) | 726 | 50.43 | 703.57 | 75.66 | 790.17 | 74.58 | 814.40 | 63.12 |
Hindlimb (grams force) | 338.23 | 31.68 | 335.47 | 58.65 | 367.70 | 55.31 | 322.63 | 35.34 |
Table 12: Mean Motor Activity Record Main Study
Dose (mg/kg body weight) | 0 | 30 | 120 | 360 | ||||
Sex: Male | ||||||||
Group (N) | G1 (10) | G2 (10) | G3 (10) | G4 (10) | ||||
Parameter | Mean | SD | Mean | SD | Mean | SD | Mean | SD |
DT(cm) | 2065.90 | 337.07 | 1581.20 | 556.19 | 1884.20 | 597.67 | 2025.10 | 971.27 |
RT(sec) | 142.60 | 24.72 | 198.70 | 61.79 | 172.80 | 63.42 | 191.80 | 103.03 |
ST (sec) | 140.30 | 19.01 | 136.80 | 22.95 | 119.50 | 9.23 | 107.70↓ | 23.79 |
AT(sec) | 317.10 | 34.89 | 264.50 | 67.08 | 307.70 | 62.39 | 300.50 | 101.72 |
BSM | 96.20 | 6.92 | 92.90 | 13.44 | 86.20 | 7.33 | 76.10↓↓ | 16.24 |
HC | 1873.80 | 300.30 | 1441.00 | 427.19 | 1441.50 | 505.78 | 1421.20↓ | 636.95 |
AC | 1244.50 | 246.28 | 893.90 | 323.26 | 1045.20 | 346.45 | 1141.00 | 553.19 |
HB | 1.00 | 0.00 | 1.20 | 1.75 | 0.60 | 0.52 | 1.20 | 0.63 |
CR | 34.50 | 8.44 | 21.60 | 9.16 | 26.00 | 11.93 | 24.90 | 13.88 |
CCR | 26.30 | 6.11 | 23.90 | 12.64 | 26.60 | 11.06 | 26.40 | 13.33 |
Sex: Female | ||||||||
Group (N) | G1 (10) | G2 (10) | G3 (10) | G4 (10) | ||||
Parameter | Mean | SD | Mean | SD | Mean | SD | Mean | SD |
DT(cm) | 1372.30 | 610.33 | 2052.80 | 417.37 | 2197.40↑ | 732.81 | 2405.50↑↑ | 789.97 |
RT(sec) | 253.60 | 90.56 | 152.50 | 35.11 | 167.80 | 56.42 | 144.70↓ | 76.18 |
ST (sec) | 131.30 | 36.03 | 139.40 | 21.70 | 128.50 | 18.34 | 124.00 | 28.81 |
AT(sec) | 215.10 | 75.46 | 308.10↑↑ | 37.43 | 303.70↑ | 63.44 | 331.30↑↑↑ | 77.94 |
BSM | 85.80 | 17.54 | 89.00 | 9.15 | 91.50 | 9.95 | 88.40 | 16.71 |
HC | 1207.00 | 580.22 | 1821.30↑ | 390.25 | 1804.70↑ | 572.57 | 1886.70↑ | 575.24 |
AC | 752.70 | 419.97 | 1206.70↑ | 265.43 | 1243.60↑ | 453.04 | 1293.00 | 433.25 |
HB | 0.70 | 1.57 | 0.00 | 0.00 | 0.10 | 0.32 | 0.40 | 0.52 |
CR | 15.30 | 10.03 | 27.50 | 11.35 | 26.10 | 10.89 | 27.80↑ | 11.39 |
CCR | 17.20 | 13.82 | 26.50 | 10.42 | 29.80 | 12.99 | 32.30↑ | 12.70 |
Keys: DT=Distance travelled (cm), RT=Resting time (sec), ST=Stereotypic time (sec), AT=Ambulatory time (sec), BSM= Burst of stereotypic movements, HC= Horizontal counts, AC=Ambulatory count, HB=Horizontal break, CR=Clockwise rotation and CCR=Counter Clockwise rotation and N = Number of animals in the group, SD= Standard Deviation, cm= Centimeter, sec= seconds, SD= standard deviation, N=No. of animals.
Table 13: Absolute Organ Weight (g)
Sex: Male
Group | G1 | G2 | |||
Dose (mg/Kg) | 0 | 30 | |||
Organ | No. of Animals | Mean | SD | Mean | SD |
Body weight | 10 | 391.5000 | 38.5379 | 417.9000 | 53.8092 |
Brain | 10 | 2.0882 | 0.1281 | 2.1372 | 0.1427 |
Adrenals | 10 | 0.0767 | 0.0164 | 0.0747 | 0.0140 |
Testes | 10 | 3.5679 | 0.3882 | 3.3821 | 0.3621 |
Epididymis | 10 | 1.5378 | 0.1356 | 1.5228 | 0.1829 |
Prostate and Seminal vesicle | 10 | 2.8429 | 0.6506 | 2.7116 | 0.4172 |
Heart | 10 | 1.2697 | 0.1102 | 1.3115 | 0.1528 |
Liver | 10 | 11.7107 | 2.2078 | 13.5756 | 2.3159 |
Kidneys | 10 | 2.9739 | 0.3727 | 3.1869 | 0.4982 |
Spleen | 10 | 0.7115 | 0.1168 | 0.8922↑ | 0.1468 |
Thymus | 10 | 0.3512 | 0.0626 | 0.3465 | 0.0726 |
Thyroid/Parathyroid | 10 | 0.0214 | 0.0041 | 0.0205 | 0.0037 |
Pitutary | 10 | 0.0130 | 0.0015 | 0.0125 | 0.0022 |
Group | G3 | G4 | |||
Dose (mg/Kg) | 120 | 360 | |||
Organ | No. of Animals | Mean | SD | Mean | SD |
Body weight | 10 | 407.3000 | 46.7001 | 403.4000 | 39.5171 |
Brain | 10 | 2.0857 | 0.0803 | 2.1232 | 0.1253 |
Adrenals | 10 | 0.0806 | 0.0137 | 0.0843 | 0.0168 |
Testes | 10 | 3.3317 | 0.3805 | 3.2489 | 0.2375 |
Epididymis | 10 | 1.5165 | 0.1356 | 1.4912 | 0.1524 |
Prostate and Seminal vesicle | 10 | 2.4167 | 0.3886 | 2.4522 | 0.3900 |
Heart | 10 | 1.2707 | 0.1252 | 1.2870 | 0.1101 |
Liver | 10 | 13.1193 | 1.6177 | 12.2915 | 1.2275 |
Kidneys | 10 | 2.9434 | 0.2905 | 2.9161 | 0.2700 |
Spleen | 10 | 0.7984 | 0.1654 | 0.7876 | 0.1144 |
Thymus | 10 | 0.4017 | 0.0912 | 0.3508 | 0.1163 |
Thyroid/Parathyroid | 10 | 0.0217 | 0.0020 | 0.0234 | 0.0022 |
Pitutary | 10 | 0.0128 | 0.0011 | 0.0128 | 0.0016 |
Table 13: Absolute Organ Weight (g) (Continued)
Sex: Female
Group | G1 | G2 | |||
Dose (mg/Kg) | 0 | 30 | |||
Organ | No. of Animals | Mean | SD | Mean | SD |
Body weight | 10 | 231.0000 | 22.1761 | 242.0000 | 16.3231 |
Brain | 10 | 1.9132 | 0.1084 | 1.9524 | 0.1050 |
Adrenals | 10 | 0.0843 | 0.0145 | 0.0811 | 0.0152 |
Uterus | 10 | 0.5679 | 0.1807 | 0.8057↑ | 0.2359 |
Ovaries | 10 | 0.1420 | 0.0256 | 0.1307 | 0.0125 |
Heart | 10 | 0.7963 | 0.0855 | 0.8812 | 0.1278 |
Liver | 10 | 7.1794 | 0.9708 | 8.0657 | 1.0710 |
Kidneys | 10 | 1.7949 | 0.1986 | 1.9129 | 0.1409 |
Spleen | 10 | 0.5625 | 0.1341 | 0.5736 | 0.1356 |
Thymus | 10 | 0.3260 | 0.0600 | 0.3310 | 0.0536 |
Thyroid/Parathyroid | 10 | 0.0189 | 0.0022 | 0.0189 | 0.0025 |
Pitutary | 10 | 0.0156 | 0.0013 | 0.0165 | 0.0020 |
Group | G3 | G4 | |||
Dose (mg/Kg) | 120 | 360 | |||
Organ | No. of Animals | Mean | SD | Mean | SD |
Body weight | 10 | 251.50 | 16.2361 | 233.5000 | 18.9400 |
Brain | 10 | 1.9440 | 0.1430 | 1.9657 | 0.0647 |
Adrenals | 10 | 0.0856 | 0.0126 | 0.0936 | 0.0152 |
Uterus | 10 | 0.6685 | 0.1457 | 0.6158 | 0.2409 |
Ovaries | 10 | 0.1581 | 0.0363 | 0.1616 | 0.0251 |
Heart | 10 | 0.9002 | 0.0981 | 0.8553 | 0.0620 |
Liver | 10 | 8.0313 | 1.4899 | 7.4647 | 0.8717 |
Kidneys | 10 | 1.8903 | 0.1614 | 1.9918 | 0.1619 |
Spleen | 10 | 0.6030 | 0.1399 | 0.5464 | 0.0717 |
Thymus | 10 | 0.3631 | 0.0616 | 0.3333 | 0.0657 |
Thyroid/Parathyroid | 10 | 0.0186 | 0.0019 | 0.0174 | 0.0032 |
Pitutary | 10 | 0.0177 | 0.0035 | 0.0143 | 0.0021 |
Key: SD= Standard deviation. ,↑: increased as compared to control (P<0.05)
Table 14: Organ Weight Relative to Body Weight (g %)
Sex: Male
Group | G1 | G2 | |||
Dose (mg/Kg) | 0 | 30 | |||
Organ | No. of Animals | Mean | SD | Mean | SD |
Body weight | 10 | 391.5000 | 38.5379 | 417.9000 | 53.8092 |
Brain | 10 | 0.5357 | 0.0289 | 0.5161 | 0.0475 |
Adrenals | 10 | 0.0200 | 0.0058 | 0.0179 | 0.0024 |
Testes | 10 | 0.9159 | 0.1034 | 0.8233 | 0.1491 |
Epididymis | 10 | 0.3936 | 0.0200 | 0.3671 | 0.0428 |
Prostate and Seminal vesicle | 10 | 0.7241 | 0.1411 | 0.6501 | 0.0683 |
Heart | 10 | 0.3263 | 0.0330 | 0.3151 | 0.0227 |
Liver | 10 | 2.9739 | 0.3388 | 3.2384 | 0.2156 |
Kidneys | 10 | 0.7583 | 0.0411 | 0.7615 | 0.0524 |
Spleen | 10 | 0.1824 | 0.0282 | 0.2152 | 0.0342 |
Thymus | 10 | 0.0906 | 0.0187 | 0.0832 | 0.0156 |
Thyroid/Parathyroid | 10 | 0.0055 | 0.0013 | 0.0049 | 0.0009 |
Pitutary | 10 | 0.0033 | 0.0003 | 0.0030 | 0.0006 |
Group | G3 | G4 | |||
Dose (mg/Kg) | 120 | 360 | |||
Organ | No. of Animals | Mean | SD | Mean | SD |
Body weight | 10 | 407.3000 | 46.7001 | 403.4000 | 39.5171 |
Brain | 10 | 0.5174 | 0.0547 | 0.5310 | 0.0616 |
Adrenals | 10 | 0.0199 | 0.0034 | 0.0213 | 0.0055 |
Testes | 10 | 0.8239 | 0.1014 | 0.8113 | 0.0891 |
Epididymis | 10 | 0.3754 | 0.0423 | 0.3709 | 0.0325 |
Prostate and Seminal vesicle | 10 | 0.5975↓ | 0.1004 | 0.6110 | 0.0990 |
Heart | 10 | 0.3130 | 0.0171 | 0.3201 | 0.0216 |
Liver | 10 | 3.2244 | 0.2040 | 3.0738 | 0.4486 |
Kidneys | 10 | 0.7244 | 0.0346 | 0.7268 | 0.0765 |
Spleen | 10 | 0.1966 | 0.0377 | 0.1970 | 0.0377 |
Thymus | 10 | 0.0996 | 0.0241 | 0.0866 | 0.0260 |
Thyroid/Parathyroid | 10 | 0.0054 | 0.0009 | 0.0059 | 0.0007 |
Pitutary | 10 | 0.0032 | 0.0003 | 0.0032 | 0.0005 |
Keys: SD = Standard deviation, N = Number of animals in group ↓: Decreased as compared to control (P<0.05)
Table 14: Organ Weight Relative to Body Weight (g %)
Sex: Female
Group | G1 | G2 | |||
Dose (mg/Kg) | 0 | 30 | |||
Organ | No. of Animals | Mean | SD | Mean | SD |
Body weight | 10 | 231.0000 | 22.1761 | 242.0000 | 16.3231 |
Brain | 10 | 0.8361 | 0.1002 | 0.8097 | 0.0654 |
Adrenals | 10 | 0.0367 | 0.0070 | 0.0335 | 0.0055 |
Uterus | 10 | 0.2457 | 0.0743 | 0.3350↑ | 0.1008 |
Ovaries | 10 | 0.0615 | 0.0102 | 0.0542 | 0.0063 |
Heart | 10 | 0.3450 | 0.0235 | 0.3635 | 0.0383 |
Liver | 10 | 3.0998 | 0.1962 | 3.3299 | 0.3724 |
Kidneys | 10 | 0.7765 | 0.0346 | 0.7928 | 0.0699 |
Spleen | 10 | 0.2408 | 0.0378 | 0.2370 | 0.0541 |
Thymus | 10 | 0.1416 | 0.0252 | 0.1368 | 0.0205 |
Thyroid/Parathyroid | 10 | 0.0082 | 0.0007 | 0.0078 | 0.0011 |
Pitutary | 10 | 0.0068 | 0.0008 | 0.0069 | 0.0012 |
Group | G3 | G4 | |||
Dose (mg/Kg) | 120 | 360 | |||
Organ | No. of Animals | Mean | SD | Mean | SD |
Body weight | 10 | 251.5000 | 16.2361 | 233.5000 | 18.9400 |
Brain | 10 | 0.7735 | 0.0396 | 0.8463 | 0.0662 |
Adrenals | 10 | 0.0340 | 0.0043 | 0.0401 | 0.0057 |
Uterus | 10 | 0.2678 | 0.0663 | 0.2625 | 0.0946 |
Ovaries | 10 | 0.0626 | 0.0120 | 0.0697 | 0.0125 |
Heart | 10 | 0.3584 | 0.0361 | 0.3668 | 0.0145 |
Liver | 10 | 3.1924 | 0.5331 | 3.1913 | 0.1807 |
Kidneys | 10 | 0.7520 | 0.0510 | 0.8559 | 0.0722 |
Spleen | 10 | 0.2389 | 0.0494 | 0.2344 | 0.0283 |
Thymus | 10 | 0.1439 | 0.0186 | 0.1430 | 0.0263 |
Thyroid/Parathyroid | 10 | 0.0074 | 0.0007 | 0.0075 | 0.0012 |
Pitutary | 10 | 0.0071 | 0.0014 | 0.0062 | 0.0008 |
Keys: SD = Standard deviation, N = Number of animals in group ↑: increased as compared to control (P<0.05)
Sex: Male
Group (N) | G1 (10) | G2 (10) | G3 (10) | G4 (10) | ||||
Dose (mg/kg bw) | 0 | 30 | 120 | 360 | ||||
Parameter | Mean | SD | Mean | SD | Mean | SD | Mean | SD |
Total Erythrocyte Count (RBC) | 8.86 | 0.32 | 8.61 | 0.36 | 8.66 | 0.40 | 8.43 | 0.23 |
Hematocrit (HCT) | 41.17 | 0.81 | 40.66 | 1.60 | 40.72 | 1.59 | 41.97 | 1.19 |
Mean Corpuscular Volume (MCV) | 46.51 | 1.45 | 47.20 | 1.32 | 46.99 | 1.20 | 49.81↑↑↑ | 1.47 |
Hemoglobin (HGB) | 14.66 | 0.31 | 14.47 | 0.55 | 14.51 | 0.60 | 14.84 | 0.52 |
MCH | 16.54 | 0.47 | 16.81 | 0.47 | 16.77 | 0.46 | 17.64↑↑↑ | 0.60 |
MCHC | 35.63 | 0.22 | 35.65 | 0.29 | 35.68 | 0.23 | 35.56 | 0.25 |
Platelet Count (PLT) | 653.70 | 64.77 | 616.70 | 87.52 | 615.60 | 101.28 | 589.20 | 81.79 |
Total Leukocyte count (WBC) | 9.72 | 3.06 | 10.50 | 2.32 | 9.13 | 2.88 | 9.71 | 2.40 |
Neutrophil | 27.50 | 2.37 | 27.70 | 1.89 | 27.50 | 2.27 | 27.80 | 2.62 |
Lymphocyte | 72.00 | 2.49 | 71.80 | 1.93 | 71.80 | 1.81 | 71.70 | 2.36 |
Monocyte | 0.30 | 0.48 | 0.30 | 0.48 | 0.40 | 0.52 | 0.30 | 0.48 |
Eosinophil | 0.20 | 0.42 | 0.20 | 0.42 | 0.30 | 0.48 | 0.20 | 0.42 |
Basophil | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
Prothrombin Time (PT) | 18.28 | 1.42 | 19.27 | 1.32 | 17.72 | 1.08 | 19.11 | 2.29 |
(aPTT) | 29.01 | 1.73 | 28.35 | 1.43 | 27.69 | 1.79 | 28.32 | 1.84 |
Reticulocyte | 1.18 | 0.11 | 1.18 | 0.08 | 1.19 | 0.07 | 1.19 | 0.06 |
Keys: SD = Standard deviation, N = Number of animals in group, MCH; Mean Corpuscular Hemoglobin, MCHC; Mean Corpuscular Hemoglobin Concentration, aPTT; Activated Partial thromboplastin Time.: ↑↑↑ increased as compared to control (P<0.001),
Table 15:Hematology (Continued)
Sex: Female
Group (N) | G1 (10) | G2 (10) | G3 (10) | G4 (10) | ||||
Dose (mg/kg bw) | 0 | 30 | 120 | 360 | ||||
Parameter | Mean | SD | Mean | SD | Mean | SD | Mean | SD |
Total Erythrocyte Count (RBC) | 7.93 | 0.41 | 8.14 | 0.32 | 7.82 | 0.31 | 7.96 | 0.34 |
Hematocrit (HCT) | 39.97 | 2.63 | 41.55 | 0.96 | 40.51 | 1.66 | 41.56 | 1.29 |
Mean Corpuscular Volume (MCV) | 50.36 | 2.22 | 51.06 | 1.73 | 51.84 | 2.03 | 52.19 | 1.19 |
Hemoglobin (HGB) | 13.65 | 0.56 | 13.73 | 0.43 | 13.27 | 0.54 | 13.76 | 0.35 |
MCH | 17.24 | 0.48 | 16.87 | 0.55 | 16.99 | 0.52 | 17.28 | 0.39 |
MCHC | 34.30 | 1.31 | 33.09 | 0.57 | 32.79 | 0.53 | 33.13 | 0.37 |
Platelet Count (PLT) | 583.00 | 65.23 | 640.30 | 99.76 | 602.40 | 63.52 | 608.80 | 74.95 |
Total Leukocyte count (WBC) | 6.72 | 2.70 | 6.03 | 1.14 | 6.09 | 1.74 | 6.75 | 0.79 |
Neutrophil | 27.30 | 2.41 | 27.30 | 2.45 | 27.60 | 2.41 | 27.70 | 3.13 |
Lymphocyte | 72.20 | 2.57 | 72.20 | 2.15 | 72.00 | 2.21 | 71.80 | 3.26 |
Monocyte | 0.30 | 0.48 | 0.30 | 0.48 | 0.20 | 0.42 | 0.30 | 0.48 |
Eosinophil | 0.20 | 0.42 | 0.20 | 0.42 | 0.20 | 0.42 | 0.20 | 0.42 |
Basophil | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
Prothrombin Time (PT) | 18.60 | 1.20 | 17.48 | 0.49 | 19.08 | 0.95 | 18.76 | 0.70 |
(aPTT) | 28.25 | 0.68 | 28.52 | 1.46 | 27.47 | 1.36 | 28.95 | 1.32 |
Reticulocyte | 1.16 | 0.12 | 1.15 | 0.07 | 1.18 | 0.08 | 1.17 | 0.07 |
Keys: SD = Standard deviation, N = Number of animals in group ↓: decreased as compared to control (P<0.05) MCH; Mean Corpuscular Hemoglobin, MCHC; Mean Corpuscular Hemoglobin Concentration, aPTT; Activated Partial thromboplastin Time
Sex: Male
Group (N) | G1 (10) | G2 (10) | ||
Dose (mg/kg bw) | 0 | 30 | ||
Parameter | Mean | SD | Mean | SD |
Glucose (mg/dl) | 85.08 | 11.24 | 84.82 | 21.40 |
Cholesterol (mg/dl) | 55.76 | 6.91 | 56.91 | 7.72 |
Triglyceride (mg/dl) | 76.32 | 19.10 | 74.81 | 20.93 |
ALT (U/l) | 90.28 | 11.21 | 79.67 | 29.14 |
AST (U/l) | 125.98 | 18.40 | 136.12 | 28.19 |
GGT(U/I) | 1.04 | 0.57 | 1.22 | 0.83 |
Creatinine (mg/dl) | 0.48 | 0.07 | 0.53 | 0.03 |
Albumin (g/dl) | 3.48 | 0.12 | 3.39 | 0.16 |
Total Protein (g/dl) | 6.57 | 0.22 | 6.52 | 0.37 |
Total Bilirubin (mg/dl) | 0.22 | 0.06 | 0.34 | 0.48 |
Urea (mg/dl) | 34.22 | 3.49 | 33.59 | 4.04 |
Phosphorus(mg/dl) | 4.92 | 0.45 | 5.29 | 0.37 |
Calcium (mg/dl) | 10.40 | 0.42 | 10.76 | 1.43 |
ALP (U/l) | 307.93 | 61.81 | 294.20 | 68.66 |
Bile Acid (µmol/l) | 22.00 | 8.79 | 35.95 | 51.36 |
HDL Cholesterol (mg/dl) | 24.74 | 3.90 | 26.39 | 4.42 |
LDL Cholesterol (mg/dl) | 20.56 | 2.38 | 20.97 | 3.86 |
BUN (mg/dl) | 16.07 | 1.80 | 16.00 | 2.60 |
A/G Ratio | 1.14 | 0.11 | 1.09 | 0.08 |
Globulin (g/dl) | 3.08 | 0.23 | 3.13 | 0.30 |
Na (mmol/l) | 142.33 | 0.87 | 144.70↑ | 2.26 |
K (mmol/l) | 5.02 | 0.27 | 5.12 | 0.34 |
Cl (mmol/l) | 85.08 | 11.24 | 103.90 | 2.08 |
Keys: SD = Standard deviation, N = Number of animals in group.↑: increased as compared to control (P<0.05)
Table 16: Clinical Chemistry (Continued)
Sex: Male
Group (N) | G3 (10) | G4 (10) | ||
Dose (mg/kg bw) | 0 | 30 | ||
Parameter | Mean | SD | Mean | SD |
Glucose (mg/dl) | 90.08 | 14.16 | 77.97 | 17.90 |
Cholesterol (mg/dl) | 64.26 | 9.35 | 54.89 | 6.81 |
Triglyceride (mg/dl) | 71.13 | 26.32 | 67.66 | 25.04 |
ALT (U/l) | 72.36 | 18.47 | 80.02 | 14.12 |
AST (U/l) | 143.43 | 25.17 | 134.08 | 21.34 |
GGT(U/I) | 0.93 | 0.43 | 1.32 | 0.42 |
Creatinine (mg/dl) | 0.52 | 0.05 | 0.56↑↑↑ | 0.04 |
Albumin (g/dl) | 3.48 | 0.14 | 3.49 | 0.11 |
Total Protein (g/dl) | 6.60 | 0.26 | 6.64 | 0.26 |
Total Bilirubin (mg/dl) | 0.18 | 0.04 | 0.22 | 0.04 |
Urea (mg/dl) | 27.99↓↓ | 4.72 | 33.29 | 4.39 |
Phosphorus(mg/dl) | 4.88 | 1.59 | 5.37 | 0.63 |
Calcium (mg/dl) | 10.28 | 0.35 | 10.51 | 0.40 |
ALP (U/l) | 290.46 | 74.14 | 289.10 | 75.17 |
Bile Acid (µmol/l) | 19.57 | 4.08 | 26.01 | 5.99 |
HDL Cholesterol (mg/dl) | 31.97↑↑ | 6.89 | 27.61 | 3.97 |
LDL Cholesterol (mg/dl) | 22.17 | 3.78 | 20.50 | 2.23 |
BUN (mg/dl) | 13.08↓↓ | 2.21 | 15.55 | 2.05 |
A/G Ratio | 1.12 | 0.06 | 1.11 | 0.10 |
Globulin (g/dl) | 3.12 | 0.17 | 3.16 | 0.24 |
Na (mmol/l) | 147.90↑↑↑ | 2.13 | 151.30↑↑↑ | 1.64 |
K (mmol/l) | 5.07 | 0.35 | 5.33 | 0.50 |
Cl (mmol/l) | 122.90↑↑↑ | 17.35 | 115.80↑↑↑ | 1.69 |
Keys: SD = Standard deviation, N = Number of animals in group, bw= Body weight, ↑: increased as compared to control (P<0.05), .: ↑↑↑ increased as compared to control (P<0.001), ↓↓ decreased as compared to control (P<0.01), ↑↑ increased as compared to control (P<0.01).
Table 16: Clinical Chemistry (Continued)
Sex: Female
Group (N) | G1 (10) | G2 (10) | ||
Dose (mg/kg bw) | 0 | 30 | ||
Parameter | Mean | SD | Mean | SD |
Glucose (mg/dl) | 76.85 | 15.83 | 72.48 | 10.70 |
Cholesterol (mg/dl) | 45.91 | 6.21 | 50.53 | 6.25 |
Triglyceride (mg/dl) | 48.15 | 12.70 | 69.79 | 24.09 |
ALT (U/l) | 78.58 | 11.26 | 90.45 | 23.94 |
AST (U/l) | 128.53 | 23.81 | 145.36 | 31.66 |
GGT(U/I) | 1.40 | 0.84 | 2.20 | 2.28 |
Creatinine (mg/dl) | 0.60 | 0.04 | 0.55 | 0.05 |
Albumin (g/dl) | 3.55 | 0.16 | 3.74 | 0.08 |
Total Protein (g/dl) | 6.67 | 0.24 | 6.82 | 0.26 |
Total Bilirubin (mg/dl) | 0.19 | 0.03 | 0.21 | 0.06 |
Urea (mg/dl) | 37.14 | 5.69 | 33.22 | 4.13 |
Phosphorus(mg/dl) | 4.27 | 0.99 | 4.09 | 0.63 |
Calcium (mg/dl) | 10.32 | 0.32 | 10.59 | 0.37 |
ALP (U/l) | 194.62 | 88.57 | 159.10 | 50.78 |
Bile Acid (µmol/l) | 18.45 | 3.98 | 20.68 | 5.38 |
HDL Cholesterol (mg/dl) | 28.95 | 4.78 | 32.37 | 5.00 |
LDL Cholesterol (mg/dl) | 9.52 | 1.53 | 9.94 | 1.35 |
BUN (mg/dl) | 17.38 | 2.68 | 15.52 | 1.93 |
A/G Ratio | 1.14 | 0.11 | 1.23 | 0.10 |
Globulin (g/dl) | 3.13 | 0.24 | 3.07 | 0.25 |
Na (mmol/l) | 141.90 | 1.85 | 144.40 | 1.26 |
K (mmol/l) | 4.68 | 0.47 | 4.66 | 0.46 |
Cl (mmol/l) | 91.90 | 31.99 | 105.30↑↑↑ | 0.67 |
Keys: SD = Standard deviation, N = Number of animals in group,↑↑↑: significant increase as at P<0.001.
Table 16: Clinical Chemistry (Continued)
Sex: Female
Group (N) | G3 (10) | G4 (10) | ||
Dose (mg/kg bw) | 0 | 30 | ||
Parameter | Mean | SD | Mean | SD |
Glucose (mg/dl) | 77.48 | 15.37 | 86.68 | 11.91 |
Cholesterol (mg/dl) | 55.38 | 9.25 | 51.64 | 7.02 |
Triglyceride (mg/dl) | 54.47 | 18.26 | 50.72 | 15.64 |
ALT (U/l) | 75.17 | 33.13 | 70.36 | 31.72 |
AST (U/l) | 138.94 | 34.33 | 159.53 | 62.76 |
GGT(U/I) | 1.10 | 0.42 | 1.65 | 0.94 |
Creatinine (mg/dl) | 0.57 | 0.09 | 0.56 | 0.08 |
Albumin (g/dl) | 3.88 | 0.26 | 3.78 | 0.15 |
Total Protein (g/dl) | 7.01 | 0.31 | 6.93 | 0.37 |
Total Bilirubin (mg/dl) | 2.20 | 6.32 | 0.16 | 0.05 |
Urea (mg/dl) | 32.39 | 7.03 | 27.69↓↓↓ | 4.82 |
Phosphorus(mg/dl) | 4.16 | 0.55 | 4.27 | 0.59 |
Calcium (mg/dl) | 10.67 | 0.37 | 10.67 | 0.39 |
ALP (U/l) | 154.54 | 48.64 | 195.27 | 90.77 |
Bile Acid (µmol/l) | 18.36 | 4.71 | 15.53 | 2.85 |
HDL Cholesterol (mg/dl) | 35.13 | 7.35 | 31.19 | 5.78 |
LDL Cholesterol (mg/dl) | 11.20 | 2.25 | 12.57↑↑↑ | 1.90 |
BUN (mg/dl) | 15.14 | 3.29 | 12.88↓↓↓ | 2.35 |
A/G Ratio | 2.18 | 2.86 | 1.21 | 0.09 |
Globulin (g/dl) | 3.13 | 0.29 | 3.15 | 0.28 |
Na (mmol/l) | 146.70↑↑ | 2.36 | 150.90↑↑↑ | 2.77 |
K (mmol/l) | 4.88 | 0.15 | 5.02↑ | 0.43 |
Cl (mmol/l) | 111.90↑↑↑ | 2.13 | 110.00↑↑↑ | 2.05 |
Keys: SD = Standard deviation, N = Number of animals in group,↑: increased as compared to control (P<0.05). ,↑: increased as compared to control (P<0.05), .: ↑↑↑ increased as compared to control (P<0.001), ↑↑ decreased as compared to control (P<0.01), ↓↓↓ increased as compared to control (P<0.001)
Table 17: Summary of Urine Analysis Physiochemical Parameters
Physiochemical parameters | ||||||||||||
Parameters | Unit | Observation | Group 1 | Group 2 | Group 3 | Group 4 | ||||||
Mean | SD | Mean | SD | Mean | SD | Mean | SD | |||||
pH | - | - | 6.40 | 0.210 | 6.00 | 0.470 | 6.05 | 0.550 | 6.05 | 0.640 | ||
Sp. Gravity | - | - | 1.020 | 0.003 | 1.020 | 0.002 | 1.020 | 0.003 | 1.020 | 0.003 | ||
Volume | ml | - | 8.42 | 5.205 | 7.76 | 4.692 | 6.39 | 4.077 | 7.83 | 4.464 | ||
Male | ||||||||||||
Total No. of Observations | ||||||||||||
Color | Pos | Neg | Pos | Neg | Pos | Neg | Pos | Neg | ||||
Pale Yellow | - | - | 10 | 0 | 10 | 0 | 10 | 0 | 10 | 0 | ||
Yellow | - | - | 0 | 10 | 0 | 10 | 0 | 10 | 0 | 10 | ||
Dark yellow | - | - | 0 | 10 | 0 | 10 | 0 | 10 | 0 | 10 | ||
Yellowish Brown | - | - | 0 | 10 | 0 | 10 | 0 | 10 | 0 | 10 | ||
Brown | - | - | 0 | 10 | 0 | 10 | 0 | 10 | 0 | 10 | ||
Appearance | Clear | - | - | 10 | 0 | 10 | 0 | 10 | 0 | 10 | 0 | |
Turbid | - | - | 0 | 10 | 0 | 10 | 0 | 10 | 0 | 10 | ||
Hazy | - | - | 0 | 10 | 0 | 10 | 0 | 10 | 0 | 10 | ||
Blood / Blood Cell | RBC/µl | - | 0 | 10 | 0 | 10 | 0 | 10 | 0 | 10 | ||
Bilirubin | mg/dl | - | 0 | 10 | 0 | 10 | 0 | 10 | 0 | 10 | ||
Urobilinogen | mg/dl | - | 0 | 10 | 0 | 10 | 0 | 10 | 0 | 10 | ||
Ketone | mg/dl | - | 0 | 10 | 0 | 10 | 0 | 10 | 0 | 10 | ||
Protein | mg/dl | - | 0 | 10 | 0 | 10 | 0 | 10 | 0 | 10 | ||
Nitrite | - | - | 0 | 10 | 0 | 10 | 0 | 10 | 0 | 10 | ||
Glucose | mg/dl | - | 0 | 10 | 0 | 10 | 0 | 10 | 0 | 10 | ||
Leucocytes | WBC/µl | - | 0 | 10 | 0 | 10 | 0 | 10 | 0 | 10 | ||
Table 17: Summary of Urine Analysis Physiochemical Parameters (Continued)
Physiochemical parameters | |||||||||||||||
Parameters | Unit | Observation | Group 1 | Group 2 | Group 3 | Group 4 | |||||||||
Mean | SD | Mean | SD | Mean | SD | Mean | SD | ||||||||
pH | - | - | 6.40 | 0.210 | 6.00 | 0.470 | 6.05 | 0.550 | 6.05 | 0.640 | |||||
Sp. Gravity | - | - | 1.020 | 0.003 | 1.020 | 0.002 | 1.020 | 0.003 | 1.020 | 0.003 | |||||
Volume | ml | - | 3.39 | 2.165 | 5.20 | 3.198 | 4.55 | 1.613 | 4.83 | 2.063 | |||||
Female | |||||||||||||||
Total No. of Observations | |||||||||||||||
Colour | Pos | Neg | Pos | Neg | Pos | Neg | Pos | Neg | |||||||
Pale Yellow | - | - | 10 | 0 | 10 | 0 | 10 | 0 | 10 | 0 | |||||
Yellow | - | - | 0 | 10 | 0 | 10 | 0 | 10 | 0 | 10 | |||||
Dark yellow | - | - | 0 | 10 | 0 | 10 | 0 | 10 | 0 | 10 | |||||
Yellowish Brown | - | - | 0 | 10 | 0 | 10 | 0 | 10 | 0 | 10 | |||||
Brown | - | - | 0 | 10 | 0 | 10 | 0 | 10 | 0 | 10 | |||||
Appearance | Clear | - | - | 10 | 0 | 10 | 0 | 10 | 0 | 10 | 0 | ||||
Turbid | - | - | 0 | 10 | 0 | 10 | 0 | 10 | 0 | 10 | |||||
Hazy | - | - | 0 | 10 | 0 | 10 | 0 | 10 | 0 | 10 | |||||
Blood / Blood Cell | RBC/µl | - | 0 | 10 | 0 | 10 | 0 | 10 | 0 | 10 | |||||
Bilirubin | mg/dl | - | 0 | 10 | 0 | 10 | 0 | 10 | 0 | 10 | |||||
Urobilinogen | mg/dl | - | 0 | 10 | 0 | 10 | 0 | 10 | 0 | 10 | |||||
Ketone | mg/dl | - | 0 | 10 | 0 | 10 | 0 | 10 | 0 | 10 | |||||
Protein | mg/dl | - | 0 | 10 | 0 | 10 | 0 | 10 | 0 | 10 | |||||
Nitrite | - | - | 0 | 10 | 0 | 10 | 0 | 10 | 0 | 10 | |||||
Glucose | mg/dl | - | 0 | 10 | 0 | 10 | 0 | 10 | 0 | 10 | |||||
Leucocytes | WBC/µl | - | 0 | 10 | 0 | 10 | 0 | 10 | 0 | 10 | |||||
Table 17: Summary of Urine Analysis Microscopic Parameters
Parameters | Sex | Unit/ | Group 1 | Group 2 | Group 3 | Group 4 | |
Epithelial cells | Absent | Male | / h.p.f. | 8 | 7 | 7 | 7 |
1-2 | 2 | 3 | 3 | 3 | |||
3-6 | 0 | 0 | 0 | 0 | |||
6-12 | 0 | 0 | 0 | 0 | |||
>12 | 0 | 0 | 0 | 0 | |||
N | 10 | 10 | 10 | 10 | |||
Pus cells | Absent | / h.p.f. | 4 | 5 | 5 | 8 | |
1-2 | 6 | 5 | 5 | 2 | |||
3-6 | 0 | 0 | 0 | 0 | |||
6-12 | 0 | 0 | 0 | 0 | |||
>12 | 0 | 0 | 0 | 0 | |||
N | 10 | 10 | 10 | 10 | |||
RBCs | Absent | / h.p.f. | 10 | 9 | 10 | 10 | |
1-2 | 0 | 0 | 0 | 0 | |||
3-6 | 0 | 0 | 0 | 0 | |||
6-12 | 0 | 0 | 0 | 0 | |||
>12 | 0 | 0 | 0 | 0 | |||
N | 10 | 9 | 10 | 10 | |||
Epithelial cast | Absent | / h.p.f. | 7 | 8 | 6 | 7 | |
1-2 | 3 | 2 | 4 | 3 | |||
3-6 | 0 | 0 | 0 | 0 | |||
>6 | 0 | 0 | 0 | 0 | |||
N | 10 | 10 | 10 | 10 | |||
White cell cast | Absent | / h.p.f. | 10 | 10 | 10 | 10 | |
1-2 | Male | 0 | 0 | 0 | 0 | ||
3-6 | 0 | 0 | 0 | 0 | |||
>6 | 0 | 0 | 0 | 0 | |||
N | 10 | 10 | 10 | 10 | |||
Red cell cast | Absent |
/ h.p.f. | 10 | 10 | 10 | 10 | |
1-2 | 0 | 0 | 0 | 0 | |||
3-6 | 0 | 0 | 0 | 0 | |||
>6 | 0 | 0 | 0 | 0 | |||
N | 10 | 10 | 10 | 10 | |||
Waxy cast | Absent |
/ h.p.f. | 10 | 10 | 10 | 10 | |
1-2 | 0 | 0 | 0 | 0 | |||
3-6 | 0 | 0 | 0 | 0 | |||
>6 | 0 | 0 | 0 | 0 | |||
N | 10 | 10 | 10 | 10 | |||
Granularcells | Absent |
/ h.p.f. | 7 | 8 | 6 | 6 | |
1-2 | 3 | 2 | 4 | 4 | |||
3-6 | 0 | 0 | 0 | 0 | |||
>6 | 0 | 0 | 0 | 0 | |||
N | 10 | 10 | 10 | 10 | |||
hyaline cast | Absent | / h.p.f. | 10 | 10 | 10 | 10 | |
1-2 | 0 | 0 | 0 | 0 | |||
3-6 | 0 | 0 | 0 | 0 | |||
>6 | 0 | 0 | 0 | 0 | |||
N | 10 | 10 | 10 | 10 | |||
Triple Phosphate | Absent | / h.p.f. | 6 | 5 | 2 | 10 | |
OCC | 0 | 0 | 0 | 0 | |||
Few | Male | 0 | 0 | 0 | 0 | ||
Many | 0 | 0 | 0 | 0 | |||
N | 6 | 5 | 2 | 10 | |||
Calcium Oxalate | Absent |
/ h.p.f. | 7 | 5 | 8 | 5 | |
OCC | 0 | 0 | 0 | 0 | |||
Few | 0 | 0 | 0 | 0 | |||
Many | 0 | 0 | 0 | 0 | |||
N | 7 | 5 | 8 | 5 | |||
Calcium Carbonate | Absent |
/ h.p.f. | 10 | 10 | 10 | 10 | |
OCC | 0 | 0 | 0 | 0 | |||
Few | 0 | 0 | 0 | 0 | |||
Many | 0 | 0 | 0 | 0 | |||
N | 10 | 10 | 10 | 10 | |||
Sperm | Absent |
/ h.p.f. | 3 | 2 | 2 | 1 | |
OCC | 0 | 0 | 0 | 0 | |||
Few | 0 | 0 | 0 | 0 | |||
Many | 0 | 0 | 0 | 0 | |||
N | 3 | 2 | 2 | 1 | |||
Bacteria | Absent | / h.p.f. | 10 | 10 | 10 | 10 | |
OCC | 0 | 0 | 0 | 0 | |||
Few | 0 | 0 | 0 | 0 | |||
Many | 0 | 0 | 0 | 0 | |||
N | 10 | 10 | 10 | 10 | |||
Table 17: Summary of Urine Analysis Microscopic Parameters (Continued)
Parameters | Sex | Unit | Group 1 | Group 2 | Group 3 | Group 4 | |
Epithelial cells | Absent | Female
| / h.p.f. | 6 | 8 | 5 | 5 |
1-2 | 4 | 2 | 5 | 5 | |||
3-6 | 0 | 0 | 0 | 0 | |||
6-12 | 0 | 0 | 0 | 0 | |||
>12 | 0 | 0 | 0 | 0 | |||
N | 10 | 10 | 10 | 10 | |||
Pus cells | Absent | / h.p.f. | 6 | 8 | 4 | 4 | |
1-2 | 4 | 2 | 6 | 6 | |||
3-6 | 0 | 0 | 0 | 0 | |||
6-12 | 0 | 0 | 0 | 0 | |||
>12 | 0 | 0 | 0 | 0 | |||
N | 10 | 10 | 10 | 10 | |||
RBCs | Absent | / h.p.f. | 10 | 10 | 10 | 10 | |
1-2 | 0 | 0 | 0 | 0 | |||
3-6 | 0 | 0 | 0 | 0 | |||
6-12 | 0 | 0 | 0 | 0 | |||
>12 | 0 | 0 | 0 | 0 | |||
N | 10 | 10 | 10 | 10 | |||
Epithelial cast | Absent | / h.p.f. | 6 | 8 | 6 | 6 | |
1-2 | 4 | 2 | 4 | 4 | |||
3-6 | 0 | 0 | 0 | 0 | |||
>6 | 0 | 0 | 0 | 0 | |||
N | 10 | 10 | 10 | 10 | |||
White cell cast | Absent | / h.p.f. | 10 | 10 | 10 | 10 | |
1-2 | 0 | 0 | 0 | 0 | |||
3-6 | 0 | 0 | 0 | 0 | |||
>6 | Female | 0 | 0 | 0 | 0 | ||
N | 10 | 10 | 10 | 10 | |||
Red cell cast | Absent | / h.p.f. | 10 | 10 | 10 | 10 | |
1-2 | 0 | 0 | 0 | 0 | |||
3-6 | 0 | 0 | 0 | 0 | |||
>6 | 0 | 0 | 0 | 0 | |||
N | 10 | 10 | 10 | 10 | |||
Waxy cast | Absent | / h.p.f. | 10 | 10 | 10 | 10 | |
1-2 | 0 | 0 | 0 | 0 | |||
3-6 | 0 | 0 | 0 | 0 | |||
>6 | 0 | 0 | 0 | 0 | |||
N | 10 | 10 | 10 | 10 | |||
Granularcells | Absent | / h.p.f. | 7 | 9 | 6 | 8 | |
1-2 | 3 | 1 | 4 | 2 | |||
3-6 | 0 | 0 | 0 | 0 | |||
>6 | 0 | 0 | 0 | 0 | |||
N | 10 | 10 | 10 | 10 | |||
hyaline cast | Absent | / h.p.f. | 10 | 10 | 10 | 10 | |
1-2 | 0 | 0 | 0 | 0 | |||
3-6 | 0 | 0 | 0 | 0 | |||
>6 | 0 | 0 | 0 | 0 | |||
N | 10 | 10 | 10 | 10 | |||
Triple Phosphate | Absent | / h.p.f. | 3 | 4 | 4 | 4 | |
OCC | 0 | 0 | 0 | 0 | |||
Few | 0 | 0 | 0 | 0 | |||
Many | Female | 0 | 0 | 0 | 0 | ||
N | 3 | 4 | 4 | 4 | |||
Calcium Oxalate | Absent | / h.p.f. | 5 | 7 | 6 | 8 | |
OCC | 0 | 0 | 0 | 0 | |||
Few | 0 | 0 | 0 | 0 | |||
Many | 0 | 0 | 0 | 0 | |||
N | 5 | 7 | 6 | 8 | |||
Calcium Carbonate | Absent | / h.p.f. | 10 | 10 | 10 | 10 | |
OCC | 0 | 0 | 0 | 0 | |||
Few | 0 | 0 | 0 | 0 | |||
Many | 0 | 0 | 0 | 0 | |||
N | 10 | 10 | 10 | 10 | |||
Sperm | Absent | / h.p.f. | 10 | 10 | 10 | 10 | |
OCC | 0 | 0 | 0 | 0 | |||
Few | 0 | 0 | 0 | 0 | |||
Many | 0 | 0 | 0 | 0 | |||
N | 10 | 10 | 10 | 10 | |||
Bacteria | Absent | / h.p.f. | 10 | 10 | 10 | 10 | |
OCC | 0 | 0 | 0 | 0 | |||
Few | 0 | 0 | 0 | 0 | |||
Many | 0 | 0 | 0 | 0 | |||
N | 10 | 10 | 10 | 10 | |||
Table 18: Mean Hormone levels (T3, T4 & TSH)
Male | |||||
Group | Dose (mg/kg) |
| T3 (pg/ml) | T4 (ng/ml) | TSH (µIU/ml) |
G1 | 0 | Mean | 329.45 | 12.87 | 0.42 |
SD | 108.62 | 3.75 | 0.06 | ||
N | 10 | 10 | 10 | ||
G2 | 30 | Mean | 329.00 | 11.92 | 0.43 |
SD | 120.56 | 1.63 | 0.05 | ||
N | 10 | 10 | 10 | ||
G3 | 120 | Mean | 383.10 | 12.24 | 0.49↑ |
SD | 130.19 | 1.13 | 0.06 | ||
N | 10 | 10 | 10 | ||
G4 | 360 | Mean | 371.60 | 11.97 | 0.43 |
SD | 178.48 | 1.69 | 0.04 | ||
N | 10 | 10 | 10 | ||
Key: SD: Standard deviation; N: No. of animals, ↑=significant increase at p<0.05
Female | |||||
Group | Dose (mg/kg) |
| T3 (pg/ml) | T4 (ng/ml) | TSH (µIU/ml) |
G1 | 0 | Mean | 418.70 | 12.54 | 0.46 |
SD | 174.62 | 1.46 | 0.06 | ||
N | 10 | 10 | 10 | ||
G2 | 30 | Mean | 374.20 | 11.45 | 0.53 |
SD | 60.18 | 1.70 | 0.17 | ||
N | 10 | 10 | 10 | ||
G3 | 120 | Mean | 416.60 | 12.13 | 0.47 |
SD | 151.02 | 1.13 | 0.05 | ||
N | 10 | 10 | 10 | ||
G4 | 360 | Mean | 379.05 | 12.55 | 0.46 |
SD | 186.92 | 1.12 | 0.08 | ||
N | 10 | 10 | 10 | ||
Key: SD: Standard deviation; N: No. of animals
Table 19 Summary of Gross Pathology Observation
(Male and female)
Group | G1 | G2 | G3 | G4 | G1 | G2 | G3 | G4 |
Dose (mg/kg) | 0 | 30 | 120 | 360 | 0 | 30 | 120 | 360 |
Sex | Male | Female | ||||||
Total Number of Animals Observed | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
Organ & Lesion | ||||||||
Testes | ||||||||
Reduced size,minimal | 0 | 1 | 0 | 0 | ./. | ./. | ./. | ./. |
Thymus | ||||||||
Haemorrhage, minimal | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 |
Note: All organs/tissues as per study plan were observed.
Table 20 Microscopic Pathology Observation
Group | G1 | G2 | G3 | G4 | G1 | G2 | G3 | G4 | ||||
Dose (mg/kg) | 0 | 30 | 120 | 360 | 0 | 30 | 120 | 360 | ||||
Sex | Male | Female | ||||||||||
Total Number of Animals Observed | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | ||||
No abnormality detected | 8 | 9 | 6 | 1 | 5 | 10 | 6 | 0 | ||||
Liver | ||||||||||||
Microgranuloma, minimal | 1 | NA | NA | 3 | 3 | NA | NA | 1 | ||||
Single cell necrosis, minimal | 1 | NA | NA | 0 | 0 | NA | NA | 0 | ||||
MNC infiltration, minimal | 0 | NA | NA | 1 | 2 | NA | NA | 4 | ||||
Kidneys | ||||||||||||
Interstitial haemorrhages, minimal | 0 | 0 | 4 | 0 | 0 | 0 | 3 | 3 | ||||
Interstitial haemorrhages, mild | 0 | 0 | 0 | 6 | 0 | 0 | 0 | 2 | ||||
Interstitial MNC infiltration, minimal | 0 | 0 | 0 | 3 | 1 | 0 | 1 | 0 | ||||
Tubular cast, minimal | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | ||||
Tubular cast, mild | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 4 | ||||
Gross Lesions | ||||||||||||
Testes | ||||||||||||
Tubular degeneration and atrophy, minimal | NA | 1 | NA | NA | NA | NA | NA | NA | ||||
Thymus | ||||||||||||
Haemorrhage, minimal | 0 | NA | NA | 0 | NA | NA | 1 | NA | ||||
Note: All of the preserved organs of groups G1 and G4 and gross observation of G2 and G3 were observed and the organs with abnormality are listed.
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- LOAEL
- 30 mg/kg bw/day
- Study duration:
- subchronic
- Species:
- rat
- Quality of whole database:
- Klimisch 1
Repeated dose toxicity: inhalation - systemic effects
Link to relevant study records
- Endpoint:
- repeated dose toxicity: inhalation, other
- Data waiving:
- other justification
- Justification for data waiving:
- other:
Reference
Endpoint conclusion
- Endpoint conclusion:
- no study available
Repeated dose toxicity: inhalation - local effects
Endpoint conclusion
- Endpoint conclusion:
- no study available
Repeated dose toxicity: dermal - systemic effects
Link to relevant study records
- Endpoint:
- repeated dose toxicity: dermal, other
- Data waiving:
- other justification
- Justification for data waiving:
- other:
Reference
Endpoint conclusion
- Endpoint conclusion:
- no study available
Repeated dose toxicity: dermal - local effects
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Justification for classification or non-classification
In the OECD 408 study, the substance did not produce any significant or severe toxicological effects up to 120 mg/kg bw/day (mkd). In male rats, observed effects at 120 mkd included significant changes in clinical chemistry (electrolyte levels) and histopathological findings in the kidneys (i.e. interstitial haemorrhage of minimal severity in 4 out of 10 males). In the female rats, observed effects at 120 mkd included significant changes in clinical chemistry (electrolyte levels) that could be corroborated with significant changes in the functional observatory battery (indicative of hyperactivity) and histopathological findings in the kidneys (i.e. interstitial haemorrhage of minimal severity in 3 out of 10 females). According to CLP guidance values, a justification for STOT RE can be considered if there are significant or severe toxicological effects observed at a dose level below 300 mkd in a 28-days repeated dose toxicity study or, by applying Haber’s rule, below 100 mkd in a 90-days repeated dose toxicity study (see Section 3.9.2.9.6 in Regulation EC No. 1272/2008). Based on the NOAEL value of 1000 mkd from the OECD 407 study and the lack of significant or severe toxicological effects at 120 mkd in the OECD 408 study, the substance is regarded to be classified as Not Classified for STOT RE as per Regulation EC No. 1272/2008.
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