2,2'-(octylimino)bisethanol

Endpoint summary

Description of key information

OECD 408 -90 day repeated dose toxicity:

Oral administration of Genamin 3920 [2,2’-(Octylimino)bisethanol] for 90 consecutive days followed by 28 days of recovery period was found to be well tolerated in Wistar rats up to 350 mg/kg b.w./day without any obvious signs of toxicity.

On the basis of the study findings, the No Observed Adverse Effect Level (NOAEL) of Genamin 3920 [2,2’-(Octylimino)bisethanol] following 90-day repeated dose Oral administration in Wistar rats was 350 mg/kg b.w./day under the tested conditions.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

DECEMBER 2020 to JULY 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted in 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Batch No. ESD0033823

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Historically, the rat has been found to be a suitable model for pre-clinical safety evaluation studies and is recommended by regulatory agencies. The Wistar rat has shown to be sensitive to toxic effects of a variety of drugs and other chemicals. Therefore, the species and strain is a reasonable alternative to larger mammals for toxicity testing with respect to human safety assessment. This study is designed to use the fewest number of animals possible, consistent with the objectives of the study, contemporary scientific needs and standards, and in consideration of regulatory requirements.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Temperature : 19.5 to 24.7 °C
Relative humidity : 40 - 69%
Light/dark cycle (photoperiod) : 12 h light and 12 h dark cycle
Fresh air changes : Minimum 12 air-changes/hour

Route of administration:

oral: gavage

Details on route of administration:

The dose formulations of vehicle/test item were administered to respective groups by oral route once daily for 90 days at the dose volume of 5 mL/kg b.w. Dose was administered using a calibrated disposable syringe attached to 16 gauge stainless steel ball tipped oral gavage needle. Dose volumes were calculated based on the most recently recorded body weight of individual animal.

Vehicle:

corn oil

Details on oral exposure:

The test item dose formulations and vehicle were administered orally by gavage.

Dose selection rationale: A 28-day repeated dose oral toxicity study of Genamin 3920 in Wistar rats conducted at 20, 100 and 500 mg/kg (ECHA, 2020). Mortality was observed at 500 mg/kg b.w. Maximum Tolerated Dose and No Observed Effect Level were 100 and 20 mg/kg b.w. respectively (REACH, EC number: 239-555-0 | CAS number: 15520-05-5). On the basis of published data, the highest dose (350 mg/kg b.w.) selected in this study is expected to produce toxicity. Further, lower doses of 25, 75 and 150 mg/kg b.w. were selected in order to produce a gradation of toxic effects, if any. Concurrent control group animals will be treated with vehicle alone.
In a prenatal developmental study (according to OECD 414) pregnant Wistar rats were administered the test item at the dose levels of 75, 150 and 350 through oral route, once daily, from gestation days (GD) 5 to 19. As mortality was observed at the dose level of 350 mg/kg/day (high dose) during different gestation days, the dose was reduced to 300 mg/kg/day for this group.
Based on all available data summerizd here the use of 350 mg/kg bw per day as highest dose in the OECD 408 study is scientifcally justified and in line with animal welfare consideration.

Analytical verification of doses or concentrations:

yes

Remarks:

The results revealed homogenous distribution in the dose formulation. The observed concentrations at pre-dose, during 6th week and last week of treatment were in the range between 94.0% - 105.7% as compared to the nominal concentrations

Details on analytical verification of doses or concentrations:

The results revealed homogenous distribution in the dose formulation. The observed concentrations at pre-dose, during 6th week and last week of treatment were in the range between 94.0% - 105.7% as compared to the nominal concentrations

Duration of treatment / exposure:

90 consecutive days

Frequency of treatment:

Once daily

Dose / conc.:

350 mg/kg bw/day (nominal)

Remarks:

Dose selection rationale: A 28-day repeated dose oral toxicity study of Genamin 3920 in Wistar rats conducted at 20, 100 and 500 mg/kg (ECHA, 2020). Mortality was observed at 500 mg/kg b.w. Maximum Tolerated Dose and No Observed Effect Level were 100 and 20 mg/kg b.w. respectively (REACH, EC number: 239-555-0 | CAS number: 15520-05-5- Study No 134233).
In a prenatal developmental study (according to OECD 414) pregnant Wistar rats were administered the test item at the dose levels of 75, 150 and 350 through oral route, once daily, from gestation days (GD) 5 to 19. As mortality was observed at the dose level of 350 mg/kg/day (high dose) during different gestation days, the dose was reduced to 300 mg/kg/day for this group.

On the basis of all published data, the highest dose (350 mg/kg b.w.) selected in this study is expected to produce toxicity. Further, lower doses of 25, 75 and 150 mg/kg b.w. were selected in order to produce a gradation of toxic effects, if any. Concurrent control group animals will be treated with vehicle alone.

Based on all available data summerizd here the use of 350 mg/kg bw per day as highest dose in the OECD 408 study is scientifcally justified and in line with animal welfare consideration.

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

75 mg/kg bw/day (nominal)

Dose / conc.:

25 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 m and 10 f in each main dose group; 5 m and 5 f in each recovery dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A 28-day repeated dose oral toxicity study of Genamin 3920 in Wistar rats conducted at 20, 100 and 500 mg/kg (ECHA, 2020). Mortality was observed at 500 mg/kg b.w. Maximum Tolerated Dose and No Observed Effect Level were 100 and 20 mg/kg b.w. respectively (REACH, EC number: 239-555-0 | CAS number: 15520-05-5). On the basis of published data, the highest dose (350 mg/kg b.w.) selected in this study is expected to produce toxicity. Further, lower doses of 25, 75 and 150 mg/kg b.w. were selected in order to produce a gradation of toxic effects, if any. Concurrent control group animals will be treated with vehicle alone. The doses were selected based on available literature and in consultation with the sponsor’s suggestions.
- Rationale for animal assignment (if not random): Animals were randomised into four treatment and one control groups with 10 rats/sex/group. Additionally, 5 /sex/group were maintained for control and high dose recovery. The animals were identified by accession numbers and randomised into treatment and control groups based on body weight. The body weight variation did not exceed ± 20% of the mean body weight range.
- Fasting period before blood sampling for clinical biochemistry:Blood was collected from overnight fasted (water allowed) animals via retro-orbital plexus under isoflurane anaesthesia:
- Rationale for selecting satellite groups: to check for reversibility or occurrence of delayed toxic effects.
- Post-exposure recovery period in satellite groups: 28 days post 90-day treatment

Observations and examinations performed and frequency:

Clinical Observation
Routine cage-side observations were made for all animals once a day throughout the study period for general signs of toxicity and twice a day to check morbidity and mortality.

Detailed Clinical Examination
Detailed clinical examination was carried out once in a week (± 1 day) with exception during last week of treatment and recovery periods. During detailed clinical examination, all animals were observed for changes in skin, fur, eyes, mucous membrane, occurrence of secretions and excretions, autonomic activity such as lacrimation, piloerection, pupil size, unusual respiratory pattern, changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes and bizarre behaviour.

Ophthalmoscopic Examination
Ophthalmologic examination for all group animals was carried out once prior to randomisation for all animals and during the last week of treatment period, for control (G1) and test item high dose group (G5) animals. As no abnormality was observed at test item high dose (G5), the examination was not extended to lower and recovery groups. Mydriasis was induced before examination by instilling 1% Tropicamide.

Body Weight
Individual animal body weights for all the groups of animals were recorded prior to initiation of treatment and at weekly intervals thereafter.

Feed Consumption
Feed consumption was measured at weekly intervals.

Neurobehavioral Examination / FOB (functional observation battery)
Animals in the cage were observed for posture and convulsions.
The animals were observed for the ease of removal from the cage. During handling observation, the animals were observed for ease of handling, palpebral closure, lacrimation, eye examination, piloerection and salivation.
Animals were placed in an open field arena with a clean absorbent paper underneath. Animals were observed for approximately 2 minutes for gait, mobility, arousal, respiration, tonic and clonic movements, stereotypic behaviour, bizarre behaviour, urination, defecation, vocalizations and rearing.
Sensory reactivity and neuromuscular measurements were performed on all animals belonging to the main and recovery groups during the last week of exposure/recovery periods respectively.
Sensory reactivity of all animals were assessed by subjecting them to approach response, touch response, click response, tail pinch response and pupil response measurements.
The animal was held in a supine position and dropped from a height of approximately 30 cm. The ease and uprightness of landing was recorded in a single score as normal or abnormal.
The fore limb and hind limb grip strengths (g) of the animals were measured with the help of a grip strength meter (Orchid Scientific). The fore and hind limb grip strength of each animal was measured 3 consecutive times and average values were calculated.
The tarsal joint pad of each hind foot of the animals was marked with non-permanent non-toxic ink. The animals were suspended in a prone position and dropped from a height of approximately 30 cm on to a recording sheet. The procedure was repeated thrice and the average was calculated.
Motor activity (total and ambulatory activities) were evaluated for all main and recovery group animals during the last week of exposure and recovery periods respectively using OPTO VARIMEX and automated animal activity measuring system (Columbus Instruments, USA) equipped with a computer analyser. Animals were monitored for 3 consecutive ten-minute intervals allowing examination of exploratory and acclimatization activity levels. Stereotypic activity was calculated by subtracting ambulatory activity from total activity.

Clinical Pathology
Clinical Pathology investigations were performed for all animals of main groups and recovery groups on Day 91 and Day 119 respectively. Blood was collected from overnight fasted (water allowed) animals via retro-orbital plexus under isoflurane anaesthesia:
Blood (~0.5 mL) was collected in tubes containing 10% w/v K2EDTA and samples were analysed for following haematological parameters as listed below using haematology analyser (ADVIA, 2120, Siemens).
Red Blood Cell count (RBC)
White Blood Cell count (WBC)
Haemoglobin (HGB)
Haematocrit (HCT)
Mean Corpuscular Haemoglobin Concentration (MCHC)
Mean Corpuscular Volume (MCV)
Mean Corpuscular Haemoglobin (MCH)
Platelet Count (PLT)
Mean Platelet Volume (MPV)
Red Cell Distribution Width (RDW)
Reticulocyte Count (RET)
Differential Leukocyte Count (DLC)
Neutrophils (Neu)
Lymphocytes (Lymp)
Basophils (Baso)
Eosinophils (Eosi)
Monocytes (Mono)

Blood (~1 mL) for coagulation parameters were collected in tubes containing 3.2% w/v Sodium Citrate (Approximately 1:9 ratio of anticoagulant to blood). Following coagulation parameters as listed below were analysed using automatic coagulometer (Diagnostica Start-4, Stago, France).
Prothrombin Time (PT)
Activated Partial Thromboplastin Time (APTT)

Blood (~2 mL) was collected in tubes without anticoagulant. Serum was collected by centrifugation at 3500 rpm for 10 min at 4 ºC. Samples were analysed with an automatic biochemical analyser (Vitros 4600, Ortho Clinical Diagnostics) using standard reagent kits and laboratory methodology. The following parameters were analysed.
Glucose (GLU)
Total Protein (TP)
Albumin (Alb)
Globulin (Glo)
Albumin/Globulin ratio (A/GR) (Calculated)
Alanine Aminotransferase (ALT)
Aspartate Aminotransferase (AST)
Alkaline Phosphatase (ALP)
Total Bilirubin (BIL)
Blood Urea Nitrogen (BUN)
Creatinine (Crea)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Phosphorus (P)
Triglycerides (TRG)
Total Cholesterol (CHO)
HDL Cholesterol (HDL)
LDL Cholesterol (LDL)
Total bile acids
Urea
T3
T4
TSH

Urine output (24 hour ± 30 min) was measured for all surviving animals during last week of treatment and recovery periods.
Urine was collected from all surviving animals using metabolic cages on experimental Days 91 and 119 for main and recovery group animals respectively. Urine was analysed for following parameters using urine analyser (Uriscan Pro II).
Timed Urine Volume Collection#
Colour
Appearance/Clarity*
Specific Gravity
pH
Protein
Glucose
Ketone Bodies
Urobilinogen
Bilirubin
Leucocytes
Nitrites
Blood/Blood cells
An aliquot of the urine sample was centrifuged and the resulting deposit spread on a microscope slide. The deposit was examined for the presence of Epithelial cells (E), Crystals (Cr) and other abnormal constituents (A).

Sacrifice and pathology:

All animals of main and recovery groups were euthanized on Day 91 and Day 119 respectively by CO2 asphyxiation followed by exsanguination. Before scheduled euthanasia, the animals were fasted overnight (water allowed) and terminal body weights were recorded. The animals were subjected to detailed gross pathological observation during necropsy.
All the organs as listed and specified below were collected, weighed and preserved.
All gross lesions
Adrenal glands*
Aorta
Brain - Cerebrum, Cerebellum, Mid brain, & Medulla
Epididymides*
Eyes with optic nerve
Femur with bone marrow
Heart
Kidneys*
Large intestine (Caecum, Colon and Rectum)
Liver*
Lungs with bronchi and bronchioles
Lymph node (Mesenteric and Mandibular)
Oesophagus
Ovaries*
Pancreas
Pituitary#
Prostate with Seminal vesicles and coagulating glands
Salivary glands- Submandibular, Parotid, Sublingual
Sciatic Nerve
Skeletal muscle
Skin along with Mammary Glands
Small intestine (Duodenum, Jejunum, Ileum with Peyer’s Patch)
Spinal Cord (Cervical, Thoracic, Lumbar)
Spleen
Stomach
Testes*
Thymus
Thyroid and parathyroid gland#
Trachea
Urinary bladder
Uterus with cervix
Vagina

Lungs were preserved by inflation with fixative.
*- Paired organs were weighed together. #-Weighed after partial fixation
Relative organ weights were calculated for those organs weighed using the following formula.
Relative organ weight (%) = absolute organ weight (g)/terminal body weight (g) X 100
The organs except testes and eyes were preserved in 10% neutral buffered formalin. Testes were preserved in Modified Davidson’s fixative and eyes were preserved in Davidson’s fixative.

Bone marrow smears were prepared for all animals of main and recovery groups at the time of necropsy by paint brush technique and stained with Giemsa stain. As there was no treatment related changes observed in haematological parameters, examination of bone marrow smears was not performed.

At the time of necropsy, progressive sperm motility was determined manually in vas deferens sperm samples for all the males. Additionally, sperm smears were also prepared from vas deferens of all males and fixed in methanol. Sperm morphology evaluation was restricted to control and high-dose males. As there was no treatment related changes observed in high dose group, the evaluation was not extended to lower dose and recovery high dose groups. The oestrus cycle of all females were determined by taking vaginal smears before terminal sacrifice.

Histopathology examination was carried out on all dead animals, gross lesions and preserved organs of vehicle control and high dose main group animals. As there was a histophatological changes observed in stomach, trachea and kidneys in high dose group/found dead animals, the evaluation was extended to lower dose and recovery high dose groups.
Tissues selected for histopathological evaluation were processed, embedded and sections were cut at 3 - 5 μ. Sections were placed on grease-free slides and stained with Haematoxylin and Eosin. A 5-step grading system of minimum, mild, moderate, marked and severe was used to rank microscopic findings for comparison among groups. Data was compiled based on incidences and severity of changes.

Statistics:

The data was analysed using Graphpad Prism, version 4.00. Statistical comparisons were carried out for all continuous data between treatment and control groups using parametric (one-way analysis of variance (ANOVA), Dunnett's t-Test) or non-parametric (Kruskal-Wallis test, Mann Whitney’s Test) test procedures. The choice of parametric or non-parametric test was based on whether the groups satisfy the homogeneity of variance as evaluated by Bartlett’s test. Statistical significance was evaluated at p ≤ 0.05 and/or p ≤ 0.01. All quantitative data was summarized and expressed as Mean ± SD. Comparison between recovery groups was done by Student’s t-test.
Note: Statistical analysis of Bilirubin and LDL parameters was not performed as the results of the parameters for most of the animals were below detectable range.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Animals from mid high (150 mg/kg bw per day) (Male: 3/10; Female: 9/10) and high (350 mg/kg bw per day) (Male: 9/10; Female: 8/10) dose groups displayed transient salivation from Days 51 to 60.

Weekly detailed clinical observations did not reveal any abnormalities in animals treated with test item as well as vehicle control. One male animal from high dose (350 mg/kg bw per day)exhibited bronchial rales and one female animal from vehicle control was having mild alopecia-bilateral from Week 9 to Week 13. Due to the isolated incidences these effects are not considered to be of toxicological relevance.
For more details see attached table

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One animal each from low (T107/023) and mid high (T107/067) dose groups were found dead on Day 41 presumed to be gavaging error.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no significant changes in weekly body weights of the all test item treated animals when compared to the vehicle control.
Statistically significant increase in body weight was recorded on Day 43 and Day 50 in recovery high dose females. However, no correlating changes were observed in feed consumption and normal body weight gain was observed at the end of treatment period and was considered as incidental finding
For more details see attached table

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No treatment related changes were recorded in the average daily feed consumption of the test item treated groups when compared to vehicle control throughout the treatment and recovery periods. An isolated marginal (~12%) decrease in feed consumption of high dose recovery females was observed on Day 29 when compared to recovery vehicle control animals.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ocular abnormalities were detected in ophthalmoscopic examinations carried out during pre-dose observations in all animals and during the last week of treatment period.

Haematological findings:

no effects observed

Description (incidence and severity):

No treatment related changes were observed in any of the haematology parameters in test item treated groups including recovery groups as compared with respective vehicle controls. Significant increase in MCH in low dose females, HCT in recovery high dose males and decrease in relative eosinophil count in mid and mid high dose group females were considered not related to test item as the values are within historical control ranges and because of absence of dose relationship or associated changes in other parameters.
For more details see attached table

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Significant increase in triglycerides and calcium was observed in high dose females. In absence of associated changes in other parameters, it was not attributed to test item effect. Significant decrease in BUN and urea in low and high dose females were not dose dependent. Significant increase in cholesterol, BUN, HDL, T3 and urea was observed in recovery high dose males and significant increase in globulin, cholesterol, HDL, chloride and bile acids was observed in recovery high dose females when compared to concurrent control groups. These changes were within the normal biological range, hence were considered as incidental findings and not related to test item treatment.
For more details see attached table

Endocrine findings:

no effects observed

Urinalysis findings:

no effects observed

Description (incidence and severity):

No treatment related significant changes were observed in any of the urinalysis parameters in main and recovery group animals in both sexes when compared to respective vehicle controls. Significant increase in pH in high dose males, decrease in pH in low, mid and mid high dose group females and increase in volume in recovery high dose males were observed. In absence of associated histopathological changes/dose dependency, these changes were considered not related to test item treatment. Furthermore, all the changes observed are within historical control ranges.
For more details see attached table

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No relevant effects were observed in any of the parameters of the functional observation battery performed at the end of the treatment and recovery periods.
Statistically significant decrease in foot splay and increase in stereotypic behaviour was observed in recovery high dose females and males respectively. The animals from the recovery high dose were apparently healthy and in absence of test item related changes in other neurobehavioral observations, these changes were considered incidental.

Sensory Reactivity Measurements
All animals of test item treated groups as well as the control groups displayed normal approach, tail pinch, touch, click and pupil responses.


Air Righting Reflex
Air righting reflex of all the animals of both the control and test item treated groups were found to be normal.


Grip Strength
No alterations in the fore and hind limb grip strength were recorded in any of the treated group animals as compared to concurrent control groups

Hind Limb Foot Splay
The hind limb foot splay of all test item treated groups were comparable to concurrent control groups. Statistically significant decrease in foot splay was observed in recovery high dose females. The observed difference was minimal and in absence of test item related changes in other neurobehavioral observations, the observed change did not have any toxicological significance.

Motor Activity
Administration of test item did not cause any significant alterations in the total, ambulatory and stereotypic activities of treated rats as compared to respective control group animalsStatistically significant increase in stereotypic behaviour was observed in recovery high dose males when compared to recovery vehicle control. The animals from the recovery high dose were apparently healthy, in absence of test item related changes in other neurobehavioral observations the observed change did not have any toxicological significance


For more details see attached table

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No test item related changes were observed in both absolute and relative organ weight in main and recovery group animals in both sexes when compared to respective vehicle controls. However, a significant decrease was observed in relative weight of brain in high dose females and heart and thyroid in recovery high dose females when compared to respective vehicle controls. Absence of related histopathology finding in high dose animals, the decreased relative weights were considered not related to test item treatment. Furthermore, all the changes observed are within historical control ranges

Gross pathological findings:

no effects observed

Description (incidence and severity):

Distended and empty gastro intestinal tract were recorded in one found dead animal each from low (T107/023) and mid high (T107/067) dose groups. However, correlated histopathology finding in GI tract was not observed. Bronchial rales and dyspnea which were observed before the death of these animals could be an indication for misgavaging. Small unilateral seminal vesicle observed in one control male and enlarged bilateral testes observed in one 150 mg/kg test item treated male. Correlated histopathology findings observed in seminal vesicles (atrophy) and testes (dilatation) revealed that the observed findings were considered as spontaneous/incidental.
For more details see attached table

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

One male (T107/081) sacrificed terminally from 350 mg/kg dose group showed multifocal necrosis in kidneys together with squamous cell hyperplasia in non-glandular stomach. In addition, two males from the same group showed mononuclear cell infiltration in non-glandular stomach. However, in absence correlating changes in other parameters, it was considered as test item related non-adverse finding and due to the irritating properties of the test item.As the kidney findings were limited to one animal each from low dose and high dose groups it was considered as incidental not related to test item. However, histopathology findings observed in stomach were considered as test item related.
No histopathological changes were observed in stomach and kidneys of recovery high dose group animals.
Other histopathology findings observed in liver (inflammatory cell infiltrate), kidneys (casts, basophilc tubule and inflammatory cell infiltrate), spleen (EMH), thymus (Ectopic tissue), testes (tubular dilatation and tubular degeneration), epididymides (cell debris), prostate (atrophy and inflammatory cell infiltrate), seminal vesicles (atrophy), trachea (inflammatory cell infiltrate) and heart (inflammatory cell infiltrate) across groups and the sex were considered as spontaneous/incidental.
For more details see attached table

Histopathological findings: neoplastic:

no effects observed

Details on results:

No test item related mortalities were observed during study period. One animal each from low (T107/023) and mid high (T107/067) dose groups were found dead on Day 41. Bronchial rales and dyspnea were observed before the death of these animals. On microscopic evaluation, erosion in trachea was observed in mid high dose group animal which was considered as an indicative of regurgitation/wrong dosing. Further, multifocal necrosis in kidneys along with mild to marked atrophic changes were noted in spleen and thymus of low dose group animal. Despite the aforesaid changes in low dose, there were no other microscopic observations that explained the cause of death. These deaths are presumed to be incidental and due to gavaging error as brochniail rales and dyspenea occurred after application of the test item and before the death. Forthemore these deaths occured in a low or mid high dose animals and there were no adverse findings in any other rats at any dose level.
Animals from mid high (Male: 3/10; Female: 9/10) and high (Male: 9/10; Female: 8/10) dose groups displayed transient salivation from Days 51 to 60. Transient salivation is clinical finding frequently observed after oral gavaging of local irritant effect of test item and is not assumed to be a sign of systemic toxicity (REACH, EC number: 239-555-0 | CAS number: 15520-05-5).
In addition, few isolated incidences of bronchial rales (Male - 25, 150 and 350 mg/kg b.w.: 1/10), dyspnea (Male - 25 and 150 mg/kg b.w.: 1/10), alopecia (Female - 0 mg/kg b.w.: 1/10) and hyperactivity (Female - 75 mg/kg b.w.: 2/10) were observed in different experimental groups.


Detailed Clinical Observations

Weekly detailed clinical observations did not reveal any abnormalities in animals treated with test item as well as vehicle control. One male animal from high dose exhibited bronchial rales and one female animal from vehicle control was having mild alopecia-bilateral from Week 9 to Week 13. Due to the isolated incidences these effects are not considered to be of toxicological relevance.

The data was analysed using Graphpad Prism, version 4.00. Statistical comparisons were carried out for all continuous data between treatment and control groups using parametric (one-way analysis of variance (ANOVA), Dunnett's t-Test) or non-parametric (Kruskal-Wallis test, Mann Whitney’s Test) test procedures. The choice of parametric or non-parametric test was based on whether the groups satisfy the homogeneity of variance as evaluated by Bartlett’s test. Statistical significance was evaluated at p ≤ 0.05 and/or p ≤ 0.01. All quantitative data was summarized and expressed as Mean ± SD. Comparison between recovery groups was done by Student’s t-test.
Note: Statistical analysis of Bilirubin and LDL parameters was not performed as the results of the parameters for most of the animals were below detectable range.

Key result

Dose descriptor:

NOAEL

Effect level:

350 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Summary of Detailed Clinical Examination

Gender: Male            No. of Animals/Group/Sex: 10      

Group & Dose (mg/kg b.w.)	Clinical Signs	No. of Animals Showing Clinical Signs
		Week
		#	1	2	3	4	5	6
G1 & 0	No Abnormality Detected	10	10	10	10	10	10	10
G2 & 25	No Abnormality Detected	10	10	10	10	10	10	9
G3 & 75	No Abnormality Detected	10	10	10	10	10	10	10
G4 & 150	No Abnormality Detected	10	10	10	10	10	10	9
G5 & 350	No Abnormality Detected	10	10	10	10	10	10	9
	Bronchial Rales	-	-	-	-	-	-	1

 

Group & Dose (mg/kg b.w.)	Clinical Signs	No. of Animals Showing Clinical Signs
		Week
		7	8	9	10	11	12
G1 & 0	No Abnormality Detected	10	10	10	10	10	10
G2 & 25	No Abnormality Detected	9	9	9	9	9	9
G3 & 75	No Abnormality Detected	10	10	10	10	10	10
G4 & 150	No Abnormality Detected	9	9	9	9	9	9
G5 & 350	No Abnormality Detected	10	10	10	10	10	10

        Gender: Female        No. of Animals/Group/Sex: 10                

Group & Dose (mg/kg b.w.)	Clinical Signs	No. of Animals Showing Clinical Signs
		Week
		#	1	2	3	4	5	6
G1 & 0	No Abnormality Detected	10	10	10	10	10	10	10
G2 & 25	No Abnormality Detected	10	10	10	10	10	10	10
G3 & 75	No Abnormality Detected	10	10	10	10	10	10	10
G4 & 150	No Abnormality Detected	10	10	10	10	10	10	10
G5 & 350	No Abnormality Detected	10	10	10	10	10	10	10

Group & Dose (mg/kg b.w.)	Clinical Signs	No. of Animals Showing Clinical Signs
		Week
		7	8	9	10	11	12
G1 & 0	No Abnormality Detected	10	10	9	9	9	9
	Alopecia (1, bilateral)	-	-	1	1	1	1
G2 & 25	No Abnormality Detected	10	10	10	10	10	10
G3 & 75	No Abnormality Detected	10	10	10	8	10	10
	Hyperactive	-	-	-	2	-	-
G4 & 150	No Abnormality Detected	10	10	10	10	10	10
G5 & 350	No Abnormality Detected	10	10	10	10	10	10

Summary of Neurobehavioral Examination

Gender: Male            No. of Animals/Group/Sex: 10                

Parameters	Group & Dose (mg/kg b.w.)
	G1 (0)	G2 (25)	G3 (75)	G4 (150)	G5 (350)
Home Cage Observations
Posture
Sitting or standing alert, watching	8	6	6	2	5
Curled up often asleep	0	1	0	0	1
Rearing	2	2	4	7	4
Convulsions
Absent	10	9	10	9	10
Handling Observations
Ease of Removal
Very easy	8	8	9	9	10
Easy	2	1	1	0	0
Handling Reactivity
Easy	10	9	10	9	10
Palpebral Closure
Wide open	10	9	10	9	10
Lacrimation
No external lacrimation	10	9	10	9	10
Eye examination
Normal	10	9	10	9	10
Piloerection
Absent	10	9	10	9	10
Salivation
No external Salivation	10	9	10	9	10

Parameters		Group & Dose (mg/kg b.w.)
		G1 (0)	G2 (25)	G3 (75)	G4 (150)	G5 (350)
Open Field Observations
Gait
Normal		10	9	10	9	10
Mobility
Normal		10	9	10	9	10
Arousal
Low		10	9	10	8	10
High		0	0	0	1	0
Respiration
Normal		10	9	10	9	10
Tonic Movement
Absent		10	9	10	9	10
Clonic Movement
Absent		10	9	10	9	10
Stereotype
Absent		10	9	10	9	10
Bizzare Behaviour
Absent		10	9	10	9	10
No. of Rears (Mean ± S.D.)		9.3 ± 3.2	9.67±3.28	8± 3.71	12.44±3.64	10.1±2.8
No. of Vocalisations	0	10	9	10	9	10
No. of Urine Pools	0	10	5	9	8	9
	1 to 3	0	4	1	1	1
No. of Faecal Bolus	0	9	7	9	8	9
	1 to 3	0	1	0	1	1
	4 to 6	1	1	1	0	0

Parameters	Group & Dose (mg/kg b.w.)
	G6 (0)	G7 (350)
Home Cage Observations
Posture
Sitting or standing alert, watching	3	4
Curled up often asleep	0	1
Rearing	2	0
Convulsions
Absent	5	5
Handling Observations
Ease of Removal
Very easy	5	5
Handling Reactivity
Easy	4	4
Moderately difficult	1	1
Palpebral Closure
Wide open	5	5
Lacrimation
No external lacrimation	5	5
Eye examination
Normal	5	5
Piloerection
Absent	5	5
Salivation
No external Salivation	5	5

Parameters		Group & Dose (mg/kg b.w.)
		G6 (0)	G7 (350)
Open Field Observations
Gait
Normal		5	5
Mobility
Normal		5	5
Arousal
Low		4	5
High		1	0
Respiration
Normal		5	5
Tonic Movement
Absent		5	5
Clonic Movement
Absent		5	5
Stereotype
Absent		5	5
Bizzare Behaviour
Absent		5	5
N° of Rears (Mean ± S.D.)		10 ± 2.35	9.4 ± 2.7
N° of Vocalisations	0	5	5
N° of Urine Pools	0	3	4
	1 to 3	2	1
N° of Faecal Bolus	0	4	4
	1 to 3	1	1

Gender: Female        No. of Animals/Group/Sex: 10                

Parameters	Group & Dose (mg/kg b.w.)
	G1 (0)	G2 (25)	G3 (75)	G4 (150)	G5 (350)
Home Cage Observations
Posture
Sitting or standing alert, watching	2	4	3	6	2
Curled up often asleep	1	0	1	1	7
Rearing	7	6	6	3	1
Convulsions
Absent	10	10	10	10	10
Handling Observations
Ease of Removal
Very easy	10	10	10	10	10
Handling Reactivity
Easy	10	10	10	10	10
Palpebral Closure
Wide open	10	10	10	10	10
Lacrimation
No external lacrimation	10	10	10	10	10
Eye examination
Normal	10	10	10	10	10
Piloerection
Absent	10	10	10	10	10
Salivation
No external Salivation	10	10	10	10	10

Parameters		Group & Dose (mg/kg b.w.)
		G1 (0)	G2 (25)	G3 (75)	G4 (150)	G5 (350)
Open Field Observations
Gait
Normal		10	10	10	10	10
Mobility
Normal		10	10	10	10	10
Arousal
Low		9	8	8	10	9
High		1	1	2	0	1
Very high		0	1	0	0	0
Respiration
Normal		10	10	10	10	10
Tonic Movement
Absent		10	10	10	10	10
Clonic Movement
Absent		10	10	10	10	10
Stereotype
Absent		10	10	10	10	10
Bizzare Behaviour
Absent		10	10	10	10	10
No. of Rears (Mean ± S.D.)		15.9±4.82	15.7±5.72	16.2±4.52	14.8±3.97	13.1±3.84
No. of Vocalisations	0	10	10	10	10	10
No. of Urine Pools	0	10	9	9	7	0
	1 to 3	0	1	1	3	0
No. of Faecal Bolus	0	10	8	10	9	10
	1 to 3	0	2	0	1	0
	4 to 6	0	0	0	0	0

Gender: Female        No. of Animals/Group/Sex: 5                 

Parameters	Group & Dose (mg/kg b.w.)
	G6 (0)	G7 (350)
Home Cage Observations
Posture
Sitting or standing alert, watching	3	2
Rearing	2	3
Convulsions
Absent	5	5
Handling Observations
Ease of Removal
Very easy	5	5
Handling Reactivity
Easy	5	5
Palpebral Closure
Wide open	5	5
Lacrimation
No external lacrimation	5	5
Eye examination
Normal	5	5
Piloerection
Absent	5	5
Salivation
No external Salivation	5	5

Parameters		Group & Dose (mg/kg b.w.)
		G6 (0)	G7 (350)
Open Field Observations
Gait
Normal		5	5
Mobility
Normal		5	5
Arousal
Low		4	4
High		1	1
Respiration
Normal		5	5
Tonic Movement
Absent		5	5
Clonic Movement
Absent		5	5
Stereotype
Absent		5	5
Bizzare Behaviour
Absent		5	5
N° of Rears (Mean ± S.D.)		11.4 ± 1.14	11.2 ± 2.28
N° of Vocalisations	0	5	5
N° of Urine Pools	0	5	5
N° of Faecal Bolus	0	5	5

Summary of Motor Activity, Grip Strength and Hind Limb Foot Splay Record

 Gender: Male

Parameter	Group & Dose (mg/kg b. w.)
	G1 & 0			G2 & 25			G3 & 75
	Mean	SD	N	Mean	SD	N	Mean	SD	N
Motor Activity (number of counts)
Total Count	5299.90	1484.45	10	5292.89	1549.11	9	5219.10	1981.30	10
Ambulatory Count	4025.80	1275.65	10	4102.78	1361.38	9	3995.70	1722.22	10
Stereotypic Count	1274.10	242.20	10	1190.11	235.26	9	1223.40	274.84	10
Grip Strength (g)
Fore limb	1040.75	54.89	10	1037.06	106.74	9	991.16	29.63	10
Hind limb	722.96	56.94	10	714.46	51.90	9	756.58	55.75	10
Hind Limb Foot Splay (mm)
Foot Splay	81.23	26.49	10	85.85	23.12	9	89.83	18.35	10

Parameter	Group & Dose (mg/kg b. w.)
	G4 & 150			G5 & 350
	Mean	SD	N	Mean	SD	N
Motor Activity (number of counts)
Total Count	6127.22	1236.41	9	6197.50	1095.19	10
Ambulatory Count	4725.56	1019.83	9	4806.50	978.48	10
Stereotypic Count	1401.67	259.08	9	1391.00	159.55	10
Grip Strength (g)
Fore limb	1047.68	43.45	9	1080.83	94.71	10
Hind limb	762.12	32.64	9	755.29	43.63	10
Hind Limb Foot Splay (mm)
Foot Splay	79.85	20.05	9	71.20	16.97	10

Parameter	Group & Dose (mg/kg b. w.)
	G6 & 0			G7 & 350
	Mean	SD	N	Mean	SD	N
Motor Activity (number of counts)
Total Count	4013.20	1428.58	5	5820.80	1452.61	5
Ambulatory Count	2991.00	1263.13	5	4500.60	1374.62	5
Stereotypic Count	1022.20	181.41	5	1320.20↑	139.23	5
Grip Strength (g)
Fore limb	1000.19	58.94	5	1008.91	24.57	5
Hind limb	782.49	43.70	5	796.49	48.99	5
Hind Limb Foot Splay (mm)
Foot Splay	83.93	13.07	5	88.73	22.36	5

Gender: Female                                                                            

Parameter	Group & Dose (mg/kg b. w.)
	G1 & 0			G2 & 25			G3 & 75
	Mean	SD	N	Mean	SD	N	Mean	SD	N
Motor Activity (number of counts)
Total Count	8118.20	2843.48	10	7783.40	1882.50	10	6820.70	1624.90	10
Ambulatory Count	6464.30	2585.44	10	6179.40	1665.90	10	5288.90	1397.31	10
Stereotypic Count	1653.90	295.89	10	1604.00	284.55	10	1531.80	244.00	10
Grip Strength (g)
Fore limb	937.49	72.88	10	946.14	63.27	10	918.25	56.61	10
Hind limb	630.03	35.42	10	641.39	52.37	10	618.03	29.23	10
Hind Limb Foot Splay (mm)
Foot Splay	59.90	10.52	10	57.27	15.71	10	56.07	10.77	10

Parameter	Group & Dose (mg/kg b. w.)
	G4 & 150			G5 & 350
	Mean	SD	N	Mean	SD	N
Motor Activity (number of counts)
Total Count	7798.70	1763.36	10	6845.20	1855.11	10
Ambulatory Count	6178.90	1597.72	10	5301.20	1680.68	10
Stereotypic Count	1619.80	265.97	10	1544.00	206.96	10
Grip Strength (g)
Fore limb	931.21	68.27	10	976.50	77.00	10
Hind limb	680.39	44.45	10	666.03	59.90	10
Hind Limb Foot Splay (mm)
Foot Splay	61.70	14.49	10	70.20	18.82	10

Parameter	Group & Dose (mg/kg b. w.)
	G6 & 0			G7 & 350
	Mean	SD	N	Mean	SD	N
Motor Activity (number of counts)
Total Count	6356.80	1757.20	5	6242.60	1088.20	5
Ambulatory Count	5053.20	1419.08	5	4971.40	808.83	5
Stereotypic Count	1303.60	347.21	5	1271.20	289.55	5
Grip Strength (g)
Fore limb	785.32	31.37	5	749.11	26.82	5
Hind limb	655.05	13.07	5	651.53	16.99	5
Hind Limb Foot Splay (mm)
Foot Splay	77.67	4.28	5	65.27↓	6.72	5

Summary of Ophthalmologic Examination

Gender: Male            No. of Animals/Group/Sex: 10                

Group & Dose (mg/kg b.w.)	Observation	No. of Animals
		Pre-dose		Day 90
		Right Eye	Left Eye	Right Eye	Left Eye
G1 & 0	NAD	10	10	10	10
G2 & 25	NAD	10	10	-	-
G3 & 75	NAD	10	10	-	-
G4 & 150	NAD	10	10	-	-
G5 & 350	NAD	10	10	10	10

Gender: Male            No. of Animals/Group/Sex: 5                

Group & Dose  (mg/kg b.w.)	Observation	No. of Animals
		Pre-dose
		Right Eye	Left Eye
G6 & 0	NAD	5	5
G7 & 350	NAD	5	5

Gender: Female        No. of Animals/Group/Sex: 10                

Group & Dose (mg/kg b.w.)	Observation	No. of Animals
		Pre-dose		Day 90
		Right Eye	Left Eye	Right Eye	Left Eye
G1 & 0	NAD	10	10	10	10
G2 & 25	NAD	10	10	-	-
G3 & 75	NAD	10	10	-	-
G4 & 150	NAD	10	10	-	-
G5 & 350	NAD	10	10	10	10

Gender: Female        No. of Animals/Group/Sex: 5                

Group & Dose (mg/kg b.w.)	Observation	No. of Animals
		Pre-dose
		Right Eye	Left Eye
G6 & 0	NAD	5	5
G7 & 350	NAD	5	5

Summary of Body Weight (g)

Gender: Male

Week	Group & Dose (mg/kg b.w.)
	G1 & 0			G2 & 25			G3 & 75
	Mean	SD	N	Mean	SD	N	Mean	SD	N
#	229.72	16.44	10	225.23	15.34	10	227.51	18.08	10
1	294.92	16.56	10	291.27	16.78	10	295.09	20.10	10
2	344.04	24.91	10	342.71	15.72	10	348.01	25.01	10
3	378.54	28.87	10	382.38	17.30	10	386.88	29.36	10
4	414.05	34.07	10	420.20	18.12	10	426.68	33.97	10
5	439.53	36.89	10	449.89	17.99	10	455.62	37.46	10
6	466.59	41.96	10	479.97	19.43	9	486.43	44.48	10
7	490.86	46.10	10	505.08	20.18	9	510.48	45.66	10
8	513.79	50.72	10	524.50	20.54	9	531.03	52.25	10
9	528.00	53.85	10	540.23	19.44	9	549.84	52.90	10
10	543.64	54.30	10	559.33	21.80	9	569.97	57.59	10
11	557.18	58.33	10	574.04	27.06	9	584.60	61.26	10
12	564.12	57.39	10	582.16	30.53	9	591.63	65.50	10
13	562.49	59.04	10	580.37	30.58	9	591.20	58.72	10

Week	Group & Dose (mg/kg b.w.)
	G4 & 150			G5 & 350
	Mean	SD	N	Mean	SD	N
#	229.26	19.45	10	227.10	13.54	10
1	290.86	22.98	10	282.87	17.29	10
2	340.26	26.81	10	331.55	24.82	10
3	377.59	31.75	10	372.70	27.82	10
4	412.19	33.71	10	408.75	25.47	10
5	438.66	37.30	10	432.88	31.67	10
6	457.71	40.50	9	454.31	37.23	10
7	481.00	40.88	9	479.47	38.51	10
8	501.26	38.99	9	496.20	38.44	10
9	519.37	38.23	9	509.71	40.85	10
10	536.40	39.78	9	527.20	39.06	10
11	547.19	40.74	9	534.26	41.72	10
12	553.20	42.08	9	541.53	43.73	10
13	554.40	42.39	9	542.59	44.77	10

Week	Group & Dose (mg/kg b.w.)
	G6 & 0			G7 & 350
	Mean	SD	N	Mean	SD	N
#	218.58	21.88	5	228.77	16.73	5
1	285.05	16.72	5	286.63	25.47	5
2	328.47	19.29	5	325.36	10.60	5
3	362.05	25.69	5	355.97	12.24	5
4	395.06	29.04	5	398.61	18.67	5
5	419.38	29.82	5	433.96	22.02	5
6	447.37	28.05	5	457.80	28.18	5
7	468.48	34.04	5	481.53	31.78	5
8	488.26	37.57	5	504.12	39.05	5
9	500.80	36.17	5	521.07	46.06	5
10	517.28	34.11	5	537.99	51.84	5
11	534.47	35.78	5	554.43	63.25	5
12	535.76	35.73	5	566.11	55.76	5
13	534.81	35.05	5	570.83	52.13	5
14	539.14	33.59	5	583.30	52.95	5
15	550.37	38.03	5	595.36	47.03	5
16	560.09	37.47	5	607.78	51.94	5
17	566.34	38.99	5	612.79	48.53	5

Gender: Female

Week	Group & Dose (mg/kg b.w.)
	G1 & 0			G2 & 25			G3 & 75
	Mean	SD	N	Mean	SD	N	Mean	SD	N
#	175.18	12.76	10	177.64	10.15	10	177.04	11.95	10
1	198.75	12.62	10	201.45	15.43	10	199.43	11.91	10
2	217.40	16.54	10	218.77	14.74	10	218.45	20.44	10
3	234.33	20.82	10	234.86	15.91	10	232.76	21.69	10
4	250.53	23.47	10	254.86	21.01	10	250.55	21.08	10
5	260.99	21.18	10	263.11	20.28	10	263.04	21.67	10
6	273.17	20.35	10	273.63	21.28	10	275.16	21.86	10
7	283.67	21.46	10	281.84	18.81	10	280.22	22.00	10
8	288.77	23.58	10	289.63	21.21	10	289.39	24.33	10
9	297.13	22.83	10	295.48	22.64	10	298.61	26.47	10
10	289.11	21.59	10	291.85	23.10	10	291.52	25.86	10
11	296.71	23.97	10	299.90	23.19	10	302.36	22.27	10
12	301.81	27.81	10	304.91	23.44	10	308.68	29.20	10
13	304.28	24.29	10	306.78	23.74	10	308.83	25.41	10

Week	Group & Dose (mg/kg b.w.)
	G4 & 150			G5 & 350
	Mean	SD	N	Mean	SD	N
#	177.52	13.41	10	174.11	9.54	10
1	202.28	17.27	10	201.10	13.28	10
2	224.23	22.47	10	224.57	11.16	10
3	237.10	21.41	10	243.60	14.74	10
4	252.36	20.38	10	258.59	19.39	10
5	264.68	20.41	10	270.98	21.62	10
6	277.40	19.45	10	290.59	21.15	10
7	280.01	24.51	10	298.53	22.39	10
8	289.18	24.48	10	308.41	22.27	10
9	295.98	25.73	10	311.72	28.57	10
10	293.72	25.30	10	314.90	23.55	10
11	303.45	27.77	10	327.46	29.31	10
12	310.49	27.54	10	335.74	27.88	10
13	310.66	27.23	10	332.97	30.76	10

Week	Group & Dose (mg/kg b.w.)
	G4 & 150			G5 & 350
	Mean	SD	N	Mean	SD	N
#	166.76	8.73	5	170.32	9.49	5
1	194.85	11.35	5	192.54	11.11	5
2	206.87	9.34	5	218.35	7.76	5
3	223.74	11.47	5	234.27	13.95	5
4	239.58	12.45	5	245.79	16.19	5
5	246.22	12.13	5	258.85	17.20	5
6	250.85	10.31	5	274.56*+	16.17	5
7	259.28	14.34	5	284.44*+	17.02	5
8	270.32	13.09	5	286.48	22.54	5
9	275.96	11.39	5	292.72	18.70	5
10	271.23	12.52	5	296.72	24.75	5
11	281.96	12.43	5	299.61	22.68	5
12	288.01	16.17	5	309.40	23.10	5
13	291.32	10.73	5	307.54	24.41	5
14	292.02	14.56	5	313.79	22.39	5
15	296.77	11.83	5	313.50	17.56	5
16	299.05	11.87	5	316.83	20.21	5
17	298.05	13.13	5	316.68	22.36	5

Summary of Haematology Parameters

Gender: Male

Parameters	Group & Dose (mg/kg b.w.)
	G1 & 0			G2 & 25			G3 & 75
	Mean	SD	N	Mean	SD	N	Mean	SD	N
RBC (10^6cells/µL)	8.75	0.32	10	8.69	0.44	9	8.46	0.30	10
HGB (g/dL)	14.61	0.44	10	14.59	0.61	9	14.29	0.75	10
HCT (%)	44.91	1.26	10	44.67	1.54	9	43.61	1.89	10
MCV (fL)	51.34	1.15	10	51.46	1.60	9	51.58	1.83	10
MCH (pg)	16.70	0.43	10	16.78	0.60	9	16.91	0.82	10
MCHC (g/dL)	32.54	0.36	10	32.62	0.59	9	32.75	0.61	10
RDW (%)	13.17	0.48	10	12.91	0.20	9	12.92	0.51	10
RET(x10^9 cells/L)	54.61	20.75	10	50.09	10.99	9	57.59	9.46	10
PLT (10^3cells/µL)	1140.30	144.08	10	1087.78	108.08	9	1058.20	151.22	10
MPV (fL)	6.72	0.28	10	6.72	0.13	9	6.80	0.41	10
WBC (10^3cells/µL)	9.77	2.68	10	8.21	1.83	9	8.80	1.62	10
NEU (10^3cells/µL)	2.34	1.18	10	1.80	0.74	9	2.03	0.49	10
LYM (10^3cells/µL)	6.70	2.68	10	5.86	1.09	9	6.17	1.36	10
MON (10^3cells/µL)	0.43	0.34	10	0.24	0.05	9	0.31	0.13	10
EOS (10^3cells/µL)	0.15	0.08	10	0.15	0.04	9	0.16	0.06	10
BAS (10^3cells/µL)	0.02	0.01	10	0.02	0.01	9	0.02	0.01	10
NEU (%)	24.64	14.57	10	21.47	4.34	9	23.38	5.80	10
LYM (%)	67.63	17.91	10	71.87	4.90	9	69.98	6.42	10
MON (%)	4.68	4.39	10	3.03	0.65	9	3.40	0.99	10
EOS (%)	1.43	0.61	10	1.88	0.32	9	1.77	0.46	10
BAS (%)	0.25	0.07	10	0.22	0.10	9	0.18	0.08	10

Parameters	Group & Dose (mg/kg b.w.)
	G4 & 150			G5 & 350
	Mean	SD	N	Mean	SD	N
RBC (10^6cells/µL)	8.97	0.43	9	8.69	0.36	10
HGB (g/dL)	14.80	0.58	9	14.37	0.44	10
HCT (%)	45.21	1.33	9	44.48	1.60	10
MCV (fL)	50.43	1.32	9	51.21	1.13	10
MCH (pg)	16.52	0.58	9	16.56	0.53	10
MCHC (g/dL)	32.73	0.64	9	32.33	0.78	10
RDW (%)	12.77	0.74	9	12.77	0.18	10
RET(x10^9 cells/L)	49.28	18.86	9	55.78	6.77	10
PLT (10^3cells/µL)	1148.00	143.92	9	1095.00	109.41	10
MPV (fL)	6.89	0.29	9	6.82	0.38	10
WBC (10^3cells/µL)	7.93	1.79	9	8.30	0.90	10
NEU (10^3cells/µL)	1.94	0.75	9	2.15	0.80	10
LYM (10^3cells/µL)	5.49	1.16	9	5.67	1.05	10
MON (10^3cells/µL)	0.26	0.09	9	0.22	0.08	10
EOS (10^3cells/µL)	0.13	0.03	9	0.13	0.03	10
BAS (10^3cells/µL)	0.01	0.01	9	0.02	0.01	10
NEU (%)	24.13	4.78	9	26.12	9.72	10
LYM (%)	69.43	4.44	9	68.29	9.91	10
MON (%)	3.21	0.91	9	2.71	0.90	10
EOS (%)	1.78	0.60	9	1.60	0.30	10
BAS (%)	0.18	0.08	9	0.18	0.08	10

Parameters	Group & Dose (mg/kg b.w.)
	G6 & 0			G7 & 350
	Mean	SD	N	Mean	SD	N
RBC (10^6cells/µL)	8.57	0.30	5	8.84	0.16	5
HGB (g/dL)	14.22	0.40	5	14.76	0.35	5
HCT (%)	43.40	1.16	5	45.20↑	1.02	5
MCV (fL)	50.64	1.52	5	51.10	1.23	5
MCH (pg)	16.62	0.61	5	16.68	0.41	5
MCHC (g/dL)	32.78	0.33	5	32.66	0.29	5
RDW (%)	13.46	0.21	5	13.56	0.34	5
RET(x10^9 cells/L)	146.68	22.82	5	160.88	22.67	5
PLT (10^3cells/µL)	977.20	106.61	5	1032.00	93.78	5
MPV (fL)	7.84	0.28	5	7.76	0.11	5
WBC (10^3cells/µL)	9.03	1.40	5	8.02	1.95	5
NEU (10^3cells/µL)	1.83	0.36	5	1.50	0.23	5
LYM (10^3cells/µL)	6.63	1.29	5	5.97	1.64	5
MON (10^3cells/µL)	0.26	0.06	5	0.25	0.08	5
EOS (10^3cells/µL)	0.18	0.05	5	0.16	0.05	5
BAS (10^3cells/µL)	0.02	0.00	5	0.02	0.01	5
NEU (%)	20.36	3.32	5	19.20	2.85	5
LYM (%)	73.14	4.03	5	74.18	3.20	5
MON (%)	3.04	1.24	5	3.04	0.53	5
EOS (%)	1.98	0.51	5	2.00	0.32	5
BAS (%)	0.22	0.04	5	0.18	0.04	5

Parameters	Group & Dose (mg/kg b.w.)
	G1 & 0			G2 & 25			G3 & 75
	Mean	SD	N	Mean	SD	N	Mean	SD	N
RBC (10^6cells/µL)	7.80	0.37	10	7.57	0.24	10	7.78	0.33	10
HGB (g/dL)	14.01	0.69	10	13.84	0.35	10	13.53	0.62	10
HCT (%)	42.04	2.22	10	41.39	1.26	10	40.94	1.64	10
MCV (fL)	53.92	0.89	10	54.67	1.27	10	52.64	0.95	10
MCH (pg)	17.97	0.55	10	18.28*+	0.43	10	17.39	0.43	10
MCHC (g/dL)	33.35	0.82	10	33.46	0.81	10	33.02	0.37	10
RDW (%)	12.14	0.47	10	11.70	0.42	10	12.10	0.66	10
RET(x10^9 cells/L)	58.60	15.09	10	51.10	14.58	10	61.42	18.96	10
PLT (10^3cells/µL)	991.70	249.59	10	975.20	148.69	10	987.70	113.01	10
MPV (fL)	7.08	0.29	10	7.09	0.32	10	7.13	0.38	10
WBC (10^3cells/µL)	6.13	2.44	10	4.25	1.23	10	5.00	1.53	10
NEU (10^3cells/µL)	1.27	0.67	10	0.78	0.25	10	0.96	0.47	10
LYM (10^3cells/µL)	4.39	1.63	10	3.17	0.96	10	3.66	0.99	10
MON (10^3cells/µL)	0.17	0.08	10	0.11	0.05	10	0.23	0.24	10
EOS (10^3cells/µL)	0.20	0.17	10	0.11	0.06	10	0.09	0.04	10
BAS (10^3cells/µL)	0.01	0.01	10	0.01	0.01	10	0.01	0.01	10
NEU (%)	20.13	4.73	10	18.62	4.08	10	18.62	3.37	10
LYM (%)	72.58	5.38	10	74.65	4.88	10	74.04	4.41	10
MON (%)	2.74	0.58	10	2.53	0.61	10	4.33	4.14	10
EOS (%)	3.10	1.50	10	2.59	1.10	10	1.82*-	0.90	10
BAS (%)	0.18	0.09	10	0.14	0.10	10	0.13	0.05	10

Key: N = No. of Animals, *+ = significantly high at p ≤0.05 as compared to G1, *- = significantly low at p ≤0.05 as compared to G1

Gender: Female                           

Parameters	Group & Dose (mg/kg b.w.)
	G4 & 150			G5 & 350
	Mean	SD	N	Mean	SD	N
RBC (10^6cells/µL)	7.71	0.28	10	7.63	0.50	10
HGB (g/dL)	13.68	0.41	10	13.55	0.92	10
HCT (%)	41.36	1.03	10	40.96	2.41	10
MCV (fL)	53.64	1.06	10	53.70	1.33	10
MCH (pg)	17.75	0.34	10	17.76	0.57	10
MCHC (g/dL)	33.05	0.35	10	33.09	0.45	10
RDW (%)	11.93	0.45	10	12.10	0.35	10
RET(x10^9 cells/L)	51.30	12.26	10	51.32	10.97	10
PLT (10^3cells/µL)	971.10	118.01	10	917.90	69.74	10
MPV (fL)	7.10	0.26	10	7.15	0.21	10
WBC (10^3cells/µL)	4.82	0.94	10	4.60	1.02	10
NEU (10^3cells/µL)	0.93	0.27	10	1.15	0.56	10
LYM (10^3cells/µL)	3.55	0.81	10	3.12	1.21	10
MON (10^3cells/µL)	0.18	0.11	10	0.16	0.13	10
EOS (10^3cells/µL)	0.09	0.04	10	0.10	0.06	10
BAS (10^3cells/µL)	0.01	0.01	10	0.01	0.01	10
NEU (%)	19.45	4.62	10	26.27	16.14	10
LYM (%)	73.50	4.88	10	66.20	19.03	10
MON (%)	4.02	3.41	10	3.93	4.29	10
EOS (%)	1.76*-	0.85	10	2.09	1.05	10
BAS (%)	0.13	0.05	10	0.15	0.08	10

Key: N = No. of Animals, *- = significantly low at p ≤0.05 as compared to G1

Parameters	Group & Dose (mg/kg b.w.)
	G6 & 0			G7 & 350
	Mean	SD	N	Mean	SD	N
RBC (10^6cells/µL)	7.65	0.22	5	7.75	0.30	5
HGB (g/dL)	14.10	0.40	5	14.30	0.59	5
HCT (%)	42.24	1.41	5	41.84	1.31	5
MCV (fL)	55.22	1.63	5	54.02	1.60	5
MCH (pg)	18.44	0.54	5	18.44	0.51	5
MCHC (g/dL)	33.40	0.43	5	34.18	0.68	5
RDW (%)	11.90	0.51	5	11.98	0.90	5
RET(x10^9 cells/L)	110.92	10.45	5	107.18	23.95	5
PLT (10^3cells/µL)	911.40	65.54	5	921.00	116.55	5
MPV (fL)	7.76	0.32	5	7.92	0.35	5
WBC (10^3cells/µL)	3.55	0.72	5	4.23	1.48	5
NEU (10^3cells/µL)	0.68	0.19	5	0.75	0.29	5
LYM (10^3cells/µL)	2.62	0.50	5	3.21	1.10	5
MON (10^3cells/µL)	0.09	0.05	5	0.11	0.07	5
EOS (10^3cells/µL)	0.10	0.02	5	0.10	0.04	5
BAS (10^3cells/µL)	0.01	0.00	5	0.01	0.01	5
NEU (%)	19.24	2.79	5	17.70	2.84	5
LYM (%)	73.86	2.20	5	76.08	3.25	5
MON (%)	2.48	1.24	5	2.48	0.64	5
EOS (%)	2.80	0.60	5	2.48	0.69	5
BAS (%)	0.20	0.12	5	0.10	0.00	5

Summary of Coagulation Parameters

Gender: Male                                                                                               

Parameters	Group & Dose (mg/kg b.w.)
	G1 & 0			G2 & 25			G3 & 75
	Mean	SD	N	Mean	SD	N	Mean	SD	N
PT (Sec)	20.05	1.61	10	20.77	1.75	9	18.51	2.22	10
APTT (Sec)	18.48	2.60	10	20.87	2.65	9	18.85	2.87	10

Parameters	Group & Dose (mg/kg b.w.)
	G4 & 150			G5 & 350
	Mean	SD	N	Mean	SD	N
PT (Sec)	18.87	2.13	9	18.81	2.02	10
APTT (Sec)	18.30	1.75	9	18.75	2.21	10

Parameters	Group & Dose (mg/kg b.w.)
	G6 & 0			G7 & 350
	Mean	SD	N	Mean	SD	N
PT (Sec)	20.98	2.79	5	19.96	1.30	5
APTT (Sec)	15.24	2.43	5	16.28	1.37	5

Gender: Female                                                                                            

Parameters	Group & Dose (mg/kg b.w.)
	G1 & 0			G2 & 25			G3 & 75
	Mean	SD	N	Mean	SD	N	Mean	SD	N
PT (Sec)	16.04	1.16	10	15.87	1.19	10	16.91	1.67	10
APTT (Sec)	19.20	4.39	10	18.13	3.64	10	18.67	4.63	10

Parameters	Group & Dose (mg/kg b.w.)
	G4 & 150			G5 & 350
	Mean	SD	N	Mean	SD	N
PT (Sec)	15.79	1.35	10	16.02	1.22	10
APTT (Sec)	17.55	2.55	10	18.07	2.83	10

Parameters	Group & Dose (mg/kg b.w.)
	G6 & 0			G7 & 350
	Mean	SD	N	Mean	SD	N
PT (Sec)	20.54	0.47	5	18.16↓	1.95	5
APTT (Sec)	16.60	1.35	5	17.08	0.81	5

Key: N = Number of animals, ↓ = significantly low at p ≤0.05 as compared to G6

Summary of Clinical Chemistry Parameters

Gender: Male        

Parameters	Group & Dose (mg/kg b.w.)
	G1 & 0			G2 & 25			G3 & 75
	Mean	SD	N	Mean	SD	N	Mean	SD	N
GLU (mg/dL)	117.70	6.85	10	116.00	7.87	9	123.50	26.07	10
TP (g/dL)	6.39	0.12	10	6.38	0.19	9	6.25	0.22	10
ALB (g/dL)	3.86	0.13	10	3.87	0.16	9	3.73	0.22	10
GLO (g/dL)	2.53	0.09	10	2.51	0.08	9	2.50	0.09	10
A/G Ratio	1.53	0.07	10	1.53	0.07	9	1.49	0.11	10
TRG (mg/dL)	89.30	30.25	10	103.67	36.60	9	112.10	34.01	10
CHO(mg/dL)	70.80	11.84	10	68.11	11.39	9	57.52	11.75	10
ALT (U/L)	35.50	6.93	10	32.33	7.97	9	43.10	33.85	10
AST (U/L)	97.00	20.34	10	85.89	22.87	9	117.10	96.79	10
ALP (U/L)	61.90	10.31	10	66.67	12.01	9	70.60	14.18	10
BUN (mg/dL)	14.10	2.69	10	14.78	2.64	9	13.90	2.42	10
CRE (mg/dL)	0.60	0.09	10	0.62	0.07	9	0.62	0.06	10
Na (mmol/L)	149.00	1.33	10	149.44	1.24	9	148.90	1.52	10
K (mmol/L)	4.48	0.23	10	4.44	0.33	9	4.47	0.43	10
Cl (mmol/L)	117.70	1.83	10	118.00	2.06	9	117.20	2.70	10
Ca (mg/dL)	11.56	0.19	10	11.54	0.26	9	11.66	0.32	10
P (mg/dL)	7.14	0.49	10	6.94	0.45	9	6.99	0.68	10
HDL (mg/dL)	39.47	8.26	10	41.42	7.75	9	33.86	7.70	10
Total bile acids (µmol/L)	16.55	10.24	10	16.44	10.95	9	18.36	16.12	10
Urea (mg/dL)	30.39	5.60	10	31.62	5.64	9	29.75	5.19	10
T3 (ng/dL)	61.43	10.23	10	68.16	15.67	9	62.33	9.40	10
T4 (µg/dL)	4.30	1.07	10	4.09	1.16	9	4.02	0.82	10
TSH (µIU/mL)	2.74	1.64	10	1.88	0.98	9	1.19	0.63	10

Key: N = No. of Animals

Note: The results of Bilirubin and LDL for majority of animals were below detectable range and hence mean and SD could not be calculated

Parameters	Group & Dose (mg/kg b.w.)
	G4 & 150			G5 & 350
	Mean	SD	N	Mean	SD	N
GLU (mg/dL)	122.11	14.26	9	122.70	20.30	10
TP (g/dL)	6.41	0.22	9	6.34	0.24	10
ALB (g/dL)	3.86	0.16	9	3.86	0.20	10
GLO (g/dL)	2.56	0.11	9	2.50	0.08	10
A/G Ratio	1.51	0.12	9	1.54	0.08	10
TRG (mg/dL)	109.33	35.31	9	102.40	23.63	10
CHO(mg/dL)	69.83	11.87	9	69.54	9.55	10
ALT (U/L)	49.67	45.04	9	34.60	11.69	10
AST (U/L)	115.22	92.74	9	87.50	25.78	10
ALP (U/L)	67.78	16.68	9	70.80	17.69	10
BUN (mg/dL)	14.22	2.28	9	14.40	2.99	10
CRE (mg/dL)	0.61	0.06	9	0.61	0.03	10
Na (mmol/L)	149.78	1.64	9	149.10	2.28	10
K (mmol/L)	4.66	0.23	9	4.68	0.35	10
Cl (mmol/L)	118.22	2.22	9	118.40	2.72	10
Ca (mg/dL)	11.73	0.34	9	11.47	0.47	10
P (mg/dL)	7.02	0.37	9	6.87	0.55	10
HDL (mg/dL)	39.79	7.16	9	40.60	4.99	10
Total bile acids (µmol/L)	7.77	4.70	9	7.17	2.87	10
Urea (mg/dL)	30.44	4.88	9	30.82	6.40	10
T3 (ng/dL)	56.95	7.54	9	54.22	10.54	10
T4 (µg/dL)	3.63	0.64	9	3.40	0.94	10
TSH (µIU/mL)	1.48	0.43	9	1.84	1.04	10

Parameters	Group & Dose (mg/kg b.w.)
	G6 & 0			G7 & 350
	Mean	SD	N	Mean	SD	N
GLU (mg/dL)	147.40	8.96	5	134.60	18.85	5
TP (g/dL)	6.22	0.26	5	6.36	0.23	5
ALB (g/dL)	3.68	0.29	5	3.76	0.19	5
GLO (g/dL)	2.54	0.09	5	2.56	0.13	5
A/G Ratio	1.46	0.11	5	1.48	0.08	5
TRG (mg/dL)	110.20	29.13	5	98.00	22.02	5
CHO(mg/dL)	59.26	2.43	5	76.74 ↑	15.24	5
ALT (U/L)	40.00	7.11	5	41.80	10.89	5
AST (U/L)	113.80	36.42	5	110.60	26.35	5
ALP (U/L)	70.00	11.64	5	82.20	9.83	5
BUN (mg/dL)	14.60	1.67	5	17.20 ↑	1.10	5
CRE (mg/dL)	0.58	0.04	5	0.60	0.07	5
Na (mmol/L)	140.80	1.48	5	140.40	0.55	5
K (mmol/L)	4.74	0.30	5	4.64	0.19	5
Cl (mmol/L)	114.40	1.82	5	114.40	1.14	5
Ca (mg/dL)	10.96	0.42	5	11.16	0.21	5
P (mg/dL)	6.48	0.20	5	6.96	0.54	5
HDL (mg/dL)	30.51	2.67	5	42.37 ↑	8.37	5
Total bile acids (µmol/L)	20.38	19.37	5	12.90	8.19	5
Urea (mg/dL)	31.24	3.58	5	36.81↑	2.34	5
T3 (ng/dL)	31.97	2.14	5	39.71↑↑	3.12	5
T4 (µg/dL)	3.14	0.43	5	3.64	0.34	5
TSH (µIU/mL)	2.87	1.30	5	3.58	1.65	5

 

Key: N = No. of Animals, ­ = significantly high at p ≤0.05 as compared to G6, ­­ = significantly high at p ≤0.01 as compared to G6

Note: The results of Bilirubin and LDL for majority of animals were below detectable range and hence mean and SD could not be calculated

Gender: Female                  

Parameters	Group & Dose (mg/kg b.w.)
	G1 & 0			G2 & 25			G3 & 75
	Mean	SD	N	Mean	SD	N	Mean	SD	N
GLU (mg/dL)	118.50	15.63	10	127.70	29.12	10	107.80	22.13	10
TP (g/dL)	6.28	0.30	10	6.24	0.34	10	6.16	0.25	10
ALB (g/dL)	4.09	0.28	10	4.05	0.31	10	3.98	0.18	10
GLO (g/dL)	2.20	0.12	10	2.19	0.09	10	2.16	0.12	10
A/G Ratio	1.86	0.16	10	1.86	0.12	10	1.83	0.12	10
TRG (mg/dL)	58.20	11.75	10	68.40	14.41	10	66.00	13.52	10
CHO(mg/dL)	75.96	17.61	10	71.76	9.11	10	66.56	14.39	10
ALT (U/L)	38.10	18.88	10	27.30	2.87	10	28.30	3.40	10
AST (U/L)	118.50	46.78	10	86.10	20.01	10	91.60	17.18	10
ALP (U/L)	39.80	13.60	10	36.10	5.72	10	35.40	8.14	10
BUN (mg/dL)	19.20	2.57	10	17.40 *-	0.52	10	18.90	3.14	10
CRE (mg/dL)	0.60	0.07	10	0.65	0.07	10	0.64	0.07	10
Na (mmol/L)	145.20	1.32	10	146.40	1.43	10	145.90	3.25	10
K (mmol/L)	4.35	0.27	10	4.06 *-	0.24	10	4.03 *-	0.12	10
Cl (mmol/L)	115.90	2.38	10	116.70	2.45	10	116.70	3.47	10
Ca (mg/dL)	11.31	0.32	10	11.28	0.33	10	11.19	0.28	10
P (mg/dL)	5.80	0.64	10	5.51	0.59	10	5.75	0.76	10
HDL (mg/dL)	50.52	11.63	10	48.62	5.17	10	46.00	10.25	10
Total bile acids (µmol/L)	15.80	7.48	10	16.48	10.96	10	13.76	7.11	10
Urea (mg/dL)	41.09	5.51	10	37.24 *-	1.11	10	40.45	6.73	10
T3 (ng/dL)	58.74	20.81	10	61.79	16.68	10	53.74	18.26	10
T4 (µg/dL)	2.37	0.74	10	2.51	0.68	10	2.36	0.62	10
TSH (µIU/mL)	1.34	1.08	10	1.20	1.09	10	1.43	1.46	10

 Key: N = No. of Animals, *- = significantly low at p ≤0.05 as compared to G1

Note: The results of Bilirubin and LDL for majority of animals were below detectable range and hence mean and SD could not be calculated.

Parameters	Group & Dose (mg/kg b.w.)
	G4 & 150			G5 & 350
	Mean	SD	N	Mean	SD	N
GLU (mg/dL)	127.50	26.22	10	130.00	19.24	10
TP (g/dL)	6.21	0.48	10	6.40	0.38	10
ALB (g/dL)	4.00	0.46	10	4.16	0.32	10
GLO (g/dL)	2.23	0.11	10	2.22	0.15	10
A/G Ratio	1.79	0.23	10	1.89	0.16	10
TRG (mg/dL)	71.40	24.98	10	95.80 **+	29.86	10
CHO(mg/dL)	74.64	19.15	10	81.74	13.89	10
ALT (U/L)	33.00	8.15	10	28.20	3.85	10
AST (U/L)	99.70	33.37	10	89.30	38.13	10
ALP (U/L)	38.20	4.61	10	43.50	4.90	10
BUN (mg/dL)	17.00	2.16	10	16.10*-	2.18	10
CRE (mg/dL)	0.68 *+	0.06	10	0.59	0.07	10
Na (mmol/L)	145.50	1.43	10	144.70	2.54	10
K (mmol/L)	4.14	0.24	10	4.28	0.31	10
Cl (mmol/L)	116.00	2.26	10	116.50	2.88	10
Ca (mg/dL)	11.40	0.44	10	11.77*+	0.42	10
P (mg/dL)	5.88	0.46	10	5.64	0.74	10
HDL (mg/dL)	50.06	10.97	10	55.71	9.80	10
Total bile acids (µmol/L)	18.73	10.96	10	10.37	7.28	10
Urea (mg/dL)	36.38	4.62	10	34.45 *-	4.67	10
T3 (ng/dL)	59.18	10.97	10	64.20	14.86	10
T4 (µg/dL)	2.44	0.59	10	2.57	0.75	10
TSH (µIU/mL)	1.82	2.08	10	0.69	0.43	10

 

Key: N = No. of Animals, *+ or *- = significantly high or low at p ≤0.05 as compared to G1, **- = significantly low at p ≤0.01 as compared to G1, **+ = significantly high at p ≤0.01 as compared to G1

Note: The results of Bilirubin and LDL for majority of animals were below detectable range and hence mean and SD could not be calculated.

Parameters	Group & Dose (mg/kg b.w.)
	G6 & 0			G7 & 350
	Mean	SD	N	Mean	SD	N
GLU (mg/dL)	130.80	13.22	5	121.40	14.38	5
TP (g/dL)	6.24	0.13	5	6.24	0.25	5
ALB (g/dL)	3.96	0.13	5	3.82	0.16	5
GLO (g/dL)	2.28	0.08	5	2.40 ↑	0.07	5
A/G Ratio	1.72	0.11	5	1.58	0.08	5
TRG (mg/dL)	77.40	28.26	5	67.60	6.39	5
CHO(mg/dL)	59.03	7.33	5	79.73 ↑↑	5.36	5
ALT (U/L)	27.80	3.11	5	27.20	6.57	5
AST (U/L)	76.20	6.76	5	76.00	9.35	5
ALP (U/L)	32.00	5.87	5	39.00	3.74	5
BUN (mg/dL)	16.80	1.92	5	17.80	2.39	5
CRE (mg/dL)	0.56	0.05	5	0.62	0.08	5
Na (mmol/L)	138.00	1.41	5	138.60	0.55	5
K (mmol/L)	3.90	0.25	5	4.02	0.08	5
Cl (mmol/L)	112.20	1.64	5	116.00 ↑↑	1.22	5
Ca (mg/dL)	10.86	0.25	5	10.84	0.18	5
P (mg/dL)	4.48	0.67	5	4.68	0.22	5
HDL (mg/dL)	38.07	4.79	5	48.87 ↑↑	3.90	5
Total bile acids (µmol/L)	7.40	2.31	5	13.95 ↑	5.92	5
Urea (mg/dL)	35.95	4.12	5	38.09	5.11	5
T3 (ng/dL)	41.30	8.30	5	47.33	13.84	5
T4 (µg/dL)	2.68	0.70	5	2.94	0.63	5
TSH (µIU/mL)	0.52	0.25	5	0.54	0.20	5

Key: N = No. of Animals, ­ = significantly high at p ≤0.05 as compared to G6, ­­ = significantly high at p ≤0.01 as compared to G6

Note: The results of Bilirubin and LDL for majority of animals were below detectable range and hence mean and SD could not be calculated

Summary of Urinalysis Parameters

Gender: Male No. of Animals/Group/Sex: 10    

Parameters		Group & Dose (mg/kg b.w.)
		G1 & 0		G2 & 25		G3 & 75
Urine Volume (mL)
Mean ± SD		13.40 ± 4.55		12.44 ± 3.84		11.10 ± 2.73
Colour
Light Yellow		9		8		10
Yellow		1		1		-
Appearance/Clarity
Clear		6		4		6
Slightly cloudy		4		5		4
Specific Gravity
Mean ± SD		1.01 ± 0.00		1.02 ± 0.00		1.01 ± 0.00
Nitrite
Negative		6		4		8
Positive		4		5		2
pH
Mean ± SD		7.05 ± 0.80		7.78 ± 1.03		7.20 ± 1.01
Protein (mg/dL)
Negative		6		3		5
10		1		1		2
30		1		1		3
100		2		4		-
Glucose (mg/dL)
Negative		10		9		10
Ketone bodies (mg/dL)
Negative		1		1		4
5		6		3		3
10		3		5		3
Urobilinogen (EU/dL)
Normal		10		9		10
Bilirubin (mg/dL)
Negative		10		9		10
Blood (RBC/µL)
Negative		10		9		10
Leu (WBC/µL)
Negative	3		1		1
10	3		5		5
25	3		3		4
75	1		-		-

Parameters	Group & Dose (mg/kg b.w.)
	G1 & 0		G2 & 25		G3 & 75
Microscopy
Epithelial Cells
0 (0/hpf)		9		5		7
1+ (1-2/hpf)		1		4		3
Crystals
1+ (1-5/hpf)		5		1		1
2+ (5-10/hpf)		-		2		6
3+ (10-15/hpf)		5		2		2
4+ (>15/hpf)		-		4		1
Leucocytes (W)
0 (0/hpf)		6		4		9
1+(1-2/hpf)		4		5		1
Erythrocytes (R)
0 (0/hpf)		10		9		10
Other Abnormal  Constituents (A)
0(0/hpf)		4		4		4
1+*(1-2/hpf)		6		3		6
1+(1-2/hpf)		-		1		-
2+*(3-4/hpf)		-		1		-
Cast
0(0/hpf)		10		9		10

Parameters	Group & Dose (mg/kg b.w.)
	G4 & 150	G5 & 350
Urine Volume (mL)
Mean ± SD	12.00 ± 3.00	12.60 ± 4.99
Colour
Light Yellow	9	9
Yellow	-	1
Appearance
Clear	6	8
Slightly cloudy	3	2
Specific Gravity
Mean ± SD	1.02 ± 0.01	1.01 ± 0.00
Nitrite
Negative	8	6
Positive	1	4
pH
Mean ± SD	7.33 ± 1.00	8.50 ± 0.47 **+
Protein (mg/dL)
Negative	4	4
10	1	1
30	1	3
100	3	1
300	-	1
Glucose (mg/dL)
Negative	9	10
Ketone bodies (mg/dL)
Negative	2	5
5	2	2
10	5	3
Urobilinogen (EU/dL)
Normal	9	10
Bilirubin (mg/dL)
Negative	9	10
Blood (RBC/µL)
Negative	9	10
Leu (WBC/µL)
Negative	2	2
10	3	5
25	2	2
75	2	1

Parameters	Group & Dose (mg/kg b.w.)
	G4 & 150	G5 & 350
Microscopy
Epithelial Cells
0 (0/hpf)	8	8
1+ (1-2/hpf)	1	2
Crystals
1+ (1-5/hpf)	2	-
2+ (5-10/hpf)	3	1
3+(10-15/hpf)	3	3
4+ (>15/hpf)	1	6
Leucocytes (W)
0 (0/hpf)	4	3
1+(1-2/hpf)	5	7
Erythrocytes (R)
0 (0/hpf)	9	10
Other Abnormal  Constituents (A)
0(0/hpf)	5	4
1+*(1-2/hpf)	4	4
1+(1-2/hpf)	-	1
2+*	-	1
Casts
0(0/hpf)	9	10

Key: hpf = high power field, * = Spermatozoa

Gender: Male No. of Animals/Group/Sex: 5

Parameters	Group & Dose (mg/kg b.w.)
	G6 & 0	G7 & 350
Urine Volume (mL)
Mean ± SD	11.40 ± 3.29	19.20 ± 5.76↑
Colour
Light Yellow	5	5
Appearance
Clear	3	3
Slightly cloudy	2	2
Specific Gravity
Mean ± SD	1.01 ± 0.00	1.02 ± 0.00
Nitrite
Negative	3	4
Positive	2	1
pH
Mean ± SD	8.60 ± 0.55	7.90 ± 1.14
Protein (mg/dL)
Negative	3	-
10	1	3
30	-	1
100	1	1
Glucose (mg/dL)
Negative	5	5
Ketone bodies (mg/dL)
Negative	3	1
5	2	3
10	-	1
Urobilinogen (EU/dL)
Normal	5	5
Bilirubin (mg/dL)
Negative	5	5
Blood (RBC/µL)
Negative	5	5
Leu (WBC/µL)
Negative	3	1
10	-	2
25	2	2

Parameters	Group & Dose (mg/kg b.w.)
	G6 & 0	G7 & 350
Microscopy
Epithelial Cells
0 (0/hpf)	4	2
1+ (1-2/hpf)	1	3
Crystals
0(0/hpf)	1	1
1+ (1-5/hpf)	3	3
2+ (5-10/hpf)	1	1
Leucocytes (W)
0 (0/hpf)	5	4
1+(1-2/hpf)	-	1
Erythrocytes (R)
0 (0/hpf)	5	5
Other Abnormal  Constituents (A)
0(0/hpf)	5	5
Casts
0(0/hpf)	2	1
1+ (1-5/hpf)	2	3
2+ (5-10/hpf)	1	1

Gender: Female         No. of Animals/Group/Sex: 10    

Parameters		Group & Dose (mg/kg b.w.)
		G1 & 0		G2 & 25	G3 & 75
Urine Volume (mL)
Mean ± SD		18.80 ± 7.67		18.00 ± 6.68	19.20 ± 7.80
Colour
Light Yellow		10		10	9
Yellow		-		-	1
Appearance/Clarity
Clear		7		8	7
Slightly cloudy		3		2	3
Specific Gravity
Mean ± SD		1.01 ± 0.00		1.01 ± 0.01	1.02 ± 0.00 (N=9)
<=1.030		-		-	1/10
Nitrite
Negative		4		9	4
Positive		6		1	6
pH
Mean ± SD		8.25 ± 0.79		7.25 ± 1.09 *-	6.95 ± 1.07 **-
Protein (mg/dL)
Negative		10		10	9
100					1
Glucose (mg/dL)
Negative		10		9	10
100		-		1
Ketone bodies (mg/dL)
Negative		10		10	10
5
Urobilinogen (EU/dL)
Normal		10		10	10
Bilirubin (mg/dL)
Negative		10		10	10
Blood (RBC/µL)
Negative		10		10	10
Leu (WBC/µL)
Negative	10		10			9
10	-		-			1

Key: *- = significantly high at p ≤0.05 as compared to G1, **- = significantly high at p ≤0.01 as compared to G1

Parameters		Group & Dose (mg/kg b.w.)
		G1 & 0		G2 & 25	G3 & 75
Microscopy
Epithelial Cells
0 (0/hpf)	9		9		8
1+ (1-2/hpf)	1		1		2
Crystals
1+ (1-5/hpf)	5		6		7
2+ (5-10/hpf)	-		2		-
3+ (10-15/hpf)	2				2
4+ (>15/hpf)	3		2		1
Leucocytes (W)
0 (0/hpf)	9		8		8
1+(1-2/hpf)	1		2		2
Erythrocytes (R)
0 (0/hpf)	10		10		10
Other Abnormal  Constituents
0(0/hpf)	10		10		10

Parameters	Group & Dose (mg/kg b.w.)
	G4 & 150	G5 & 350
Urine Volume (mL)
Mean ± SD	23.10 ± 8.74	19.70 ± 7.20
Colour
Light Yellow	10	10
Appearance
Clear	7	8
Slightly cloudy	3	2
Specific Gravity
Mean ± SD	1.01 ± 0.00	1.01 ± 0.00
Nitrite
Negative	4	5
Positive	6	5
pH
Mean ± SD	7.30 ± 1.03 *-	8.75 ± 0.35
Protein (mg/dL)
Negative	9	6
10	-	1
30	-	2
100	1	1
Glucose (mg/dL)
Negative	10	10
Ketone bodies (mg/dL)
Negative	10	10
Urobilinogen (EU/dL)
Normal	10	10
Bilirubin (mg/dL)
Negative	10	10
Blood (RBC/µL)
Negative	8	9
5	1	1
10	1	-
Leu (WBC/µL)
Negative	9	8
10	-	1
25	1	1

Key: *- = significantly high at p ≤0.05 as compared to G1

Parameters	Group & Dose (mg/kg b.w.)
	G4 & 150	G5 & 350
Microscopy
Epithelial Cells
0 (0/hpf)	9	10
1+ (1-2/hpf)	1	-
Crystals
1+ (1-5/hpf)	5	2
2+ (5-10/hpf)	3	2
3+ (10-15/hpf)	1	1
4+ (>15/hpf)	1	5
Leucocytes (W)
0 (0/hpf)	9	9
1+(1-2/hpf)	1	1
Erythrocytes (R)
0 (0/hpf)	10	10
Other Abnormal  Constituents (A)
Nil	10	10
Casts
0 (0/hpf)	10	10

Parameters	Group & Dose (mg/kg b.w.)
	G6 & 0	G7 & 350
Urine Volume (mL)
Mean ± SD	14.00 ± 5.15	15.40 ± 2.70
Colour
Light Yellow	5	5
Appearance
Clear	5	5
Specific Gravity
Mean ± SD	1.01 ± 0.00	1.01 ± 0.00
Nitrite
Negative	3	3
Positive	2	2
pH
Mean ± SD	8.30 ± 0.76	8.40 ± 0.55
Protein (mg/dL)
Negative	4	5
10	1	-
Glucose (mg/dL)
Negative	5	5
Ketone bodies (mg/dL)
Negative	5	5
Urobilinogen (EU/dL)
Normal	5	5
Bilirubin (mg/dL)
Negative	5	5
Blood (RBC/µL)
Negative	5	5
Leu (WBC/µL)
Negative	5	5

Parameters	Group & Dose (mg/kg b.w.)
	G6 & 0	G7 & 350
Microscopy
Epithelial Cells
0 (0/hpf)	2	4
1+ (1-2/hpf)	3	1
Crystals
0(0/hpf)	-	1
1+ (1-5/hpf)	5	4
Leucocytes (W)
0 (0/hpf)	5	2
1+(1-2/hpf)	-	3
Erythrocytes (R)
0 (0/hpf)	5	5
Other Abnormal  Constituents (A)
0(0/hpf)	3	2
1+ (1-5/hpf)	2	3

Summary of Absolute Organ Weight (g)

Gender: Male

Parameters	Group & Dose (mg/kg b.w.)
	G1 & 0			G2 & 25			G3 & 75
	Mean	SD	N	Mean	SD	N	Mean	SD	N
Terminal Body Weight	535.58	54.84	10	555.95	30.41	9	566.87	60.87	10
Adrenals	0.0717	0.0108	10	0.0775	0.0057	9	0.0719	0.0079	10
Heart	1.5903	0.1637	10	1.6907	0.0752	9	1.6192	0.1422	10
Brain	2.1604	0.1634	10	2.2375	0.0783	9	2.1984	0.1440	10
Liver	15.7950	2.0402	10	16.9435	0.8424	9	16.2539	2.1613	10
Thymus	0.3095	0.0659	10	0.3378	0.0777	9	0.2800	0.0599	10
Lungs	2.3101	0.4229	10	2.5017	0.6207	9	2.1007	0.2754	10
Kidneys	3.4621	0.4637	10	3.7290	0.2813	9	3.5481	0.4357	10
Spleen	0.9547	0.1824	10	0.9992	0.1821	9	1.0409	0.1964	10
Thyroid & parathyroid gland	0.0298	0.0040	10	0.0302	0.0064	9	0.0324	0.0038	10
Pituitary gland	0.0141	0.0024	10	0.0132	0.0025	9	0.0139	0.0012	10
Testes	4.4119	1.0029	10	4.1513	0.3101	9	4.0648	0.2139	10
Epididymides	1.5261	0.1960	10	1.6832	0.2377	9	1.5935	0.1436	10
Prostate + Seminal vesicles with coagulating glands	3.1671	0.4558	10	3.5278	0.4532	9	3.5052	0.4755	10

Parameters	Group & Dose (mg/kg b.w.)
	G4 & 150			G5 & 350
	Mean	SD	N	Mean	SD	N
Terminal Body Weight	532.27	41.02	9	519.68	43.01	10
Adrenals	0.0694	0.0101	9	0.0746	0.0070	10
Heart	1.5509	0.1731	9	1.5296	0.1333	10
Brain	2.1773	0.1368	9	2.1959	0.1209	10
Liver	16.2525	2.6461	9	16.2705	2.5194	10
Thymus	0.3555	0.0694	9	0.3023	0.1031	10
Lungs	2.1881	0.5015	9	2.1901	0.3364	10
Kidneys	3.4915	0.2802	9	3.4773	0.2991	10
Spleen	1.0166	0.2100	9	1.0029	0.1427	10
Thyroid & parathyroid gland	0.0307	0.0042	9	0.0316	0.0063	10
Pituitary gland	0.0134	0.0019	9	0.0136	0.0008	10
Testes	4.3310	1.0704	9	3.9988	0.2779	10
Epididymides	1.5470	0.2330	9	1.6937	0.2346	10
Prostate + Seminal vesicles with coagulating glands	3.4367	0.2823	9	3.2567	0.5621	10

Parameters	Group & Dose (mg/kg b.w.)
	G6 & 0			G7 & 350
	Mean	SD	N	Mean	SD	N
Terminal Body Weight	538.85	38.08	5	585.82	46.59	5
Adrenals	0.0613	0.0079	5	0.0783 ↑↑	0.0062	5
Heart	1.5328	0.1331	5	1.7112	0.1429	5
Brain	2.2654	0.1048	5	2.2796	0.0682	5
Liver	16.2545	2.6403	5	18.1434	2.3783	5
Thymus	0.3722	0.1547	5	0.4774	0.2768	5
Lungs	2.0333	0.2463	5	2.4074	0.5404	5
Kidneys	3.4059	0.2255	5	3.7194 ↑	0.1603	5
Spleen	0.8864	0.1558	5	1.0839	0.2516	5
Thyroid & parathyroid gland	0.0431	0.0037	5	0.0453	0.0022	5
Pituitary gland	0.0181	0.0040	5	0.0203	0.0009	5
Testes	4.1354	0.3255	5	4.7030	0.6757	5
Epididymides	1.5524	0.1296	5	1.7272	0.1535	5
Prostate + Seminal vesicles with coagulating glands	3.7056	0.1763	5	4.2455 ↑	0.4182	5

Key: N = No. of Animals, ­ = significantly high at p ≤0.05 as compared to G6, ­­= significantly high at p ≤0.01 as compared to G6

Gender: Female

Parameters	Group & Dose (mg/kg b.w.)
	G1 & 0			G2 & 25			G3 & 75
	Mean	SD	N	Mean	SD	N	Mean	SD	N
Terminal Body Weight	283.81	23.49	10	287.95	22.26	10	291.46	25.10	10
Adrenals	0.0949	0.0131	10	0.0941	0.0092	10	0.0948	0.0116	10
Heart	0.9761	0.1058	10	0.9971	0.0651	10	1.0068	0.0698	10
Brain	2.0215	0.1350	10	2.0392	0.1293	10	2.0322	0.0860	10
Liver	9.1084	1.7731	10	9.2187	1.2790	10	9.4593	1.7691	10
Thymus	0.3256	0.0546	10	0.3268	0.0938	10	0.3197	0.0590	10
Lungs	1.7148	0.3918	10	2.2858	0.8802	10	2.0765	1.0813	10
Kidneys	1.9401	0.2188	10	2.0218	0.2004	10	1.9487	0.1447	10
Spleen	0.6860	0.0789	10	0.7286	0.1355	10	0.7278	0.1363	10
Thyroid & parathyroid gland	0.0333	0.0051	10	0.0324	0.0049	10	0.0358	0.0069	10
Pituitary gland	0.0166	0.0042	10	0.0156	0.0025	10	0.0178	0.0020	10
Ovaries	0.1703	0.0407	10	0.1740	0.0295	10	0.1861	0.0243	10
Uterus With Cervix	0.8594	0.1955	10	0.8988	0.2890	10	0.8386	0.1534	10

Parameters	Group & Dose (mg/kg b.w.)
	G4 & 150			G5 & 350
	Mean	SD	N	Mean	SD	N
Terminal Body Weight	290.34	26.07	10	314.68	29.23	10
Adrenals	0.0992	0.0109	10	0.1000	0.0191	10
Heart	1.0288	0.1166	10	1.0333	0.0602	10
Brain	1.9816	0.0996	10	1.9867	0.1068	10
Liver	10.2137	1.7890	10	10.3750	2.0584	10
Thymus	0.3453	0.0776	10	0.3687	0.0633	10
Lungs	2.0250	0.8353	10	2.1813	1.1673	10
Kidneys	2.0794	0.2028	10	2.1206	0.2829	10
Spleen	0.7289	0.1188	10	0.6857	0.1094	10
Thyroid & parathyroid gland	0.0293	0.0037	10	0.0363	0.0043	10
Pituitary gland	0.0171	0.0035	10	0.0180	0.0034	10
Ovaries	0.1757	0.0258	10	0.1725	0.0158	10
Uterus With Cervix	0.7545	0.1607	10	0.7457	0.1612	10

Parameters	Group & Dose (mg/kg b.w.)
	G6 & 0			G7 & 350
	Mean	SD	N	Mean	SD	N
Terminal Body Weight	278.21	13.85	5	296.37	19.35	5
Adrenals	0.0984	0.0114	5	0.0886	0.0115	5
Heart	1.0666	0.0724	5	1.0354	0.0809	5
Brain	2.1188	0.0870	5	2.1277	0.1737	5
Liver	9.1984	0.6984	5	8.7385	0.6148	5
Thymus	0.2971	0.0412	5	0.3822	0.0833	5
Lungs	1.7501	0.4023	5	1.6007	0.2190	5
Kidneys	1.9079	0.0305	5	1.9734	0.1472	5
Spleen	0.6183	0.0561	5	0.7034	0.0861	5
Thyroid & parathyroid gland	0.0418	0.0060	5	0.0328 ↓↓	0.0032	5
Pituitary gland	0.0187	0.0016	5	0.0171	0.0015	5
Ovaries	0.1852	0.0682	5	0.1708	0.0164	5
Uterus With Cervix	0.6583	0.1979	5	0.5827	0.1437	5

Key: N = No. of Animals, ↓↓ = significantly high at p ≤0.01 as compared to G6

Summary of Relative Organ Weight (%)

Gender: Male

Parameters	Group & Dose (mg/kg b.w.)
	G1 & 0			G2 & 25			G3 & 75
	Mean	SD	N	Mean	SD	N	Mean	SD	N
Adrenals	0.0135	0.0019	10	0.0140	0.0011	9	0.0128	0.0017	10
Heart	0.2976	0.0208	10	0.3049	0.0205	9	0.2871	0.0235	10
Brain	0.4050	0.0245	10	0.4035	0.0262	9	0.3901	0.0308	10
Liver	2.9451	0.1663	10	3.0532	0.1865	9	2.8637	0.1947	10
Thymus	0.0585	0.0146	10	0.0610	0.0140	9	0.0499	0.0118	10
Lungs	0.4344	0.0840	10	0.4511	0.1113	9	0.3706	0.0325	10
Kidneys	0.6465	0.0555	10	0.6718	0.0542	9	0.6261	0.0455	10
Spleen	0.1778	0.0260	10	0.1808	0.0371	9	0.1837	0.0289	10
Pituitary gland	0.0026	0.0004	10	0.0024	0.0005	9	0.0025	0.0003	10
Thyroid & parathyroid gland	0.0056	0.0010	10	0.0054	0.0012	9	0.0058	0.0010	10
Testes	0.8214	0.1515	10	0.7489	0.0705	9	0.7232	0.0726	10
Epididymides	0.2850	0.0233	10	0.3038	0.0487	9	0.2833	0.0337	10
Prostate + Seminal vesicles with coagulating glands	0.5934	0.0873	10	0.6379	0.1022	9	0.6275	0.1255	10

Parameters	Group & Dose (mg/kg b.w.)
	G4 & 150			G5 & 350
	Mean	SD	N	Mean	SD	N
Adrenals	0.0130	0.0016	9	0.0144	0.0015	10
Heart	0.2911	0.0178	9	0.2948	0.0207	10
Brain	0.4116	0.0451	9	0.4238	0.0234	10
Liver	3.0434	0.3220	9	3.1324	0.4396	10
Thymus	0.0674	0.0161	9	0.0574	0.0150	10
Lungs	0.4090	0.0709	9	0.4253	0.0826	10
Kidneys	0.6571	0.0439	9	0.6697	0.0349	10
Spleen	0.1902	0.0326	9	0.1928	0.0197	10
Pituitary gland	0.0025	0.0004	9	0.0026	0.0003	10
Thyroid & parathyroid gland	0.0058	0.0007	9	0.0061	0.0013	10
Testes	0.8180	0.2039	9	0.7726	0.0672	10
Epididymides	0.2928	0.0529	9	0.3258	0.0355	10
Prostate + Seminal vesicles with coagulating glands	0.6484	0.0654	9	0.6325	0.1237	10

Parameters	Group & Dose (mg/kg b.w.)
	G6 & 0			G7 & 350
	Mean	SD	N	Mean	SD	N
Adrenals	0.0114	0.0010	5	0.0135	0.0020	5
Heart	0.2847	0.0187	5	0.2922	0.0131	5
Brain	0.4214	0.0222	5	0.3916	0.0405	5
Liver	3.0004	0.2964	5	3.0900	0.2037	5
Thymus	0.0690	0.0270	5	0.0802	0.0428	5
Lungs	0.3788	0.0528	5	0.4085	0.0659	5
Kidneys	0.6324	0.0147	5	0.6374	0.0474	5
Spleen	0.1641	0.0249	5	0.1845	0.0356	5
Pituitary gland	0.0033	0.0005	5	0.0035	0.0004	5
Thyroid & parathyroid gland	0.0080	0.0004	5	0.0078	0.0007	5
Testes	0.7709	0.0840	5	0.8072	0.1319	5
Epididymides	0.2889	0.0277	5	0.2961	0.0342	5
Prostate + Seminal vesicles with coagulating glands	0.6904	0.0586	5	0.7242	0.0254	5

Gender: Female

Parameters	Group & Dose (mg/kg b.w.)
	G1 & 0			G2 & 25			G3 & 75
	Mean	SD	N	Mean	SD	N	Mean	SD	N
Adrenals	0.0335	0.0045	10	0.0329	0.0042	10	0.0326	0.0031	10
Heart	0.3446	0.0328	10	0.3472	0.0226	10	0.3466	0.0243	10
Brain	0.7156	0.0659	10	0.7112	0.0628	10	0.7023	0.0704	10
Liver	3.1993	0.4497	10	3.1999	0.3539	10	3.2307	0.3996	10
Thymus	0.1152	0.0211	10	0.1133	0.0303	10	0.1097	0.0176	10
Lungs	0.6033	0.1229	10	0.7902	0.2823	10	0.6999	0.3174	10
Kidneys	0.6839	0.0575	10	0.7019	0.0403	10	0.6706	0.0466	10
Spleen	0.2418	0.0197	10	0.2540	0.0502	10	0.2500	0.0428	10
Pituitary gland	0.0058	0.0011	10	0.0054	0.0009	10	0.0061	0.0008	10
Thyroid & parathyroid gland	0.0118	0.0022	10	0.0112	0.0014	10	0.0123	0.0024	10
Ovaries	0.0599	0.0128	10	0.0606	0.0106	10	0.0644	0.0108	10
Uterus With Cervix	0.3022	0.0590	10	0.3120	0.0932	10	0.2905	0.0640	10

Parameters	Group & Dose (mg/kg b.w.)
	G4 & 150			G5 & 350
	Mean	SD	N	Mean	SD	N
Adrenals	0.0344	0.0046	10	0.0316	0.0042	10
Heart	0.3545	0.0252	10	0.3305	0.0315	10
Brain	0.6858	0.0481	10	0.6357 *-	0.0597	10
Liver	3.5109	0.4465	10	3.2846	0.4869	10
Thymus	0.1181	0.0190	10	0.1181	0.0234	10
Lungs	0.6944	0.2591	10	0.6811	0.3185	10
Kidneys	0.7168	0.0366	10	0.6743	0.0704	10
Spleen	0.2517	0.0398	10	0.2173	0.0229	10
Pituitary gland	0.0059	0.0009	10	0.0057	0.0010	10
Thyroid & parathyroid gland	0.0101	0.0011	10	0.0116	0.0013	10
Ovaries	0.0615	0.0136	10	0.0551	0.0063	10
Uterus With Cervix	0.2610	0.0567	10	0.2382	0.0513	10

Key: N = No. of Animals, *- = significantly low at p ≤0.05 as compared to G1

Parameters	Group & Dose (mg/kg b.w.)
	G6 & 0			G7 & 350
	Mean	SD	N	Mean	SD	N
Adrenals	0.0353	0.0028	5	0.0301	0.0054	5
Heart	0.3838	0.0264	5	0.3492 ↓	0.0115	5
Brain	0.7626	0.0382	5	0.7182	0.0391	5
Liver	3.3163	0.3358	5	2.9531	0.1941	5
Thymus	0.1071	0.0164	5	0.1283	0.0226	5
Lungs	0.6268	0.1277	5	0.5416	0.0781	5
Kidneys	0.6874	0.0418	5	0.6656	0.0154	5
Spleen	0.2234	0.0300	5	0.2368	0.0177	5
Pituitary gland	0.0067	0.0008	5	0.0058	0.0006	5
Thyroid & parathyroid gland	0.0151	0.0028	5	0.0111↓	0.0016	5
Ovaries	0.0670	0.0252	5	0.0580	0.0090	5
Uterus With Cervix	0.2369	0.0732	5	0.1978	0.0537	5

Key: N = No. of Animals, ↓ = significantly low at p ≤0.05 as compared to G6

Summary of Vaginal Smear Examination

Gender: Female

Observation	Group & Dose (mg/kg b.w.)
	No. of animals
	G1 & 0	G2 & 25	G3 & 75	G4 & 150	G5 & 30
Proestrus	6	6	4	2	3
Estrus	3	3	2	4	6
Metestrus	-	1	4	4	1
Diestrus	1	-	-	-	-

Observation	Group & Dose (mg/kg b.w.)
	No. of animals
	G6 & 0	G7 & 350
Proestrus	1	3
Estrus	1	-
Metestrus	1	2
Diestrus	2	-

Summary of Gross Pathology Findings

Gender: Male

Gross Pathology observation (s)	Number of animals with or without lesion (s)/ Numbers of animals observed
	G1	G2	G3	G4	G5	G6	G7
Seminal vesicle – Small, unilateral	1/10	0/10	0/10	0/10	0/10	0/5	0/5
Gastro intestinal tract – Distended and empty	0/10	1/10^#	0/10	1/10^#	0/10	0/5	0/5
Testes – Enlarged, bilateral	0/10	0/10	0/10	1/10	0/10	0/5	0/5
No Abnormality Detected	9/10	9/10	10/10	8/10	10/10	5/5	5/5

Gender: Female

Gross Pathology observation (s)	Number of animals with or without lesion (s)/ Numbers of animals observed
	G1	G2	G3	G4	G5	G6	G7
No Abnormality Detected	10/10	10/10	10/10	10/10	10/10	5/5	5/5

Summary of Histopathology Findings

Gender: Male                                         

Organs/ Histopathology	No. of tissues showing lesion / no. of tissues examined
	Group & Dose (mg/kg b.w.)
	G1 (0)	G2* (25)	G3* (75)	G4* (150)	G5 (350)	G6* (0)	G7* (350)
LIVER (N)	8/10	1/1^#	-	0/1^#	9/10	-	-
Infiltrate, mononuclear cell, multifocal	2/10	-	-	0/1^#	1/10	-	-
Infiltrate, poly morpho mononuclear cell, multifocal	0/10	-	-	1/1^#	0/10	-	-
LUNGS (with bronchi and bronchiole) (N)	10/10	1/1^#	-	1/1^#	10/10	-	-
KIDNEYS (N)	7/10	8/10	4/10	5/10	6/10	4/5	2/5
Cast, focal, bilateral	2/10	0/10	0/10	1/10	2/10	0/5	0/5
Cast, focal, unilateral	0/10	2/10	2/10	1/10	4/10	1/5	2/5
Basophilia, tubule, focal, bilateral	0/10	0/10	0/10	1/10	0/10	0/5	0/5
Basophilia, tubule, focal, unilateral	1/10	0/10	3/10	2/10^#	2/10	1/5	1/5
Basophilia, tubule, multifocal, bilateral	0/10	0/10	1/10	0/10	0/10	0/5	0/5
Necrosis, multifocal, bilateral	0/10	1/10^#	0/10	0/10	1/10	0/5	0/5
Infiltrate, inflammatory cell, focal, cortex, unilateral	0/10	0/10	0/10	1/10	0/10	0/5	0/5
URINARY BLADDER (N)	10/10	1/1^#	-	1/1^#	10/10	-	-
SPLEEN (N)	10/10	0/1^#	-	1/1^#	10/10	-	-
Atrophy	0/10	1/1^#	-	0/1^#	0/10	-	-
EMH, multifocal	0/10	0/1^#	-	0/1^#	0/10	-	-
THYMUS (N)	10/10	0/1^#	-	1/1^#	9/10	-	-
Atrophy	0/10	1/1^#	-	0/1^#	0/10	-	-
Ectopic tissue, focal	0/10	0/1^#	-	0/1^#	1/10	-	-
MESENTERIC LYMPHNODES (N)	10/10	1/1^#	-	1/1^#	10/10	-	-
PITUITARY GLAND (N)	10/10	1/1^#	-	1/1^#	10/10	-	-

Key: N = within normal histological limit; * = only target organs (Stomach, trachea and kidneys) were evaluated; ^# = Found dead animal.

Organs/ Histopathology	No. of tissues showing lesion / no. of tissues examined
	Group & Dose (mg/kg b.w.)
	G1 (0)	G2* (25)	G3* (75)	G4* (150)	G5 (350)	G6* (0)	G7* (350)
TESTES (N)	8/10	1/1^#	-	1/1^#	9/10	-	-
Dilatation, tubule, multifocal, unilateral	2/10	0/1^#	-	1/1^$	0/10	-	-
Dilatation, tubule, multifocal, bilateral	0/10	1/1^$	-	0/1^#	0/10	-	-
Degeneration, tubule, multifocal, unilateral	0/10	0/1^#	-	0/1^#	1/10	-	-
ADRENAL GLANDS (N)	10/10	1/1^#	-	1/1^#	10/10	-	-
EPIDIDYMIDES (N)	10/10	1/1^#$	-	1/1^#$	9/10	-	-
Cell debris, lumen, multifocal, unilateral	0/10	0/1^#	-	0/1^#	1/10	-	-
PROSTATE (N)	7/10	1/1^#	-	1/1^#	10/10	-	-
Atrophy, diffuse	1/10	0/1^#	-	0/1^#	10/10	-	-
Infiltrate, inflammatory cell, multifocal	2/10	0/1^#	-	0/1^#	10/10	-	-
SEMINAL VESICLES (with coagulating glands) (N)	9/10	1/1^#	-	1/1^#	10/10	-	-
Atrophy, diffuse, unilateral	1/10	0/1^#	-	0/1^#	0/10	-	-
HEART (N)	10/10	1/1^#	-	1/1^#	9/10	-	-
Infiltrate, inflammatory cell, multifocal	0/10	0/1^#	-	0/1^#	1/10	-	-
AORTA (N)	10/10	1/1^#	-	1/1^#	10/10	-	-
SALIVARY GLANDS (N)	10/10	1/1^#	-	1/1^#	10/10	-	-
MANDIBULAR LYMPHNODES (N)	10/10	1/1^#	-	1/1^#	10/10	-	-
ESOPHAGUS (N)	10/10	1/1^#	-	1/1^#	10/10	-	-
TRACHEA (N)	10/10	10/10	10/10	8/10	9/10	3/5	5/5
Erosion, diffuse	0/10	0/1^#	-	1/1^#	0/10	-	-
Infiltration, MNC/PMNC, diffuse	0/10	0/10	0/10	1/10	1/10	2/5	0/5

Key: N = within normal histological limit; MNC/PMNC = Mono nuclear cell/poly morpho nuclear cell; * = only target organs (Stomach, trachea and kidneys) were evaluated; ^# = Found dead animal; ^$ = Gross pathology organ

Organs/ Histopathology	No. of tissues showing lesion / no. of tissues examined
	Group & Dose (mg/kg b.w.)
	G1 (0)	G2* (25)	G3* (75)	G4* (150)	G5 (350)	G6* (0)	G7* (350)
THYROID (N)	10/10	1/1^#	-	1/1^#	10/10	-	-
PARATHYROID (N)	10/10	1/1^#	-	1/1^#	10/10	-	-
STOMACH (N)	10/10	10/10	9/10	10/10	8/10	5/5	5/5
Hyperplasia, squamous cell – Nonglandular stomach, diffuse	0/10	0/10	0/10	0/10	1/10	0/5	0/5
Inflammation – Nonglandular stomach, diffuse	0/10	0/10	0/10	0/10	2/10	0/5	0/5
PANCREAS (N)	10/10	1/1^#	-	1/1^#	10/10	-	-
DUODENUM (N)	10/10	1/1^#	-	1/1^#	10/10	-	-
JEJUNUM (N)	10/10	1/1^#	-	1/1^#	10/10	-	-
ILEUM (with peyer's patches) (N)	10/10	1/1^#	-	1/1^#	10/10	-	-
CECUM (N)	10/10	1/1^#	-	1/1^#	10/10	-	-
COLON (N)	10/10	1/1^#	-	1/1^#	10/10	-	-
RECTUM (N)	10/10	1/1^#	-	1/1^#	10/10	-	-
BRAIN (N)	10/10	1/1^#	-	1/1^#	10/10	-	-
SCIATIC NERVE (N)	10/10	1/1^#	-	1/1^#	10/10	-	-
EYES (with optic nerve) (N)	10/10	1/1^#	-	1/1^#	10/10	-	-
SKIN (N)	10/10	1/1^#	-	1/1^#	10/10	-	-
MAMMARY GLANDS (N)	10/10	1/1^#	-	1/1^#	10/10	-	-
SKELETAL MUSCLE (N)	10/10	1/1^#	-	1/1^#	10/10	-	-
SPINAL CORD (N)	10/10	1/1^#	-	1/1^#	10/10	-	-
FEMUR WITH JOINT (N)	10/10	1/1^#	-	1/1^#	10/10	-	-

Key: N = within normal histological limit; * = only target organs (Stomach, trachea and kidneys) were evaluated; ^# = Found dead animal.

Gender: Female                                         

Organs/ Histopathology	No. of tissues showing lesion / no. of tissues examined
	Group & Dose (mg/kg b.w.)
	G1 (0)	G2* (25)	G3* (75)	G4* (150)	G5 (350)	G6* (0)	G7* (350)
LIVER (N)	10/10	-	-	-	10/10	-	-
LUNGS (with bronchi and bronchiole) (N)	10/10	-	-	-	10/10	-	-
KIDNEYS (N)	9/10	7/10	9/10	10/10	8/10	5/5	5/5
Cast, focal, unilateral	0/10	2/10	1/10	0/10	1/10	0/5	0/5
Cast, multifocal, bilateral	0/10	0/10	0/10	0/10	1/10	0/5	0/5
Basophilia, tubule, focal, unilateral	0/10	1/10	0/10	0/10	0/10	0/5	0/5
Infiltrate, inflammatory cell, multifocal, urethelium, bilateral	1/10	0/10	0/10	0/10	0/10	0/5	0/5
URINARY BLADDER (N)	10/10	-	-	-	10/10	-	-
SPLEEN (N)	9/10	-	-	-	9/10	-	-
EMH, multifocal	1/10	-	-	-	1/10	-	-
THYMUS (N)	10/10	-	-	-	9/10	-	-
Ectopic tissue, focal	0/10	-	-	-	1/10	-	-
MESENTERIC LYMPHNODES (N)	10/10	-	-	-	10/10	-	-
PITUITARY GLAND (N)	10/10	-	-	-	10/10	-	-
OVARIES  (N)	10/10	-	-	-	10/10	-	-
ADRENAL GLANDS (N)	10/10	-	-	-	10/10	-	-
UTERUS (with cervix and vagina) (N)	10/10	-	-	-	10/10	-	-
HEART (N)	10/10	-	-	-	10/10	-	-
AORTA (N)	10/10	-	-	-	10/10	-	-
SALIVARY GLANDS (N)	10/10	-	-	-	10/10	-	-
MANDIBULAR LYMPHNODES (N)	10/10	-	-	-	10/10	-	-
ESOPHAGUS (N)	10/10	-	-	-	10/10	-	-

Key: N = within normal histological limit; * = only target organs (Stomach, trachea and kidneys) were evaluated

Organs/ Histopathology	No. of tissues showing lesion / no. of tissues examined
	Group & Dose (mg/kg b.w.)
	G1 (0)	G2* (25)	G3* (75)	G4* (150)	G5 (350)	G6* (0)	G7* (350)
TRACHEA (N)	10/10	8	10/10	10/10	10/10	5/5	5/5
Infiltration, MNC/PMNC, diffuse	0/10	2	0/10	0/10	0/10	0/5	0/5
THYROID (N)	10/10	-	-	-	10/10	-	-
PARATHYROID (N)	10/10	-	-	-	10/10	-	-
STOMACH (N)	10/10	10/10	10/10	10/10	10/10	5/5	5/5
PANCREAS (N)	10/10	-	-	-	10/10	-	-
DUODENUM (N)	10/10	-	-	-	10/10	-	-
JEJUNUM (N)	10/10	-	-	-	10/10	-	-
ILEUM (with peyer's patches) (N)	10/10	-	-	-	10/10	-	-
CECUM (N)	10/10	-	-	-	10/10	-	-
COLON (N)	10/10	-	-	-	10/10	-	-
RECTUM (N)	10/10	-	-	-	10/10	-	-
BRAIN (N)	10/10	-	-	-	10/10	-	-
SCIATIC NERVE (N)	10/10	-	-	-	10/10	-	-
EYES (with optic nerve) (N)	10/10	-	-	-	10/10	-	-
SKIN (N)	10/10	-	-	-	10/10	-	-
MAMMARY GLANDS (N)	10/10	-	-	-	10/10	-	-
SKELETAL MUSCLE (N)	10/10	-	-	-	10/10	-	-
SPINAL CORD (N)	10/10	-	-	-	10/10	-	-
FEMUR WITH JOINT (N)	10/10	-	-	-	10/10	-	-

 

Key: N = within normal histological limit; * = only target organs (Stomach, trachea and kidneys) were evaluated; MNC/PMNC = Mono nuclear cell/ Poly morpho nuclear cell

Results of Dose Formulation Analysis

Formulation Analysis - Pre-Dose

Group	Nominal concentration (mg/ml)	Found Concentration (mg/ml)		Average (mg/ml)	Observed Concentration (%)	Average (%)	SD	Deviation (%)
G1 & G6	0.0	-	-	-	-	-	-	-
G2	5.0	Top	5.0009	4.7503	99.9	94.9	4.3405	-5.4
		Mid	4.6402		92.7
		Bottom	4.6098		92.1
G3	15.0	Top	14.3155	14.2758	95.4	95.2	1.6623	-5.1
		Mid	14.5083		96.7
		Bottom	14.0037		93.4
G4	30.0	Top	28.0970	29.7599	93.6	99.2	4.8590	-0.8
		Mid	30.6789		102.3
		Bottom	30.5040		101.7
G5 & G7	70.0	Top	71.7697	72.4562	102.5	103.5	0.9165	3.4
		Mid	73.0347		104.3
		Bottom	72.5641		103.7

Formulation Analysis - 6^th Week

Group	Nominal concentration (mg/ml)	Found Concentration (mg/ml)		Average (mg/ml)	Observed Concentration (%)	Average (%)	SD	Deviation (%)
G1 & G6	0.0	-	-	-	-	-	-	-
G2	5.0	Top	4.9834	5.0111	99.6	100.2	0.6658	0.2
		Mid	5.0033		100.0
		Bottom	5.0464		100.9
G3	15.0	Top	15.4202	15.5604	102.8	103.7	1.0693	3.6
		Mid	15.5313		103.5
		Bottom	15.7296		104.9
G4	30.0	Top	31.8328	31.7250	106.1	105.7	0.3215	5.4
		Mid	31.6800		105.6
		Bottom	31.6622		105.5
G5 & G7	70.0	Top	71.2798	71.5079	101.8	102.1	1.2342	2.1
		Mid	70.7613		101.1
		Bottom	72.4826		103.5

Formulation Analysis - 13^th Week

Group	Nominal concentration (mg/ml)	Found Concentration (mg/ml)		Average (mg/ml)	Observed Concentration (%)	Average (%)	SD	Deviation (%)
G1 & G6	0.0	-	-	-	-	-	-	-
G2	5.0	Top	5.0273	5.0282	100.5	100.5	0.0577	0.5
		Mid	5.0253		100.5
		Bottom	5.0321		100.6
G3	15.0	Top	15.0679	15.0358	100.5	100.2	0.2517	0.2
		Mid	15.0057		100.0
		Bottom	15.0338		100.2
G4	30.0	Top	30.5151	30.8564	101.7	102.8	2.3180	2.8
		Mid	30.3908		101.3
		Bottom	31.6634		105.5
G5 & G7	70.0	Top	68.5567	68.8393	97.9	98.3	0.5132	-1.7
		Mid	68.7349		98.2
		Bottom	69.2264		98.9

                                                  

Conclusions:

Oral administration of Genamin 3920 [2,2’-(Octylimino)bisethanol] for 90 consecutive days followed by 28 days of recovery period was found to be well tolerated in Wistar rats up to 350 mg/kg b.w./day without any obvious signs of toxicity.
On the basis of the study findings, the No Observed Adverse Effect Level (NOAEL) of Genamin 3920 [2,2’-(Octylimino)bisethanol] following 90-day repeated dose Oral administration in Wistar rats was 350 mg/kg b.w./day under the tested conditions.

Executive summary:

The aim of this study was to assess the possible health hazards which could arise from repeated exposure of Genamin 3920 via oral administration to rats over a period of 90 days.

The test item was administered daily in graduated doses to 4 groups of test animals, one dose level per group for a treatment period of 90 days. Animals of an additional control group were handled identically as the dose groups but received vehicle alone. The 5 groups comprised of 10 male and 10 female Wistar rats. To detect possible/ delayed occurrence or persistence of, or recovery from toxic effects, animals in the control and high dose recovery groups were observed for a period of 28 days

The vehicle and test item formulations were subjected to active ingredient analyses prior to treatment, during the 6th week and 13th week of the treatment period. The concentrations of Genamin 3920 [2,2’-(Octylimino)bisethanol] at all tested concentrations were within 100±10% of nominal concentrations, thereby meeting the acceptance criteria. The test item in formulation was found to be stable up to 48 hours.

Assessment of toxicity was based on mortality, clinical signs, body weights, feed consumption, neurobehavioral observations, clinical and anatomic pathology.

Body weight and food consumption were measured weekly.

At the conclusion of the treatment period, all animals were sacrificed and subjected to necropsy. The wet weight of organs of all animals were taken and preserved for histopathological observations.

A full histopathological evaluation of the tissues was performed on high dose and control animals. These examinations were extended to animals of all other dosage groups for treatment-related changes that were observed in the high dose group. Only organs and tissues of the other dosage groups showing changes in the high dose group were examined.

These examinations were also extended to animals subjected to necropsy at the end of the recovery period.

The following doses were evaluated:

Control:                                   0 mg/kg body weight

Low Dose:                              25 mg/kg body weight

Medium Dose:                        75 mg/kg body weight

Medium High Dose:               150 mg/kg body weight

High Dose:                              350 mg/kg body weight

The test item formulation was prepared on basis of stability data (48h).

Summary Results:

Weekly body weight and feed consumption did not reveal treatment related changes and were comparable to concurrent vehicle controls.

No treatment related changes were observed in any of the haematology, clinical chemistry, coagulation and urinalysis parameters in test item treated groups including recovery groups as compared with respective vehicle controls.

No treatment related organ weight changes were observed in any of the organ in test item treated groups including recovery groups as compared with respective vehicle controls.

No test item related changes were observed in sperm motility and morphology of main and recovery groups as compared with respective vehicle controls.

On histopathological examination, one male sacrificed terminally from 350 mg/kg dose group showed multifocal necrosis in kidneys together with squamous cell hyperplasia in non-glandular stomach. In addition, two males from the same group showed mononuclear cell infiltration in non-glandular stomach. As the kidneys findings were limited to one animal from high dose group, it was considered as incidental not related to test item. However, histopathology findings observed in stomach were considered as test item related non-adverse finding due to the irritating properties of the test item. Moreover, no histopathological changes were observed in stomach and kidneys of recovery high dose group animals.

Conclusion:

On the basis of this 90-Day Repeated Dose Oral Toxicity study with Genamin 3920 in male and female Wistar rats with dose levels of 25, 75, 150, and 350 mg/kg bw/d, the following conclusions can be made:

Histopathological lesions (in stomach) which were attributable to treatment with the test item were considered to be the results of local irritant effects due to the physicochemical character of the test item.

No signs of systemic toxicity of Genamin 3920 were found at dose levels of 25, 75, 150 and 350 mg/kg/d. Thus, the NOAEL is assumed to be 350 mg/kg/d in both genders.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

350 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

1 reliable without restriction; Guideline stuy under GLP

Repeated dose toxicity: inhalation - systemic effects

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

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

During a 28 day oral gavage study in rats local irritant effects in the forestomach including secondary changes thereof were observed.

All treatment related histopathological lesions were considered to be the results of local irritant effects due to the physicochemical character of the test item.

The forestomach tissue is not present in humans, and the elicited lesions resulted from a peracute or a continuous local stimulus to this structure of the rat by gavage. There is therefore no relevance for human.

Mortality at 500 mg/kg bw/d and clinical findings, attenuated body weight development and food consumption at 100 and 500 mg/kg bw/d are assumed to be related to the local irritant effect of the test item.

Thus, the no-observed effect level (NOEL) of this study could be established at 20 mg/kg bw/day in both genders.

No signs of systemic toxicity of the test material were found at dose levels of 20, 100 and 500 mg/kg/d. Thus, the NOAEL(systemic) is assumed to be 500 mg/kg/d in both genders.

In a 90 day repaed dose toxicity study according to OECD TG 408:

Oral administration of Genamin 3920 [2,2’-(Octylimino)bisethanol] for 90 consecutive days followed by 28 days of recovery period was found to be well tolerated in Wistar rats up to 350 mg/kg b.w./day without any obvious signs of toxicity.

On the basis of the study findings, the No Observed Adverse Effect Level (NOAEL) of Genamin 3920 [2,2’-(Octylimino)bisethanol] following 90-day repeated dose Oral administration in Wistar rats was 350 mg/kg b.w./day under the tested conditions.

Justification for classification or non-classification

no classification due to systemic effects; local effects are cosidered elsewhere.