Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24-06- 2014 - 01-09-2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from study report

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Principles of method if other than guideline:
The Repeated Dose 28-day Oral Toxicity study was designed and conducted to determine the toxicity profile of the test chemical when administered daily for 28 days in the Sprague Dawley rats.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of the test chemical: 2,2,2-TRICHLORO-1-PHENYLETHYL ACETATE
- Molecular formula: C10H9Cl3O2
- Molecular weight: 267.538 g/mol
- Substance type: Organic

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
1) In order to meet the regulatory requirement for testing in a rodent species

2) Widely used in as a species of choice for pre-clinical toxicological studies.

3) This strain is widely used throughout the industry in the non-clinical laboratory studies.

4) This study is intended to provide information on the health hazards likely to arise from exposure to the test item.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: National Institute of Biosciences, Pune
- Age at study initiation: 6 to 8 weeks
- Weight at study initiation:
Males: 132.8 g to 165.2 g
Females: 129.4 g to 156.4 g
- Fasting period before study: No Data
- Housing: The rats were housed in polycarbonate cages with paddy husk as bedding.
- Diet (e.g. ad libitum): Rodent feed supplied by the Nutrivet Life Sciences, Pune was provided ad libitum from individual feeders on cage top.
- Water (e.g. ad libitum): Water was provided ad libitum from individual bottles attached to the cages. Water was from a local source and passed through the reverse osmosis membrane before use.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Room temperature was maintained at 22 ± 3°C (actual range, 19.6 °C to 21.4 °C).
- Humidity (%): Room humidity was maintained at 30% to 70% (actual range, 54.3% to 60.6%).
- Air changes (per hr): At least 10 Air changes per hour
- Photoperiod (hrs dark / hrs light): An artificial light and dark cycle of 12 hours each was provided to the room.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG 400
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was suspended in Polyethylene Glycol-400 for preparation of suspension(s). The suspension(s) of test item was made at volumes suitable for daily use for 28 days. The solution(s) were prepared at concentrations of 0, 25, 50 and 100 mg/ml such that dosage of 0 (vehicle), 250, 500 and 1000 mg/kg body weight respectively were administered. The concentration of the test item was varied so as to maintain the dose volume constant at or upto 10 ml/kg body weight.


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

VEHICLE
- Justification for use and choice of vehicle (if other than water): Polyethylene glycol
- Concentration in vehicle: 0 (vehicle), 250, 500 and 1000 mg/kg body weight
- Amount of vehicle (if gavage): 10 mL/Kg
- Lot/batch no. (if required): No Data
- Purity: No Data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item prepared formulation analysis for Concentration, homogeneity and stability were conducted at Subcontracted Laboratory.


Duration of treatment / exposure:
28 Days consecutively and 2 week recovery period.
Frequency of treatment:
Once Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control Group (G1)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
Low Dose Group (G2)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
Mid Dose Group (G3)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
High Dose Group (G4)
No. of animals per sex per dose:
Control (0 mg/kg bw/day): 6 males and 6 females
Control Recovery (0 mg/kg bw/day): 6 males and 6 females
Low dose (250 mg/Kg/Day): 6 males and 6 females.
Mid Dose (500 mg/Kg/Day): 6 males and 6 females
High Dose (1000 mg/Kg/Day): 6 males and 6 females
High Dose Recovery(1000 mg/Kg/Day): 6 males and 6 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses were selected based on the results of the Dose Range Finder study conducted.The study was conducted at the dose levels of 0 mg/kg, 50 mg/kg, 100 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg body weight.

1) All the male and female animals from control and all the treated dose groups up to 1000 mg/kg survived throughout the dosing period of 14 days.

2) Male and female animals from control and all the treated dose groups exhibited normal body weight gain at the end of the dosing period of 14 days.

3) Daily clinical observations did not reveal any signs of toxicity in male and female animals from different dose groups during the dosing period of 14 days.

4) Gross pathological examination did not reveal any abnormality attributable to the treatment.

Based on these results, the 28 day study dose levels were finalized as 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg body weight and animals were exposed to the treatment, every day, for a period of 28 days.

- Rationale for animal assignment (if not random): Animals were randomized on the basis of sex and body weights. A total of 48 animals (24 males + 24 females) were selected and randomly distributed into four groups with 6 animals/sex/group and 3/sex/cage.

- Rationale for selecting satellite groups: No data
- Post-exposure recovery period in satellite groups: No data
- Section schedule rationale (if not random): No Data
Positive control:
No Data Available

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed twice daily for mortality. Rats were examined once daily for clinical signs.

- Cage side observations checked in table [No.?] were included. Viability, clinical signs such as skin and fur changes, eye and mucous membrane changes, respiratory, circulatory and general changes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were performed once before the start of dose administration and once a week thereafter until scheduled sacrifice.
a) Home Cage Observations:
In home cage, rats were observed for behavior, alterations, vocalizations, respiration and palpebral closer
1) Behavior in Home cage:
The behavior of the rat was observed in home cage upon initial approach by the observer and description of behavior in home cage was recorded as:
1 = apparently sleeping.
2 = Awake, but immobile; apparently normal posture.
3 = Engaged in apparently normal movement such as rearing, drinking, or grooming.
4 = Immobile, with unusual posture or tonic convulsion (lying on side with legs extended, flattened body or arched back. (opisthotonos or emprosthonos).
5 = Unusual behaviour or muscular patterns (stereotyped behaviour such as head bobbing or weaving, circling, repetitive licking or grooming, bizarre behaviour such as self mutilation, writhing or retropulsion, unusual muscular patterns such as tremors, spasms or clonic convulsion).
2) Alterations Home cage:
The alterations of the rat were observed in home cage upon initial approach by the observer and description of alterations in home cage was recorded as:
1 = No alterations in behaviour or posture (Normal).
2 = Stereotyped behaviour pattern (head bobbing or weaving, circling, repetitive licking or grooming).
3 = Bizarre behaviours such as self-mutilation, writhing or retropulsion.
4 = Twitches or tremors in the limbs or repetitive movements of the mouth or jaws.
5 = Whole body tremors or spasms.
6 = Unusual posture (opisthotonos, emprosthotonos, tonic extension, head tilt, straub tail).
7 = Tonic-clonic seizure.
3) Vocalizations:
The occurrence of spontaneous or unprovoked vocalization was recorded as:
1= No vocalization/ Normal
2= Vocalization noted
If vocalization observed the actual number was recorded.
4) Respiration:
The observations for respiration were recorded as:
1 = Normal
2 = Abnormal
The type of abnormal respiration e.g. bradypnea, hyperpnea, dyspnea, rals was recorded in the clinical signs.
5) Palpebral closer:
The degree of closure of the eyelids was recorded as:
1 = Eyelids wide open (Normal).
2 = Eyelids slightly closed.
3 = Eyelids dropping, approximately half-closed.
4 = Eyelids completely shut.
b) Handling Observations:
After completing home cage observations, the rat was observed for reaction to removal, reaction to handling, urination, defecation, prominence of eye, lacrimation, salivation, piloerection, examination of mucous membrane, examination of skin / fur, examination of natural orifices and animal appearance.
1) Reaction to Removal:
The reaction of the rat removed from home cage was recorded as:
1 = Sits quietly and is easily removed.
2 = Vocalization without resistance to being picked up.
3 = Runs around cage with or without vocalization, or freezes or rears, following the investigator’s hand.
4 = Tail and throat rattles, may attack.
2) Reaction to handling:

The reaction of the rat to handling from home cage was recorded as:
1 = Quiet with no resistance.
2 = Vocalization without resistance.
3 = Tense or rigid.
4 = Squirming and twisting, may attempt to bite.

3) Urination:
The frequency of urination was recorded as:
0 = No urination during the observation period.
1 = Urine present; quantity is not excessive.
2 = the amount of urination is excessive.

4) Defecation:

The frequency of defecation was recorded as:
0 = No defecation during the observation period.
1 = Fecal boluses have normal consistency.
2 = Soft or liquid feces.

5) Prominence of Eye:

The eyes were examined for prominence of eye and observation was recorded as:
1 = Normal
2 = Exophthalmos
3 = Enophthalmos

6) Lacrimation:

The degree of lacrimation was recorded as:
1 = No excess lacrimation (normal).
2 = Excess moisture at the margin of the eyelid.
3 = Persistent dampness at the margin of the eyelid
4 = Dampness extends beyond the margin of the eyelid.

7) Salivation:

The degree of salivation was recorded as:
1 = No excess salivation (normal).
2 = Margin of mouth wet.
3 = Wet zone ¼ to ½ of submandibular area.
4 = Wet zone extends to the entire submandibular area.

8) Piloerection:
Piloerection was differentiated from a scruffy or ungroomed coat by patting the back of the animal in a rostral to caudal direction. Piloerection was considered in case the animal hairs were erect after patting. The observation for piloerection was recorded as:
0 = Absent
1 = Present
9) Examination of Mucous Membrane:
The observation for visual mucous membrane was recorded as:
¬¬¬1 = Normal
2 = Abnormal (discolouration)
10) Examination of Skin / Fur:
The observation for skin / fur examination was recorded as:
1 = Normal
2 = Abnormal
11) Examination of Natural Orifices:
The observation for natural orifices examination was recorded as:
1 = Normal
2 = Abnormal
12) Animal Appearance:
The observation for appearance of animal was recorded as:
1 = Clean and groomed.
2 = Unkempt (with scruffy and ungroomed coat)
3 = Stained by urine and/or feces.
c) Open field observation:
The animal was placed in the open field and its appearance and behavior were observed. The following observations were made and recorded:
1) Stereotype Behaviour:
The stereotype behaviour can be defined as the pronounced repetition of specific gesture or movements i.e. presence of excessive or repetitive behaviour that appears purposeless to the observer. The observation was recorded as:
0 = Absent
1 = Excessive grooming / licking / head bobbing or weaving
2 = Circling movements
2) Bizarre Behaviour:
The bizarre behaviour includes any unusual behaviour that will not be normally observed in the test species. The observation was recorded as:
0 = Absent
1 = Retropulsion
2 = Biting of cage
3 = Biting to other animal(s)
4 = Self destructive biting or mutilation
3) Rearing (Rears):
The number of times the rat raises both forelimbs off the surfaces is considered as rearing. The number of these actions was counted and total number of rearing was recorded.
4) Movements:
In the open field, each animal was observed for presence of clonic and tonic movements:
4.1) Clonic Movements:
The observation for clonic movement was recorded as:
0 = None /Normal
1 = repetitive mouth/jaw motion, such as, Chewing, clones of jaw
2 = Mild clonic tremors of whole body
3 = Repetitive clonic tremors/ seizure of whole body
4.2) Tonic Movements:
The observation for tonic movement was recorded as:
0 = None /Normal
1 = Contractions of limbs
2 =Unusual posture (Opisthotonos, Emprosthotonous, tonic extension, head tilt, straub tail).
3= seizure

5) Gait Pattern:
The gait pattern was evaluated by observing the movement of the rat in the open field and the observation was recorded as:
1 = Normal
2 = Ataxic
3 = Hind limbs or forelimbs show exaggerated or overcompensated movements, drag or appear splayed.
4 = Spastic
5 = Duck walk
6 = Scissor
7 = Hunched back
6) Mobility Score:
A measure of the ability of the animal to locomote despite gait abnormalities was recorded. The ranking of the degree of impairment of locomotion was recorded as:
1 = Normal
2 = Slightly impaired
3 = Totally impaired

7) Severity of Gait:
The severity of the gait abnormalities is graded and documented as follows:
1 = Slight gait abnormality
2 = Moderate gait abnormality
3 = Extreme gait abnormality

8) Pupillary response:
The animal eyes were briefly covered for 30 seconds with hand/cloth and then the penlight was pointed and the response to penlight was recorded as:

1 = Response
2 = No response

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on the day of randomization, day of first dosing, weekly thereafter and a fasting body weight at scheduled sacrifice on day 29 and day 43.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, The quantity of feed consumed by control and different treatment groups was recorded on commencement of treatment and weekly thereafter until scheduled sacrifice and the feed consumption per animal was calculated for each group.

- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

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

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The eyes of all the animals were examined prior to the initiation of the dosing and at scheduled sacrifice.
- Dose groups that were examined: All animals were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected at terminations.
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes, Food was withheld overnight from all rats prior to sampling.
- How many animals: Blood samples were collected from all rats from each group.
- Parameters checked in table [No.?] were examined. Hb, RBC, HCT, MCV, MCH, MCHC, WBC, N, L, E, M, B, Pt

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected at respective terminations.
- Animals fasted: Yes, Food was withheld overnight from all rats prior to sampling.
- How many animals: Blood samples were collected from all rats from each group.
- Parameters checked in table [No.?] were examined. Total Protein , Blood Urea Nitrogen , Urea Nitrogen, ALT, AST, ALP, GGT, Glucose, Ca, P, Albumin, Total bilirubin, creatinine, total cholesterol, triglycerides, globulin, Na, K, Cl

URINANALYSIS: No data
- Time schedule for collection of urine: No data
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked in table [No.?] were examined. No data

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Towards the end of the exposure period of 28 days and towards the end of the recovery period on day 42.
- Dose groups that were examined: All animals from all groups.
- Battery of functions tested:
Sensory activity: Towards the end of the exposure period, sensory reactivity to stimuli of different types (e.g., auditory, visual and proprioceptive stimuli) was conducted for all the animals.

1) Arousal level:
The activity/arousal level of the animal was described during the observation period and is documented as follows:
1 = Very low (stuporous, comatose)
2 = Low (sluggish, some exploratory movement possible).
3 = Apparently normal (alert with exploratory movement).
4 = High (sudden startle, darting or freezing without apparent stimuli).
5 = Very high (sudden bouts of running, freezing with spontaneous vocalization).

2) Sensory Activity:
Sensory activity was assessed by following methods:
2.1) Assessment of Visual Response: A blunt probe was held approximately 4 cm away from front of the face / eye and moved away steadily and reaction was documented.
2.2) Touch Response: Avoiding the animal’s field of vision, the rump was gently touched with blunt probe. The animals reaction to this stimulus was observed and was documented.
2.3) Auditory Response: Using a clicker approximately 5 cm above the back of the animal sudden sound was made. The animals reaction to this stimulus was observed and was documented.
2.4) Tail Pinch Response: The reaction to a tail pinch was rated (The tail pinch was applied by the blunt forceps at approximately 3 cm from the tip of tail. The reaction to tail pinch was observed and was documented.
All above four responses were graded as follows:
1 = No reaction.
2 = orientating response: Slowly turns towards the stimulus or walks away.
3 = Startle response or freezing reaction.
4 = More energetic response than “2” or “3”.
5 = Jumps at or away, attack or bites.
3) Visual Placing Response:
The animal was removed from its cage and held at the base of the tail (holding the tail more distally can strip off the skin) then slowly lowered forward towards the edge of the observation area or another raised edge (such as the rim of an overturned cage). The visual placing response is graded and documented:
1 = Early extension of forelimbs to reach for the screen.
2 = Extends limbs only after contact with the vibrissae or nose.
3 = No extension even after contact with nose.
4) Air righting response:
Holding the animal in a supine position, it was dropped from approximately 30cm and the righting response was rated:
1 = Lands with all feet on the ground.
2 = Uncoordinated landing or lands on side.
3 = Lands on back.

Grip strength: Grip strength of fore limbs was measured with a digital grip strength meter (Columbus Instruments International Corporation, Ohio, USA) to determine the ability of the rat to grasp and hold on the mesh platform. The grip strength of each rat was measured in Kilogram (Kg) for 3 consecutive times and average of the three grip strength values was calculated.

Motor activity: Motor activity of each animal was monitored using an automated animal activity measuring system (Columbus Instruments, OPTO-M3, Ohio, USA). Animals were monitored for three consecutive 10 minutes intervals allowing for examination of both exploratory and acclimation activity levels. During this period, total and ambulatory activity of the animal was recorded. Stereotypic activity was calculated by subtracting ambulatory activity from total activity.
Other: No Data
Sacrifice and pathology:
SACRIFICE: The rats were sacrificed by CO2 asphyxiation, after 28 consecutive days of oral administration, all surviving study rats were sacrificed on day 29 (Group I, III, IV, V).

GROSS PATHOLOGY: Yes, All the rats survived through the dosing period of 28 days and were sacrificed and gross lesions were noted.

ORGAN WEIGHTS: Liver, Kidneys, Adrenals, Epididymides, Prostate + Seminal Vesicle with Coagulation gland as whole, Thymus, Spleen, Brain, Heart, Lungs, Uterus, Testes/Ovaries were dissected free of fat and weighed. The paired organs were weighed together.

HISTOPATHOLOGY: Yes, From each rat, samples or the whole of the tissues listed below were preserved. All tissues were fixed in 10% neutral buffered formalin except, eyes and testes of all animals were preserved in Davidson’s solution for 24 hours and transferred to 10% neutral buffered formalin. Procedure for preparation of slides of tissues of various organs from the rats of various dose groups were performed.

Following tissue samples of organs from control and animals treated at different dose groups were preserved and those from control and treated at the highest dose level of 1000 mg/kg were subjected to histopathological examination:
Adrenals, Aorta, Brain (cerebrum, cerebellum and pons), Caecum, Cervix, Colon, Duodenum, Epididymides, Eyes, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs, Mesenteric Lymphnodes, Oesophagus, Ovaries, Pancreas, Pituitary gland, Pharyngeal Lymphnodes, Prostate, Rectum, Skeletal Muscles, Skin with Mammary Gland, Spleen, Sternum with bone marrow, Sciatic Nerve, Spinal Cord (Cervical, mid thoracic and lumbar), Stomach, Seminal Vesicles with Coagulation Gland, Testes, Thymus, Thyroid, Trachea, Vagina, Urinary Bladder, Uterus.
Other examinations:
No Data Available
Statistics:
Raw data was processed and analyzed for reporting group means and standard deviations with significance between the controls and treated groups, using in-house developed and validated MS-Excel 2003 based statistical software. All the parameters characterized by continuous data such as body weight, per cent body weight change, feed consumption, organ weight, relative organ weight, haematological and clinical chemistry data were subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analyses of Variance (ANOVA) and Dunnett’s t-test. Where the data did not meet the homogeneity of variance, Student’s t-test was performed to calculate significance.

Significance was calculated at 1% as well as 5% level and indicated in the summary tables as follows:

* = Significant than control at 95% level of confidence (p≤ 0.05).
** = Significant than control at 99% level of confidence (p≤ 0.01).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs of toxicity were observed in the animals throughout the dosing period of 28 days
Mortality:
no mortality observed
Description (incidence):
All the animals from control and all the treated dose groups survived throughout the dosing period of 28 days.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Animals from control and different dose groups exhibited normal body weight gain throughout the dosing period of 28 days.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Animals from control and all treated dose groups exhibited normal feed consumption at the end of the dosing period of 28 days.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Haematological investigations conducted at the end of dosing period on day 29, revealed following significant changes in the values of different parameters studied when compared with that of respective controls.

Male :
MCV (p≤0.01), MCH (p≤0.01) and Total WBC (P≤0.05) : Increased values were obtained for animals from 250 mg/kg dose group,
Platelets : Increased values were obtained for animals from 250 mg/kg and 1000 mg/kg dose groups (p≤0.05),
Hb and HCT : Decreased values were obtained for animals from 250 mg/kg (p≤0.05), 500 mg/kg (p≤0.01) and 1000 mg/kg (p≤0.01) dose groups,
Total RBC : Decreased values were obtained for animals from 250 mg/kg and 500 mg/kg dose groups (p≤0.01) and
MCV and MCH : Decreased values were obtained for animals from 1000 mg/kg dose group (p≤0.01).

Female :
Platelets : Increased values were obtained for animals from 500 mg/kg dose group (p≤0.01),
Hb : Decreased values were obtained for animals from 250 mg/kg (p≤0.05) and 500 mg/kg (p≤0.01) dose groups,
Total RBC (p≤0.01), HCT (p≤0.05) and Total WBC (P≤0.05) : Decreased values were obtained for animals from 500 mg/kg dose group,
MCV : Decreased values were obtained for animals from 1000 mg/kg dose group (p≤0.01) and
MCH and MCHC : Decreased values were obtained for animals from 250 mg/kg (p≤0.05) and 1000 mg/kg (p≤0.01) dose groups.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Biochemical investigations conducted at the end of dosing period on day 29, revealed following significant changes in the values of different parameters studied when compared with that of respective controls.

Male :
Total Protein : Elevated levels were observed in animals from 500 mg/kg dose group (p≤0.05),
Creatinine : Elevated levels were observed in animals from 1000 mg/kg dose group (p≤0.05),
Cholesterol : Elevated levels were observed in animals from 500 mg/kg and 1000 mg/kg dose groups (p≤0.05),
Bilirubin : Decreased levels were observed in animals from 250 mg/kg (p≤0.05) and 500 mg/kg (p≤0.01) dose groups and
Chloride : Decreased levels were observed in animals from 500 mg/kg dose group (p≤0.01).

Female :
Sodium : Elevated levels were observed in animals from 250 mg/kg, 500 mg/kg and 1000 mg/kg dose groups (p≤0.01),
Alkaline Phosphatase : Decreased levels were observed in animals from 500 mg/kg dose group (p≤0.01) and
Chloride : Decreased levels were observed in animals from 1000 mg/kg dose group (p≤0.05).
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Sensory Reactivity Observations:
All animals from control and different dose groups showed normal arousal level, visual response, touch response, auditory response, tail pinch response and visual placing response. Normal air righting reflex was observed in all animals from control and different dose groups in week 4.

Grip Strength:
Grip strength values observed in male and female animals for control and different dose groups were comparable.

Motor Activity:
Higher values were observed in male animals from 250 mg/kg, 500 mg/kg and 1000 mg/kg dose groups for second interval (p≤0.05). Lower values were observed in female animals from 1000 mg/kg dose group for first interval (p≤0.01) and for second interval (p≤0.05).

These changes were within laboratory range and were considered to be of no toxicological importance
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Male -
In comparison with controls organ weight data of male animals sacrificed on day 29, revealed decreased relative weight of testes of animals from 500 mg/kg dose group (p≤0.05).

Although significant changes in organ weights were observed in male animals from intermediate dose group, no related gross pathological or histological changes were seen and hence considered to be of no toxicological importance.

Female -
In comparison with controls on day 29, organ weight data of female animals from different dose groups was found to be comparable.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Gross pathological examination in male and female animals from control and different treatment groups did not reveal any abnormality.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related histopathological changes were evident in male and female rats from control and high dose groups.
Histopathological examination revealed minimal focal to multifocal periportal mononuclear cell infiltration in the liver; minimal interstitial haemorrhages and/or tubular dilatation in the kidneys; minimal alveolar haemorrhages and/or alveolar histiocytosis in the lungs; minimal multifocal haemosiderosis and/or diffused congestion in spleen; minimal eosinophilic cells infiltration and/or luminal dilatation and/or endometrial gland dilatation in uterus; minimal luminal seminal coagulum in the urinary bladder; minimal dilatation of zona reticularis and/or vacuolation in zona fasiculata in adrenals; minimal multifocal haemorrhages in thymus; presence of ultimobranchial cysts in thyroid; in male or female animals from control and high dose group. All the changes observed in the control and high dose treatment group animals were similar, incidental and mode of death related, physiological and are covered in the facility historical data of the histopathology findings.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
ophthalmological examination
haematology
clinical biochemistry
urinalysis
behaviour (functional findings)
organ weights and organ / body weight ratios
gross pathology
neuropathology
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

VIABILITY

 

Dose

Mortality

Group

mg/kg

Males

Females

Number

Male

Female

Absolute

Relative %

Absolute

Relative %

I

0

0

0/6

0

0/6

0

II

250

250

0/6

0

0/6

0

III

500

500

0/6

0

0/6

0

IV

1000

1000

0/6

0

0/6

0

GROUP MEAN BODY WEIGHT (g)

 

Sex - Male

Group

Dose

 

Day

Number

mg/kg bw

 

0

1

8

15

22

28

I

0

Mean

154.23

159.17

214.55

266.05

299.30

323.63

±SD

11.21

11.28

14.09

19.19

20.96

19.45

II

250

Mean

152.37

157.73

207.88

261.47

292.90

316.53

±SD

12.54

12.53

12.50

15.07

14.41

14.22

III

500

Mean

153.25

159.88

201.80

248.33

285.63

306.90

±SD

11.25

11.82

11.18

11.82

13.66

13.97

IV

1000

Mean

155.10

161.08

205.98

260.42

291.13

311.83

±SD

9.84

9.88

7.48

16.30

12.05

10.81

Sex - Female

Group

Dose

 

Day

Number

mg/kg bw

 

0

1

8

15

22

28

I

0

Mean

138.18

141.05

179.03

204.52

220.50

234.18

±SD

9.51

9.69

8.67

10.74

11.85

11.00

II

250

Mean

138.95

142.18

169.42

192.18

210.32

224.70

±SD

8.80

8.94

4.88

7.42

15.75

15.72

III

500

Mean

139.28

142.22

169.40

189.75

206.50

221.45

±SD

9.47

9.97

5.81

5.78

12.09

12.83

IV

1000

Mean

140.22

143.20

169.85

187.55

202.20

218.48

±SD

10.30

10.14

7.09

6.25

10.41

8.98

GROUP MEAN FEED CONSUMPTION (g/animal/day)

Sex - Male

Group

Dose

 

WEEK

Number

mg/kg

 

0 Day

1

2

3

4

I

0

Mean

15.73

17.73

19.65

21.50

24.18

II

250

Mean

15.46

17.63

19.43

21.82

23.62

III

500

Mean

15.60

17.96

18.58

20.97

23.38

IV

1000

Mean

15.55

17.68

19.32

21.02

23.32

Sex - Female

Group

Dose

 

WEEK

Number

mg/kg

 

0 Day

1

2

3

4

I

0

Mean

11.76

13.38

15.53

16.85

19.62

II

250

Mean

11.88

13.55

15.38

16.70

19.21

III

500

Mean

11.62

13.22

15.03

16.48

18.62

IV

1000

Mean

12.03

13.58

15.22

16.22

18.58

SUMMARY OF CLINICAL OBSERVATIONS

AND GENERAL APPEARANCE

Sex : Male

 

Group

Number

Dose mg/kg

Observed Signs

Total Number of

Animals

Animal Nos.

Period of signs

in days

from - to

Mortality

I

0

Nil

6

1 - 6

1 - 28

0/6

II

250

Nil

6

13 - 18

1 - 28

0/6

III

500

Nil

6

25 - 30

1 - 28

0/6

IV

1000

Nil

6

37 - 42

1 - 28

0/6

Sex : Female

 

Group

Number

Dose mg/kg

Observed Signs

Total Number of

Animals

Animal Nos.

Period of signs

in days

from - to

Mortality

I

0

Nil

6

7 - 12

1 - 28

0/6

II

250

Nil

6

19 - 24

1 - 28

0/6

III

500

Nil

6

31 - 36

1 - 28

0/6

IV

1000

Nil

6

43 - 48

1 - 28

0/6

GROUP MEAN HAEMATOLOGY

Sex : Male

Day : 29

 

Group

Dose

 

Hb

Total RBC

HCT

MCV

MCH

MCHC

Number

mg/kg

 

(g/dL)

(x 106/µL)

(%)

(fL)

(pg)

(g/dL)

I

0

Mean

17.62

8.05

43.97

54.63

21.92

40.07

±SD

0.31

0.08

0.55

0.45

0.38

0.42

II

250

Mean

16.75*

7.40**

41.75*

56.48**

22.68**

40.12

±SD

0.48

0.28

1.31

0.79

0.18

0.52

III

500

Mean

15.62**

7.21**

39.40**

54.77

21.72

39.68

±SD

0.74

0.51

2.13

2.84

1.10

0.28

IV

1000

Mean

16.17**

7.91

40.68**

51.48**

20.43**

39.70

±SD

0.47

0.33

1.27

0.89

0.46

0.40

 

 

Group

Dose

 

Platelets

Total WBC

Differential %

Pt.

Number

mg/kg

 

(x 103/ µL)

(x 103/µL)

N

L

E

M

B

(Sec.)

I

0

Mean

305.33

8.08

19.33

78.00

1.67

1.00

0.00

14.67

±SD

7.15

1.19

2.73

3.63

0.82

0.89

0.00

3.56

II

250

Mean

321.00*

13.20*

18.67

79.00

1.17

1.17

0.00

15.83

±SD

15.58

4.02

2.34

1.90

1.17

0.75

0.00

2.86

III

500

Mean

341.50

7.92

18.67

79.00

1.33

1.00

0.00

15.67

±SD

45.31

3.91

2.34

2.97

1.21

0.89

0.00

3.14

IV

1000

Mean

338.17*

9.07

18.67

79.17

1.17

1.00

0.00

13.50

±SD

32.50

1.77

2.16

2.79

0.75

0.89

0.00

1.87

 

Hb         : Hemoglobin                    RBC     : Red Blood Corpuscules                         

HCT      : Hematocrit                       MCV    : Mean Corpuscular Volume

MCH    : Mean Corpuscular Hemoglobin        MCHC  : Mean Corpuscular Hemoglobin Concentration

WBC    : White Blood Corpuscles                  Pt.        : Prothrombin time

N          : Neutrophils                      L          : Lymphocytes

E          : Eosinophils                      M         : Monocytes

B          : Basophils

*  = Significant at 95% level of confidence (p≤0.05)

** = Significant at 99% level of confidence (p≤0.01)

Sex : Female

Day : 29

 

Group

Dose

 

Hb

Total RBC

HCT

MCV

MCH

MCHC

Number

mg/kg

 

(g/dL)

(x 106/µL)

(%)

(fL)

(pg)

(g/dL)

I

0

Mean

17.43

8.00

43.10

53.85

21.80

40.47

±SD

0.31

0.30

1.14

0.72

0.49

0.41

II

250

Mean

16.40*

8.07

41.30

51.32

20.40*

39.78*

±SD

0.79

0.39

2.03

3.53

1.45

0.22

III

500

Mean

15.18**

6.94**

37.90*

54.58

21.90

40.10

±SD

0.54

0.25

1.43

0.37

0.17

0.54

IV

1000

Mean

16.63

8.12

42.25

52.07**

20.48**

39.38**

±SD

0.85

0.52

2.32

0.96

0.37

0.35

 

 

Group

Dose

 

Platelets

Total WBC

Differential %

Pt.

Number

mg/kg

 

(x 103/ µL)

(x 103/µL)

N

L

E

M

B

(Sec.)

I

0

Mean

295.83

8.57

17.83

80.33

1.17

0.67

0.00

15.33

±SD

30.90

2.43

2.32

2.34

0.75

0.52

0.00

3.08

II

250

Mean

308.17

11.80

17.83

79.50

1.67

1.00

0.00

16.33

±SD

59.19

3.96

2.99

3.33

0.82

0.63

0.00

2.80

III

500

Mean

372.33**

5.48*

17.33

80.83

1.17

0.67

0.00

16.17

±SD

18.48

0.17

2.88

2.48

0.75

0.52

0.00

2.79

IV

1000

Mean

318.00

8.33

17.67

79.83

1.00

1.50

0.00

15.33

±SD

39.65

2.37

3.01

2.93

0.63

0.55

0.00

2.16

 

Hb         : Hemoglobin                    RBC     : Red Blood Corpuscules                         

HCT      : Hematocrit                       MCV    : Mean Corpuscular Volume

MCH    : Mean Corpuscular Hemoglobin        MCHC  : Mean Corpuscular Hemoglobin Concentration

WBC    : White Blood Corpuscles                  Pt.        : Prothrombin time

N          : Neutrophils                      L          : Lymphocytes

E          : Eosinophils                      M         : Monocytes

B          : Basophils

*  = Significant at 95% level of confidence (p≤0.05)

** = Significant at 99% level of confidence (p≤0.01)

GROUP MEAN CLINICAL BIOCHEMISTRY

 Sex : Male

Day : 29

 

Group Number

Dose      mg/kg

 

Total Protein

(g/dL)

BUN

(mg/dL)

Urea

Nitrogen

(mg/dL)

ALT

(U/L)

AST

(U/L)

ALP

(U/L)

Glucose

(mg/dL)

I

0

Mean

6.57

14.17

30.88

48.17

101.50

171.50

74.33

±SD

0.29

1.72

3.75

3.25

17.95

37.13

6.53

II

250

Mean

6.77

16.00

34.88

46.67

92.17

172.67

76.50

±SD

0.29

1.26

2.76

7.45

13.12

34.20

8.92

III

500

Mean

7.04*

15.17

33.06

48.17

104.33

185.50

78.50

±SD

0.19

1.47

3.21

9.87

24.20

33.12

5.21

IV

1000

Mean

6.77

14.83

32.34

48.83

104.00

193.67

65.33

±SD

0.28

2.48

5.41

4.79

8.46

22.26

5.43

 

Group Number

Dose mg/kg

 

Calcium

(mmol/L)

Phospho-rous

(mg/dL)

GGT

(U/L)

Total Bilirubin

(mg/dL)

Albumin

(g/dL)

Globulin

(g/dL)

Creatinine

(mg/dL)

I

0

Mean

3.59

7.62

6.50

0.31

1.17

5.40

0.45

±SD

0.11

0.83

1.22

0.04

0.13

0.39

0.03

II

250

Mean

3.69

7.48

6.33

0.20*

1.28

5.48

0.46

±SD

0.12

0.88

0.52

0.09

0.09

0.29

0.05

III

500

Mean

3.76

8.03

5.17

0.13**

1.21

5.82

0.47

±SD

0.10

0.89

0.75

0.02

0.09

0.17

0.04

IV

1000

Mean

3.68

8.27

5.67

0.27

1.27

5.48

0.52*

±SD

0.10

0.82

0.82

0.02

0.10

0.25

0.04

 

Group Number

Dose

mg/kg

 

Sodium

(mmol/L)

Potassium

(mmol/L)

Chloride

(mmol/L)

Total Cholesterol

(mg/dL)

Triglycerides

(mg/dL)

I

0

Mean

146.34

4.22

109.07

31.33

74.17

±SD

1.30

0.19

1.26

8.31

20.54

II

250

Mean

145.43

4.54

107.77

41.00

66.67

±SD

0.80

0.26

1.67

7.16

14.46

III

500

Mean

146.76

4.67

105.66**

43.33*

81.17

±SD

1.46

0.43

1.90

6.38

23.66

IV

1000

Mean

146.40

4.23

108.67

42.83*

77.33

±SD

1.91

0.32

1.01

8.01

30.87

 

BUN : Blood Urea Nitrogen           

ALT : Alanine Aminotransferase       

AST : Aspartate Transaminase

ALP : Alkaline Phosphatase

 

*  = Significant at 95% level of confidence (p≤0.05)

** = Significant at 99% level of confidence (p≤0.01)

GROUP MEAN CLINICAL BIOCHEMISTRY

 

Laboratory Test Item Code : TAS/122/001

Test System : Sprague Dawley Rat

Sex : Female

Day : 29

 

Group Number

Dose      mg/kg

 

Total Protein

(g/dL)

BUN

(mg/dL)

Urea

Nitrogen

(mg/dL)

ALT

(U/L)

AST

(U/L)

ALP

(U/L)

Glucose

(mg/dL)

I

0

Mean

6.53

15.17

33.06

44.83

107.33

164.00

72.83

±SD

0.31

2.23

4.86

4.31

15.58

20.07

4.58

II

250

Mean

6.48

15.17

33.06

45.50

113.33

132.00

64.00

±SD

0.39

1.60

3.49

9.03

23.59

30.57

8.92

III

500

Mean

6.53

13.67

29.79

42.50

90.83

99.00**

70.50

±SD

0.43

0.82

1.78

6.60

8.98

20.42

6.41

IV

1000

Mean

6.42

15.33

33.43

41.50

95.67

125.83

71.67

±SD

0.44

3.72

8.12

5.86

9.07

33.47

7.45

 

Group Number

Dose mg/kg

 

Calcium

(mmol/L)

Phospho-rous

(mg/dL)

GGT

(U/L)

Total Bilirubin

(mg/dL)

Albumin

(g/dL)

Globulin

(g/dL)

Creatinine

(mg/dL)

I

0

Mean

3.71

6.57

6.50

0.11

1.29

5.25

0.55

±SD

0.11

0.64

1.05

0.02

0.15

0.29

0.05

II

250

Mean

3.71

7.48

6.67

0.13

1.19

5.32

0.53

±SD

0.11

0.75

1.37

0.06

0.17

0.33

0.04

III

500

Mean

3.68

7.17

5.17

0.12

1.29

5.23

0.51

±SD

0.04

0.84

0.75

0.02

0.10

0.31

0.05

IV

1000

Mean

3.71

6.77

6.33

0.11

1.22

5.20

0.55

±SD

0.04

0.94

1.75

0.02

0.13

0.40

0.06

 

Group Number

Dose

mg/kg

 

Sodium

(mmol/L)

Potassium

(mmol/L)

Chloride

(mmol/L)

Total Cholesterol

(mg/dL)

Triglycerides

(mg/dL)

I

0

Mean

144.61

4.34

108.89

52.33

76.00

±SD

0.66

0.40

1.06

5.09

15.49

II

250

Mean

146.38**

4.43

107.33

48.17

63.17

±SD

0.77

0.22

2.43

8.91

19.01

III

500

Mean

146.32**

4.25

108.89

47.83

83.33

±SD

0.54

0.14

1.53

7.88

25.73

IV

1000

Mean

146.45**

4.21

106.32*

50.17

88.00

±SD

1.42

0.27

1.21

5.85

28.71

 

BUN : Blood Urea Nitrogen           

ALT : Alanine Aminotransferase       

AST : Aspartate Transaminase

ALP : Alkaline Phosphatase

 

*  = Significant at 95% level of confidence (p≤0.05)

** = Significant at 99% level of confidence (p≤0.01)

 

GROUP MEAN ABSOLUTE ORGAN WEIGHTS (g)

 Sex : Male

Day : 29

 

GroupNumber

Dose mg/kg

 

Body Weight (g)

 

Brain

        Liver

 

 Kidneys

 

 

Adrenals

 

 Testes

Prostate + Seminal

Vesicle with

Coagulation gland

I

0

Mean

304.27

2.033

13.192

2.429

0.0607

3.182

1.491

±SD

19.75

0.144

1.775

0.195

0.0133

0.139

0.284

II

250

Mean

298.33

1.936

12.352

2.244

0.0545

3.077

1.258

±SD

15.62

0.154

1.338

0.202

0.0028

0.318

0.154

III

500

Mean

288.80

1.975

12.340

2.162

0.0523

2.594

1.133

±SD

13.01

0.078

0.903

0.409

0.0067

0.276

0.246

IV

1000

Mean

294.33

1.999

12.163

2.347

0.0474

2.964

1.213

±SD

10.28

0.086

1.233

0.214

0.0070

0.249

0.122

 

GroupNumber

Dose mg/kg

 

Heart

Spleen

Lungs

Thymus

Epididymides

I

0

Mean

1.364

1.404

1.760

0.430

0.764

±SD

0.162

0.247

0.238

0.100

0.058

II

250

Mean

1.231

1.349

1.623

0.372

0.671

±SD

0.045

0.413

0.188

0.068

0.048

III

500

Mean

1.233

1.411

1.817

0.363

0.658

±SD

0.072

0.227

0.441

0.066

0.032

IV

1000

Mean

1.133

1.488

1.531

0.343

0.673

±SD

0.144

0.513

0.315

0.081

0.076

 

 

GROUP MEAN ABSOLUTE ORGAN WEIGHTS (g)

Sex : Female

Day : 29

 

GroupNumber

Dose mg/kg

 

Body Weight (g)

 

Brain

        Liver

 

 

Kidneys

 

 

Adrenals

 

 

Ovaries

 

I

0

Mean

217.20

1.908

8.040

1.646

0.0559

0.0822

±SD

11.85

0.098

0.889

0.136

0.0087

0.0105

II

250

Mean

207.97

1.849

7.651

1.475

0.0556

0.0669

±SD

16.31

0.111

1.027

0.176

0.0078

0.0095

III

500

Mean

206.17

1.813

7.788

1.523

0.0558

0.0757

±SD

11.88

0.124

1.110

0.261

0.0044

0.0192

IV

1000

Mean

201.28

1.828

7.225

1.478

0.0512

0.0703

±SD

8.75

0.069

0.878

0.150

0.0082

0.0076

 

Group No.

Dose mg/kg

 

Heart

Spleen

Lungs

Thymus

Uterus

I

0

Mean

0.955

1.130

1.394

0.435

0.346

±SD

0.085

0.271

0.361

0.097

0.077

II

250

Mean

0.881

1.396

1.462

0.386

0.313

±SD

0.088

0.605

0.204

0.147

0.106

III

500

Mean

0.848

1.028

1.632

0.363

0.293

±SD

0.113

0.198

0.181

0.085

0.079

IV

1000

Mean

0.815

0.834

1.322

0.345

0.293

±SD

0.048

0.149

0.217

0.050

0.150

GROUP MEAN RELATIVE ORGAN WEIGHTS (%)

Sex : Male

Day : 29

 

GroupNumber

Dose mg/kg

 

Body Weight (g)

 

Brain

 

Liver

 

 Kidneys

 

 

Adrenals

 

 Testes

Prostate + Seminal

Vesicle with

Coagulation gland

I

0

Mean

304.27

0.668

4.326

0.798

0.0199

1.048

0.490

±SD

19.75

0.029

0.404

0.034

0.0039

0.043

0.082

II

250

Mean

298.33

0.650

4.138

0.753

0.0183

1.033

0.423

±SD

15.62

0.055

0.368

0.066

0.0012

0.120

0.057

III

500

Mean

288.80

0.685

4.280

0.752

0.0182

0.899*

0.393

±SD

13.01

0.036

0.381

0.153

0.0025

0.099

0.085

IV

1000

Mean

294.33

0.680

4.131

0.798

0.0161

1.008

0.412

±SD

10.28

0.031

0.379

0.072

0.0026

0.083

0.041

 

GroupNumber

Dose mg/kg

 

Heart

Spleen

Lungs

Thymus

Epididymides

I

0

Mean

0.448

0.461

0.578

0.141

0.251

±SD

0.042

0.068

0.063

0.031

0.015

II

250

Mean

0.413

0.448

0.546

0.125

0.225

±SD

0.014

0.115

0.080

0.022

0.018

III

500

Mean

0.428

0.491

0.631

0.126

0.228

±SD

0.041

0.095

0.162

0.025

0.014

IV

1000

Mean

0.385

0.505

0.521

0.116

0.228

±SD

0.048

0.173

0.110

0.026

0.022

*= Significant at 95% level of confidence

 

GROUP MEAN RELATIVE ORGAN WEIGHTS (%)

Sex : Female

Day : 29

 

GroupNumber

Dose mg/kg

 

Body Weight (g)

 

Brain

 

Liver

 

 

Kidneys

 

 

Adrenals

 

 

Ovaries

 

I

0

Mean

217.20

0.880

3.695

0.758

0.0257

0.0378

±SD

11.85

0.047

0.262

0.038

0.0031

0.0047

II

250

Mean

207.97

0.892

3.672

0.709

0.0270

0.0324

±SD

16.31

0.060

0.323

0.055

0.0049

0.0055

III

500

Mean

206.17

0.879

3.766

0.736

0.0272

0.0368

±SD

11.88

0.032

0.372

0.095

0.0029

0.0094

IV

1000

Mean

201.28

0.910

3.588

0.734

0.0253

0.0349

±SD

8.75

0.053

0.396

0.066

0.0032

0.0030

 

Group No.

Dose mg/kg

 

Heart

Spleen

Lungs

Thymus

Uterus

I

0

Mean

0.439

0.519

0.641

0.201

0.160

±SD

0.022

0.118

0.159

0.047

0.041

II

250

Mean

0.423

0.673

0.709

0.183

0.149

±SD

0.024

0.304

0.131

0.057

0.041

III

500

Mean

0.410

0.496

0.792

0.177

0.142

±SD

0.041

0.081

0.089

0.045

0.038

IV

1000

Mean

0.405

0.413

0.654

0.171

0.146

±SD

0.015

0.060

0.084

0.021

0.076

 

 SUM

MARY OF GROSS PATHOLOGY FINDINGS

Sex : Male

 

Site and lesion observed

Group

I

II

III

IV

Dose (mg/kg)

0

250

500

1000

 

No Abnormality Detected

 

1 - 6

13 - 18

25 - 30

37 - 42

 

SUMMARY OF GROSS PATHOLOGY FINDINGS

Sex : Female

 

Site and lesion observed

Group

I

II

III

IV

Dose (mg/kg)

0

250

500

1000

 

No Abnormality Detected

 

7 - 12

19 - 24

31 - 36

43 - 48

 

 

                                         

 

 

 

 

 

Applicant's summary and conclusion

Conclusions:
The No Observed Adverse Effect Level (NOAEL) for the test chemical in Sprague Dawley Rats, via oral gavage, for 28 days was considered to be 1000 mg/kg bw/day.
Executive summary:

The Repeated Dose 28-day Oral Toxicity study was designed and conducted to determine the toxicity profile of the test chemical when administered daily for 28 days in the Sprague Dawley rats. The test chemical was dissolved in Polyethylene glycol and used at dose level of 0, 250 mg/kg, 500 mg/kg and 1000 mg/kg body weight. During the study period, the treated animals were observed or mortality, cllinical signs, body weight and food consumption changes, hematology and clinical chemistry, neurobehaviour, urinalysis, ophthalmology and were subjected to gross and histopathology. All the male and female animals from control and all the treated dose groups up to 1000 mg/kg survived throughout the dosing period of 28 days. Male and female animals from control and all the treated dose groups exhibited normal body weight gain and food intake at the end of the dosing period of 28 days. Daily and detailed (weekly) clinical observations did not reveal any signs of toxicity in male and female animals from different dose groups during the dosing period of 28 days. Towards the end of the exposure period in week 4, functional observation battery such as sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli) revealed no abnormalities attributable to the treatment. Grip strength values observed in male and female animals for control and different dose groups were comparable. Higher values for motor activity were observed in male animals from 250 mg/kg, 500 mg/kg and 1000 mg/kg dose groups for second interval. Lower values for motor activity were observed in female animals from 1000 mg/kg dose group for first and second interval. These changes were within laboratory range and were considered to be of no toxicological importance. Haematological analysis performed on 29thday revealed statistically significant increase in the values of MCV, MCH and Total WBC of male rats dosed at 250 mg/kg, increase values of Platelets of male rats dosed at 250 mg/kg and 1000 mg/kg and increased values of Platelets of female rats dosed at 500 mg/kg. In addition, statistically significant decrease was observed in the values of Hb and HCT of male rats dosed at 250 mg/kg, 500 mg/kg and 1000 mg/kg, decreased values of Total RBC of male rats dosed at 250 mg/kg and 500 mg/kg, decreased values of MCV and MCH of male rats dosed at 1000 mg/kg, decreased values of Hb of female rats dosed at 250 mg/kg and 500 mg/kg, decreased values of Total RBC, HCT and Total WBC of female rats dosed at 500 mg/kg, decreased values of MCV of female rats dosed at 1000 mg/kg, decreased values of MCH and MCHC of female rats dosed at 250 mg/kg and 1000 mg/kg, the increase / decrease in the values of different parameters were marginal and within the normal laboratory limits. Statistically significant increase of Total Protein (in male rats at 500 mg/Kg/day), Creatinine (in male rats at 1000 mg/Kg/day), Cholesterol (in male rats at 500 and 1000 mg/Kg/day), Sodium (in female rats at 250, 500 and 1000 mg/Kg/day) and statistically significant decrease in Bilirubin (in male rats at 250 and 500 mg/Kg/day), Chloride and Alkaline Phosphatase (in female rats at 500 mg/Kg/day) was observed. Although there was an increase/decrease in the values of various biochemical parameters, the deviations were marginal and within the range of normal laboratory limits. Organ weight data of male animals sacrificed on day 29, revealed decreased relative weights of testes of animals from 500 mg/kg dose group. Although significant changes in organ weights were observed in male animals from intermediate dose group, no related gross pathological or histological changes were seen and hence considered to be of no toxicological importance. Organ weight data of female animals sacrificed on day 29, was found to be comparable with that of controls. Gross pathological examination conducted in rats of all dose groups during necropsy did not reveal any abnormality attributable to the treatment. Histopathological examination conducted in rats of control and high dose groups did not reveal any abnormality attributable to the treatment. Hence based on the observations made, the No Observed Adverse Effect Level (NOAEL) for the test chemical in Sprague Dawley Rats, via oral gavage, for 28 days was considered to be 1000 mg/kg bw/day.