Zinc, O,O-mixed (iso-Bu), (iso-Pr), (pentyl) phosphorodithioate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 22nd, 2022 to March 18th, 2023

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

other: DRF study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

DRF for the OECD 408

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From September 22nd, 2022 to March 18th, 2023

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

other: main study OECD 408

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rattus norvegicus

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
- Females (if applicable) nulliparous and non-pregnant: yes
- Housing: 2 and 3 rats/sex/cage throughout the experimental period in sterilised polypropylene rat cages. Each cage was fitted with a stainless-steel top grille having provision for keeping rodent pellet feed and a polypropylene water bottle with stainless steel drinking nozzle. Cages were placed on 5 tier racks. The bottom of each cage was layered with clean sterilised rice (paddy) husk as the bedding material. Rats were provided wooden chew blocks as an environmental enrichment material in each cage. Cages, bedding, and enrichment materials were changed alternate day throughout the experiment period. Water bottles were refilled daily and changed twice a week during experimental period. Cage racks were cleaned daily with cloth soaked with disinfectant solution throughout the study period. Cage lids were changed once during study period.
- Diet (e.g. ad libitum): standard rodent pellet feed (Teklad Certified Global 14% Protein Rodent Diet) ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days prior to the randomisation

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 23 °C
- Humidity (%): 65 to 66
- Air changes (per hr): 21
- Photoperiod (hrs dark / hrs light): 12 h light and 12 h dark; light hours were 06.00 - 18.00 h which was maintained by fluorescent tube lights attach to an autotimer. Light intensity 217 Lux

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Required amount of the test item was received for each dose level separately. The test item was mixed with the corn oil to prepare dose formulations of required concentrations. The prepared dose formulations were thoroughly mixed using a magnetic stirrer before dosing and with cannula intermittently during the dosing. The dose formulations were prepared on each day of the dosing and was administered to rats immediately, after preparation.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil was selected as a vehicle based on solubility test performed
- Concentration in vehicle: see details in section 8

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

see details in section 8

Duration of treatment / exposure:

14 days

Frequency of treatment:

daily exposure

Dose / conc.:

400 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

0 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: During 7-day DRF study, there were mortalities observed at dose level 500 and 1000 mg/kg b. wt./day and 250 mg/kg b. wt./day was well tolerated on repeated administration. Therefore, three dose levels for the test item (low dose - 100, mid-dose - 200, and high dose - 400 mg/kg b. wt./day) were selected based on the result of maximum tolerated dose with 7-day dose range finding study

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day

BODY WEIGHT: Yes
- Time schedule for examinations: recorded for individual rats on days 1 (before commencement of treatment), 4, 8, 11 and 14. Rats was weighed on the day of necropsy i.e., on day 15 (fasted body weight).

FOOD CONSUMPTION:
- Food consumption: Yes. A weighed amount of feed was offered to rats in each cage on day 1, 4, 8, and 11. Leftover feed was weighed for rats in each cage on day 4, 8, 11 and 14.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of treatment (on day 15)
- Anaesthetic used for blood collection: Yes, isoflurane anesthesia
- Animals fasted: Yes, overnight (with ad libitum supply of drinking water)
- Parameters checked: see "any other information on materials"

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of treatment (on day 15)
- Animals fasted: Yes
- Parameters checked: see "any other information on materials"

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, At scheduled sacrifice (on day 15), rats were euthanised by carbon dioxide asphyxiation. All terminally sacrificed and found dead rats were subjected to a full gross necropsy under the supervision of a veterinary pathologist. Rats were examined carefully for external abnormalities. The cranial, thoracic, and abdominal cavities were cut, opened, and a thorough examination of organs was carried out to detect abnormalities.
Organs were weighed from all surviving rats. Adherent adipose tissue from organs were trimmed off and the wet weight of the organs were recorded for liver, kidneys, adrenals, testes, epididymides, prostate with seminal vesicles and coagulating glands, thymus, heart, brain, spleen, uterus with cervix, ovaries with oviduct and thyroid with parathyroid. The paired organ was weighed together, and combined weight were presenting together. All above mentioned organs were preserved in 10% neutral buffered formalin solution except testes were preserved in modified Davidson’s fluid. The organ weight ratios as percentage of the terminal body weight were determined. Histopathology of preserved organ was not performed considering preliminary dose range finding study in consultation with the Sponsor. Preserved tissues were discarded at the time of finalisation of the report.

Description (incidence and severity):

All rats were normal, throughout the dosing period in vehicle control and treatment groups except mild salivation was observed intermittently during the treatment period in male and female rats of low, mid, and high dose groups. It is well known that salivation is often observed in gavage studies and may be a reaction to the taste or irritation of the test item rather than an indication of toxicity (Wook-Joon Yu at al, 2011). Therefore, it was considered that the transient salivation observed in this study was of no toxicological significance

Description (incidence):

No mortality or morbidity was observed during the treatment period in vehicle control and treatment groups except one female rat (rat number 39) was found dead on day 12 in high dose group. Mortality observed in high dose was associated with increase in thickness of stomach and considered as effect of test item treatment

Description (incidence and severity):

Mean body weight of male and female rats from treatment groups was comparable with that of vehicle control group. Following few statistical variations in body weight change were observed.
A statistically significant decrease in mean body weight change was observed on day 11 and 14 in male rats of high dose group when compared with that of the vehicle control group.
A statistically significant increase in mean body weight change was observed on day 11 in female rats of low, mid, and high dose groups when compared with that of the vehicle control group.
The reduction in body weight gain was minimal, inconsistent, and dose independent. Hence, it was not considered as effect of test item treatment

Description (incidence and severity):

Mean food consumption of male and female rats from low and mid dose groups was comparable with that of vehicle control group.
A statistically significant decrease in mean food consumption was observed during days 1-4 (36% of control) and days 4-8 (24% of control) in male rats of high dose group and during days 11-14 (21% of control) in female rats of high dose group. Reduction of food consumption in high dose group male rats was observed only in initial days of dosing and recovered later. Hence, it was not considered toxicologically significant.
A statistically significant increase in food consumption was noted during days 1-4 (10.5% of control) in female rats of mid dose group. Increase in food consumption of mid dose female rats was observed at one interval and considered as biological variation

Description (incidence and severity):

Statistically significant increase in absolute lymphocyte was observed in male rats of mid dose group and high dose group male and female rats when compared with that of vehicle control group. These changes lead to an increase in WBC value with or without statistical significance. These changes were considered as an effect of test item treatment

Description (incidence and severity):

Test item treatment was associated with the following statistical changes
• Increase in mean creatinine kinase of mid dose (male rats) and high dose groups (male and female rats).
• Decrease in mean albumin and total protein of high dose male rats.
• Decrease in mean urea and BUN of high dose female rats.
All the above changes in mainly observed in mid and high dose groups and interrelated. Hence, it could be considered as an effect of test item treatment.
Other effects were not considered as related to test item treatment (increase in potassium of mid and high dose male rats and decrease in chloride of mid dose male rats) as values of all rats were within historical ranges

Description (incidence and severity):

Test item treatment was associated with the following statistical changes
• Decrease in mean terminal body weight of high dose male rats
• Decrease in mean absolute and relative weight of the prostate and seminal vesicles with coagulating glands of mid dose and high dose male rats
• Decrease in mean absolute and relative weights of the thymus of high dose female rats
All the above-mentioned changes were dose dependent and considered as effect of test item treatment.
Other alterations noted in organ weight (increase in relative weight of adrenals of mid dose males and decrease in the relative weight of ovaries with oviducts of low dose and mid dose females) were not considered as related to the test item treatment in absence of dose dependency

Description (incidence and severity):

Gross examination revealed increased thickness in stomach [G3 (M: 2/5; F: 1/5), G4 (M: 5/5; F: 4/5)]. Findings observed in stomach could be due to lower pH of test item and also similar findings observed in structurally related chemicals. Therefore, it was considered as an effect of test item treatment. Unilateral absence of testis and epididymis observed in one rat of low dose group was considered incidental/spontaneous and not related to the test item treatment

Dose descriptor:

LOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

Critical effects observed:

yes

Lowest effective dose / conc.:

200 mg/kg bw/day (nominal)

System:

male reproductive system

Organ:

seminal vesicle

other: prostate

Treatment related:

yes

Critical effects observed:

yes

Lowest effective dose / conc.:

200 mg/kg bw/day (nominal)

System:

immune system

Organ:

thymus

Treatment related:

yes

Conclusions:

The dose selected for the 90 day study are 75, 150 and 300 mg/kg bw

Executive summary:

This study was conducted to evaluate the potential toxicity of the substance in Wistar rats following daily administration through oral gavage for 90 consecutive days as per the OECD Guideline 408.

The prepared dose formulations of the test item in the vehicle were administered orally, by gavage, at three graduated dose levels (G2 - low dose, G3 - mid-dose, and G4 - high dose) to male and female Wistar rats for 90 consecutive days. Rats in the concurrent vehicle control group (G1) received vehicle only.

To assess reversibility, persistence, or delayed occurrence of toxic effects, an additional group (G6 – high dose recovery) were treated at the high dose level for 90 days and then observed further for a period of 28 days without any treatment. For comparison purposes, a vehicle control recovery group (G5) was also included and was given vehicle alone over the equivalent period (90 days) and observed further for 28 days.

During the experimental period, all rats were observed for signs of toxicity, morbidity, and mortality. Body weight was recorded at weekly intervals. Body weight change and food consumption were calculated at weekly intervals. An ophthalmological examination was performed before treatment and sacrifices. Neurobehavioral observations were performed prior to treatment and at weekly intervals thereafter. Functional observational battery was performed during 12^th week of the treatment period and last week of recovery period. Vaginal smear was examined on the day of necropsy in all surviving female rats. At the end of the treatment and recovery periods, clinical pathology evaluations were performed on all surviving rats. All surviving rats were sacrificed and subjected to gross pathological examination. The weight of selected organs was recorded for each rat. Microscopic examination of tissues and organs was performed in vehicle control (G1) and high dose (G4) groups.

 

Dose Formulation Analysis: The a.i. concentration and homogeneity results of dose formulation samples collected on days 1, 29, 57, and 85 were within the acceptable range of ±15% of nominal concentration and %CV < 10, respectively.

Mortality and Morbidity: No mortality or morbidity was observed throughout the treatment and recovery periods in male and female rats of control and low dose groups.

Mortality was observed at different interval in rats treated with mid i.e., 150 mg/kg b. wt./day [Male – 2/10; female – 1/10] and high dose i.e., 300 mg/kg b. wt./day [Male – 7/15; female – 2/15] groups. Mortality observed in treatment groups was associated with clinical sign like, gasping, weakness and salivation. Incidence of mortalities was stopped on reduction on doses i.e., 25, 50 and 100 mg/kg b. wt./day from day 37 of male rats and day 36 of female rats. Hence, it was considered as an effect of test item treatment.

Clinical Observations: All rats were normal throughout the dosing period in control and low dose groups. Mild to moderate salivation was observed intermittently during treatment days 6 to 90 in male and female rats of mid and high dose treated groups. It is well known that salivation is often observed in gavage studies and may be a reaction to the taste or irritation of the test item rather than an indication of toxicity (Wook-Joon Yu et al, 2011). Therefore, it was considered that the transient salivation observed in this study was of doubtful or no toxicological significance.

Apart from salivation, there was weakness (on days 35 to 37) and gasping (days 62 to 68) also observed in male and female rats of mid and high dose groups. These signs were associated with mortalities. Hence, they were considered as effect of test item treatment and adverse in nature.

Ophthalmological Examination: Ophthalmological examination of all rats did not reveal any abnormalities.

Neurobehavioural Observations: No treatment-related changes were observed in neurobehavioural observations performed in male and female rats from the treatment groups when compared to those respective vehicle control groups.

Functional Observational Battery: No treatment-related changes were observed in functional observational battery parameters performed in male and female rats from treatment groups compared to those respective vehicle control groups.

Body Weight and Body Weight Change: No treatment related changes were observed in the mean body weight and mean body weight change of male and female rats of low and mid dose groups compared to that of vehicle control groups. In high dose groups, significant reduction in body weight and body weight change were observed at different interval in male rats. Body weight gain reduction in male rats of high dose groups was consistent and more than 10% of control groups. It was supported with reduction in food consumption also. Hence, reduction in body weight was considered as an effect of test item and adverse in nature.

Food Consumption: No treatment related changes were observed in the mean food consumption of female rats of treatment groups compared to that of vehicle control group. A significant decrease in food consumption was observed at different interval in male and female rats of low, mid and high dose groups when compared with those respective vehicle control groups.

 

Reduction in food consumption of male rats treated with low and mid dose groups was inconsistent and marginal. Hence, it was considered as an effect of test item but not adverse in nature. Reduction of food consumption at high dose was observed in both sexes and was consistent. In addition, this reduction was observed in more that 10% of control groups and associated with reduction in body weight. Therefore, these changes were considered as effect of test item and adverse in nature.

Hematology and Coagulation: No treatment-related change was observed in hematology and coagulation parameters of rats from treatment groups compared to the respective vehicle control groups.

Clinical Chemistry: No treatment-related change was observed in clinical chemistry parameters of rats from treatment groups compared to the respective vehicle control groups.

Hormone Analysis: No treatment-related change was observed in serum level of T3, T4 and TSH of rats from treatment groups compared to those respective vehicle control groups.

Organ Weight: No treatment-related change was observed in absolute and relative weight of organs in male and female rats of treatment groups compared to those respective vehicle control groups.

Macroscopic Examination: Macroscopic examination did not reveal any treatment related lesion in treatment groups.

Microscopic Examination: Microscopic examination did not reveal any treatment related lesion in treatment groups.

Conclusion:

Initial doses i.e., 150 and 300 mg/kg b. wt./day had produced mortality in male and female rats during dosing days 15 to 36. Additionally, mortality was also noticed after the reduction of doses i.e., 100 mg/kg b. wt./day level (during days 38 to 60). These mortalities were associated with toxic signs like weakness and gasping. Also, the high dose i.e., 300/100 mg/kg b. wt./day was associated with significant reduction in body weight as well as food consumption. All these changes were considered as adverse effects of test item treatment.

Terminal parameters like clinical pathology, organ weight and microscopic examination did not reveal any treatment related adverse changes at any of the dose level.

The No Observed Adverse Effect Level (NOAEL) of CAS # 101747-77-7 was hence concluded as 50 mg/kg b. wt./day under the conditions and procedures followed in this study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Report date:

2023

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Zinc, O,O-mixed (iso-Bu), (iso-Pr), (pentyl) phosphorodithioate

EC Number:

820-225-5

Cas Number:

101747-77-7

Molecular formula:

C12-20H28-44O4P2S4Zn

IUPAC Name:

Zinc, O,O-mixed (iso-Bu), (iso-Pr), (pentyl) phosphorodithioate

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rat was selected as a test system because it has been historically shown to be a suitable model for repeated dose toxicity studies and is recommended in OECD Test Guidelines. The ECHA, EFSA, EPA and other regulatory authorities also recommend it. Wistar strain has been chosen for this study because of its widespread use as a rodent model in toxicology studies and well-established historical information available about its genetic and physiologic background. The laboratory has extensive in-house historical control data available for this species. The results of the study may be of value in predicting the toxicity of the test item to human beings.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animal Breeding Facility (ABF), testing facility
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6-7 weeks old
- Weight at study initiation:
- Fasting period before study:
- Housing: Rats were housed in groups of 2 to 3 rats/cage/sex during the study period in sterilised polypropylene rat cages. Rats with more than 400 g (3 rats/cage) and 500 g (2 rats/cage) body weight during the study period, were removed from original cage and housed in a new cage. Each cage was fitted with a stainless-steel top grille having provision for keeping rodent pellet feed and a polypropylene water bottle with stainless steel drinking nozzle. Cages were placed on 5 tier racks and cage rotation was performed at weekly intervals in each cage rack which ensures almost similar environmental conditions for rats from different groups. The bottom of the cage was layered with clean sterilised paddy (rice) husk as the bedding material. Rats were provided wooden chew blocks as an environment enrichment material in each cage. Cages, bedding, and enrichment materials were changed on alternate days throughout the study period. Water bottles were refilled daily and changed twice a week during the experimental period. Cage racks were cleaned daily with cloth soaked with disinfectant solution, throughout the study period. Cage lids were changed once every two weeks throughout the study period. Contaminant analysis (chemical and microbial) of the bedding and enrichment material samples are routinely performed.
- Diet: sterile standard rodent pellet feed ad libitum (Teklad Certified Global 14% Protein Rodent Diet, Envigo, USA)
- Water: unlimited supply of clean and filtered drinking water (Reverse Osmosis water filtration system)
- Acclimation period: A total of 56 male and 56 female rats were received in the experimental room and acclimatised for a period of 5 days prior to randomisation. During acclimatisation, rats were observed twice daily for clinical signs, mortality, and morbidity. After randomisation, 50 male and 50 female rats were housed for 2 (male) to 3 (female) days pre-treatment period, under experimental room conditions before commencement of dosing. During the pre-treatment period, baseline NBO and ophthalmological examination were performed on rats from all groups. Rats from all groups were also observed twice daily for mortality, morbidity, and clinical signs.

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 64-67
- Air changes (per hr): 21
- Photoperiod: 12 hrs dark / 12 hrs light

Experimental Room Sanitation: Every day, the floor of the experiment room was cleaned and mopped with surface disinfectant solution (2.5 % v/v Dettol and 1 % v/v Virkon S). All worktops were also cleaned with cloth wetted with the same disinfectant solution.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The route of vehicle and test item administration was oral though gavage and selected as it is the most common route of exposure.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was mixed with the vehicle to prepare dose formulations of required concentrations. The prepared dose formulations were thoroughly mixed using magnetic stirrer before dosing and with cannula intermittently during dosing. The dose formulations were prepared on each day of the dosing and was administered to rats immediately, after preparation.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil was selected as vehicle based on solubility test performed at the laboratory. Test item was well miscible with corn oil and formed solution

The dose formulations were administered once daily by oral gavage, using an intubation cannula attached to a graduated syringe. A constant dose volume of 4 mL/kg b. wt. was used, and individual doses were adjusted according to the most recently recorded body weight of each rat. Rats from the vehicle control group received the vehicle only

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method for stability, active ingredient (a.i.) concentration, and homogeneity of dose formulation was validated at the Department of Chemistry. Based on the stability results, the stability of the test item in the formulation is 24 h.
Two sets of samples (10 samples per set) were collected by sampling three aliquots (upper, middle, and lower layers) from each concentration except vehicle control (only one middle aliquot). The sampling was performed prior to initiation of dosing and at monthly intervals thereafter, for dose formulation analysis. One set of samples was used for analyses and the other set of samples was stored in the frozen condition. The second set of samples will be disposed of during report finalization. The mean concentration was determined and compared to the nominal value and the coefficient of variation calculated. The acceptance criteria used for analysis was ±15% from nominal value and %CV < 10.

Duration of treatment / exposure:

90 days for test groups
Rats allocated to the recovery groups were maintained untreated for a period of 28 days after the treatment for 90 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

from day 37 of male and day 36 in female rats

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

from day 37 of male and day 36 in female rats

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

from day 37 of male and day 36 in female rats

Doses / concentrations 4

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

up to day 37 of male and day 36 in female rats

Doses / concentrations 5

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

up to day 37 of male and day 36 in female rats

Doses / concentrations 6

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

up to day 37 of male and day 36 in female rats

No. of animals per sex per dose:

10/sex/dose for test groups
6/sex/dose for recovery groups

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Three dose levels (low dose – 75, mid-dose – 150 and high dose - 300 mg/kg b. wt.) were selected based on results of repeated dose 14-day DRF study performed. There were mortalities noticed from days 32 to 36 in mid and high dose group. Hence, doses were reduced as low dose – 25, mid-dose – 50 and high dose - 100 mg/kg b. wt. from day 37 of male and day 36 in female rats. Based on initial and revised doses, prorated doses in male/female for the study period are: 45/44, 90/89 and 180/178 mg/kg b. wt./day respectively at low dose, mid dose and high dose
- Fasting period before blood sampling for clinical biochemistry: yes, overnight fasting
- Post-exposure recovery period in satellite groups: 28 days
- Dose range finding studies: 14-days study (included in current IUCLID-see cross reference)

Positive control:

no

Examinations

Observations and examinations performed and frequency:

1. Mortality and Morbidity: All rats were observed twice daily for mortality and signs of morbidity.
2. linical Observations: All rats were observed twice daily for clinical signs.
3. Ophthalmological Examination: An ophthalmological examination was performed on all rats prior to the commencement of dosing and prior to sacrifices (terminal and recovery) in surviving rats. Eye drops of mydriatic agent (Homatropine hydrobromide) were used to dilate pupils approximately 20 minutes before the eye examination. Both eyes of each rat were examined using a direct ophthalmoscope.
4. Neurobehavioural Observations: To assess the behavioural and neurological status of each rat, the following NBO parameters were evaluated prior to initiation of treatment and at weekly intervals, thereafter.
4.1 Home Cage Observations: In the home cage, rats were observed for posture, clonic convulsion, and tonic convulsion.
4.2 Handling Observations: After completion of home cage observations, the rat was picked up by the observer and observed for the below-mentioned parameters: Ease of removing rat from cage, Handling reactivity, Palpebral closure, Lacrimation, Eye examination, Piloerection, Skin examination, Salivation
4.3 Open Field Observations: For open-field observations, rats were placed (one at a time) in an open arena (size: 495 × 495 × 280 mm) on a flat surface covered with clean absorbent paper and observed for a period of 2 minutes. Each rat was observed for below mentioned parameters: Gait, Mobility, Arousal, Vocalizations, Rears, Respiration, Clonic movement, Tonic movement, Urination, Defecation, Stereotypy, Bizarre behavior
5.Functional Observational Battery: In addition to NBO, the below mentioned FOB parameters were performed during the 12th week of treatment (G1 to G4) and last week of the recovery period (G5 and G6) for surviving rats.
5.1 Motor Activity: Motor activity was evaluated for rats using an automated photobeam activity system equipped with validated PAS software. Rats were monitored for three consecutive 10 minutes intervals (total 30 minutes for each rat) allowing for examination of both exploratory and acclimation activity levels. The motor activity parameters including total activity, ambulatory activity, and fine activity.
5.2 Sensory Reactivity Measurements: For sensory reactivity measurements, rats were placed in an open arena (size: 495 × 495 × 203 mm) with a flat surface covered with clean absorbent paper. The below mentioned parameters were performed and recorded for each rat: Approach Response, touch response, click response, pupil response, tail-pinch response, air righting reflex.
5.3 Grip strength: Grip strength of both forelimb and hindlimb was measured with a grip strength meter to determine the ability of the rat to grasp and hold on the mesh platform. The grip strength of each rat was measured 3 consecutive times; results were averaged separately for the forelimb and hindlimb.
5.4 Foot Splay: The landing hind limb feet of each rat were marked with non-permanent, non-toxic ink just prior to testing. The rat was suspended in a prone position and then dropped onto a recording sheet from a height of approximately 30 cm. This procedure was repeated thrice. The distance between the two footprints was measured and an average of three-foot splay values was calculated.
5.5 Body Weight: The body weight of all rats was recorded at the beginning of treatment and at weekly intervals, thereafter. Rats from all groups were also weighed on the day of necropsy (fasted body weight) and at death. The percentage body weight change compared to pre-treatment body weight was calculated.
5.6 Food Consumption: A weighed amount of feed was offered weekly. Leftover feed was measured in each cage once at weekly intervals during the study period.
5.7 Estrous Cycle Evaluation: On the day of necropsy, a vaginal smear was examined from all surviving female rats to determine the stage of the estrous cycle.
5.8 Clinical Pathology: At the end of treatment and recovery periods, blood samples were collected from all surviving rats under light isoflurane anesthesia by orbital plexus puncture using a fine heparinized capillary tube. Rats were fasted overnight (with ad libitum supply of drinking water) prior to blood collection (approximately 3 mL blood). Blood samples were collected for hematology (in vials containing 4% EDTA anticoagulant for whole blood), coagulation parameters (in vials containing 3.2% sodium citrate anticoagulant for plasma separation), and clinical chemistry analysis (in plain vials for serum separation). Serum thyroid hormones T3 and T4 were analyzed using a validated bioanalytical method by LC-MS/MS and serum TSH was analyzed by ELISA method. Urine was collected individually from all surviving rats at the end of the treatment and recovery periods in urine collection tubes. For urine collection, rats were housed overnight in metabolic cages during which feed was withheld but water was provided ad libitum. Plasma samples were stored at -70 ± 10 °C until the analysis of coagulation parameters while, analysis of hematology, clinical chemistry, and urine parameters was performed on the same day of sample collection. Retained samples of serum and plasma will be discarded during report finalization.
5.8.1. Hematology and Coagulation Parameters: Haematocrit, Haemoglobin, Mean Corpuscular Haemoglobin, Mean Corpuscular Haemoglobin Concentration, Mean Corpuscular Volume, Platelets Count, Erythrocyte Count (Red Blood Cells), Total Leukocyte Count (White Blood Cells), Differential Leukocyte Count, Reticulocyte Count, Prothrombin Time, Activated Partial Thromboplastin Time
5.8.2 Clinical Chemistry Parameters: Alanine Aminotransferase, Albumin, Alkaline Phosphatase, Aspartate Aminotransferase, Bile Acids, Calcium, Creatinine, Creatine Kinase, Gamma Glutamyl Transpeptidase, Glucose, Lactate Dehydrogenase, Inorganic Phosphorous, Total Cholesterol, Total Protein, Total Bilirubin, Triglycerides, Urea, High-density lipoproteins, Low-density lipoproteins, Albumin: Globulin ratio, Globulin, Blood Urea Nitrogen, Chloride, Potassium, Sodium
5.8.3. Urinalysis Parameters: Appearance, Color, Volume, Sediment evaluation (Microscopic examination), Specific gravity, pH, Protein, Glucose, Ketone, Blood, Bilirubin, Urobilinogen

Sacrifice and pathology:

Gross Pathology
At scheduled terminal and recovery sacrifices, all surviving rats were euthanized by carbon dioxide asphyxiation. Found dead rats were subjected to post-mortem examination. Rats were subjected to a full gross necropsy under the supervision of a veterinary pathologist. All rats were examined carefully for external abnormalities. The cranial, thoracic, and abdominal cavities were cut, opened and a thorough examination of the organs was carried out to detect abnormalities. Descriptions of all macroscopic abnormalities seen at necropsy were recorded.

Organ/Tissue Collection
The organs and tissues, as applicable, from male and female rats of main and recovery groups, were collected, weighed, and preserved. Adherent adipose tissue from the organs was trimmed off and wet weights of organs were recorded. All organs were preserved in 10% neutral buffered formalin solution except the testes and eyes which were collected in modified Davidson’s fluid and Davidson’s fluid, respectively. Organs were weighed from all surviving rats. The paired organs were weighed together, and combined weights were presented. The weights of the thyroid with parathyroid and pituitary were determined after fixation. The organ weight ratios as a percentage of the terminal body weight were calculated. Bone marrow smears were collected from femur bone of rats from all groups and stained by May Grunwald-Giemsa stain. The slides were examined from G1 and G4 for different cell counts and myeloid:erythroid ratio were calculated.
The following organs were weighed: Adrenal, Brain, Epididymides, Heart, Kidneys, Liver, Lungs, Ovaries, Pituitary, Prostate + seminal vesicle with coagulating gland, Spleen, Testes, Thymus, Thyroid with parathyroid, Uterus with cervix,

Microscopic Examination
Histopathological examination was carried out for the preserved organs and tissues of rats from vehicle control (G1) and high dose (G4) groups. All organs and tissue samples, were processed, embedded, and cut at a thickness of 3 to 5 micrometers and stained with haematoxylin and eosin.
The following tissues were examined: Adrenal, Brain, Epididymides, Heart, Kidneys, Liver, Lungs, Ovaries, Pituitary, Prostate + seminal vesicle with coagulating gland, Spleen, Testes, Thymus, Thyroid with parathyroid, Uterus with cervix,, aorta, Bone with marrow (femur with joint), Eyes, Larynx, Lymph nodes – prescapular, Lymph nodes – mesenteric, Large intestine (cecum, colon, and rectum), Mammary glands
Nose, Oesophagus, Optic nerve, Pharynx, Pancreas, Salivary glands, Sciatic nerves, Skeletal muscle, Skin, Small intestine (duodenum, jejunum, and ileum with Peyer’s patches), Spinal cord, Stomach, Trachea, Urinary bladder, Vagina

Statistics:

Data were recorded in standard formats and validated Pristima® software and summarised in tabular form. Data were processed to get group means and standard deviations. All parametric and non-parametric data was analyzed statistically,
Type of Data:
Parametric Body weight, body weight change, food consumption, FOB parameters (grip strength and foot splay), absolute organ weight, relative organ weight, hematology and coagulation, clinical chemistry, thyroid hormones, and some of the urinalysis parameters
Non-parametric NBO and FOB parameters (urination, defecation, rearing count, vocalization, and motor activity)
Significance Level:
Parametric At 1 and 5% level between vehicle control and treated groups
Non-parametric At 5% level between vehicle control and treated groups

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All rats were normal throughout the dosing period in control and low dose groups.
Mild to moderate salivation was observed intermittently during dosing days 6 to 90 in male and female rats of mid and high dose treated groups. It is well known that salivation is often observed in gavage studies and may be a reaction to the taste or irritation of the test item rather than an indication of toxicity (Wook-Joon Yu et al, 2011). Therefore, it was considered that the transient salivation observed in this study was of doubtful or no toxicological significance.
Apart from salivation, there was weakness (on day 35 to 37) and gasping also observed in male and female rats of mid and high dose groups during treatment days 62 to 68. These signs were associated with mortalities. Hence, they were considered as effect of test item treatment and adverse in nature.

Mortality:

mortality observed, treatment-related

Description (incidence):

No mortality or morbidity was observed throughout the treatment and recovery periods in male and female rats of control and low dose groups.
Mortality was observed at different interval in rats treated with mid i.e., 150 mg/kg b. wt./day [Male – on days 27 (1/10) and 36 (1/9); female – on day 35 (1/10)] and high dose i.e., 300 mg/kg b. wt./day [Male – on days 29 (1/15), 36 (2/14), 38 (1/12), 42 (1/11), 43 (1/10), and 60 (1/9); female – on day 15 (1/15) and 32 (1/14)] groups. Mortality observed in treatment groups was associated with clinical sign like, gasping, weakness and salivation. Incidence of mortalities was stopped on reduction on doses i.e., 25, 50 and 100 mg/kg b. wt./day from day 37 of male rats and day 36 of female rats. Hence, it was considered as effect of test item treatment

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight and mean body weight change of male and female rats of treatment groups were comparable with those of respective vehicle control groups except following statistical variations.
• Significant decrease in mean body weight on dosing days 50 to 85 and recovery days 1 to 28 in male rats of high dose recovery group.
• Significant decrease in mean body weight change on dosing days 71 to 85 and recovery days 1 to 28 in male rats of high dose recovery group.
Body weight gain reduction in male rats of high dose groups was consistent and more than 10% of control groups. It was supported with reduction in food consumption also. Hence, reduction in body weight was considered as an effect of test item and adverse in nature

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption of female rats of treatment groups were comparable with those of respective vehicle control groups. Following statistical variations were noted mainly in male rats and some of female rats
• Significant decrease in mean food consumption during days 1-8, 71-78, and 85-90 in male rats of low dose group.
• Significant decrease in mean food consumption during days 64-71, 71-78, 78-85, and 85-90 in male rats and during days 50-57 in female rats of mid dose group.
• Significant decrease in mean food consumption during days 1-8, 36-43, 50-57, 57-64, 64-71, 71-78, 78-85, and 85-90 in male rats of high dose group.
• Significant decrease in mean food consumption dosing days 15-22, 36-43, 43-50, 71-78, 78-85 and 85-91 and during recovery days 1-8, 8-15, 18-22 and 22-28 in male rats of high dose recovery group.
• Significant decrease in mean food consumption during dosing days 1-8, 8-15, 15-22, and 22-29 in female rats of high dose recovery group.
Reduction in food consumption of male rats treated with low and mid dose groups was inconsistent and marginal. Hence, it was considered as an effect of test item but non adverse in nature. Reduction of food consumption was observed in both sexes and consistent at high dose level. Also, it was more that 10% of control groups and associated with reduction in body weight gain. Therefore, these changes of high dose groups were considered as effect of test item and adverse in nature .

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The ophthalmological examination of rats from all groups did not reveal any abnormalities

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematological examination did not reveal any treatment related effect.
Statistically significant decrease was noted in MCV (mean corpuscular volume) and MCH (mean corpuscular haemoglobin) in male rats of high dose group. It was not interpreted as treatment related effect, due to lack of significant changes in the values of haemoglobin, haematocrit and RBC. Statistically significant decreases in APTT in males and reticulocytes in females and increase in the RBC in males of high dose recovery group. Alterations were not related to the test item treatment due to absence of similar alterations at the end of treatment and lack of consistency between sexes.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant decrease was noted in the urea and BUN in male rats of low, mid and high dose groups. Alterations were related to the test item treatment and not considered as adverse, in absence of histopathological findings in kidneys and complete recovery of the effect after recovery period.
A statistically significant increase was noted in the AST in male rats of low and high dose groups. It was not related to the test item treatment, as values of subjected groups were within historical control ranges and lack of dose dependency.
Statistically significant decrease was noted in the phosphorus in male rats of mid dose group, while statistically significant increase was noted in the triglyceride in female rats of low dose groups. Alterations were not considered as the treatment related, due to lack of dose dependency.
In high dose recovery group, statistically significant increase was noted in LDL in males and potassium in females, while decrease was observed in the phosphorus in females. These alterations noted in the recovery group could not be considered as treatment related due to absence of similar findings at terminal sacrifice and lack of consistency between sexes.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

Mean serum TSH levels of male and female rats of treatment groups were comparable with those of respective vehicle control groups.
Statistically, significant decrease in the serum T4 level was observed in male rats of mid and high dose recovery groups when compared with those of the respective vehicle control groups. Statistically, significant increase in the serum T3 level was observed in female rats of high dose recovery group when compared with that of the vehicle control recovery group. All these variations were inconsistent and dose independent. In absence of microscopic changes in thyroid and parathyroid, these changes were considered as not related to test item treatment

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant decrease in the urine specific gravity was noted in male rats of low, mid and high dose groups and pH in female rats mid and high dose groups. Alterations were related to the test item treatment and considered as non-adverse due to absence of histopathological findings in kidneys and complete recovery of the effect after recovery period.
Statistically significant increase was noted in the urine volume in mid dose (G3) females. It was not considered related to the test item treatment in absence of dose dependency.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Home Cage Observations
In the home cage, all rats from the treatment (G2, G3, G4 and G6) and vehicle control groups (G1 and G5) showed normal posture, asleep (curled up often asleep), sitting A (sitting but with head hung down), sitting B (sitting normally, feet tucked in), sitting C (sitting or standing alert, watching) and rearing. Clonic and tonic convulsions were absent in the home cage during NBO.

Handling Observations
NBO performed during the handling of rats did not reveal any abnormalities. All rats revealed normal behavior during removal (very easy - rats sit quietly) and handling (easy - alert, limbs put against the body). No rats showed salivation, lacrimation, and piloerection. Eyelids were wide open in all rats. The eye and skin examinations did not reveal any abnormalities in any rat

Open Field Observations
In the open field, all rats from treatment and vehicle control groups showed normal gait, mobility, arousal, and respiration during the ‘two-minute observation period’. Conic and tonic movements, vocalization, stereotypy, and bizarre behavior were absent in this assessment. The rearing, urination, and defecation count of male and female rats from treatment groups were comparable with those of the respective vehicle control groups except the following statistical variations.
• Significant increase in rearing count in male rats of low (on day 68 and 75) and mid dose (on day 75) groups.
• Significant decrease in rearing count on day 20 in female rats of high dose recovery group.
These findings were intermittent and not dose dependent. Hence, it was not considered as test item related effect

Functional Observational Battery
Motor Activity: Mean motor activity data of rats from the treatment groups were comparable with those of the respective vehicle control groups
Sensory Reactivity Measurements: Sensory reactivity parameters, viz., approach response, touch response, click response, tail pinch response, pupil response, and air righting reflex in rats of the treatment groups were comparable with those of the respective vehicle control groups
Grip Strength: Mean grip strength values (forelimb and hindlimb) of treated rats were comparable with those of the respective vehicle control groups
Foot Splay: Mean hindlimb foot splay values of treated rats were comparable with those of the respective vehicle control groups

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Terminal:
Statistically significant decrease was noted in the absolute and relative weights of prostate and seminal vesicles with coagulating glands in males rats of mid dose group. It was not related to the test item treatment in absence of dose dependency.
Statistically significant decrease was noted in absolute weight of adrenals in female rats of high dose group. This effect was not considered as adverse, as treatment related histopathological alterations were not observed in adrenals.
Recovery:
In males, statistically significant and marked decrease was noted in the terminal body weight, and statistically significant decreases were noted in absolute and relative weights of adrenals and thymus, and absolute weights of epididymides, kidneys, liver, spleen, and prostate and seminal vesicles with coagulating glands. Similarly, statistically significant increases were noted in relative weights of brain and testes in males. These alterations in organ weights were considered to be owed to decrease in the terminal body weight.
In females, statistically significant increase was noted in absolute weight of spleen. It was not related to the test item treatment in absence of similar effect at the end of treatment

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Gross examination of the animals of either sex across various groups did not reveal any treatment related abnormality. However, smaller size of testes (Animal N° 28, G2 Male), enlarged and hard lungs lobe (Animal N° 67, G4 Male), and cyst on ovary and unilateral absence of uterine horn (Animal N° 32, G2 Female) was observed. These findings were not related to the test item treatment and considered as incidental or congenital due to lower incidence

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Microscopic examination did not reveal any lesion related to the test item treatment.
Microscopic examination of organs/tissues from found dead rats of high dose group (Animal N° 61, 63, 65, 68, 70 and 72) did not reveal any abnormality of pathological significance, hence cause of death could not be determined.

Other effects:

no effects observed

Description (incidence and severity):

Bone marrow Examination
No treatment related alterations were noted in myeloid: erythroid ratio after bone marrow examination

Estrous Cycle Status of Termination
The test item did not show any adverse effects on the estrous cycle of female rats at any dose level. The pattern and number of females showing estrous cycle stages indicates the normal cyclicity of females in all study groups (G1 to G6). This was further supported by normal histopathological observations in the uterus, cervix, and vagina of G1 and G4 females.

Effect levels

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

mortality

Remarks on result:

other: 50 mg/kg b. wt./day (prorated dose: males – 90 and females – 89 mg/kg b. wt./day)

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Active Ingredient Concentration and Homogeneity Analysis

Results of a.i. concentration and homogeneity of formulated dose samples for low dose, mid dose, and high dose are summarised below:

 

Sample Collected on Day	Concentration (mg/mL)
	Low dose – 18.75		Mid dose – 37.50		High Dose – 75.00
	% Mean Recovery	% CV	% Mean Recovery	% CV	% Mean Recovery	% CV
1	98.77	0.45	99.66	0.21	99.73	0.62
29	96.76	1.43	102.49	0.62	104.00	0.13

 

Sample Collected on Day	Concentration (mg/mL)
	Low dose – 6.25		Mid dose – 12.50		High Dose – 25.00
	% Mean Recovery	% CV	% Mean Recovery	% CV	% Mean Recovery	% CV
57	108.32	0.09	108.52	0.46	90.49	0.25
85	108.20	0.15	114.49	0.47	100.77	0.40

The a.i. concentration and homogeneity of the test item in dose formulations were within the allowable limits of ±15% of the nominal concentration and %CV < 10

Applicant's summary and conclusion

Conclusions:

NOAEL = 50 mg/kg b. wt./day (prorated dose: males – 90 and females – 89 mg/kg b. wt./day) .

Executive summary:

This study was conducted to evaluate the potential toxicity of the substance in Wistar rats following daily administration through oral gavage for 90 consecutive days as per the OECD Guideline 408.

The prepared dose formulations of the test item in the vehicle were administered orally, by gavage, at three graduated dose levels (G2 - low dose, G3 - mid-dose, and G4 - high dose) to male and female Wistar rats for 90 consecutive days. Rats in the concurrent vehicle control group (G1) received vehicle only.

To assess reversibility, persistence, or delayed occurrence of toxic effects, an additional group (G6 – high dose recovery) were treated at the high dose level for 90 days and then observed further for a period of 28 days without any treatment. For comparison purposes, a vehicle control recovery group (G5) was also included and was given vehicle alone over the equivalent period (90 days) and observed further for 28 days.

During the experimental period, all rats were observed for signs of toxicity, morbidity, and mortality. Body weight was recorded at weekly intervals. Body weight change and food consumption were calculated at weekly intervals. An ophthalmological examination was performed before treatment and sacrifices. Neurobehavioral observations were performed prior to treatment and at weekly intervals thereafter. Functional observational battery was performed during 12^th week of the treatment period and last week of recovery period. Vaginal smear was examined on the day of necropsy in all surviving female rats. At the end of the treatment and recovery periods, clinical pathology evaluations were performed on all surviving rats. All surviving rats were sacrificed and subjected to gross pathological examination. The weight of selected organs was recorded for each rat. Microscopic examination of tissues and organs was performed in vehicle control (G1) and high dose (G4) groups.

 

Dose Formulation Analysis: The a.i. concentration and homogeneity results of dose formulation samples collected on days 1, 29, 57, and 85 were within the acceptable range of ±15% of nominal concentration and %CV < 10, respectively.

Mortality and Morbidity: No mortality or morbidity was observed throughout the treatment and recovery periods in male and female rats of control and low dose groups.

Mortality was observed at different interval in rats treated with mid i.e., 150 mg/kg b. wt./day [Male – 2/10; female – 1/10] and high dose i.e., 300 mg/kg b. wt./day [Male – 7/15; female – 2/15] groups. Mortality observed in treatment groups was associated with clinical sign like, gasping, weakness and salivation. Incidence of mortalities was stopped on reduction on doses i.e., 25, 50 and 100 mg/kg b. wt./day from day 37 of male rats and day 36 of female rats. Hence, it was considered as an effect of test item treatment.

Clinical Observations: All rats were normal throughout the dosing period in control and low dose groups. Mild to moderate salivation was observed intermittently during treatment days 6 to 90 in male and female rats of mid and high dose treated groups. It is well known that salivation is often observed in gavage studies and may be a reaction to the taste or irritation of the test item rather than an indication of toxicity (Wook-Joon Yu et al, 2011). Therefore, it was considered that the transient salivation observed in this study was of doubtful or no toxicological significance.

Apart from salivation, there was weakness (on days 35 to 37) and gasping (days 62 to 68) also observed in male and female rats of mid and high dose groups. These signs were associated with mortalities. Hence, they were considered as effect of test item treatment and adverse in nature.

Ophthalmological Examination: Ophthalmological examination of all rats did not reveal any abnormalities.

Neurobehavioural Observations: No treatment-related changes were observed in neurobehavioural observations performed in male and female rats from the treatment groups when compared to those respective vehicle control groups.

Functional Observational Battery: No treatment-related changes were observed in functional observational battery parameters performed in male and female rats from treatment groups compared to those respective vehicle control groups.

Body Weight and Body Weight Change: No treatment related changes were observed in the mean body weight and mean body weight change of male and female rats of low and mid dose groups compared to that of vehicle control groups. In high dose groups, significant reduction in body weight and body weight change were observed at different interval in male rats. Body weight gain reduction in male rats of high dose groups was consistent and more than 10% of control groups. It was supported with reduction in food consumption also. Hence, reduction in body weight was considered as an effect of test item and adverse in nature.

Food Consumption: No treatment related changes were observed in the mean food consumption of female rats of treatment groups compared to that of vehicle control group. A significant decrease in food consumption was observed at different interval in male and female rats of low, mid and high dose groups when compared with those respective vehicle control groups. Reduction in food consumption of male rats treated with low and mid dose groups was inconsistent and marginal. Hence, it was considered as an effect of test item but not adverse in nature. Reduction of food consumption at high dose was observed in both sexes and was consistent. In addition, this reduction was observed in more that 10% of control groups and associated with reduction in body weight. Therefore, these changes were considered as effect of test item and adverse in nature.

Hematology and Coagulation: No treatment-related change was observed in hematology and coagulation parameters of rats from treatment groups compared to the respective vehicle control groups.

Clinical Chemistry: No treatment-related change was observed in clinical chemistry parameters of rats from treatment groups compared to the respective vehicle control groups.

Hormone Analysis: No treatment-related change was observed in serum level of T3, T4 and TSH of rats from treatment groups compared to those respective vehicle control groups.

Organ Weight: No treatment-related change was observed in absolute and relative weight of organs in male and female rats of treatment groups compared to those respective vehicle control groups.

Macroscopic Examination: Macroscopic examination did not reveal any treatment related lesion in treatment groups.

Microscopic Examination: Microscopic examination did not reveal any treatment related lesion in treatment groups.

Conclusion:

Initial doses i.e., 150 and 300 mg/kg b. wt./day had produced mortality in male and female rats during dosing days 15 to 36. Additionally, mortality was also noticed after the reduction of doses i.e., 100 mg/kg b. wt./day level (during days 38 to 60). These mortalities were associated with toxic signs like weakness and gasping. Also, the high dose i.e., 300/100 mg/kg b. wt./day was associated with significant reduction in body weight as well as food consumption. All these changes were considered as adverse effects of test item treatment.

Terminal parameters like clinical pathology, organ weight and microscopic examination did not reveal any treatment related adverse changes at any of the dose level.

The No Observed Adverse Effect Level (NOAEL) of the test material was hence concluded as 50 mg/kg b. wt./day under the conditions and procedures followed in this study.