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Administrative data

Description of key information

Key Study: Sub-chronic repeated (90-day) toxicity study in rats. OECD 408, GLP study. The only test item-related effect found was minimal microscopic changes in thymus at the high dose of 1000 mg/kg bw/day in males (G4M). As this change was minimal in severity and was found to be reversible, it was considered as non-adverse effect. Thus, the NOAEL of the test item for both sexes was determined to be 1000 mg/kg bw/day.


Supporting Study: Dose Range Finding Study in rats, OECD 407, non GLP. The oral administration of test item by gavage to Sprague Dawley Rats for a period of 28 consecutive days did not produce test item-related effects in any of the parameters examined at the dose levels of 100, 300 and 1000 mg/kg bw/day under experimental conditions employed and the same are then considered appropriate for a subsequent 90-day repeated dose toxicity study in rats.


 

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 August 2020 to 24 December 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
One deviation was recorded for administering 3.1 mL on Day 57 instead of 3.2 mL for Re7208 of G3M. This marginal variation in dose volume does not have any impact on the outcome of the study as variation was only 0.1 mL and also in mid dose group only.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: In-house
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7-8 weeks
- Weight at study initiation: Males: 152.08-153.74 g (Mean), Females: 139.62- 141.52 g (Mean)
- Fasting period before study: no
- Housing: Two animals of same sex were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted feed and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
- Diet (e.g. ad libitum): Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to the animals throughout the experimental period.
- Water (e.g. ad libitum): Water was provided ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: six days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7°C to 22.9°C
- Humidity (%): 46% to 65%
- Air changes (per hr): 12-15 Air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle

IN-LIFE DATES: From: 14.08.2020 To: 16.12.2020
Route of administration:
oral: gavage
Details on route of administration:
The test formulations and vehicle was administered through oral gavage using oral intubation cannula attached to disposable syringe
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5%
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item formulations were prepared before dose administration. The required quantity of test item was weighed separately and transferred, mixed, and triturated well in a mortar. A small quantity of vehicle was added and triturated until a homogenous suspension was formed and thereafter the entire quantity of the formulation was transferred to measuring cylinder. A small quantity of vehicle was added to rinse the mortar and this was transferred into the measuring cylinder. The rinsing procedure of mortar and pestle was repeated (many times) to ensure complete transfer of the contents to the measuring cylinder. Finally, the volume was adjusted to required mark in measuring cylinder with vehicle to get a desired concentration of 10, 30 and 100 mg/mL of test item for low, mid, and high dose groups, respectively.

VEHICLE
- Justification for use and choice of vehicle (if other than water): homogenous suspension with test item
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg
- Lot/batch no. (if required): BCBN1690V (Sigma Aldrich)
- Expiry Date: 9 March 2024
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Verification of concentration and homogonetiy of the test item was conduced by BIONEEDS INDIA PRIVATE LIMITED DEVARAHOSAHALLY, SOMPURA HOBLI, NELAMANGALA TALUK, BANGALORE RURAL DISTRICT, PIN - 562 111, KARNATAKA, INDIA which hold the Good Laboratory Practice Certificate (GLP). 0.1 mL of sample was pipetted out into a digestion tube, a volume of 6 mL of concentrated nitric acid was added and placed in microwave sample preparation system and digested by the selected method. After digestion, the digestion tubes were cooled to room temperature and the contents were transferred to suitable volumetric flasks and diluted with Milli-Q water.


TECHNIQUE AND TEST METHOD: ICP-MS


TEST PARAMETERS: Timings Sweeps : 30 Seconds, Reading : 1, Replicates : 3, Analyte : Aluminium, Mass : 26.9815, Sample Flush : 35 seconds, Read Delay : 15 Seconds, Wash : 45 seconds, Mode : KED, Flow (Helium) : 4.0 mL, Vehicle: 0.5% Carboxymethyl Cellulose


ACCEPTANCE CRITERIA: Homogeneity and dose formulation analysis of prepared test formulations for the dose concentration verification were performed during week 1, month 2 and month 3 of the dosing period and the obtained mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is ≤10%.
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
G1- control group
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
G2
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
G3
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
G4
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
G1R- control recovery
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
G4R- high dose recovery
No. of animals per sex per dose:
10 males and 10 females per group for the main groups, 5 males and 5 females for the recovery groups (G1R and G4R).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses of 100, 300 and 1000 mg/kg/day were selected for test item based on the results obtained from the dose range finding study in which no adverse effects were found up to the dose of 1000 mg/kg/day.
- Fasting period before blood sampling for clinical biochemistry: overnight
- Post-exposure recovery period in satellite groups: 28 days
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily for clinical signs of toxicity and twice daily for mortality and morbidity
- Cage side observations checked: clinical signs of toxicity, mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before initiation of the treatment and at weekly intervals thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: on the Day 1 before test item administration and weekly thereafter

FOOD CONSUMPTION :
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, weekly.

FOOD EFFICIENCY: No
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during Week 12 for vehicle control and high dose main group animals (G1, and G4) and during Week 17 for recovery group animals (G1R and G4R).
- Dose groups that were examined: G1, G4, G1R and G4R

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on experimental day 91 and 119 for main and recovery groups respectively
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: Yes
- How many animals: all of them
- Parameters checked: Hemoglobin concentration, hematocrit, erythrocyte count, total leukocyte count, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, mean platelet volume, reticulocyte count, absolute reticulocyte count, differential leucocytes count, absolute differential leucocytes count, prothrombin time and
activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on experimental day 91 and 119 for main and recovery groups respectively
- Animals fasted: Yes
- How many animals: all of them
- Parameters checked: Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, cholinesterase, total protein, albumin, total bilirubin, glucose, total cholesterol, creatinine, urea, triglycerides, phosphorous, calcium, high density lipoprotein, low density lipoprotein, blood urea nitrogen, globulin, albumin/globulin ratio, sodium, potassium and chloride.

PLASMA/SERUM HORMONES/LIPIDS: Yes (Thyroid Hormones T3 (Triiodothyronine), T4 (Thyroxine) and Thyroid Stimulating Hormone (TSH))
- Time of blood sample collection: at termination.
- Animals fasted: Not specified
- How many animals: all of them

URINALYSIS: Yes
- Time schedule for collection of urine: on experimental day 91 and 119 for main and recovery groups respectively
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Blood, bilirubin, urobilinogen, ketones, protein, leuocytes, microalbumin, pH, nitrite and specific gravity.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during Week 12 for vehicle control and high dose main group animals (G1 and G4) and during Week 17 for recovery group animals (G1R and G4R).
- Dose groups that were examined: G1, G4, G1R and G4R
- Battery of functions tested: sensory activity / grip strength / motor activity / other: home cage observations, handling observations, open field observations, neuromuscular observations, physiological observation (rectal temperature)

IMMUNOLOGY: No

OTHER:
-Vaginal Smear Examination from all the females on the day of sacrifice.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
On day 91, all the animals of groups G1, G2, G3 and G4 were subjected to necropsy and detailed gross pathological examination. All the animals from recovery groups (G1R and G4R) were subjected to necropsy and detailed gross pathological examination on day 119. The animals were fasted overnight (water was provided ad libitum) prior to scheduled necropsy. On the day of terminal sacrifice, the body weight of all the fasted animals were recorded prior to exsanguination. The animals were euthanized using carbon dioxide asphyxiation exsanguination.
Necropsy included an examination of external surfaces, external orifices, abdominal, thoracic and cranial cavities, organs and tissues. All the animals were sacrificed as per random numbers generated for the study and complete gross pathological examination was performed

TISSUE COLLECTION, ORGAN WEIGHTS, PRESERVATION AND HISTOPATHOLOGY: Yes (see table 1)
On the day of scheduled necropsy, a list of organs were collected from all animals and were trimmed of any adherent tissue and fat, as appropriate and weighed wet as soon as possible to avoid drying.
Organ weight ratio was calculated against fasting body weight. The organs and tissues were collected, trimmed off any adherent tissue, as appropriate. All the tissues mentioned below, from all animals, were preserved in 10% v/v Neutral Buffered Formalin and Modified Davidson’s fixative for eyes and testes.

HISTOPATHOLOGY: Yes
Histopathological examination was conducted on the tissues from the vehicle control (G1) and high dose (G4). Further, thymus from lower and recovery group males were processed and subjected for histopathological evaluation as it was found to be target organ of toxicity in high dose males.
All organs and tissue samples were processed, embedded in paraffin, cut at a thickness of 4 to 6 micrometers and stained with hematoxylin and eosin. The bone marrow smear was fixed in methanol and stained with Giemsa Stain. Testes were sectioned at 3-4 micrometers thickness and addition to hematoxylin and eosin stain, Periodic acid –Schiff and hematoxylin stain was used for spermatogenesis evaluation.
A detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen or any cell or stage specificity of testicular findings

GROSS PATHOLOGY: Yes
All animals were fasted overnight (water allowed) and on the day of necropsy (day 91 for main groups, day 119 for recovery groups) all animals were weighed and then subjected to necropsy. Some organs (see Table 1) were collected, weighed and preserved for histopathology.
Necropsy included an examination of external surfaces, external orifices, abdominal, thoracic and cranial cavities, organs and tissues.

HISTOPATHOLOGY: Yes
Histopathological examination was carried out on the preserved organs of the vehicle control (G1) and high dose (G4) group animals.
Other examinations:
BONE MARROW SMEAR EXAMINATION:
Bone marrow smear was prepared for all the animals at the time of necropsy. As there were no treatment related changes in haematology parameters or microscopic changes in organ like spleen, a cytologic evaluation of the bone marrow was not carried out. In thymus, minimal, decreased cellularity of lymphocytes was noted in high dose males (5/10) only and was found reversible and hence considered non-adverse finding.
Statistics:
The data was subjected to statistical analysis. The computer printout of the data was verified with the original raw data. After verification, the data was subjected to statistical analysis using SPSS software version 22. Body weight, percent change in body weight (with respect to Day 1 body weight), feed consumption, organ weights and ratios, hematological and clinical chemistry estimations and urine analysis parameters (pH, urine volume, specific gravity and urobilinogen), FOB parameters (Open field observations, Body temperature, Hind Limb Footsplay, Grip strength and Movement Counts) were subjected to statistical analysis. One way ANOVA followed by Dunnett’s post test was done for different treatment and recovery groups comparing with the respective vehicle control group data. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05). Any other statistical tests used for the analysis of data was mentioned in the final report. Statistically significant changes obtained from the aforementioned tests was designated by the superscripts throughout the report as stated below:
*: Statistically significant (P<0.05)
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant lower feed consumption was noted during week 13 in G4RF. The noted change is considered as incidental in the absence of any concomitant changes in body weights.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Increase in percent eosinophils (G4M), absolute neutrophils (G2F), absolute monocytes (G2F), decrease in MCHC (G3F, G4F), percent lymphocytes (G2F) were noted. The noted variation in differential counts is considered incidental in the absence of dose responsiveness. Decrease in MCHC without any variation in haemoglobin concentration is considered incidental. In recovery groups, increase in percent neutrophils (G4RM), decrease in percent lymphocytes (G4RM), absolute monocytes (G4RF), absolute basophils (G4RF) were noted. The observed changes in differential counts without any variation in total leucocyte count is considered incidental.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Increase in glucose (G2M), calcium (G4M), sodium (G2M, G4M), phosphorus (G3F, G4F) was noted. The noted changes are considered incidental in the absence of dose responsiveness.
In recovery groups, increase in total cholesterol (G4RF), high density lipoprotein (G4RF), cholinesterase (G4RF), decrease in ALP (G4RF) was noted. The noted variations are considered incidental as similar variation was not noted in other sex nor at the end of treatment period.
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistical significance decrease in T3 (G2M, G4F) and increase in TSH (G4F) was noted which is considered incidental in the absence of associated microscopic (or histopathological) or macroscopic changes in thyroid in the high dose group and dose responsiveness.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant increase in specific gravity (G2M), decrease in pH (G4RF) was noted. Increase in specific gravity is considered incidental in the absence of dose responsiveness. Decrease in pH is also considered incidental as no such variation was noted in other sex or at the end of treatment period.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Decreased movement counts (G4M, G4F); increased rearing (G4M), increase in hind limb footsplay (G4F) was noted. The noted variations are considered incidental in the absence of dose responsiveness and changes in other neuromuscular parameters. Also, no clinical signs were noted during the in-life phase of the study.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
- Absolute weights: Decrease in weight of thymus (G2M, G4M), brain (G2M, G3M, G4M, G3F), adrenals (G2F, G3F), uterus with cervix (G2F, G3F), kidneys (G4RM); increase in weight of spleen (G3M), pituitary (G2M, G3M), uterus with cervix (G4F), heart (G4F), liver (G3F, G4F) was noted
- Relative weights (relative to body weight): Decrease in weight of thymus (G2M, G4M), brain (G2M, G3M, G3F), adrenals (G2F, G3F), ovaries with oviduct (G4F), uterus with cervix (G2F, G3F); increase in weight of spleen (G3M), uterus with cervix (G4F), heart (G4F), liver (G3F) was noted.

Variation in thymus was associated with microscopic changes like minimal, decreased cellularity of lymphocytes only in high dose male rats (G4M) and was characterized by paler staining of cortex and/or decreased cortico-medullary ratio with prominent stromal cells.

All other changes noted are considered incidental and not related to treatment in the absence of dose responsiveness and/or absence of microscopic changes in the high dose main groups.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In thymus, minimal, decreased cellularity of lymphocytes was observed only in high dose male rats and was characterized by, paler staining of cortex and/or decreased cortico-medullary ratio with prominent stromal cells. This change was found to be reversible at the end of 28-day treatment free recovery period as similar changes were not observed in recovery group. Further, as this change was minimal in severity and was found to be reversible, it can be considered as non-adverse finding. Few other microscopic findings observed in this study such as ultimobranchial cyst/s in thyroid gland, age related involution in thymus, dilatation of sinus in mandibular lymph node, dilatation of lumen in uterus in females and all other findings were considered incidental as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats of this age.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Vaginal smear examination revealed normal stages of estrous cyclicity both in main and recovery females.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effect observed up to highest dose tested.
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
no

Table 2. Summary of clinical signs of toxicity, detailed clinical examination and mortality record. 



















































































Group, Sex & Dose
(mg/kg/day)
No. of
Animals
Clinical Signs of ToxicityMortality (No. of Incidences/
No. of Animals)
G1, M & 010N0/10
G2, M & 10010N0/10
G3, M & 30010N0/10
G4, M & 100010N0/10
G1R, M & 05N0/5
G4R, M & 10005N0/5
G1, F & 010N0/10
G2, F & 10010N0/10
G3, F & 30010N0/10
G4, F & 100010N0/10
G1R, F & 05N0/5
G4R, F & 10005N0/5

M: Male; F: Female; R: Recovery; N: Normal


 


Table 3. Summary of body weights (g) record






























































































































































































































































































































































































































Group, Sex & Dose (mg/kg/day)

 Body Weight (g) on Days
1815222936435057647178859097104111118
G1, M & 0Mean152.16187.81231.45263.58286.93315.51337.43349.15367.58375.99385.73392.00397.84402.11----
±SD9.2517.9817.1719.7324.8629.5233.0735.8542.0645.5249.1849.7750.5351.40----
n1010101010101010101010101010----
G2, M & 100Mean152.87193.51241.12277.16302.54332.20356.41370.52386.05391.59400.71412.23423.57428.98----
±SD6.4314.0418.8722.2625.1630.1034.6737.2041.7944.1042.6240.0934.6936.14----
n1010101010101010101010101010----
G3, M & 300Mean152.08188.22228.08258.40282.81304.21324.53331.28344.80352.32371.32382.99387.58394.26----
±SD4.576.467.7213.0016.2422.7326.0329.2227.7732.9236.5941.3744.4644.33----
n1010101010101010101010101010----
G4, M & 1000Mean152.98188.39223.67248.07274.56301.28316.91326.15329.98346.73359.82369.54378.32382.55----
±SD5.339.179.408.8714.5117.2917.5225.0027.0323.8726.0128.7827.3627.34----
n1010101010101010101010101010----
G1R, M & 0Mean152.18194.66238.35267.84291.02317.59331.21349.10358.18370.26381.91390.39395.88399.93405.61409.78413.43420.70
±SD6.199.8315.8818.5427.8523.9424.5519.2825.2019.6817.4327.4931.0030.1731.6030.5330.9630.04
n555555555555555555
G4R, M & 1000Mean153.74190.61223.75255.61279.31299.54321.26348.73355.75371.42379.62379.68390.13397.15401.61406.95410.49417.01
±SD3.938.9216.2518.2223.3917.7721.7421.2324.4428.9636.4433.5123.9524.2224.2822.9721.6919.12
n555555555555555555

 






























































































































































































































































































































































































































Group, Sex & Dose (mg/kg/day)

 Body Weight (g) on Days
1815222936435057647178859097104111118
G1, F & 0Mean141.52160.85181.40190.98203.02212.63219.84229.52236.87239.87243.51248.30249.90252.75----
±SD6.196.595.876.566.896.518.5013.7012.1813.8013.2113.3512.2112.06----
n1010101010101010101010101010----
G2, F & 100Mean141.12162.21185.03196.49207.05218.11228.67234.34235.96240.35246.69250.90255.08258.00----
±SD7.589.3611.0512.5112.3513.6015.4515.4119.9222.2923.3521.0519.8319.03----
n1010101010101010101010101010----
G3, F & 300Mean140.21161.44179.53190.24202.33210.43216.74224.85233.31236.41242.13248.07250.59253.67----
±SD6.968.019.2911.5912.0813.9312.1613.5513.2711.9212.5913.9413.2413.44----
n1010101010101010101010101010----
G4, F & 1000Mean140.93163.88185.30198.32211.24217.91226.25238.18242.60245.33249.75254.92256.56263.73----
±SD5.889.3612.3812.0419.2613.7216.3217.1120.4121.6519.8022.4323.6730.30----
n1010101010101010101010101010----
G1R, F & 0Mean139.62160.30178.74186.58197.38206.32214.49224.07236.58232.92234.08236.88241.77245.46250.64254.42258.80264.37
±SD6.246.385.536.639.219.079.909.8212.7512.7514.4613.6616.5315.9615.1313.5113.6912.39
n555555555555555555
G4R, F & 1000Mean140.20165.41185.44201.64212.04213.72225.32235.60242.69246.06250.82255.23254.56264.07267.85272.42276.82282.60
±SD5.0915.9312.8815.7716.9920.4523.6623.8725.5927.2426.1626.2727.8629.9130.4430.2531.4530.45
n555555555555555555

 


Table 4. Summary of feed consumption (g/rat/day) record



























































































































































































































Group, Sex & Dose (mg/kg/day)


 

Week 1



Week 2



Week 3



Week 4



Week 5



Week 6



Week 7



Week 8



Week 9



Week 10



Week 11



Week 12



Week 13



G1, M & 0



Mean



18.27



19.62



24.04



25.20



25.54



25.44



25.28



25.01



25.27



25.42



24.68



24.56



25.07



±SD



2.71



1.83



1.66



1.57



1.76



0.57



1.79



0.89



1.69



2.16



2.11



2.04



3.42



n



5



5



5



5



5



5



5



5



5



5



5



5



5



G2, M & 100



Mean



20.09



21.19



24.69



25.45



25.82



24.52



24.76



25.40



24.75



25.92



26.18



26.51



23.95



±SD



0.85



0.50



1.44



1.08



2.53



1.35



2.35



0.25



2.08



1.50



1.25



0.43



2.80



n



5



5



5



5



5



5



5



5



5



5



5



5



5



G3, M & 300



Mean



18.78



21.38



22.61



24.93



25.70



25.60



24.79



25.88



24.40



26.48



25.96



26.39



23.11



±SD



1.53



1.28



1.39



1.06



0.82



0.95



0.52



0.90



1.45



0.61



0.85



0.62



1.48



n



5



5



5



5



5



5



5



5



5



5



5



5



5



G4, M & 1000



Mean



19.09



21.43



23.72



25.51



25.45



25.68



25.38



25.52



24.69



25.97



25.84



25.69



24.61



±SD



0.81



0.34



0.88



1.17



0.28



0.45



1.15



1.00



1.43



0.88



1.13



1.41



2.60



n



5



5



5



5



5



5



5



5



5



5



5



5



5



 



























































































































































































































Group, Sex & Dose (mg/kg/day)


 

Week 1



Week 2



Week 3



Week 4



Week 5



Week 6



Week 7



Week 8



Week 9



Week 10



Week 11



Week 12



Week 13



G1, F & 0



Mean



16.68



17.05



17.63



18.49



18.96



19.89



18.40



17.17



17.90



19.22



17.92



18.24



19.41



±SD



0.57



0.88



1.46



1.64



1.83



2.01



2.62



1.29



3.00



1.38



1.81



2.76



1.96



n



5



5



5



5



5



5



5



5



5



5



5



5



5



G2, F & 100



Mean



15.87



17.40



17.77



18.28



19.15



21.22



17.07



17.43



17.62



17.80



18.14



18.51



19.13



±SD



0.60



2.03



2.34



2.06



1.54



1.23



2.90



2.61



0.70



5.90



1.10



0.68



3.15



n



5



5



5



5



5



5



5



5



5



5



5



5



5



G3, F & 300



Mean



15.85



17.07



17.13



16.83



19.67



18.31



16.37



17.61



17.42



18.93



16.31



16.52



15.38



±SD



1.52



1.32



1.37



2.44



2.57



3.03



4.82



1.29



0.49



3.79



0.64



0.59



2.85



n



5



5



5



5



5



5



5



5



5



5



5



5



5



G4, F & 1000



Mean



15.67



17.77



18.25



18.54



19.40



20.32



17.18



18.22



18.55



19.15



19.09



19.38



17.29



±SD



0.85



1.01



1.08



1.32



2.26



2.02



1.62



1.93



1.47



1.55



2.35



2.14



2.61



n



5



5



5



5



5



5



5



5



5



5



5



5



5



 




















































































































































































Group, Sex & Dose (mg/kg/day)


 

Week 1



Week 2



Week 3



Week 4



Week 5



Week 6



Week 7



Week 8



Week 9



Week 10



G1R, M & 0



Mean



20.58



20.95



25.45



26.34



27.08



26.12



25.02



25.63



25.27



24.85



±SD



1.13



1.13



1.89



1.61



0.63



0.32



0.88



0.80



1.42



2.11



n



3



3



3



3



3



3



3



3



3



3



G4R, M & 1000



Mean



18.74



21.75



24.84



26.41



26.75



25.82



25.65



25.59



27.37



26.17



±SD



1.56



0.40



1.07



1.14



1.52



0.98



1.06



0.43



1.98



4.06



n



3



3



3



3



3



3



3



3



3



3



G1R, F & 0



Mean



16.55



16.72



16.76



19.72



19.07



19.22



16.95



15.25



15.96



17.46



±SD



0.99



1.65



1.65



2.66



1.36



1.16



0.67



1.84



1.42



0.40



n



3



3



3



3



3



3



3



3



3



3



G4R, F & 1000



Mean



16.98



17.56



17.74



17.83



17.96



18.67



16.08



17.96



17.65



18.05



±SD



0.72



2.06



0.36



0.37



0.88



5.08



3.53



3.66



2.97



2.75



n



3



3



3



3



3



3



3



3



3



3



 













































































































































Group, Sex & Dose (mg/kg/day)


 

Week 11



Week 12



Week 13



Week 14



Week 15



Week 16



Week 17



G1R, M & 0



Mean



25.58



24.85



24.74



21.59



21.48



22.24



22.28



±SD



1.26



1.51



2.85



2.17



1.78



2.00



1.64



n



3



3



3



3



3



3



3



G4R, M & 1000



Mean



23.27



20.69



24.57



23.02



21.84



22.13



22.28



±SD



3.79



4.99



1.33



3.35



2.17



1.96



1.93



n



3



3



3



3



3



3



3



G1R, F & 0



Mean



16.35



19.60



18.03



17.70



16.21



16.30



16.70



±SD



1.39



2.96



0.91



3.24



1.66



1.62



1.19



n



3



3



3



3



3



3



3



G4R, F & 1000



Mean



16.52



18.42



15.60*



20.19



14.61



14.82



16.53



±SD



1.04



1.37



0.71



1.57



0.48



0.73



1.52



n



3



3



3



3



3



3



3



*: Statistically significant (P<0.05).


 


Table 5. Summary of neurological/functional observation battery (FOB) record











































































































































































































Week 12



Parameters↓



Group & Sex



G1 & M



G4 & M



G1 & F



G4 & F



Dose (mg/kg/day)



0



1000



0



1000



Number of Animals



10



10



10



10



Open field Observation



  Mobility



1



1



1



1



 Gait



1



1



1



1



 Arousal



3



3



3



3



Number of Rearing



Mean



7.0



8.2*



6.9



7.4



±SD



1.3



0.9



1.4



1.2



Numbers of Urination



Mean



1.5



1.2



1.5



1.1



±SD



0.7



0.4



0.5



0.6



Number of Defecation



Mean



1.1



1.1



1.2



1.3



±SD



0.6



0.6



0.4



0.5



Clonic involuntary movement



1



1



1



1



Tonic involuntary movement



1



1



1



1



Stereotype Behaviour



1



1



1



1



Excessive Grooming



Mean



4.5



3.8



4.2



4.6



±SD



0.8



0.9



0.8



0.8



Sensory Observations



Approach Response



2



2



2



2



Auditory Response



2



2



2



2



Touch Response  



2



2



2



2



Pupil Reflex



2



2



2



2



Tail Pinch Response



2



2



2



2



Righting Reflex



1



1



1



1



Physiological observation



 



Body temperature (°F)



Mean



98.6



98.6



99.3



99.4



±SD



0.2



0.2



0.3



0.2



Open field Observation:  Mobility - 1=Normal, Gait - 1=Normal, Arousal - 3=Normal, Clonic involuntary movements - 1=None/Normal, Tonic involuntary movements - 1=None/Normal, Stereotype Behaviour - 1=Absent,  Sensory Observations: Approach response - 2=Normal,  Auditory Response - 2=Normal, Touch Response - 2=Normal, Pupil Reflex - 2=Normal, Tail Pinch Response - 2=Normal, Righting Reflex - 1=Present



*: Statistically significant (P<0.05).


 


Table 6. Summary of actimeter reading record























































































































































#Week 12/17


 

Group, Sex & Dose (mg/kg/day)


 

Actimeter Readings


 

Movement Counts (no.)


 
 

G1, M & 0



Mean



2098.6


 

±SD



53.0


 

n



10


 

G4, M & 1000



Mean



2024.8*


 

±SD



9.9


 

n



10


 

G1, F & 0



Mean



2441.0


 

±SD



94.2


 

n



10


 

G4, F & 1000



Mean



2279.5*


 

±SD



59.6


 

n



10


 

G1R, M & 0



Mean



2208.6


 

±SD



37.9


 

n



5


 

G4R, M & 1000



Mean



2107.6


 

±SD



94.5


 

n



5


 

G1R, F & 0



Mean



2087.8


 

±SD



53.6


 

n



5


 

G4R, F & 1000



Mean



2090.6


 

±SD



63.9


 

n



5


 

*: Statistically significant (P<0.05)


#: Week 12 for main group and Week 17 for recovery group.


 


Table 7. Summary of haematology record





























































































































































































































Group, Sex & Dose


(mg/kg/day)



 



Total Leucocyte Count



Total Erythrocyte Count 



Hemoglobin



Haematocrit



Mean Corpuscular Volume



Mean Corpuscular Hemoglobin



Mean Corpuscular Hemoglobin Concentration 



  (WBC)        



 (RBC)    



(HGB)      



 (HCT)   



  (MCV)     



(MCH)



(MCHC)      



(103 cells/µL)



(106 cells/µL)



(g/dL)



(%)



(fL)



(pg)



(g/dL)



G1, M & 0



Mean



9.44



8.37



14.26



43.20



51.72



17.06



33.01



±SD



1.77



0.53



0.69



2.10



2.32



0.80



0.81



n



10



10



10



10



10



10



10



G2,M & 100



Mean



9.02



8.77



15.01



45.02



51.41



17.16



33.34



±SD



2.38



0.59



0.98



2.75



1.92



0.80



0.59



n



10



10



10



10



10



10



10



G3, M & 300



Mean



9.85



8.49



14.82



44.03



51.87



17.47



33.65



±SD



4.22



0.74



1.45



4.01



1.80



0.75



0.46



n



10



10



10



10



10



10



10



G4, M & 1000



Mean



8.33



8.44



14.59



44.04



52.32



17.31



33.11



±SD



1.97



0.58



0.52



2.02



1.91



0.76



0.44



n



10



10



10



10



10



10



10



G1R, M & 0



Mean



11.61



8.70



14.16



43.42



49.96



16.28



32.58



±SD



3.10



0.76



1.26



3.19



0.92



0.34



0.66



n



5



5



5



5



5



5



5



G4R, M & 1000



Mean



12.43



8.88



14.54



45.32



51.10



16.42



32.12



±SD



5.12



0.92



1.18



3.92



1.81



0.38



0.69



n



5



5



5



5



5



5



5



 


















































































































































































































































Group, Sex & Dose


 (mg/kg/day)



 



Platelet Count



Mean Platelet Volume



Reticulocyte Count



Neutrophils



Lymphocytes



Monocytes



Eosinophils



Basophils



  (PLT)



(MPV)   



(Retic)



(Neut)



(Lymph)



(Mono)



(Eos)



(Baso)



(103 cells/µL)



(fL)



(%)



(%)



         (%)



(%)



(%)



(%)



G1, M & 0



Mean



749.40



6.33



2.26



24.09



68.41



4.60



1.70



0.35



±SD



149.67



0.45



0.58



5.76



5.59



1.71



0.81



0.20



n



10



10



10



10



10



10



10



10



G2, M & 100



Mean



844.70



6.90



1.96



33.13



58.74



4.89



1.88



0.36



±SD



143.74



0.82



0.86



18.28



20.11



2.43



0.79



0.13



n



10



10



10



10



10



10



10



10



G3, M & 300



Mean



699.10



6.39



1.84



22.59



70.29



4.09



1.67



0.35



±SD



192.74



0.33



0.64



5.83



7.16



1.38



0.68



0.13



n



10



10



10



10



10



10



10



10



G4, M & 1000



Mean



668.20



6.75



1.76



35.52



55.17



4.79



3.24*



0.25



±SD



114.49



0.48



0.57



13.98



14.97



1.41



1.92



0.08



n



10



10



10



10



10



10



10



10



G1R, M & 0



Mean



768.40



6.74



2.51



27.82



63.46



5.08



2.00



0.64



±SD



82.34



0.46



1.07



2.53



4.90



2.51



0.37



0.27



n



5



5



5



5



5



5



5



5



G4R, M & 1000



Mean



710.80



6.82



2.41



38.28*



52.12*



4.88



2.38



1.46



±SD



114.11



0.26



0.74



7.21



7.50



0.74



1.37



0.75



n



5



5



5



5



5



5



5



5



 


















































































































































































































































Group, Sex & Dose


(mg/kg/day)



 



Absolute Reticulocyte Count  



Absolute Neutrophils   



Absolute Lymphocytes  



Absolute Monocytes



Absolute Eosinophils



Absolute  Basophils       



Prothrombin Time  



Activated Prothrombin Time       



(Retic)



(Neut)



(Lymph)



(Mono)



(Eos)



(Baso)



(PT)



(APTT)



(109 cells/L)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(Seconds)



(Seconds)



G1, M & 0



Mean



187.71



2.32



6.44



0.42



0.15



0.03



18.11



27.45



±SD



41.99



0.97



1.16



0.14



0.06



0.02



1.76



1.51



n



10



10



10



10



10



10



10



10



G2, M & 100



Mean



168.40



2.76



5.55



0.42



0.18



0.03



18.87



25.77



±SD



64.88



1.04



2.76



0.18



0.10



0.02



1.44



1.88



n



10



10



10



10



10



10



10



10



G3, M & 300



Mean



155.56



2.06



7.11



0.40



0.15



0.04



18.02



26.21



±SD



56.32



0.53



3.69



0.20



0.05



0.02



1.24



2.51



n



10



10



10



10



10



10



10



10



G4, M & 1000



Mean



147.18



3.10



4.43



0.41



0.26



0.02



18.44



26.61



±SD



43.50



2.08



1.38



0.21



0.17



0.01



1.89



5.77



n



10



10



10



10



10



10



10



10



G1R, M & 0



Mean



213.10



3.26



7.34



0.60



0.24



0.07



16.36



23.24



±SD



73.94



1.05



1.88



0.38



0.11



0.02



0.56



3.64



n



5



5



5



5



5



5



5



5



G4R, M & 1000



Mean



209.40



4.91



6.39



0.60



0.27



0.17



16.30



23.68



±SD



49.97



2.62



2.36



0.24



0.11



0.10



0.80



3.05



n



5



5



5



5



5



5



5



5



 





























































































































































































































Group, Sex & Dose


(mg/kg/day)



 



Total Leucocyte Count



Total Erythrocyte Count 



Hemoglobin



Haematocrit



Mean Corpuscular Volume



Mean Corpuscular Hemoglobin



Mean Corpuscular Hemoglobin Concentration 



  (WBC)        



 (RBC)    



(HGB)      



 (HCT)   



  (MCV)     



(MCH)



(MCHC)      



(103 cells/µL)



(106 cells/µL)



(g/dL)



(%)



(fL)



(pg)



(g/dL)



G1, F & 0



Mean



6.32



7.82



14.15



41.13



52.60



18.11



34.42



±SD



1.81



0.36



0.87



2.39



1.80



0.52



0.73



n



10



10



10



10



10



10



10



G2, F & 100



Mean



7.93



7.82



14.16



41.30



52.91



18.15



34.24



±SD



1.65



0.38



0.57



1.42



1.90



0.74



0.45



n



10



10



10



10



10



10



10



G3, F & 300



Mean



7.09



7.76



13.77



40.69



52.52



17.76



33.82*



±SD



1.94



0.49



0.92



2.45



1.33



0.47



0.55



n



10



10



10



10



10



10



10



G4, F & 1000



Mean



6.84



7.86



14.38



42.55



54.11



18.30



33.82*



±SD



1.99



0.26



0.58



1.70



1.97



0.61



0.38



n



10



10



10



10



10



10



10



G1R, F & 0



Mean



10.28



8.23



14.18



42.70



51.98



17.28



33.26



±SD



4.28



0.83



0.89



2.93



2.23



0.80



0.40



n



5



5



5



5



5



5



5



G4R, F & 1000



Mean



6.03



8.24



14.54



43.74



53.06



17.66



33.26



±SD



1.22



0.66



1.26



3.92



1.34



0.38



0.39



n



5



5



5



5



5



5



5



 


















































































































































































































































Group, Sex & Dose


(mg/kg/day)



 



Platelet Count



Mean Platelet Volume



Reticulocyte Count



Neutrophils



Lymphocytes



Monocytes



Eosinophils



Basophils



  (PLT)



(MPV)   



(Retic)



(Neut)



(Lymph)



(Mono)



(Eos)



(Baso)



(103 cells/µL)



(fL)



(%)



(%)



(%)



(%)



(%)



(%)



G1, F & 0



Mean



665.30



6.93



1.73



24.24



68.58



4.30



1.69



0.28



±SD



200.24



0.68



0.44



6.40



6.87



1.39



0.52



0.09



n



10



10



10



10



10



10



10



10



G2, F & 100



Mean



642.80



7.02



1.82



30.49



60.64*



5.07



2.40



0.34



±SD



215.90



0.54



0.59



8.74



9.35



1.34



0.89



0.10



n



10



10



10



10



10



10



10



10



G3, F & 300



Mean



712.10



6.75



1.88



23.22



68.76



4.41



2.41



0.29



±SD



99.59



0.42



0.70



3.40



3.62



0.76



0.94



0.11



n



10



10



10



10



10



10



10



10



G4, F & 1000



Mean



750.90



6.61



1.61



21.03



71.80



3.98



1.96



0.28



±SD



182.92



0.53



0.56



5.68



6.05



1.67



0.45



0.08



n



10



10



10



10



10



10



10



10



G1R, F & 0



Mean



815.80



6.48



1.87



25.62



66.14



4.48



1.88



0.56



±SD



244.30



0.54



0.46



3.97



2.80



1.74



0.90



0.19



n



5



5



5



5



5



5



5



5



G4R, F & 1000



Mean



785.20



6.72



2.13



27.92



65.50



3.96



1.56



0.38



±SD



211.29



0.37



0.34



3.94



4.21



0.63



0.21



0.13



n



5



5



5



5



5



5



5



5



 


















































































































































































































































Group, Sex & Dose


(mg/kg/day)



 



Absolute Reticulocyte Count  



Absolute Neutrophils   



Absolute Lymphocytes  



Absolute Monocytes



Absolute Eosinophils



Absolute  Basophils       



Prothrombin Time  



Activated Prothrombin Time       



(Retic)



(Neut)



(Lymph)



(Mono)



(Eos)



(Baso)



(PT)



(APTT)



(109 cells/L)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(Seconds)



(Seconds)



G1, F & 0



Mean



135.83



1.49



4.38



0.27



0.11



0.02



19.38



26.55



±SD



36.29



0.47



1.53



0.11



0.04



0.01



1.34



2.45



n



10



10



10



10



10



10



10



10



G2, F & 100



Mean



141.99



2.42*



4.82



0.39*



0.18*



0.03



19.17



24.77



±SD



45.52



0.93



1.37



0.10



0.06



0.01



2.53



2.10



n



10



10



10



10



10



10



10



10



G3, F & 300



Mean



146.06



1.64



4.88



0.32



0.16



0.02



21.70



23.74



±SD



54.79



0.54



1.34



0.09



0.06



0.01



4.28



1.87



n



10



10



10



10



10



10



10



10



G4, F & 1000



Mean



126.36



1.39



4.95



0.28



0.14



0.02



19.90



26.27



±SD



43.63



0.37



1.70



0.16



0.06



0.01



2.21



3.67



n



10



10



10



10



10



10



10



10



G1R, F & 0



Mean



151.84



2.68



6.81



0.43



0.18



0.06



17.12



23.36



±SD



29.59



1.35



2.80



0.15



0.10



0.02



1.03



2.24



n



5



5



5



5



5



5



5



5



G4R, F & 1000



Mean



176.44



1.66



3.98



0.24*



0.10



0.02*



15.86



26.58



±SD



34.63



0.27



0.97



0.05



0.03



0.01



2.03



6.69



n



5



5



5



5



5



5



5



5



*: Statistically significant (P<0.05).


 


Table 8. Summary of clinical chemistry record

















































































































































































































































































































Group, Sex & Dose


(mg/kg/day)



 



Glucose



Urea



Creatinine



Total Cholesterol



Triglycerides



High-density lipoprotein



Low-density lipoprotein



Total Protein



Albumin



Alanine aminotransferase



Aspartate aminotransferase



 (GLU)


 

 (CRE)



(CHO)



(TRI)



(HDL)



(LDL)



(TPR)



  (ALB)



(ALT)



(AST)



(mg/dL)



(mg/dL)



 (mg/dL)



(mg/dL)



(mg/dL)



 (mg/dL)



(mg/dL)



  g/dL)



  (g/dL)



 (U/L)



 (U/L)



G1, M & 0



Mean



98.40



30.41



0.48



39.60



39.70



8.17



4.94



6.17



2.76



65.90



101.00



±SD



11.07



3.55



0.03



6.29



11.25



1.28



1.15



0.33



0.14



9.09



12.42



n



10



10



10



10



10



10



10



10



10



10



10



G2, M & 100



Mean



115.10*



32.91



0.46



41.30



33.20



8.54



4.62



6.19



2.85



59.70



95.20



±SD



12.55



4.22



0.03



8.62



7.91



1.28



1.00



0.39



0.15



8.77



11.06



n



10



10



10



10



10



10



10



10



10



10



10



G3, M & 300



Mean



111.40



33.57



0.49



37.20



31.20



7.65



4.82



6.36



2.82



64.50



114.40



±SD



14.83



4.86



0.03



7.97



8.66



2.40



1.65



0.23



0.19



15.37



19.84



n



10



10



10



10



10



10



10



10



10



10



10



G4, M & 1000



Mean



108.30



31.36



0.46



41.70



36.30



9.49



4.81



6.45



2.92



93.10



133.40



±SD



11.56



5.51



0.03



10.20



9.68



2.01



1.15



0.26



0.14



102.51



95.73



n



10



10



10



10



10



10



10



10



10



10



10



G1R, M & 0



Mean



101.20



36.28



0.52



32.00



35.00



8.16



4.72



6.84



2.90



63.40



115.60



±SD



7.60



5.44



0.03



5.52



7.35



1.19



0.73



0.15



0.17



10.92



13.52



n



5



5



5



5



5



5



5



5



5



5



5



G4R, M & 1000



Mean



100.80



34.92



0.54



38.60



36.00



9.94



4.30



6.90



3.00



60.40



112.40



±SD



12.64



4.31



0.03



11.87



5.70



1.90



0.70



0.35



0.12



13.63



17.81



n



5



5



5



5



5



5



5



5



5



5



5



 

















































































































































































































































































































Group, Sex & Dose


(mg/kg/day)



 



Alkaline phosphatase



Total Bilirubin



Calcium



Phosphorous



Cholinesterase



Globulin



Albumin/


Globulin ratio



Blood Urea Nitrogen



Sodium



Potassium



Chloride



(ALP)



(BIT)



(CAL)



(PHO)



(CHE)



(GLO)



(A/G Ratio)



(BUN)



 (Na)



 (K)  



 (CLO)



 (U/L)



(mg/dL)



(mg/dL)



(mg/dL)



 (U/L)



(g/dL)


 

(mg/dL)



(mmol/L)



 (mmol/L)



(mmol/L)



G1, M & 0



Mean



151.50



0.09



9.07



4.84



80.90



3.41



0.81



14.19



143.16



3.63



111.69



±SD



30.16



0.03



0.41



0.27



11.97



0.30



0.08



1.66



1.01



0.22



1.71



n



10



10



10



10



10



10



10



10



10



10



10



G2, M & 100



Mean



141.30



0.09



9.26



4.65



95.50



3.34



0.86



15.36



144.47*



3.72



113.48



±SD



38.30



0.01



0.29



0.36



27.81



0.35



0.10



1.97



0.71



0.18



1.12



n



10



10



10



10



10



10



10



10



10



10



10



G3, M & 300



Mean



157.50



0.09



9.41



4.96



87.80



3.54



0.80



15.67



143.86



3.53



112.33



±SD



70.32



0.01



0.26



0.34



18.03



0.13



0.06



2.27



1.02



0.19



1.73



n



10



10



10



10



10



10



10



10



10



10



10



G4, M & 1000



Mean



142.90



0.08



9.55*



4.99



84.90



3.53



0.84



14.64



144.95*



3.49



113.02



±SD



45.17



0.01



0.28



0.46



15.93



0.35



0.12



2.57



1.26



0.16



2.46



n



10



10



10



10



10



10



10



10



10



10



10



G1R, M & 0



Mean



108.20



0.05



9.06



4.84



128.80



3.94



0.74



16.93



142.92



3.31



111.22



±SD



31.59



0.01



0.26



0.53



32.06



0.14



0.06



2.54



1.06



0.22



1.36



n



5



5



5



5



5



5



5



5



5



5



5



G4R, M & 1000



Mean



120.60



0.04



9.14



4.42



99.80



3.90



0.77



16.30



142.64



3.48



111.40



±SD



17.34



0.02



0.30



0.72



30.26



0.26



0.04



2.01



0.94



0.15



1.36



n



5



5



5



5



5



5



5



5



5



5



5



 

















































































































































































































































































































Group, Sex & Dose


(mg/kg/day)



 



Glucose



Urea



Creatinine



Total Cholesterol



Triglycerides



High-density lipoprotein



Low-density lipoprotein



Total Protein



Albumin



Alanine aminotransferase



Aspartate aminotransferase



 (GLU)


 

 (CRE)



(CHO)



(TRI)



(HDL)



(LDL)



(TPR)



  (ALB)



(ALT)



(AST)



(mg/dL)



(mg/dL)



 (mg/dL)



(mg/dL)



(mg/dL)



 (mg/dL)



(mg/dL)



  (g/dL)



  (g/dL)



 (U/L)



 (U/L)



G1, F & 0



Mean



129.20



28.82



0.52



58.00



48.00



14.70



3.07



6.87



3.30



50.00



94.70



±SD



16.65



4.29



0.03



6.85



24.38



1.91



0.41



0.32



0.15



14.01



14.29



n



10



10



10



10



10



10



10



10



10



10



10



G2, F & 100



Mean



126.80



31.81



0.53



55.00



42.40



13.19



3.10



7.06



3.42



45.40



89.60



±SD



8.80



4.25



0.04



10.30



10.25



2.64



0.81



0.48



0.33



6.62



8.54



n



10



10



10



10



10



10



10



10



10



10



10



G3, F & 300



Mean



125.50



32.11



0.51



53.10



38.50



12.75



3.37



6.90



3.25



46.20



97.00



±SD



15.10



3.26



0.04



11.95



10.32



2.62



1.04



0.30



0.24



13.31



18.68



n



10



10



10



10



10



10



10



10



10



10



10



G4, F & 1000



Mean



135.70



28.50



0.51



51.60



37.50



12.45



3.13



7.12



3.29



39.30



91.00



±SD



16.83



3.42



0.04



10.72



8.11



2.31



0.86



0.46



0.20



3.77



10.00



n



10



10



10



10



10



10



10



10



10



10



10



G1R, F & 0



Mean



117.00



35.32



0.59



44.60



36.60



13.62



2.88



7.22



3.49



44.80



101.40



±SD



12.39



5.31



0.03



8.53



12.88



1.91



0.75



0.26



0.21



9.04



13.32



n



5



5



5



5



5



5



5



5



5



5



5



G4R, F & 1000



Mean



118.20



32.08



0.58



66.80*



43.00



19.68*



3.68



7.38



3.77



41.00



113.40



±SD



15.69



4.87



0.02



18.58



14.53



4.61



0.58



0.47



0.37



10.91



25.71



n



5



5



5



5



5



5



5



5



5



5



5



 

















































































































































































































































































































Group, Sex & Dose


(mg/kg/day)



 



Alkaline phosphatase



Total Bilirubin



Calcium



Phosphorous



Cholinesterase



Globulin



Albumin/


Globulin ratio



Blood Urea Nitrogen



Sodium



Potassium



Chloride



(ALP)



(BIT)



(CAL)



(PHO)



(CHE)



(GLO)



(A/G Ratio)



(BUN)



 (Na)



 (K)  



 (CLO)



 (U/L)



(mg/dL)



(mg/dL)



(mg/dL)



 (U/L)



(g/dL)


 

(mg/dL)



 mmol/L)



 (mmol/L)



 mmol/L)



G1, F & 0



Mean



114.90



0.07



9.95



3.69



634.40



3.57



0.93



13.45



141.55



3.28



110.48



±SD



33.14



0.03



0.25



0.43



175.96



0.31



0.10



2.00



0.58



0.16



0.92



n



10



10



10



10



10



10



10



10



10



10



10



G2, F & 100



Mean



90.80



0.09



9.94



3.77



611.40



3.64



0.96



14.85



141.48



3.52



110.36



±SD



34.32



0.01



0.39



0.60



205.38



0.43



0.17



1.98



0.73



0.31



1.40



n



10



10



10



10



10



10



10



10



10



10



10



G3, F & 300



Mean



103.80



0.09



10.07



4.88*



636.20



3.65



0.90



14.98



142.00



3.38



110.23



±SD



77.06



0.02



0.30



0.34



278.72



0.30



0.12



1.52



0.85



0.23



1.38



n



10



10



10



10



10



10



10



10



10



10



10



G4, F & 1000



Mean



82.10



0.07



9.97



4.59*



686.70



3.83



0.87



13.30



141.61



3.43



110.88



±SD



19.28



0.03



0.32



0.77



251.66



0.33



0.07



1.60



0.95



0.55



1.38



n



10



10



10



10



10



10



10



10



10



10



10



G1R, F & 0



Mean



83.80



0.03



9.48



3.42



612.40



3.73



0.94



16.48



142.20



3.41



112.38



±SD



25.57



0.02



0.41



0.65



98.35



0.17



0.07



2.48



0.73



0.16



1.28



n



5



5



5



5



5



5



5



5



5



5



5



G4R, F & 1000



Mean



46.20*



0.05



9.50



3.62



995.20*



3.61



1.05



14.97



142.58



3.43



112.32



±SD



9.73



0.01



0.45



0.43



232.70



0.25



0.12



2.27



0.93



0.22



2.21



n



5



5



5



5



5



5



5



5



5



5



5



*: Statistically significant (P<0.05).


 


Table 9. Summary of urinalysis record












































































































































































































































































































































































































Examination



Group & Sex



G1 & M



G2 & M



G3 & M



G4 & M



G1R & M



G4R & M



Dose (mg/kg/day)



0



100



300



1000



0



1000



Number of Animals



10



10



10



10



5



5



Physical



Colour



Pale Yellow



8



10



8



8



5



5



Yellow



2



-



2



2



-



-



Appearance



Clear



9



8



9



9



4



5



Turbid



1



2



1



1



1



-



Volume (mL)



Mean



5.8



5.2



5.5



5.3



8.0



6.5



±SD



1.5



1.7



0.8



1.1



2.3



0.9



Chemical



pH



Mean



8.6



8.5



8.6



8.5



7.3



7.4



±SD



0.2



0.0



0.2



0.0



0.4



0.4



Specific Gravity



Mean



1.007



1.009*



1.006



1.007



1.013



1.009



±SD



0.002



0.002



0.002



0.002



0.004



0.002



Urobilinogen (mg/dL)



Mean



1.3



1.4



1.3



1.1



0.2



0.2



±SD



1.2



0.5



0.6



0.7



0.0



0.0



Bilirubin (mg/dL)



Neg



-



-



-



-



5



5



1



9



8



9



9



-



-



3



1



2



1



1



-



-



Ketones (mg/dL)



Neg



10



10



10



10



5



5



Blood (Ery/µL)



Neg



10



10



10



9



-



-



Ca10



-



-



-



1



2



1



Ca25



-



-



-



-



1



3



Ca80



-



-



-



-



2



1



 


Proteins (mg/dL)


 



Neg



-



-



-



-



5



4



Trace



-



-



-



-



-



1



>=300



10



10



10



10



-



-



Nitrite



Neg



10



10



10



10



3



5



Pos



-



-



-



-



2



-



Leucocytes (Leu/µL)



Ca70



7



6



6



6



-



-



Ca15



1



-



-



1



5



5



Ca125



1



4



4



3



-



-



>=Ca500



1



-



-



-



-



-



Glucose (mg/dL)



Neg



10



10



10



10



5



5



Micro albumin (MALB) (mg/dL)



Neg



-



-



-



-



-



2



15



-



-



-



-



5



2



>15



10



10



10



10



-



1



Microscopic



Epithelial cells



0



6



6



7



7



3



2



0-1



3



2



2



2



1



2



1-2



1



2



1



1



1



1



Casts



Absent



10



10



10



10



5



5



Crystals



Present



10



10



10



10



5



5



 






























































































































































































































































































































































































































      Examination



Group & Sex



G1 & F



G2 & F



G3 & F



G4 & F



G1R & F



G4R & F



Dose (mg/kg/day)



0



100



300



1000



0



1000



Number of Animals



10



10



10



10



5



5



Physical



Colour



Pale Yellow



7



8



6



9



5



4



Yellow



3



2



4



1



-



1



Appearance



Clear



9



7



10



8



5



4



Turbid



1



3



-



2



-



1



Volume (mL)



Mean



5.9



5.4



5.2



5.5



9.2



7.8



±SD



1.4



0.9



0.8



1.2



1.2



2.4



Chemical



pH



Mean



8.6



8.5



8.5



8.6



8.3



7.5*



±SD



0.2



0.0



0.0



0.2



0.3



0.0



Specific Gravity



Mean



1.007



1.008



1.008



1.009



1.006



1.010



±SD



0.003



0.003



0.003



0.002



0.002



0.006



Urobilinogen (mg/dL)



Mean



1.1



1.1



1.0



1.6



0.2



0.2



±SD



0.7



0.8



0.6



1.0



0.0



0.0



Bilirubin (mg/dL)



Neg



-



-



-



-



5



5



1



8



7



8



5



-



-



3



2



3



2



5



-



-



Ketones (mg/dL)



5



-



-



-



-



-



3



Neg



10



10



10



10



5



2



Blood (Ery/µL)



Neg



10



10



10



10



-



-



Ca10



-



-



-



-



1



-



Ca80



-



-



-



-



2



3



>=Ca200



-



-



-



-



2



2



 


Proteins (mg/dL)



Neg



-



-



-



-



-



1



Trace



-



-



-



-



5



3



30



-



-



-



-



-



1



>=300



10



10



10



10



-



-



Nitrite



Neg



10



10



10



10



5



4



Pos



-



-



-



-



-



1



Leucocytes (Leu/µL)



Neg



-



-



-



-



2



-



Ca70



5



3



6



3



-



4



Ca15



-



1



-



-



3



1



Ca125



4



6



4



6



-



-



>=Ca500



1



-



-



1



-



-



Glucose (mg/dL)



Neg



10



10



10



10



5



5



Micro albumin (MALB) (mg/dL)



15



-



-



-



-



-



1



>15



10



10



10



10



5



4



Microscopic



Epithelial cells



0



6



6



7



5



2



3



0-1



3



2



3



4



2



1



1-2



1



2



-



1



1



1



Casts



Absent



10



10



10



10



5



5



Crystals



Present



10



10



10



10



5



5



*: Statistically significant (P<0.05).


 


Table 10. Summary of absolute organ weights (g) record










































































































































































































































































































Group, Sex & Dose


(mg/kg/day)



 



Adrenals



Thymus



Spleen



Testes



Epididymides



Heart



Kidneys



Brain



Liver



Prostate+Seminal vesicles with coagulating glands (PSC)



Thyroid along with parathyroid



Pituitary



G1, M & 0



Mean



0.0607



0.3146



1.0518



3.4852



1.4990



1.3322



2.4524



2.2109



10.1183



3.4192



0.0311



0.0118



±SD



0.0087



0.0286



0.0837



0.1792



0.0745



0.0992



0.1857



0.1872



0.7609



0.3992



0.0071



0.0013



n



10



10



10



10



10



10



10



10



10



10



10



10



G2, M & 100



Mean



0.0725



0.2701*



1.0986



3.3317



1.5310



1.3420



2.6263



2.0286*



10.1493



3.3746



0.0301



0.0137*



±SD



0.0150



0.0408



0.1116



0.1725



0.1082



0.0555



0.2540



0.0802



0.6137



0.4279



0.0058



0.0015



n



10



10



10



10



10



10



10



10



10



10



10



10



G3, M & 300



Mean



0.0657



0.2874



1.4000*



3.4346



1.5825



1.3618



2.4372



1.9158*



9.6114



3.1373



0.0296



0.0135*



±SD



0.0155



0.0511



0.2514



0.2645



0.1377



0.0573



0.1292



0.0736



0.2494



0.4327



0.0058



0.0013



n



10



10



10



10



10



10



10



10



10



10



10



10



G4, M & 1000



Mean



0.0614



0.1822*



1.0159



3.4937



1.5284



1.3548



2.3200



2.0210*



10.0858



3.1556



0.0309



0.0126



±SD



0.0056



0.0226



0.0657



0.1809



0.1050



0.0449



0.2220



0.0586



0.3796



0.4620



0.0073



0.0018



n



10



10



10



10



10



10



10



10



10



10



10



10



G1R, M & 0



Mean



0.0879



0.4786



1.3579



3.5237



1.8048



1.4099



3.2460



2.1860



11.1301



3.3972



0.0320



0.0131



±SD



0.0073



0.3408



0.4710



0.2732



0.3187



0.1364



0.0254



0.1077



0.9636



0.5847



0.0062



0.0009



n



5



5



5



5



5



5



5



5



5



5



5



5



G4R, M & 1000



Mean



0.0821



0.2844



1.1994



3.5459



1.5579



1.6577



3.0017*



2.1412



10.6402



3.2913



0.0341



0.0124



±SD



0.0161



0.0765



0.1641



0.2108



0.1186



0.3634



0.1140



0.2365



0.0840



0.3513



0.0088



0.0011



n



5



5



5



5



5



5



5



5



5



5



5



5



 























































































































































































































































































Group, Sex & Dose


(mg/kg/day)



 



Adrenals



Thymus



Spleen



Ovaries


With oviduct



Uterus with cervix



Heart



Kidneys



Brain



Liver



Thyroid along with parathyroid



Pituitary



G1, F & 0



Mean



0.0899



0.2970



0.7721



0.1834



0.7494



0.8778



1.5773



2.0595



6.6549



0.0269



0.0151



±SD



0.0162



0.0343



0.0695



0.0188



0.1149



0.0673



0.1618



0.1884



0.2953



0.0041



0.0020



n



10



10



10



10



10



10



10



10



10



10



10



G2, F & 100



Mean



0.0723*



0.3263



0.7774



0.1799



0.5693*



0.9394



1.6403



1.9483



7.1163



0.0250



0.0153



±SD



0.0110



0.0500



0.1080



0.0296



0.0691



0.0671



0.1486



0.0834



0.5037



0.0042



0.0027



n



10



10



10



10



10



10



10



10



10



10



10



G3, F & 300



Mean



0.0642*



0.3099



0.6830



0.1684



0.5218*



0.9030



1.6191



1.8763*



7.5492*



0.0251



0.0146



±SD



0.0082



0.0525



0.0854



0.0169



0.0676



0.0764



0.1134



0.0461



0.4368



0.0042



0.0027



n



10



10



10



10



10



10



10



10



10



10



10



G4, F & 1000



Mean



0.0814



0.3217



0.8393



0.1640



0.9336*



1.0134*



1.6846



1.9466



7.2948*



0.0288



0.0161



±SD



0.0114



0.0749



0.0765



0.0155



0.1281



0.0682



0.1570



0.0859



0.7540



0.0048



0.0024



n



10



10



10



10



10



10



10



10



10



10



10



G1R, F & 0



Mean



0.0791



0.2851



0.6121



0.1990



0.8530



1.0021



1.8805



1.9020



7.9569



0.0240



0.0146



±SD



0.0149



0.0544



0.2953



0.0449



0.2254



0.0448



0.0744



0.0497



0.8616



0.0038



0.0020



n



5



5



5



5



5



5



5



5



5



5



5



G4R, F & 1000



Mean



0.0870



0.2979



0.7606



0.2072



0.9065



1.0576



2.0242



1.9721



8.5214



0.0271



0.0148



±SD



0.0094



0.0570



0.1371



0.0731



0.2404



0.0867



0.3460



0.1723



1.2695



0.0041



0.0019



n



5



5



5



5



5



5



5



5



5



5



5



*: Statistically significant (P<0.05).


 


Table 11. Summary of organ weight relative to fasting body weight (%) record





























































































































































































































































































































Group, Sex & Dose


(mg/kg/day)


 

Fasting Body Weight (g)



Adrenals



Thymus



Spleen



Testes



Epididymides



Heart



Kidneys



Brain



Liver



Prostate+Seminal vesicles with coagulating glands (PSC)



Thyroid along with parathyroid



Pituitary



G1, M & 0



Mean



373.20



0.0166



0.0852



0.2859



0.9494



0.4081



0.3611



0.6641



0.6007



2.7473



0.9309



0.0085



0.0032



±SD



52.77



0.0036



0.0096



0.0398



0.1314



0.0542



0.0395



0.0650



0.0780



0.3336



0.1595



0.0023



0.0003



n



10



10



10



10



10



10



10



10



10



10



10



10



10



G2, M & 100



Mean



400.65



0.0182



0.0678*



0.2757



0.8382



0.3845



0.3378



0.6582



0.5111*



2.5453



0.8478



0.0076



0.0034



±SD



36.40



0.0037



0.0107



0.0309



0.0914



0.0390



0.0367



0.0628



0.0625



0.1836



0.1243



0.0018



0.0005



n



10



10



10



10



10



10



10



10



10



10



10



10



10



G3, M & 300



Mean



367.64



0.0182



0.0788



0.3840*



0.9459



0.4348



0.3751



0.6728



0.5282*



2.6488



0.8609



0.0081



0.0037



±SD



43.85



0.0050



0.0150



0.0759



0.1297



0.0560



0.0468



0.0974



0.0686



0.3334



0.1311



0.0012



0.0006



n



10



10



10



10



10



10



10



10



10



10



10



10



10



G4, M & 1000



Mean



353.38



0.0175



0.0517*



0.2888



0.9967



0.4355



0.3852



0.6587



0.5749



2.8672



0.8989



0.0088



0.0036



±SD



28.14



0.0023



0.0065



0.0258



0.1200



0.0503



0.0284



0.0638



0.0458



0.2068



0.1517



0.0024



0.0006



n



10



10



10



10



10



10



10



10



10



10



10



10



10



G1R, M & 0



Mean



398.51



0.0222



0.1227



0.3461



0.8897



0.4542



0.3541



0.8177



0.5503



2.8181



0.8557



0.0081



0.0033



±SD



29.21



0.0025



0.0951



0.1364



0.1160



0.0875



0.0265



0.0540



0.0402



0.4468



0.1614



0.0020



0.0004



n



5



5



5



5



5



5



5



5



5



5



5



5



5



G4R, M & 1000



Mean



394.91



0.0210



0.0719



0.3041



0.9012



0.3943



0.4168



0.7614



0.5447



2.7003



0.8361



0.0086



0.0031



±SD



21.49



0.0052



0.0181



0.0404



0.0920



0.0147



0.0715



0.0397



0.0761



0.1373



0.1055



0.0021



0.0004



n



5



5



5



5



5



5



5



5



5



5



5



5



5



 










































































































































































































































































































Group, Sex & Dose


(mg/kg/day)



 



Fasting Body Weight (g)



Adrenals



Thymus



Spleen



Ovaries


With oviduct



Uterus with cervix



Heart



Kidneys



Brain



Liver



Thyroid along with parathyroid



Pituitary



G1, F & 0



Mean



234.52



0.0385



0.1268



0.3304



0.0785



0.3212



0.3744



0.6737



0.8808



2.8411



0.0115



0.0064



±SD



11.76



0.0074



0.0151



0.0383



0.0097



0.0578



0.0241



0.0739



0.0982



0.1359



0.0020



0.0010



n



10



10



10



10



10



10



10



10



10



10



10



10



G2, F & 100



Mean



240.63



0.0301*



0.1370



0.3256



0.0749



0.2382*



0.3922



0.6833



0.8132



2.9690



0.0104



0.0064



±SD



19.68



0.0044



0.0268



0.0565



0.0116



0.0379



0.0367



0.0594



0.0567



0.2456



0.0018



0.0015



n



10



10



10



10



10



10



10



10



10



10



10



10



G3, F & 300



Mean



236.62



0.0273*



0.1312



0.2902



0.0713



0.2221*



0.3827



0.6859



0.7958*



3.2042*



0.0107



0.0061



±SD



13.56



0.0044



0.0238



0.0449



0.0077



0.0378



0.0370



0.0583



0.0576



0.3139



0.0021



0.0009



n



10



10



10



10



10



10



10



10



10



10



10



10



G4, F & 1000



Mean



246.80



0.0334



0.1307



0.3423



0.0671*



0.3836*



0.4144*



0.6883



0.7978



2.9773



0.0118



0.0066



±SD



31.05



0.0059



0.0280



0.0297



0.0089



0.0732



0.0415



0.0789



0.0869



0.3381



0.0025



0.0013



n



10



10



10



10



10



10



10



10



10



10



10



10



G1R, F & 0



Mean



245.78



0.0321



0.1164



0.2524



0.0810



0.3505



0.4087



0.7667



0.7758



3.2335



0.0097



0.0059



±SD



12.20



0.0052



0.0244



0.1240



0.0186



0.1093



0.0312



0.0503



0.0517



0.2736



0.0014



0.0008



n



5



5



5



5



5



5



5



5



5



5



5



5



G4R, F & 1000



Mean



263.62



0.0332



0.1130



0.2880



0.0792



0.3460



0.4033



0.7697



0.7558



3.2390



0.0104



0.0056



±SD



30.13



0.0042



0.0180



0.0349



0.0280



0.0945



0.0345



0.1156



0.1024



0.3873



0.0018



0.0007



n



5



5



5



5



5



5



5



5



5



5



5



5



*: Statistically significant (P<0.05).


 


Table 12. Incidences of test-item related changes in thymus in male rats.
































Organ/FindingG1G2G3G4G1RG4R
Number examined1010101055
Thymus : Cellularity, decreased, lymphocytes---5--

Key: - = No incidence, G = Group, R: Recovery

Conclusions:
In a 90-day repeated dose toxicity study conducted in Sprague Dawley Rats, the NOAEL of the test item for both sexes is 1000 mg/kg bw/day.
Executive summary:

A study was conducted to assess the toxic potential of the test item when administered for a period of 90 consecutive days repeatedly to Sprague Dawley rats, according to OECD 408 Guideline (GLP study). Four groups (G1, G2, G3 and G4) of 10 animals of each sex and two recovery groups (G1R and G4R) were tested, being G1 and G1R the vehicle control groups (0.5% w/v CMC). The treatment groups had the following concentrations: 100, 300 and 1000 mg/kg bw. The vehicle and test item formulations were administered orally by gavage to the animals at an equivolume of 10 mL/kg bw. 


No clinical signs of toxicity and no mortality/morbidity were noted, as well as no changes in body weight, feed consumption and gross pathology in all groups. Besides, no effects in the ophthalmological and neurological/functional examinations were observed. No treatment related changes were observed in hematology, coagulation, clinical chemistry and urinalysis parameters, as well as no treatment related changes were observed in thyroid hormones (T3, T4 and TSH) and in vaginal smear examination. No gross pathological changes were observed. Decrease in weight of thymus in high dose males (G4M) was noted which was correlated with microscopic changes like minimal, decreased cellularity of lymphocytes. This change was found to be reversible at the end of 28-day treatment free recovery period as similar changes were not observed in recovery group. Further, as this change was minimal in severity and was found to be reversible, it is considered as non-adverse finding.


Therefore, based on the obtained results from this 90-day repeated dose toxicity study conducted in Sprague Dawley Rats, the NOAEL is estimated as 1000 mg/kg bw/day under experimental conditions employed and the NOEL is 300 mg/kg bw/day for males considering the non-adverse reversible minimal microscopic changes in thymus at 1000 mg/kg/day dose males.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
12 March 2020 to 19 May 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: In-house
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7 weeks
- Weight at study initiation: Males: 166.04-168.65 g (Mean), Females: 147.46- 148.12 g (Mean)
- Fasting period before study: no
- Housing: Two animals of same sex were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted feed and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
- Diet (e.g. ad libitum): Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to the animals throughout the the acclimatization and experimental period.
- Water (e.g. ad libitum): Water was provided ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: six days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.0°C to 23.6°C
- Humidity (%): 46% to 66%
- Air changes (per hr): 12-15 Air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle

IN-LIFE DATES: From: 12.03.2020 To: 29.04.2020
Route of administration:
oral: gavage
Details on route of administration:
The test item was administered through oral route using stainless steel (gavage) cannula.
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5%
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item formulations were prepared daily before dose administration. The required quantity of test item was weighed separately and transferred, mixed, and triturated well in a mortar. A small quantity of vehicle was added and triturated until a homogenous suspension was formed and thereafter the entire quantity of the formulation was transferred to measuring cylinder. A small quantity of vehicle was added to rinse the mortar and this was transferred into the measuring cylinder. The rinsing procedure of mortar and pestle was repeated (many times) to ensure complete transfer of the contents to the measuring cylinder. Finally, the volume was adjusted to required mark in measuring cylinder with vehicle to get a desired concentration of 10, 30 and 100 mg/mL of test item for low, mid, and high dose groups, respectively.

VEHICLE
- Justification for use and choice of vehicle (if other than water): homogenous suspension with test item
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg
- Lot/batch no. (if required): BCBN1690V (Sigma Aldrich)
- Expiry Date: 9 March 2024
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
The stability and homogeneity of the test item in dose formulations were not established under this dose range finding study. However, freshly prepared test item formulations were administered to the animals and homogeneity was achieved by thorough stirring.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
G1- control group
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
G2
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
G3
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
G4
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
G1R- control recovery
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
G4R- high dose recovery
No. of animals per sex per dose:
5 males and 5 females for the main and recovery groups.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses of 100, 300 and 1000 mg/kg/day were selected based on available data.
- Fasting period before blood sampling for clinical biochemistry: overnight
- Post-exposure recovery period in satellite groups: 14 days
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily for clinical signs of toxicity and twice daily for mortality and morbidity
- Cage side observations checked: clinical signs, mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before initiation of the treatment and at weekly intervals thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: on the Day 1 before test item administration and weekly thereafter

FOOD CONSUMPTION :
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, weekly

FOOD EFFICIENCY: No
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before start of treatment and during Week 4 for vehicle control and high dose main group animals (G1, and G4) and during Week 6 for recovery group animals (G1R and G4R).
- Dose groups that were examined: G1, G4, G1R and G4R

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on experimental day 29 and 43 for main and recovery groups respectively
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: Yes
- How many animals: all of them
- Parameters checked: Hemoglobin concentration, hematocrit, erythrocyte count, total leukocyte count, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, mean platelet volume, reticulocyte count, absolute reticulocyte count, differential leucocytes count, absolute differential leucocytes count, prothrombin time and
activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on experimental day 29 and 43 for main and recovery groups respectively
- Animals fasted: Yes
- How many animals: all of them
- Parameters checked: Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, cholinesterase, total protein, albumin, total bilirubin, glucose, total cholesterol, creatinine, urea, triglycerides, phosphorous, calcium, high density lipoprotein, low density lipoprotein, blood urea nitrogen, globulin, albumin/globulin ratio, sodium, potassium and chloride.

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: Yes
- Time schedule for collection of urine: on experimental day 29 and 43 for main and recovery groups respectively
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Blood, bilirubin, urobilinogen, ketones, protein, glucose, leuocytes, pH, nitrite and specific gravity.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during Week 4 for vehicle control and high dose main group animals (G1 and G4) and during Week 6 for recovery group animals (G1R and G4R).
- Dose groups that were examined: G1, G4, G1R and G4R
- Battery of functions tested: sensory activity / grip strength / motor activity / other: home cage observations, handling observations, open field observations, neuromuscular observations, physiological observation (rectal temperature)

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were fasted overnight (water allowed) and on the day of necropsy (day 29 for main groups, day 43 for recovery groups) all animals were weighed and then subjected to necropsy. Some organs (see Table 1) were collected, weighed and preserved for histopathology.
Necropsy included an examination of external surfaces, external orifices, abdominal, thoracic and cranial cavities, organs and tissues.

HISTOPATHOLOGY: Yes
Histopathological examination was carried out on the preserved organs of the vehicle control (G1) and high dose (G4) group animals.
Statistics:
The data was subjected to statistical analysis. The computer printout of the data (in the form of appendix) was verified with the original raw data. After verification, the data was subjected to statistical analysis using SPSS software version 22. Body weight, body weight gain (percent change in body weight with respect to Day 1 body weight), feed consumption, organ weights and ratios, haematological and clinical chemistry estimations and urine analysis parameters (urobilinogen, pH and specific gravity), FOB parameters (rearing, urination, defecation, excessive grooming, body temperature, grip strength, motor activity and hind limb foot splay) were subjected to statistical analysis. One-way ANOVA followed by Dunnett’s post-test was done for different treatment groups comparing with the control group data. Comparison of means between recovery groups was done using student ‘t’ test. All analysis and comparisons were evaluated at the 95% level of confidence (p<0.05).
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
A non-dose dependent statistically significant increase in mean body weight was observed in G3 females (Day 22) and considered as incidental due to lack of dose dependency. In G4R females, statistically significant decrease in percent change in body weight with respect to Day 1 (during Days 1-8) is considered incidental as the mean body weights of high dose groups are unaffected in either sex.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In main group males, non-dose dependent statistically significant increase in absolute and percent basophils in G2 and decrease in APTT in G3 were observed but are considered as incidental due to the absence of dose responsiveness.
In G4R males, statistically significant decrease in PT was noted which is considered incidental in the absence of similar change in other sex.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In G4R females, decrease in urea and BUN and increase in cholinesterase were noted. All these changes noted are considered as incidental in the absence of similar changes in other sex.
Endocrine findings:
not examined
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Absolute Organ Weight: In main groups, statistically significant increase in thymus (G2M), increase in prostate+seminal vesicles and coagulating glands (G3M and G4M), increase in epididymis (G4M) and increase in brain weight (G3F) was observed.
Organ Weight Relative to body weight: In main groups, statistically significant increase in thymus (G2M), increase in prostate+seminal vesicles and coagulating glands (G3M and G4M) and increase in epididymis (G3M and G4M) was observed.
All these changes are considered incidental and not related to test item as there were no associated histopathological or gross pathological changes noted in high dose group animals.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Few microscopic findings were observed in this study such as ultimobranchial cyst in thyroid gland, cast(s) in the kidneys and all other findings. However, these effects noted were considered incidental as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats of this age.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed up to the highest dose tested
Key result
Critical effects observed:
no

Table 2. Summary of clinical signs of toxicity, detailed clinical examination and mortality record. 




















































































Group, Sex & Dose (mg/kg/day)



No. of


Animals



Clinical Signs of Toxicity



Mortality                                (No. of Incidences/


No. of Animals)



G1, M & 0



5



N



0/5



G2, M & 100



5



N



0/5



G3, M & 300



5



N



0/5



G4, M & 1000



5



N



0/5



G1, F & 0



5



N



0/5



G2, F & 100



5



N



0/5



G3, F & 300



5



N



0/5



G4, F & 1000



5



N



0/5



G1R, M & 0



5



N



0/5



G4R, M & 1000



5



N



0/5



G1R, F & 0



5



N



0/5



G4R, F & 1000



5



N



0/5



M: Male; F: Female; R: Recovery; N: Normal


 


Table 3. Summary of body weights (g) record























































































































































































































































Group, Sex & Dose


(mg/kg/day)


 

Body Weight (g) on Days


 
 

1



8



15



22



28


 

G1, M & 0



Mean



166.80



201.74



236.57



281.66



301.51


 

±SD



10.86



12.52



18.92



23.04



26.55


 

n



5



5



5



5



5


 

G2, M & 100



Mean



166.36



206.35



239.31



279.76



293.08


 

±SD



9.80



7.43



12.68



18.50



20.72


 

n



5



5



5



5



5


 

G3, M & 300



Mean



166.69



205.95



234.17



271.71



283.77


 

±SD



11.12



14.88



16.17



16.46



18.39


 

n



5



5



5



5



5


 

G4, M & 1000



Mean



166.04



203.02



238.30



281.55



292.37


 

±SD



10.44



16.29



21.52



20.35



20.73


 

n



5



5



5



5



5


 

G1, F & 0



Mean



147.46



169.25



181.46



204.32



213.73


 

±SD



7.75



6.07



5.68



8.37



8.93


 

n



5



5



5



5



5


 

G2, F & 100



Mean



147.92



164.33



177.30



205.17



212.26


 

±SD



7.84



9.01



5.70



7.83



8.13


 

n



5



5



5



5



5


 

G3, F & 300



Mean



148.12



170.99



190.39



217.20*



223.11


 

±SD



6.58



7.16



6.88



4.53



5.73


 

n



5



5



5



5



5


 

G4, F & 1000



Mean



147.52



166.86



176.27



205.05



210.28


 

±SD



6.53



7.73



8.55



9.57



9.58


 

n



5



5



5



5



5


 

 
















































































































































Group, Sex & Dose


(mg/kg/day)


 

Body Weight (g) on Days



1



8



15



22



28



35



42



G1R, M & 0



Mean



166.46



203.57



231.27



274.77



289.08



300.24



312.84



±SD



8.27



10.92



11.50



15.46



17.72



17.05



16.92



n



5



5



5



5



5



5



5



G4R, M & 1000



Mean



168.65



201.65



227.56



268.95



278.78



291.24



303.39



±SD



7.77



11.01



13.43



19.37



20.40



20.70



21.25



n



5



5



5



5



5



5



5



G1R, F & 0



Mean



146.98



172.87



188.75



215.04



222.92



230.84



238.60



±SD



4.91



9.91



14.50



13.22



13.25



12.95



12.84



n



5



5



5



5



5



5



5



G4R, F & 1000



Mean



147.97



165.96



179.73



203.68



210.24



219.45



227.09



±SD



4.83



6.07



5.52



9.94



11.06



11.54



11.08



n



5



5



5



5



5



5



5



*: Statistically significant (p<0.05).


 


Table 4. Summmary of percent change in body weight (%) with respect to day 1 record






























































































































































































































Group, Sex & Dose


(mg/kg/day)


 

Percent Change in Body Weight (%) during Days


 
 

1 to 8



1 to 15



1 to 22



1 to 28


 

G1, M & 0



Mean



21.06



41.99



68.99



80.89


 

±SD



5.08



9.95



10.78



12.75


 

n



5



5



5



5


 

G2, M & 100



Mean



24.20



44.00



68.36



76.33


 

±SD



3.94



6.45



10.25



10.80


 

n



5



5



5



5


 

G3, M & 300



Mean



23.52



40.48



63.15



70.40


 

±SD



1.01



2.51



6.09



7.60


 

n



5



5



5



5


 

G4, M & 1000



Mean



22.17



43.39



69.51



76.02


 

±SD



2.57



5.97



3.58



2.70


 

n



5



5



5



5


 

G1, F & 0



Mean



147.46



169.25



181.46



204.32


 

±SD



7.75



6.07



5.68



8.37


 

n



5



5



5



5


 

G2, F & 100



Mean



147.92



164.33



177.30



205.17


 

±SD



7.84



9.01



5.70



7.83


 

n



5



5



5



5


 

G3, F & 300



Mean



148.12



170.99



190.39



217.20


 

±SD



6.58



7.16



6.88



4.53


 

n



5



5



5



5


 

G4, F & 1000



Mean



147.52



166.86



176.27



205.05


 

±SD



6.53



7.73



8.55



9.57


 

n



5



5



5



5


 

 



































































































































Group, Sex & Dose


(mg/kg/day)


 

Percent Change in Body Weight (%) during Days



1 to 8



1 to 15



1 to 22



1 to 28



1 to 35



1 to 42



G1R, M & 0



Mean



22.32



39.06



65.36



73.97



80.74



88.34



±SD



3.84



6.58



12.07



13.36



14.11



14.52



n



5



5



5



5



5



5



G4R, M & 1000



Mean



19.55



34.92



59.42



65.25



72.66



79.87



±SD



3.10



4.79



7.66



8.19



8.65



8.85



n



5



5



5



5



5



5



G1R, F & 0



Mean



146.98



172.87



188.75



215.04



222.92



230.84



±SD



4.91



9.91



14.50



13.22



13.25



12.95



n



5



5



5



5



5



5



G4R, F & 1000



Mean



147.97*



165.96



179.73



203.68



210.24



219.45



±SD



4.83



6.07



5.52



9.94



11.06



11.54



n



5



5



5



5



5



5



*: Statistically significant (p<0.05).


 


Table 5. Summmary of haematology record


















































































































































































































































Group, Sex & Dose (mg/kg/day)



 



Total Leucocyte Count



Total Erythrocyte Count 



Hemoglobin



Haematocrit



Mean Corpuscular Volume



Mean Corpuscular Hemoglobin



Mean Corpuscular Hemoglobin Concentration 



Platelet Count



  (WBC)        



 (RBC)    



(HGB)      



 (HCT)   



  (MCV)     



(MCH)



(MCHC)      



  (PLT)



(103 cells/µL)



(106 cells/µL)



(g/dL)



(%)



(fL)



(pg)



(g/dL)



(103 cells/µL)



G1, M & 0



Mean



10.85



7.23



15.02



46.20



64.08



20.86



32.54



847.80



±SD



1.20



0.62



0.89



2.66



3.03



0.88



0.17



94.52



n



5



5



5



5



5



5



5



5



G2, M & 100



Mean



13.28



7.27



14.94



45.92



63.56



20.70



32.56



877.80



±SD



3.08



0.88



0.96



3.21



4.31



1.29



0.54



141.16



n



5



5



5



5



5



5



5



5



G3, M & 300



Mean



10.67



7.00



14.54



44.52



63.62



20.78



32.66



842.40



±SD



1.30



0.41



0.55



1.90



1.21



0.55



0.67



104.14



n



5



5



5



5



5



5



5



5



G4, M & 1000



Mean



9.22



7.36



14.98



45.88



62.30



20.32



32.62



917.80



±SD



2.77



0.40



0.93



2.57



0.44



0.38



0.54



152.11



n



5



5



5



5



5



5



5



5



G1R, M & 0



Mean



10.34



7.68



14.50



44.34



58.08



18.94



32.64



741.80



±SD



1.94



0.70



1.01



2.95



5.93



1.70



0.49



198.71



n



5



5



5



5



5



5



5



5



G4R, M & 1000



Mean



8.61



7.80



14.86



45.44



58.44



19.12



32.72



752.60



±SD



2.37



0.59



0.68



1.30



4.01



1.16



0.80



184.01



n



5



5



5



5



5



5



5



5



 


















































































































































































































































Group, Sex & Dose (mg/kg/day)



 



Mean Platelet Volume



Reticulocyte Count



Neutrophils



Lymphocytes



Monocytes



Eosinophils



Basophils



Absolute Reticulocyte Count  



(MPV)   



(Retic)



(Neut)



(Lymph)



(Mono)



(Eos)



(Baso)



(Retic)



(fL)



(%)



(%)



(%)



(%)



(%)



(%)



(109 cells/L)



G1, M & 0



Mean



10.24



3.56



19.80



74.68



2.96



1.18



0.50



251.02



±SD



0.21



1.36



6.16



6.18



0.34



0.58



0.20



73.84



n



5



5



5



5



5



5



5



5



G2, M & 100



Mean



10.20



4.21



18.18



75.84



3.32



0.94



0.80*



290.50



±SD



0.16



2.83



4.69



4.96



0.89



0.66



0.22



153.34



n



5



5



5



5



5



5



5



5



G3, M & 300



Mean



10.24



4.12



17.50



77.82



2.54



0.88



0.46



288.68



±SD



0.55



1.22



5.44



6.32



0.74



0.55



0.09



86.81



n



5



5



5



5



5



5



5



5



G4, M & 1000



Mean



10.06



2.29



23.44



71.16



3.42



0.74



0.50



167.52



±SD



0.54



0.29



2.77



2.46



0.58



0.30



0.14



15.42



n



5



5



5



5



5



5



5



5



G1R, M & 0



Mean



7.50



1.96



18.14



75.40



4.46



0.94



0.32



147.74



±SD



0.43



0.63



4.34



4.55



0.81



0.40



0.13



41.46



n



5



5



5



5



5



5



5



5



G4R, M & 1000



Mean



7.60



1.90



19.34



74.02



4.34



1.22



0.30



146.00



±SD



0.56



0.68



10.77



10.97



0.98



0.45



0.07



47.02



n



5



5



5



5



5



5



5



5



 





























































































































































































































Group, Sex & Dose (mg/kg/day)



 



Absolute Neutrophils   



Absolute Lymphocytes  



Absolute Monocytes



Absolute Eosinophils



Absolute  Basophils       



Prothrombin Time  



Activated Prothrombin Time       



(Neut)



(Lymph)



(Mono)



(Eos)



(Baso)



(PT)



(APTT)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(Seconds)



(Seconds)



G1, M & 0



Mean



2.11



8.14



0.32



0.13



0.06



23.80



28.44



±SD



0.58



1.39



0.06



0.05



0.01



1.61



1.37



n



5



5



5



5



5



5



5



G2, M & 100



Mean



2.39



10.08



0.45



0.13



0.10*



23.26



28.52



±SD



0.69



2.55



0.17



0.12



0.04



2.68



4.17



n



5



5



5



5



5



5



5



G3, M & 300



Mean



1.92



8.25



0.27



0.10



0.05



23.00



24.16*



±SD



0.85



0.50



0.11



0.06



0.01



0.96



1.28



n



5



5



5



5



5



5



5



G4, M & 1000



Mean



2.22



6.51



0.30



0.07



0.04



21.38



24.48



±SD



0.88



1.78



0.09



0.03



0.01



1.34



2.24



n



5



5



5



5



5



5



5



G1R, M & 0



Mean



1.88



7.79



0.47



0.09



0.03



25.24



16.80



±SD



0.63



1.45



0.16



0.03



0.02



2.53



4.72



n



5



5



5



5



5



5



5



G4R, M & 1000



Mean



1.51



6.53



0.38



0.11



0.03



17.40*



21.66



±SD



0.40



2.38



0.14



0.06



0.01



2.27



2.36



n



5



5



5



5



5



5



5



 


















































































































































































































































Group, Sex & Dose (mg/kg/day)



 



Total Leucocyte Count



Total Erythrocyte Count 



Hemoglobin



Haematocrit



Mean Corpuscular Volume



Mean Corpuscular Hemoglobin



Mean Corpuscular Hemoglobin Concentration 



Platelet Count



  (WBC)        



 (RBC)    



(HGB)      



 (HCT)   



  (MCV)     



(MCH)



(MCHC)      



  (PLT)



(103 cells/µL)



(106 cells/µL)



(g/dL)



(%)



(fL)



(pg)



(g/dL)



(103 cells/µL)



G1, F & 0



Mean



8.52



7.20



14.86



44.48



61.82



20.66



33.40



920.40



±SD



2.54



0.24



0.47



1.74



2.79



0.69



0.42



163.75



n



5



5



5



5



5



5



5



5



G2, F & 100



Mean



10.67



6.27



13.18



41.52



66.64



20.82



31.48



808.60



±SD



4.41



0.95



2.51



3.87



5.70



1.59



3.68



208.67



n



5



5



5



5



5



5



5



5



G3, F & 300



Mean



8.86



6.68



13.90



42.96



65.38



20.98



32.36



906.00



±SD



1.38



0.87



0.88



2.38



11.14



2.20



1.85



135.69



n



5



5



5



5



5



5



5



5



G4, F & 1000



Mean



8.79



6.82



13.52



42.80



63.50



19.84



31.54



1021.20



±SD



3.56



0.82



1.53



0.92



7.51



1.12



3.18



90.67



n



5



5



5



5



5



5



5



5



G1R, F & 0



Mean



6.99



7.01



13.76



41.82



59.80



19.72



33.00



819.40



±SD



2.56



0.55



0.53



2.10



3.55



1.13



0.48



69.72



n



5



5



5



5



5



5



5



5



G4R, F & 1000



Mean



7.13



7.19



14.08



42.70



59.56



19.62



32.96



798.00



±SD



1.94



0.51



0.51



1.25



3.29



0.96



0.71



86.47



n



5



5



5



5



5



5



5



5



 


















































































































































































































































Group, Sex & Dose (mg/kg/day)



 



Mean Platelet Volume



Reticulocyte Count



Neutrophils



Lymphocytes



Monocytes



Eosinophils



Basophils



Absolute Reticulocyte Count  



(MPV)   



(Retic)



(Neut)



(Lymph)



(Mono)



(Eos)



(Baso)



(Retic)



(fL)



(%)



(%)



(%)



(%)



(%)



(%)



(109 cells/L)



G1, F & 0



Mean



9.86



2.49



24.12



70.70



2.78



1.02



0.50



180.16



±SD



0.44



0.79



14.34



13.84



0.65



0.40



0.16



61.01



n



5



5



5



5



5



5



5



5



G2, F & 100



Mean



10.78



4.75



18.62



76.36



2.58



0.92



0.74



282.24



±SD



0.99



2.61



3.95



4.32



0.47



0.28



0.66



133.39



n



5



5



5



5



5



5



5



5



G3, F & 300



Mean



10.28



4.56



17.88



77.10



2.72



1.12



0.40



287.20



±SD



0.54



2.75



5.70



5.77



0.29



0.24



0.19



139.86



n



5



5



5



5



5



5



5



5



G4, F & 1000



Mean



10.66



5.49



18.40



77.32



2.02



0.84



0.36



343.76



±SD



0.54



5.08



3.40



3.29



0.58



0.32



0.09



262.77



n



5



5



5



5



5



5



5



5



G1R, F & 0



Mean



7.42



2.27



18.78



75.40



3.34



1.28



0.32



153.90



±SD



0.68



1.63



6.44



7.63



1.18



0.83



0.11



97.40



n



5



5



5



5



5



5



5



5



G4R, F & 1000



Mean



7.52



1.81



25.32



67.66



3.60



1.34



0.22



127.82



±SD



0.68



0.68



4.51



2.35



0.97



0.64



0.04



38.55



n



5



5



5



5



5



5



5



5



 





























































































































































































































Group, Sex & Dose (mg/kg/day)



 



Absolute Neutrophils   



Absolute Lymphocytes  



Absolute Monocytes



Absolute Eosinophils



Absolute  Basophils       



Prothrombin Time  



Activated Prothrombin Time       



(Neut)



(Lymph)



(Mono)



(Eos)



(Baso)



(PT)



(APTT)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(Seconds)



(Seconds)



G1, F & 0



Mean



1.97



6.10



0.24



0.09



0.04



25.00



24.92



±SD



1.07



2.46



0.10



0.05



0.02



0.45



1.15



n



5



5



5



5



5



5



5



G2, F & 100



Mean



1.91



8.19



0.27



0.09



0.10



26.76



28.12



±SD



0.67



3.53



0.13



0.04



0.12



3.35



4.03



n



5



5



5



5



5



5



5



G3, F & 300



Mean



1.57



6.85



0.24



0.10



0.04



24.72



25.28



±SD



0.53



1.22



0.02



0.03



0.02



1.23



1.11



n



5



5



5



5



5



5



5



G4, F & 1000



Mean



1.67



6.75



0.18



0.07



0.03



25.16



27.22



±SD



0.97



2.54



0.07



0.03



0.01



0.60



2.90



n



5



5



5



5



5



5



5



G1R, F & 0



Mean



1.26



5.33



0.25



0.08



0.02



22.04



19.74



±SD



0.51



2.01



0.17



0.05



0.02



1.81



4.86



n



5



5



5



5



5



5



5



G4R, F & 1000



Mean



1.77



4.83



0.25



0.10



0.02



22.68



19.72



±SD



0.49



1.38



0.10



0.06



0.01



0.95



5.37



n



5



5



5



5



5



5



5



*: Statistically significant (p<0.05).


 


Table 6. Summmary of clinical chemistry record




























































































































































































































































































Group, Sex & Dose (mg/kg/day)



 



Glucose



Urea



Creatinine



Total Cholesterol



Triglycerides



Total Protein



Albumin



Alanine aminotransferase



Aspartate aminotransferase



Alkaline phosphatase



 (GLU)


 

 (CRE)



(CHO)



(TRI)



(TPR)



  (ALB)



(ALT)



(AST)



(ALP)



(mg/dL)



(mg/dL)



 (mg/dL)



(mg/dL)



(mg/dL)



  (g/dL)



  (g/dL)



 (U/L)



 (U/L)



 (U/L)



G1, M & 0



Mean



76.40



37.88



0.41



49.00



56.60



6.22



3.23



64.20



114.00



180.60



±SD



6.80



2.32



0.03



11.40



10.41



0.43



0.11



12.30



27.92



27.25



n



5



5



5



5



5



5



5



5



5



5



G2, M & 100



Mean



79.00



34.68



0.40



55.20



53.80



6.08



3.24



58.00



100.80



193.00



±SD



7.25



1.95



0.05



12.70



10.96



0.33



0.21



6.63



7.60



37.01



n



5



5



5



5



5



5



5



5



5



5



G3, M & 300



Mean



93.60



36.14



0.39



49.80



39.20



5.88



3.17



60.00



106.20



178.00



±SD



16.01



3.92



0.04



11.56



13.14



0.39



0.21



11.47



8.41



40.79



n



5



5



5



5



5



5



5



5



5



5



G4, M & 1000



Mean



89.40



37.10



0.42



53.60



57.80



6.30



3.25



57.20



95.20



162.40



±SD



10.06



3.03



0.05



10.43



15.35



0.29



0.21



9.63



5.76



27.62



n



5



5



5



5



5



5



5



5



5



5



G1R, M & 0



Mean



96.60



33.90



0.49



43.00



47.20



7.50



3.09



63.00



126.00



180.60



±SD



25.65



3.78



0.03



5.79



8.23



0.27



0.11



12.63



34.78



35.45



n



5



5



5



5



5



5



5



5



5



5



G4R, M & 1000



Mean



84.00



34.80



0.51



47.80



61.20



7.60



3.27



60.60



129.40



159.40



±SD



18.23



6.97



0.05



4.66



13.97



0.48



0.23



6.95



22.88



54.55



n



5



5



5



5



5



5



5



5



5



5



 




























































































































































































































































































Group, Sex & Dose (mg/kg/day)



 



Total Bilirubin



Calcium



Phosphorous



Cholinesterase



Globulin



Albumin/


Globulin ratio



Blood Urea Nitrogen



Sodium



Potassium



Chloride



(BIT)



(CAL)



(PHO)



(CHE)



(GLO)



(A/G Ratio)



(BUN)



 (Na)



 (K)  



 (CLO)



(mg/dL)



(mg/dL)



(mg/dL)



 (U/L)



(g/dL)



 



(mg/dL)



mmol/L)



 (mmol/L)



(mmol/L)



G1, M & 0



Mean



0.00



9.70



6.94



116.40



2.99



1.09



17.68



145.46



3.05



107.46



±SD



0.00



0.16



0.63



11.33



0.36



0.12



1.09



1.16



0.22



1.48



n



5



5



5



5



5



5



5



5



5



5



G2, M & 100



Mean



0.02



9.76



6.86



118.00



2.84



1.14



16.19



145.50



3.22



108.76



±SD



0.02



0.26



0.46



25.40



0.17



0.07



0.91



1.32



0.22



1.55



n



5



5



5



5



5



5



5



5



5



5



G3, M & 300



Mean



0.01



9.46



6.54



116.40



2.71



1.18



16.87



145.72



3.20



109.14



±SD



0.01



0.24



0.53



13.67



0.22



0.08



1.83



1.14



0.28



1.99



n



5



5



5



5



5



5



5



5



5



5



G4, M & 1000



Mean



0.01



9.82



6.68



135.40



3.05



1.07



17.31



146.72



3.27



109.84



±SD



0.01



0.25



0.38



30.34



0.18



0.09



1.42



1.04



0.34



1.59



n



5



5



5



5



5



5



5



5



5



5



G1R, M & 0



Mean



0.00



9.02



6.66



110.40



4.41



0.70



15.82



143.62



3.47



106.38



±SD



0.00



0.31



0.36



28.59



0.23



0.04



1.76



0.45



0.29



0.85



n



5



5



5



5



5



5



5



5



5



5



G4R, M & 1000



Mean



0.01



9.04



6.54



124.40



4.33



0.77



16.24



143.58



3.46



106.30



±SD



0.02



0.48



0.66



30.38



0.56



0.13



3.25



0.28



0.56



1.91



n



5



5



5



5



5



5



5



5



5



5



 




























































































































































































































































































Group, Sex & Dose (mg/kg/day)



 



Glucose



Urea



Creatinine



Total Cholesterol



Triglycerides



Total Protein



Albumin



Alanine aminotransferase



Aspartate aminotransferase



Alkaline phosphatase



 (GLU)


 

 (CRE)



(CHO)



(TRI)



(TPR)



  (ALB)



(ALT)



(AST)



(ALP)



(mg/dL)



(mg/dL)



 (mg/dL)



(mg/dL)



(mg/dL)



  (g/dL)



  (g/dL)



 (U/L)



 (U/L)



 (U/L)



G1, F & 0



Mean



107.60



36.22



0.45



58.20



35.20



6.58



3.39



45.00



91.00



98.80



±SD



20.32



2.62



0.06



8.67



8.23



0.16



0.19



1.73



7.18



15.99



n



5



5



5



5



5



5



5



5



5



5



G2, F & 100



Mean



107.00



40.02



0.43



54.20



31.60



6.52



3.31



50.20



93.80



94.00



±SD



14.76



7.23



0.05



6.38



7.80



0.28



0.12



5.67



12.70



31.72



n



5



5



5



5



5



5



5



5



5



5



G3, F & 300



Mean



105.00



34.14



0.44



58.60



24.40



6.52



3.28



95.00



88.40



103.00



±SD



19.27



3.72



0.04



13.32



5.55



0.44



0.23



119.10



10.01



31.29



n



5



5



5



5



5



5



5



5



5



5



G4, F & 1000



Mean



97.40



39.04



0.42



52.40



26.00



6.62



3.20



41.80



91.40



92.20



±SD



5.81



3.50



0.01



5.64



4.74



0.43



0.12



6.72



9.58



18.61



n



5



5



5



5



5



5



5



5



5



5



G1R, F & 0



Mean



115.40



46.34



0.61



52.80



45.80



7.62



3.37



38.00



99.80



74.00



±SD



8.68



3.36



0.06



16.08



22.69



0.35



0.17



4.06



7.19



21.70



n



5



5



5



5



5



5



5



5



5



5



G4R, F & 1000



Mean



104.20



37.30*



0.56



74.00



63.00



8.00



3.57



40.20



107.60



64.60



±SD



15.69



2.89



0.02



15.78



34.69



0.49



0.12



4.66



13.11



10.81



n



5



5



5



5



5



5



5



5



5



5



 




























































































































































































































































































Group, Sex & Dose (mg/kg/day)



 



Total Bilirubin



Calcium



Phosphorous



Cholinesterase



Globulin



Albumin/Globulin ratio



Blood Urea Nitrogen



Sodium



Potassium



Chloride



(BIT)



(CAL)



(PHO)



(CHE)



(GLO)



(A/G Ratio)



(BUN)



 (Na)



 (K)  



 (CLO)



(mg/dL)



(mg/dL)



(mg/dL)



(U/L)



(g/dL)



 



(mg/dL)



mmol/L)



 (mmol/L)



(mmol/L)



G1, F & 0



Mean



0.01



9.50



5.74



339.20



3.19



1.07



16.90



146.80



3.26



109.98



±SD



0.00



0.22



0.90



153.87



0.15



0.10



1.22



0.96



0.25



1.11



n



5



5



5



5



5



5



5



5



5



5



G2, F & 100



Mean



0.04



9.64



6.08



339.20



3.21



1.04



18.68



145.80



3.25



110.44



±SD



0.04



0.30



0.23



156.45



0.18



0.04



3.37



0.82



0.28



2.17



n



5



5



5



5



5



5



5



5



5



5



G3, F & 300



Mean



0.03



9.52



6.18



392.80



3.24



1.02



15.93



146.08



3.25



109.42



±SD



0.03



0.44



0.76



103.24



0.36



0.13



1.74



0.31



0.27



0.91



n



5



5



5



5



5



5



5



5



5



5



G4, F & 1000



Mean



0.02



9.58



5.82



295.60



3.42



0.95



18.22



146.80



3.35



112.30



±SD



0.02



0.13



0.48



44.11



0.41



0.13



1.63



1.36



0.23



2.17



n



5



5



5



5



5



5



5



5



5



5



G1R, F & 0



Mean



0.00



9.24



4.96



363.80



4.25



0.79



21.63



143.12



3.13



107.76



±SD



0.00



0.15



0.35



128.23



0.26



0.06



1.57



0.77



0.24



1.19



n



5



5



5



5



5



5



5



5



5



5



G4R, F & 1000



Mean



0.00



9.36



4.82



522.00*



4.43



0.81



17.41*



143.14



3.13



106.60



±SD



0.00



0.21



0.47



50.04



0.39



0.05



1.35



0.80



0.20



0.90



n



5



5



5



5



5



5



5



5



5



5



*: Statistically significant (p<0.05).


 


Table 7. Summmary of absolute organ weights (g) record










































































































































































































































































































Group, Sex & Dose (mg/kg/day)



 



Adrenals



Thymus



Spleen



Testes



Epididymes



Heart



Kidneys



Brain



Liver



Prostate+Seminal vesicles with coagulating glands (PSC)



Lungs



Thyroid along with parathyroid



G1, M & 0



Mean



0.0650



0.3827



1.1071



2.9619



0.9208



1.0555



2.2293



1.8932



9.4504



1.8162



2.2357



0.0265



±SD



0.0054



0.0237



0.1226



0.1131



0.0658



0.0642



0.1133



0.0368



0.5226



0.0533



0.1654



0.0046



n



5



5



5



5



5



5



5



5



5



5



5



5



G2, M & 100



Mean



0.0653



0.4345*



1.1480



3.1239



0.9164



1.0255



2.3303



1.9193



9.1267



1.8468



2.2892



0.0272



±SD



0.0055



0.0241



0.1758



0.1064



0.0498



0.0535



0.1783



0.0600



0.2846



0.0482



0.2341



0.0030



n



5



5



5



5



5



5



5



5



5



5



5



5



G3, M & 300



Mean



0.0640



0.3655



1.1745



3.0821



0.9945



1.1462



2.2259



1.9611



9.9354



2.0696*



2.0302



0.0257



±SD



0.0052



0.0398



0.1221



0.2433



0.0593



0.1155



0.1601



0.1184



0.2256



0.1255



0.0603



0.0022



n



5



5



5



5



5



5



5



5



5



5



5



5



G4, M & 1000



Mean



0.0624



0.3582



1.0955



3.0368



1.0421*



1.0209



2.0452



1.9481



9.2955



1.9882*



2.1237



0.0268



±SD



0.0034



0.0183



0.0852



0.1892



0.0658



0.0426



0.0693



0.0429



0.2726



0.0750



0.1233



0.0036



n



5



5



5



5



5



5



5



5



5



5



5



5



G1R, M & 0



Mean



0.0562



0.4038



1.0462



3.2560



1.0915



1.1330



2.9957



1.9890



10.7320



1.9709



2.2137



0.0261



±SD



0.0042



0.0567



0.1133



0.1275



0.1171



0.1165



0.3451



0.0392



0.7802



0.0667



0.2298



0.0023



n



5



5



5



5



5



5



5



5



5



5



5



5



G4R, M & 1000



Mean



0.0570



0.4199



1.0465



3.2101



1.0799



1.1174



2.8470



2.0440



11.0235



2.0310



2.2273



0.0278



±SD



0.0035



0.0530



0.1095



0.1234



0.1159



0.0848



0.0877



0.0629



0.7546



0.0963



0.1983



0.0027



n



5



5



5



5



5



5



5



5



5



5



5



5



 























































































































































































































































































Group, Sex & Dose (mg/kg/day)



 



Adrenals



Thymus



Spleen



Ovaries



Uterus



Heart



Kidneys



Brain



Liver



Lungs



Thyroid along with parathyroid



G1, F & 0



Mean



0.0622



0.3508



1.0520



0.1481



0.4742



0.9289



1.7274



1.7529



7.0155



1.8666



0.0276



±SD



0.0039



0.0347



0.0962



0.0138



0.0575



0.0411



0.0521



0.0789



0.3258



0.0876



0.0019



n



5



5



5



5



5



5



5



5



5



5



5



G2, F & 100



Mean



0.0634



0.3558



1.1011



0.1561



0.4584



0.8925



1.6806



1.8289



7.0099



1.8620



0.0261



±SD



0.0054



0.0186



0.1395



0.0144



0.0387



0.0083



0.1311



0.1068



0.1685



0.1193



0.0032



n



5



5



5



5



5



5



5



5



5



5



5



G3, F & 300



Mean



0.0663



0.3936



1.1317



0.1510



0.4627



0.9646



1.8377



1.9906*



7.0515



1.9899



0.0269



±SD



0.0037



0.0364



0.0792



0.0127



0.0401



0.0411



0.0692



0.1342



0.4025



0.0822



0.0022



n



5



5



5



5



5



5



5



5



5



5



5



G4, F & 1000



Mean



0.0642



0.3658



1.0344



0.1570



0.4702



0.8786



1.7861



1.8945



7.1033



1.9446



0.0264



±SD



0.0043



0.0323



0.0956



0.0099



0.0442



0.0647



0.0829



0.0896



0.5884



0.0886



0.0037



n



5



5



5



5



5



5



5



5



5



5



5



G1R, F & 0



Mean



0.0576



0.3131



0.9075



0.1519



0.4456



0.9248



1.6678



1.9168



8.1132



1.9274



0.0271



±SD



0.0044



0.0277



0.0520



0.0081



0.0508



0.0546



0.0420



0.0464



0.5800



0.0870



0.0027



n



5



5



5



5



5



5



5



5



5



5



5



G4R, F & 1000



Mean



0.0588



0.3065



0.8669



0.1479



0.4234



0.9631



1.6538



1.9619



8.2348



2.0209



0.0264



±SD



0.0027



0.0271



0.0677



0.0129



0.0465



0.0408



0.0402



0.0415



0.5318



0.0818



0.0023



n



5



5



5



5



5



5



5



5



5



5



5



*: Statistically significant (p<0.05).


 


Table 8. Summmary of organ weight relative to fasting body weight (%) record





























































































































































































































































































































Group, Sex & Dose (mg/kg/day)



 



Fasting Body Weight (g)



Adrenals



Thymus



Spleen



Testes



Epididymes



Heart



Kidneys



Brain



Liver



Prostate+Seminal vesicles with coagulating glands (PSC)



Lungs



Thyroid along with parathyroid



G1, M & 0



Mean



279.87



0.0234



0.1372



0.3983



1.0670



0.3304



0.3801



0.8010



0.6810



3.4091



0.6523



0.8004



0.0096



±SD



25.26



0.0025



0.0086



0.0573



0.1271



0.0302



0.0475



0.0732



0.0657



0.4682



0.0491



0.0399



0.0022



n



5



5



5



5



5



5



5



5



5



5



5



5



5



G2, M & 100



Mean



269.30



0.0243



0.1616*



0.4278



1.1635



0.3408



0.3822



0.8658



0.7152



3.4039



0.6893



0.8523



0.0102



±SD



19.10



0.0025



0.0074



0.0710



0.0692



0.0145



0.0320



0.0397



0.0493



0.2852



0.0635



0.0948



0.0018



n



5



5



5



5



5



5



5



5



5



5



5



5



5



G3, M & 300



Mean



259.43



0.0248



0.1410



0.4538



1.1933



0.3849*



0.4430



0.8596



0.7590



3.8403



0.7984*



0.7851



0.0099



±SD



16.58



0.0031



0.0140



0.0516



0.1363



0.0382



0.0496



0.0674



0.0739



0.2224



0.0343



0.0560



0.0005



n



5



5



5



5



5



5



5



5



5



5



5



5



5



G4, M & 1000



Mean



268.67



0.0233



0.1343



0.4104



1.1339



0.3889*



0.3816



0.7661



0.7289



3.4819



0.7428*



0.7945



0.0100



±SD



20.08



0.0023



0.0162



0.0520



0.0850



0.0267



0.0302



0.0822



0.0667



0.3720



0.0518



0.0794



0.0012



n



5



5



5



5



5



5



5



5



5



5



5



5



5



G1R, M & 0



Mean



287.50



0.0196



0.1415



0.3647



1.1388



0.3823



0.3970



1.0451



0.6941



3.7494



0.6891



0.7769



0.0091



±SD



20.48



0.0003



0.0258



0.0386



0.1172



0.0563



0.0611



0.1263



0.0417



0.3818



0.0659



0.1303



0.0009



n



5



5



5



5



5



5



5



5



5



5



5



5



5



G4R, M & 1000



Mean



277.44



0.0206



0.1528



0.3771



1.1625



0.3932



0.4037



1.0326



0.7416



3.9934



0.7380



0.8046



0.0101



±SD



22.94



0.0010



0.0282



0.0213



0.0938



0.0675



0.0286



0.1030



0.0750



0.4191



0.0926



0.0691



0.0009



n



5



5



5



5



5



5



5



5



5



5



5



5



5



 










































































































































































































































































































Group, Sex & Dose (mg/kg/day)



 



Fasting Body Weight (g)



Adrenals



Thymus



Spleen



Ovaries



Uterus



Heart



Kidneys



Brain



Liver



Lungs



Thyroid along with parathyroid



G1, F & 0



Mean



196.99



0.0316



0.1780



0.5341



0.0756



0.2403



0.4719



0.8788



0.8904



3.5629



0.9479



0.0140



±SD



9.45



0.0020



0.0133



0.0429



0.0105



0.0221



0.0195



0.0549



0.0335



0.1130



0.0285



0.0011



n



5



5



5



5



5



5



5



5



5



5



5



5



G2, F & 100



Mean



194.16



0.0327



0.1833



0.5695



0.0804



0.2365



0.4603



0.8655



0.9435



3.6131



0.9595



0.0135



±SD



8.42



0.0035



0.0072



0.0893



0.0072



0.0228



0.0184



0.0521



0.0724



0.0906



0.0573



0.0014



n



5



5



5



5



5



5



5



5



5



5



5



5



G3, F & 300



Mean



206.72



0.0321



0.1903



0.5477



0.0731



0.2240



0.4669



0.8900



0.9639



3.4135



0.9633



0.0130



±SD



6.14



0.0018



0.0154



0.0386



0.0058



0.0214



0.0221



0.0505



0.0761



0.2224



0.0488



0.0010



n



5



5



5



5



5



5



5



5



5



5



5



5



G4, F & 1000



Mean



193.26



0.0334



0.1892



0.5374



0.0813



0.2430



0.4550



0.9265



0.9822



3.6818



1.0104



0.0137



±SD



10.49



0.0035



0.0120



0.0675



0.0038



0.0111



0.0296



0.0673



0.0624



0.3381



0.0969



0.0022



n



5



5



5



5



5



5



5



5



5



5



5



5



G1R, F & 0



Mean



222.25



0.0261



0.1416



0.4093



0.0686



0.2017



0.4183



0.7529



0.8654



3.6658



0.8710



0.0122



±SD



13.52



0.0032



0.0181



0.0288



0.0060



0.0311



0.0484



0.0540



0.0630



0.3921



0.0829



0.0012



n



5



5



5



5



5



5



5



5



5



5



5



5



G4R, F & 1000



Mean



208.00



0.0284



0.1474



0.4181



0.0714



0.2039



0.4644



0.7982



0.9465



3.9816



0.9740



0.0128



±SD



12.28



0.0023



0.0108



0.0430



0.0083



0.0233



0.0342



0.0655



0.0718



0.4821



0.0650



0.0015



n



5



5



5



5



5



5



5



5



5



5



5



5



*: Statistically significant (p<0.05).


 


Table 9. Summmary of gross pathology findings































































































































Sex



Male



Female



Route of administration



Oral Gavage



Treatment



Vehicle Control



Ultramarine Violet



Vehicle


Control



Ultramarine Violet



Group



G1



G2



G3



G4



G1R



G4R



G1



G2



G3



G4



G1R



G4R



Dose (mg/kg/day)



0



100



300



1000



0



1000



0



100



300



1000



0



1000



Number of animals



5



5



5



5



5



5



5



5



5



5



5



5



No. of dead rats during treatment



0



0



0



0



0



0



0



0



0



0



0



0



No. of moribund sacrificed rats



0



0



0



0



0



0



0



0



0



0



0



0



No. of terminally sacrificed rats



5



5



5



5



5



5



5



5



5



5



5



5



No of rats showing gross pathology



0



0



0



0



0



0



0



0



0



0



0



0



 


Table 10. Summmary of histopathology findings

















































































































































































































































































































































































































Route of administration



Oral Gavage



Treatment



Vehicle Control



Ultramarine Violet



Dose (mg/kg/day)



0



1000



Group



G1



G4



Sex



M



F



M



F



Number of Animals



5



5



5



5



Adrenals



Number examined



5



5



5



5



Within normal limits



5



5



5



5



Brain



Number examined



5



5



5



5



Within normal limits



5



5



5



5



Cervix



Number examined



X



5



X



5



Within normal limits



-



5



-



5



Coagulating glands



Number examined



5



X



5



X



Within normal limits



5



-



5



-



Epididymides



Number examined



5



X



5



X



Within normal limits



5



-



5



-



Eyes



Number examined



5



5



5



5



Within normal limits



5



5



5



5



Heart



Number examined



5



5



5



5



Within normal limits



4



5



5



5



Infiltrations, mononuclear cells



Minimal



1



-



-



-



Kidneys



Number examined



5



5



5



5



Within normal limits



4



5



5



5



Cast(s)



Minimal



1



-



-



-



Liver



Number examined



5



5



5



5



Within normal limits



3



5



4



5



Infiltrations, mononuclear cells



Minimal



2



-



1



-



Lungs



Number examined



5



5



5



5



Within normal limits



5



5



5



5



Ovaries



Number examined



X



5



X



5



Within normal limits



-



5



-



5



Parathyroid



Number examined



5



5



5



5



Within normal limits



5



5



5



5



Prostate gland



Number examined



5



X



5



X



Within normal limits



5



-



5



-



Seminal vesicles



Number examined



5



X



5



X



Within normal limits



5



-



5



-



Skin



Number examined



5



5



5



5



Within normal limits



5



5



5



5



Spinal cord



Number examined



5



5



5



5



Within normal limits



5



5



5



5



Spleen



Number examined



5



5



5



5



Within normal limits



5



5



5



5



Testes



Number examined



5



X



5



X



Within normal limits



5



-



5



-



Thymus



Number examined



5



5



5



5



Within normal limits



5



5



5



5



Thyroid



Number examined



5



5



5



5



Within normal limits



5



5



5



3



Ultimobranchial cyst



Present



-



-



-



2



Uterus



Number examined



X



5



X



5



Within normal limits



-



5



-



5



Vagina



Number examined



X



5



X



5



Within normal limits



-



5



-



5



 -: No incidence; X: Not applicable.


 

Conclusions:
In a 28-day repeated dose toxicity study conducted in Sprague Dawley Rats, the NOAEL of the test item for both sexes is 1000 mg/kg bw/day.
Executive summary:

To establish doses for a subsequent sub-chronic toxicity study on the test substance, a dose range finding study was performed for a period of 28 consecutive days according to OECD 407 Guideline (GLP study) using Sprague Dawley rats. Four groups (G1, G2, G3 and G4) of 5 animals of each sex and two recovery groups (G1R and G4R) were tested, being G1 and G1R the vehicle control groups (0.5% w/v CMC). The treatment groups had the following concentrations: 100, 300 and 1000 mg/kg bw. The vehicle and test item formulations were administered orally by gavage to the animals at an equivolume of 10 mL/kg bw. 


No mortality and clinical signs were observed throughout the dosing and recovery period. No test item-related changes in body weight, percent change in body weight and feed consumption were observed throughout the experimental period. No test item-related neurological/functional abnormalities and no ocular changes were observed during examination. No test item related changes were noted in haematology, coagulation, clinical chemistry and urinalysis parameters. There were no test item-related effects observed in absolute and relative organ weights. No test item related gross and histopathological changes were observed up to the highest dose tested in both the sexes.


Therefore, based on the obtained results from this 28-day repeated dose toxicity study conducted in Sprague Dawley Rats, the NOAEL is estimated as 1000 mg/kg bw/day under experimental conditions employed. Therefore, the dose levels of 100, 300 and 1000 mg/kg/day can be selected for subsequent 90-day repeat dose toxicity study in rats.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
1 key study is available with a Klimisch score of 1.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the available data, the substance is not classified for specific target organ toxicity by repeated exposure (STOT-RE) according to CLP Regulation (EC) no 1272/2008.