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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 October 2019 – 19 November 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to other study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 February 2020 – 22 July 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
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 bred animals.
- Females (if applicable) nulliparous and non-pregnant: yes
- Weight at study initiation: 343.19-343.99 g (range of average values of each group of males at first day of dosing); 254.10-255.22 g (range of average values of each group of females at first day of dosing)
- Housing: Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with a stainless-steel mesh top grill having facilities for holding pelleted food and drinking water in a water bottle fitted with a stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
Acclimatization: maximum of 2 animals of same sex per cage.
Pre mating: 2 animals of the same sex and group per cage.
Mating: 2 animals (one male and one female) of the same group per cage.
Post mating: After confirming presence of sperm in the vaginal smear (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually. Sterilized paper shreds were provided as a nesting material from gestation day 20 onwards.
Recovery animals: 2 animals of same sex per cage.
- 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 experimental period. Deep bore-well water passed through a Reverse Osmosis Unit was provided in plastic water bottles with stainless-steel sipper tubes.
- Acclimation period: at least 5 days. Females were screened for normal oestrous cycles (4 to 5 days) in a two weeks pre-treatment period before initiation of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6-23.2ºC
- Humidity (%): 46-65 %
- Air changes (per hr): 12-15
- Photoperiod: 12 hrs dark / 12 hrs light
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The required quantity of test item was weighed into a clean beaker and a little volume of vehicle was added into the beaker and mixed well with glass rod. The formulation was transferred into a measuring cylinder, the beaker was rinsed with some more amount of vehicle also transferred into the measuring cylinder. This procedure was repeated until the entire quantity of the test item formulation was transferred into the measuring cylinder. Finally, the volume was made up to the required quantity with vehicle to get the desired concentration for the different dose levels.
Test item formulations were prepared daily before administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item was found insoluble in distilled water but formed uniform suspension with corn oil at the concentration of 1.9 mg/mL (considering highest dose of 9.5 mg/kg bw with a dose volume of 5 mL/kg bw) based on the in-house suspendibility test results. Hence, corn oil was selected as vehicle for test item formulation preparations. Corn oil is universally accepted and routinely used vehicle in oral toxicity studies.
- Concentration in vehicle: 0.4, 0.86 and 1.9 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): L12017004, L32011001.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test item in dose formulations was established in a preliminary study. The test item formulations were stable at room temperature for 6 hours at the concentrations of 0.4 mg/mL and 1.9 mg/mL in corn oil. Prepared test item formulations were administered to the animals within established stability conditions.
Homogeneity and dose formulation analysis for dose concentration verification was done by a validated HPLC method (study nº BIO-ANM 1513). Sampling and analysis of formulations were performed during week 1 (16-03-2020) and week 5 (14-04-2020) of the treatment. Samples were collected in duplicates from top, middle and bottom layers from low, mid and high dose concentrations and in duplicates from single layer from vehicle control. Formulations were considered acceptable, as the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) was ≤10%.
Duration of treatment / exposure:
Main group males: two weeks pre-mating, during mating and up to the day before sacrifice during the post-mating period (total of 37 days of treatment).
Main group females: two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13.
Recovery animals: same period as for the main group females until the first scheduled sacrifice of dams and kept without treatment for a further 14 days observation.
Frequency of treatment:
Once a day
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
G1 - Vehicle control + G1R - Vehicle Control Recovery Group
Dose / conc.:
2 mg/kg bw/day (actual dose received)
Remarks:
G2 - Low dose
Dose / conc.:
4.3 mg/kg bw/day (actual dose received)
Remarks:
G3 - Mid dose
Dose / conc.:
9.5 mg/kg bw/day (actual dose received)
Remarks:
G4 - High dose + G4R - High dose Recovery Group
No. of animals per sex per dose:
Main Group: 12
Recovery Group: 5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Doses of 0, 50, 100 and 300 mg/kg bw /day were selected in a first DRF study (BIO-CTX 139) based on the available results for an acute oral toxicity study conducted as per OECD 423 guideline. Treatment related effects including mortalities were found at the lowest level tested. Thus, it was not possible to properly decide on doses for the main study and a second DRF study (BIO-CTX 171) was performed at doses of 0, 2.7, 8.3 and 25 mg/kg bw /day. The dose of 25 mg/kg bw /d was the lowest dose level at which treatment related adverse effects were found and based on these results the doses 2, 4.3 and 9.5 mg/kg bw /day were considered for present main study.
- Fasting period before blood sampling for clinical biochemistry: yes, overnight (water allowed).
- Post-exposure recovery period in satellite groups: 14 days

Positive control:
None
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.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the treatment on day 1 and weekly thereafter during treatment for all the animals. Signs noted were included, but not be limited to, changes in skin, fur, eyes and mucous membranes, occurrence of secretions and excretions and autonomic activity such as lacrimation, piloerection, pupil size, and unusual respiratory pattern.

BODY WEIGHT: Yes
- Time schedule for examinations: The main group animals were weighed at receipt, on the first day of dosing, weekly thereafter and at termination. The main group females were weighed on gestation days 0, 7, 14 and 20 during pregnancy and on days 1, 4, 7 and 13 during lactation period. The recovery group animals (both males and females) were weighed at receipt, on the first day of dosing, weekly thereafter and at termination.

FOOD CONSUMPTION: yes
-feed consumption was measured for main group animals (both males and females) once a week during premating and weekly once for main group males during the post mating period. Feed consumption was not measured during the mating period for both males and females. Thereafter, feed consumption for main group females was recorded during gestation days 0 to 7, 7 to 14 and 14 to 20 and on lactation days 1 to 4, 4 to 7 and 7 to 13. Feed consumption was measured for recovery group animals (both males and females) once a week throughout the experimental period. Average feed intake per rat (g/rat/day) was calculated using the amount of feed given and left over in each cage and the number of rats surviving in each cage.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once before treatment for all animals, at the end of the dosing period for males (shortly prior to scheduled sacrifice) and during the lactation period for females (shortly prior to scheduled sacrifice) of vehicle control and high dose main group animals and during the last week for the recovery group animals.
- Dose groups that were examined: vehicle control and high dose main group.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled terminal sacrifice (for main group males on day 38, for main group females on lactation day 14 and for recovery group animals on the day 66).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 5 males and 5 females randomly selected from each main group and all recovery group animals.
- Parameters checked: Haemoglobin concentration (HGB), Haematocrit (HCT), Erythrocyte count (RBC), Total leukocyte count (WBC), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet count (PLT), Mean platelet volume (MPV), Reticulocyte count (Retic), Absolute reticulocyte count, Differential leucocytes count (DLC), Absolute differential leucocytes count (DLC). Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) were estimated by a coagulation analyzer.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled terminal sacrifice (for main group males on day 38, for main group females on lactation day 14 and for recovery group animals on the day 66).
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 5 males and 5 females randomly selected from each main group and all recovery group animals.
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Cholinesterase, Total Protein, Albumin, Total bilirubin, Glucose, Total Cholesterol, Creatinine, Urea, Blood urea nitrogen (BUN), Triglycerides, Phosphorous, Calcium, Globulin. Sodium (mmol/L), Potassium (mmol/L) and Chloride (mmol/L) were estimated using a Na/K/Cl analyzer.

URINALYSIS: Yes
- Time schedule for collection of urine: on the day of scheduled terminal sacrifice.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 5 randomly selected males of each main group and all recovery animals.
- Parameters checked: Blood, Bilirubin, Urobilinogen, Ketones, Protein, Glucose, Microalbumin, Leucocytes. In addition pH, nitrite and specific gravity were also analyzed. After analysis of the above parameters, the urine was subjected for centrifugation at 1500 rpm for 3 minutes. Then the urine was subjected for microscopic examination for urine sediments.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Towards the end of the dosing period for males (on day 37) and during lactation period for females (on lactation day 13). Towards the end of the recovery period (shortly prior to scheduled sacrifice on day 65) for the recovery group.
- Dose groups that were examined: all main and recovery groups.
- Battery of functions tested: Home Cage Observations (convulsions, tremors and palpebral closure), Handling Observations, Open Field Observations, Sensory Observations, Neuromuscular Observations, Physiological Observation (Rectal temperature), Grip strength assessment and Motor activity assessment.

OTHER:

Oestrous Cyclicity: Estrous cycles were monitored during the acclimatization to evaluate its normal oestrous cyclicity (4 to 5 days). Only females with normal oestrous cyclicity were selected for the treatment. For the main group females, the vaginal smears were monitored daily from the beginning of the treatment period until evidence of mating. Oestrous cyclicity was also monitored on the day of sacrifice for main group females.

Litter Observation: The number of pups born (dead and live) in a litter, sex and external observations were recorded at birth. Individual body weight of live pups on lactation day 1 (within 24 hours of parturition), 4, 7 and 13 was recorded. The anogenital distance of each pup was measured on postnatal day 4 (lactation day 4). The number of nipples/areolae in male pups was counted on postnatal day 13 (lactation day 13). Fertility index for dams and sires, pup survival index and sex ratio at birth were calculated.

Thyroxine Hormone (T4) Level estimation: Blood samples were collected for measurement of serum T4 levels on the following schedule:
a. Two pups per litter on lactation day 4 based on the following conditions:
- Two female pups in order to retain more male pups for nipple retention on PND 13.
- No pups were eliminated when litter size dropped below 10 pups/litter.
- Only one pup was eliminated and used for blood collection in case of only one pup was available above ten.
b. All main group females (dams) at termination (lactation day 14).
c. Two pups per litter (same sex) at termination (lactation day 13).
d. All adult main group males, at termination (after completion of 37 days of treatment).






Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see tables 1 and 2)

HISTOPATHOLOGY: Yes (see tables 1 and 2)
Other examinations:
Indices calculation (see table 3)


Statistics:
After verification, the data was subjected to statistical analysis using SPSS software, version 22. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05). The statistical analysis was followed to the parameters as mentioned in the table 4 below.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were no clinical signs of toxicity observed at groups G1/G1R, G2 and G3 animals of either sex throughout the experimental period.

Animals in groups G4/G4R were noted with test item-related clinical signs of toxicity such as, lethargic, rough hair coat, wet perineum, chromodacryorrhea, hair thinning or blood stains around vagina. However, all the animals from group G4R started to recover from the above mentioned clinical signs during recovery period and found normal at termination. In group G4 all the animals did not reveal any clinical signs till day 23 of treatment period. However, from day 24 onwards the animals started to reveal clinical signs. In group G4R all the animals did not reveal any clinical signs till day 25 of treatment period. However, from day 26 onwards the animals started to reveal clinical signs.
Mortality:
no mortality observed
Description (incidence):
There were no mortality/morbidity observed at any dose group during the experimental period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were no changes noted in mean body weight and percent change in mean body weight gain with respect to day 1 at groups G2 and G3 in both sexes throughout the experimental period including gestation and lactation periods.

In group G4, there were no changes noted in mean body weight and percent change in mean body weight gain with respect to day 1 till week 3 in both sexes. However, reduction in mean body weight and percent change in mean body weight gain was noted from week 4 onwards and continued till termination. Even though these reductions were not statistically significant, these changes are considered as test item-related due to noted test item-related clinical signs and test item-related effects in neurological observations during these periods.

In group G4R, there were no changes noted in mean body weight and percent change in mean body weight gain with respect to day 1 till week 3 in both sexes. However, reduction in mean body weight and percent change in mean body weight gain was noted from week 4 onwards and continued till end of dosing period. Furthermore, some of these reductions were found to be statistically significant compared to the control group. These changes are considered as test item-related due to noted test item-related clinical signs and test item-related decreased mean feed consumption during these periods. However, the body weight and body weight gains were started to recover during recovery period in both sexes.

In group G4, reduction in mean body weight and percent change in mean body weight gain was noted throughout the gestation period which is statitistically significant on gestation day (GD) 14 and 20 for mean body weight and statitistically significant during GD 7 to 14 and 14 to 20 for percent change in mean body weight gain when compared with vehicle control group. These reductions are considered as test item-related due to noted test item-related clinical signs and test item-related reduction in feed consumption during these periods.

In group G4, reduction in mean body weight and percent change in mean body weight gain was noted throughout the lactation period which is statitistically significant on lactation day (LD) 4, 7 and 13 for mean body weight and statitistically significant during LD 4 to 7 and 7 to 13 for percent change in mean body weight gain when compared with vehicle control group. These reductions are considered as test item-related due to noted test item-related clinical signs and test item-related reduction in feed consumption during these periods.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There were no changes noted in mean feed consumption at groups G2 and G3 in both sexes during pre-mating, gestation and lactation periods when compared with vehicle control group.

In group G4, there were no changes noted in mean feed consumption in both sexes during pre-mating period when compared with vehicle control group. However, reductions in mean feed consumption were noted during gestation and lactation periods which are statitistically significant during GD 14 to 20 and during LD 1 to 4, 4 to 7 and 7 to 13, when compared with vehicle control group. These reductions are considered as test item-related due to noted test item-related clinical signs and test item-related reduction in body weights during these periods.

In group G4R, there were no changes noted in mean feed cosnumption till week 3 in both sexes. However, reduction in mean feed consumption was noted from week 4 onwards and continued till end of dosing period. These reductions are statistically significant during week 5 to 9 in G4R males; statistically significant during week 6 in G4R females when compared with respective vehicle control group. These changes are considered as test item-related due to noted test item-related clinical signs, decreased mean body weight and or percent body weight gain during these periods in both sexes. However, the mean feed consumption was started to recover during recovery period in both sexes.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no ocular changes observed in any of the animals from all the tested dose and vehicle control main groups in both sexes during ophthalmological examination conducted towards end of the dosing period. Likewise, there were no ocular changes observed in any of the animals from both recovery groups in either sex during ophthalmological examination conducted towards end of the recovery period.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
There were no changes noted in mean haematology parameters in groups G2 and G3 of both sexes when compared with vehicle control group. In group G4/G4R, the following statistically significant changes were noted: decrease in mean absolute differential leucocyte count (nuetrophills, lymphocytes, monocytes, eosinophils, basophils), eosinophils (%), WBC count of group G4 males; decrease in mean RBC count, HGB, HCT, monocytes (absolute and %), prothrombin time of group G4 females; increase in mean reticulocyte count (absolute and %), eosinophils (absolute and %) and monocytes (%), decrease in mean lymphocytes (%) and prothrombin time of group G4R males; and decrease in RBC count and Activated Prothrombin Time, increase in MCV, MCH, reticulocyte count (absolute and %) of group G4R females.


Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
There were no test item-related changes noted in mean clinical chemistry parameters of groups G2 and G3 in both sexes when compared with vehicle control group. In group G4/G4R, the following statistically significant changes were noted: decrease in mean alanine aminotransferase (ALT), aspartate aminotransferase (AST) levels of group G4 males; decrease in mean urea and blood urea nitrogen levels of group G4 females; decrease in mean creatinine levels and increase in mean calcium and phosphorous levels of group G4R males; increase in mean creatinine, sodium and total bilirubin levels and decrease in mean total cholesterol levels of group G4R females.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related changes observed in urinalysis parameters in any of the tested dose levels of both main and recovery groups when compared with vehicle control group. Some of the sporadically occurring statistically significant differences like, increase in mean urine pH levels and decrease in urine specific gravity levels in group G4R females were considered incidental and therefore of no toxicological relevance.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
There were no changes noted in neurological/functional examinations like home cage/ handling/open-field/sensory/neuromuscular (hind limb foot splay)/physiological observations and grip strength or motor activity assessments in any of the animals from groups G2 and G3 when compared with vehicle control groups.

In group G4, there were no changes noted in home cage/handling/open-field/sensory/ neuromuscular (hind limb foot splay)/physiological observations when compared with vehicle control group in both sexes. However, a slight (non-statistically significant) reduction in mean movements count during motor activity assessment in G4 males, statistically significant reduction in mean fore/hind limb grip strengths in G4 males were noted when compared with vehicle control group. These changes can be considered as secondary effects to the test item exposure, noted due to test item-related clinical signs, decreased mean body weight, percent body weight gain and mean feed consumption during this period.

There were no changes noted in neurological/functional examinations like home cage/ handling/open-field/sensory/neuromuscular (hind limb foot splay)/physiological observations and grip strength or motor activity assessments in any of the animals from group G4R in both sexes when compared with vehicle control group.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There were no changes noted in both mean absolute and relative organ weights of groups G2 and G3 in either sex when compared with vehicle control group.

In group G4, statistically significant decrease in mean thymus weight (absolute and relative) of males, statistically significant decrease in mean spleen weight (absolute), mean liver weight (absolute) and thymus weight (absolute and relative) of females was noted when compared with vehicle control group. These observed changes can be considered as test item-related due to noted mild decrease in lymphocytes in white pulp of spleen and cortical area of thymus during microscopic examination of these organs at this dose level. These changes can also correlate with test item-related and statistically significant reduction in mean terminal body weight at this dose level.

Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
There were no gross pathological changes observed in any of the adult animals from groups G1/G1R, G2 and G3.

The animals of group G4 were noted with test item related external gross pathological changes like wet pernineum (4 males and 7 females), wet pernineum along with blood stains around vagina (2 females) and test item related internal gross pathological changes like uterus with resorptions/dead fetuses during necropsy. However, the group G4R animals of both sexes treated with high dose did not reveal any external or internal gross pathological changes during necropsy. These observations are because of recovery of animals from clinical signs during recovery period and hence all the animals were found normal at termination.

At necropsy, the dead pups which were found dead by birth and during lactation period were noted with pale discoloration and red discoloration at nasal region from group G4 litters. No gross pathological changes were noted in any of the survived pups from group G4 and all pups from other dose group litters.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related changes were observed in spleen, thymus and bone marrow of femur in both sexes at group G4. The microscopic changes were characterized by mild decrease in lymphocytes in white pulp of spleen, cortical area of thymus and also mild decrease in marrow cellularity of femoral bone. Similar changes were not observed in lower and recovery group of animals. All these changes in thymus, spleen and femoral bone marrow were considered to be reversible, secondary effects of reduced food consumption and decreased body weight gain (Everds et.al., 2013). Few of the other microscopic findings observed in the study such as accessory cortical tissue in adrenal glands, ultimobranchial cysts(s) in thyroid gland 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.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Oestrus cycle: There were no irregularities observed in the oestrus cyclicity of main group females during pre-mating period at any of the tested dose groups. The mean length of oestrus cycle (days) per dam during pre-mating period was unaffected at all the tested dose groups when compared with vehicle control group.

Litter observations: In group G4, test item-related reduced gestation index, increased post-implantation loss, increased postnatal loss, reduced litter size, reduced live birth index per litter, increased number of dead pups at birth / during postnatal period and reduced pup survival index were noted. These noted changes are considered as test item-related due to noted test item-related clinical signs, test item-related reduction in maternal body weights and feed consumption during gestation and lactation periods.

Serum Thyroxine (T4) Levels: There were no changes noted in T4 levels of males and dams of main groups and PND 13 pups when compared with vehicle control group. The obtained T4 levels are within the normal range of this species and strain. Also, there were no changes noted in T4 levels of PND 4 pups from groups G2 and G3 when compared with vehicle control group. In group G4, T4 levels of PND 4 pups was not conducted as there were no surplus pups remained from this dose level on PND 4. However, the obtained T4 levels of PND 4 pups from groups G2 and G3 are within the normal range of this species and strain.
Key result
Dose descriptor:
LOAEL
Effect level:
9.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOAEL
Effect level:
4.3 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Remarks on result:
other: Effects observed at the highest tested dose of 9.5 mg/kg bw/day.
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
9.5 mg/kg bw/day (actual dose received)
System:
immune system
Organ:
bone marrow
spleen
thymus

Table 5.1. Summary of clinical signs of toxicity, detailed clinical examination and mortality record. Males

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

Total No. of Animals

Clinical Signs of Toxicitya:

Observation
(No. of Animals revealed)

Detailed Clinical Examinationb:

Observation
(No. of Animals revealed)

Mortalityc:

No. of Mortalities
(Total No. of Animals)

G1, M & 0

12

N (12)

NAD (12)

0 (12)

G2, M & 2

12

N (12)

NAD (12)

0 (12)

G3, M & 4.3

12

N (12)

NAD (12)

0 (12)

G4, M & 9.5

12

116 (7); 121 (1); 110+ (7); 82+ (5)

Rough hair coat (4);

Hair thinning (1);

Wet perineum (6);

Chromodacryorrhea (5)

0 (12)

M: Male; N: Normal; NAD: No Abnormality Detected; 82: Chromodacryorrhea; 110: Wet perineum; 116: Rough hair coat; 121: Hair thinning; +: Slight

a: observed daily once; b: observed weekly once; c: observed twice daily

Table 5.2. Summary of clinical signs of toxicity, detailed clinical examination and mortality record. Females

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

Total No. of Animals

Clinical Signs of Toxicitya:

Observation
(No. of Animals revealed)

Detailed Clinical Examinationb:

Observation
(No. of Animals revealed)

Mortalityc:

No. of Mortalities
(Total No. of Animals)

G1, F & 0

12

N (12)

NAD (12)

0 (12)

G2, F & 2

12

N (12)

NAD (12)

0 (12)

G3, F & 4.3

12

N (12)

NAD (12)

0 (12)

G4, F & 9.5

12

116 (4); 121 (1); 110+ (9); 82+ (3); 145 (1); 1 (1)

Rough hair coat (3);

Wet perineum (8);

Chromodacryorrhea (3);

Hair thinning (1)

0 (12)

F: Female; N: Normal; NAD: No Abnormality Detected; 1: Lethargy; 82: Chromodacryorrhea; 110: Wet perineum; 116: Rough hair coat; 121: Hair thinning; 145: Blood stains around vaginal region +: Slight

a: observed daily once; b: observed weekly once; c: observed twice daily

Table 5.3. Summary of clinical signs of toxicity, detailed clinical examination and mortality record. Recovery groups

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

Total No. of Animals

Clinical Signs of Toxicitya:

Observation
(No. of Animals revealed)

Detailed Clinical Examinationb:

Observation
(No. of Animals revealed)

Mortalityc:

No. of Mortalities
(Total No. of Animals)

G1R, M & 0

5

N (5)

NAD (5)

0 (5)

G4R, M & 9.5

5

1 (4); 82+ (4); 110+ (5); 116 (4)

Rough hair coat (4);

Chromodacryorrhea (4);

Wet perineum (5);

Lethargy (4)

0 (5)

G1R, F & 0

5

N (5)

NAD (5)

0 (5)

G4R, F & 9.5

5

1 (1); 2 (1); 82+ (2); 110+ (3);

116 (2)

Rough hair coat (2);

Chromodacryorrhea (2);

Wet perineum (3);

Lethargy (1);

Dehydration (1)

0 (5)

M: Male; F: Female; R: Recovery; N: Normal; 1: Lethargy; 2: Dehydration; 82: Chromodacryorrhea; 110: Wet perineum; 116: Rough hair coat; +: Slight

a: observed daily once; b: observed weekly once; c: observed twice daily

Table 6.1. Summary of body weight (g) record. Males

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

Body Weight (g) on Day

1

8

15

22

28

35

G1, M & 0

Mean

343.80

354.30

372.89

381.94

392.62

399.38

±SD

28.76

30.14

27.43

28.36

29.44

33.00

n

12

12

12

12

12

12

G2, M & 2

Mean

343.19

355.48

375.06

382.19

395.24

404.80

±SD

27.66

28.50

31.44

30.40

32.57

34.95

n

12

12

12

12

12

12

G3, M & 4.3

Mean

343.22

352.18

369.94

377.53

393.85

401.39

±SD

28.59

31.35

34.05

37.72

41.80

46.65

n

12

12

12

12

12

12

G4, M & 9.5

Mean

343.99

356.69

372.18

376.97

374.98

376.93

±SD

29.28

27.87

30.98

33.95

37.26

41.44

n

12

12

12

12

12

12

Table 6.2. Summary of body weight (g) record. Females

Group, Sex & Dose

(mg/kg body weight/day)

Body Weight (g) on Day

1

8

15

22#

28#

G1, F & 0

Mean

254.53

259.57

269.83

275.95

256.16

±SD

20.28

18.04

19.70

19.89

-

n

12

12

12

7

1

G2, F & 2

Mean

254.62

262.12

271.75

263.95

268.77

±SD

19.45

18.11

17.30

20.71

-

n

12

12

12

4

1

G3, F & 4.3

Mean

255.22

260.53

267.92

267.32

326.14

±SD

20.49

21.14

21.57

27.80

-

n

12

12

12

6

1

G4, F & 9.5

Mean

254.10

259.09

269.40

274.25

253.66

±SD

18.53

19.62

19.05

19.13

-

n

12

12

12

4

1

#: Presented mean values are obtained from the females in cohabitation period. Data of females confirmed with mating considered for gestation body weight

Table 6.3. Summary of body weight (g) record. Recovery groups

Group, Sex & Dose

(mg/kg body weight/day)

Body Weight (g) on Days

1

8

15

22

28

35

42

49

56

63

G1R, M & 0

Mean

338.71

351.28

367.49

378.53

397.05

408.94

415.18

422.50

436.74

442.08

±SD

23.47

21.70

23.01

23.71

28.55

27.70

28.66

28.44

35.50

33.17

n

5

5

5

5

5

5

5

5

5

5

G4R, M & 9.5

Mean

337.91

342.76

361.70

368.88

370.56

362.34*

357.64*

366.45*

383.76*

394.64*

±SD

25.62

25.46

28.70

28.16

24.16

21.46

24.58

23.15

28.63

27.68

n

5

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

251.59

257.19

267.20

273.45

278.38

282.59

284.20

287.64

296.38

301.07

±SD

18.16

21.21

23.27

23.59

22.25

23.55

24.39

23.98

25.02

26.17

n

5

5

5

5

5

5

5

5

5

5

G4R, F & 9.5

Mean

252.36

258.10

264.50

268.36

259.94

255.07

255.73

262.66

267.59

272.77

±SD

16.35

15.94

16.15

18.71

22.25

33.33

34.22

27.97

24.17

21.70

n

5

5

5

5

5

5

5

5

5

5

* Statistically significant (P<0.05) change than the vehicle control group

Table 7.1. Summary of percent change in body weight (%) with respect to day 1. Males

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

Percent Change in Body Weight (%) during Day

1 to 8

1 to 15

1 to 22

1 to 28

1 to 35

G1, M & 0

Mean

3.05

8.56

11.25

14.38

16.34

±SD

1.74

2.38

4.35

5.07

6.23

n

12

12

12

12

12

G2, M & 2

Mean

3.62

9.33

11.53

15.46

18.22

±SD

2.94

4.45

6.66

9.26

9.48

n

12

12

12

12

12

G3, M & 4.3

Mean

2.58

7.74

9.92

14.67

16.81

±SD

1.95

3.18

4.80

6.45

7.68

n

12

12

12

12

12

G4, M & 9.5

Mean

3.76

8.25

9.64

9.03

9.51

±SD

2.02

2.98

4.86

6.23

6.82

n

12

12

12

12

12

Table 7.2. Summary of percent change in body weight (%) with respect to day 1. Females

Group, Sex & Dose

(mg/kg body weight/day)

Percent Change in Body Weight (%) during Day

1 to 8

1 to 15

1 to 22#

1 to 28#

G1, F & 0

Mean

2.06

6.09

8.43

14.57

±SD

1.65

2.32

3.45

-

n

12

12

7

1

G2, F & 2

Mean

3.04

6.92

10.13

13.02

±SD

3.27

5.09

4.96

-

n

12

12

4

1

G3, F & 4.3

Mean

2.08

4.99

7.49

12.14

±SD

1.02

1.93

2.35

-

n

12

12

6

1

G4, F & 9.5

Mean

1.95

6.05

7.49

10.12

±SD

1.46

1.88

1.97

-

n

12

12

4

1

#: Presented mean values are obtained from the females in cohabitation period. Data of females confirmed with mating considered for gestation body weight

Table 7.3. Summary of percent change in body weight (%) with respect to day 1. Recovery groups

Group, Sex & Dose

(mg/kg body weight/day)

Percent Change in Body Weight (%) during Day

1 to 8

1 to 15

1 to 22

1 to 28

1 to 35

1 to 42

1 to 49

1 to 56

1 to 63

G1R, M & 0

Mean

3.76

8.55

11.82

17.38

20.84

22.64

24.82

28.97

30.57

±SD

1.04

2.02

2.35

6.75

5.06

3.90

4.35

5.83

5.25

n

5

5

5

5

5

5

5

5

5

G4R, M & 9.5

Mean

1.45*

7.03

9.17

9.77

7.40*

6.06*

8.77*

13.86*

17.14*

±SD

1.21

1.26

1.72

3.41

4.44

6.75

8.10

8.93

9.39

n

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

2.17

6.12

8.63

10.61

12.26

12.89

14.27

17.72

19.57

±SD

1.45

2.33

2.95

1.82

2.15

2.64

2.59

1.84

2.37

n

5

5

5

5

5

5

5

5

5

G4R, F & 9.5

Mean

2.30

4.85

6.34

2.90*

0.75*

1.00*

3.87*

5.90*

8.01*

±SD

2.11

2.47

3.04

3.19

7.96

8.55

5.45

3.79

2.69

n

5

5

5

5

5

5

5

5

5

* Statistically significant (P<0.05) change than the vehicle control group.

Table 8.1. Summary of average feed consumption (g/animal/day) record. Males

Group, Sex & Dose

(mg/kg body weight/day)

Feed Consumption (g/animal/day) during Pre-mating Period

Week 1

Week 2

G1, M & 0

Mean

23.99

25.23

±SD

2.20

1.91

n

12

12

G2, M & 2

Mean

23.14

24.85

±SD

1.75

2.04

n

12

12

G3, M & 4.3

Mean

22.64

24.69

±SD

2.63

2.06

n

12

12

G4, M & 9.5

Mean

22.08

24.28

±SD

1.03

1.54

n

12

12

Table 8.2. Summary of average feed consumption (g/animal/day) record. Females

Group, Sex & Dose

(mg/kg body weight/day)

Feed Consumption (g/animal/day) during Pre-mating Period

Week 1

Week 2

G1, F & 0

Mean

16.20

20.55

±SD

1.64

2.35

n

12

12

G2, F & 2

Mean

17.23

21.03

±SD

1.43

1.88

n

12

12

G3, F & 4.3

Mean

16.76

20.68

±SD

1.06

1.29

n

12

12

G4, F & 9.5

Mean

16.10

20.32

±SD

1.31

1.91

n

12

12

Table 8.3. Summary of average feed consumption (g/animal/day) record. Recovery groups

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

Feed Consumption (g/animal/day)

Week 1

Week 2

Week 3

Week 4

Week 5

Week 6

Week 7

Week 8

Week 9

G1R, M & 0

Mean

22.48

24.88

26.99

28.00

29.15

29.76

30.17

30.58

29.57

±SD

1.31

3.64

3.32

4.22

3.85

3.92

4.25

2.57

1.61

n

5

5

5

5

5

5

5

5

5

G4R, M & 9.5

Mean

22.21

24.46

24.95

25.44

20.32*

17.90*

18.99*

21.66*

24.12*

±SD

1.12

1.06

3.06

3.83

3.10

1.83

0.51

0.87

1.76

n

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

17.98

21.28

22.56

23.52

25.78

27.50

27.69

27.87

29.37

±SD

2.32

1.53

2.90

3.00

4.55

4.72

3.16

6.88

3.86

n

5

5

5

5

5

5

5

5

5

G4R, F & 9.5

Mean

17.99

20.95

20.90

19.32

17.90

16.07*

20.93

23.17

25.33

±SD

2.18

0.91

1.75

3.17

3.77

3.21

4.54

2.65

2.58

n

5

5

5

5

5

5

5

5

5

* Statistically significant (P<0.05) change than the vehicle control group.

Table 9.1. Summary of neurological/functional observation battery record. Males

Day 37

Parameters↓

Group & Sex

G1 & M

G2 & M

G3 & M

G4 & M

Dose (mg/kg body weight/day)

0

2

4.3

9.5

Number of Animals (randomly selected)

5

5

5

5

Home Cage Observations

Home cage posture

1

1

1

1

Respiratory pattern

1

1

1

1

Clonic involuntary movements

1

1

1

1

Tonic involuntary movements

1

1

1

1

Vocalization

1

1

1

1

Palpebral closure

1

1

1

1

Handling Observations

a. Ease of removal from the cage

2

2

2

2

b. Ease of handling animal in hand

2

2

2

2

Red or crusty deposits

Eyes

1

1

1

1

Nose

1

1

1

1

Mouth

1

1

1

1

Lacrimation

1

1

1

1

Salivation

1

1

1

1

Fur appearance

1

1

1

1

Piloerection

1

1

1

1

Eye prominence

1

1

1

1

Muscle tone

1

1

1

1

Home Cage Observations:Home cage posture - 1=Normal; Respiratory Pattern - 1=Normal; Clonic involuntary movements - 1=Normal; Tonic involuntary movements - 1=Normal; Vocalization - 1=Present; Palpebral closure - 1=Normal; Handling Observations: Ease of removal from the cage - 2=Normal;
Ease of handling animal in hand - 2=Normal; Red or crusty depsits - 1=Absent; Lacrimation - 1=None; Salivation- 1=Normal; Fur appearance - 1=Normal; Piloerection - 1=None; Eye Prominence - 1=Normal; Muscle tone - 1=Normal

Day 37

Parameters↓

Group & Sex

G1 & M

G2 & M

G3 & M

G4 & M

Dose (mg/kg body weight/day)

0

2

4.3

9.5

Number of Animals (randomly selected)

5

5

5

5

Open Field Observations

Mobility

1

1

1

1

Gait

1

1

1

1

Arousal

3

3

3

3

Number of rearing

Mean

4.6

4.6

4.6

3.8

±SD

0.9

0.9

1.1

1.6

Number of urination

Mean

2.4

2.4

2.2

2.2

±SD

0.5

0.5

0.4

0.8

Number of defecation

Mean

1.4

2.6

1.8

2.2

±SD

0.5

0.9

0.8

0.8

Clonic involuntary movement

1

1

1

1

Tonic involuntary movement

1

1

1

1

Stereotype behaviour

1

1

1

1

Excessive grooming

Mean

2.2

2.2

2.4

2.2

±SD

0.4

0.4

0.5

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

98.3

98.2

98.4

98.6

±SD

0.5

0.7

0.6

0.6

Open Field Observations: Mobility- 1=Normal;Gait- 1=Normal;Arousal- 3=Normal;Clonic involuntary movement- 1=None;Tonic involuntary movement -1=None;Stereotype behavior -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

Table 9.2. Summary of neurological/functional observation battery record. Females

Lactation Day 13

Parameters↓

Group & Sex

G1 & F

G2 & F

G3 & F

G4 & F

Dose (mg/kg body weight/day)

0

2

4.3

9.5

Number of Animals (randomly selected)

5

5

5

5

Home Cage Observations

Home cage posture

1

1

1

1

Respiratory pattern

1

1

1

1

Clonic involuntary movements

1

1

1

1

Tonic involuntary movements

1

1

1

1

Vocalization

1

1

1

1

Palpebral closure

1

1

1

1

Handling Observations

a. Ease of removal from the cage

2

2

2

2

b. Ease of handling animal in hand

2

2

2

2

Red or crusty deposits

Eyes

1

1

1

1

Nose

1

1

1

1

Mouth

1

1

1

1

Lacrimation

1

1

1

1

Salivation

1

1

1

1

Fur appearance

1

1

1

1

Piloerection

1

1

1

1

Eye prominence

1

1

1

1

Muscle tone

1

1

1

1

Home Cage Observations:Home cage posture - 1=Normal; Respiratory Pattern - 1=Normal; Clonic involuntary movements - 1=Normal; Tonic involuntary movements - 1=Normal; Vocalization - 1=Present; Palpebral closure - 1=Normal; Handling Observations: Ease of removal from the cage - 2=Normal;
Ease of handling animal in hand - 2=Normal; Red or crusty depsits - 1=Absent; Lacrimation - 1=None; Salivation- 1=Normal; Fur appearance - 1=Normal; Piloerection - 1=None; Eye Prominence - 1=Normal; Muscle tone - 1=Normal

Lactation Day 13

Parameters↓

Group & Sex

G1 & F

G2 & F

G3 & F

G4 & F

Dose (mg/kg body weight/day)

0

2

4.3

9.5

Number of Animals (randomly selected)

5

5

5

5

Open Field Observations

Mobility

1

1

1

1

Gait

1

1

1

1

Arousal

3

3

3

3

Number of rearing

Mean

3.8

3.6

3.2

4.0

±SD

0.8

0.9

0.4

1.2

Numbers of urination

Mean

3.0

2.8

3.2

2.4

±SD

0.7

0.4

0.4

0.9

Number of defecation

Mean

2.4

2.2

2.4

2.0

±SD

0.5

0.4

0.5

0.7

Clonic involuntary movement

 1

 1

 1

 1

Tonic involuntary movement

 1

 1

 1

 1

Stereotype behaviour

 1

 1

 1

 1

Excessive grooming

Mean

3.0

2.6

2.8

2.4

±SD

0.7

0.5

0.8

0.5

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

98.5

98.3

98.7

±SD

0.7

0.6

0.5

0.6

Open Field Observations: Mobility- 1=Normal;Gait- 1=Normal;Arousal- 3=Normal;Clonic involuntary movement- 1=None;Tonic involuntary movement -1=None;Stereotype behavior -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

Table 9.3. Summary of neurological/functional observation battery record. Recovery groups

Day 64

Parameters↓

Group & Sex

G1R & M

G4R & M

G1R & F

G4R & F

Dose (mg/kg body weight/day)

0

9.5

0

9.5

Number of Animals

5

5

5

5

Home Cage Observations

Home cage posture

1

1

1

1

Respiratory pattern

1

1

1

1

Clonic involuntary movements

1

1

1

1

Tonic involuntary movements

1

1

1

1

Vocalization

1

1

1

1

Palpebral closure

1

1

1

1

Handling Observations

a. Ease of removal from the cage

2

2

2

2

b. Ease of handling animal in hand

2

2

2

2

Red or crusty deposits

Eyes

1

1

1

1

Nose

1

1

1

1

Mouth

1

1

1

1

Lacrimation

1

1

1

1

Salivation

1

1

1

1

Fur appearance

1

1

1

1

Piloerection

1

1

1

1

Eye prominence

1

1

1

1

Muscle tone

1

1

1

1

Home Cage Observations:Home cage posture - 1=Normal; Respiratory Pattern - 1=Normal; Clonic involuntary movements - 1=Normal; Tonic involuntary movements - 1=Normal; Vocalization - 1=Present; Palpebral closure - 1=Normal; Handling Observations: Ease of removal from the cage - 2=Normal;
Ease of handling animal in hand - 2=Normal; Red or crusty depsits - 1=Absent; Lacrimation - 1=None; Salivation- 1=Normal; Fur appearance - 1=Normal; Piloerection - 1=None; Eye Prominence - 1=Normal; Muscle tone - 1=Normal

Day 64

Parameters↓

Group & Sex

G1R & M

G4R & M

G1R & F

G4R & F

Dose (mg/kg body weight/day)

0

9.5

0

9.5

Number of Animals

5

5

5

5

Open Field Observations

Mobility

1

1

1

1

Gait

1

1

1

1

Arousal

3

3

3

3

Number of rearing

Mean

3.6

3.6

3.6

3.6

±SD

0.9

0.9

0.9

0.9

Numbers of urination

Mean

2.4

2.8

2.8

2.6

±SD

0.5

0.4

0.4

0.5

Number of defecation

Mean

2.2

2.2

2.4

2.4

±SD

0.4

0.4

0.5

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

2.6

2.6

2.4

2.4

±SD

0.5

0.5

0.5

0.5

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

98.2

98.3

98.2

98.5

±SD

0.5

0.7

0.4

0.6

Open Field Observations: Mobility- 1=Normal;Gait- 1=Normal;Arousal- 3=Normal;Clonic involuntary movement- 1=None;Tonic involuntary movement -1=None;Stereotype behavior -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

Table 10.1. Summary of haematology record. Males

Group, Sex

& Dose

(mg/kg body weight/day)

Total Leucocyte Count

Total Erythrocyte Count

Haemoglobin

Haematocrit

Mean Corpuscular Volume

Mean Corpuscular Haemoglobin

(WBC)

(RBC)

(HGB)

(HCT)

(MCV)

(MCH)

(103cells/µL)

(106cells/µL)

(g/dL)

(%)

(fL)

(pg)

G1, M & 0

Mean

12.73

6.77

13.22

43.04

64.62

19.64

±SD

2.46

1.11

1.95

2.92

9.06

1.37

n

5

5

5

5

5

5

G2, M & 2

Mean

10.23

7.61

14.96

45.34

59.66

19.72

±SD

2.21

0.24

0.83

2.47

4.43

1.47

n

5

5

5

5

5

5

G3, M & 4.3

Mean

11.00

6.01

11.76

39.98

72.14

20.42

±SD

2.18

2.34

2.97

4.89

18.42

3.01

n

5

5

5

5

5

5

G4, M & 9.5

Mean

6.55*

6.85

13.50

41.42

61.24

19.94

±SD

1.26

1.11

1.52

4.10

6.33

2.03

n

5

5

5

5

5

5

Group, Sex

& Dose

(mg/kg body weight/day)

Mean Corpuscular Haemoglobin Concentration

Platelet Count

Mean Platelet Volume

Reticulocyte Count

Neutrophils

Lymphocytes

(MCHC)

(PLT)

(MPV)

(Retic)

(Neut)

(Lymph)

(g/dL)

(103cells/µL)

(fL)

(%)

(%)

(%)

G1, M & 0

Mean

30.66

824.60

6.40

7.61

18.72

75.92

±SD

3.05

116.04

1.54

8.75

4.41

4.70

n

5

5

5

5

5

5

G2, M & 2

Mean

33.06

761.80

6.00

2.01

22.02

72.58

±SD

0.38

97.20

0.54

0.84

3.04

2.52

n

5

5

5

5

5

5

G3, M & 4.3

Mean

29.04

862.80

8.10

24.46

17.38

75.96

±SD

3.76

176.98

1.92

23.34

6.86

8.05

n

5

5

5

5

5

5

G4, M & 9.5

Mean

32.60

616.60

6.64

2.63

14.48

79.88

±SD

0.92

256.07

0.45

2.28

2.65

2.12

n

5

5

5

5

5

5

Group, Sex

& Dose

(mg/kg body weight/day)

Monocytes

Eosinophils

Basophils

Absolute Reticulocyte

Count

Absolute Neutrophils

Absolute Lymphocytes

(Mono)

(Eos)

(Baso)

(Retic)

(Neut)

(Lymph)

(%)

(%)

(%)

(109cells/L)

(103cells/µL)

(103cells/µL)

G1, M & 0

Mean

2.92

1.24

0.34

440.52

2.34

9.70

±SD

0.43

0.57

0.05

465.83

0.57

2.17

n

5

5

5

5

5

5

G2, M & 2

Mean

2.82

1.46

0.28

152.56

2.28

7.41

±SD

0.96

0.34

0.04

62.48

0.70

1.52

n

5

5

5

5

5

5

G3, M & 4.3

Mean

4.28

1.02

0.44

1064.60

1.97

8.29

±SD

1.53

0.82

0.05

939.34

1.03

1.49

n

5

5

5

5

5

5

G4, M & 9.5

Mean

1.90

0.10*

0.38

163.70

0.92*

5.25*

±SD

0.73

0.00

0.08

126.00

0.09

1.12

n

5

5

5

5

5

5

Group, Sex

& Dose

(mg/kg body weight/day)

Absolute Monocytes

Absolute Eosinophils

Absolute Basophils

Prothrombin Time

Activated Prothrombin Time

(Mono)

(Eos)

(Baso)

(PT)

(APTT)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(Seconds)

(Seconds)

G1, M & 0

Mean

0.37

0.15

0.05

24.46

28.66

±SD

0.11

0.08

0.02

1.53

4.14

n

5

5

5

5

5

G2, M & 2

Mean

0.27

0.15

0.03

21.38

24.48

±SD

0.05

0.06

0.01

2.84

1.76

n

5

5

5

5

5

G3, M & 4.3

Mean

0.46

0.12

0.05

21.98

32.94

±SD

0.18

0.10

0.01

1.84

8.33

n

5

5

5

5

5

G4, M & 9.5

Mean

0.12*

0.00*

0.02*

23.48

26.02

±SD

0.04

0.00

0.01

1.59

2.09

n

5

5

5

5

5

* Statistically significant (P<0.05) change than the vehicle control group.

Table 10.2. Summary of haematology record. Females

Group, Sex

& Dose

(mg/kg body weight/day)

Total Leucocyte Count

Total Erythrocyte Count

Haemoglobin

Haematocrit

Mean Corpuscular Volume

Mean Corpuscular Haemoglobin

(WBC)

(RBC)

(HGB)

(HCT)

(MCV)

(MCH)

(103cells/µL)

(106cells/µL)

(g/dL)

(%)

(fL)

(pg)

G1, F & 0

Mean

11.60

6.87

14.36

43.18

62.96

20.92

±SD

2.26

0.31

0.49

1.83

3.42

1.17

n

5

5

5

5

5

5

G2, F & 2

Mean

12.52

7.15

14.40

43.18

60.48

20.14

±SD

3.54

0.22

0.85

1.83

3.13

1.25

n

5

5

5

5

5

5

G3, F & 4.3

Mean

10.27

7.14

14.34

42.84

60.12

20.12

±SD

2.88

0.55

0.81

2.51

2.83

1.02

n

5

5

5

5

5

5

G4, F & 9.5

Mean

7.47

6.08*

13.06*

39.00*

64.46

21.54

±SD

3.50

0.58

0.86

1.79

4.95

1.22

n

5

5

5

5

5

5

Group, Sex

& Dose

(mg/kg body weight/day)

Mean Corpuscular

Haemoglobin Concentration

Platelet Count

Mean Platelet Volume

Reticulocyte Count

Neutrophils

Lymphocytes

(MCHC)

(PLT)

(MPV)

(Retic)

(Neut)

(Lymph)

(g/dL)

(103cells/µL)

(fL)

(%)

(%)

(%)

G1, F & 0

Mean

33.26

812.20

7.38

2.76

28.22

64.68

±SD

0.64

162.21

0.38

0.43

10.10

10.49

n

5

5

5

5

5

5

G2, F & 2

Mean

33.34

935.00

7.02

2.62

32.80

60.48

±SD

0.71

212.99

0.36

1.01

11.16

9.68

n

5

5

5

5

5

5

G3, F & 4.3

Mean

33.48

899.60

7.22

2.78

33.38

60.14

±SD

0.70

126.40

0.59

1.16

8.27

7.36

n

5

5

5

5

5

5

G4, F & 9.5

Mean

33.42

792.00

7.32

5.15

23.78

72.76

±SD

0.75

148.99

0.51

5.10

14.07

14.59

n

5

5

5

5

5

5

Group, Sex

& Dose

(mg/kg body weight/day)

Monocytes

Eosinophils

Basophils

Absolute Reticulocyte

Count

Absolute Neutrophils

Absolute Lymphocytes

(Mono)

(Eos)

(Baso)

(Retic)

(Neut)

(Lymph)

(%)

(%)

(%)

(109cells/L)

(103cells/µL)

(103cells/µL)

G1, F & 0

Mean

4.20

1.28

0.84

188.92

3.33

7.46

±SD

1.74

0.94

0.65

27.49

1.44

1.69

n

5

5

5

5

5

5

G2, F & 2

Mean

3.54

1.14

1.02

187.04

4.30

7.39

±SD

0.98

0.33

0.95

71.65

2.38

1.37

n

5

5

5

5

5

5

G3, F & 4.3

Mean

3.94

1.16

0.58

193.26

3.45

6.13

±SD

1.66

0.63

0.24

62.25

1.17

1.77

n

5

5

5

5

5

5

G4, F & 9.5

Mean

1.88*

0.46

0.28

291.02

1.79

5.43

±SD

0.83

0.26

0.15

253.48

1.26

2.97

n

5

5

5

5

5

5

Group, Sex

& Dose

(mg/kg body weight/day)

Absolute Monocytes

Absolute Eosinophils

Absolute Basophils

Prothrombin Time

Activated Prothrombin Time

(Mono)

(Eos)

(Baso)

(PT)

(APTT)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(Seconds)

(Seconds)

G1, F & 0

Mean

0.48

0.14

0.10

30.36

36.14

±SD

0.19

0.11

0.07

6.22

27.82

n

5

5

5

5

5

G2, F & 2

Mean

0.44

0.15

0.12

28.44

32.34

±SD

0.16

0.08

0.09

4.96

22.35

n

5

5

5

5

5

G3, F & 4.3

Mean

0.43

0.12

0.06

23.96

24.44

±SD

0.26

0.07

0.03

4.32

9.12

n

5

5

5

5

5

G4, F & 9.5

Mean

0.13*

0.03

0.02

21.74*

23.10

±SD

0.05

0.01

0.02

4.86

7.56

n

5

5

5

5

5

* Statistically significant (P<0.05) change than the vehicle control group.

Table 10.3. Summary of haematology record. Recovery groups

Group, Sex

& Dose

(mg/kg body weight/day)

Total Leucocyte Count

Total Erythrocyte Count

Haemoglobin

Haematocrit

Mean Corpuscular Volume

Mean Corpuscular Haemoglobin

(WBC)

(RBC)

(HGB)

(HCT)

(MCV)

(MCH)

(103cells/µL)

(106cells/µL)

(g/dL)

(%)

(fL)

(pg)

G1R, M & 0

Mean

11.36

7.73

14.72

44.62

58.24

19.22

±SD

3.13

1.07

1.05

3.22

6.09

1.83

n

5

5

5

5

5

5

G4R, M & 9.5

Mean

9.83

6.95

14.86

44.30

63.90

21.44

±SD

0.83

0.36

0.48

1.88

3.17

1.40

n

5

5

5

5

5

5

G1R, F & 0

Mean

8.85

7.17

13.64

40.04

55.86

19.04

±SD

1.97

0.18

0.69

1.47

1.99

0.88

n

5

5

5

5

5

5

G4R, F & 9.5

Mean

7.63

6.26*

13.14

38.70

61.96*

21.06*

±SD

2.11

0.42

0.40

1.14

3.38

1.24

n

5

5

5

5

5

5

Group, Sex

& Dose

(mg/kg body weight/day)

Mean Corpuscular

Haemoglobin Concentration

Platelet Count

Mean Platelet Volume

Reticulocyte Count

Neutrophils

Lymphocytes

(MCHC)

(PLT)

(MPV)

(Retic)

(Neut)

(Lymph)

(g/dL)

(103cells/µL)

(fL)

(%)

(%)

(%)

G1R, M & 0

Mean

33.02

891.20

6.92

1.96

22.78

71.40

±SD

0.33

99.39

0.22

0.44

2.28

2.88

n

5

5

5

5

5

5

G4R, M & 9.5

Mean

33.54

873.80

7.22

3.99*

23.88

65.46*

±SD

0.76

313.03

1.07

1.20

3.45

3.76

n

5

5

5

5

5

5

G1R, F & 0

Mean

34.06

1009.00

6.72

1.59

20.66

73.00

±SD

0.50

181.21

0.25

0.48

1.31

1.95

n

5

5

5

5

5

5

G4R, F & 9.5

Mean

33.96

941.20

6.66

3.34*

20.76

72.64

±SD

0.49

112.29

0.11

0.77

8.11

8.76

n

5

5

5

5

5

5

Group, Sex

& Dose

(mg/kg body weight/day)

Monocytes

Eosinophils

Basophils

Absolute Reticulocyte Count

Absolute Neutrophils

Absolute Lymphocytes

(Mono)

(Eos)

(Baso)

(Retic)

(Neut)

(Lymph)

(%)

(%)

(%)

(109cells/L)

(103cells/µL)

(103cells/µL)

G1R, M & 0

Mean

3.30

1.52

0.44

150.92

2.58

8.12

±SD

1.08

0.44

0.18

33.84

0.73

2.32

n

5

5

5

5

5

5

G4R, M & 9.5

Mean

4.96*

4.46*

0.52

274.24*

2.37

6.42

±SD

0.81

1.28

0.11

69.40

0.50

0.32

n

5

5

5

5

5

5

G1R, F & 0

Mean

3.28

1.74

0.30

113.82

1.82

6.47

±SD

0.54

0.38

0.10

34.81

0.40

1.48

n

5

5

5

5

5

5

G4R, F & 9.5

Mean

3.20

2.32

0.34

208.76*

1.46

5.68

±SD

0.76

0.97

0.09

46.00

0.19

1.97

n

5

5

5

5

5

5

Group, Sex

& Dose

(mg/kg body weight/day)

Absolute Monocytes

Absolute Eosinophils

Absolute Basophils

Prothrombin Time

Activated Prothrombin Time

(Mono)

(Eos)

(Baso)

(PT)

(APTT)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(Seconds)

(Seconds)

G1R, M & 0

Mean

0.38

0.17

0.05

27.68

17.52

±SD

0.18

0.04

0.03

1.22

0.68

n

5

5

5

5

5

G4R, M & 9.5

Mean

0.49

0.44*

0.05

24.36*

18.76

±SD

0.08

0.14

0.01

1.93

2.16

n

5

5

5

5

5

G1R, F & 0

Mean

0.29

0.15

0.03

22.62

22.30

±SD

0.09

0.04

0.01

2.08

2.43

n

5

5

5

5

5

G4R, F & 9.5

Mean

0.24

0.18

0.03

24.36

18.16*

±SD

0.06

0.10

0.01

3.63

2.21

n

5

5

5

5

5

* Statistically significant (P<0.05) change than the vehicle control group.

Table 11.1. Summary of clinical chemistry record. Males

Group, Sex

& Dose

(mg/kg body

weight/day)

Glucose

Urea

Creatinine

Total Cholesterol

Triglycerides

(GLU)

-

(CRE)

(CHO)

(TRI)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

G1, M & 0

Mean

94.80

29.14

0.61

46.60

49.00

±SD

3.27

1.24

0.07

6.69

6.28

n

5

5

5

5

5

G2, M & 2

Mean

100.00

34.74*

0.60

47.20

58.80

±SD

10.46

4.18

0.03

8.29

26.44

n

5

5

5

5

5

G3, M & 4.3

Mean

92.20

33.88*

0.59

36.40

48.60

±SD

13.86

1.59

0.02

6.35

14.08

n

5

5

5

5

5

G4, M & 9.5

Mean

92.60

29.56

0.59

40.60

68.80

±SD

9.45

3.12

0.04

5.22

15.99

n

5

5

5

5

5

Group, Sex

& Dose

(mg/kg body

weight/day)

Total Protein

Albumin

Alanine aminotransferase

Aspartate aminotransferase

Alkaline
phosphatase

(TPR)

(ALB)

(ALT)

(AST)

(ALP)

(g/dL)

(g/dL)

(U/L)

(U/L)

(U/L)

G1, M & 0

Mean

6.66

3.12

72.60

122.40

136.20

±SD

0.34

0.17

17.11

14.12

28.86

n

5

5

5

5

5

G2, M & 2

Mean

6.64

3.10

50.00*

106.20

145.00

±SD

0.29

0.12

11.66

16.48

52.78

n

5

5

5

5

5

G3, M & 4.3

Mean

6.72

3.01

56.20

106.40

160.00

±SD

0.23

0.17

9.47

14.17

68.60

n

5

5

5

5

5

G4, M & 9.5

Mean

6.52

3.07

41.20*

84.60*

134.60

±SD

0.29

0.16

7.26

4.93

70.21

n

5

5

5

5

5

Group, Sex

& Dose

(mg/kg body weight/day)

Total Bilirubin

Calcium

Phosphorous

Globulin

Blood Urea Nitrogen

(BIT)

(CAL)

(PHO)

(GLO)

(BUN)

(mg/dL)

(mg/dL)

(mg/dL)

(g/dL)

(mg/dL)

G1, M & 0

Mean

0.07

7.68

6.06

3.54

13.60

±SD

0.08

0.64

0.63

0.21

0.58

n

5

5

5

5

5

G2, M & 2

Mean

0.02

7.92

6.20

3.54

16.21*

±SD

0.04

0.44

0.72

0.29

1.95

n

5

5

5

5

5

G3, M & 4.3

Mean

0.08

7.76

6.38

3.71

15.81*

±SD

0.08

0.35

0.78

0.30

0.75

n

5

5

5

5

5

G4, M & 9.5

Mean

0.04

8.02

6.14

3.45

13.79

±SD

0.02

0.64

0.64

0.21

1.46

n

5

5

5

5

5

Group, Sex

& Dose

(mg/kg body

weight/day)

Sodium

Potassium

Chloride

(Na)

(K)

(CLO)

(mmol/L)

(mmol/L)

(mmol/L)

G1, M & 0

Mean

144.60

3.71

109.66

±SD

1.13

0.20

2.99

n

5

5

5

G2, M & 2

Mean

145.24

3.88

110.10

±SD

1.07

0.11

1.34

n

5

5

5

G3, M & 4.3

Mean

144.92

3.70

109.76

±SD

1.32

0.31

2.00

n

5

5

5

G4, M & 9.5

Mean

145.84

3.91

112.00

±SD

0.86

0.27

1.01

n

5

5

5

* Statistically significant (P<0.05) change than the vehicle control group.

Table 11.2. Summary of clinical chemistry record. Females

Group, Sex

& Dose

(mg/kg body

weight/day)

Glucose

Urea

Creatinine

Total Cholesterol

Triglycerides

(GLU)

(CRE)

(CHO)

(TRI)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

G1, F & 0

Mean

85.40

49.18

0.52

65.80

48.60

±SD

12.95

15.76

0.06

25.09

19.99

n

5

5

5

5

5

G2, F & 2

Mean

92.00

47.12

0.60

73.00

114.20*

±SD

23.79

10.64

0.07

14.68

45.25

n

5

5

5

5

5

G3, F & 4.3

Mean

91.20

42.90

0.52

67.00

101.00

±SD

14.62

9.17

0.07

7.65

34.52

n

5

5

5

5

5

G4, F & 9.5

Mean

106.20

30.72*

0.54

71.00

70.80

±SD

15.85

7.22

0.04

19.17

25.88

n

5

5

5

5

5

Group, Sex

& Dose

(mg/kg body

weight/day)

Total Protein

Albumin

Alanine aminotransferase

Aspartate aminotransferase

Alkaline
phosphatase

(TPR)

(ALB)

(ALT)

(AST)

(ALP)

(g/dL)

(g/dL)

(U/L)

(U/L)

(U/L)

G1, F & 0

Mean

6.72

3.29

84.40

102.00

243.20

±SD

0.65

0.38

29.25

18.75

138.36

n

5

5

5

5

5

G2, F & 2

Mean

6.56

3.41

120.20

129.80

388.00

±SD

0.29

0.35

36.46

42.74

302.67

n

5

5

5

5

5

G3, F & 4.3

Mean

6.32

3.16

124.20

127.20

385.80

±SD

0.55

0.35

29.95

37.41

67.22

n

5

5

5

5

5

G4, F & 9.5

Mean

6.50

3.41

91.20

111.60

278.80

±SD

0.45

0.40

66.95

57.69

67.20

n

5

5

5

5

5

Group, Sex

& Dose

(mg/kg body

weight/day)

Total Bilirubin

Calcium

Phosphorous

Globulin

Blood Urea Nitrogen

(BIT)

(CAL)

(PHO)

(GLO)

(BUN)

(mg/dL)

(mg/dL)

(mg/dL)

(g/dL)

(mg/dL)

G1, F & 0

Mean

0.13

8.84

6.10

3.43

22.95

±SD

0.10

0.73

1.26

0.35

7.36

n

5

5

5

5

5

G2, F & 2

Mean

0.10

9.40

6.66

3.15

21.99

±SD

0.08

0.77

2.01

0.26

4.97

n

5

5

5

5

5

G3, F & 4.3

Mean

0.09

9.10

6.50

3.16

20.02

±SD

0.09

0.54

0.83

0.36

4.28

n

5

5

5

5

5

G4, F & 9.5

Mean

0.16

9.48

5.28

3.09

14.33*

±SD

0.14

0.69

1.65

0.22

3.37

n

5

5

5

5

5

Group, Sex

& Dose

(mg/kg body weight/day)

Sodium

Potassium

Chloride

(Na)

(K)

(CLO)

(mmol/L)

(mmol/L)

(mmol/L)

G1, F & 0

Mean

141.36

3.27

103.92

±SD

3.01

0.54

4.31

n

5

5

5

G2, F & 2

Mean

140.12

3.34

103.34

±SD

2.51

0.37

3.78

n

5

5

5

G3, F & 4.3

Mean

139.92

3.21

102.52

±SD

1.74

0.29

2.83

n

5

5

5

G4, F & 9.5

Mean

141.28

3.44

103.80

±SD

2.63

0.38

4.86

n

5

5

5

* Statistically significant (P<0.05) change than the vehicle control group.

Table 11.3. Summary of clinical chemistry record. Recovery groups

Group, Sex

& Dose

(mg/kg body

weight/day)

Glucose

Urea

Creatinine

Total Cholesterol

Triglycerides

(GLU)

-

(CRE)

(CHO)

(TRI)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

G1R, M & 0

Mean

95.80

31.34

0.49

43.40

30.60

±SD

6.83

2.80

0.01

6.35

3.65

n

5

5

5

5

5

G4R, M & 9.5

Mean

98.60

28.08

0.45*

40.40

35.20

±SD

20.60

3.19

0.02

9.61

5.02

n

5

5

5

5

5

G1R, F & 0

Mean

106.60

32.32

0.54

65.60

34.00

±SD

9.02

4.63

0.06

5.86

6.63

n

5

5

5

5

5

G4R, F & 9.5

Mean

107.40

27.32

0.75*

53.00*

47.20

±SD

11.37

2.46

0.17

4.30

12.99

n

5

5

5

5

5

Group, Sex

& Dose

(mg/kg body

weight/day)

Total Protein

Albumin

Alanine aminotransferase

Aspartate aminotransferase

Alkaline
phosphatase

(TPR)

(ALB)

(ALT)

(AST)

(ALP)

(g/dL)

(g/dL)

(U/L)

(U/L)

(U/L)

G1R, M & 0

Mean

6.56

3.05

69.40

113.80

120.40

±SD

0.23

0.16

12.54

9.98

28.59

n

5

5

5

5

5

G4R, M & 9.5

Mean

6.68

3.07

66.80

110.60

152.00

±SD

0.36

0.13

12.07

12.88

44.86

n

5

5

5

5

5

G1R, F & 0

Mean

7.04

3.61

47.60

105.80

57.80

±SD

0.28

0.16

15.68

25.06

18.77

n

5

5

5

5

5

G4R, F & 9.5

Mean

6.98

3.76

44.40

89.80

65.80

±SD

0.61

0.30

11.01

16.90

29.42

n

5

5

5

5

5

Group, Sex

& Dose

(mg/kg body

weight/day)

Total Bilirubin

Calcium

Phosphorous

Globulin

Blood Urea Nitrogen

(BIT)

(CAL)

(PHO)

(GLO)

(BUN)

(mg/dL)

(mg/dL)

(mg/dL)

(g/dL)

(mg/dL)

G1R, M & 0

Mean

0.15

9.12

4.98

3.51

14.63

±SD

0.10

0.19

0.23

0.35

1.31

n

5

5

5

5

5

G4R, M & 9.5

Mean

0.17

9.78*

5.60*

3.61

13.10

±SD

0.09

0.40

0.34

0.23

1.49

n

5

5

5

5

5

G1R, F & 0

Mean

0.11

10.04

4.22

3.43

15.08

±SD

0.08

0.42

0.41

0.34

2.16

n

5

5

5

5

5

G4R, F & 9.5

Mean

0.22*

9.94

4.16

3.22

12.75

±SD

0.05

0.54

0.25

0.37

1.15

n

5

5

5

5

5

Group, Sex

& Dose

(mg/kg body

weight/day)

Sodium

Potassium

Chloride

(Na)

(K)

(CLO)

(mmol/L)

(mmol/L)

(mmol/L)

G1R, M & 0

Mean

143.74

3.37

108.70

±SD

1.15

0.16

1.86

n

5

5

5

G4R, M & 9.5

Mean

143.40

3.40

106.86

±SD

0.69

0.19

1.67

n

5

5

5

G1R, F & 0

Mean

142.80

3.33

108.14

±SD

0.79

0.42

0.31

n

5

5

5

G4R, F & 9.5

Mean

144.44*

3.12

108.10

±SD

0.80

0.23

1.02

n

5

5

5

* Statistically significant (P<0.05) change than the vehicle control group.

Table 12. Summary of urinalysis record

Examination

Group, Sex & Dose

(mg/kg body weight/day)

G1,

M & 0

G2,

M & 2

G3,

M & 4.3

G4,

M & 9.5

Number of Animals

(randomly selected)

5

5

5

5

Physical Examination

Color

Pale Yellow

3

4

3

4

Yellow

2

1

2

1

Appearance

Clear

3

4

3

3

Turbidity

2

1

2

2

Volume (mL)

Mean

8.9

9.2

7.8

5.9

±SD

2.2

1.4

2.8

2.3

Chemical Examination

pH

Mean

8.1

8.4

8.4

8.5

±SD

0.5

0.2

0.2

0.0

Specific Gravity

Mean

1.007

1.006

1.006

1.006

±SD

0.003

0.002

0.002

0.002

Urobilinogen (mg/dL)

Mean

0.2

0.2

0.6

0.4

±SD

0.0

0.0

0.8

0.4

Bilirubin (mg/dL)

Neg

5

5

4

5

1

-

-

1

-

Ketones (mg/dL)

5

2

3

3

3

Neg

3

2

2

2

Blood (Ery/µL)

Ca25

-

2

1

-

Neg

2

2

2

3

>=Ca200

2

1

1

1

Ca10

1

-

1

1

Protein (mg/dL)

Trace

-

3

1

1

100

2

1

-

3

>=300

-

1

2

-

30

1

-

2

1

Neg

2

-

-

-

Nitrite

Neg

2

1

3

1

Pos

3

4

2

4

Leucocytes

Neg

3

3

3

2

Ca15

2

1

2

2

Ca70

-

1

-

1

Glucose (mg/dL)

Neg

5

5

5

5

Microalbumin

(mg/dL)

15

1

-

-

-

>15

3

5

5

5

Neg

1

-

-

-

Microscopic Examination

Epi Cells

0

4

2

4

4

0-1

1

2

-

-

1-2

-

1

1

1

Casts

Absent

5

5

5

5

Crystals

Present

5

5

5

5

Examination

Group, Sex & Dose

(mg/kg body weight/day)

G1R,

M & 0

G4R,

M & 9.5

G1R,

F & 0

G4R,

F & 9.5

Number of Animals

5

5

5

5

Physical Examination

Color

Pale Yellow

5

4

5

5

Yellow

-

1

-

-

Appearance

Clear

2

3

5

4

Turbidity

3

2

-

1

Volume (mL)

Mean

7.6

8.1

7.5

7.8

±SD

2.3

1.8

1.3

3.4

Chemical Examination

pH

Mean

8.4

8.3

7.0

8.1*

±SD

0.2

0.4

0.5

0.4

Specific gravity

Mean

1.007

1.006

1.017

1.007*

±SD

0.004

0.002

0.003

0.003

Urobilinogen (mg/dL)

Mean

0.2

0.2

0.2

0.2

±SD

0.0

0.0

0.0

0.0

Bilirubin (mg/dL)

Neg

5

5

5

5

Ketones (mg/dL)

5

4

2

1

1

Neg

1

3

4

4

Blood (Ery/µL)

Ca25

-

2

2

2

Ca80

-

2

-

2

Neg

-

-

-

1

>=Ca200

3

1

1

-

Ca10

2

-

2

-

Protein (mg/dL)

Trace

-

3

2

-

30

3

2

-

2

100

1

-

-

 

Neg

1

-

3

3

Nitrite

Neg

2

1

4

2

Pos

3

4

1

3

Leucocytes

Neg

5

3

5

5

Ca15

-

2

-

-

Glucose (mg/dL)

Neg

5

5

5

5

Microalbumin

(mg/dL)

15

-

-

3

2

>15

4

5

2

2

Neg

1

-

-

1

Microscopic Examination

Epi Cells

0

3

3

3

3

0-1

1

1

2

1

1-2

1

1

-

1

Casts

Absent

5

5

5

5

Crystals

Present

5

5

5

5

M: Male; F: Female; R: Recovery; SD: Standard Deviation; Pos: Positive; Neg: Negative              

Ca: Calculated; >=: Greater than or equal to; >: Greater than

Table 13. Summary of vaginal smear examination for determination of oestrus cyclicity

Determination of Oestrus Cyclicity during Pre-mating Treatment Period

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

Total No. of

Females Evaluated

Females with

Complete Regular Cycles

Females with at least one Irregular Oestrus Cycle

Average Length of

Oestrous Cycle (Days)

G1, F & 0

12

n

12

0

Mean

4.96

%

100.00

0.00

±SD

0.14

n

12

G2, F & 2

12

n

12

0

Mean

4.92

%

100.00

0.00

±SD

0.19

n

12

G3, F & 4.3

12

n

12

0

Mean

4.96

%

100.00

0.00

±SD

0.14

n

12

G4, F & 9.5

12

n

12

0

Mean

5.00

%

100.00

0.00

±SD

0.00

n

12

Table 14. Summary record of gestation body weight (g)

Group, Sex & Dose

(mg/kg body weight/day)

Body Weight (g) on Gestation Day (GD)

0

7

14

20

G1, F & 0

Mean

278.31

293.77

322.21

382.79

±SD

23.53

26.12

26.55

33.83

n

11

11

11

11

G2, F & 2

Mean

275.69

292.67

325.49

388.21

±SD

16.80

17.63

23.01

23.29

n

12

12

12

12

G3, F & 4.3

Mean

268.09

286.56

316.82

375.04

±SD

16.98

18.05

21.33

24.66

n

11

11

11

11

G4, F & 9.5

Mean

268.90

279.30

292.77*

315.74*

±SD

20.52

21.48

24.39

28.38

n

9

9

9

9

n: Number of Litters

*: Statistically significant (P<0.05) change than the vehicle control group

Table 15. Summary record of percent change in body weight (%) during gestation period

Group, Sex & Dose

(mg/kg body weight/day)

Percent Change in Body Weight (%) during Gestation Day (GD)

0 to 7

7 to 14

14 to 20

G1, F & 0

Mean

5.52

9.76

18.81

±SD

1.01

2.30

3.72

n

11

11

11

G2, F & 2

Mean

6.18

11.17

19.41

±SD

1.61

2.33

4.18

n

12

12

12

G3, F & 4.3

Mean

6.90

10.55

18.48

±SD

1.54

1.80

5.04

n

11

11

11

G4, F & 9.5

Mean

3.87

4.81*

7.82*

±SD

1.93

2.73

3.18

n

9

9

9

n: Number of Litters

*: Statistically significant (P<0.05) change than the vehicle control group

Table 16. Summary of lactation body weight (g) record

Group, Sex & Dose

(mg/kg body weight/day)

Body Weight (g) on Lactation Day (LD)

1

4

7

13

G1, F & 0

Mean

307.86

310.70

318.55

331.25

±SD

27.73

29.72

30.50

29.30

n

11

11

11

11

G2, F & 2

Mean

311.03

317.21

323.91

335.95

±SD

23.47

23.94

23.20

21.92

n

12

12

12

12

G3, F & 4.3

Mean

302.13

309.03

317.06

331.58

±SD

21.59

22.31

22.33

23.37

n

11

11

11

11

G4, F & 9.5

Mean

282.79

280.88*

281.10*

282.55*

±SD

28.29

29.58

30.91

30.44

n

9

9

9

9

n: Number of Litters

*: Statistically significant (P<0.05) change than the vehicle control group

Table 17. Summary of percent change in body weight (%) during lactation

Group, Sex & Dose

(mg/kg body weight/day)

Percent Change in Body Weight (%) during Lactation Day (LD)

1 to 4

4 to 7

7 to 13

G1, F & 0

Mean

0.89

2.53

4.05

±SD

1.38

1.20

1.25

n

11

11

11

G2, F & 2

Mean

2.00

2.15

3.76

±SD

1.89

1.26

1.26

n

12

12

12

G3, F & 4.3

Mean

2.29

2.61

4.58

±SD

1.28

0.84

1.06

n

11

11

11

G4, F & 9.5

Mean

-0.70

0.04*

0.55*

±SD

2.21

1.41

1.02

n

9

9

9

n: Number of dams

* Statistically significant (P<0.05) change than the vehicle control group.

Table 18.1. Summary of absolute organ weights (g) record. Males

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

Adrenals

Thymus

Spleen

Testes

Epididymes

Heart

Kidneys

Brain

Liver

G1, M & 0

Mean

0.0573

0.3336

1.2240

3.4297

1.4279

1.3142

2.4930

1.9968

9.5906

±SD

0.0059

0.0745

0.1263

0.1785

0.1078

0.1285

0.2223

0.1735

0.9855

n

5

5

5

12

12

5

5

5

5

G2, M & 2

Mean

0.0580

0.3343

1.3110

3.4437

1.4561

1.4206

2.6853

2.0335

10.7475

±SD

0.0015

0.0258

0.1323

0.2288

0.1167

0.1636

0.2341

0.1507

1.1692

n

5

5

5

12

12

5

5

5

5

G3, M & 4.3

Mean

0.0573

0.3630

1.2304

3.5510

1.4691

1.3602

2.5710

2.0455

10.0981

±SD

0.0052

0.0915

0.3067

0.2816

0.1595

0.1507

0.2624

0.0927

0.8477

n

5

5

5

12

12

5

5

5

5

G4, M & 9.5

Mean

0.0622

0.2103*

1.0243

3.2469

1.4406

1.3828

2.4998

2.0719

10.2933

±SD

0.0058

0.0219

0.1794

0.3584

0.1728

0.1683

0.3065

0.1339

1.7572

n

5

5

5

12

12

5

5

5

5

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

Prostate

Seminal vesicles with coagulating glands

Lungs

Thyroid along with parathyroidW

G1, M & 0

Mean

1.4901

1.6335

2.2906

0.0201

±SD

0.2713

0.3206

0.2613

0.0023

n

12

12

5

12

G2, M & 2

Mean

1.4601

1.7087

2.3882

0.0211

±SD

0.2423

0.4194

0.2229

0.0042

n

12

12

5

12

G3, M & 4.3

Mean

1.3257

1.7352

2.3277

0.0208

±SD

0.3358

0.3346

0.2665

0.0032

n

12

12

5

12

G4, M & 9.5

Mean

1.4384

1.5831

2.4727

0.0215

±SD

0.3947

0.4769

0.3709

0.0031

n

12

12

5

12

n: Number of Animals (Adrenals, Thymus, Spleen, Heart, Kidneys, Brain & Liver weighed for 5 randomly selected animals; Testes, Epididymis, Prostate, Seminal vesicles with coagulating glands & Thyroid along with parathyroid weighed for all animals); w: Weighed post fixation.

*: Statistically significant (P<0.05) change than the vehicle control group

Table 18.2. Summary of absolute organ weights (g) record. Females

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

Adrenals

Thymus

Spleen

Heart

Kidneys

Brain

Liver

Lungs

Thyroid along with parathyroidW

G1, F & 0

Mean

0.0901

0.2217

1.1327

1.2036

2.3634

2.1352

12.9287

2.1487

0.0205

±SD

0.0102

0.0339

0.0932

0.1093

0.2518

0.0693

1.7899

0.2039

0.0029

n

5

5

5

5

5

5

5

5

11

G2, F & 2

Mean

0.0820

0.2382

1.1168

1.2173

2.4303

2.0116

13.3214

1.9219

0.0215

±SD

0.0058

0.0402

0.1109

0.0309

0.2635

0.1630

1.8291

0.0902

0.0027

n

5

5

5

5

5

5

5

5

12

G3, F & 4.3

Mean

0.0840

0.2104

1.1447

1.1633

2.4926

2.1440

13.5957

2.0602

0.0221

±SD

0.0040

0.0316

0.0650

0.1158

0.2005

0.1109

0.4648

0.1476

0.0023

n

5

5

5

5

5

5

5

5

11

G4, F & 9.5

Mean

0.0764

0.1436*

0.9023*

1.1161

2.1686

2.0183

10.0050*

2.1364

0.0189

±SD

0.0139

0.0455

0.1127

0.2021

0.2380

0.1481

2.1592

0.2409

0.0020

n

5

5

5

5

5

5

5

5

9

n: Number of Animals (Adrenals, Thymus, Spleen, Heart, Kidneys, Brain & Liver weighed for 5 randomly selected animals; Thyroid along with parathyroid weighed for all pregnant females); w: Weighed post fixation

*: Statistically significant (P<0.05) change than the vehicle control group

Table 19.1. Summary of terminal body weight (g) and organ weight (%) relative to terminal body weight record. Males

Group, Sex

& Dose

(mg/kg body weight/day)

Fasting Body Weight (g)

Adrenals

Thymus

Spleen

Testes

Epididymes

Heart

Kidneys

Brain

Liver

G1, M & 0

Mean

379.18

0.0157

0.0911

0.3352

0.9098

0.3791

0.3603

0.6816

0.5463

2.6199

±SD

34.78

0.0022

0.0183

0.0346

0.0763

0.0419

0.0397

0.0398

0.0368

0.1617

n

12

5

5

5

12

12

5

5

5

5

G2, M & 2

Mean

383.55

0.0150

0.0870

0.3398

0.9040

0.3819

0.3663

0.6946

0.5267

2.7859

±SD

35.09

0.0012

0.0119

0.0384

0.0940

0.0396

0.0209

0.0523

0.0418

0.3293

n

12

5

5

5

12

12

5

5

5

5

G3, M & 4.3

Mean

380.70

0.0161

0.1003

0.3408

0.9494

0.3900

0.3788

0.7206

0.5730

2.8115

±SD

46.81

0.0025

0.0192

0.0772

0.1640

0.0541

0.0333

0.1154

0.0672

0.1383

n

12

5

5

5

12

12

5

5

5

5

G4, M & 9.5

Mean

358.52

0.0170

0.0571*

0.2763

0.9101

0.4031

0.3738

0.6767

0.5630

2.7772

±SD

38.07

0.0022

0.0054

0.0266

0.0953

0.0406

0.0148

0.0472

0.0368

0.2655

n

12

5

5

5

12

12

5

5

5

5

Group, Sex & Dose

(mg/kg body weight/day)

Prostate

Seminal vesicles with coagulating glands

Lungs

Thyroid along with parathyroid

G1, M & 0

Mean

0.3949

0.4328

0.6252

0.0053

±SD

0.0759

0.0845

0.0408

0.0006

n

12

12

5

12

G2, M & 2

Mean

0.3843

0.4516

0.6204

0.0056

±SD

0.0834

0.1252

0.0815

0.0012

n

12

12

5

12

G3, M & 4.3

Mean

0.3497

0.4565

0.6463

0.0055

±SD

0.0816

0.0722

0.0253

0.0010

n

12

12

5

12

G4, M & 9.5

Mean

0.4027

0.4475

0.6694

0.0060

±SD

0.1107

0.1531

0.0767

0.0007

n

12

12

5

12

*: Statistically significant than the control group (p<0.05)

Table 19.2. Summary of terminal body weight (g) and organ weight (%) relative to terminal body weight record. Females

Group, Sex

& Dose

(mg/kg body weight/day)

Fasting Body Weight (g)

Adrenals

Thymus

Spleen

Heart

Kidneys

Brain

Liver

Lungs

Thyroid along with parathyroid

G1, F & 0

Mean

317.65

0.0288

0.0711

0.3637

0.3847

0.7545

0.6852

4.1349

0.6897

0.0065

±SD

29.29

0.0020

0.0125

0.0444

0.0254

0.0484

0.0561

0.5037

0.0817

0.0008

n

11

5

5

5

5

5

5

5

5

11

G2, F & 2

Mean

322.35

0.0255

0.0739

0.3468

0.3788

0.7587

0.6249

4.1373

0.5973

0.0067

±SD

22.14

0.0019

0.0103

0.0247

0.0152

0.1145

0.0370

0.4862

0.0087

0.0006

n

12

5

5

5

5

5

5

5

5

12

G3, F & 4.3

Mean

317.65

0.0263

0.0655

0.3581

0.3625

0.7794

0.6706

4.2480

0.6447

0.0070

±SD

23.71

0.0033

0.0083

0.0381

0.0309

0.0934

0.0684

0.3391

0.0744

0.0007

n

11

5

5

5

5

5

5

5

5

11

G4, F & 9.5

Mean

266.69*

0.0275

0.0508*

0.3207

0.3946

0.7701

0.7192

3.5311

0.7648

0.0071

±SD

25.13

0.0066

0.0149

0.0294

0.0507

0.0517

0.0559

0.4982

0.1233

0.0009

n

9

5

5

5

5

5

5

5

5

9

*: Statistically significant than the control group (p<0.05)

Table 20. Summary of serum thyroxine (T4) hormone levels (ng/mL) record - adults

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

Serum T4 Levels (ng/mL)

G1, M & 0

Mean

51.383

±SD

6.023

n

12

G2, M & 2

Mean

51.333

±SD

3.501

n

12

G3, M & 4.3

Mean

52.324

±SD

5.705

n

12

G4, M & 9.5

Mean

55.676

±SD

7.908

n

12

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

Serum T4 Levels (ng/mL)

G1, F & 0

Mean

45.222

±SD

3.897

n

11

G2, F & 2

Mean

43.690

±SD

5.786

n

12

G3, F & 4.3

Mean

45.895

±SD

3.977

n

11

G4, F & 9.5

Mean

50.490

±SD

7.106

n

9

Table 21. Summary of serum thyroxine (T4) hormone levels (ng/ml) record - PND 4 pups

Group, Sex & Dose

(mg/kg body weight/day)

Serum Thyroxine Hormone (T4) Levels
(ng/mL)

G1, F & 0

Mean

34.666

±SD

8.503

n

4

G2, F & 2

Mean

29.118

±SD

3.612

n

5

G3, F & 4.3

Mean

31.232

±SD

4.869

n

7

G4, F & 9.5

Mean

-

±SD

-

n

-

n: Number of Litters.

No pups were sacrificed from group G4, as the litter size is equal to or less than 10.

Table 22. Summary of serum thyroxine (T4) hormone levels (ng/ml) record - PND 13 pups

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

Serum T4 Levels (ng/mL)

G1, F & 0

Mean

42.055

±SD

3.692

n

11

G2, F & 2

Mean

44.330

±SD

4.328

n

12

G3, F & 4.3

Mean

43.180

±SD

4.290

n

11

G4, F & 9.5

Mean

46.427

±SD

11.722

n

4

n: Number of Litters.

Table 23. Summary of gross pathology findings: adults

Sex

Males

Females

Route of administration

Oral Gavage

Vehicle/Test item

Vehicle Control

Test item

Vehicle Control

Test item

Vehicle Control

Test item

Vehicle Control

Test item

Parameters

Group

G1

G2

G3

G4

G1R

G4R

G1

G2

G3

G4

G1R

G4R

Dose (mg/kg body weight/day)

0

2

4.3

9.5

0

9.5

0

2

4.3

9.5

0

9.5

Number of animals

12

12

12

12

5

5

12

12

12

12

5

5

No. of dead rats during treatment or recovery period

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

12

12

12

12

5

5

12

12

12

12

5

5

No. of rats showing external gross pathologyfindings

0

0

0

4

0

0

0

0

0

9

0

0

Wet perineum

-

-

-

4

-

-

-

-

-

7

-

-

Wet perineum with blood stains around vaginal region

-

-

-

-

-

-

-

-

-

2

-

-

 

No. of rats showing internal gross pathologyfindings

0

0

0

0

0

0

0

0

0

2

0

0

Uterus with resorptions/dead fetuses

-

-

-

-

-

-

-

-

-

2

-

-

Table 24. Summary of gross pathology findings :pups

Group

G1

G2

G3

G4

Dose (mg/kg body weight/day)

0

2

4.3

9.5

Sex

Male

Female

Male

Female

Male

Female

Male

Female

Total No. of Live Pups at Birth

61

56

55

70

56

63

36

31

 

a.     Total No. of Dead Pups at Birth

1

-

1

 

1

-

3

3

External Gross Pathology Findings

NAD

1

-

-

-

1

-

1

-

Cannibalized

-

-

1

-

-

-

-

-

Pale Colored

-

-

-

-

-

-

2

3

Internal Gross Pathology Findings

NAD

1

-

NA

-

1

-

3

3

 

b.     Total No. of Dead Pups during postnatal Period

-

-

-

-

-

-

19

23

External Gross Pathology Findings

NAD

-

-

-

-

-

-

3

5

Cannibalized

-

-

-

-

-

-

-

1

Pale Colored

-

-

-

-

-

-

16

16

Nose Discoloration, red

-

-

-

-

-

-

-

1

Internal Gross Pathology Findings

NAD

-

-

-

-

-

-

19

23

 

c.   Total No. of pups sacrificed on PND 4

-

6

-

9

-

11

-

-

External Gross Pathology Findings

NAD

-

6

-

9

-

11

-

-

Internal Gross Pathology Findings

NAD

-

6

-

9

-

11

-

-

 

d.   Total No. of pups sacrificed on PND 13

61

50

55

61

56

52

17

8

External Gross Pathology Findings

NAD

61

50

55

61

56

52

17

8

Internal Gross Pathology Findings

NAD

61

50

55

61

56

52

17

8

NAD: No Abnormality Detected; PND: Post-natal Day; - : No incidence; NA: Not Applicable

Table 25. Summary of histopathology findings: Adult animals (randomly selected animals)

Route of Administration

Oral Gavage

Vehicle /Test item

Control

 Test item

Dose (mg/kg body weight/day)

0

9.5

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

4

4

5

5

Accessory cortical tissue

Present

1

-

-

-

Angiectasis

Minimal

-

1

-

-

Brain

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Caecum

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

-

Cowper’s glands

Number examined

5

X

5

X

Within normal limits

5

-

5

-

Colon

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Duodenum

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Epididymides

Number examined

5

X

5

X

Within normal limits

5

-

5

-

Eyes with Optic nerve

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Femur bone with joint

Number examined

5

5

5

5

Within normal limits

5

5

-

-

Cellularity, decreased, bone marrow

Mild

-

-

5

5

Glans penis

Number examined

5

X

5

X

Within normal limits

5

-

5

-

Heart

Number examined

5

5

5

5

Within normal limits

3

3

5

5

Infiltrations, mononuclear cells, Ventricle

Minimal

2

2

-

-

Ileum with Peyer’s patches

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Jejunum

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Kidneys

Number examined

5

5

5

5

Within normal limits

4

5

3

4

Mineralization, Cortex

Minimal

1

-

-

-

Infiltrations, mononuclear cells, interstitial

Minimal

-

-

1

-

Dilatation, tubules

Minimal

-

-

-

1

Cast(s), medulla

Minimal

-

-

1

-

Levator ani plus bulbocavernous muscle complex

Number examined

5

X

5

X

Within normal limits

5

-

5

-

Route of administration

Oral Gavage

Vehicle /Test item

Control

 Test item

Dose (mg/kg body weight/day)

0

9.5

Group

G1

G4

Sex

M

F

M

F

Number of Animals

5

5

5

5

Liver

Number examined

5

5

5

5

Within normal limits

2

3

4

3

Infiltrations, mononuclear cells

Minimal

3

2

1

2

Lungs

Number examined

5

5

5

5

Within normal limits

3

4

5

5

Macrophages, increased, alveoli

Minimal

1

-

-

-

Infiltrations, mononuclear cells

Minimal

1

1

-

-

Mammary glands

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Mandibular lymph nodes

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Mesenteric lymph nodes

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

Pituitary gland

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

-

Rectum

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Sciatic nerve

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Seminal vesicles

Number examined

5

X

5

X

Within normal limits

5

-

5

-

Skeletal muscle

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

3

3

Cellularity, decreased, white pulp

Mild

-

-

2

2

Stomach

Number examined

5

5

5

5

Within normal limits

4

4

4

4

Dilatation, gastric glands, glandular

Minimal

1

1

1

1

Testes

Number examined

5

X

5

X

Within normal limits

5

-

5

-

Route of administration

Oral Gavage

Vehicle /Test item

Control

 Test item

Dose (mg/kg body weight/day)

0

9.5

Group

G1

G4

Sex

M

F

M

F

Number of Animals

5

5

5

5

Thymus

Number examined

5

5

5

5

Within normal limits

5

5

3

3

Cellularity, decreased, lymphocyte, cortex

Mild

-

-

2

2

Thyroid

Number examined

5

5

5

5

Within normal limits

4

4

3

5

Ultimobranchial cyst (s)

Present

1

1

2

-

Trachea

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Urinary bladder

Number examined

5

5

5

5

Within normal limits

5

5

5

5

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.

Table 26. Summary of histopathology findings: Males. Reproductive organs and thyroid with parathyroid glands

Route of administration

Oral Gavage

Vehicle /Test item

Control

Test item

Dose (mg/kg body weight/day)

0

9.5

Group

G1

G4

Number of Animals

7

7

Thyroid

Number examined

7

7

Within normal limits

4

7

Ultimobranchial cyst(s)

Present

3

-

Parathyroid

Number examined

7

7

Within normal limits

7

7

Coagulating glands

Number examined

7

7

Within normal limits

7

7

Cowper’s glands

Number examined

7

7

Within normal limits

7

7

Glans penis

Number examined

7

7

Within normal limits

7

7

Levator ani plus bulbocavernous muscle complex

Number examined

7

7

Within normal limits

7

7

Epididymides

Number examined

7

7

Within normal limits

7

7

Prostate gland

Number examined

7

7

Within normal limits

7

7

Seminal vesicles

Number examined

7

7

Within normal limits

7

7

Testes

Number examined

7

7

Within normal limits

7

7

-: no incidence

Table 27. Summary of histopathology findings: Females. Reproductive organs and thyroid with parathyroid glands

Route of administration

Oral Gavage

Vehicle /Test item

Control

 Test item

Dose (mg/kg body weight/day)

0

9.5

Group

G1

G4

Number of Animals

7

7

Thyroid

Number examined

7

7

Within normal limits

5

3

Ultimobranchial cyst(s)

Present

2

4

Parathyroid

Number examined

7

7

Within normal limits

7

7

Ovaries

Number examined

7

7

Within normal limits

7

7

Uterus with Cervix

Number examined

7

7

Within normal limits

7

7

Table 28. Summary of histopathology findings: Record of lower doses and recovery groups

Route of administration

Oral Gavage

Sex

Male

Female

Dose (mg/kg body weight/day)

2

4.3

0

9.5

2

4.3

0

9.5

Group

G2

G3

G1R

G4R

G2

G3

G1R

G4R

Number of Animals

5

5

5

5

5

5

5

5

Spleen

Number examined

5

5

5

5

5

5

5

5

Within normal limits

5

5

5

5

5

5

5

5

Thymus

Number examined

5

5

5

5

5

5

5

5

Within normal limits

5

5

5

5

5

5

5

5

Femur bone with joint

Number examined

5

5

5

5

5

5

5

5

Within normal limits

5

5

5

5

5

5

5

5

Conclusions:
Based on the findings of this study, the NOAEL of the test substance after a repeated oral exposure of 37 days in males and 64 days in females was determined to be 4.3 mg/kg bw/day.





Executive summary:

A combined repeated dose toxicity study with the reproduction /developmental toxicity screening test of the test substance POLY-U by oral administration in rats was conducted according to OECD guideline 422, in accordance with GLP principles. A total of 116 (58 males + 58 females) Sprague Dawley rats were distributed to four main groups and two recovery groups to detect delayed occurrence and recovery from toxic effects. Each main group (G1, G2, G3 and G4) consisted of 12 males and 12 females and each recovery group (G1R and G4R) consisted of 5 males and 5 females. The animals in G1/G1R group were administered with vehicle (Corn oil), and the animals in G2, G3 and G4/G4R groups were administered with test item at the dose levels of 2, 4.3 and 9.5 mg/kg bw/d, based on the results of two consecutive dose range finding studies performed with the same species for a period of 14 days at doses of 50, 100 and 300 mg/kg bw /d and 2.7, 8.3 and 25 mg/kg bw /d respectively. The test item was administered to the main group males for a period of 37 days (two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period), to the main group females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13 and to the recovery group animals for a period of 50 days with a 14 days recovery. The vehicle and test item formulations were administered orally by gavage at the dose volume of 5 mL/kg body weight. The stability of test item formulations in corn oil was established before initiation of the treatment. Dose formulation analysis for homogeneity and concentration verification was performed during weeks 1 and 5 of the treatment period and the results were within acceptable limits.

All the main group and recovery group animals were observed for clinical signs, mortality and morbidity, detailed clinical examination, body weight, feed consumption and ophthalmological and neurological/functional examinations. The clinical pathological (haematology, clinical chemistry and urinalysis) examinations were conducted for 5 randomly selected animals from each group per sex for main group and from all the animals for recovery group at termination. The gross pathology and organ weighing were performed on the day of termination for all the main and recovery group animals. All the main group females were observed for maternal body weight and feed consumption during gestation and lactation. The main group females were evaluated for oestrus cyclicity. Each dam was observed for mating performance, fertility performance, gestation length, litter size, number and percentage of live/dead pups, live birth index, sex ratio and observation of litter throughout the lactation period. Histopathological examination was conducted on all the tissues collected from the main group vehicle control and high dose group animals sacrificed at termination with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. The pups were observed once daily for clinical signs and external examinations, weighed individually on postnatal day (PND) 1, 4, 7 and 13, measured for anogenital distance on PND 4, observed for retention of any nipples/areolae in male pups on PND 13 and observed for gross pathological observations at termination. The analysis of thyroxine hormone (T4) levels in serum collected at termination was performed for main group males and for PND 4 (from available pups) and PND 13 pups.

In groups G2 and G3, there were no indication of test item-related effects in any of the parameters assessed for systemic or reproductive toxicity.

In group G4/G4R, the animals were noted with test item-related clinical signs of toxicity such as, lethargic, rough hair coat, wet perineum, chromodacryorrhea, hair thinning or blood stains around vagina, as well as test item-related reduction in body weight and feed consumption. Also, test item-related reduced motor activity assessments, effects on clinical pathology investigations and reduced thymus spleen and liver weights were found at the dose level of 9.5 mg/kg bw/d. During gross pathology, test item-related changes like wet pernineum (4/12 males and 7/12 females), wet pernineum along with blood stains around vagina (2/12 females) and uterus with resorptions/dead fetuses (2/12 females) were noted from group G4. Microscopically, test item-related changes were observed in spleen, thymus and bone marrow of femur in both sexes at this dose level. Thus, the investigations were extended to the lower dose and recovery groups for these organs. Effects were not observed in these groups. All these changes in thymus, spleen and femoral bone marrow were considered to be reversible, secondary effects of reduced food consumption and decreased body weight gain (Everds et.al., 2013). The high dose recovery group animals treated with 9.5 mg/kg bw/day were recovered from the systemic toxicity effects during recovery period.

In group G4, test item-related reduced gestation index, increased post-implantation loss, increased postnatal loss, reduced litter size, reduced live birth index per litter, increased number of dead pups at birth / during postnatal period and reduced pup survival index were noted. Also, test item-related external anomalies in pups, reduced mean pup weight and ano-genital distance ratio per litter in either sex were noted. However, these reproductive effects were considered as secondary non-specific effects occurred as a result of systemic toxicity due to absence of adverse effects or no effects noted on major reproductive end points at this dose.

Therefore, based on the above results discussed, the NOAEL for the test item POLY-U is considered to be 4.3 mg/kg bw/day.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
(The study period was only 14 days as it was conducted as a dose range finding study for a subsequent more extensive toxicity study)
GLP compliance:
no
Remarks:
DRF study performed without GLP
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
N,N'-(methylenedi-p-phenylene)bis(aziridine-1-carboxamide)
EC Number:
231-034-6
EC Name:
N,N'-(methylenedi-p-phenylene)bis(aziridine-1-carboxamide)
Cas Number:
7417-99-4
Molecular formula:
C19H20N4O2
IUPAC Name:
N-[4-({4-[(aziridine-1-carbonyl)amino]phenyl}methyl)phenyl]aziridine-1-carboxamide
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: In-house bred animals.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks (at receipt)
- Weight at study initiation: 253.87-255.78 g (range of average values of each group of males); 237.34-238.38 g (range of average values of each group of females)
- Fasting period before study: No
- Housing: Maximum of 3 animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with a stainless-steel mesh top grill having facilities for holding pelleted food and drinking water in a water bottle fitted with a 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 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 a Reverse Osmosis Unit was provided in plastic water bottles with stainless-steel sipper tubes.
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY:
Analyses of feed and water were performed before starting the study. The corresponding certificates are annexed to the test report.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6-22.9 ºC
- Humidity (%): 46-68 %
- Air changes (per hr): 12-15
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 31 October 2019 To: 19 November 2019

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item formulations were freshly prepared before dose administration on each treatment day. The required quantity of test item was weighed and triturated well in a mortar with a small quantity of vehicle until a homogenous suspension was formed and thereafter the entire quantity of the formulation was transferred into measuring cylinder. A small quantity of vehicle was added to rinse the mortar and this was transferred into the measuring cylinder. Rinsing procedure was repeated until complete transfer of test item formulation into the measuring cylinder. Finally, the volume was made up to the required quantity with vehicle to get the desired concentration. The test formulations were maintained under stirring conditions using magnetic stirrer to maintain homogeneity of the test item formulations.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test substance was tested in a solubility test with distilled water and also in a suspendibility test with 0.5% w/v carboxy methyl cellulose. Finally, the test item was found to form uniform suspension with corn oil at the concentration of 60 mg/mL (the highest dose concentration selected for the study considering the dose volume of 5 mL/kg body weight). Hence, corn oil was selected as vehicle for test item formulation preparations. Corn oil is universally accepted and routinely used vehicle in oral toxicity studies.
- Concentration in vehicle: 10, 20 and 60 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): N2192016
- Purity: N/A
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 was 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 using magnetic stirrer. The actual dose volume for each animal was calculated based on the most recent body weight.
Duration of treatment / exposure:
14 days
Frequency of treatment:
Once a day
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
G1 (vehicle only)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
G2
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
G3
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
G4
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
According to ECHA registration dossier, in an acute oral toxicity study (OECD guideline 423), Wistar female rats were treated at a dose of 300 mg/kg bw and 2000 mg/kg bw at single dose. There were no mortalities or clinical signs noted in female rats at 300 mg/kg bw and noted 100% mortalities with treatment related clinical signs at 2000 mg/kg bw. Based on these results it was concluded that the LD50 of the test item was lower than 2000 mg/kg bw and the LD50 cut-off value could be considered around 500 mg/kg bw. Hence, the doses of 0, 50, 100 and 300 mg/kg bw/day were selected as control, low, mid and high dose groups.
- Fasting period before blood sampling for clinical biochemistry: The animals were fasted overnight before blood collection. Water was provided ad libitum during fasting period.
Positive control:
None

Examinations

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.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the treatment on day 1 and weekly thereafter during treatment for all the animals. Signs noted were included, but not be limited to, changes in skin, fur, eyes and mucous membranes, occurrence of secretions and excretions and autonomic activity such as lacrimation, piloerection, pupil size, and unusual respiratory pattern.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual animal body weight was recorded on the day of randomization, on day of treatment (Day 1) prior to treatment and weekly thereafter.

FOOD CONSUMPTION: yes
-feed consumption was measured at weekly intervals. Average feed intake per rat (g/rat/day) was calculated using the amount of feed given and left over in each cage and the number of rats surviving in each cage.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once during acclimatization (Pre-treatment) and at week 2 of treatment.
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day of necropsy (day 15)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (water provided ad libitum)
- How many animals: all surviving animals
- Parameters checked: Haemoglobin concentration (HGB), Haematocrit (HCT), Erythrocyte count (RBC), Total leukocyte count (WBC), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet count (PLT), Mean platelet volume (MPV), Reticulocyte count (Retic), Absolute reticulocyte count, Differential leucocytes count (DLC), Absolute differential leucocytes count (DLC). Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) were estimated by coagulation analyzer.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of necropsy (day 15)
- Animals fasted: Yes (water provided ad libitum)
- How many animals: all surviving animals
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Cholinesterase, Total Protein, Albumin, Total bilirubin, Glucose, Total Cholesterol, Creatinine, Urea, Triglycerides, Phosphorous, Calcium, Blood Urea Nitrogen, Globulin, Albumin/Globulin ratio. Sodium (mmol/L), Potassium (mmol/L) and Chloride (mmol/L) were estimated using Prolyte Na/K/Cl analyzer (Diamond Diagnostics).

URINALYSIS: Yes
- Time schedule for collection of urine: on the day of necropsy (day 15)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Blood, Bilirubin, Urobilinogen, Ketones, Protein, Glucose, Leucocytes. In addition pH, nitrite and specific gravity were also analyzed. After analysis of the above parameters, the urine was subjected for centrifugation at 1500 rpm for 3 minutes. Then the urine was subjected for microscopic examination for urine sediments.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All the surviving animals and animals found dead were subjected to necropsy and detailed gross pathological examination of external surfaces, external orifices, abdominal, thoracic and cranial cavities, as well as organs and tissues of each animal with special emphasis on reproductive organs.
The following organs from all animals at the scheduled sacrifices were weighed wet as soon as possible to avoid drying: Kidneys, Adrenals, Spleen, Heart, Liver, Thymus, Brain, Lungs Testes/Ovaries, Epididymides/Uterus, Prostate along with seminal vesicles with coagulating gland. Relative organ weights were calculated against fasting body weight.

-HISTOPATHOLOGY: No
Statistics:
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, hematological, clinical chemistry estimations, urinalysis parameters (whichever applicable), absolute and relative organ weights 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. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Group G2 (50 mg/kg bw/d) males and females were noted with treatment related clinical signs like lethargy, soft stools/diarrhoea, blood stains around anal region, ataxia/changes in gait, abdominal breathing/unusual respiratory pattern, chromodacryorrhea, wet perineum/urine staining, rough hair coat and piloerection from day 3 and continued till termination.
Group G3 (100 mg/kg bw/d) males and females were noted with treatment related clinical signs like lethargy, salivation, soft stools/diarrhoea, ataxia, abdominal breathing/unusual respiratory pattern, chromodacryorrhea, wet perineum/urine staining, rough hair coat and piloerection from day 2 and continued till termination/death.
Group G4 (300 mg/kg bw/d) males and females were noted with test item related clinical signs like lethargy, salivation, chromodacryorrhea, wet perineum, rough hair coat and piloerection from day 1 and continued till termination/death.
Mortality:
mortality observed, treatment-related
Description (incidence):
A total of 3/5 males and 3/5 females were found dead in group G2, and all the males and females were found dead in groups G3 and G4.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In group G2 males and females, statistically significant reductions in body weight were found on day 7 & 14 (mean body weight) and during day 1 to 7 & 7 to 14 (percent change in mean body weight).
In group G3 males and females, statistically significant reductions in body weight were found on day 7 (mean body weight) and during day 1 to 7 (percent change in mean body weight) in both sexes. The mean body weight on day 14 and percent change in mean body weight on day 14 with respect to day 1 was not available from both sexes due to noted mortalities.
In group G4 males, the mean body weight and percent change in mean body weight with respect to day 1 were not available due to noted mortalities. For females statistically significant reductions in body weight were found on day 7 (mean body weight) and during day 1 to 7 (percent change in mean body weight) in both the sex. The mean body weight on day 14 and percent change in mean body weight on day 14 with respect to day 1 was not available from both sexes due to noted mortalities.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In group G2 males and females, statistically significant reductions in mean feed consumption were found during week 1 and 2.
In group G3 males and females, statistically significant reductions in mean feed consumption were found during week 1 in both sexes. The mean feed consumption was not available from both sexes during week 2 due to noted mortalities.
In group G4 males, the mean feed consumption was not available due to noted mortalities. For females statistically significant reductions in mean feed consumption were found during week 1 and the mean feed consumption during week 2 was not available due to noted mortalities.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no ocular changes observed during the opthalmoscopic examination carried out during week 2 (day 14) for all group G1 males and females, and for all surviving group G2 males and females. A total of 2 males and 2 females from group G2 was not subjected to opthalmoscopic examination due to mortalities.
The group G3 and G4 males and females were either dead or moribund sacrificed before scheduled sacrifice. Hence, there was no data on opthalmoscopic examination available from these dose groups.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In group G2 test item related changes in haematology parameters were noticed which were analysed on the day of termination (day 15) for all surviving animals.
The group G3 and G4 males and females were either dead or moribund sacrificed before scheduled sacrifice. Hence, there was no data available on heamatolgy parameters from these groups.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In group G2 test item related changes in clinical chemistry parameters were noticed which were analysed on the day of termination (day 15) for all surviving animals.
The group G3 and G4 males and females were either dead or moribund sacrificed before scheduled sacrifice. Hence, there was no data available on chemistry parameters from these dose groups.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
There were no changes observed in urinalysis parameters analysed on the day of termination (day 15) in group G2 males and females when compared with concurrent control group.
The group G3 and G4 males and females were either dead or moribund sacrificed before scheduled sacrifice. Hence, there was no data available on urinalysis parameters from these groups.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In group G2, test item related changes in all of the organs weighed in both the sex were noted when compared with concurrent control group.
The group G3 and G4 males and females were either dead or moribund sacrificed before scheduled sacrifice. Hence, there was no data available on absolute / relative organ weights from these groups.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
In group G2, test item related gross pathological changes like wet perineum (all males and females) externally and reduced thymus size (4 males and 2 females), reduced thymus along with spleen and testes size (1 male) and reduced thymus along with spleen size (3 females) were noted in both sexes.
In group G3 all 5 males and 5 females were found dead during treatment period and subjected to gross pathology. The external gross pathological observation revealed with wet perineum (all males and females) and internal gross pathological observation noted with reduced thymus along with spleen size (all males and females).
In group G4 all 5 males and 5 females were found dead during treatment period and subjected to gross pathology. The external gross pathological observations revealed with wet perineum (all males and 2 females), wet perineum along with wet mouth (3 females). The internal gross pathological observation noted with reduced thymus along with spleen size (all males and females).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
< 50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Treatment related effects observed at the lowest dose tested (50 mg/kg bw).

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Table 1.1. Summary of clinical signs of toxicity, detailed clinical examination and mortality record. Males

Group, Sex & Dose

(mg/kg body weight/day)

No. of

Animals

Clinical Signs of Toxicity: Observation
(No. of Animals noted)

Detailed Clinical Examination:
Observation
(No. of Animals noted)

Mortality

(No. of incidence/

No. of animals)

G1, M & 0

5

N (5)

No Abnormality Detected (5)

0/5

G2, M & 50

5

1 (5); 37 (5); 38 (1); 47(3); 71 (2); 82 (5); 110 (5); 116 (5); 117(1)

Urine staining (5); Diarrhoea (3);

Piloerection (3); Unusual respiratory pattern (1);

Changes in gait (3); Chromodacryorrhea (5);

Piloerectiion (3); Lethargy (1);

Rough hair coat (1)

3/5

G3, M & 100

5

1 (5); 6 (2); 37 (5); 71 (4);

82 (5);110 (5); 116 (5); 117 (1);

Urine staining (5); Diarrhoea (2);

Unusual respiratory pattern (1); Chromodacryorrhea (5);

Lethargy (5); Rough hair coat (3)

5/5

G4, M & 300

5

1 (5); 6 (5); 82 (5); 110 (5); 116 (5); 117 (1)

-

5/5

M: Male; N: Normal; 1: Lethargy; 6: Salivation; 37: Soft stool; 38: Blood stains around anal region; 47: Ataxia; 71: Abdominal breathing; 82: Chromodacryorrhea; 110: Wet perineum; 116: Rough hair coat; 117: Piloerection.

Note: G2 males did not reveal any clinical signs till Day 3 and started to reveal treatment related clinical signs from Day 4 onwards.

         G3 males did not reveal any clinical signs till Day 2 and started to reveal treatment related clinical signs from Day 3 onwards.

         G4 males did not reveal any clinical signs after first day of dose administration, started to reveal treatment related clinical signs from Day 2 onwards.

       

Table 1.2. Summary of clinical signs of toxicity, detailed clinical examination and mortality record. Females

Group, Sex & Dose

(mg/kg body weight/day)

No. of

Animals

Clinical Signs of Toxicity:
Observation
(No. of Animals noted)

Detailed Clinical Examination:
Observation
(No. of Animals noted)

Mortality

(No. of incidence/

No. of animals)

G1, F & 0

5

N (5)

No Abnormality Detected (5)

0/5

G2, F & 50

5

1 (5); 37 (4); 47 (1); 71 (3); 82 (5); 110 (5); 116 (5); 117 (1)

Urine Staining (5); Diarrhoea (4);

Piloerection (1); Unusual repiratory pattern (2);

Changes in gait (2); Chromodacryorrhea (4);

Lethargy (5); Piloerection (1)

3/5

G3, F & 100

5

1 (5); 6 (1); 37 (5); 47 (1); 71 (3); 82 (5); 110 (5); 116 (3)

Urine Staining (5); Diarrhoea (1);

Chromodacryorrhea (4); Lethargy (5);

Rough hair coat (2)

5/5

G4, F & 300

5

1 (5); 6 (3); 37 (5); 71 (2); 82 (5); 110 (5); 116 (5); 117 (1)

Salivation (2); Urine staining (4);

Diarrhoea (4); Piloerection (1);

Unusual repiratory pattern (1); Chromodacryorrhea (4); Rough hair coat (3)

5/5

F: Female; N: Normal; 1: Lethargy; 6: Salivation; 37: Soft stool; 47: Ataxia; 71: Abdominal breathing; 82: Chromodacryorrhea; 110: Wet perineum; 116: Rough hair coat; 117: Piloerection.

Note: G2 females did not reveal any clinical signs till Day 4 and started to reveal treatment related clinical signs from Day 5 onwards.

         G3 females did not reveal any clinical signs till Day 2 and started to reveal treatment related clinical signs from Day 3 onwards.

         G4 females did not reveal any clinical signs after first day of dose administration, started to reveal treatment related clinical signs from Day 2 onwards.

Table 2.1. Summary of body weight (g) record. Males

Group, Sex & Dose

(mg/kg body weight/day)

 

Body Weight (g) on Day

1

7

14

G1, M & 0

Mean

255.49

293.79

314.10

±SD

13.01

19.26

28.70

n

5

5

5

G2, M & 50

Mean

253.87

232.28*

186.34*

±SD

17.37

13.95

2.88

n

5

5

3

G3, M & 100

Mean

254.79

206.73*

-

±SD

17.47

17.37

-

n

5

5

-

G4, M & 300

Mean

255.78

-

-

±SD

13.49

-

-

n

5

-

-

M: Male; SD: Standard deviation; n: Number of animals; -: Not applicable

*: Statistically significant (P<0.05) change than the vehicle control group

Table 2.2. Summary of body weight (g) record. Females

Group, Sex & Dose

(mg/kg body weight/day)

 

Body Weight (g) on Day

1

7

14

G1, F & 0

Mean

237.34

245.39

253.88

±SD

14.08

13.78

12.54

n

5

5

5

G2, F & 50

Mean

238.25

220.84*

177.09*

±SD

13.67

11.41

20.29

n

5

5

3

G3, F & 100

Mean

238.38

216.09*

-

±SD

13.57

8.98

-

n

5

5

-

G4, F & 300

Mean

238.09

182.91*

-

±SD

11.91

14.31

-

n

5

4

-

F: Female; SD: Standard deviation; n: Number of animals; -: Not applicable

*: Statistically significant (P<0.05) change than the vehicle control group

Table 3.1. Summary of percent change in body weight (%) with respect to day 1. Males

Group, Sex & Dose

(mg/kg body weight/day)

 

Percent Change in Body Weight (%) during Day

1 to 7

1 to 14

G1, M & 0

Mean

14.92

22.75

±SD

2.21

5.61

n

5

5

G2, M & 50

Mean

-8.43*

-56.86*

±SD

2.80

39.50

n

5

5

G3, M & 100

Mean

-18.74*

-

±SD

6.60

-

n

5

-

M: Male; SD: Standard deviation; n: Number of animals; -: Not applicable

*: Statistically significant (P<0.05) change than the vehicle control group

Table 3.2. Summary of percent change in body weight (%) with respect to day 1. Females

Group, Sex & Dose

(mg/kg body weight/day)

 

Percent Change in Body Weight (%) during Days

1 to 7

1 to 14

G1, F & 0

Mean

3.44

7.04

±SD

2.55

2.50

n

5

5

G2, F & 50

Mean

-7.17*

-54.44*

±SD

5.05

42.25

n

5

5

G3, F & 100

Mean

-9.19*

-

±SD

4.91

-

n

5

-

G4, F & 300

Mean

-38.69*

-

±SD

34.36

-

n

5

-

F: Female; SD: Standard deviation; n: Number of animals; -: Not applicable

*: Statistically significant (P<0.05) change than the vehicle control group

Table 4.1. Summary of average feed consumption (g/animal/day) record. Males

Group, Sex & Dose

(mg/kg body weight/day)

 

Week 1

Feed Consumption

Week 2

Feed Consumption

G1, M & 0

Mean

20.64

21.53

±SD

3.47

4.88

n

5

5

G2, M & 50

Mean

10.68*

4.65*

±SD

1.17

1.21

n

5

5

G3, M & 100

Mean

7.31*

-

±SD

1.36

-

n

5

-

M: Male; SD: Standard deviation; n: Number of animals; -: Not applicable

*: Statistically significant (P<0.05) change than the vehicle control group

Table 4.2. Summary of average feed consumption (g/animal/day) record. Females

Group, Sex & Dose

(mg/kg body weight/day)

 

Week 1

Feed Consumption

Week 2

Feed Consumption

G1, F & 0

Mean

14.19

16.13

±SD

1.19

0.89

n

5

5

G2, F & 50

Mean

8.13*

3.39*

±SD

2.01

1.85

n

5

5

G3, F & 100

Mean

8.22*

-

±SD

1.58

-

n

5

-

G4, F & 300

Mean

2.51*

-

±SD

0.19

-

n

5

-

F: Female; SD: Standard deviation; n: Number of animals; -: Not applicable

*: Statistically significant (P<0.05) change than the vehicle control group

Table 5. Summary of haematology record.

Group, Sex &

Dose (mg/kg body weight/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)

(103cells/µL)

(106cells/µL)

(g/dL)

(%)

(fL)

(pg)

(g/dL)

(103cells/µL)

G1, M & 0

Mean

9.45

7.48

13.98

45.02

60.18

18.66

31.06

703.00

±SD

1.38

0.16

0.52

1.53

1.40

0.63

0.40

192.08

n

5

5

5

5

5

5

5

5

G2, M & 50

Mean

1.45*

5.72

10.75

33.25

57.85

18.70

32.40*

6.00*

±SD

0.78

1.67

3.46

10.96

2.33

0.57

0.28

2.83

n

2

2

2

2

2

2

2

2

G1, F & 0

Mean

10.60

7.11

12.76

40.68

57.28

17.98

31.38

828.60

±SD

3.91

0.41

0.44

1.58

1.20

0.50

0.33

130.66

n

5

5

5

5

5

5

5

5

G2, F & 50

Mean

4.59*

6.65

11.80

36.00

54.10*

17.80

32.90

71.50*

±SD

1.11

0.92

1.41

5.09

0.14

0.42

0.85

30.41

n

2

2

2

2

2

2

2

2

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

 

Mean Platelet Volume

Reticulocyte Count

Neutrophils

Lymphocytes

Monocytes

Eosinophils

Basophils

(MPV)   

(Retic)

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(fL)

(%)

(%)

(%)

(%)

(%)

(%)

G1, M & 0

Mean

6.56

2.20

21.30

72.80

3.40

1.04

0.30

±SD

0.11

0.31

5.76

5.57

0.79

0.43

0.10

n

5

5

5

5

5

5

5

G2, M & 50

Mean

10.35

0.02*

4.95*

93.15*

1.35*

0.05*

0.25

±SD

3.04

0.01

1.91

3.04

1.20

0.07

0.07

n

2

2

2

2

2

2

2

G1, F & 0

Mean

6.60

1.98

21.14

73.66

2.70

0.90

0.26

±SD

0.19

0.45

7.31

7.70

0.69

0.37

0.11

n

5

5

5

5

5

5

5

G2, F & 50

Mean

8.25

0.03*

51.05

48.15

0.05*

0.05*

0.25

±SD

1.63

0.00

68.94

68.09

0.07

0.07

0.07

n

2

2

2

2

2

2

2

Group, Sex &

Dose (mg/kg body weight/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)

(109cells/L)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(Seconds)

(Seconds)

G1, M & 0

Mean

164.88

2.00

6.90

0.32

0.10

0.03

21.70

19.74

±SD

25.87

0.56

1.29

0.08

0.05

0.01

5.44

4.54

n

5

5

5

5

5

5

5

5

G2, M & 50

Mean

0.75*

0.07*

1.36*

0.02*

0.00*

0.00*

17.30

29.55

±SD

0.07

0.01

0.78

0.01

0.00

0.00

1.98

7.14

n

2

2

2

2

2

2

2

2

G1, F & 0

Mean

140.72

2.07

8.00

0.27

0.09

0.03

17.30

31.62

±SD

33.65

0.47

3.67

0.05

0.03

0.02

2.09

9.25

n

5

5

5

5

5

5

5

5

G2, F & 50

Mean

2.00*

1.97

2.59

0.01*

0.00*

0.02

16.85

29.00

±SD

0.00

2.60

3.66

0.01

0.00

0.01

1.06

2.83

n

2

2

2

2

2

2

2

2

*: Statistically significant (P<0.05) change than the vehicle control group

Table 6. Summary of clinical chemistry record.

Group, Sex

& Dose

(mg/kg body weight/day)

 

Glucose

Urea

Creatinine

Total Cholesterol

Triglycerides

Total Protein

Albumin

 (GLU)

 (CRE)

(CHO)

(TRI)

(TPR)

 (ALB)

 (mg/dL)

(mg/dL)

 (mg/dL)

(mg/dL)

(mg/dL)

 (g/dL)

 (g/dL)

G1, M & 0

Mean

97.20

33.60

0.52

45.80

38.00

6.20

3.15

±SD

21.32

6.72

0.04

8.47

10.39

0.16

0.14

n

5

5

5

5

5

5

5

G2, M & 50

Mean

129.50

46.75

0.48

27.00*

35.50*

5.15

2.64*

±SD

27.58

11.53

0.00

1.41

4.95

0.21

0.05

n

2

2

2

2

2

2

2

G1, F & 0

Mean

90.60

38.06

0.63

57.40

26.40

7.26

3.57

±SD

11.48

4.31

0.05

6.27

5.90

0.26

0.27

n

5

5

5

5

5

5

5

G2, F & 50

Mean

65.50*

167.20

1.82

48.50

45.00

6.70

3.34

±SD

9.19

192.05

1.80

14.85

26.87

0.42

0.15

n

2

2

2

2

2

2

2

Group, Sex

& Dose

(mg/kg body weight/day)

 

Alanine

aminotransferase

Aspartate aminotransferase

Alkaline phosphatase

Total Bilirubin

Calcium

Phosphorous

(ALT)

(AST)

(ALP)

(BIT)

(CAL)

(PHO)

 (U/L)

 (U/L)

 (U/L)

(mg/dL)

(mg/dL)

(mg/dL)

G1, M & 0

Mean

55.00

98.00

235.20

0.11

9.28

6.12

±SD

14.20

9.30

70.69

0.00

0.40

0.47

n

5

5

5

5

5

5

G2, M & 50

Mean

37.50

96.50

241.00

0.25

8.45

5.35

±SD

14.85

19.09

291.33

0.05

0.49

0.64

n

2

2

2

2

2

2

G1, F & 0

Mean

41.00

103.60

116.80

0.09

9.46

4.90

±SD

7.78

41.69

63.35

0.04

0.30

0.32

n

5

5

5

5

5

5

G2, F & 50

Mean

37.50

82.50

84.00

0.20*

10.65*

9.05

±SD

7.78

3.54

53.74

0.00

0.49

7.14

n

2

2

2

2

2

2

Group, Sex

& Dose

(mg/kg body

weight/day)

 

Cholinesterase

Globulin

Albumin/

Globulin ratio

Blood Urea Nitrogen

Sodium

Potassium

Chloride

(CHE)

(GLO)

(A/G Ratio)

(BUN)

 (Na)

 (K)  

 (CLO)

 (U/L)

(g/dL)

(mg/dL)

 (mmol/L)

 (mmol/L)

 (mmol/L)

G1, M & 0

Mean

155.00

3.05

1.03

15.68

146.56

3.52

115.18

±SD

55.51

0.13

0.08

3.14

1.42

0.69

1.76

n

5

5

5

5

5

5

5

G2, M & 50

Mean

136.00

2.52*

1.06

21.82

142.25*

4.19

112.25

±SD

25.46

0.26

0.13

5.38

1.06

0.22

3.61

n

2

2

2

2

2

2

2

G1, F & 0

Mean

830.00

3.69

0.97

17.76

146.06

3.09

115.82

±SD

313.93

0.20

0.11

2.01

1.38

0.16

1.45

n

5

5

5

5

5

5

5

G2, F & 50

Mean

521.50

3.37

1.00

78.03

151.20

5.50

120.75

±SD

169.00

0.28

0.04

89.63

6.93

2.44

7.28

n

2

2

2

2

2

2

2

*: Statistically significant (P<0.05) change than the vehicle control group

Table 7.1. Summary of urinalysis record. Males

Examination

Group & Sex

G1 & M

G2 & M

Dose (mg/kg body weight/day)

0

50

Number of animals

5

2

Physical

Colour

Pale yellow

5

2

Appearance

Clear

4

2

Turbid

1

-

Volume (mL)

Mean

4.9

4.3

±SD

1.0

0.4

Chemical

pH

Mean

7.9

8.5

±SD

0.9

0.0

Specific Gravity

Mean

1.013

1.005

±SD

0.010

0.000

Urobilinogen (mg/dL)

Mean

0.6

0.2

±SD

0.8

0.0

Bilirubin (mg/dL)

Neg

4

1

3

1

-

1

-

1

Ketones (mg/dL)

Neg

5

2

Blood (Ery/µL)

Neg

5

-

>=Ca200

-

2

Proteins (mg/dL)

Neg

2

-

Trace

1

-

100

-

1

30

1

-

>=300

1

1

Nitrite

Pos

5

2

Leucocytes (Leu/µL)

Neg

4

1

Ca70

1

1

Glucose (mg/dL)

Neg

5

2

Microscopic

Epithelial cells

0

2

1

0-1

1

1

1-2

1

-

2-3

1

-

Casts

Absent

5

2

Crystals

Present

5

2

Table 7.2. Summary of urinalysis record. Females

Examination

Group & Sex

G1 & F

G2 & F

Dose (mg/kg body weight/day)

0

50

Number of animals

5

2

Physical

Colour

Pale yellow

3

2

Yellow

2

-

Appearance

Clear

3

2

Turbid

2

-

Volume (mL)

Mean

4.6

5.8

±SD

1.0

1.1

Chemical

pH

Mean

8.0

8.5

±SD

0.5

0.0

Specific Gravity

Mean

1.012

1.008

±SD

0.004

0.004

Urobilinogen (mg/dL)

Mean

0.2

0.2

±SD

0.0

0.0

Bilirubin (mg/dL)

Neg

5

2

Ketones (mg/dL)

Neg

5

2

Blood (Ery/µL)

Neg

3

2

Ca10

2

-

Proteins (mg/dL)

Neg

3

-

Trace

1

1

>=300

1

1

Nitrite</