Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Key study: Test method OECD 422. GLP study. The NOAEL of the test substance was determined to be 4.3 mg/kg bw/day in a repeated dose toxicity study at doses of 2, 4.3 and 9.5 mg/kg bw/day.

Key study: Dose range finding study according to test method OECD 407. DRF nº1: After an oral exposure of the test substance at doses of 50, 100 and 300 mg/kg bw /day for 14 days in males and females rats, 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 another dose range finding study was considered to be performed.

Key study: Dose range finding study according to test method OECD 407. DRF nº2: The tested dose of 25 mg/kg bw /d was the lowest dose level at which treatment related adverse effects were found in a second DRF perfomed at doses of 2.7, 8.3 and 25 mg/kg bw /day for 14 days in the same conditions as DRF nº1.

Key value for chemical safety assessment

Toxic effect type:
concentration-driven

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

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
Reason / purpose for cross-reference:
reference to other study
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
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
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
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
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).
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
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).
Key result
Critical effects observed:
no

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

Neg

2

-

Pos

3

2

Leucocytes (Leu/µL)

Neg

5

2

Glucose (mg/dL)

Neg

5

2

Microscopic

Epithelial cells

0

3

1

0-1

2

1

Casts

Absent

5

2

Crystals

Present

5

2

Table 8.1. 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

Prostate+Seminal vesicles with coagulating glands (PSC)

Lungs

G1, M & 0

Mean

0.0666

0.4265

0.6070

3.1496

1.9348

1.1320

2.2874

2.0027

9.8748

2.3939

2.0915

±SD

0.0225

0.0675

0.0719

0.4489

1.5506

0.0205

0.2215

0.1075

1.2209

0.2261

0.2787

n

5

5

5

5

5

5

5

5

5

5

5

G2, M & 50

Mean

0.0904

0.1271*

0.3547*

2.6744

0.8543

0.8612*

1.8948

1.9436

8.3810

1.0995*

1.4195*

±SD

0.0115

0.0011

0.0154

0.0822

0.0748

0.0507

0.2379

0.0167

0.1577

0.0587

0.0660

n

2

2

2

2

2

2

2

2

2

2

2

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

Table 8.2. Summary of absolute organ weights (g) record. Females

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

 

Adrenals

Thymus

Spleen

Ovaries

Uterus

Heart

Kidneys

Brain

Liver

Lungs

G1, F & 0

Mean

0.0767

0.3629

0.5325

0.1519

0.8539

0.8522

1.6618

1.8790

7.1617

1.7757

±SD

0.0115

0.0255

0.0640

0.0121

0.3953

0.1163

0.3867

0.0718

0.7306

0.2630

n

5

5

5

5

5

5

5

5

5

5

G2, F & 50

Mean

0.1050

0.2220*

0.3407

0.1148

0.4232

0.7189

1.6481

1.9438

6.5787

1.5437

±SD

0.0339

0.0347

0.2169

0.0431

0.2209

0.1127

0.2593

0.3224

0.7404

0.3969

n

2

2

2

2

2

2

2

2

2

2

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

Table 9.1. Summary of fasting body weight (g) and organ weight relative to fasting 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

Prostate+Seminal vesicles with coagulating glands (PSC)

Lungs

G1, M & 0

Mean

283.58

0.0236

0.1501

0.2154

1.1150

0.6753

0.4017

0.8092

0.7116

3.4832

0.8477

0.7384

±SD

26.08

0.0079

0.0187

0.0312

0.1608

0.5107

0.0346

0.0759

0.0799

0.3013

0.0879

0.0834

n

5

5

5

5

5

5

5

5

5

5

5

5

G2, M & 50

Mean

176.09*

0.0514*

0.0722*

0.2015

1.5190*

0.4853

0.4892*

1.0756*

1.1038*

4.7594*

0.6246*

0.8063

±SD

1.31

0.0069

0.0011

0.0103

0.0579

0.0461

0.0324

0.1271

0.0013

0.0542

0.0380

0.0435

n

2

2

2

2

2

2

2

2

2

2

2

2

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

Table 9.2. Summary of fasting body weight (g) and organ weight relative to fasting body weight (%) record. Females

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

 

Fasting Body Weight (g)

Adrenals

Thymus

Spleen

Ovaries

Uterus

Heart

Kidneys

Brain

Liver

Lungs

G1, F & 0

Mean

233.10

0.0331

0.1556

0.2282

0.0655

0.3656

0.3645

0.7121

0.8070

3.0738

0.7636

±SD

12.51

0.0059

0.0047

0.0229

0.0075

0.1616

0.0332

0.1547

0.0297

0.2826

0.1191

n

5

5

5

5

5

5

5

5

5

5

5

G2, F & 50

Mean

155.40*

0.0674

0.1430

0.2200

0.0737

0.2731

0.4630

1.0615*

1.2520

4.2361*

0.9947

±SD

1.58

0.0211

0.0238

0.1418

0.0270

0.1449

0.0772

0.1776

0.2202

0.5195

0.2655

n

2

2

2

2

2

2

2

2

2

2

2

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

Table 10.1. Summary of gross pathology record. Males

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

G1, M & 0

G2, M & 50

G3, M & 100

G4, M & 300

Total No. of Animals

5

5

5

5

No. of Animals Euthanized Terminally

5

2

0

0

No. of Animals Found Dead

0

3

5

5

External Gross pathological Observation

No Abnormality Detected

5

0

0

0

Wet perineum

0

5

5

0

Wet perineum and wet mouth

0

0

0

5

Internal Gross pathological Observation

No Abnormality Detected

5

0

0

0

Thymus: Small in size

0

4

0

0

Thymus: Small in size; Spleen: Small in size; Testes: Small in size,unilateral

0

1

0

0

Thymus: Small in size; Spleen: Small in size

0

0

5

5

Table 10.2. Summary of gross pathology record. Females

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

G1, F & 0

G2, F & 50

G3, F & 100

G4, F & 300

Total No. of Animals

5

5

5

5

No. of Animals Euthanized Terminally

5

2

0

0

No. of Animals Euthanized for Moribund

0

0

0

1

No. of Animals Found Dead

0

3

5

4

External Gross pathological Observation

No Abnormality Detected

5

0

0

0

Wet perineum

0

5

5

2

Wet perineum and wet mouth

0

0

0

3

Internal Gross pathological Observation

No Abnormality Detected

5

0

0

0

Thymus: Small in size

0

2

0

0

Thymus: Small in size; Spleen: Small in size

0

3

5

5

Conclusions:
In this dose range finding study, after an oral exposure of the test substance for 14 days in males and females rats, treatment related effects including mortalities were found at the lowest level tested (50 mg/kg bw /day). Thus, it is not possible to properly decide on doses for the main study and another dose range finding study is considered to be performed.







Executive summary:

A dose range finding study was performed for the test substance in accordance with OECD guideline 407 in order to evaluate its toxic potential after repeated exposure and select the appropriate doses for a subsequent toxicity study. The test item was diluted in corn oil (vehicle) and added to Sprague-Dawley rats (5 per sex per dose) at doses of 0 (vehicle only), 50, 100 and 300 mg/kg bw/day for a period of 14 days. The vehicle and test item formulations were administered orally (gavage) at a dose volume of 5 mL/kg body weight. Freshly prepared test item formulations were administered to the animals as soon as possible after preparation.

All animals for each group were observed for clinical signs of toxicity once daily, mortality and morbidity twice daily, and detailed clinical examination, body weight and feed consumption weekly. Ophthalmological examination was carried out during week 2 (day 14) for all group animals. Clinical pathology (haematology, clinical chemistry analysis and urinalysis) was conducted for all the animals sacrificed at termination.  All the animals were subjected to gross pathological examination and the organs were weighed, collected and preserved.

The dose of 50 mg/kg body weight revealed test item related clinical signs of toxicity and mortalities (60%). The doses of 100 and 300 mg/kg body weight revealed test item related clinical signs of toxicity and mortalities (100%). At all dose levels, test item related reduced body weight and feed consumption and test item related gross pathological changes in both sexes were noted. At dose of 50

mg/kg body weight, test item related effects in clinical pathological parameters, absolute / relative organ weights were noted. At doses of 100 and 300 mg/kg body weight, data on clinical pathological parameters and absolute / relative organ weights were not available because all animals were either dead or moribund sacrificed before scheduled sacrifice. In the dose of 50 mg/kg body weight, gross pathological findings were wet perineum (all males and females), 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). In the dose of 100 mg/kg body weight, gross pathological findings were wet perineum (all males and females) and reduced thymus along with spleen size (all males and females). In the dose of 300 mg/kg body weight, gross pathological findings were wet perineum (all males and 2 females), wet perineum along with wet mouth (3 females) and reduced thymus along with spleen size (all males and females). Based on these results, it is not possible to properly decide on doses for the main study and another dose range finding study is considered to be performed.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 December 2019 – 08 January 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Reason / purpose for cross-reference:
reference to other study
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
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: 283.88-289.46 g (range of average values of each group of males); 245.80-247.72 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): 19.4-22.8 ºC
- Humidity (%): 47-66 %
- Air changes (per hr): 12-15
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 20 December 2019 To: 08 January 2020
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 solubility/suspendibility test for the test item was assessed in a previous dose range finding study with the following results: 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: 0.54, 1.66 and 5 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
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
G1 (vehicle only)
Dose / conc.:
2.7 mg/kg bw/day (actual dose received)
Remarks:
G2
Dose / conc.:
8.3 mg/kg bw/day (actual dose received)
Remarks:
G3
Dose / conc.:
25 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 for a first dose range finding study.
The dose of 50 mg/kg bw/d revealed test item related clinical signs of toxicity and mortalities (60%) and test item related effects in clinical pathology parameters and organ weights. Doses of 100 and 300 mg/kg bw/d revealed test item related clinical signs of toxicity and mortalities (100%). All doses revealed test-item related reduced body weight and feed consumption and test item related gross pathological changes in both sexes. In view of these results, it was not possible to properly decide on doses for the main study, thus the present dose range finding study was considered to be performed with doses of 0, 2.7, 8.3 and 25 mg/kg bw/day.

- 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
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).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In group G2 (2.7 mg/kg bw/day), there were no clinical signs of toxicity noted and no changes were noted in detailed clinical examination.
In group G3 (8.3 mg/kg bw/day), there were no adverse test item-related effects noted in any of the parameters of both sexes, except noted wet perineum in one female (recovered later).
In group G4 (25 mg/kg bw/day), the animals of both sexes were noted with test item-related clinical signs like, lethargy, chromodacryorrhea, wet perineum, rough hair coat during the treatment period.
Mortality:
no mortality observed
Description (incidence):
No mortalities were found in any dose level.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In group G2 there were no changes noted in mean body weight and percent change in mean body weight gain.
In group G3 and G4 test item-related slight reduction in mean body weight/body weight gain was noted in both sexes.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In group G2 there were no changes noted in mean feed consumption.
In group G3 and G4 test item-related slight reduction in mean feed consumption was noted 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):
The opthalmoscopic examination did not reveal any changes in any dose group.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In group G2 the haematology assessment did not reveal any test item-related changes in both sexes.
In group G3 the haematological examination revealed test item-related reduced platelet count in males and reduced absolute/percent eosinophils in both males and females.
In group G4 test item-related effects were noted in haematology parameters of either sex.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In group G2 the clinical chemistry assessment did not reveal any test item-related changes in both sexes.
In group G3 the clinical chemistry did not reveal any test item-related changes in both sexes.
In group G4 test item-related effects were noted in clinical chemistry parameters of either sex.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Urinalysis assessment did not reveal any test item-related changes in any dose group. However, statistically significant increase in mean pH of urine at group G3 and G4 females was noted when compared with vehicle control group. This change is considered as incidental and unrelated to treatment as the change was not occurred in dose dependent manner and also no effects were noted on other urinalysis parameters at these dose levels.
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):
There were no test item-related changes observed in mean absolute and relative organ weights at group G2 and G3 in both sexes when compared with the control. However, statistically significant decrease in mean absolute and relative thymus weight at group G3 female was noted when compared with the control.
In group G4 statistically significant reduction in mean absolute thymus, relative thymus, absolute PSC, relative adrenals, relative testes, relative brain weights were noted from males; statistically significant reduction in mean absolute thymus, relative thymus, absolute spleen, relative spleen, absolute uterus, relative uterus, absolute liver, relative brain weights were noted from females. These noted changes are considered as test item-related due to noted clinical signs, reduced body weight and noted gross pathological changes at this dose level.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
There were no external and internal gross pathological changes observed in group G1, G2 and G3 animals of both sexes during necropsy.
In group G4 there were no external gross pathological changes noted in either sex. Internal gross pathological observations found were reduced thymus size (2 males and 4 females), reduced thymus size along with spleen and PCS size (1 male), reduced thymus along with spleen size (1 male and 1 female) and reduced thymus along with PCS size (1 male). These changes are considered as test item-related due to noted significant reduction in absolute or relative weights of the same organs at this dose level.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
8.3 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
organ weights and organ / body weight ratios
Remarks on result:
other: Treatment related adverse effects observed at the dose level of 25 mg/kg bw /d.
Key result
Critical effects observed:
no

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

Day 1 to 15: N (5)

No Abnormality Detected (5)

0/5

G2, M & 2.7

5

Day 1 to 15: N (5)

No Abnormality Detected (5)

0/5

G3, M & 8.3

5

Day 1 to 15: N (5)

No Abnormality Detected (5)

0/5

G4, M & 25

5

-    Day 1 to 5:N (5)

-    Day 6:N (1); 82+ (4)

-    Day 8:N (1); 82+ (3); 1,82+,116 (1)

-    Day 9:N (1); 82+ (2);1,82+(1);

1,82+,116, 110+(1)

-    Day 10 & 11:N (1); 82+ (1);

-    1,82+ (2); 1,82+,110+,116(1)

-    Day 12:N (1);82+ (1);1,82+ (1);

1,82+,110+ (1); 1,82+,110+,116(1)

-    Day 13 & 15:1,82+,110+,116(2);

116(1);82+(1);1,82+(1)

Day 7:No Abnormality Detected (1); Chromodacryorrhea (4)

Day 14:Chromodacryorrhea,

Urine staining/Wet perineum,

Rough hair coat (2); Rough hair coat (1);

Chromodacryorrhea (2),

0/5

M: Male; N: Normal; 1: Lethargy; 82: Chromodacryorrhea; 110: Wet perineum; 116: Rough hair coat; +: Slight

       

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

Day 1 to 15: N (5)

No Abnormality Detected (5)

0/5

G2, F & 2.7

5

Day 1 to 15: N (5)

No Abnormality Detected (5)

0/5

G3, F & 8.3

5

-    Day 1 to 5:N (5)

-    Day 6 to 8:N (4); 110+ (1)

-    Day 9 to 15:N (5)

Day 7:No Abnormality Detected (4);

Urine staining/Wet perineum(1)

Day 14:No Abnormality Detected (5)

0/5

G4, F & 25

5

-    Day 1 to 5:N (5)

-    Day 6:N (4); 82+ (1)

-    Day 8:N (2); 1,116(2);1,82+(1)

-    Day 9 to 11:N (2); 1,110+,116(1);

1,110++,116(1);1,82+,110+,116(1)

-    Day 12:N (2); 1,110++,116(1);

1,110+,116(1);1,82+,116(1)

-    Day 13 & 15:N (2); 1,110++,116(1);

1,116(1);1,82+,116(1)

Day 7:No Abnormality Detected (4);

Chromodacryorrhea (1)

Day 14:No Abnormality Detected (2);
Urine staining/
Wet perineum,

Rough Hair Coat (1); Chromodacryorrhea,

Rough Hair Coat (1); Rough Hair Coat (1)

0/5

F: Female; N: Normal; 1: Lethargy; 82: Chromodacryorrhea; 110: Wet perineum; 116: Rough hair coat; +: Slight; ++: Moderate

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

285.46

300.43

325.69

±SD

18.62

24.79

32.43

n

5

5

5

G2, M & 2.7

Mean

283.88

295.31

321.63

±SD

21.02

27.37

33.83

n

5

5

5

G3, M & 8.3

Mean

289.46

295.90

318.17

±SD

20.50

28.36

39.12

n

5

5

5

G4, M & 25

Mean

286.90

265.40

234.77*

±SD

19.93

16.83

28.67

n

5

5

5

M: Male; SD: Standard deviation; n: Number of animals;

*: 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

247.72

259.41

268.45

±SD

13.16

18.07

18.21

n

5

5

5

G2, F & 2.7

Mean

245.80

253.42

262.64

±SD

16.67

14.87

14.77

n

5

5

5

G3, F & 8.3

Mean

246.32

248.25

252.76

±SD

20.18

17.48

20.72

n

5

5

5

G4, F & 25

Mean

246.86

218.96*

214.85*

±SD

19.67

26.97

51.46

n

5

5

5

F: Female; SD: Standard deviation; n: Number of animals;

*: 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

5.15

13.93

±SD

2.58

5.84

n

5

5

G2, M & 2.7

Mean

3.94

13.20

±SD

2.91

5.87

n

5

5

G3, M & 8.3

Mean

2.13

9.67

±SD

4.22

7.46

n

5

5

G4, M & 25

Mean

-7.45*

-18.12*

±SD

1.74

8.72

n

5

5

M: Male; SD: Standard deviation; n: Number of animals;

*: 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 Day

1 to 7

1 to 14

G1, F & 0

Mean

4.68

8.33

±SD

3.51

3.41

n

5

5

G2, F & 2.7

Mean

3.16

6.94

±SD

2.02

2.88

n

5

5

G3, F & 8.3

Mean

0.89

2.67

±SD

3.10

3.84

n

5

5

G4, F & 25

Mean

-11.52*

-13.76*

±SD

4.70

14.61

n

5

5

F: Female; SD: Standard deviation; n: Number of animals;

*: 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)

 

Average Feed consumption(g/rat/day)

Week 1

Week 2

G1, M & 0

Mean

21.41

21.91

±SD

3.71

3.70

n

5

5

G2, M & 2.7

Mean

19.89

20.70

±SD

2.81

2.36

n

5

5

G3, M & 8.3

Mean

19.09

20.46

±SD

3.58

1.24

n

5

5

G4, M & 25

Mean

13.31

7.62*

±SD

0.31

2.48

n

5

5

M: Male; SD: Standard deviation; n: Number of animals;

*: 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)

 

Average Feed consumption(g/rat/day)

Week 1

Week 2

G1, F & 0

Mean

18.06

19.39

±SD

3.54

4.93

n

5

5

G2, F & 2.7

Mean

16.66

17.19

±SD

0.18

0.35

n

5

5

G3, F & 8.3

Mean

15.71

16.18

±SD

1.56

1.57

n

5

5

G4, F & 25

Mean

8.07*

9.76

±SD

3.17

6.63

n

5

5

F: Female; SD: Standard deviation; n: Number of animals;

*: 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

Mean Platelet Volume

 (WBC)        

 (RBC)    

(HGB)      

 (HCT)   

 (MCV)     

(MCH)

(MCHC)      

 (PLT)

(MPV)   

(103cells/µL)

(106cells/µL)

(g/dL)

(%)

(fL)

(pg)

(g/dL)

(103cells/µL)

(fL)

G1, M & 0

Mean

10.56

7.41

13.50

44.98

61.06

18.32

30.06

797.20

8.32

±SD

2.80

0.56

0.45

1.19

5.94

1.46

1.74

109.08

2.34

n

5

5

5

5

5

5

5

5

5

G2, M & 2.7

Mean

8.56

7.67

14.12

46.90

61.36

18.46

30.16

772.80

8.04

±SD

1.40

0.62

0.66

2.39

4.95

1.09

1.99

182.93

2.24

n

5

5

5

5

5

5

5

5

5

G3, M & 8.3

Mean

8.82

7.19

13.46

44.88

63.82

18.82

30.08

564.40*

8.16

±SD

2.59

1.03

1.32

1.26

12.59

1.20

3.54

174.14

1.39

n

5

5

5

5

5

5

5

5

5

G4, M & 25

Mean

3.79*

4.81*

9.26*

29.76*

64.70

19.58

30.50

43.40*

18.54

±SD

2.16

2.13

3.84

9.73

8.14

1.57

2.82

27.86

14.25

n

5

5

5

5

5

5

5

5

5

Group, Sex & Dose

(mg/kg body weight/day)

 

Reticulocyte Count

Neutrophils

Lymphocytes

Monocytes

Eosinophils

Basophils

(Retic)

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(%)

(%)

(%)

(%)

(%)

(%)

G1, M & 0

Mean

2.88

20.46

71.36

6.38

1.08

0.26

±SD

0.79

2.19

1.37

3.12

0.41

0.11

n

5

5

5

5

5

5

G2, M & 2.7

Mean

3.07

24.74

66.88

6.42

1.12

0.32

±SD

0.79

2.24

5.61

3.45

0.34

0.08

n

5

5

5

5

5

5

G3, M & 8.3

Mean

3.17

16.34

76.32

5.82

0.26*

0.34

±SD

1.89

3.86

4.14

1.10

0.11

0.09

n

5

5

5

5

5

5

G4, M & 25

Mean

0.23*

5.50*

93.18*

0.52*

0.30*

0.14

±SD

0.34

3.66

4.16

0.35

0.42

0.13

n

5

5

5

5

5

5

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

211.22

2.19

7.55

0.63

0.10

0.03

19.22

30.30

±SD

50.39

0.74

2.10

0.32

0.03

0.02

3.49

7.63

n

5

5

5

5

5

5

5

5

G2, M & 2.7

Mean

232.20

2.14

5.67

0.58

0.10

0.03

19.30

30.82

±SD

45.06

0.55

0.53

0.38

0.05

0.01

1.15

6.69

n

5

5

5

5

5

5

5

5

G3, M & 8.3

Mean

221.56

1.46

6.74

0.50

0.02*

0.03

17.38

26.80

±SD

128.95

0.62

2.05

0.11

0.01

0.01

2.46

7.05

n

5

5

5

5

5

5

5

5

G4, M & 25

Mean

8.02*

0.18*

3.57*

0.01*

0.00*

0.01*

20.10

24.32

±SD

11.19

0.17

2.14

0.01

0.01

0.01

4.60

2.50

n

5

5

5

5

5

5

5

5

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

Mean Platelet

Volume

 (WBC)        

 (RBC)    

(HGB)      

 (HCT)   

 (MCV)     

(MCH)

(MCHC)      

 (PLT)

(MPV)   

(103cells/µL)

(106cells/µL)

(g/dL)

(%)

(fL)

(pg)

(g/dL)

(103cells/µL)

(fL)

G1, F & 0

Mean

10.14

7.09

13.50

42.28

59.62

19.04

31.90

847.20

6.82

±SD

2.55

0.28

1.16

2.90

2.35

1.07

0.63

82.33

0.28

n

5

5

5

5

5

5

5

5

5

G2, F & 2.7

Mean

10.69

7.26

13.94

42.58

58.66

19.24

32.78

836.00

6.74

±SD

4.77

0.46

0.75

3.10

1.60

0.43

0.80

97.22

0.22

n

5

5

5

5

5

5

5

5

5

G3, F & 8.3

Mean

9.46

7.35

14.02

43.46

59.04

19.04

32.22

837.40

6.82

±SD

2.67

0.40

0.88

3.00

1.36

0.30

0.38

106.09

0.28

n

5

5

5

5

5

5

5

5

5

G4, F & 25

Mean

5.37

6.30*

11.64*

35.34*

56.20*

18.52

32.94*

106.60*

8.18*

±SD

1.67

0.53

0.88

2.27

2.07

0.45

0.57

85.69

0.97

n

5

5

5

5

5

5

5

5

5

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

 

Reticulocyte Count

Neutrophils

Lymphocytes

Monocytes

Eosinophils

Basophils

(Retic)

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(%)

(%)

(%)

(%)

(%)

(%)

G1, F & 0

Mean

1.91

20.50

73.62

3.72

1.20

0.30

±SD

0.18

5.04

4.17

1.33

0.59

0.00

n

5

5

5

5

5

5

G2, F & 2.7

Mean

2.04

18.08

76.72

3.46

1.00

0.28

±SD

0.52

2.70

2.88

0.23

0.46

0.11

n

5

5

5

5

5

5

G3, F & 8.3

Mean

1.93

14.22

80.62

3.68

0.28*

0.32

±SD

0.56

4.76

5.86

1.08

0.19

0.08

n

5

5

5

5

5

5

G4, F & 25

Mean

0.11*

3.72*

94.28*

0.64*

0.12*

0.14*

±SD

0.13

2.50

4.46

0.71

0.08

0.05

n

5

5

5

5

5

5

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, F & 0

Mean

135.36

2.07

7.48

0.36

0.13

0.03

20.80

28.84

±SD

15.95

0.72

1.95

0.13

0.09

0.01

4.42

5.02

n

5

5

5

5

5

5

5

5

G2, F & 2.7

Mean

148.76

1.85

8.28

0.36

0.10

0.03

18.18

30.84

±SD

42.97

0.61

3.94

0.16

0.06

0.02

1.29

12.02

n

5

5

5

5

5

5

5

5

G3, F & 8.3

Mean

141.26

1.29

7.70

0.33

0.03*

0.03

18.16

24.54

±SD

38.31

0.44

2.60

0.08

0.02

0.01

2.05

3.25

n

5

5

5

5

5

5

5

5

G4, F & 25

Mean

6.68*

0.19*

5.07

0.03*

0.00*

0.01*

19.28

24.34

±SD

7.70

0.15

1.66

0.04

0.01

0.00

3.29

4.53

n

5

5

5

5

5

5

5

5

*: 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

84.20

32.72

0.47

42.80

38.00

5.98

2.89

±SD

13.59

8.79

0.02

5.54

17.48

0.15

0.06

n

5

5

5

5

5

5

5

G2, M & 2.7

Mean

83.00

34.44

0.51

50.80

30.40

6.24

3.00

±SD

10.42

6.49

0.05

7.05

6.80

0.33

0.15

n

5

5

5

5

5

5

5

G3, M & 8.3

Mean

98.00

32.24

0.49

46.00

46.00

6.18

3.03

±SD

23.95

6.69

0.05

10.84

10.34

0.31

0.13

n

5

5

5

5

5

5

5

G4, M & 25

Mean

100.40

43.08

0.45

26.40*

27.20

5.48

2.55

±SD

7.20

23.31

0.05

2.97

5.89

0.63

0.41

n

5

5

5

5

5

5

5

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

48.80

92.20

164.60

0.05

9.22

8.92

±SD

12.93

13.18

39.37

0.03

0.29

1.45

n

5

5

5

5

5

5

G2, M & 2.7

Mean

52.00

96.40

194.60

0.08

9.38

7.22*

±SD

4.95

5.03

24.00

0.04

0.29

0.73

n

5

5

5

5

5

5

G3, M & 8.3

Mean

41.40

94.80

238.20

0.12

9.24

7.54

±SD

6.43

28.03

89.83

0.16

0.27

0.46

n

5

5

5

5

5

5

G4, M & 25

Mean

36.20

73.20

95.00

0.15

8.70*

7.22*

±SD

12.76

24.68

37.24

0.09

0.24

1.00

n

5

5

5

5

5

5

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

113.00

3.09

0.94

15.27

146.54

3.54

109.44

±SD

20.72

0.15

0.05

4.11

0.88

0.27

0.71

n

5

5

5

5

5

5

5

G2, M & 2.7

Mean

135.40

3.24

0.92

16.07

146.60

3.84

109.56

±SD

47.95

0.20

0.03

3.03

1.22

0.21

1.59

n

5

5

5

5

5

5

5

G3, M & 8.3

Mean

123.80

3.15

0.97

15.05

146.30

4.15

110.46

±SD

38.58

0.32

0.11

3.12

1.73

1.07

1.97

n

5

5

5

5

5

5

5

G4, M & 25

Mean

79.80

2.93

0.88

19.98

146.20

3.97

112.40*

±SD

16.48

0.41

0.16

10.62

0.86

0.32

2.11

n

5

5

5

5

5

5

5

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, F & 0

Mean

96.20

35.78

0.54

57.80

22.80

6.62

3.17

±SD

16.81

9.49

0.02

10.80

4.38

0.42

0.06

n

5

5

5

5

5

5

5

G2, F & 2.7

Mean

87.00

30.58

0.51

50.00

27.20

6.48

3.26

±SD

16.61

5.09

0.04

9.03

5.07

0.16

0.11

n

5

5

5

5

5

5

5

G3, F & 8.3

Mean

101.20

30.90

0.53

52.60

28.80

6.64

3.40

±SD

19.66

2.65

0.04

7.06

10.47

0.44

0.30

n

5

5

5

5

5

5

5

G4, F & 25

Mean

120.40

30.20

0.50

36.20*

29.20

5.82*

2.95

±SD

29.14

6.01

0.06

15.51

15.99

0.61

0.28

n

5

5

5

5

5

5

5

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, F & 0

Mean

45.20

87.00

145.00

0.04

9.32

6.94

±SD

8.01

15.78

76.97

0.01

0.25

0.64

n

5

5

5

5

5

5

G2, F & 2.7

Mean

45.20

84.40

136.80

0.04

9.32

7.24

±SD

4.27

10.06

48.87

0.02

0.23

1.34

n

5

5

5

5

5

5

G3, F & 8.3

Mean

39.40

82.20

100.60

0.06

9.44

9.02

±SD

6.27

4.97

14.98

0.01

0.19

4.07

n

5

5

5

5

5

5

G4, F & 25

Mean

36.60

86.60

113.60

0.09*

8.90*

5.84

±SD

3.91

12.54

59.95

0.02

0.27

1.07

n

5

5

5

5

5

5

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, F & 0

Mean

483.80

3.45

0.93

16.70

145.16

3.76

110.48

±SD

120.58

0.39

0.09

4.42

4.34

1.07

3.60

n

5

5

5

5

5

5

5

G2, F & 2.7

Mean

488.40

3.22

1.02

14.27

147.20

3.27

110.60

±SD

99.60

0.23

0.11

2.37

0.73

0.18

1.60

n

5

5

5

5

5

5

5

G3, F & 8.3

Mean

443.20

3.24

1.05

14.42

147.34

3.15

111.32

±SD

127.83

0.25

0.11

1.23

1.63

0.28

1.47

n

5

5

5

5

5

5

5

G4, F & 25

Mean

347.20

2.87*

1.04

14.09

146.06

3.73

111.58

±SD

254.50

0.38

0.10

2.80

2.74

0.61

2.28

n

5

5

5

5

5

5

5

*: 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

G3, M

G4, M

 Dose (mg/kg body weight/day)

0

2.7

8.3

25

Number of Animals

5

5

5

5

Physical Examination

Color

Pale Yellow

5

5

4

5

Yellow

0

0

1

0

Apperance

Clear

4

5

4

4

Turbidity

1

0

1

1

Volume (mL)

Mean

4.8

6.2

6.0

4.4

±SD

0.7

0.8

0.9

1.1

Chemical Examination

pH

Mean

7.3

7.4

7.5

7.9

±SD

0.4

0.2

0.4

0.7

Specific gravity (SG)

Mean

1.020

1.015

1.016

1.014

±SD

0.008

0.004

0.009

0.007

Urobilinogen (UBG) (mg/dL)

Mean

0.2

0.2

0.2

0.2

±SD

0.0

0.0

0.0

0.0

Bilirubin (BIL) (mg/dL)

Neg

5

5

5

5

1

0

0

0

0

Ketones (KET) (mg/dL)

5

5

3

2

3

15

0

0

1

0

Neg

0

2

2

2

Blood (BLD) (Ery/µL)

Ca10

0

0

0

1

Ca80

4

1

3

3

Ca25

0

0

1

0

>=Ca200

1

4

1

1

Protein (PRO) (mg/dL)

Trace

4

1

2

1

Neg

0

4

2

1

30

1

0

1

2

100

0

0

0

1

Nitrite (NIT)

Neg

2

5

2

4

Pos

3

0

3

1

Leucocytes (LEU) (Leu/µL)

Neg

5

5

2

4

Ca15

0

0

3

1

Glucose(GLU) (mg/dL)

Neg

5

5

5

5

Microscopic Examination

Epi Cells

0

4

3

4

3

0-1

0

2

0

1

1-2

1

0

1

0

2-3

0

0

0

1

Casts

Absent

5

5

5

5

Crystals

Present

5

5

5

5

M: Male; Neg: Negative; Pos: Positive; Ca: Calculated approximately; SD: Standard deviation; >= more than equal to

Table 7.2. Summary of urinalysis record. Females

Examination

 

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

G1, F

G2, F

G3, F 

G4, F 

Dose (mg/kg body weight/day)

 0

2.7

8.3

25

Number of Animals

5

5

5

5

Physical Examination

Color

Pale Yellow

5

5

5

5

Apperance

Clear

5

5

5

5

Volume (mL)

Mean

5.3

5.6

6.1

5.8

±SD

1.5

1.1

2.0

1.0

Chemical Examination

pH

Mean

6.8

7.3

7.8*

7.6*

±SD

0.3

0.4

0.4

0.2

Specific gravity (SG)

Mean

1.015

1.016

1.014

1.013

±SD

0.004

0.004

0.004

0.003

Urobilinogen (UBG) (mg/dL)

Mean

0.2

0.2

0.2

0.2

±SD

0.0

0.0

0.0

0.0

Bilirubin (BIL) (mg/dL)

Neg

5

5

5

5

Ketones (KET) (mg/dL)

5

0

0

1

0

Neg

5

5

4

5

Blood (BLD) (Ery/µL)

Ca10

2

1

3

1

Ca25

0

0

0

1

Ca80

0

0

0

2

>=Ca200

0

0

0

1

Neg

3

4

2

0

Protein (PRO) (mg/dL)

Trace

1

0

0

0

Neg

4

5

4

5

100

0

0

1

0

Nitrite (NIT)

Neg

5

5

3

1

Pos

0

0

2

4

Leucocytes (LEU) (Leu/µL)

Neg

5

5

5

5

Glucose(GLU)(mg/dL)

Neg

5

5

5

5

Microscopic Examination

Epi Cells

0

4

4

3

3

0-1

1

1

2

1

1-2

0

0

0

1

Casts

Absent

5

5

5

5

Crystals

Present

5

5

5

5

F: Female; Neg: Negative; Pos: Positive; Ca: Calculated approximately; >= more than equal to; SD: Standard deviation;

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

Table 8.1. Summary of absolute organ weights (g) record. Males

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

 

Adrenals

Thymus

Spleen

Testes

Epididymis

Heart

Kidneys

Brain

Liver

Prostate+Seminal vesicles with coagulating glands

Lungs

G1, M & 0

Mean

0.0518

0.3537

1.0897

3.2889

1.0565

1.1689

2.4151

1.9058

10.0892

2.2410

2.2864

±SD

0.0089

0.0873

0.3362

0.3593

0.1060

0.2408

0.4920

0.1369

1.9523

0.3177

0.6103

n

5

5

5

5

5

5

5

5

5

5

5

G2, M & 2.7

Mean

0.0518

0.3044

1.2695

3.2207

0.9811

1.0864

2.1333

1.9364

9.2042

2.2023

2.4908

±SD

0.0034

0.1268

0.3010

0.6065

0.1643

0.3076

0.4350

0.1494

1.8123

0.2864

0.9321

n

5

5

5

5

5

5

5

5

5

5

5

G3, M & 8.3

Mean

0.0483

0.2635

1.0093

3.2752

1.0884

1.1165

2.2240

1.9264

9.3631

2.3537

2.4398

±SD

0.0084

0.0552

0.5907

0.3214

0.0988

0.1110

0.3576

0.1069

1.3706

0.2593

0.2491

n

5

5

5

5

5

5

5

5

5

5

5

G4, M & 25

Mean

0.0564

0.0994*

0.6824

3.0020

0.9072

0.9152

1.8692

1.8565

7.9519

1.2656*

1.8941

±SD

0.0122

0.0324

0.3706

0.2183

0.0792

0.1738

0.1842

0.0640

1.1102

0.4555

0.3959

n

5

5

5

5

5

5

5

5

5

5

5

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

Table 8.2. Summary of absolute organ weights (g) record. Females

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

 

Adrenals

Thymus

Spleen

Ovaries

Uterus

Heart

Kidneys

Brain

Liver

Lungs

G1, F & 0

Mean

0.0662

0.3965

0.6406

0.1213

0.7294

0.9054

1.6172

1.9606

7.6942

2.1865

±SD

0.0057

0.1017

0.1435

0.0295

0.2004

0.1340

0.0606

0.0663

0.5108

0.2787

n

5

5

5

5

5

5

5

5

5

5

G2, F & 2.7

Mean

0.0671

0.3437

0.5401

0.1200

0.5062

0.9308

1.6759

1.9225

7.6816

2.1255

±SD

0.0043

0.0650

0.0689

0.0209

0.1498

0.0711

0.2399

0.0820

0.5841

0.4252

n

5

5

5

5

5

5

5

5

5

5

G3, F & 8.3

Mean

0.0689

0.2658*

0.4884

0.1133

0.4804

0.9128

1.5927

1.8693

7.7629

1.9623

±SD

0.0023

0.0386

0.0602

0.0085

0.1262

0.0749

0.0321

0.0587

0.3641

0.3719

n

5

5

5

5

5

5

5

5

5

5

G4, F & 25

Mean

0.0616

0.1210*

0.3617*

0.0991

0.3365*

0.8106

1.4502

1.9254

6.3781*

1.7117

±SD

0.0098

0.0593

0.1010

0.0279

0.2478

0.1277

0.2116

0.1213

1.3336

0.1206

n

5

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 fasting body weight (g) and organ weight relative to fasting body weight (%) record. Males

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

 

Fasting

(Terminal) Body Weight (g)

Adrenals

Thymus

Spleen

Testes

Epididymes

Heart

Kidneys

Brain

Liver

Prostate+Seminal vesicles with coagulating glands

Lungs

G1, M & 0

Mean

298.76

0.0175

0.1168

0.3745

1.1068

0.3549

0.3884

0.8026

0.6409

3.3658

0.7526

0.7647

±SD

31.45

0.0036

0.0199

0.1454

0.1269

0.0283

0.0508

0.0942

0.0464

0.4342

0.0929

0.1740

n

5

5

5

5

5

5

5

5

5

5

5

5

G2, M & 2.7

Mean

296.54

0.0177

0.1031

0.4353

1.0866

0.3328

0.3630

0.7172

0.6570

3.0872

0.7538

0.8221

±SD

39.24

0.0025

0.0443

0.1302

0.1705

0.0581

0.0609

0.0782

0.0441

0.2126

0.1522

0.1828

n

5

5

5

5

5

5

5

5

5

5

5

5

G3, M & 8.3

Mean

292.42

0.0165

0.0895

0.3370

1.1285

0.3764

0.3841

0.7591

0.6638

3.2043

0.8226

0.8430

±SD

36.73

0.0017

0.0104

0.1673

0.1278

0.0546

0.0343

0.0509

0.0519

0.2836

0.1871

0.1264

n

5

5

5

5

5

5

5

5

5

5

5

5

G4, M & 25

Mean

217.93*

0.0263*

0.0465*

0.3217

1.3941*

0.4182

0.4184

0.8599

0.8592*

3.6619

0.5764

0.8629

±SD

23.72

0.0074

0.0182

0.1877

0.2039

0.0370

0.0524

0.0620

0.0880

0.5003

0.1844

0.0900

n

5

5

5

5

5

5

5

5

5

5

5

5

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

Table 9.2. Summary of fasting body weight (g) and organ weight relative to fasting body weight (%) record. Females

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

 

Fasting

(Terminal) Body Weight (g)

Adrenals

Thymus

Spleen

Ovaries

Uterus

Heart

Kidneys

Brain

Liver

Lungs

G1, F & 0

Mean

247.71

0.0267

0.1590

0.2594

0.0487

0.2933

0.3662

0.6549

0.7942

3.1091

0.8899

±SD

16.90

0.0015

0.0344

0.0589

0.0103

0.0712

0.0515

0.0444

0.0564

0.1455

0.1652

n

5

5

5

5

5

5

5

5

5

5

5

G2, F & 2.7

Mean

242.24

0.0278

0.1414

0.2226

0.0494

0.2111

0.3848

0.6918

0.7952

3.1742

0.8775

±SD

15.94

0.0021

0.0215

0.0189

0.0067

0.0705

0.0262

0.0875

0.0390

0.2013

0.1657

n

5

5

5

5

5

5

5

5

5

5

5

G3, F & 8.3

Mean

232.28

0.0298

0.1145*

0.2103

0.0489

0.2074

0.3958

0.6913

0.8119

3.3683

0.8452

±SD

22.96

0.0027

0.0117

0.0153

0.0033

0.0522

0.0470

0.0720

0.0930

0.3709

0.1298

n

5

5

5

5

5

5

5

5

5

5

5

G4, F & 25

Mean

197.16*

0.0320

0.0587*

0.1815*

0.0509

0.1603*

0.4167

0.7509

1.0096*

3.2569

0.8914

±SD

42.13

0.0071

0.0184

0.0135

0.0120

0.0827

0.0432

0.1288

0.2107

0.3711

0.1371

n

5

5

5

5

5

5

5

5

5

5

5

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

Table 10.1. Summary of gross pathology record. Males

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

G1, M & 0

G2, M & 2.7

G3, M & 8.3

G4, M & 25

Total No. of Animals

5

5

5

5

No. of Animals Euthanized Terminally

5

5

5

5

External Gross pathological Observation

No Abnormality Detected

5

5

5

5

Internal Gross pathological Observation

No Abnormality Detected

5

5

5

0

Thymus: Small in size

0

0

0

2

Thymus: Small in size; Spleen: Small in size; PSC: Small in size

0

0

0

1

Thymus: Small in size; Spleen: Small in size

0

0

0

1

Thymus: Small in size; PSC: Small in size

0

0

0

1

PSC: Prostate+Seminal vesicles with coagulating glands

Table 10.2. Summary of gross pathology record. Females

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

G1, F & 0

G2, F & 2.7

G3, F & 8.3

G4, F & 25

Total No. of Animals

5

5

5

5

No. of Animals Euthanized Terminally

5

5

5

5

External Gross pathological Observation

No Abnormality Detected

5

5

5

5

Internal Gross pathological Observation

No Abnormality Detected

5

5

5

0

Thymus: Small in size

0

0

0

4

Thymus-small, Spleen-small

0

0

0

1

Conclusions:
In this dose range finding study, after an oral exposure of the test substance for 14 days in males and females rats, treatment related adverse effects were found at the top tested dose level of 25 mg/kg bw /d.









Executive summary:

A second dose range finding study was performed for the test substance in accordance with OECD guideline 407 in order to select the appropriate doses for a subsequent toxicity study. The test item was diluted in corn oil (vehicle) and added to Sprague-Dawley rats (5 per sex per dose) at doses of 0 (vehicle only), 2.7, 8.3 and 25 mg/kg bw/day for a period of 14 days. The vehicle and test item formulations were administered orally (gavage) at a dose volume of 5 mL/kg bw. Freshly prepared test item formulations were administered to the animals as soon as possible after preparation.

All animals for each group were observed for clinical signs of toxicity once daily, mortality and morbidity twice daily, and detailed clinical examination, body weight and feed consumption weekly. Ophthalmological examination was carried out during week 2 (day 14) for all group animals. Clinical pathology (haematology, clinical chemistry analysis and urinalysis) was conducted for all the animals sacrificed at termination.  All the animals were subjected to gross pathological examination and the organs were weighed, collected and preserved.

At dose of 2.7 mg/kg bw/d there were no test item-related effects noted in any parameter of both sexes throughout the treatment period and no gross pathological changes noted during necropsy. At dose of 8.3 mg/kg bw/d there were no adverse test item-related effects noted in any parameter of both sexes throughout the treatment period, except noted wet perineum in one female (recovered later), slight reduction in mean body weight/body weight gain, mean feed consumption and reduced platelet counts and eosinophils. There were no gross pathological changes noted during necropsy. At dose of 25 mg/kg bw/d, animals of both sexes were noted with test item-related clinical signs, significant reduction in body weight/body weight gain, feed consumption, significant effects in haematology / clinical chemistry parameters and reduced absolute / relative organ weights. The gross pathological examination revealed test item-related significant reduction in size of thymus, spleen and prostate along with seminal vesicles with coagulating gland. No ophthalmological effects were found in any dose group.

Based on these results, it can be concluded that the tested dose of 25 mg/kg bw /d was the lowest dose level at which treatment related adverse effects were found.

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)