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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 August 2021 – 15 December 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to other study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
17 April 2021 – 22 June 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
(number of animals per group was only 6 males and 6 females as the study was conducted as a dose range finding study for a subsequent complete OECD 422 study)
GLP compliance:
yes
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: 262.34-264.14 g (range of average values of each group of males at first day of dosing); 245.14-247.67 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.
- 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): 19.6-22.9ºC
- Humidity (%): 46-65 %
- Air changes (per hr): 12-15
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 17 April 2021 To: 22 June 2021
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
at a concentration of 0.5% (w/v)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required quantity of test item was weighed in a beaker and ground well in a mortar using pestle with a small quantity of vehicle until a homogenous suspension was formed. Thereafter the entire quantity of test formulation was transferred into measuring cylinder. A small quantity of vehicle was added to rinse the mortar and transferred into the measuring cylinder. The rinsing procedure of mortar and pestle was repeated many times to ensure the transfer of contents to the measuring cylinder. Finally, the volume was made up to required quantity with vehicle to get 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 insoluble in distilled water and uniformly suspended in 0.5% w/v Carboxymethyl Cellulose at the concentration of 100 mg/mL (the highest dose concentration selected for the study considering the dose volume of 10 mL/kg body weight) as per laboratory solubility/suspendibility test results.
Hence, 0.5% w/v Carboxymethyl Cellulose was used as vehicle for test item formulations as it is a routinely used and universally accepted vehicle of choice for the oral toxicity studies.
- Concentration in vehicle: 6.4, 16, 40 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. (if required): SLCD3996, BCBN1690V.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy.
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol: No
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:
Males: two weeks pre-mating, during mating and up to the day before sacrifice during the post-mating period (total of 29 days of treatment).
Females: two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13.
Frequency of treatment:
Once a day
Details on study schedule:
F1 animals were not mated.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
G1 - Vehicle control
Dose / conc.:
64 mg/kg bw/day (nominal)
Remarks:
G2 - Low dose
Dose / conc.:
160 mg/kg bw/day (nominal)
Remarks:
G3 - Low-mid dose
Dose / conc.:
400 mg/kg bw/day (nominal)
Remarks:
G4 - High-mid dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
G5 - High dose
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
There was no toxicity information available on the test item, strontium hydrogen phosphate. Some available toxicity information of structurally similar or analogue substances for the test item was consulted for the dose selection.
- Fasting period before blood sampling for clinical biochemistry: yes, overnight (water allowed).

Positive control:
None
Parental animals: Observations and examinations:
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.

BODY WEIGHT: Yes
- Time schedule for examinations:
Males were weighed at receipt, on the first day of dosing, weekly thereafter and at termination. Females were weighed at receipt, on the first day of dosing, weekly thereafter during pre-mating and until confirmation of mating. Pregnant animals were weighed on gestation days GD 0, 7, 14 and 20 and on lactation days 1, 4, 7, 13 and 14 (terminal body weight).

FOOD CONSUMPTION: yes
-Feed consumption (cage wise) was measured for all males and females once in a week during premating period coinciding with body weight recording. Feed consumption was measured for all pregnant animals during GD 0 to 7, 7 to 14 and 14 to 20, during LD 1 to 4, 4 to 7 and 7 to 13.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once before treatment (on the day of randomization) and at the end of the dosing period for all males and females (shortly prior to scheduled sacrifice).
- Dose groups that were examined: all groups.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled terminal sacrifice (for males on day 30 and for littered females on lactation day 14).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (water provided ad libitum)
- How many animals: all animals (males and females).
- 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 males on day 30 and for littered females on lactation day 14).
- Animals fasted: Yes (water provided ad libitum)
- How many animals: all animals (males and females).
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), 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: all males.
- 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 (shortly prior to scheduled sacrifice).
- Dose groups that were examined: all the animals (males and females) from all the dose 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.

Oestrous cyclicity (parental animals):
Oestrus cycles were monitored for two weeks during pre-mating treatment period to evaluate its normal oestrus cyclicity (4 to 5 days). Vaginal smears were monitored daily from the beginning of the treatment period until evidence of mating.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Individual pup from each litter was observed for external behavioral changes and for mortalities twice daily. Individual live pup weight from each litter was recorded on PND 1 (within 24 hours of parturition), 4, 7 and 13. 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.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for gross abnormalities with particular attention to the external reproductive genitals.

Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after completion of 29 days of treatment.
- Maternal animals: All surviving animals on lactation day 14.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.

HISTOPATHOLOGY / ORGAN WEIGHTS
The organs indicated in Table 1 were prepared for microscopic examination and weighed.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring was sacrificed on lactation day 13.
- These animals were subjected to postmortem examinations (macroscopic examination).

GROSS NECROPSY
- Gross necropsy consisted of examinajtion of gross abnormalities with particular attention to the external reproductive genitals.
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 2 below.
Reproductive indices:
Mating index, fertility index, gestation index, parturation index, pregnancy index, sex ratio, live birth index, post implantation loss (%).
Offspring viability indices:
Pup survival index on lactation day 4/7/13, sex ratio on lactation day 4/7/13, post natal loss (%), AGD ratio.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs of toxicity noted in any of the tested dose group animals of both sexes throughout the experimental period.
Dermal irritation (if dermal study):
not examined
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:
no effects observed
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 in all the tested dose groups of both sexes when compared with vehicle control group throughout the experimental period.

Also, there were no changes noted in mean gestational or lactation body weight and percent change in mean gestational or lactation body weight gain in all the tested dose groups when compared with vehicle control group during gestation or lactation period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no changes noted in mean feed consumption in all the tested dose groups measured during pre-mating period, gestational or lactation periods (for prenant females) when compared with the vehicle control group.
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 noted in any of the animals of both sexes from all the tested dose and vehicle control groups during ophthalmological examination conducted towards the end of dosing period.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In males the following mean haematological values were noted with statistically significant differences when compared with vehicle control groups:
- decrease in total leucocyte count and absolute lymphocytes in group G3 males;
- increase in percentage eosinophils, absolute and percentage basophils in group G4 males;
- increase in absolute neutrophils in group G2 males

The above mentioned statistically significant changes are considered as incidental and unrelated to treatment as these changes did not occur in a dose dependant manner and also the obtained mean values are within laboratory historical control range of same species and strain.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The following mean clinical chemistry values were noted with statistically significant changes in the tested dose groups when compared with vehicle control groups.
- decrease in creatinine levels in group G5 males;
- increase in potassium levels in groups G2, G4 and G5 males;
- increase in sodium levels in group G4 females.

The above mentioned statistically significant changes are considered as incidental and unrelated to treatment as these changes did not occur in a dose dependant manner and also the obtained mean values are within laboratory historical control range of same species and strain.
Endocrine findings:
not examined
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no changes noted in urine physical, chemical and microscopic examination parameters in all the tested dose groups (males) when compared with the vehicle control group.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no changes noted in any of the neurological/functional examination end points in all the tested dose groups of both sexes when compared with the vehicle control group.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
There were no irregularities observed in the oestrus cyclicity of females from any of the tested dose groups and vehicle control group during determined treatment period until confirmed as mated. The mean length of oestrus cycle (days) per dam was unaffected in all the tested dose groups when compared with vehicle control group.

However, a statistically significant decrease in mean oestrus cycle length in group G5 was noted when compared with vehicle control group. The noted statistically significant change is considered as incidental and unrelated to treatment as there were no irregularities in oestrus cycle noted in any of the females and also no effects were noted in any of the reproductive endpoints at this dose level.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Male Fertility Index (%): A total of 5 (out of 6), 5 (out of 6), 6 (out of 6), 5 (out of 6) and 5 (out of 6) males were confirmed as impregnating a female with a fertility index of 83.3%, 83.3%, 100.0%, 83.3% and 83.3% from groups G1, G2, G3, G4 and G5, respectively. There were no statistically significant differences noted in male fertility index (no. of males impregnating females) from any of the tested dose groups when compared with vehicle control group.

Female Fertility Index (%): A total of 5 (out of 6), 5 (out of 6), 6 (out of 6), 5(out of 6) and 5 (out of 6) females were confirmed with presence of live or dead pups/evidence of parturition with a fertility index of 83.3%, 83.3%, 100.0%, 83.3% and 83.3% from groups G1, G2, G3, G4 and G5, respectively. There were no statistically significant differences noted in female fertility index from any of all the tested dose groups when compared with vehicle control group.

Gestation Index (%): A total of 5 (out of 5), 5 (out of 5), 6 (out of 6), 5 (out of 5) and 5 (out of 5), pregnant females were confirmed with live born pups during parturition from groups G1, G2, G3, G4 and G5, respectively with a gestation index of 100.0%.

Post-Implantation Loss (%): A mean number of 0.40, 0.20, 0.50, 0.20 & 0.40 post-implantation losses per dam with a percentage of 2.87%, 1.67%, 3.85%, 1.67% and 3.21%, were noted from groups G1, G2, G3, G4 and G5, respectively. There were no statistically significant differences noted for this end point in any of the tested dose groups when compared with the vehicle control group.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects found up to the highest dose tested (1000 mg/kg bw/day)
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
There were no changes noted in daily observation of pups at all the tested and vehicle control groups.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The noted pup mortalities at birth across the tested dose groups including control group are incidences with lack of dose correlation and gross findings. Hence, these incidences are considered as spontaneous findings without any relation to administration of test item.

A mean number of 0.00, 0.00, 0.00, 0.20 & 0.00 post-natal losses per dam with a percentage of 0.00%, 0.00%, 0.00%, 1.18% and 0.00%, were noted from groups G1, G2, G3, G4 and G5, respectively. There were no statistically significant differences noted for this end point in any of the tested dose groups when compared with the vehicle control group.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related changes noted in mean pup (both male and female) weight per litter recorded on postnatal day (PND) 1, 4, 7 and 13 from all the tested dose groups when compared with vehicle control group.

The noted statistically significant increase in mean male pup weight in group G2 during postnatal day 1, 4 and 7 is considered as incidental and unrelated to treatment.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
There were no changes observed in mean pup (both male and female) anogenital distance measurement (mm) and ratio per litter recorded on PND 4 in any of the tested dose groups when compared with vehicle control group.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
There were no occurrences of nipple/arealaes in any of the male pups examined on their PND 13 from any of the tested dose group litters and vehicle control group litters.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross pathological changes observed in any of the pups found dead at birth, cannibalized or terminally sacrificed from all the tested dose groups and vehicle control group litters during conduct of necropsy.
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects found up to the highest dose tested (1000 mg/kg bw/day)
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Table 3. Summary of the study

























































































































































































































































 



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




Parameters ↓



G1 & 0



G2 & 64



G3 & 160



G4 & 400



G5 & 1000



Reproductive Indices



No. of Pairs started



6



6



6



6



6



Reproductive Performance / Indices (males)



Total No. of males cohabited



6



6



6



6



6



No. of males with confirmed mating



6



6



6



6



6



No. of males impregnating/siring a female



5



5



6



5



5



Male Mating Index (%)



100.0



100.0



100.0



100.0



100.0



Male Fertility Index (%)



83.3



83.3



100.0



83.3



83.3



 



Reproductive Performance and Indices (females)



Mean Oestrus cycle length (days)



Mean



4.83



4.96



4.93



4.90



4.36



±SD



0.26



0.10



0.12



0.15



0.25



No. of females with regular oestrus cycle



6



6



6



6



6



Total No. of females cohabited



6



6



6



6



6



No. of sperm-positive females



6



6



6



6



6



No. of females with evidence of pregnancy



5



5



6



5



5



No. of females with live born pups



5



5



6



5



5



No. of females with conceiving days 1 to 5



2



3



3



3



2



No. of females with conceiving days ≥6



4



3



3



3



4



Pre-coital Interval (Days)



Mean



5.83



5.83



7.17



5.83



6.50



±SD



2.93



3.19



4.45



4.17



3.27



Female Mating Index (%)



100.0



100.0



100.0



100.0



100.0



Female Fertility Index (%)



83.3



83.3



100.0



83.3



83.3



No. of females with Pregnancy≤21 days



-



1



-



-



1



No. of females with Pregnancy = 22 days



4



4



5



5



3



No. of females with Pregnancy≥23 days



1



-



1



-



1



Gestation Length (Days)



Mean



22.20



21.80



22.17



22.00



22.00



±SD



0.45



0.45



0.41



0.00



0.71



Gestation Index (%)



100.0



100.0



100.0



100.0



100.0



Female Fecundity or Pregnancy Index (%)



83.3



83.3



100.0



83.3



83.3



Parturition index (%)



100.0



100.0



100.0



100.0



100.0



















































































































































































































































































 



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




Parameters ↓



G1 & 0



G2 & 64



G3 & 160



G4 & 400



G5 & 1000



Delivery and Litter Observations



No. of Dams with live young born



5



5



6



5



5



No. of Dams with live young at PND 4



5



5



6



5



5



No. of Dams with live young at PND 7



5



5



6



5



5



No. of Dams with live young at PND 13



5



5



6



5



5



No. of Implants/dam



Mean



13.80



11.60



13.50



14.00



13.40



±SD



1.10



0.89



0.55



2.35



1.14



No. of Live pups/dam at birth



Mean



13.40



11.40



13.00



13.80



13.00



±SD



1.14



0.89



1.26



2.59



1.58



No. of Live pups/dam at PND 4



Mean



13.40



11.40



13.00



13.60



13.00



±SD



1.14



0.89



1.26



2.30



1.58



No. of Live pups/dam at PND 7



Mean



13.40



11.40



13.00



13.60



13.00



±SD



1.14



0.89



1.26



2.30



1.58



No. of Live pups/dam at PND 13



Mean



13.40



11.40



13.00



13.60



13.00



±SD



1.14



0.89



1.26



2.30



1.58



Live Birth Index/dam (%)



Mean



98.67



100.00



98.72



100.00



98.46



±SD



2.98



0.00



3.14



0.00



3.44



Pup Survival Index/dam on PND 4 (%)



Mean



100.00



100.00



100.00



98.82



100.00



±SD



0.00



0.00



0.00



2.63



0.00



Pup Survival Index/dam on PND 7 (%)



Mean



100.00



100.00



100.00



100.00



100.00



±SD



0.00



0.00



0.00



0.00



0.00



Pup Survival Index/dam on PND 13 (%)



Mean



100.00



100.00



100.00



100.00



100.00



±SD



0.00



0.00



0.00



0.00



0.00



Sex ratio (m/f)/dam At Birth



Mean



1.08



0.99



1.01



1.17



1.19



±SD



0.31



0.26



0.43



0.53



0.78



Sex ratio (m/f)/dam at PND 4



Mean



1.08



0.99



1.01



1.21



1.19



±SD



0.31



0.26



0.43



0.53



0.78



Sex ratio (m/f)/dam at PND 7



Mean



1.08



0.99



1.01



1.21



1.19



±SD



0.31



0.26



0.43



0.53



0.78



Sex ratio (m/f)/dam at PND 13



Mean



1.08



0.99



1.01



1.21



1.19



±SD



0.31



0.26



0.43



0.53



0.78





















































































































































































































































 



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



Parameters ↓



G1 & 0



G2 & 64



G3 & 160



G4 & 400



G5 & 1000



Delivery and Litter Observations



Mean Male


Pup weight (g)/dam



At birth



Mean



6.65



6.80



6.67



6.69



6.71



±SD



0.08



0.06



0.06



0.11



0.05



PND 4



Mean



10.75



11.04



10.74



10.77



10.83



±SD



0.11



0.18



0.10



0.23



0.19



PND 7



Mean



14.77



15.11



14.78



14.83



14.84



±SD



0.09



0.18



0.12



0.29



0.15



PND 13



Mean



27.99



28.36



28.02



27.89



27.99



±SD



0.18



0.25



0.38



0.49



0.21



Mean Female


Pup weight (g)/dam



At Birth



Mean



6.22



6.28



6.26



6.23



6.23



±SD



0.05



0.10



0.23



0.13



0.10



PND 4



Mean



10.29



10.38



10.36



10.29



10.30



±SD



0.09



0.09



0.20



0.12



0.07



PND 7



Mean



14.34



14.42



14.37



14.31



14.33



±SD



0.09



0.09



0.22



0.16



0.10



PND 13



Mean



27.24



27.37



27.34



27.28



27.31



±SD



0.13



0.14



0.26



0.23



0.11



Mean Male Pup AGD Measurement/


dam (mm) on PND 4



Mean



4.95



5.02



4.93



4.94



4.97



±SD



0.07



0.04



0.09



0.09



0.06



Mean Male Pup AGD ratio/


dam on PND 4



Mean



2.24



2.25



2.23



2.24



2.25



±SD



0.03



0.01



0.03



0.03



0.02



Mean Female Pup AGD Measurement/


dam (mm) on PND 4



Mean



2.52



2.58



2.50



2.54



2.57



±SD



0.04



0.07



0.06



0.08



0.11



Mean Female Pup AGD ratio/


dam on PND 4



Mean



1.16



1.18



1.15



1.17



1.18



±SD



0.02



0.03



0.03



0.03



0.05



Male Pup Nipple Retention/


dam on PND 13



Mean



0.00



0.00



0.00



0.00



0.00



±SD



0.00



0.00



0.00



0.00



0.00



















































































































































 



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




Parameters ↓



G1 & 0



G2 & 64



G3 & 160



G4 & 400



G5 & 1000



ABNORMAL PUPS



No. of Dams with 0 abnormal pups



5



5



6



5



5



LOSS OF OFFSPRING



Pre-natal or post-implantation loss (implantations minus live births)



No. of Females with 0 post-implantation loss



3



4



4



4



3



No. of Females with 1 post-implantation loss



2



1



1



1



2



No. of Females with 2 post-implantation losses



-



-



1



-



-



Post-implantation Loss (No.)



Mean



0.40



0.20



0.50



0.20



0.40



±SD



0.55



0.45



0.84



0.45



0.55



Post-implantation Loss (%)



Mean



2.87



1.67



3.85



1.67



3.21



±SD



3.95



3.73



6.44



3.73



4.39



Post-natal loss (live births minus alive at PND 13)



No. of Females with 0



5



5



6



4



5



No. of Females with 1



-



-



-



1



-



Post-natal loss (No.)



Mean



0.00



0.00



0.00



0.20



0.00



±SD



0.00



0.00



0.00



0.45



0.00



Post-natal Loss (%)



Mean



0.00



0.00



0.00



1.18



0.00



±SD



0.00



0.00



0.00



2.63



0.00



Table 4: Summary of body weight (g) record















































































































































Group, Sex


& Dose
(mg/kg body


weight/day)



 



Body Weight (g) on Day



1



7



14



21



28



G1, M & 0



Mean



262.52



282.71



303.24



317.21



332.13



±SD



16.18



19.53



26.79



29.63



33.17



n



6



6



6



6



6



G2, M & 64



Mean



262.34



278.27



298.68



315.95



338.36



±SD



17.03



13.17



19.44



23.40



23.06



n



6



6



6



6



6



G3, M & 160



Mean



263.15



284.15



306.00



313.95



330.07



±SD



16.95



21.11



28.13



29.79



32.85



n



6



6



6



6



6



G4, M & 400



Mean



263.48



278.90



302.96



316.24



338.90



±SD



17.49



20.05



21.62



24.60



27.23



n



6



6



6



6



6



G5, M & 1000



Mean



264.14



287.58



307.47



321.94



340.00



±SD



13.98



13.09



21.09



24.58



25.03



n



6



6



6



6



6
































































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Body Weight (g) on Day



1



7



14



21#



G1, F & 0



Mean



246.55



253.53



256.86



281.37



±SD



15.16



18.29



19.49



14.77



n



6



6



6



2



G2, F & 64



Mean



245.14



252.03



255.34



264.76



±SD



16.38



15.51



17.50



28.67



n



6



6



6



2



G3, F & 160



Mean



245.47



254.78



260.42



278.48



±SD



15.97



17.26



17.75



19.67



n



6



6



6



2



G4, F & 400



Mean



246.81



253.27



259.45



256.13



±SD



16.32



19.44



22.76



5.10



n



6



6



6



2



G5, F & 1000



Mean



247.67



249.71



254.23



264.36



±SD



14.08



13.72



13.68



18.09



n



6



6



6



3



#: The mean, standard deviation and number of animals for day 21 are calculated from data of females under cohabitation. The data was not subjected to statistical analysis due to uneven number of variables.


The data of mated females is excluded under this summary table and presented under summary of gestational body weight record.


 


Table 5: Summary record of gestation body weight (g)































































































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Body Weight (g) on Gestation Day (GD)



0



7



14



20



G1, F & 0



Mean



258.80



271.39



301.12



357.10



±SD



20.92



20.92



19.29



26.19



n



5



5



5



5



G2, F & 64



Mean



265.35



279.89



308.45



358.98



±SD



19.09



19.85



18.88



18.88



n



5



5



5



5



G3, F & 160



Mean



265.95



280.06



309.75



371.29



±SD



18.18



18.95



20.42



26.59



n



6



6



6



6



G4, F & 400



Mean



263.21



276.48



306.39



365.95



±SD



25.83



25.27



22.86



27.14



n



5



5



5



5



G5, F & 1000



Mean



261.97



274.76



305.21



363.83



±SD



15.76



17.23



19.81



21.71



n



5



5



5



5



G1: 1 out of 6 females was non-pregnant; G2: 1 out of 6 females was non-pregnant;


G4: 1 out of 6 females was non-pregnant; G5: 1 out of 6 females was non-pregnant.


Note: The data of non-pregnant females was excluded for mean calculations and statistical analysis, but presented in individual animal body weight record.


 


Table 6: Summary record of lactation body weight (g)































































































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Body Weight (g) on Lactation Day (LD)



1



4



7



13



G1, F & 0



Mean



269.59



274.65



282.68



297.51



±SD



20.35



17.90



18.44



16.64



n



5



5



5



5



G2, F & 64



Mean



280.41



287.52



296.63



311.17



±SD



20.98



20.78



20.89



21.76



n



5



5



5



5



G3, F & 160



Mean



283.97



287.50



295.63



310.30



±SD



24.63



27.60



28.81



28.87



n



6



6



6



6



G4, F & 400



Mean



276.10



277.83



284.03



295.36



±SD



23.41



24.87



25.26



23.62



n



5



5



5



5



G5, F & 1000



Mean



277.95



281.88



288.50



302.22



±SD



14.08



11.32



11.48



12.79



n



5



5



5



5



G1: 1 out of 6 females was non-pregnant; G2: 1 out of 6 females was non-pregnant;


G4: 1 out of 6 females was non-pregnant; G5: 1 out of 6 females was non-pregnant.


 


Table 7: Summary of haematology record


































































































































































































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Total


Leucocyte


Count



Total


Erythrocyte


Count



Haemoglobin



Haematocrit



Mean


Corpuscular Volume



Mean


Corpuscular Haemoglobin



Mean Corpuscular Haemoglobin Concentration



Platelet


Count



Mean


Platelet


Volume



(WBC)



(RBC)



(HGB)



(HCT)



(MCV)



(MCH)



(MCHC)



(PLT)



(MPV)



(103 cells/µL)



(106 cells/µL)



(g/dL)



(%)



(fL)



(pg)



(g/dL)



(103 cells/µL)



(fL)



G1, M & 0



Mean



9.75



8.11



15.35



47.57



59.35



19.07



32.27



844.83



7.37



±SD



0.88



0.98



0.97



1.10



7.22



1.33



1.62



90.45



0.35



n



6



6



6



6



6



6



6



6



6



G2, M & 64



Mean



11.45



8.06



16.22



48.08



59.92



20.22



33.77



938.50



7.18



±SD



1.54



0.76



0.84



2.84



3.67



1.16



0.96



61.10



0.15



n



6



6



6



6



6



6



6



6



6



G3, M & 160



Mean



7.46*



8.76



16.23



48.48



55.40



18.53



33.50



834.17



6.93



±SD



1.73



0.29



0.44



1.74



2.61



0.63



0.50



74.05



0.23



n



6



6



6



6



6



6



6



6



6



G4, M & 400



Mean



9.20



7.62



15.02



46.25



60.92



19.70



32.58



851.17



7.32



±SD



1.35



0.52



1.38



1.67



4.89



0.92



3.83



166.32



0.61



n



6



6



6



6



6



6



6



6



6



G5, M & 1000



Mean



10.24



8.41



16.03



47.97



57.28



19.13



33.43



974.17



7.10



±SD



0.76



0.77



0.80



2.01



3.49



0.97



0.48



97.08



0.21



n



6



6



6



6



6



6



6



6



6



 












































































































































































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



7.46



20.68



74.42



2.73



0.83



0.33



±SD



5.70



4.71



4.98



0.67



0.60



0.10



n



6



6



6



6



6



6



G2, M & 64



Mean



5.28



25.92



69.00



2.88



1.00



0.40



±SD



1.16



3.56



3.92



1.26



0.26



0.09



n



6



6



6



6



6



6



G3, M & 160



Mean



3.09



25.97



68.78



2.75



1.20



0.55



±SD



1.01



4.24



5.25



0.68



0.53



0.24



n



6



6



6



6



6



6



G4, M & 400



Mean



9.24



22.32



72.22



2.23



1.93*



0.63*



±SD



5.96



1.93



2.82



1.05



1.19



0.27



n



6



6



6



6



6



6



G5, M & 1000



Mean



4.64



23.15



71.62



3.15



0.92



0.38



±SD



2.34



6.70



7.32



0.93



0.39



0.08



n



6



6



6



6



6



6



 
















































































































































































































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)



(109 cells/L)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(Seconds)



(Seconds)



G1, M & 0



Mean



559.63



2.02



7.25



0.27



0.09



0.03



16.68



23.65



±SD



366.46



0.47



0.74



0.08



0.06



0.00



2.17



2.17



n



6



6



6



6



6



6



6



6



G2, M & 64



Mean



418.72



3.01*



7.86



0.33



0.12



0.05



15.07



22.27



±SD



61.62



0.75



0.75



0.15



0.03



0.01



0.61



1.54



n



6



6



6



6



6



6



6



6



G3, M & 160



Mean



269.50



1.89



5.19*



0.20



0.09



0.04



16.57



22.25



±SD



82.77



0.26



1.57



0.05



0.02



0.01



1.88



4.83



n



6



6



6



6



6



6



6



6



G4, M & 400



Mean



681.83



2.06



6.64



0.21



0.18



0.06*



16.80



23.25



±SD



414.31



0.37



0.93



0.12



0.13



0.02



1.90



2.65



n



6



6



6



6



6



6



6



6



G5, M & 1000



Mean



376.43



2.39



7.31



0.32



0.10



0.04



16.85



26.03



±SD



165.51



0.85



0.73



0.09



0.05



0.01



1.30



3.73



n



6



6



6



6



6



6



6



6



 


































































































































































































































Group, Sex


& Dose


(mg/kg body


weight/day)



 



Total


Leucocyte Count



Total Erythrocyte Count 



Haemoglobin



Haematocrit



Mean Corpuscular Volume



Mean Corpuscular Haemoglobin



Mean Corpuscular Haemoglobin Concentration 



Platelet


Count



Mean


Platelet


Volume



  (WBC)        



 (RBC)    



(HGB)      



 (HCT)   



  (MCV)     



(MCH)



(MCHC)      



  (PLT)



(MPV)   



(103 cells/µL)



(106 cells/µL)



(g/dL)



(%)



(fL)



(pg)



(g/dL)



(103 cells/µL)



(fL)



G1, F & 0



Mean



10.26



7.78



15.62



48.55



62.48



20.12



32.17



719.33



7.92



±SD



2.20



0.60



0.71



2.98



1.85



0.79



1.21



200.28



1.28



n



6



6



6



6



6



6



6



6



6



G2, F & 64



Mean



9.42



7.26



14.92



46.12



64.20



20.72



32.30



694.50



7.52



±SD



2.52



0.90



0.70



1.41



6.72



1.79



0.87



186.56



0.48



n



6



6



6



6



6



6



6



6



6



G3, F & 160



Mean



9.78



7.73



15.14



49.50



64.08



20.72



32.33



529.50



7.88



±SD



1.86



0.94



0.75



6.10



2.18



1.17



0.84



268.77



0.81



n



6



6



5



6



6



6



6



6



6



G4, F & 400



Mean



9.99



7.40



14.80



46.57



63.08



20.03



31.77



867.17



7.35



±SD



3.22



0.52



0.51



1.62



3.40



0.77



0.74



200.07



0.57



n



6



6



6



6



6



6



6



6



6



G5, F & 1000



Mean



9.93



7.62



14.83



46.00



60.32



19.48



32.32



868.83



7.18



±SD



2.89



0.35



1.02



4.16



3.89



0.93



0.79



216.14



0.62



n



6



6



6



6



6



6



6



6



6



 












































































































































































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



3.63



34.73



58.78



3.45



1.77



0.30



±SD



1.57



4.36



4.27



1.30



1.58



0.32



n



6



6



6



6



6



6



G2, F & 64



Mean



4.63



33.18



59.82



3.92



1.70



0.27



±SD



1.63



8.57



6.02



1.42



1.29



0.18



n



6



6



6



6



6



6



G3, F & 160



Mean



5.64



36.52



56.92



3.82



1.38



0.38



±SD



2.20



13.55



13.54



0.95



1.56



0.31



n



6



6



6



6



6



6



G4, F & 400



Mean



3.72



30.47



62.80



3.72



1.82



0.38



±SD



1.46



8.85



9.79



0.78



1.30



0.21



n





6



6



6



6



6



G5, F & 1000



Mean



3.23



31.63



59.48



4.53



1.62



0.32



±SD



0.76



9.65



12.58



1.73



1.70



0.08



n



6



6



6



6



6



6



 
















































































































































































































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)



(109 cells/L)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(Seconds)



(Seconds)



G1, F & 0



Mean



277.30



3.51



6.09



0.35



0.17



0.03



20.40



20.53



±SD



103.74



0.57



1.62



0.13



0.14



0.03



5.73



8.47



n



6



6



6



6



6



6



6



6



G2, F & 64



Mean



328.53



3.07



5.66



0.37



0.17



0.03



16.18



18.97



±SD



105.58



0.86



1.85



0.20



0.14



0.01



3.00



4.79



n



6



6



6



6



6



6



6



6



G3, F & 160



Mean



424.40



3.60



5.57



0.37



0.12



0.04



17.45



25.60



±SD



136.76



1.72



1.76



0.07



0.12



0.02



3.64



9.10



n



6



6



6



6



6



6



6



6



G4, F & 400



Mean



275.86



2.93



6.40



0.38



0.16



0.04



19.27



20.75



±SD



94.83



1.03



2.46



0.17



0.08



0.02



4.96



8.71



n





6



6



6



6



6



6



6



G5, F & 1000



Mean



246.25



3.22



5.89



0.43



0.15



0.03



17.55



25.58



±SD



63.13



1.47



1.88



0.12



0.12



0.01



2.44



7.11



n



6



6



6



6



6



6



6



6



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


°: Outliered values are excluded for mean calculations and statistical analysis.


 


Table 8: 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



105.00



38.80



0.56



41.33



29.17



7.28



3.11



±SD



17.58



4.19



0.03



7.45



13.47



0.31



0.20



n



6



6



6



6



6



6



6



G2, M & 64



Mean



122.83



38.53



0.53



42.17



27.83



6.93



3.04



±SD



29.14



2.64



0.02



4.62



5.23



0.36



0.16



n



6



6



6



6



6



6



6



G3, M & 160



Mean



114.33



38.78



0.53



46.00



34.00



7.20



2.99



±SD



10.75



4.66



0.02



7.29



5.83



0.24



0.19



n



6



6



6



6



6



6



6



G4, M & 400



Mean



123.67



40.63



0.53



45.67



29.00



7.12



3.17



±SD



11.67



5.22



0.01



8.21



8.37



0.26



0.11



n



6



6



6



6



6



6



6



G5, M & 1000



Mean



122.00



44.03



0.51*



44.00



33.50



7.00



3.05



±SD



15.36



7.08



0.02



7.07



14.12



0.35



0.14



n



6



6



6



6



6



6



6



 












































































































































































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



60.83



114.50



167.17



0.06



9.05



5.65



±SD



10.26



13.90



38.59



0.02



0.46



0.60



n



6



6



6



6



6



6



G2, M & 64



Mean



73.17



122.00



190.83



0.06



9.07



5.78



±SD



6.05



13.61



19.70



0.03



0.41



0.79



n



6



6



6



6



6



6



G3, M & 160



Mean



68.83



111.33



215.00



0.03



9.03



5.73



±SD



24.86



27.00



80.19



0.01



0.38



0.68



n



6



6



6



6



6



6



G4, M & 400



Mean



70.83



121.83



215.00



0.05



9.27



5.53



±SD



23.74



37.28



43.74



0.03



0.27



0.47



n



6



6



6



6



6



6



G5, M & 1000



Mean



56.83



99.83



223.40



0.04



9.27



5.43



±SD



7.31



15.68



40.23



0.03



0.33



0.46



n



6



6





6



6



6



 












































































































































































Group, Sex


& Dose


(mg/kg body


weight/day)



 



Globulin



Albumin/


Globulin ratio



Blood Urea Nitrogen



Sodium



Potassium



Chloride



(GLO)



(A/G Ratio)



(BUN)



 (Na)



 (K)  



 (CLO)



(g/dL)



-



(mg/dL)



 (mmol/L)



 (mmol/L)



 (mmol/L)



G1, M & 0



Mean



4.17



0.75



18.11



142.25



3.23



108.65



±SD



0.26



0.07



1.95



1.16



0.29



1.17



n



6



6



6



6



6



6



G2, M & 64



Mean



3.89



0.79



17.98



142.57



3.73*



108.15



±SD



0.31



0.07



1.23



1.51



0.39



2.60



n



6



6



6



6



6



6



G3, M & 160



Mean



4.21



0.71



18.10



142.97



3.57



109.03



±SD



0.27



0.08



2.18



0.62



0.20



1.03



n



6



6



6



6



6



6



G4, M & 400



Mean



3.95



0.80



18.96



142.08



3.85*



109.38



±SD



0.25



0.06



2.44



0.62



0.29



1.20



n



6



6



6



6



6



6



G5, M & 1000



Mean



3.96



0.77



20.55



140.55



3.95*



106.18



±SD



0.25



0.04



3.31



2.81



0.29



3.79



n



6



6



6



6



6



6



 






























































































































































































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



103.83



55.70



0.58



71.67



87.33



7.37



3.61



±SD



22.53



16.06



0.04



13.53



44.99



0.56



0.16



n



6



6



6



6



6



6



6



G2, F & 64



Mean



117.00



52.22



0.60



60.00



56.20



7.70



3.61



±SD



17.93



10.73



0.11



18.57



14.10



0.24



0.21



n



6



6



6



6





6



6



G3, F & 160



Mean



103.83



57.90



0.62



71.67



125.60



7.68



3.56



±SD



15.68



10.51



0.09



21.30



60.71



0.50



0.14



n



6



6



6



6





6



6



G4, F & 400



Mean



98.00



63.53



0.60



75.00



166.20



7.78



3.66



±SD



20.87



9.60



0.05



8.79



145.01



0.33



0.24



n



6



6



6



6





6



6



G5, F & 1000



Mean



103.83



52.10



0.61



56.67



89.20



7.78



3.62



±SD



27.23



7.11



0.07



13.17



52.83



0.53



0.23



n



6



6



6



6





6



6



 












































































































































































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



118.33



156.17



286.17



0.01



9.88



6.88



±SD



55.50



82.99



189.92



0.01



0.67



2.46



n



6



6



6



6



6



6



G2, F & 64



Mean



124.50



164.50



263.00



0.03



9.55



5.87



±SD



60.35



81.65



179.91



0.03



1.89



2.33



n



6



6



6



6



6



6



G3, F & 160



Mean



173.83



171.75



431.83



0.01



10.23



8.25



±SD



61.25



64.07



201.54



0.02



1.59



1.68



n



6





6



6



6



6



G4, F & 400



Mean



125.83



124.00



393.50



0.01



10.60



6.62



±SD



31.40



29.48



281.23



0.01



1.37



1.28



n



6



6



6



6



6



6



G5, F & 1000



Mean



114.00



127.33



266.83



0.01



10.05



7.12



±SD



55.40



19.82



162.09



0.02



1.27



1.68



n



6



6



6



6



6



6



 












































































































































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Globulin



Albumin/


Globulin ratio



Blood Urea Nitrogen



Sodium



Potassium



Chloride



(GLO)



(A/G Ratio)



(BUN)



(Na)



(K)



(CLO)



(g/dL)



-



(mg/dL)



(mmol/L)



(mmol/L)



(mmol/L)



G1, F & 0



Mean



3.76



0.98



26.00



139.00



3.58



102.13



±SD



0.54



0.14



7.50



1.92



0.54



3.67



n



6



6



6



6



6



6



G2, F & 64



Mean



4.09



0.89



24.37



140.77



3.64



103.85



±SD



0.21



0.08



5.01



1.02



0.61



3.31



n



6



6



6



6



6



6



G3, F & 160



Mean



4.13



0.87



27.02



139.18



3.73



98.65



±SD



0.42



0.08



4.90



2.24



0.42



2.96



n



6



6



6



6



6



6



G4, F & 400



Mean



4.13



0.90



29.65



141.72*



3.57



100.83



±SD



0.40



0.12



4.48



2.41



0.39



4.22



n



6



6



6



6



6



6



G5, F & 1000



Mean



4.17



0.87



24.31



140.75



3.92



98.65



±SD



0.38



0.06



3.32



0.72



0.80



3.07



n



6



6



6



6



6



6



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


°: Outliered values are excluded for mean calculations and statistical analysis.


 


Table 9: Summary record of vaginal smear examination for determination of oestrus cyclicity





















































































































Determination of Oestrus Cyclicity during Treatment Period



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



Total No. of


Females Evaluated


 

Females with


Complete Regular Cycles



Females with at least one Irregular Oestrus Cycle



Average Length of


Oestrous Cycle (Days)



G1, F & 0



6



n



6



0



Mean



4.83



%



100.00



0.00



±SD



0.26



n



6



G2, F & 64



6



n



6



0



Mean



4.96



%



100.00



0.00



±SD



0.10



n



6



G3, F & 160



6



n



6



0



Mean



4.93



%



100.00



0.00



±SD



0.12



n



6



G4, F & 400



6



n



6



0



Mean



4.90



%



100.00



0.00



±SD



0.15



n



6



G5, F & 1000



6



n



6



0



Mean



4.36*



%



100.00



0.00



±SD



0.25



n



6



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


 


Table 10: Summary record of mean pup weight (g) per litter






























































































































































































































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

PND 1


 

PND 4


 

PND 7


 

PND 13



Mean Pup Weight (g)



 



Mean Pup Weight (g)



 



Mean Pup Weight (g)



 



Mean Pup Weight (g)



Male



Female



 



Male



Female



 



Male



Female



 



Male



Female



G1, F & 0



Mean



6.65



6.22


 

10.75



10.29


 

14.77



14.34


 

27.99



27.24



±SD



0.08



0.05


 

0.11



0.09


 

0.09



0.09


 

0.18



0.13



n



5 (34)



5 (33)


 

5 (34)



5 (33)


 

5 (34)



5 (33)


 

5 (34)



5 (33)



G2, F & 64



Mean



6.80*



6.28


 

11.04*



10.38


 

15.11*



14.42


 

28.36



27.37



±SD



0.06



0.10


 

0.18



0.09


 

0.18



0.09


 

0.25



0.14



n



5 (28)



5 (29)


 

5 (28)



5 (29)


 

5 (28)



5 (29)


 

5 (28)



5 (29)



G3, F & 160



Mean



6.67



6.26


 

10.74



10.36


 

14.78



14.37


 

28.02



27.34



±SD



0.06



0.23


 

0.10



0.20


 

0.12



0.22


 

0.38



0.26



n



6 (38)



6 (40)


 

6 (38)



6 (40)


 

6 (38)



6 (40)


 

6 (38)



6 (40)



G4, F & 400



Mean



6.69



6.23


 

10.77



10.29


 

14.83



14.31


 

27.89



27.28



±SD



0.11



0.13


 

0.23



0.12


 

0.29



0.16


 

0.49



0.23



n



5 (36)



5 (33)


 

5 (36)



5 (32)


 

5 (36)



5 (32)


 

5 (36)



5 (32)



G5, F & 1000



Mean



6.71



6.23


 

10.83



10.30


 

14.84



14.33


 

27.99



27.31



±SD



0.05



0.10


 

0.19



0.07


 

0.15



0.10


 

0.21



0.11



n



5 (32)



5 (33)


 

5 (32)



5 (33)


 

5 (32)



5 (33)


 

5 (32)



5 (33)



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

Conclusions:
In a dose range finding study for a further study on reproductive/ developmental toxicity, the oral administration of the test item in Sprague Dawley rats did not produce any indication of systemic, reproduction and developmental toxicity up to the dose level of 1000 mg/kg bw/d and therefore doses of 100, 300 and 1000 mg/kg bw/d are considered appropriate for the definitive test.



Executive summary:

To establish doses for a definitive OECD 422 study, a dose range finding study non GLP was performed using Sprague Dawley Rats. A total of 60 (30 males and 30 females) rats were allocated to five different groups. Each group (G1, G2, G3, G4 and G5) consisted of 6 males and 6 females. The animals from group G1 were administered with vehicle (0.5% w/v Carboxymethyl Cellulose), whereas the animals from groups G2, G3, G4 and G5 were administered with test item at the dose levels of 64, 160, 400 and 1000 mg/kg bw/d. The vehicle and test item formulations were administered to the animals by oral gavage route with the dose volume of 10 mL/kg bw. Males were treated for two weeks pre-mating (14-days), during mating and up to the day before sacrifice during post-mating period with a total period of 29 days. The females were treated for two weeks during pre-mating period (14-days), mating (maximum of 13 days), pregnancy (gestation) and up to lactation day 13, ranging from 49 to 61 days. For systemic toxicity assessment, animals were observed for clinical signs, mortality and morbidity, body weight and feed consumption (including during gestation and lactation periods), ophthalmological examination, neurological/functional observations, clinical pathological examinations (haematology, clinical chemistry and urinalysis) and organ weighing. For reproductive toxicity assessment, males and dams were evaluated for reproductive performances such as, mating index, fertility index, gestation index, parturition index, pre-coital interval, gestation length, post-implantation loss and postnatal loss. At birth and during lactation, parameters such as, number of live/survived/dead pups, litter size, sex ratio and live birth/survival index per litter were observed. For developmental toxicity assessment, all survived pups from each litter were observed once daily for external examinations and twice daily for mortality till termination (PND13). All pups from each litter were weighed individually on PND 1, 4, 7 and 13 and measured for AGD on PND 4. All male pups were observed for retention of nipples on PND 13. All adults and pups were subject to detailed gross pathological examination with special emphasis towards reproductive organs. There were no treatment related effects observed for systemic, reproduction or developmental toxicity up to the dose level of 1000 mg/kg bw/d. Based on these results, it is concluded that the doses of 100, 300 and 1000 mg/kg bw/d can be selected as low, mid and high dose levels for the subsequent “Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test”.

Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 August 2021 – 15 December 2021
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 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: 371.37-373.02 g (range of average values of each group of males at first day of dosing); 252.64-253.66 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 group: maximum of 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): 19.9-22.9ºC
- Humidity (%): 48-63 %
- Air changes (per hr): 12-15
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 19 August 2021 To: 11 November 2021
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
at a concentration of 0.5% (w/v)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required quantity of test item was weighed in a beaker and ground well in a mortar using pestle with a small quantity of vehicle until a homogenous suspension was formed. Thereafter the entire quantity of test formulation was transferred into measuring cylinder. A small quantity of vehicle was added to rinse the mortar and transferred into the measuring cylinder. The rinsing procedure of mortar and pestle was repeated many times to ensure the transfer of contents to the measuring cylinder. Finally, the volume was made up to required quantity with vehicle to get desired concentration for the different dose levels.
Test item formulations were administered to the animals within established stability conditions.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item was insoluble in distilled water and uniformly suspended in 0.5% w/v Carboxymethyl Cellulose at the concentration of 100 mg/mL (the highest dose concentration selected for the study considering the dose volume of 10 mL/kg body weight) as per laboratory solubility/suspendibility test results.
Hence, 0.5% w/v Carboxymethyl Cellulose was used as vehicle for test item formulations as it is a routinely used and universally accepted vehicle of choice for the oral toxicity studies.
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. (if required): SLCH1520
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy.
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol: No
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 followed by 48 hours at the concentrations of 5 mg/mL and 100 mg/mL in 0.5% w/v Carboxymethyl cellulose. Prepared test item formulations were administered to the animals within these established stability conditions.
Homogeneity and dose formulation analysis for dose concentration verification was done by a validated analytical method ICP-MS (study nº BIO-ANM 1742). Sampling and analysis of formulations were performed during week 1 (07/09/2021) and week 5 (05/10/2021) of the treatment. Samples (5 mL approx.) 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 (total of 29 days).
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 15 days observation.
Frequency of treatment:
Once a day
Details on study schedule:
F1 animals were not mated.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
G1 - Vehicle control + G1R - Vehicle Control Recovery Group
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
G2 - Low dose
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
G3 - Mid dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
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 were decided based on the results of a dose range finding study in which there were no effects for systemic, reproductive and developmental toxicity up to the dose level of 1000 mg/kg bw/day.
- Fasting period before blood sampling for clinical biochemistry: yes, overnight (water allowed).
Positive control:
None
Parental animals: Observations and examinations:
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.

BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed at receipt, on the first day of dosing, weekly thereafter and at termination. Females were weighed at receipt, on the first day of dosing, weekly thereafter during pre-mating and until confirmation of mating. Pregnant animals were weighed on gestation days GD 0, 7, 14 and 20 and on lactation days 1, 4, 7, 13 and 14 (terminal body weight).

FOOD CONSUMPTION: yes
-Feed consumption (cage wise) was measured for all males and females once in a week during premating period coinciding with body weight recording. Feed consumption was measured for all pregnant animals during GD 0 to 7, 7 to 14 and 14 to 20, during LD 1 to 4, 4 to 7 and 7 to 13.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once before treatment (on the day of randomization) and at the end of the dosing period for all males and females (shortly prior to scheduled sacrifice).
- Dose groups that were examined: all groups.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled terminal sacrifice (for males on day 29 and for littered females on lactation day 14).
- 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 males on day 29 and for littered females on lactation day 14).
- 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), Total Protein, Albumin, 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 29) and during lactation period for females (on lactation day 13). Towards the end of the recovery period (shortly prior to scheduled sacrifice on day 64) for the recovery group.
- Dose groups that were examined: 5 males and 5 females randomly selected from each main group and all recovery group animals.
- 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:
Thyroxine Hormone (T4) Level estimation: Blood samples were collected from all main group animals 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 available above normal litter size.
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 29 days of treatment).
Oestrous cyclicity (parental animals):
Oestrus cyclicity was monitored for two weeks after five days of acclimatization to evaluate the normal oestrus cycle (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.
Sperm parameters (parental animals):
Testes and epididymis were collected and weighed for all animals in all dose groups. Histopathology was initially performed for all animals in the control and high dose groups. A detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen or any cell or stage specificity of testicular findings.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
The number of male/female pups born (live/dead/cannibalized) per litter, sex ratio (m/f) and live birth index per litter at the day of littering. Number of pups survived/dead/cannibalized per litter during lactation period and pup survival index (%) per litter between LD 1 to 4, 4 to 7 and 7 to 13. Sex ratio (m/f) on LD 4, 7 and 13. External deformities or clinical signs once daily and for mortalities twice daily in all pups. Individual live pup weight from each litter on PND 1 (within 24 hours of parturition), 4, 7 and 13. anogenital distance (AGD) on PND 4 in all pups. Retention of nipples/areolae on PND 13 in all male pups.
Also, blood samples were collected for measurement of serum T4 levels on the following schedule:
1. 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 the normal litter size.
2. Two pups per litter (same sex) at termination (lactation day 13).

GROSS EXAMINATION OF DEAD PUPS:
Yes, for gross abnormalities with particular attention to the external reproductive genitals.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after completion of 29 days of treatment.
- Maternal animals: All surviving animals on lactation day 14.
- Recovery animals: All surviving animals after completion of 14 days observation from the first scheduled sacrifice of dams.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.

HISTOPATHOLOGY / ORGAN WEIGHTS
The organs indicated in Tables 1 and 2 were prepared for microscopic examination and weighed.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring was sacrificed on lactation day 13.
- These animals were subjected to postmortem examinations (macroscopic examination).

GROSS NECROPSY
- Gross necropsy consisted of examinajtion of gross abnormalities with particular attention to the external reproductive genitals.
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 3 below.
Reproductive indices:
Mating index, fertility index, gestation index, parturation index, pregnancy index, sex ratio, live birth index, post implantation loss (%).
Offspring viability indices:
Pup survival index on lactation day 4/7/13, sex ratio on lactation day 4/7/13, post natal loss (%), AGD ratio.



Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs of toxicity noted in any of the tested dose group animals of both sexes throughout the experimental period.
Dermal irritation (if dermal study):
not examined
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, non-treatment-related
Description (incidence and severity):
There were no test item related changes noted in mean body weight and percent change in mean body weight gain with respect to day 1 in all the tested dose groups (main and recovery) of both sexes throughout the experimental period when compared with vehicle control group. Also, there were no test item related changes noted in mean gestational body weight and percent change in mean gestational body weight gain in all the tested dose groups when compared with the vehicle control group. Finally, there were no changes in mean lactation body weight and percent change in mean lactation body weight gain in all the tested dose groups when compared with the vehicle control group.

The noted statistically significant increase in mean percent change in body weight on day 28 with respect to day 1 in group G3 males is considered as incidental and unrelated to treatment as there were no changes noted in mean body weight and mean feed consumption during this period.

The noted statistically significant increase in mean percent change in gestation body weight gain during day 7 to 14 in groups G2, G3 and G4 is considered as incidental and toxicologically not significant.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were no changes in mean feed consumption (including during gestation period) in all the tested dose groups (main and recovery) of both sexes when compared with the vehicle control group. Also, there were no test item related changes in mean feed consumption during lactation period in all the tested dose groups when compared with the vehicle control group.

The noted statistically significant increase in mean lactation feed consumption during day 7 to 13 in group G4 is considered as incidental and toxicologically not significant.
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 or vehicle control groups (main and recovery) of both sexes.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related changes noted in the obtained mean haematological values in all the tested dose groups (main and recovery) of both sexes when compared with the vehicle control group.
The noted statistically significant decrease in mean prothrombin time in group G4 males and decrease in mean corpuscular hemoglobin concentration in group G2 females is considered as incidental and unrelated to treatment, as these changes did not occur in a dose dependant manner and also the obtained mean values are within laboratory historical control range of same species and strain.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related changes in mean clinical chemistry values in all the tested dose groups (main and recovery) of both sexes when compared with the vehicle control groups.
The noted statistically significant increase in glucose and triglycerides levels in group G3 males, calcium levels in groups G3 and G4 males, Albumin/Globulin Ratio in groups G2 and G4 females; decrease in creatinine levels in group G4 males, total protein levels in groups G2, G3 and G4 females, albumin levels in group G3 females, globulin levels in groups G2 and G4 females are considered as incidental and unrelated to treatment, as these changes did not occur in a dose dependant manner and also the obtained mean values are within laboratory historical control range of same species and strain.
Endocrine findings:
no effects observed
Description (incidence and severity):
There were no changes noted in mean serum T4 levels in any of the tested dose groups (main group males) when compared with the vehicle control group.

The assessment of serum T4 levels was performed for adult males and
PND 13 pups. Further assessment of T4 levels in serum samples from the dams and PND 4 pups was not assessed as there were no treatment-related effects observed in the T4 levels of adult males or PND 13 pups.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no changes in mean urinalysis values in all the tested dose groups (main group males and recovery group males and females) when compared with the vehicle control group.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
The neurological/functional observations such as, home cage, handling, open-field, sensory, physiological observations did not reveal any changes in any of the animals of both sexes from all the tested dose groups performed towards end of the dosing period for main groups and towards end of recovery period for recovery groups.
There were no changes in mean fore/hind limb grip strengths, mean motor activity assessments and mean hind limb foot splay in all tested dose groups of both sexes when compared with vehicle control groups during conduct of neurological / functional examinations.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic findings in the study.
Few microscopic findings observed in this study such as ultimobranchial cyst in thyroid glands, epithelial cyst in thymus 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:
no effects observed
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related irregularities observed in oestrus cyclicity in any of the tested dose group animals during pre-mating and mating treatment periods. The mean length of oestrus cycle per female during pre-mating and mating treatment period was unaffected by the test item administration in any of the tested dose groups and the mean length was comparable with the vehicle control group.

The noted statistically significant decrease in mean oestrus cycle length in group G3 is considered as incidental and toxicologically not significant as all the females were noted with normal oestrus cycle length (4 to 5 days).
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No effects were detected during examination of spermatogenesis stages in the male gonads and histopathology of interstitial testicular cell structure.
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no changes observed in birth parameters such as, total litter size, number of live pups born, sex ratio (m/f) and live birth index in all the tested dose groups when compared with the vehicle control group.

Male Fertility Index (%): A total of 10 (out of 12), 11 (out of 12), 10 (out of 12) and 10 (out of 12) mated males were confirmed as fertile by impregnating a female or siring a litter with a fertility index of 83.3%, 91.7%, 83.3% and 83.3% from the tested dose groups G1, G2, G3 and G4 respectively. There were no statistically significant differences noted for male fertility index in any of the tested dose groups when compared with the vehicle control group.

Female Fertility Index (%): A total of 10 (out of 12), 11 (out of 12), 10 (out of 12) and 10 (out of 12) mated females were confirmed with presence of implantations / presence of live or dead pups / evidence of parturition with a fertility index of 83.3%, 91.7%, 83.3% and 83.3% from groups G1, G2, G3 and G4 respectively. There were no statistically significant differences noted for female fertility index in any of the tested dose groups when compared with the vehicle control group.

Gestation Index (%): A total of 10 (out of 10), 11 (out of 11), 10 (out of 10) and 10 (out of 10) pregnant females were confirmed with live born pups with a gestation index of 100.0% for all the tested dose groups and vehicle control group.

Post-Implantation Loss (%): A mean number of 0.20, 0.09, 2.20 and 0.70 post-implantation losses with a percentage of 2.00, 0.45, 11.77 and 4.74 were noted from groups G1, G2, G3 and G4 respectively. There were no statistically significant changes as for mean number and percentage of occurred post-implantation losses in any of the tested dose groups when compared with the vehicle control group. The noted increase in mean number of post-implantation loss and its percentage in group G3 is due to one dam (Rf9936) which was noted with 13 implantation loss with a percentage of 61.9. However, this occurrence is considered as incidental finding and unrelated to treatment as there were neither such losses noted in other animals of same group nor in next dose level i.e. group G4. Also, the noted difference is not statistically significant when compared with vehicle control group (obtained p value = 0.065).
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects found up to the highest dose tested (1000 mg/kg bw/day)
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
There were no external abnormalities or behavioural changes in any of the pups during daily observation from all the tested dose groups and vehicle control group during postnatal period. All pups had normal behaviour during daily observations.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
A mean number of 0.00, 0.09, 0.00 and 0.00 post-natal losses with a percentage of 0.00, 0.48, 0.00 and 0.00 were noted from groups G1, G2, G3 and G4 respectively.
There were no statistically significant changes as for mean number and percentage of occurred post-natal losses in any of the tested dose groups when compared with the vehicle control group.


The pup survival index per litter during lactation period was unaffected by the test item in all the tested dose groups when compared with the vehicle control group.





Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related changes noted in mean pup weight per litter, both male and female, in all the tested dose groups when compared with the vehicle control group, recorded on postnatal day (PND) 1, 4, 7 and 13.

The noted statistically significant decrease in mean male pup weight on postnatal day13 in G4 group is considered as incidental and unrelated to treatment as the obtained mean values were within the historical control data range.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
There were no changes in mean pup (both male and female) anogenital distance measurement (mm) and its ratio per litter in all the tested dose groups when compared with the vehicle control group, recorded on postnatal day 4.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
There were no occurrences or evidences of retention of nipples in any of the male pups examined on the postnatal day 13 from all the tested dose groups and vehicle control group litters.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross pathological changes observed in any of the pups found dead at birth, cannibalized or terminally sacrificed from all the tested dose groups and vehicle control group litters during conduct of necropsy.
Histopathological findings:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related changes noted in mean serum T4 hormone levels of postnatal day 13 pups in any of the tested dose group litters when compared with vehicle control group litters.
The noted statistically significant decrease in mean T4 levels in group G2 is considered as incidental and unrelated to treatment as the obtained mean values were within the historical control data range.

The assessment of serum T4 levels was performed for adult males and
PND 13 pups. Further assessment of T4 levels in serum samples from the dams and PND 4 pups was not assessed as there were no treatment-related effects observed in the T4 levels of adult males or PND 13 pups.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects found up to the highest dose tested (1000 mg/kg bw/day)
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

TABLE 4. SUMMARY OF THE STUDY






























































































































































































































 



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




Parameters ↓



G1 & 0



G2 & 100



G3 & 300



G4 & 1000



Reproductive Indices



No. of Pairs started



12



12



12



12



Reproductive Performance / Indices (males)



Total No. of males cohabited



12



12



12



12



No. of males with confirmed mating



12



12



12



12



No. of males impregnating/siring a female



10



11



10



10



Male Mating Index (%)



100.0



100.0



100.0



100.0



Male Fertility Index (%)



83.3



91.7



83.3



83.3



 



Reproductive Performance and Indices (females)



Mean Oestrus cycle length (days)



Mean



4.68



4.56



4.50



4.63



±SD



0.16



0.17



0.17



0.18



No. of females with regular oestrus cycle



12



12



12



12



Total No. of females cohabited



12



12



12



12



No. of sperm-positive females



12



12



12



12



No. of females with evidence of pregnancy



10



11



10



10



No. of females with live born pups



10



11



10



10



No. of females with conceiving days 1 to 5



8



9



10



12



No. of females with conceiving days ≥6



4



3



2



-



Pre-coital Interval (Days)



Mean



5.00



3.92



2.92



1.75



±SD



4.18



3.75



2.91



0.97



Female Mating Index (%)



100.0



100.0



100.0



100.0



Female Fertility Index (%)



83.3



91.7



83.3



83.3



No. of females with Pregnancy≤21 days



1



1



1



1



No. of females with Pregnancy = 22 days



6



9



5



6



No. of females with Pregnancy≥23 days



3



1



4



3



Gestation Length (Days)



Mean



22.20



22.00



22.30



22.20



±SD



0.63



0.45



0.67



0.63



Gestation Index (%)



100.0



100.0



100.0



100.0



Female Fecundity or Pregnancy Index (%)



83.3



91.7



83.3



83.3



Parturition index (%)



100.0



100.0



100.0



100.0




















































































































































































































































 



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




Parameters ↓



G1 & 0



G2 & 100



G3 & 300



G4 & 1000



Delivery and Litter Observations



No. of Dams with live young born



10



11



10



10



No. of Dams with live young at PND 4



10



11



10



10



No. of Dams with live young at PND 7



10



11



10



10



No. of Dams with live young at PND 13



10



11



10



10



No. of Implants/dam



Mean



12.10



12.18



14.70



14.20



±SD



3.00



3.49



4.11



2.70



No. of Live pups/dam at birth



Mean



11.90



12.09



12.50



13.50



±SD



3.21



3.27



3.44



2.72



No. of Live pups/dam at PND 4



Mean



11.90



12.00



12.50



13.50



±SD



3.21



3.07



3.44



2.72



No. of Live pups/dam at PND 7



Mean



10.80



10.91



11.20



11.90



±SD



2.35



2.21



2.70



2.28



No. of Live pups/dam at PND 13



Mean



10.80



10.91



11.20



11.90



±SD



2.35



2.21



2.70



2.28



Live Birth Index/dam (%)



Mean



100.00



99.55



99.41



99.33



±SD



0.00



1.51



1.86



2.11



Pup Survival Index/dam on


PND 4 (%)



Mean



100.00



99.52



100.00



100.00



±SD



0.00



1.59



0.00



0.00



Pup Survival Index/dam on


PND 7 (%)



Mean



 



100.00



100.00



100.00



±SD



0.00



0.00



0.00



0.00



Pup Survival Index/dam on


PND 13 (%)



Mean



100.00



100.00



100.00



100.00



±SD



0.00



0.00



0.00



0.00



Sex ratio (m/f)/dam At Birth



Mean



1.43



1.30



1.14



1.21



±SD



1.07



0.50



0.58



0.54



Sex ratio (m/f)/dam at PND 4



Mean



1.43



1.29



1.14



1.21



±SD



1.07



0.51



0.58



0.54



Sex ratio (m/f)/dam at PND 7



Mean



1.85



1.79



1.42



1.80



±SD



1.27



1.16



0.74



1.24



Sex ratio (m/f)/dam at PND 13



Mean



1.85



1.79



1.42



1.80



±SD



1.27



1.16



0.74



1.24



Note: For calculation of Pup survival index (%) on PND 7 and 13, the no. of pups sacrificed for blood collection on PND 4 were excluded.


 

























































































































































































































 



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



Parameters ↓



G1 & 0



G2 & 100



G3 & 300



G4 & 1000



Delivery and Litter Observations



Mean Male


Pup weight (g)/dam



At birth



Mean



6.78



6.79



6.77



6.75



±SD



0.12



0.17



0.12



0.10



PND 4



Mean



11.12



11.33



11.18



11.28



±SD



0.50



0.40



0.40



0.42



PND 7



Mean



14.88



15.10



14.71



14.67



±SD



0.34



0.42



0.47



0.30



PND 13



Mean



28.69



28.48



28.77



27.81



±SD



0.50



0.77



0.93



0.93



Mean Female


Pup weight (g)/dam



At Birth



Mean



6.33



6.27



6.23



6.22



±SD



0.22



0.13



0.15



0.12



PND 4



Mean



10.31



10.45



10.35



10.38



±SD



0.36



0.27



0.20



0.17



PND 7



Mean



13.80



14.09



13.67



13.78



±SD



0.29



0.40



0.36



0.29



PND 13



Mean



26.90



27.52



26.93



26.24



±SD



1.08



0.69



1.12



1.62



Mean Male Pup AGD Measurement/dam (mm) on PND 4



Mean



5.00



4.97



5.02



4.97



±SD



0.15



0.08



0.11



0.09



Mean Male Pup AGD ratio/dam on PND 4



Mean



2.24



2.21



2.25



2.22



±SD



0.06



0.04



0.03



0.02



Mean Female Pup AGD Measurement/dam (mm) on PND 4



Mean



2.67



2.67



2.66



2.66



±SD



0.09



0.07



0.08



0.06



Mean Female Pup AGD ratio/dam on PND 4



Mean



1.23



1.22



1.22



1.22



±SD



0.03



0.03



0.04



0.03



Male Pup Nipple Retention/dam on PND 13



Mean



0.00



0.00



0.00



0.00



±SD



0.00



0.00



0.00



0.00

























































































































































 



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




Parameters ↓



G1 & 0



G2 & 100



G3 & 300



G4 & 1000



ABNORMAL PUPS



No. of Dams with 0 abnormal pups



10



11



10



10



LOSS OF OFFSPRING



 



No. of Females with 0 post-implantation loss



9



10



5



6



No. of Females with 1 post-implantation loss



-



1



2



3



No. of Females with 2 post-implantation losses



1



-



-



-



No. of Females with 3 post-implantation losses



-



-



1



-



No. of Females with 4 post-implantation losses



-



-



1



1



No. of Females with 13 post-implantation losses



-



-



1



-



Post-implantation Loss (No.)



Mean



0.20



0.09



2.20



0.70



±SD



0.63



0.30



4.05



1.25



Post-implantation Loss (%)



Mean



2.00



0.45



11.77



4.74



±SD



6.32



1.51



19.53



8.42



Post-natal loss (live births minus alive at PND 13)



No. of Females with 0



10



10



10



10



No. of Females with 1



-



1



-



-



Postnatal loss (No.)



Mean



0.00



0.09



0.00



0.00



±SD



0.00



0.30



0.00



0.00



Postnatal Loss (%)



Mean



0.00



0.48



0.00



0.00



±SD



0.00



1.59



0.00



0.00



Note:  The noted increase in mean number of post-implantation loss and its percentage in group G3 and G4 are not statistically significant when compared with vehicle control group and the obtained p value = 0.065.


 


TABLE 5. SUMMARY OF PERCENT CHANGE IN BODY WEIGHT GAIN (%) WITH RESPECT TO DAY 1 RECORD









































































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Percent Change in Body Weight Gain (%) during Day



1 to 8



1 to 14



1 to 21



1 to 28



G1, M & 0



Mean



4.83



10.23



17.65



23.15



±SD



1.58



1.30



2.47



3.60



n



12



12



12



12



G2, M & 100



Mean



4.56



10.84



18.21



24.05



±SD



1.45



1.83



3.22



4.06



n



12



12



12



12



G3, M & 300



Mean



4.60



11.20



19.29



26.56*



±SD



1.43



2.09



2.66



3.25



n



12



12



12



12



G4, M & 1000



Mean



4.64



11.54



19.29



24.95



±SD



1.47



2.06



1.98



2.43



n



12



12



12



12





























































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Percent Change in Body Weight Gain (%) during Day



1 to 8



1 to 14



1 to 21



G1, F & 0



Mean



3.06



6.04



12.82



±SD



1.72



2.24



3.68



n



12



12



4



G2, F & 100



Mean



3.47



5.88



12.55



±SD



2.47



2.34



3.87



n



12



12



3



G3, F & 300



Mean



4.27



7.32



12.86



±SD



1.61



2.76



-



n



12



12



1



G4, F & 1000



Mean



3.40



6.19



-



±SD



1.54



2.24



-



n



12



12



-



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


#: The data obtained from females in cohabitation only considered for mean calculations. The data of day 1 to 21 body weight was not subjected to statistical analysis due to uneven number of variables.


 
































































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Percent Change in Body Weight (%) during Day



1 to 8



1 to 14



1 to 21



1 to 28



1 to 35



1 to 42



1 to 49



1 to 56



1 to 63



G1R, M & 0



Mean



5.04



12.17



20.93



27.05



33.03



36.96



41.00



44.79



48.34



±SD



1.03



3.12



4.37



4.56



4.95



5.96



4.43



7.11



7.47



n



5



5



5



5



5



5



5



5



5



G4R, M & 1000



Mean



5.23



11.69



19.10



25.07



31.04



35.97



40.39



43.87



47.24



±SD



1.65



2.51



1.60



4.38



7.37



7.66



7.59



9.05



8.16



n



5



5



5



5



5



5



5



5



5

































































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Percent Change in Body Weight (%) during Day



1 to 8



1 to 14



1 to 21



1 to 28



1 to 35



1 to 42



1 to 48



1 to 56



1 to 63



G1R, F & 0



Mean



3.85



6.55



10.41



15.72



19.35



21.92



23.74



27.66



31.64



±SD



0.93



1.26



1.79



3.11



3.10



2.91



2.94



3.19



2.67



n



5



5



5



5



5



5



5



5



5



G4R, F & 1000



Mean



4.26



7.11



11.13



16.52



20.28



22.80



24.03



27.39



31.03



±SD



1.05



0.83



1.02



0.72



0.97



1.35



1.28



2.63



4.03



n



5



5



5



5



5



5



5



5



5



 


TABLE 6. SUMMARY RECORD OF PERCENT CHANGE IN BODY WEIGHT GAIN (%) DURING GESTATION PERIOD




























































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

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



0 to 7



7 to 14



14 to 20



G1, F & 0



Mean



5.88



11.32



18.39



±SD



1.09



1.89



5.88



n



10



10



10



G2, F & 100



Mean



6.52



13.77*



20.14



±SD



1.11



1.07



7.06



n



11



11



11



G3, F & 300



Mean



6.39



13.83*



21.28



±SD



0.73



2.48



7.30



n



10



10



10



G4, F & 1000



Mean



6.42



14.72*



23.34



±SD



1.35



1.66



5.74



n



10



10



10



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


Note: Excluded the data of non-pregnant females for mean calculations and statistical analysis, but presented in individual animal data.


 


TABLE 7. SUMMARY RECORD OF PERCENT CHANGE IN BODY WEIGHT GAIN (%) DURING LACTATION PERIOD




























































































Group, Sex


& Dose (mg/kg body weight/day)


 

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



1 to 4



4 to 7



7 to 13



G1, F & 0



Mean



2.05



2.76



5.25



±SD



0.98



1.42



2.96



n



10



10



10



G2, F & 100



Mean



1.30



1.98



5.86



±SD



0.96



0.56



2.51



n



11



11



11



G3, F & 300



Mean



1.30



1.87



4.93



±SD



1.14



0.52



2.00



n



10



10



10



G4, F & 1000



Mean



1.14



2.03



5.60



±SD



0.92



0.59



2.14



n



10



10



10



 


TABLE 8. SUMMARY OF HAEMATOLOGY RECORD





























































































































































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Total


Leucocyte


Count



Total


Erythrocyte


Count



Haemoglobin



Haematocrit



Mean


Corpuscular


Volume



Mean


Corpuscular


Haemoglobin



Mean


Corpuscular


Haemoglobin


Concentration



Platelet


Count



Mean


Platelet


Volume



(WBC)



(RBC)



(HGB)



(HCT)



(MCV)



(MCH)



(MCHC)



(PLT)



(MPV)



(103 cells/µL)



(106 cells/µL)



(g/dL)



(%)



(fL)



(pg)



(g/dL)



(103 cells/µL)



(fL)



G1, M & 0



Mean



7.93



8.93



16.58



47.88



53.76



18.62



34.62



1048.60



6.10



±SD



2.39



0.50



0.74



1.64



2.42



1.05



0.50



244.74



0.22



n



5



5



5



5



5



5



5



5



5



G2, M & 100



Mean



7.88



8.48



16.66



44.90



52.96



19.80



37.36



991.60



6.02



±SD



0.84



0.86



0.59



4.00



1.28



1.96



3.13



161.48



0.18



n



5



5



5



5



5



5



5



5



5



G3, M & 300



Mean



8.18



8.62



16.14



46.38



53.84



18.76



34.88



1104.00



5.90



±SD



1.96



0.50



0.50



2.08



1.59



0.72



1.53



232.30



0.19



n



5



5



5



5



5



5



5



5



5



G4, M & 1000



Mean



8.36



8.69



16.06



47.16



54.44



18.52



34.08



966.00



6.28



±SD



2.02



0.54



0.42



1.32



3.09



0.87



0.55



158.58



0.42



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



2.56



22.14



73.32



2.24



1.28



0.26



±SD



0.59



6.94



7.78



1.41



0.63



0.05



n



5



5



5



5



5



5



G2, M & 100



Mean



2.18



22.82



73.00



1.92



1.30



0.22



±SD



0.33



7.12



7.31



0.44



0.29



0.13



n



5



5



5



5



5



5



G3, M & 300



Mean



2.28



24.52



71.46



1.42



1.78



0.12



±SD



0.71



4.21



4.83



0.51



0.70



0.04



n



5



5



5



5



5



5



G4, M & 1000



Mean



2.33



25.68



68.98



2.78



1.24



0.36



±SD



0.53



4.92



5.76



1.01



0.80



0.26



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)



(109 cells/L)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(Seconds)



(Seconds)



G1, M & 0



Mean



228.20



1.73



5.86



0.16



0.11



0.02



16.78



25.48



±SD



52.11



0.68



2.01



0.08



0.07



0.01



2.45



2.76



n



5



5



5



5



5



5



5



5



G2, M & 100



Mean



184.94



1.80



5.75



0.15



0.10



0.02



15.92



24.00



±SD



31.52



0.58



0.82



0.05



0.03



0.01



2.26



2.42



n



5



5



5



5



5



5



5



5



G3, M & 300



Mean



195.98



2.02



5.82



0.12



0.15



0.01



13.94



22.62



±SD



56.46



0.65



1.41



0.05



0.08



0.00



1.24



4.71



n



5



5



5



5



5



5



5



5



G4, M & 1000



Mean



201.06



2.13



5.78



0.22



0.11



0.03



12.36*



21.88



±SD



40.02



0.63



1.58



0.05



0.07



0.02



0.76



1.90



n



5



5



5



5



5



5



5



5






























































































































































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Total


Leucocyte


Count



Total


Erythrocyte


Count



Haemoglobin



Haematocrit



Mean


Corpuscular Volume



Mean


Corpuscular Haemoglobin



Mean


Corpuscular Haemoglobin Concentration



Platelet


Count



Mean


Platelet


Volume



(WBC)



(RBC)



(HGB)



(HCT)



(MCV)



(MCH)



(MCHC)



(PLT)



(MPV)



(103 cells/µL)



(106 cells/µL)



(g/dL)



(%)



(fL)



(pg)



(g/dL)



(103 cells/µL)



(fL)



G1, F & 0



Mean



8.49



7.55



15.50



42.66



56.44



20.66



36.62



931.80



6.02



±SD



1.85



0.66



0.89



4.81



2.20



1.70



3.35



123.32



0.18



n



5



5



5



5



5



5



5



5



5



G2, F & 100



Mean



9.97



7.78



15.84



47.22



60.78



20.36



33.56*



858.40



6.20



±SD



3.27



0.32



0.55



2.57



3.72



0.61



1.05



294.99



0.24



n



5



5



5



5



5



5



5



5



5



G3, F & 300



Mean



9.45



7.52



15.52



45.44



60.68



20.70



34.14



1024.80



6.24



±SD



3.18



0.54



0.61



1.84



5.12



1.29



0.74



132.05



0.17



n



5



5



5



5



5



5



5



5



5



G4, F & 1000



Mean



8.45



7.75



15.72



45.94



59.30



20.30



34.28



1088.00



6.20



±SD



0.81



0.25



0.65



1.94



1.29



0.29



0.49



159.47



0.12



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



2.59



43.02



50.64



3.72



1.66



0.16



±SD



0.86



9.12



8.23



1.89



1.89



0.05



n



5



5



5



5



5



5



G2, F & 100



Mean



3.04



31.08



63.32



2.78



1.58



0.24



±SD



1.20



8.90



10.07



1.08



1.29



0.21



n



5



5



5



5



5



5



G3, F & 300



Mean



2.41



49.14



45.08



3.58



1.02



0.18



±SD



0.68



17.03



16.07



1.54



0.36



0.13



n



5



5



5



5



5



5



G4, F & 1000



Mean



2.61



36.46



56.30



4.50



1.28



0.16



±SD



0.44



4.80



5.13



0.57



0.77



0.09



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)



(109 cells/L)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(Seconds)



(Seconds)



G1, F & 0



Mean



194.26



3.69



4.25



0.33



0.13



0.01



19.66



26.14



±SD



63.78



1.22



0.96



0.22



0.14



0.01



2.45



4.20



n



5



5



5



5



5



5



5



5



G2, F & 100 



Mean



235.18



3.27



6.13



0.30



0.14



0.02



18.46



24.44



±SD



90.14



2.12



1.26



0.21



0.09



0.02



3.63



5.07



n



5



5



5



5



5



5



5



5



G3, F & 300



Mean



178.88



4.66



4.23



0.36



0.09



0.02



18.06



24.34



±SD



40.18



1.93



1.86



0.23



0.04



0.02



1.80



5.69



n



5



5



5



5



5



5



5



5



G4, F & 1000



Mean



202.20



3.08



4.76



0.38



0.11



0.01



16.80



20.76



±SD



34.70



0.45



0.64



0.04



0.07



0.00



1.71



4.92



n



5



5



5



5



5



5



5



5




















































































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Total


Leucocyte


Count



Total


Erythrocyte


Count



Haemoglobin



Haematocrit



Mean


Corpuscular Volume



Mean


Corpuscular Haemoglobin



Mean


Corpuscular Haemoglobin Concentration



Platelet


Count



Mean


Platelet


Volume



(WBC)



(RBC)



(HGB)



(HCT)



(MCV)



(MCH)



(MCHC)



(PLT)



(MPV)



(103 cells/µL)



(106 cells/µL)



(g/dL)



(%)



(fL)



(pg)



(g/dL)



(103 cells/µL)



(fL)



G1R, M & 0



Mean



8.46



9.27



16.84



49.00



52.88



18.20



34.42



1110.00



5.94



±SD



1.04



0.51



0.62



2.00



1.29



0.75



0.61



119.77



0.11



n



5



5



5



5



5



5



5



5



5



G4R, M & 1000



Mean



8.44



8.82



16.36



46.42



52.70



18.66



35.40



1194.60



5.90



±SD



1.92



1.00



0.69



4.77



2.09



1.59



2.57



167.85



0.12



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)



(%)



(%)



(%)



(%)



(%)



(%)



G1R, M & 0



Mean



2.22



24.26



71.26



2.08



1.08



0.30



±SD



0.44



9.94



8.79



0.99



0.58



0.07



n



5



5



5



5



5



5



G4R, M & 1000



Mean



2.03



22.14



73.32



1.82



2.02



0.22



±SD



0.52



5.17



6.07



0.71



1.18



0.08



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)



(109 cells/L)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(Seconds)



(Seconds)



G1R, M & 0



Mean



205.38



2.13



5.96



0.17



0.09



0.02



17.50



29.46



±SD



41.13



1.11



0.33



0.08



0.05



0.01



3.08



7.07



n



5



5



5



5



5



5



5



5



G4R, M & 1000



Mean



175.70



1.85



6.21



0.15



0.17



0.02



19.10



22.88



±SD



35.88



0.56



1.56



0.03



0.12



0.00



1.02



3.35



n



5



5



5



5



5



5



5



5




















































































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Total


Leucocyte


Count



Total


Erythrocyte


Count



Haemoglobin



Haematocrit



Mean


Corpuscular Volume



Mean


Corpuscular Haemoglobin



Mean


Corpuscular Haemoglobin Concentration



Platelet


Count



Mean


Platelet


Volume



(WBC)



(RBC)



(HGB)



(HCT)



(MCV)



(MCH)



(MCHC)



(PLT)



(MPV)



(103 cells/µL)



(106 cells/µL)



(g/dL)



(%)



(fL)



(pg)



(g/dL)



(103 cells/µL)



(fL)



G1R, F & 0



Mean



4.37



8.30



15.64



45.06



54.30



18.84



34.70



934.00



6.46



±SD



0.54



0.31



0.34



1.28



1.18



0.55



0.44



293.12



0.19



n



5



5



5



5



5



5



5



5



5



G4R, F & 1000



Mean



5.44



8.12



15.72



45.00



55.44



19.34



34.96



904.20



6.32



±SD



1.71



0.28



0.72



2.26



1.47



0.59



0.58



224.52



0.47



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)



(%)



(%)



(%)



(%)



(%)



(%)



G1R, F & 0



Mean



1.66



26.40



67.90



2.52



2.16



0.26



±SD



0.24



7.17



6.71



0.79



0.79



0.11



n



5



5



5



5



5



5



G4R, F & 1000



Mean



2.26



21.90



72.14



3.00



2.14



0.20



±SD



0.63



6.50



7.20



0.76



0.67



0.14



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)



(109 cells/L)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(Seconds)



(Seconds)



G1R, F & 0



Mean



137.10



1.15



2.98



0.11



0.09



0.01



20.08



27.18



±SD



17.41



0.30



0.53



0.04



0.03



0.00



2.24



7.03



n



5



5



5



5



5



5



5



5



G4R, F & 1000



Mean



184.06



1.14



3.99



0.16



0.11



0.01



18.06



22.48



±SD



55.31



0.26



1.53



0.05



0.03



0.01



1.64



1.95



n



5



5



5



5



5



5



5



5



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


 


TABLE 9. 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



102.60



32.56



0.50



34.40



18.00



6.68



3.49



±SD



11.33



2.27



0.02



6.19



5.39



0.19



0.06



n



5



5



5



5



5



5



5



G2, M & 100



Mean



101.40



27.56



0.49



38.60



28.60



6.88



3.58



±SD



2.19



3.58



0.03



8.53



13.01



0.22



0.13



n



5



5



5



5



5



5



5



G3, M & 300



Mean



123.60*



30.98



0.49



50.00



42.20*



6.86



3.53



±SD



13.63



5.64



0.03



20.19



18.65



0.36



0.11



n



5



5



5



5



5



5



5



G4, M & 1000



Mean



97.00



30.94



0.46*



37.80



26.20



6.56



3.42



±SD



7.97



9.12



0.03



17.77



7.46



0.11



0.10



n



5



5



5



5



5



5



5


































































































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Alanine
aminotransferase



Aspartate
aminotransferase



Alkaline
phosphatase



Calcium



Phosphorous



(ALT)



(AST)



(ALP)



(CAL)



(PHO)



(U/L)



(U/L)



(U/L)



(mg/dL)



(mg/dL)



G1, M & 0



Mean



37.80



86.00



136.40



9.16



5.88



±SD



9.04



14.78



25.93



0.23



0.31



n



5



5



5



5



5



G2, M & 100



Mean



45.20



92.40



132.60



9.42



5.84



±SD



19.11



32.00



37.96



0.16



0.36



n



5



5



5



5



5



G3, M & 300



Mean



41.40



86.00



142.40



9.64*



6.04



±SD



4.39



9.14



16.77



0.25



0.43



n



5



5



5



5



5



G4, M & 1000



Mean



46.40



93.20



155.60



9.72*



6.26



±SD



10.64



30.05



48.64



0.13



0.59



n



5



5



5



5



5

















































































































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Globulin



Albumin/


Globulin ratio



Blood Urea Nitrogen



Sodium



Potassium



Chloride



(GLO)



(A/G Ratio)



(BUN)



(Na)



(K)



(CLO)



(g/dL)



-



(mg/dL)



(mmol/L)



(mmol/L)



(mmol/L)



G1, M & 0



Mean



3.19



1.10



15.19



143.32



3.83



108.18



±SD



0.16



0.04



1.06



0.36



0.13



0.56



n



5



5



5



5



5



5



G2, M & 100



Mean



3.30



1.09



12.86



143.28



4.06



107.82



±SD



0.16



0.06



1.67



1.00



0.14



1.82



n



5



5



5



5



5



5



G3, M & 300



Mean



3.33



1.07



14.46



143.90



3.96



107.84



±SD



0.28



0.08



2.63



0.56



0.34



1.96



n



5



5



5



5



5



5



G4, M & 1000



Mean



3.14



1.09



14.44



142.58



3.80



107.64



±SD



0.07



0.04



4.26



0.97



0.21



1.19



n



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



102.20



48.60



0.55



65.80



172.80



7.48



3.69



±SD



21.92



14.79



0.07



19.04



182.83



0.46



0.25



n



5



5



5



5



5



5



5



G2, F & 100



Mean



102.20



42.64



0.54



73.80



73.80



6.36*



3.42



±SD



16.08



11.18



0.09



27.31



72.48



0.29



0.11



n



5



5



5



5



5



5



5



G3, F & 300



Mean



103.20



62.72



0.65



77.00



112.40



6.76*



3.20*



±SD



24.78



43.25



0.22



29.26



80.25



0.15



0.21



n



5



5



5



5



5



5



5



G4, F & 1000



Mean



102.40



65.28



0.57



79.80



122.00



6.48*



3.51



±SD



6.58



9.46



0.03



6.14



38.50



0.33



0.18



n



5



5



5



5



5



5



5


































































































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Alanine
aminotransferase



Aspartate
aminotransferase



Alkaline
phosphatase



Calcium



Phosphorous



(ALT)



(AST)



(ALP)



(CAL)



(PHO)



(U/L)



(U/L)



(U/L)



(mg/dL)



(mg/dL)



G1, F & 0



Mean



130.20



130.80



474.80



10.54



6.96



±SD



93.03



76.17



460.49



1.57



1.92



n



5



5



5



5



5



G2, F & 100



Mean



66.00



106.40



128.20



10.14



7.10



±SD



18.48



34.16



58.02



0.67



3.67



n



5



5



5



5



5



G3, F & 300



Mean



134.20



185.60



239.40



9.98



6.40



±SD



81.52



120.65



117.85



0.82



1.50



n



5



5



5



5



5



G4, F & 1000



Mean



83.60



98.00



210.20



10.16



7.04



±SD



18.70



11.77



67.98



1.02



1.13



n



5



5



5



5



5

















































































































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Globulin



Albumin/Globulin Ratio



Blood Urea Nitrogen



Sodium



Potassium



Chloride



(GLO)



(A/G Ratio)



(BUN)



(Na)



(K)



(CLO)



(g/dL)



-



(mg/dL)



(mmol/L)



(mmol/L)



(mmol/L)



G1, F & 0



Mean



3.79



0.98



22.68



140.60



3.95



105.40



±SD



0.38



0.12



6.90



3.12



0.32



4.55



n



5



5



5



5



5



5



G2, F & 100



Mean



2.94*



1.17*



19.90



143.44



3.89



107.56



±SD



0.25



0.11



5.22



1.74



0.73



2.80



n



5



5



5



5



5



5



G3, F & 300



Mean



3.56



0.90



29.27



139.50



4.24



105.30



±SD



0.16



0.09



20.19



3.19



1.33



4.42



n



5



5



5



5



5



5



G4, F & 1000



Mean



2.97*



1.19*



30.47



139.62



4.01



105.34



±SD



0.26



0.12



4.41



0.64



0.12



1.88



n



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)



G1R, M & 0



Mean



125.60



36.94



0.54



73.60



34.00



7.62



3.85



±SD



9.34



4.07



0.03



17.24



10.37



0.23



0.06



n



5



5



5



5



5



5



5



G4R, M & 1000



Mean



128.40



32.78



0.54



75.40



30.60



7.76



3.78



±SD



14.15



5.27



0.03



12.34



14.33



0.15



0.09



n



5



5



5



5



5



5



5
















































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Alanine


aminotransferase



Aspartate aminotransferase



Alkaline


phosphatase



Calcium



Phosphorous



(ALT)



(AST)



(ALP)



(CAL)



(PHO)



(U/L)



(U/L)



(U/L)



(mg/dL)



(mg/dL)



G1R, M & 0



Mean



38.80



80.80



106.00



10.28



5.56



±SD



4.09



8.70



22.03



0.26



0.47



n



5



5



5



5



5



G4R, M & 1000



Mean



42.40



77.60



127.00



10.22



5.48



±SD



5.41



13.07



41.26



0.19



0.64



n



5



5



5



5



5

























































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Globulin



Albumin/


Globulin ratio



Blood Urea Nitrogen



Sodium



Potassium



Chloride



(GLO)



(A/G Ratio)



(BUN)



(Na)



(K)



(CLO)



(g/dL)



-



(mg/dL)



(mmol/L)



(mmol/L)



(mmol/L)



G1R, M & 0



Mean



3.77



1.02



17.24



141.28



3.68



106.82



±SD



0.23



0.07



1.90



1.58



0.31



1.72



n



5



5



5



5



5



5



G4R, M & 1000



Mean



3.98



0.95



15.30



141.14



3.61



106.32



±SD



0.16



0.05



2.46



1.01



0.18



1.06



n



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)



G1R, F & 0



Mean



135.40



42.58



0.66



103.20



45.40



8.96



4.84



±SD



4.72



6.83



0.03



23.76



7.09



0.25



0.08



n



5



5



5



5



5



5



5



G4R, F & 1000



Mean



136.40



41.72



0.61



97.80



71.40



9.06



4.73



±SD



11.63



9.48



0.06



17.68



21.63



0.63



0.28



n



5



5



5



5



5



5



5
















































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Alanine


aminotransferase



Aspartate aminotransferase



Alkaline


phosphatase



Calcium



Phosphorous



(ALT)



(AST)



(ALP)



(CAL)



(PHO)



(U/L)



(U/L)



(U/L)



(mg/dL)



(mg/dL)



G1R, F & 0



Mean



60.20



117.40



36.00



11.18



4.00



±SD



39.10



67.06



9.35



0.32



0.60



n



5



5



5



5



5



G4R, F & 1000



Mean



59.60



103.80



50.60



11.14



4.22



±SD



38.57



50.97



11.57



0.31



0.50



n



5



5



5



5



5

























































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Globulin



Albumin/


Globulin ratio



Blood Urea Nitrogen



Sodium



Potassium



Chloride



(GLO)



(A/G Ratio)



(BUN)



(Na)



(K)



(CLO)



(g/dL)



-



(mg/dL)



(mmol/L)



(mmol/L)



(mmol/L)



G1R, F & 0



Mean



4.12



1.17



19.87



140.64



3.42



107.40



±SD



0.20



0.05



3.19



0.68



0.09



0.54



n



5



5



5



5



5



5



G4R, F & 1000



Mean



4.33



1.09



19.47



140.24



3.54



106.64



±SD



0.40



0.07



4.43



1.57



0.25



1.20



n



5



5



5



5



5



5



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


 


TABLE 10. SUMMARY OF VAGINAL SMEAR EXAMINATION RECORD FOR DETERMINATION OF OESTRUS CYCLICITY






























































































Group, Sex


& Dose
(mg/kg body weight/day)



Total No. of


Females Evaluated


 

Females with


Complete


Regular Cycles



Females with at least one Irregular


Oestrus Cycle



Average Length of


Oestrus Cycle (Days)



G1, F & 0



12



n



12



0



Mean



4.68



%



100.0



0.0



±SD



0.16



n



12



G2, F & 100



12



n



12



0



Mean



4.56



%



100.0



0.0



±SD



0.17



n



12



G3, F & 300



12



n



12



0



Mean



4.50*



%



100.0



0.0



±SD



0.17



n



12



G4, F & 1000



12



n



12



0



Mean



4.63



%



100.0



0.0



±SD



0.18



n



12



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


 


TABLE 11. SUMMARY RECORD OF REPRODUCTIVE PERFORMANCE










































Group, Sex


& Dose  
(mg/kg body weight/day)



No. of Males with Evidence of Mating
(No. of Males used for Mating)



Male Mating Index (%)


 

No. of Males Capable of Impregnating a Female
(No. of males used for Mating)



Male Fertility Index (%)



G1, M & 0



12 (12)



100.0



10 (12)



83.3



G2, M & 100



12 (12)



100.0



11 (12)



91.7



G3, M & 300



12 (12)



100.0



10 (12)



83.3



G4, M & 1000



12 (12)



100.0



10 (12)



83.3













































































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Cohabitation Record and Pre-coital Interval (Mean Time of Mating)



 



Gestational Length


(duration of pregnancy)



Pre-coital Interval (Days)


 

Conceiving Days


(1 to 5)



Conceiving Days


(More than 5 days)



Gestation Length (Days)



G1, F & 0



Mean



5.00



n



8



4



22.20



±SD



4.18



%



66.7



33.3



0.63



n



12



10



G2, F & 100



Mean



3.92



n



9



3



22.00



±SD



3.75



%



75.0



25.0



0.45



n



12



11



G3, F & 300



Mean



2.92



n



10



2



22.30



±SD



2.91



%



83.3



16.7



0.67



n



12



10



G4, F & 1000



Mean



1.75*



n



12



0



22.20



±SD



0.97



%



100.0



0.0



0.63



n



12



10















































Group, Sex


& Dose


(mg/kg body


weight/day)



Female Mating Index (%)


 

Female Fertility Index (%)



No. of Females with Evidence of Mating


(No. of Females used for Mating)



Female Mating Index (%)



No. of Females Confirmed as Fertile
(No. of Females used for Mating)



Female Fertility Index (%)



G1, F & 0



12 (12)



100.0



10 (12)



83.3



G2, F & 100



12 (12)



100.0



11 (12)



91.7



G3, F & 300



12 (12)



100.0



10 (12)



83.3



G4, F & 1000



12 (12)



100.0



10 (12)



83.3

























































Group, Sex


& Dose                                      (mg/kg body


weight/day)



Female Fecundity or Pregnancy Index (%)


 

Gestation Index (%)



No. of Pregnant Females



No. of Females


Confirmed with Mating



Female Fecundity or Pregnancy Index (%)



Females with Live Born


Pups at Parturition



No. of Females


with Evidence of Pregnancy



Gestation Index (%)



G1, F & 0



10



12



83.3



10



10



100.0



G2, F & 100



11



12



91.7



11



11



100.0



G3, F & 300



10



12



83.3



10



10



100.0



G4, F & 1000



10



12



83.3



10



10



100.0








































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



Parturition index (%)



No. of Females Littered



No. of Females with evidence of pregnancy



Parturition index (%)



G1, F & 0



10



10



100.0



G2, F & 100



11



11



100.0



G3, F & 300



10



10



100.0



G4, F & 1000



10



10



100.0


















































































































































Group, Sex


& Dose
(mg/kg body


weight/day)



 



Post-implantation Loss (%)



 



Post-natal Loss (%)



No. of


Implantations



No. of Viable


(Live)


Pups



Post-implantation Loss


(No.)



Post-implantation Loss


(%)



Total No. of Pups Found Dead/


Cannibalized during lactation period



Post-natal Loss


(%)



G1, F & 0



Mean



12.10



11.90



0.20



2.00



 



0.00



0.00



±SD



3.00



3.21



0.63



6.32



 



0.00



0.00



n



10



10



10



10



 



12



12



G2, F & 100



Mean



12.18



12.09



0.09



0.45



 



0.09



0.48



±SD



3.49



3.27



0.30



1.51



 



0.30



1.59



n



11



11



11



11



 



11



11



G3, F & 300



Mean



14.70



12.50



2.20



11.77



 



0.00



0.00



±SD



4.11



3.44



4.05



19.53



 



0.00



0.00



n



10



10



10



10



 



12



12



G4, F & 1000



Mean



14.20



13.50



0.70



4.74



 



0.00



0.00



±SD



2.70



2.72



1.25



8.42



 



0.00



0.00



n



10



10



10



10



 



12



12



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


 


TABLE 12. SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD


























































































































































Group, Sex


& Dose


(mg/kg body weight/day)


 

Adrenals



Thymus



Spleen



Testes



Epididymides



Heart



Kidneys



Brain



G1, M & 0



Mean



0.0881



0.4598



0.9731



3.5201



1.6321



1.6310



3.4141



2.4113



±SD



0.0129



0.0910



0.1241



0.2599



0.1830



0.1035



0.2100



0.1176



n



5



5



5



12



12



5



5



5



G2, M & 100



Mean



0.0797



0.4290



0.8600



3.6026



1.7102



1.6084



3.5132



2.2461



±SD



0.0054



0.0136



0.0677



0.1997



0.2026



0.1029



0.1866



0.1097



n



5



5



5



12



12



5



5



5



G3, M & 300



Mean



0.0768



0.4540



0.8366



3.3672



1.5821



1.5895



2.8478*



2.2322



±SD



0.0112



0.0917



0.1026



0.2023



0.1147



0.0982



0.0725



0.1104



n



5



5



5



12



12



5



5



5



G4, M & 1000



Mean



0.0717*



0.2907*



1.1998*



3.4860



1.5544



1.6353



3.2137



2.2388



±SD



0.0057



0.0333



0.1036



0.2722



0.1314



0.1278



0.3464



0.1312



n



5



5



5



12



12



5



5



5

































































































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Liver



Prostate



Seminal vesicles with coagulating glands



Lungs



Pituitary



Thyroid along


with parathyroid#



G1, M & 0



Mean



13.7479



1.8731



1.9272



2.4260



0.0195



0.0303



±SD



1.3993



0.5071



0.6436



0.3217



0.0012



0.0015



n



12



12



12



5



5



12



G2, M & 100



Mean



13.7213



1.7628



1.9226



2.5720



0.0180



0.0307



±SD



1.3789



0.3096



0.4410



0.0945



0.0016



0.0013



n



12



12



12



5



5



12



G3, M & 300



Mean



14.5890



1.7312



2.0113



2.5701



0.0182



0.0319*



±SD



1.7410



0.5569



0.5669



0.2502



0.0014



0.0015



n



12



12



12



5



5



12



G4, M & 1000



Mean



13.7006



1.7092



2.3425



2.6678



0.0187



0.0310



±SD



1.0406



0.4295



0.2628



0.0791



0.0010



0.0019



n



12



12



12



5



5



12

































































































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Adrenals



Thymus



Spleen



Ovaries



Uterus with Cervix



Heart



G1, F & 0



Mean



0.0549



0.2709



0.7702



0.1432



0.6125



1.2405



±SD



0.0114



0.0265



0.1546



0.0233



0.1144



0.0829



n



5



5



5



12



12



5



G2, F & 100



Mean



0.0656



0.2777



0.6426



0.1403



0.5969



1.1809



±SD



0.0141



0.0979



0.1603



0.0132



0.1126



0.0755



n



5



5



5



12



12



5



G3, F & 300



Mean



0.0667



0.1972



0.7004



0.1524



0.7271



1.3171



±SD



0.0039



0.0691



0.2512



0.0203



0.5884



0.1254



n



5



5



5



12



12



5



G4, F & 1000



Mean



0.0553



0.2097



0.6029



0.1481



0.6083



1.2362



±SD



0.0068



0.0104



0.1662



0.0163



0.2945



0.1750



n



5



5



5



12



12



5

































































































































Group, Sex


& Dose


(mg/kg body weight/day)


 

Kidneys



Brain



Liver



Lungs



Pituitary



Thyroid along


with parathyroid#



G1, F & 0



Mean



1.9762



2.0279



11.9915



1.7191



0.0170



0.0251



±SD



0.1366



0.1114



2.4375



0.2404



0.0020



0.0018



n



5



5



12



5



5



12



G2, F & 100



Mean



2.0651



2.0014



11.7354



1.9148



0.0177



0.0246



±SD



0.2024



0.0851



1.0645



0.0568



0.0010



0.0020



n



5



5



12



5



5



12



G3, F & 300



Mean



2.2289



2.0311



12.0375



2.0925*



0.0169



0.0241



±SD



0.2489



0.0980



0.7986



0.1334



0.0016



0.0019



n



5



5



12



5



5



12



G4, F & 1000



Mean



1.9525



1.9955



11.9606



2.0568*



0.0178



0.0248



±SD



0.1493



0.1300



0.9854



0.0900



0.0013



0.0016



n



5



5



12



5



5



12























































































Group, Sex


& Dose


(mg/kg body weight/day)


 

Adrenals



Thymus



Spleen



Testes



Epididymides



Heart



Kidneys



Brain



G1R, M & 0



Mean



0.0672



0.3491



0.9038



3.4753



1.6379



1.7660



2.8830



2.1857



±SD



0.0137



0.0667



0.0708



0.3153



0.1371



0.1191



0.0563



0.0899



n



5



5



5



5



5



5



5



5



G4R, M & 1000



Mean



0.0714



0.3549



0.8759



3.6831



1.6490



1.6900



2.9076



2.1591



±SD



0.0165



0.0364



0.1383



0.1164



0.1279



0.0843



0.1124



0.1103



n



5



5



5



5



5



5



5



5









































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Liver



Prostate



Seminal vesicles with coagulating glands



Lungs



Pituitary



Thyroid along


with parathyroid#



G1R, M & 0



Mean



14.7976



1.6863



1.6133



2.6073



0.0197



0.0296



±SD



0.5435



0.2738



0.5439



0.1528



0.0019



0.0012



n



5



5



5



5



5



5



G4R, M & 1000



Mean



13.9119



1.7092



2.0733



2.7021



0.0186



0.0303



±SD



1.5424



0.2676



0.7933



0.1479



0.0026



0.0015



n



5



5



5



5



5



5









































































Group, Sex


& Dose


(mg/kg body


weight/day)



 



Adrenals



Thymus



Spleen



Ovaries



Uterus with Cervix



Heart



G1R, F & 0



Mean



0.0713



0.2677



0.5367



0.1521



0.6853



1.1546



±SD



0.0092



0.0323



0.0671



0.0300



0.0535



0.0881



n



5



5



5



5



5



5



G4R, F & 1000



Mean



0.0784



0.2442



0.6058



0.1446



0.6247



1.0821



±SD



0.0025



0.0115



0.1106



0.0324



0.0814



0.0578



n



5



5



5



5



5



5









































































Group, Sex


& Dose


(mg/kg body weight/day)


 

Kidneys



Brain



Liver



Lungs



Pituitary



Thyroid along


with parathyroid#



G1R, F & 0



Mean



1.7997



2.0419



7.9208



1.7759



0.0186



0.0251



±SD



0.1899



0.1128



0.3716



0.2286



0.0008



0.0005



n



5



5



5



5



5



5



G4R, F & 1000



Mean



1.9299



1.9771



10.1686*



1.9356



0.0193



0.0262



±SD



0.1942



0.1524



1.2338



0.2208



0.0008



0.0024



n



5



5



5



5



5



5



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


 


TABLE 13. SUMMARY OF TERMINAL BODY WEIGHT (g) AND ORGAN WEIGHT RELATIVE TO TERMINAL BODY WEIGHT (%) RECORD













































































































































Group, Sex


& Dose


(mg/kg body weight/day)


 

Terminal Body Weight (g)



Adrenals



Thymus



Spleen



Testes



Epididymides



Heart



G1, M & 0



Mean



439.85



0.0198



0.1026



0.2183



0.8020



0.3724



0.3667



±SD



34.39



0.0035



0.0175



0.0291



0.0485



0.0457



0.0354



n



12



5



5



5



12



12



5



G2, M & 100



Mean



443.67



0.0186



0.1001



0.2013



0.8151



0.3878



0.3747



±SD



33.97



0.0019



0.0086



0.0277



0.0608



0.0572



0.0275



n



12



5



5



5



12



12



5



G3, M & 300



Mean



453.48



0.0164



0.0970



0.1787



0.7462



0.3506



0.3398



±SD



30.58



0.0026



0.0191



0.0209



0.0736



0.0370



0.0216



n



12



5



5



5



12



12



5



G4, M & 1000



Mean



448.80



0.0153*



0.0619*



0.2552



0.7801



0.3472



0.3479



±SD



30.87



0.0016



0.0076



0.0175



0.0831



0.0303



0.0211



n



12



5



5



5



12



12



5



























































































































































Group, Sex


& Dose


(mg/kg body weight/day)


 

Kidneys



Brain



Liver



Prostate



Seminal vesicles with coagulating glands



Lungs



Pituitary



Thyroid along


with parathyroid#



G1, M & 0



Mean



0.7695



0.5426



3.1356



0.4231



0.4431



0.5469



0.0044



0.0069



±SD



0.0950



0.0549



0.3423



0.1055



0.1632



0.0930



0.0004



0.0006



n



5



5



12



12



12



5



5



12



G2, M & 100



Mean



0.8216



0.5231



3.0993



0.3991



0.4359



0.6016



0.0042



0.0070



±SD



0.0962



0.0264



0.2982



0.0719



0.1028



0.0672



0.0003



0.0005



n



5



5



12



12



12



5



5



12



G3, M & 300



Mean



0.6090*



0.4770*



3.2356



0.3790



0.4485



0.5497



0.0039



0.0071



±SD



0.0248



0.0193



0.4756



0.1129



0.1434



0.0566



0.0003



0.0005



n



5



5



12



12



12



5



5



12



G4, M & 1000



Mean



0.6853



0.4770*



3.0592



0.3793



0.5242



0.5688



0.0040



0.0069



±SD



0.0849



0.0306



0.2242



0.0864



0.0682



0.0340



0.0002



0.0005



n



5



5



12



12



12



5



5



12














































































































































Group, Sex


& Dose


(mg/kg body weight/day)


 

Terminal Body Weight (g)



Adrenals



Thymus



Spleen



Ovaries



Uterus


with Cervix



Heart



G1, F & 0



Mean



317.06



0.0166



0.0819



0.2330



0.0454



0.1931



0.3756



±SD



19.46



0.0033



0.0072



0.0466



0.0086



0.0344



0.0311



n



12



5



5



5



12



12



5



G2, F & 100



Mean



314.41



0.0213



0.0886



0.2055



0.0447



0.1897



0.3797



±SD



16.46



0.0057



0.0285



0.0443



0.0046



0.0340



0.0202



n



12



5



5



5



12



12



5



G3, F & 300



Mean



318.10



0.0208



0.0615



0.2178



0.0483



0.2291



0.4096



±SD



15.35



0.0016



0.0221



0.0788



0.0085



0.1833



0.0345



n



12



5



5



5



12



12



5



G4, F & 1000



Mean



310.30



0.0181



0.0689



0.1981



0.0478



0.1972



0.4044



±SD



20.55



0.0012



0.0070



0.0535



0.0053



0.0973



0.0488



n



12



5



5



5



12



12



5

































































































































Group, Sex


& Dose


(mg/kg body weight/day)


 

Kidneys



Brain



Liver



Lungs



Pituitary



Thyroid along


with parathyroid#



G1, F & 0



Mean



0.5977



0.6136



3.7730



0.5201



0.0052



0.0079



±SD



0.0388



0.0359



0.6834



0.0728



0.0007



0.0004



n



5



5



12



5



5



12



G2, F & 100



Mean



0.6647



0.6445



3.7396



0.6165*



0.0056



0.0078



±SD



0.0714



0.0447



0.3749



0.0362



0.0006



0.0006



n



5



5



12



5



5



12



G3, F & 300



Mean



0.6930*



0.6318



3.7941



0.6512*



0.0053



0.0076



±SD



0.0701



0.0208



0.3315



0.0416



0.0005



0.0005



n



5



5



12



5



5



12



G4, F & 1000



Mean



0.6388



0.6544



3.8669



0.6756*



0.0059



0.0080



±SD



0.0264



0.0475



0.3622



0.0578



0.0006



0.0006



n



5



5



12



5



5



12
















































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Terminal Body Weight (g)



Adrenals



Thymus



Spleen



Testes



Epididymides



Heart



G1R, M & 0



Mean



528.23



0.0128



0.0661



0.1711



0.6582



0.3118



0.3356



±SD



36.74



0.0029



0.0115



0.0068



0.0457



0.0406



0.0321



n



5



5



5



5



5



5



5



G4R, M & 1000



Mean



516.79



0.0138



0.0687



0.1695



0.7137



0.3193



0.3272



±SD



27.43



0.0032



0.0055



0.0250



0.0303



0.0224



0.0117



n



5



5



5



5



5



5



5























































































Group, Sex


& Dose


(mg/kg body weight/day)


 

Kidneys



Brain



Liver



Prostate



Seminal vesicles with coagulating glands



Lungs



Pituitary



Thyroid along


with parathyroid#



G1R, M & 0



Mean



0.5477



0.4163



2.8092



0.3217



0.3070



0.4946



0.0037



0.0056



±SD



0.0369



0.0462



0.1649



0.0630



0.1079



0.0302



0.0001



0.0003



n



5



5



5



5



5



5



5



5



G4R, M & 1000



Mean



0.5639



0.4190



2.6870



0.3319



0.3995



0.5244



0.0036



0.0059



±SD



0.0367



0.0336



0.1856



0.0565



0.1476



0.0451



0.0004



0.0002



n



5



5



5



5



5



5



5



5
















































































Group, Sex


& Dose


(mg/kg body weight/day)



 



Terminal Body Weight (g)



Adrenals



Thymus



Spleen



Ovaries



Uterus


with Cervix



Heart



G1R, F & 0



Mean



317.31



0.0224



0.0846



0.1696



0.0479



0.2159



0.3638



±SD



9.00



0.0026



0.0121



0.0252



0.0091



0.0141



0.0246



n



5



5



5



5



5



5



5



G4R, F & 1000



Mean



307.87



0.0255



0.0795



0.1964



0.0470



0.2034



0.3518



±SD



14.43



0.0010



0.0051



0.0332



0.0105



0.0288



0.0181



n



5



5



5



5



5



5



5









































































Group, Sex


& Dose


(mg/kg body weight/day)


 

Kidneys



Brain



Liver



Lungs



Pituitary



Thyroid along


with parathyroid#



G1R, F & 0



Mean



0.5665



0.6436



2.4963



0.5590



0.0058



0.0079



±SD



0.0475



0.0338



0.0963



0.0633



0.0002



0.0002



n



5



5



5



5



5



5



G4R, F & 1000



Mean



0.6261



0.6426



3.3055*



0.6276



0.0063*



0.0085



±SD



0.0451



0.0475



0.3854



0.0529



0.0002



0.0009



n



5



5



5



5



5



5



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


 


TABLE 14. SUMMARY RECORD OF MEAN PUP WEIGHT (g) PER LITTER



















































































































































































































Group & Dose


(mg/kg body weight/day)


 

PND 1


 

PND 4


 

PND 7


 

PND 13



Mean Pup Weight (g)


 

Mean Pup Weight (g)



 



Mean Pup Weight (g)



 



Mean Pup Weight (g)



Male



Female



 



Male



Female



 



Male



Female



 



Male



Female



G1 & 0



Mean



6.78



6.33



 



11.12



10.31



 



14.88



13.80



 



28.69



26.90



±SD



0.12



0.22



 



0.50



0.36



 



0.34



0.29



 



0.50



1.08



n



10 (65)



10 (54)



 



10 (65)



10 (54)



 



10 (65)



10 (43)



 



10 (65)



10 (43)



G2 & 100



Mean



6.79



6.27



 



11.33



10.45



 



15.10



14.09



 



28.48



27.52



±SD



0.17



0.13



 



0.40



0.27



 



0.42



0.40



 



0.77



0.69



n



11 (73)



11 (60)



 



11 (72)



11 (60)



 



11 (72)



11 (48)



 



11 (72)



11 (48)



G3 & 300



Mean



6.77



6.23



 



11.18



10.35



 



14.71



13.67



 



28.77



26.93



±SD



0.12



0.15



 



0.40



0.20



 



0.47



0.36



 



0.93



1.12



n



10 (64)



10 (61)



 



10 (64)



10 (61)



 



10 (64)



10 (48)



 



10 (64)



10 (48)



G4 & 1000



Mean



6.75



6.22



 



11.28



10.38



 



14.67



13.78



 



27.81*



26.24



±SD



0.10



0.12



 



0.42



0.17



 



0.30



0.29



 



0.93



1.62



n



10 (71)



10 (64)



 



10 (71)



10 (64)



 



10 (71)



10 (48)



 



10 (71)



10 (48)



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


 


TABLE 15. SUMMARY OF SERUM T4 HORMONE LEVELS (ng/mL) RECORD - POSTNATAL DAY (PND) 13 PUPS































































Group & Dose


(mg/kg body weight/day)


 

Serum T4 Levels (ng/mL)



G1 & 0



Mean



65.057



±SD



4.071



n



10



G2 & 100



Mean



59.525*



±SD



5.558



n



11



G3 & 300



Mean



65.668



±SD



4.034



n



10



G4 & 1000



Mean



61.718



±SD



3.810



n



10



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

Conclusions:
The NOAEL of the test substance assessed in a study on reproductive/ developmental toxicity performed according to OECD TG 422 in rats by oral route was determined to be 1000 mg/kg bw/day since no indication of systemic, reproduction or developmental toxicity was observed up to the highest dose tested.




Executive summary:

A combined repeated dose toxicity study with the reproduction /developmental toxicity screening test of the test substance by oral administration in rats was conducted according to OECD guideline 422, in accordance with GLP principles. A total of 116 (58 males + 58 females) Sprague Dawley rats were distributed to four main groups and two recovery groups to detect delayed occurrence and recovery from toxic effects. Each main group (G1, G2, G3 and G4) consisted of 12 males and 12 females and each recovery group (G1R and G4R) consisted of 5 males and 5 females. The animals in G1/G1R group were administered with vehicle (0.5% w/v Carboxymethyl Cellulose), and the animals in G2, G3 and G4/G4R groups were administered with test item at the dose levels of 100, 300 and 1000 mg/kg bw/day based on the results of a dose range finding study performed with the same species at doses of 64, 160, 400 and 1000 mg/kg bw and in which no effects were observed up to the highest dose tested. The test item was administered to the main group males for a period of 29 days (two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period), to the main group females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13 and to the recovery group animals for a period of 50 days with a 15 days recovery. The vehicle and test item formulations were administered orally by gavage at the dose volume of 10 mL/kg bw. The stability of test item formulations in vehicle was established before initiation of the treatment. Dose formulation analysis for homogeneity and concentration verification was performed during weeks 1 and 5 of the treatment period and the results were within acceptable limits.


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


There were no treatment-related effects for the systemic toxicity assessment. There were no test item-related effects for clinical signs, mortality and morbidity, detailed clinical examination, body weight, feed consumption, ophthalmological examinations, clinical pathological (haematology, clinical chemistry and urinalysis) examinations and neurological/functional observations in all tested dose groups. The estimated serum T4 levels did not reveal any changes. The absolute and relative organ weights of both sexes did not reveal any changes from all tested dose groups. No gross pathological changes were observed in any of the animals from all tested dose groups during necropsy. No treatment related microscopic findings were noted in any of the tissues/organs subjected for histopathological evaluation from both high dose group and vehicle control group animals of both sexes. There was no treatment related microscopic findings noted in femur bone and liver which were subjected for histopathological evaluation in all groups.


There were no treatment-related effects for the reproductive toxicity assessment. There were no effects noted on reproductive performance of both sexes in the tested dose groups. No test item-related irregularities observed in oestrus cyclicity of the females from all tested dose groups during treatment period. No changes were noted for delivery and litter observations in all tested dose groups. No effects were noted for live birth index and pup survival index in all tested dose groups. No test item-related post-implantation losses were noted in all tested dose groups.


There were no treatment-related effects for the developmental toxicity assessment. There were no developmental/external behavioural changes noted and no test item-related mortalities noted during postnatal period in any of the pups from all tested dose group litters. The mean pup weight, mean anogenital distance and its ratio in either sex of pups per litter were unaffected by the test item in all tested dose groups. There were no incidences of retention of nipples in male pups examined on PND 13 from all tested dose and control group litters. The estimated serum T4 levels of PND 13 (from all litters) pups did not reveal any changes in all tested dose group litters. There were no gross pathological changes noted in any of the pups during scheduled sacrifice from all tested dose and control group litters.


Based on these results, the NOAEL of the test substance was determined to be 1000 mg/kg bw/day for systemic, reproduction and developmental toxicity end points.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: 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

Test material

Constituent 1
Chemical structure
Reference substance name:
Strontium hydrogen phosphate
EC Number:
236-615-8
EC Name:
Strontium hydrogen phosphate
Cas Number:
13450-99-2
Molecular formula:
SrHPO4
IUPAC Name:
strontium hydrogen phosphate
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Sprague-Dawley
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: 371.37-373.02 g (range of average values of each group of males at first day of dosing); 252.64-253.66 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 group: maximum of 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): 19.9-22.9ºC
- Humidity (%): 48-63 %
- Air changes (per hr): 12-15
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 19 August 2021 To: 11 November 2021

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
at a concentration of 0.5% (w/v)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required quantity of test item was weighed in a beaker and ground well in a mortar using pestle with a small quantity of vehicle until a homogenous suspension was formed. Thereafter the entire quantity of test formulation was transferred into measuring cylinder. A small quantity of vehicle was added to rinse the mortar and transferred into the measuring cylinder. The rinsing procedure of mortar and pestle was repeated many times to ensure the transfer of contents to the measuring cylinder. Finally, the volume was made up to required quantity with vehicle to get desired concentration for the different dose levels.
Test item formulations were administered to the animals within established stability conditions.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item was insoluble in distilled water and uniformly suspended in 0.5% w/v Carboxymethyl Cellulose at the concentration of 100 mg/mL (the highest dose concentration selected for the study considering the dose volume of 10 mL/kg body weight) as per laboratory solubility/suspendibility test results.
Hence, 0.5% w/v Carboxymethyl Cellulose was used as vehicle for test item formulations as it is a routinely used and universally accepted vehicle of choice for the oral toxicity studies.
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. (if required): SLCH1520
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 followed by 48 hours at the concentrations of 5 mg/mL and 100 mg/mL in 0.5% w/v Carboxymethyl cellulose. Prepared test item formulations were administered to the animals within these established stability conditions.
Homogeneity and dose formulation analysis for dose concentration verification was done by a validated analytical method ICP-MS (study nº BIO-ANM 1742). Sampling and analysis of formulations were performed during week 1 (07/09/2021) and week 5 (05/10/2021) of the treatment. Samples (5 mL approx.) 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%.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy.
- Any other deviations from standard protocol: no
Duration of treatment / exposure:
Main group males: two weeks pre-mating, during mating and up to the day before sacrifice (total of 29 days).
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 15 days observation.
Frequency of treatment:
daily
Duration of test:
Main group males: 29 days; main group females: from 49 to 60 days; recovery group animals: 50 days treatment plus 15 days observation.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
G1 - Vehicle control + G1R - Vehicle Control Recovery Group
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
G2 - Low dose
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
G3 - Mid dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
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 were decided based on the results of a dose range finding study in which there were no effects for systemic, reproductive and developmental toxicity up to the dose level of 1000 mg/kg bw/day.
- Fasting period before blood sampling for clinical biochemistry: yes, overnight (water allowed).

Examinations

Maternal examinations:
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.

BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed at receipt, on the first day of dosing, weekly thereafter and at termination. Females were weighed at receipt, on the first day of dosing, weekly thereafter during pre-mating and until confirmation of mating. Pregnant animals were weighed on gestation days GD 0, 7, 14 and 20 and on lactation days 1, 4, 7, 13 and 14 (terminal body weight).

FOOD CONSUMPTION: yes
-Feed consumption (cage wise) was measured for all males and females once in a week during premating period coinciding with body weight recording. Feed consumption was measured for all pregnant animals during GD 0 to 7, 7 to 14 and 14 to 20, during LD 1 to 4, 4 to 7 and 7 to 13.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once before treatment (on the day of randomization) and at the end of the dosing period for all males and females (shortly prior to scheduled sacrifice).
- Dose groups that were examined: all groups.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled terminal sacrifice (for males on day 29 and for littered females on lactation day 14).
- 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 males on day 29 and for littered females on lactation day 14).
- 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), Total Protein, Albumin, 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 29) and during lactation period for females (on lactation day 13). Towards the end of the recovery period (shortly prior to scheduled sacrifice on day 64) for the recovery group.
- Dose groups that were examined: 5 males and 5 females randomly selected from each main group and all recovery group animals.
- 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.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice:
Male animals: All surviving animals after completion of 29 days of treatment.
Maternal animals: All surviving animals on lactation day 14.
- Organs examined: The organs indicated in Tables 1 and 2 were prepared for microscopic examination and weighed.

OTHER:
Thyroxine Hormone (T4) Level estimation: Blood samples were collected from all main group animals 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 available above normal litter size.
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 29 days of treatment).
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Blood sampling:
- Plasma: Yes
- Serum: Yes
Fetal examinations:
- External examinations: Yes (all per litter)
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
- Anogenital distance of all live rodent pups: yes
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 3 below.
Indices:
Mating index, fertility index, gestation index, parturation index, pregnancy index, sex ratio, live birth index, post implantation loss (%).

Pup survival index on lactation day 4/7/13, sex ratio on lactation day 4/7/13, post natal loss (%), AGD ratio.
Historical control data:
No information reported.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs of toxicity noted in any of the tested dose group animals of both sexes throughout the experimental period.
Dermal irritation (if dermal study):
not examined
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, non-treatment-related
Description (incidence and severity):
There were no test item related changes noted in mean body weight and percent change in mean body weight gain with respect to day 1 in all the tested dose groups (main and recovery) of both sexes throughout the experimental period when compared with vehicle control group. Also, there were no test item related changes noted in mean gestational body weight and percent change in mean gestational body weight gain in all the tested dose groups when compared with the vehicle control group. Finally, there were no changes in mean lactation body weight and percent change in mean lactation body weight gain in all the tested dose groups when compared with the vehicle control group.

The noted statistically significant increase in mean percent change in body weight on day 28 with respect to day 1 in group G3 males is considered as incidental and unrelated to treatment as there were no changes noted in mean body weight and mean feed consumption during this period.

The noted statistically significant increase in mean percent change in gestation body weight gain during day 7 to 14 in groups G2, G3 and G4 is considered as incidental and toxicologically not significant.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were no changes in mean feed consumption (including during gestation period) in all the tested dose groups (main and recovery) of both sexes when compared with the vehicle control group. Also, there were no test item related changes in mean feed consumption during lactation period in all the tested dose groups when compared with the vehicle control group.

The noted statistically significant increase in mean lactation feed consumption during day 7 to 13 in group G4 is considered as incidental and toxicologically not significant.
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 or vehicle control groups (main and recovery) of both sexes.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related changes noted in the obtained mean haematological values in all the tested dose groups (main and recovery) of both sexes when compared with the vehicle control group.
The noted statistically significant decrease in mean prothrombin time in group G4 males and decrease in mean corpuscular hemoglobin concentration in group G2 females is considered as incidental and unrelated to treatment, as these changes did not occur in a dose dependant manner and also the obtained mean values are within laboratory historical control range of same species and strain.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related changes in mean clinical chemistry values in all the tested dose groups (main and recovery) of both sexes when compared with the vehicle control groups.
The noted statistically significant increase in glucose and triglycerides levels in group G3 males, calcium levels in groups G3 and G4 males, Albumin/Globulin Ratio in groups G2 and G4 females; decrease in creatinine levels in group G4 males, total protein levels in groups G2, G3 and G4 females, albumin levels in group G3 females, globulin levels in groups G2 and G4 females are considered as incidental and unrelated to treatment, as these changes did not occur in a dose dependant manner and also the obtained mean values are within laboratory historical control range of same species and strain.
Endocrine findings:
no effects observed
Description (incidence and severity):
There were no changes noted in mean serum T4 levels in any of the tested dose groups (main group males) when compared with the vehicle control group.

The assessment of serum T4 levels was performed for adult males and
PND 13 pups. Further assessment of T4 levels in serum samples from the dams and PND 4 pups was not assessed as there were no treatment-related effects observed in the T4 levels of adult males or PND 13 pups.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no changes in mean urinalysis values in all the tested dose groups (main group males and recovery group males and females) when compared with the vehicle control group.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
The neurological/functional observations such as, home cage, handling, open-field, sensory, physiological observations did not reveal any changes in any of the animals of both sexes from all the tested dose groups performed towards end of the dosing period for main groups and towards end of recovery period for recovery groups.
There were no changes in mean fore/hind limb grip strengths, mean motor activity assessments and mean hind limb foot splay in all tested dose groups of both sexes when compared with vehicle control groups during conduct of neurological / functional examinations.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related changes in mean absolute and relative organ weights at the tested dose groups G2, G3 and G4/G4R of both sexes when compared with vehicle control group.
However, statistically significant changes such as, increase in mean absolute weight of thyroid and parathyroid in group G3 males, mean absolute and relative weight of lungs at groups G3 and G4 females, mean relative weight of kidneys in group G3 females, mean relative weight of lungs in group G2 females, mean absolute and relative weight of liver in group G4R females, mean relative weight of pituitary in group G4R females; decrease in mean absolute and relative weight of kidneys in group G3 males, mean absolute and relative weight of adrenals and thymus in group G4 males, mean absolute weight of spleen in group G4 males, mean relative weight of brain in groups G3 and G4 males were noted when compared with vehicle control group.
The observed changes are considered as incidental as there were no gross or histopathological changes noted during necropsy or microscopic examinations.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross pathological changes observed in any of the animals of both sexes from all the tested dose groups and vehicle control group during conduct of necropsy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic findings in the study.
Few microscopic findings observed in this study such as ultimobranchial cyst in thyroid glands, epithelial cyst in thymus 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:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
No abortions reported.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
A mean number of 0.20, 0.09, 2.20 and 0.70 post-implantation losses with a percentage of 2.00, 0.45, 11.77 and 4.74 were noted from groups G1, G2, G3 and G4 respectively.

There were no statistically significant changes as for mean number and percentage of occurred post-implantation losses in any of the tested dose groups when compared with the vehicle control group. The noted increase in mean number of post-implantation loss and its percentage in group G3 is due to one dam which was noted with 13 implantation loss with a percentage of 61.9. However, this occurrence is considered as incidental finding and unrelated to treatment as there were no such losses noted in other animals of same group and also in next dose level i.e. group G4. Also, the noted difference is not statistically significant when compared with vehicle control group (obtained p value = 0.065).
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
A mean number of 12.10, 12.18, 14.70 and 14.20 implantation sites and mean number of 11.90, 12.09, 12.50 and 13.50 viable pups were noted from groups G1, G2, G3 and G4 respectively.

There were no statistically significant changes noted for both mean implantation sites and viable pups in any of the tested dose groups when compared with the vehicle control group.
Early or late resorptions:
not examined
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no changes observed in birth parameters such as number of live pups born and live birth index in all tested dose groups when compared with the vehicle control group.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
The mean gestation length (day of confirmed as successfully mated to day of parturition) was 22.20, 22.00, 22.30 and 22.20 days for groups G1, G2, G3 and G4 respectively.

There were no statistically significant differences noted for mean gestation length in any of the tested dose groups when compared with the vehicle control group.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
A total of 10 (out of 12), 11 (out of 12), 10 (out of 12) and 10 (out of 12), mated females were confirmed as pregnant / with evidence of implantation sites with a fecundity index of 83.3%, 91.7%, 83.3% and 83.3% from groups G1, G2, G3 and G4 respectively.

There were no statistically significant differences noted for fecundity index in any of the tested dose groups when compared with the vehicle control group.
Other effects:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No adverse effects found up to the highest dose tested (1000 mg/kg bw/day)

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related changes noted in mean pup (both male and female) weight per litter in all tested dose groups when compared with the vehicle control group, recorded on postnatal day (PND) 1, 4, 7 and 13.

The noted statistically significant decrease in mean male pup weight on postnatal day13 in G4 group is considered as incidental and unrelated to treatment as the obtained mean values were within the historical control data range.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
A mean number of 0.00, 0.09, 0.00 and 0.00 post-natal losses with a percentage of 0.00, 0.48, 0.00 and 0.00 were noted from groups G1, G2, G3 and G4 respectively.

There were no statistically significant changes as for mean number and percentage of occurred post-natal losses in any of the tested dose groups when compared with the vehicle control group.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no changes observed in birth parameters such as sex ratio (m/f) in all tested dose groups when compared with the vehicle control group.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no changes observed in birth parameters such as total litter size in all tested dose groups when compared with the vehicle control group.
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
There were no changes in mean pup (both male and female) anogenital distance measurement (mm) and its ratio per litter in all the tested dose groups when compared with the vehicle control group, recorded on postnatal day 4.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
The pup survival index per litter during lactation period was unaffected by the test item in all tested dose groups when compared with the vehicle control group.
External malformations:
no effects observed
Description (incidence and severity):
There were no gross pathological changes observed in any of the pups found dead at birth, cannibalized or terminally sacrificed from all the tested dose groups and vehicle control group litters during conduct of necropsy.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related changes noted in mean serum T4 hormone levels of postnatal day 13 pups in any of the tested dose group litters when compared with vehicle control group litters.
The noted statistically significant decrease in mean T4 levels in group G2 is considered as incidental and unrelated to treatment as the obtained mean values were within the historical control data range.

The assessment of serum T4 levels was performed for adult males and
PND 13 pups. Further assessment of T4 levels in serum samples from the dams and PND 4 pups was not assessed as there were no treatment-related effects observed in the T4 levels of adult males or PND 13 pups.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects found up to the highest dose tested (1000 mg/kg bw/day)

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

TABLE 4. SUMMARY OF THE STUDY






























































































































































































































 



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




Parameters ↓



G1 & 0



G2 & 100



G3 & 300



G4 & 1000



Reproductive Indices



No. of Pairs started



12



12



12



12



Reproductive Performance / Indices (males)



Total No. of males cohabited



12



12



12



12



No. of males with confirmed mating



12



12



12



12



No. of males impregnating/siring a female



10



11



10



10



Male Mating Index (%)



100.0



100.0



100.0



100.0



Male Fertility Index (%)



83.3



91.7



83.3



83.3



 



Reproductive Performance and Indices (females)



Mean Oestrus cycle length (days)



Mean



4.68



4.56



4.50



4.63



±SD



0.16



0.17



0.17



0.18



No. of females with regular oestrus cycle



12



12



12



12



Total No. of females cohabited



12



12



12



12



No. of sperm-positive females



12



12



12



12



No. of females with evidence of pregnancy



10



11



10



10



No. of females with live born pups



10



11



10



10



No. of females with conceiving days 1 to 5



8



9



10



12



No. of females with conceiving days ≥6



4



3



2



-



Pre-coital Interval (Days)



Mean



5.00



3.92



2.92



1.75



±SD



4.18



3.75



2.91



0.97



Female Mating Index (%)



100.0



100.0



100.0



100.0



Female Fertility Index (%)



83.3



91.7



83.3



83.3



No. of females with Pregnancy≤21 days



1



1



1



1



No. of females with Pregnancy = 22 days



6



9



5



6



No. of females with Pregnancy≥23 days



3



1



4



3



Gestation Length (Days)



Mean



22.20



22.00



22.30



22.20



±SD



0.63



0.45



0.67



0.63



Gestation Index (%)



100.0



100.0



100.0



100.0



Female Fecundity or Pregnancy Index (%)



83.3



91.7



83.3



83.3



Parturition index (%)



100.0



100.0



100.0



100.0




















































































































































































































































 



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




Parameters ↓



G1 & 0



G2 & 100



G3 & 300



G4 & 1000



Delivery and Litter Observations



No. of Dams with live young born



10



11



10



10



No. of Dams with live young at PND 4



10



11



10



10



No. of Dams with live young at PND 7



10



11



10



10



No. of Dams with live young at PND 13



10



11



10



10



No. of Implants/dam



Mean



12.10



12.18



14.70



14.20



±SD



3.00



3.49



4.11



2.70



No. of Live pups/dam at birth



Mean



11.90



12.09



12.50



13.50



±SD



3.21



3.27



3.44



2.72



No. of Live pups/dam at PND 4



Mean



11.90



12.00



12.50



13.50



±SD



3.21



3.07



3.44



2.72



No. of Live pups/dam at PND 7



Mean



10.80



10.91



11.20



11.90



±SD



2.35



2.21



2.70



2.28



No. of Live pups/dam at PND 13



Mean



10.80



10.91



11.20



11.90



±SD



2.35



2.21



2.70



2.28



Live Birth Index/dam (%)



Mean



100.00



99.55



99.41



99.33



±SD



0.00



1.51



1.86



2.11



Pup Survival Index/dam on


PND 4 (%)



Mean



100.00



99.52



100.00



100.00



±SD



0.00



1.59



0.00



0.00



Pup Survival Index/dam on


PND 7 (%)



Mean



 



100.00



100.00



100.00



±SD



0.00



0.00



0.00



0.00



Pup Survival Index/dam on


PND 13 (%)



Mean



100.00



100.00



100.00



100.00



±SD



0.00



0.00



0.00



0.00



Sex ratio (m/f)/dam At Birth



Mean



1.43



1.30



1.14



1.21



±SD



1.07



0.50



0.58



0.54



Sex ratio (m/f)/dam at PND 4



Mean



1.43



1.29



1.14



1.21



±SD



1.07



0.51



0.58



0.54



Sex ratio (m/f)/dam at PND 7



Mean



1.85



1.79



1.42



1.80



±SD



1.27



1.16



0.74



1.24



Sex ratio (m/f)/dam at PND 13



Mean



1.85



1.79



1.42



1.80



±SD



1.27



1.16



0.74



1.24



Note: For calculation of Pup survival index (%) on PND 7 and 13, the no. of pups sacrificed for blood collection on PND 4 were excluded.


 

























































































































































































































 



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



Parameters ↓



G1 & 0



G2 & 100



G3 & 300



G4 & 1000



Delivery and Litter Observations



Mean Male


Pup weight (g)/dam



At birth



Mean



6.78



6.79



6.77



6.75



±SD



0.12



0.17



0.12



0.10



PND 4



Mean



11.12



11.33



11.18



11.28



±SD



0.50



0.40



0.40



0.42



PND 7



Mean



14.88



15.10



14.71



14.67



±SD



0.34



0.42



0.47



0.30



PND 13



Mean



28.69



28.48



28.77



27.81



±SD



0.50



0.77



0.93



0.93



Mean Female


Pup weight (g)/dam



At Birth



Mean



6.33



6.27



6.23



6.22



±SD



0.22



0.13



0.15



0.12



PND 4



Mean



10.31



10.45



10.35



10.38



±SD



0.36



0.27



0.20



0.17



PND 7



Mean



13.80



14.09



13.67



13.78



±SD



0.29



0.40



0.36



0.29



PND 13



Mean



26.90



27.52



26.93



26.24



±SD



1.08



0.69



1.12



1.62



Mean Male Pup AGD Measurement/dam (mm) on PND 4



Mean



5.00



4.97



5.02



4.97



±SD



0.15



0.08



0.11



0.09



Mean Male Pup AGD ratio/dam on PND 4



Mean



2.24



2.21



2.25



2.22



±SD



0.06



0.04



0.03



0.02



Mean Female Pup AGD Measurement/dam (mm) on PND 4



Mean



2.67



2.67



2.66



2.66



±SD



0.09



0.07



0.08



0.06



Mean Female Pup AGD ratio/dam on PND 4



Mean



1.23



1.22



1.22



1.22



±SD



0.03



0.03



0.04



0.03



Male Pup Nipple Retention/dam on PND 13



Mean



0.00



0.00



0.00



0.00



±SD



0.00



0.00



0.00



0.00

























































































































































 



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




Parameters ↓



G1 & 0



G2 & 100



G3 & 300



G4 & 1000



ABNORMAL PUPS



No. of Dams with 0 abnormal pups



10



11



10



10



LOSS OF OFFSPRING



 



No. of Females with 0 post-implantation loss



9



10



5



6



No. of Females with 1 post-implantation loss



-



1



2



3



No. of Females with 2 post-implantation losses



1



-



-



-



No. of Females with 3 post-implantation losses



-



-



1



-



No. of Females with 4 post-implantation losses



-



-



1



1



No. of Females with 13 post-implantation losses



-



-



1



-



Post-implantation Loss (No.)



Mean



0.20



0.09



2.20



0.70



±SD



0.63



0.30



4.05



1.25



Post-implantation Loss (%)



Mean



2.00



0.45



11.77



4.74



±SD



6.32



1.51



19.53



8.42



Post-natal loss (live births minus alive at PND 13)



No. of Females with 0



10



10



10



10



No. of Females with 1



-



1



-



-



Postnatal loss (No.)



Mean



0.00



0.09



0.00



0.00



±SD



0.00



0.30



0.00



0.00



Postnatal Loss (%)



Mean



0.00



0.48



0.00



0.00



±SD



0.00



1.59



0.00



0.00



Note:  The noted increase in mean number of post-implantation loss and its percentage in group G3 and G4 are not statistically significant when compared with vehicle control group and the obtained p value = 0.065.


 


TABLE 5. SUMMARY OF PERCENT CHANGE IN BODY WEIGHT GAIN (%) WITH RESPECT TO DAY 1 RECORD









































































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Percent Change in Body Weight Gain (%) during Day



1 to 8



1 to 14



1 to 21



1 to 28



G1, M & 0



Mean



4.83



10.23



17.65



23.15



±SD



1.58



1.30



2.47



3.60



n



12



12



12



12



G2, M & 100



Mean



4.56



10.84



18.21



24.05



±SD



1.45



1.83



3.22



4.06



n



12



12



12



12



G3, M & 300



Mean



4.60



11.20



19.29



26.56*



±SD



1.43



2.09



2.66



3.25



n



12



12



12



12



G4, M & 1000



Mean



4.64



11.54



19.29



24.95



±SD



1.47



2.06



1.98



2.43



n



12



12



12



12





























































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Percent Change in Body Weight Gain (%) during Day



1 to 8



1 to 14



1 to 21



G1, F & 0



Mean



3.06



6.04



12.82



±SD



1.72



2.24



3.68



n



12



12



4



G2, F & 100



Mean



3.47



5.88



12.55



±SD



2.47



2.34



3.87



n



12



12



3



G3, F & 300



Mean



4.27



7.32



12.86



±SD



1.61



2.76



-



n



12



12



1



G4, F & 1000



Mean



3.40



6.19



-



±SD



1.54



2.24



-



n



12



12



-



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


#: The data obtained from females in cohabitation only considered for mean calculations. The data of day 1 to 21 body weight was not subjected to statistical analysis due to uneven number of variables.


 
































































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Percent Change in Body Weight (%) during Day



1 to 8



1 to 14



1 to 21



1 to 28



1 to 35



1 to 42



1 to 49



1 to 56



1 to 63



G1R, M & 0



Mean



5.04



12.17



20.93



27.05



33.03



36.96



41.00



44.79



48.34



±SD



1.03



3.12



4.37



4.56



4.95



5.96



4.43



7.11



7.47



n



5



5



5



5



5



5



5



5



5



G4R, M & 1000



Mean



5.23



11.69



19.10



25.07



31.04



35.97



40.39



43.87



47.24



±SD



1.65



2.51



1.60



4.38



7.37



7.66



7.59



9.05



8.16



n



5



5



5



5



5



5



5



5



5

































































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Percent Change in Body Weight (%) during Day



1 to 8



1 to 14



1 to 21



1 to 28



1 to 35



1 to 42



1 to 48



1 to 56



1 to 63



G1R, F & 0



Mean



3.85



6.55



10.41



15.72



19.35



21.92



23.74



27.66



31.64



±SD



0.93



1.26



1.79



3.11



3.10



2.91



2.94



3.19



2.67



n



5



5



5



5



5



5



5



5



5



G4R, F & 1000



Mean



4.26



7.11



11.13



16.52



20.28



22.80



24.03



27.39



31.03



±SD



1.05



0.83



1.02



0.72



0.97



1.35



1.28



2.63



4.03



n



5



5



5



5



5



5



5



5



5



 


TABLE 6. SUMMARY RECORD OF PERCENT CHANGE IN BODY WEIGHT GAIN (%) DURING GESTATION PERIOD




























































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

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



0 to 7



7 to 14



14 to 20



G1, F & 0



Mean



5.88



11.32



18.39



±SD



1.09



1.89



5.88



n



10



10



10



G2, F & 100



Mean



6.52



13.77*



20.14



±SD



1.11



1.07



7.06



n



11



11



11



G3, F & 300



Mean



6.39



13.83*



21.28



±SD



0.73



2.48



7.30



n



10



10



10



G4, F & 1000



Mean



6.42



14.72*



23.34



±SD



1.35



1.66



5.74



n



10



10



10



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


Note: Excluded the data of non-pregnant females for mean calculations and statistical analysis, but presented in individual animal data.


 


TABLE 7. SUMMARY RECORD OF PERCENT CHANGE IN BODY WEIGHT GAIN (%) DURING LACTATION PERIOD




























































































Group, Sex


& Dose (mg/kg body weight/day)


 

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



1 to 4



4 to 7



7 to 13



G1, F & 0



Mean



2.05



2.76



5.25



±SD



0.98



1.42



2.96



n



10



10



10



G2, F & 100



Mean



1.30



1.98



5.86



±SD



0.96



0.56



2.51



n



11



11



11



G3, F & 300



Mean



1.30



1.87



4.93



±SD



1.14



0.52



2.00



n



10



10



10



G4, F & 1000



Mean



1.14



2.03



5.60



±SD



0.92



0.59



2.14



n



10



10



10



 


TABLE 8. SUMMARY OF HAEMATOLOGY RECORD





























































































































































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Total


Leucocyte


Count



Total


Erythrocyte


Count



Haemoglobin



Haematocrit



Mean


Corpuscular


Volume



Mean


Corpuscular


Haemoglobin



Mean


Corpuscular


Haemoglobin


Concentration



Platelet


Count



Mean


Platelet


Volume



(WBC)



(RBC)



(HGB)



(HCT)



(MCV)



(MCH)



(MCHC)



(PLT)



(MPV)



(103 cells/µL)



(106 cells/µL)



(g/dL)



(%)



(fL)



(pg)



(g/dL)



(103 cells/µL)



(fL)



G1, M & 0



Mean



7.93



8.93



16.58



47.88



53.76



18.62



34.62



1048.60



6.10



±SD



2.39



0.50



0.74



1.64



2.42



1.05



0.50



244.74



0.22



n



5



5



5



5



5



5



5



5



5



G2, M & 100



Mean



7.88



8.48



16.66



44.90



52.96



19.80



37.36



991.60



6.02



±SD



0.84



0.86



0.59



4.00



1.28



1.96



3.13



161.48



0.18



n



5



5



5



5



5



5



5



5



5



G3, M & 300



Mean



8.18



8.62



16.14



46.38



53.84



18.76



34.88



1104.00



5.90



±SD



1.96



0.50



0.50



2.08



1.59



0.72



1.53



232.30



0.19



n



5



5



5



5



5



5



5



5



5



G4, M & 1000



Mean



8.36



8.69



16.06



47.16



54.44



18.52



34.08



966.00



6.28



±SD



2.02



0.54



0.42



1.32



3.09



0.87



0.55



158.58



0.42



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



2.56



22.14



73.32



2.24



1.28



0.26



±SD



0.59



6.94



7.78



1.41



0.63



0.05



n



5



5



5



5



5



5



G2, M & 100



Mean



2.18



22.82



73.00



1.92



1.30



0.22



±SD



0.33



7.12



7.31



0.44



0.29



0.13



n



5



5



5



5



5



5



G3, M & 300



Mean



2.28



24.52



71.46



1.42



1.78



0.12



±SD



0.71



4.21



4.83



0.51



0.70



0.04



n



5



5



5



5



5



5



G4, M & 1000



Mean



2.33



25.68



68.98



2.78



1.24



0.36



±SD



0.53



4.92



5.76



1.01



0.80



0.26



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)



(109 cells/L)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(Seconds)



(Seconds)



G1, M & 0



Mean



228.20



1.73



5.86



0.16



0.11



0.02



16.78



25.48



±SD



52.11



0.68



2.01



0.08



0.07



0.01



2.45



2.76



n



5



5



5



5



5



5



5



5



G2, M & 100



Mean



184.94



1.80



5.75



0.15



0.10



0.02



15.92



24.00



±SD



31.52



0.58



0.82



0.05



0.03



0.01



2.26



2.42



n



5



5



5



5



5



5



5



5



G3, M & 300



Mean



195.98



2.02



5.82



0.12



0.15



0.01



13.94



22.62



±SD



56.46



0.65



1.41



0.05



0.08



0.00



1.24



4.71



n



5



5



5



5



5



5



5



5



G4, M & 1000



Mean



201.06



2.13



5.78



0.22



0.11



0.03



12.36*



21.88



±SD



40.02



0.63



1.58



0.05



0.07



0.02



0.76



1.90



n



5



5



5



5



5



5



5



5






























































































































































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Total


Leucocyte


Count



Total


Erythrocyte


Count



Haemoglobin



Haematocrit



Mean


Corpuscular Volume



Mean


Corpuscular Haemoglobin



Mean


Corpuscular Haemoglobin Concentration



Platelet


Count



Mean


Platelet


Volume



(WBC)



(RBC)



(HGB)



(HCT)



(MCV)



(MCH)



(MCHC)



(PLT)



(MPV)



(103 cells/µL)



(106 cells/µL)



(g/dL)



(%)



(fL)



(pg)



(g/dL)



(103 cells/µL)



(fL)



G1, F & 0



Mean



8.49



7.55



15.50



42.66



56.44



20.66



36.62



931.80



6.02



±SD



1.85



0.66



0.89



4.81



2.20



1.70



3.35



123.32



0.18



n



5



5



5



5



5



5



5



5



5



G2, F & 100



Mean



9.97



7.78



15.84



47.22



60.78



20.36



33.56*



858.40



6.20



±SD



3.27



0.32



0.55



2.57



3.72



0.61



1.05



294.99



0.24



n



5



5



5



5



5



5



5



5



5



G3, F & 300



Mean



9.45



7.52



15.52



45.44



60.68



20.70



34.14



1024.80



6.24



±SD



3.18



0.54



0.61



1.84



5.12



1.29



0.74



132.05



0.17



n



5



5



5



5



5



5



5



5



5



G4, F & 1000



Mean



8.45



7.75



15.72



45.94



59.30



20.30



34.28



1088.00



6.20



±SD



0.81



0.25



0.65



1.94



1.29



0.29



0.49



159.47



0.12



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



2.59



43.02



50.64



3.72



1.66



0.16



±SD



0.86



9.12



8.23



1.89



1.89



0.05



n



5



5



5



5



5



5



G2, F & 100



Mean



3.04



31.08



63.32



2.78



1.58



0.24



±SD



1.20



8.90



10.07



1.08



1.29



0.21



n



5



5



5



5



5



5



G3, F & 300



Mean



2.41



49.14



45.08



3.58



1.02



0.18



±SD



0.68



17.03



16.07



1.54



0.36



0.13



n



5



5



5



5



5



5



G4, F & 1000



Mean



2.61



36.46



56.30



4.50



1.28



0.16



±SD



0.44



4.80



5.13



0.57



0.77



0.09



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)



(109 cells/L)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(Seconds)



(Seconds)



G1, F & 0



Mean



194.26



3.69



4.25



0.33



0.13



0.01



19.66



26.14



±SD



63.78



1.22



0.96



0.22



0.14



0.01



2.45



4.20



n



5



5



5



5



5



5



5



5



G2, F & 100 



Mean



235.18



3.27



6.13



0.30



0.14



0.02



18.46



24.44



±SD



90.14



2.12



1.26



0.21



0.09



0.02



3.63



5.07



n



5



5



5



5



5



5



5



5



G3, F & 300



Mean



178.88



4.66



4.23



0.36



0.09



0.02



18.06



24.34



±SD



40.18



1.93



1.86



0.23



0.04



0.02



1.80



5.69



n



5



5



5



5



5



5



5



5



G4, F & 1000



Mean



202.20



3.08



4.76



0.38



0.11



0.01



16.80



20.76



±SD



34.70



0.45



0.64



0.04



0.07



0.00



1.71



4.92



n



5



5



5



5



5



5



5



5




















































































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Total


Leucocyte


Count



Total


Erythrocyte


Count



Haemoglobin



Haematocrit



Mean


Corpuscular Volume



Mean


Corpuscular Haemoglobin



Mean


Corpuscular Haemoglobin Concentration



Platelet


Count



Mean


Platelet


Volume



(WBC)



(RBC)



(HGB)



(HCT)



(MCV)



(MCH)



(MCHC)



(PLT)



(MPV)



(103 cells/µL)



(106 cells/µL)



(g/dL)



(%)



(fL)



(pg)



(g/dL)



(103 cells/µL)



(fL)



G1R, M & 0



Mean



8.46



9.27



16.84



49.00



52.88



18.20



34.42



1110.00



5.94



±SD



1.04



0.51



0.62



2.00



1.29



0.75



0.61



119.77



0.11



n



5



5



5



5



5



5



5



5



5



G4R, M & 1000



Mean



8.44



8.82



16.36



46.42



52.70



18.66



35.40



1194.60



5.90



±SD



1.92



1.00



0.69



4.77



2.09



1.59



2.57



167.85



0.12



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)



(%)



(%)



(%)



(%)



(%)



(%)



G1R, M & 0



Mean



2.22



24.26



71.26



2.08



1.08



0.30



±SD



0.44



9.94



8.79



0.99



0.58



0.07



n



5



5



5



5



5



5



G4R, M & 1000



Mean



2.03



22.14



73.32



1.82



2.02



0.22



±SD



0.52



5.17



6.07



0.71



1.18



0.08



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)



(109 cells/L)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(Seconds)



(Seconds)



G1R, M & 0



Mean



205.38



2.13



5.96



0.17



0.09



0.02



17.50



29.46



±SD



41.13



1.11



0.33



0.08



0.05



0.01



3.08



7.07



n



5



5



5



5



5



5



5



5



G4R, M & 1000



Mean



175.70



1.85



6.21



0.15



0.17



0.02



19.10



22.88



±SD



35.88



0.56



1.56



0.03



0.12



0.00



1.02



3.35



n



5



5



5



5



5



5



5



5




















































































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Total


Leucocyte


Count



Total


Erythrocyte


Count



Haemoglobin



Haematocrit



Mean


Corpuscular Volume



Mean


Corpuscular Haemoglobin



Mean


Corpuscular Haemoglobin Concentration



Platelet


Count



Mean


Platelet


Volume



(WBC)



(RBC)



(HGB)



(HCT)



(MCV)



(MCH)



(MCHC)



(PLT)



(MPV)



(103 cells/µL)



(106 cells/µL)



(g/dL)



(%)



(fL)



(pg)



(g/dL)



(103 cells/µL)



(fL)



G1R, F & 0



Mean



4.37



8.30



15.64



45.06



54.30



18.84



34.70



934.00



6.46



±SD



0.54



0.31



0.34



1.28



1.18



0.55



0.44



293.12



0.19



n



5



5



5



5



5



5



5



5



5



G4R, F & 1000



Mean



5.44



8.12



15.72



45.00



55.44



19.34



34.96



904.20



6.32



±SD



1.71



0.28



0.72



2.26



1.47



0.59



0.58



224.52



0.47



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)



(%)



(%)



(%)



(%)



(%)



(%)



G1R, F & 0



Mean



1.66



26.40



67.90



2.52



2.16



0.26



±SD



0.24



7.17



6.71



0.79



0.79



0.11



n



5



5



5



5



5



5



G4R, F & 1000



Mean



2.26



21.90



72.14



3.00



2.14



0.20



±SD



0.63



6.50



7.20



0.76



0.67



0.14



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)



(109 cells/L)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(Seconds)



(Seconds)



G1R, F & 0



Mean



137.10



1.15



2.98



0.11



0.09



0.01



20.08



27.18



±SD



17.41



0.30



0.53



0.04



0.03



0.00



2.24



7.03



n



5



5



5



5



5



5



5



5



G4R, F & 1000



Mean



184.06



1.14



3.99



0.16



0.11



0.01



18.06



22.48



±SD



55.31



0.26



1.53



0.05



0.03



0.01



1.64



1.95



n



5



5



5



5



5



5



5



5



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


 


TABLE 9. 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



102.60



32.56



0.50



34.40



18.00



6.68



3.49



±SD



11.33



2.27



0.02



6.19



5.39



0.19



0.06



n



5



5



5



5



5



5



5



G2, M & 100



Mean



101.40



27.56



0.49



38.60



28.60



6.88



3.58



±SD



2.19



3.58



0.03



8.53



13.01



0.22



0.13



n



5



5



5



5



5



5



5



G3, M & 300



Mean



123.60*



30.98



0.49



50.00



42.20*



6.86



3.53



±SD



13.63



5.64



0.03



20.19



18.65



0.36



0.11



n



5



5



5



5



5



5



5



G4, M & 1000



Mean



97.00



30.94



0.46*



37.80



26.20



6.56



3.42



±SD



7.97



9.12



0.03



17.77



7.46



0.11



0.10



n



5



5



5



5



5



5



5


































































































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Alanine
aminotransferase



Aspartate
aminotransferase



Alkaline
phosphatase



Calcium



Phosphorous



(ALT)



(AST)



(ALP)



(CAL)



(PHO)



(U/L)



(U/L)



(U/L)



(mg/dL)



(mg/dL)



G1, M & 0



Mean



37.80



86.00



136.40



9.16



5.88



±SD



9.04



14.78



25.93



0.23



0.31



n



5



5



5



5



5



G2, M & 100



Mean



45.20



92.40



132.60



9.42



5.84



±SD



19.11



32.00



37.96



0.16



0.36



n



5



5



5



5



5



G3, M & 300



Mean



41.40



86.00



142.40



9.64*



6.04



±SD



4.39



9.14



16.77



0.25



0.43



n



5



5



5



5



5



G4, M & 1000



Mean



46.40



93.20



155.60



9.72*



6.26



±SD



10.64



30.05



48.64



0.13



0.59



n



5



5



5



5



5

















































































































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Globulin



Albumin/


Globulin ratio



Blood Urea Nitrogen



Sodium



Potassium



Chloride



(GLO)



(A/G Ratio)



(BUN)



(Na)



(K)



(CLO)



(g/dL)



-



(mg/dL)



(mmol/L)



(mmol/L)



(mmol/L)



G1, M & 0



Mean



3.19



1.10



15.19



143.32



3.83



108.18



±SD



0.16



0.04



1.06



0.36



0.13



0.56



n



5



5



5



5



5



5



G2, M & 100



Mean



3.30



1.09



12.86



143.28



4.06



107.82



±SD



0.16



0.06



1.67



1.00



0.14



1.82



n



5



5



5



5



5



5



G3, M & 300



Mean



3.33



1.07



14.46



143.90



3.96



107.84



±SD



0.28



0.08



2.63



0.56



0.34



1.96



n



5



5



5



5



5



5



G4, M & 1000



Mean



3.14



1.09



14.44



142.58



3.80



107.64



±SD



0.07



0.04



4.26



0.97



0.21



1.19



n



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



102.20



48.60



0.55



65.80



172.80



7.48



3.69



±SD



21.92



14.79



0.07



19.04



182.83



0.46



0.25



n



5



5



5



5



5



5



5



G2, F & 100



Mean



102.20



42.64



0.54



73.80



73.80



6.36*



3.42



±SD



16.08



11.18



0.09



27.31



72.48



0.29



0.11



n



5



5



5



5



5



5



5



G3, F & 300



Mean



103.20



62.72



0.65



77.00



112.40



6.76*



3.20*



±SD



24.78



43.25



0.22



29.26



80.25



0.15



0.21



n



5



5



5



5



5



5



5



G4, F & 1000



Mean



102.40



65.28



0.57



79.80



122.00



6.48*



3.51



±SD



6.58



9.46



0.03



6.14



38.50



0.33



0.18



n



5



5



5



5



5



5



5


































































































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Alanine
aminotransferase



Aspartate
aminotransferase



Alkaline
phosphatase



Calcium



Phosphorous



(ALT)



(AST)



(ALP)



(CAL)



(PHO)



(U/L)



(U/L)



(U/L)



(mg/dL)



(mg/dL)



G1, F & 0



Mean



130.20



130.80



474.80



10.54



6.96



±SD



93.03



76.17



460.49



1.57



1.92



n



5



5



5



5



5



G2, F & 100



Mean



66.00



106.40



128.20



10.14



7.10



±SD



18.48



34.16



58.02



0.67



3.67



n



5



5



5



5



5



G3, F & 300



Mean



134.20



185.60



239.40



9.98



6.40



±SD



81.52



120.65



117.85



0.82



1.50



n



5



5



5



5



5



G4, F & 1000



Mean



83.60



98.00



210.20



10.16



7.04



±SD



18.70



11.77



67.98



1.02



1.13



n



5



5



5



5



5

















































































































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Globulin



Albumin/Globulin Ratio



Blood Urea Nitrogen



Sodium



Potassium



Chloride



(GLO)



(A/G Ratio)



(BUN)



(Na)



(K)



(CLO)



(g/dL)



-



(mg/dL)



(mmol/L)



(mmol/L)



(mmol/L)



G1, F & 0



Mean



3.79



0.98



22.68



140.60



3.95



105.40



±SD



0.38



0.12



6.90



3.12



0.32



4.55



n



5



5



5



5



5



5



G2, F & 100



Mean



2.94*



1.17*



19.90



143.44



3.89



107.56



±SD



0.25



0.11



5.22



1.74



0.73



2.80



n



5



5



5



5



5



5



G3, F & 300



Mean



3.56



0.90



29.27



139.50



4.24



105.30



±SD



0.16



0.09



20.19



3.19



1.33



4.42



n



5



5



5



5



5



5



G4, F & 1000



Mean



2.97*



1.19*



30.47



139.62



4.01



105.34



±SD



0.26



0.12



4.41



0.64



0.12



1.88



n



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)



G1R, M & 0



Mean



125.60



36.94



0.54



73.60



34.00



7.62



3.85



±SD



9.34



4.07



0.03



17.24



10.37



0.23



0.06



n



5



5



5



5



5



5



5



G4R, M & 1000



Mean



128.40



32.78



0.54



75.40



30.60



7.76



3.78



±SD



14.15



5.27



0.03



12.34



14.33



0.15



0.09



n



5



5



5



5



5



5



5
















































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Alanine


aminotransferase



Aspartate aminotransferase



Alkaline


phosphatase



Calcium



Phosphorous



(ALT)



(AST)



(ALP)



(CAL)



(PHO)



(U/L)



(U/L)



(U/L)



(mg/dL)



(mg/dL)



G1R, M & 0



Mean



38.80



80.80



106.00



10.28



5.56



±SD



4.09



8.70



22.03



0.26



0.47



n



5



5



5



5



5



G4R, M & 1000



Mean



42.40



77.60



127.00



10.22



5.48



±SD



5.41



13.07



41.26



0.19



0.64



n



5



5



5



5



5

























































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Globulin



Albumin/


Globulin ratio



Blood Urea Nitrogen



Sodium



Potassium



Chloride



(GLO)



(A/G Ratio)



(BUN)



(Na)



(K)



(CLO)



(g/dL)



-



(mg/dL)



(mmol/L)



(mmol/L)



(mmol/L)



G1R, M & 0



Mean



3.77



1.02



17.24



141.28



3.68



106.82



±SD



0.23



0.07



1.90



1.58



0.31



1.72



n



5



5



5



5



5



5



G4R, M & 1000



Mean



3.98



0.95



15.30



141.14



3.61



106.32



±SD



0.16



0.05



2.46



1.01



0.18



1.06



n



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)



G1R, F & 0



Mean



135.40



42.58



0.66



103.20



45.40



8.96



4.84



±SD



4.72



6.83



0.03



23.76



7.09



0.25



0.08



n



5



5



5



5



5



5



5



G4R, F & 1000



Mean



136.40



41.72



0.61



97.80



71.40



9.06



4.73



±SD



11.63



9.48



0.06



17.68



21.63



0.63



0.28



n



5



5



5



5



5



5



5
















































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Alanine


aminotransferase



Aspartate aminotransferase



Alkaline


phosphatase



Calcium



Phosphorous



(ALT)



(AST)



(ALP)



(CAL)



(PHO)



(U/L)



(U/L)



(U/L)



(mg/dL)



(mg/dL)



G1R, F & 0



Mean



60.20



117.40



36.00



11.18



4.00



±SD



39.10



67.06



9.35



0.32



0.60



n



5



5



5



5



5



G4R, F & 1000



Mean



59.60



103.80



50.60



11.14



4.22



±SD



38.57



50.97



11.57



0.31



0.50



n



5



5



5



5



5

























































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Globulin



Albumin/


Globulin ratio



Blood Urea Nitrogen



Sodium



Potassium



Chloride



(GLO)



(A/G Ratio)



(BUN)



(Na)



(K)



(CLO)



(g/dL)



-



(mg/dL)



(mmol/L)



(mmol/L)



(mmol/L)



G1R, F & 0



Mean



4.12



1.17



19.87



140.64



3.42



107.40



±SD



0.20



0.05



3.19



0.68



0.09



0.54



n



5



5



5



5



5



5



G4R, F & 1000



Mean



4.33



1.09



19.47



140.24



3.54



106.64



±SD



0.40



0.07



4.43



1.57



0.25



1.20



n



5



5



5



5



5



5



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


 


TABLE 10. SUMMARY OF VAGINAL SMEAR EXAMINATION RECORD FOR DETERMINATION OF OESTRUS CYCLICITY






























































































Group, Sex


& Dose
(mg/kg body weight/day)



Total No. of


Females Evaluated


 

Females with


Complete


Regular Cycles



Females with at least one Irregular


Oestrus Cycle



Average Length of


Oestrus Cycle (Days)



G1, F & 0



12



n



12



0



Mean



4.68



%



100.0



0.0



±SD



0.16



n



12



G2, F & 100



12



n



12



0



Mean



4.56



%



100.0



0.0



±SD



0.17



n



12



G3, F & 300



12



n



12



0



Mean



4.50*



%



100.0



0.0



±SD



0.17



n



12



G4, F & 1000



12



n



12



0



Mean



4.63



%



100.0



0.0



±SD



0.18



n



12



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


 


TABLE 11. SUMMARY RECORD OF REPRODUCTIVE PERFORMANCE










































Group, Sex


& Dose  
(mg/kg body weight/day)



No. of Males with Evidence of Mating
(No. of Males used for Mating)



Male Mating Index (%)


 

No. of Males Capable of Impregnating a Female
(No. of males used for Mating)



Male Fertility Index (%)



G1, M & 0



12 (12)



100.0



10 (12)



83.3



G2, M & 100



12 (12)



100.0



11 (12)



91.7



G3, M & 300



12 (12)



100.0



10 (12)



83.3



G4, M & 1000



12 (12)



100.0



10 (12)



83.3













































































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Cohabitation Record and Pre-coital Interval (Mean Time of Mating)



 



Gestational Length


(duration of pregnancy)



Pre-coital Interval (Days)


 

Conceiving Days


(1 to 5)



Conceiving Days


(More than 5 days)



Gestation Length (Days)



G1, F & 0



Mean



5.00



n



8



4



22.20



±SD



4.18



%



66.7



33.3



0.63



n



12



10



G2, F & 100



Mean



3.92



n



9



3



22.00



±SD



3.75



%



75.0



25.0



0.45



n



12



11



G3, F & 300



Mean



2.92



n



10



2



22.30



±SD



2.91



%



83.3



16.7



0.67



n



12



10



G4, F & 1000



Mean



1.75*



n



12



0



22.20



±SD



0.97



%



100.0



0.0



0.63



n



12



10















































Group, Sex


& Dose


(mg/kg body


weight/day)



Female Mating Index (%)


 

Female Fertility Index (%)



No. of Females with Evidence of Mating


(No. of Females used for Mating)



Female Mating Index (%)



No. of Females Confirmed as Fertile
(No. of Females used for Mating)



Female Fertility Index (%)



G1, F & 0



12 (12)



100.0



10 (12)



83.3



G2, F & 100



12 (12)



100.0



11 (12)



91.7



G3, F & 300



12 (12)



100.0



10 (12)



83.3



G4, F & 1000



12 (12)



100.0



10 (12)



83.3

























































Group, Sex


& Dose                                      (mg/kg body


weight/day)



Female Fecundity or Pregnancy Index (%)


 

Gestation Index (%)



No. of Pregnant Females



No. of Females


Confirmed with Mating



Female Fecundity or Pregnancy Index (%)



Females with Live Born


Pups at Parturition



No. of Females


with Evidence of Pregnancy



Gestation Index (%)



G1, F & 0



10



12



83.3



10



10



100.0



G2, F & 100



11



12



91.7



11



11



100.0



G3, F & 300



10



12



83.3



10



10



100.0



G4, F & 1000



10



12



83.3



10



10



100.0








































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



Parturition index (%)



No. of Females Littered



No. of Females with evidence of pregnancy



Parturition index (%)



G1, F & 0



10



10



100.0



G2, F & 100



11



11



100.0



G3, F & 300



10



10



100.0



G4, F & 1000



10



10



100.0


















































































































































Group, Sex


& Dose
(mg/kg body


weight/day)



 



Post-implantation Loss (%)



 



Post-natal Loss (%)



No. of


Implantations



No. of Viable


(Live)


Pups



Post-implantation Loss


(No.)



Post-implantation Loss


(%)



Total No. of Pups Found Dead/


Cannibalized during lactation period



Post-natal Loss


(%)



G1, F & 0



Mean



12.10



11.90



0.20



2.00



 



0.00



0.00



±SD



3.00



3.21



0.63



6.32



 



0.00



0.00



n



10



10



10



10



 



12



12



G2, F & 100



Mean



12.18



12.09



0.09



0.45



 



0.09



0.48



±SD



3.49



3.27



0.30



1.51



 



0.30



1.59



n



11



11



11



11



 



11



11



G3, F & 300



Mean



14.70



12.50



2.20



11.77



 



0.00



0.00



±SD



4.11



3.44



4.05



19.53



 



0.00



0.00



n



10



10



10



10



 



12



12



G4, F & 1000



Mean



14.20



13.50



0.70



4.74



 



0.00



0.00



±SD



2.70



2.72



1.25



8.42



 



0.00



0.00



n



10



10



10



10



 



12



12



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


 


TABLE 12. SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD


























































































































































Group, Sex


& Dose


(mg/kg body weight/day)


 

Adrenals



Thymus



Spleen



Testes



Epididymides



Heart



Kidneys



Brain



G1, M & 0



Mean



0.0881



0.4598



0.9731



3.5201



1.6321



1.6310



3.4141



2.4113



±SD



0.0129



0.0910



0.1241



0.2599



0.1830



0.1035



0.2100



0.1176



n



5



5



5



12



12



5



5



5



G2, M & 100



Mean



0.0797



0.4290



0.8600



3.6026



1.7102



1.6084



3.5132



2.2461



±SD



0.0054



0.0136



0.0677



0.1997



0.2026



0.1029



0.1866



0.1097



n



5



5



5



12



12



5



5



5



G3, M & 300



Mean



0.0768



0.4540



0.8366



3.3672



1.5821



1.5895



2.8478*



2.2322



±SD



0.0112



0.0917



0.1026



0.2023



0.1147



0.0982



0.0725



0.1104



n



5



5



5



12



12



5



5



5



G4, M & 1000



Mean



0.0717*



0.2907*



1.1998*



3.4860



1.5544



1.6353



3.2137



2.2388



±SD



0.0057



0.0333



0.1036



0.2722



0.1314



0.1278



0.3464



0.1312



n



5



5



5



12



12



5



5



5

































































































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Liver



Prostate



Seminal vesicles with coagulating glands



Lungs



Pituitary



Thyroid along


with parathyroid#



G1, M & 0



Mean



13.7479



1.8731



1.9272



2.4260



0.0195



0.0303



±SD



1.3993



0.5071



0.6436



0.3217



0.0012



0.0015



n



12



12



12



5



5



12



G2, M & 100



Mean



13.7213



1.7628



1.9226



2.5720



0.0180



0.0307



±SD



1.3789



0.3096



0.4410



0.0945



0.0016



0.0013



n



12



12



12



5



5



12



G3, M & 300



Mean



14.5890



1.7312



2.0113



2.5701



0.0182



0.0319*



±SD



1.7410



0.5569



0.5669



0.2502



0.0014



0.0015



n



12



12



12



5



5



12



G4, M & 1000



Mean



13.7006



1.7092



2.3425



2.6678



0.0187



0.0310



±SD



1.0406



0.4295



0.2628



0.0791



0.0010



0.0019



n



12



12



12



5



5



12

































































































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Adrenals



Thymus



Spleen



Ovaries



Uterus with Cervix



Heart



G1, F & 0



Mean



0.0549



0.2709



0.7702



0.1432



0.6125



1.2405



±SD



0.0114



0.0265



0.1546



0.0233



0.1144



0.0829



n



5



5



5



12



12



5



G2, F & 100



Mean



0.0656



0.2777



0.6426



0.1403



0.5969



1.1809



±SD



0.0141



0.0979



0.1603



0.0132



0.1126



0.0755



n



5



5



5



12



12



5



G3, F & 300



Mean



0.0667



0.1972



0.7004



0.1524



0.7271



1.3171



±SD



0.0039



0.0691



0.2512



0.0203



0.5884



0.1254



n



5



5



5



12



12



5



G4, F & 1000



Mean



0.0553



0.2097



0.6029



0.1481



0.6083



1.2362



±SD



0.0068



0.0104



0.1662



0.0163



0.2945



0.1750



n



5



5



5



12



12



5

































































































































Group, Sex


& Dose


(mg/kg body weight/day)


 

Kidneys



Brain



Liver



Lungs



Pituitary



Thyroid along


with parathyroid#



G1, F & 0



Mean



1.9762



2.0279



11.9915



1.7191



0.0170



0.0251



±SD



0.1366



0.1114



2.4375



0.2404



0.0020



0.0018



n



5



5



12



5



5



12



G2, F & 100



Mean



2.0651



2.0014



11.7354



1.9148



0.0177



0.0246



±SD



0.2024



0.0851



1.0645



0.0568



0.0010



0.0020



n



5



5



12



5



5



12



G3, F & 300



Mean



2.2289



2.0311



12.0375



2.0925*



0.0169



0.0241



±SD



0.2489



0.0980



0.7986



0.1334



0.0016



0.0019



n



5



5



12



5



5



12



G4, F & 1000



Mean



1.9525



1.9955



11.9606



2.0568*



0.0178



0.0248



±SD



0.1493



0.1300



0.9854



0.0900



0.0013



0.0016



n



5



5



12



5



5



12























































































Group, Sex


& Dose


(mg/kg body weight/day)


 

Adrenals



Thymus



Spleen



Testes



Epididymides



Heart



Kidneys



Brain



G1R, M & 0



Mean



0.0672



0.3491



0.9038



3.4753



1.6379



1.7660



2.8830



2.1857



±SD



0.0137



0.0667



0.0708



0.3153



0.1371



0.1191



0.0563



0.0899



n



5



5



5



5



5



5



5



5



G4R, M & 1000



Mean



0.0714



0.3549



0.8759



3.6831



1.6490



1.6900



2.9076



2.1591



±SD



0.0165



0.0364



0.1383



0.1164



0.1279



0.0843



0.1124



0.1103



n



5



5



5



5



5



5



5



5









































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Liver



Prostate



Seminal vesicles with coagulating glands



Lungs



Pituitary



Thyroid along


with parathyroid#



G1R, M & 0



Mean



14.7976



1.6863



1.6133



2.6073



0.0197



0.0296



±SD



0.5435



0.2738



0.5439



0.1528



0.0019



0.0012



n



5



5



5



5



5



5



G4R, M & 1000



Mean



13.9119



1.7092



2.0733



2.7021



0.0186



0.0303



±SD



1.5424



0.2676



0.7933



0.1479



0.0026



0.0015



n



5



5



5



5



5



5









































































Group, Sex


& Dose


(mg/kg body


weight/day)



 



Adrenals



Thymus



Spleen



Ovaries



Uterus with Cervix



Heart



G1R, F & 0



Mean



0.0713



0.2677



0.5367



0.1521



0.6853



1.1546



±SD



0.0092



0.0323



0.0671



0.0300



0.0535



0.0881



n



5



5



5



5



5



5



G4R, F & 1000



Mean



0.0784



0.2442



0.6058



0.1446



0.6247



1.0821



±SD



0.0025



0.0115



0.1106



0.0324



0.0814



0.0578



n



5



5



5



5



5



5









































































Group, Sex


& Dose


(mg/kg body weight/day)


 

Kidneys



Brain



Liver



Lungs



Pituitary



Thyroid along


with parathyroid#



G1R, F & 0



Mean



1.7997



2.0419



7.9208



1.7759



0.0186



0.0251



±SD



0.1899



0.1128



0.3716



0.2286



0.0008



0.0005



n



5



5



5



5



5



5



G4R, F & 1000



Mean



1.9299



1.9771



10.1686*



1.9356



0.0193



0.0262



±SD



0.1942



0.1524



1.2338



0.2208



0.0008



0.0024



n



5



5



5



5



5



5



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


 


TABLE 13. SUMMARY OF TERMINAL BODY WEIGHT (g) AND ORGAN WEIGHT RELATIVE TO TERMINAL BODY WEIGHT (%) RECORD













































































































































Group, Sex


& Dose


(mg/kg body weight/day)


 

Terminal Body Weight (g)



Adrenals



Thymus



Spleen



Testes



Epididymides



Heart



G1, M & 0



Mean



439.85



0.0198



0.1026



0.2183



0.8020



0.3724



0.3667



±SD



34.39



0.0035



0.0175



0.0291



0.0485



0.0457



0.0354



n



12



5



5



5



12



12



5



G2, M & 100



Mean



443.67



0.0186



0.1001



0.2013



0.8151



0.3878



0.3747



±SD



33.97



0.0019



0.0086



0.0277



0.0608



0.0572



0.0275



n



12



5



5



5



12



12



5



G3, M & 300



Mean



453.48



0.0164



0.0970



0.1787



0.7462



0.3506



0.3398



±SD



30.58



0.0026



0.0191



0.0209



0.0736



0.0370



0.0216



n



12



5



5



5



12



12



5



G4, M & 1000



Mean



448.80



0.0153*



0.0619*



0.2552



0.7801



0.3472



0.3479



±SD



30.87



0.0016



0.0076



0.0175



0.0831



0.0303



0.0211



n



12



5



5



5



12



12



5



























































































































































Group, Sex


& Dose


(mg/kg body weight/day)


 

Kidneys



Brain



Liver



Prostate



Seminal vesicles with coagulating glands



Lungs



Pituitary



Thyroid along


with parathyroid#



G1, M & 0



Mean



0.7695



0.5426



3.1356



0.4231



0.4431



0.5469



0.0044



0.0069



±SD



0.0950



0.0549



0.3423



0.1055



0.1632



0.0930



0.0004



0.0006



n



5



5



12



12



12



5



5



12



G2, M & 100



Mean



0.8216



0.5231



3.0993



0.3991



0.4359



0.6016



0.0042



0.0070



±SD



0.0962



0.0264



0.2982



0.0719



0.1028



0.0672



0.0003



0.0005



n



5



5



12



12



12



5



5



12



G3, M & 300



Mean



0.6090*



0.4770*



3.2356



0.3790



0.4485



0.5497



0.0039



0.0071



±SD



0.0248



0.0193



0.4756



0.1129



0.1434



0.0566



0.0003



0.0005



n



5



5



12



12



12



5



5



12



G4, M & 1000



Mean



0.6853



0.4770*



3.0592



0.3793



0.5242



0.5688



0.0040



0.0069



±SD



0.0849



0.0306



0.2242



0.0864



0.0682



0.0340



0.0002



0.0005



n



5



5



12



12



12



5



5



12














































































































































Group, Sex


& Dose


(mg/kg body weight/day)


 

Terminal Body Weight (g)



Adrenals



Thymus



Spleen



Ovaries



Uterus


with Cervix



Heart



G1, F & 0



Mean



317.06



0.0166



0.0819



0.2330



0.0454



0.1931



0.3756



±SD



19.46



0.0033



0.0072



0.0466



0.0086



0.0344



0.0311



n



12



5



5



5



12



12



5



G2, F & 100



Mean



314.41



0.0213



0.0886



0.2055



0.0447



0.1897



0.3797



±SD



16.46



0.0057



0.0285



0.0443



0.0046



0.0340



0.0202



n



12



5



5



5



12



12



5



G3, F & 300



Mean



318.10



0.0208



0.0615



0.2178



0.0483



0.2291



0.4096



±SD



15.35



0.0016



0.0221



0.0788



0.0085



0.1833



0.0345



n



12



5



5



5



12



12



5



G4, F & 1000



Mean



310.30



0.0181



0.0689



0.1981



0.0478



0.1972



0.4044



±SD



20.55



0.0012



0.0070



0.0535



0.0053



0.0973



0.0488



n



12



5



5



5



12



12



5

































































































































Group, Sex


& Dose


(mg/kg body weight/day)


 

Kidneys



Brain



Liver



Lungs



Pituitary



Thyroid along


with parathyroid#



G1, F & 0



Mean



0.5977



0.6136



3.7730



0.5201



0.0052



0.0079



±SD



0.0388



0.0359



0.6834



0.0728



0.0007



0.0004



n



5



5



12



5



5



12



G2, F & 100



Mean



0.6647



0.6445



3.7396



0.6165*



0.0056



0.0078



±SD



0.0714



0.0447



0.3749



0.0362



0.0006



0.0006



n



5



5



12



5



5



12



G3, F & 300



Mean



0.6930*



0.6318



3.7941



0.6512*



0.0053



0.0076



±SD



0.0701



0.0208



0.3315



0.0416



0.0005



0.0005



n



5



5



12



5



5



12



G4, F & 1000



Mean



0.6388



0.6544



3.8669



0.6756*



0.0059



0.0080



±SD



0.0264



0.0475



0.3622



0.0578



0.0006



0.0006



n



5



5



12



5



5



12
















































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Terminal Body Weight (g)



Adrenals



Thymus



Spleen



Testes



Epididymides



Heart



G1R, M & 0



Mean



528.23



0.0128



0.0661



0.1711



0.6582



0.3118



0.3356



±SD



36.74



0.0029



0.0115



0.0068



0.0457



0.0406



0.0321



n



5



5



5



5



5



5



5



G4R, M & 1000



Mean



516.79



0.0138



0.0687



0.1695



0.7137



0.3193



0.3272



±SD



27.43



0.0032



0.0055



0.0250



0.0303



0.0224



0.0117



n



5



5



5



5



5



5



5























































































Group, Sex


& Dose


(mg/kg body weight/day)


 

Kidneys



Brain



Liver



Prostate



Seminal vesicles with coagulating glands



Lungs



Pituitary



Thyroid along


with parathyroid#



G1R, M & 0



Mean



0.5477



0.4163



2.8092



0.3217



0.3070



0.4946



0.0037



0.0056



±SD



0.0369



0.0462



0.1649



0.0630



0.1079



0.0302



0.0001



0.0003



n



5



5



5



5



5



5



5



5



G4R, M & 1000



Mean



0.5639



0.4190



2.6870



0.3319



0.3995



0.5244



0.0036



0.0059



±SD



0.0367



0.0336



0.1856



0.0565



0.1476



0.0451



0.0004



0.0002



n



5



5



5



5



5



5



5



5
















































































Group, Sex


& Dose


(mg/kg body weight/day)



 



Terminal Body Weight (g)



Adrenals



Thymus



Spleen



Ovaries



Uterus


with Cervix



Heart



G1R, F & 0



Mean



317.31



0.0224



0.0846



0.1696



0.0479



0.2159



0.3638



±SD



9.00



0.0026



0.0121



0.0252



0.0091



0.0141



0.0246



n



5



5



5



5



5



5



5



G4R, F & 1000



Mean



307.87



0.0255



0.0795



0.1964



0.0470



0.2034



0.3518



±SD



14.43



0.0010



0.0051



0.0332



0.0105



0.0288



0.0181



n



5



5



5



5



5



5



5









































































Group, Sex


& Dose


(mg/kg body weight/day)


 

Kidneys



Brain



Liver



Lungs



Pituitary



Thyroid along


with parathyroid#



G1R, F & 0



Mean



0.5665



0.6436



2.4963



0.5590



0.0058



0.0079



±SD



0.0475



0.0338



0.0963



0.0633



0.0002



0.0002



n



5



5



5



5



5



5



G4R, F & 1000



Mean



0.6261



0.6426



3.3055*



0.6276



0.0063*



0.0085



±SD



0.0451



0.0475



0.3854



0.0529



0.0002



0.0009



n



5



5



5



5



5



5



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


 


TABLE 14. SUMMARY RECORD OF MEAN PUP WEIGHT (g) PER LITTER



















































































































































































































Group & Dose


(mg/kg body weight/day)


 

PND 1


 

PND 4


 

PND 7


 

PND 13



Mean Pup Weight (g)


 

Mean Pup Weight (g)



 



Mean Pup Weight (g)



 



Mean Pup Weight (g)



Male



Female



 



Male



Female



 



Male



Female



 



Male



Female



G1 & 0



Mean



6.78



6.33



 



11.12



10.31



 



14.88



13.80



 



28.69



26.90



±SD



0.12



0.22



 



0.50



0.36



 



0.34



0.29



 



0.50



1.08



n



10 (65)



10 (54)



 



10 (65)



10 (54)



 



10 (65)



10 (43)



 



10 (65)



10 (43)



G2 & 100



Mean



6.79



6.27



 



11.33



10.45



 



15.10



14.09



 



28.48



27.52



±SD



0.17



0.13



 



0.40



0.27



 



0.42



0.40



 



0.77



0.69



n



11 (73)



11 (60)



 



11 (72)



11 (60)



 



11 (72)



11 (48)



 



11 (72)



11 (48)



G3 & 300



Mean



6.77



6.23



 



11.18



10.35



 



14.71



13.67



 



28.77



26.93



±SD



0.12



0.15



 



0.40



0.20



 



0.47



0.36



 



0.93



1.12



n



10 (64)



10 (61)



 



10 (64)



10 (61)



 



10 (64)



10 (48)



 



10 (64)



10 (48)



G4 & 1000



Mean



6.75



6.22



 



11.28



10.38



 



14.67



13.78



 



27.81*



26.24



±SD



0.10



0.12



 



0.42



0.17



 



0.30



0.29



 



0.93



1.62



n



10 (71)



10 (64)



 



10 (71)



10 (64)



 



10 (71)



10 (48)



 



10 (71)



10 (48)



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


 


TABLE 15. SUMMARY OF SERUM T4 HORMONE LEVELS (ng/mL) RECORD - POSTNATAL DAY (PND) 13 PUPS































































Group & Dose


(mg/kg body weight/day)


 

Serum T4 Levels (ng/mL)



G1 & 0



Mean



65.057



±SD



4.071



n



10



G2 & 100



Mean



59.525*



±SD



5.558



n



11



G3 & 300



Mean



65.668



±SD



4.034



n



10



G4 & 1000



Mean



61.718



±SD



3.810



n



10



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

Applicant's summary and conclusion

Conclusions:
No maternal or developmental toxicity was observed in a study performed according to OECD TG 422 with the test substance in rats by oral route. Thus, the NOAEL was determined to be the highest dose tested, i.e. 1000 mg/kg bw/day.
Executive summary:

A combined repeated dose toxicity study with the reproduction /developmental toxicity screening test of the test substance by oral administration in rats was conducted according to OECD guideline 422, in accordance with GLP principles. A total of 116 (58 males + 58 females) Sprague Dawley rats were distributed to four main groups and two recovery groups to detect delayed occurrence and recovery from toxic effects. Each main group (G1, G2, G3 and G4) consisted of 12 males and 12 females and each recovery group (G1R and G4R) consisted of 5 males and 5 females. The animals in G1/G1R group were administered with vehicle (0.5% w/v Carboxymethyl Cellulose), and the animals in G2, G3 and G4/G4R groups were administered with test item at the dose levels of 100, 300 and 1000 mg/kg bw/day based on the results of a dose range finding study performed with the same species at doses of 64, 160, 400 and 1000 mg/kg bw and in which no effects were observed up to the highest dose tested. The test item was administered to the main group males for a period of 29 days (two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period), to the main group females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13 and to the recovery group animals for a period of 50 days with a 15 days recovery. The vehicle and test item formulations were administered orally by gavage at the dose volume of 10 mL/kg bw. The stability of test item formulations in vehicle was established before initiation of the treatment. Dose formulation analysis for homogeneity and concentration verification was performed during weeks 1 and 5 of the treatment period and the results were within acceptable limits.


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


There were no treatment-related effects for the systemic toxicity assessment of the parental animals. There were no test item-related effects for clinical signs, mortality and morbidity, detailed clinical examination, body weight, feed consumption, ophthalmological examinations, clinical pathological (haematology, clinical chemistry and urinalysis) examinations and neurological/functional observations in all tested dose groups. The estimated serum T4 levels did not reveal any changes. The absolute and relative organ weights of both sexes did not reveal any changes from all tested dose groups. No gross pathological changes were observed in any of the animals from all tested dose groups during necropsy. No treatment related microscopic findings were noted in any of the tissues/organs subjected for histopathological evaluation from both high dose group and vehicle control group animals of both sexes.


There were no treatment-related effects for maternal developmental toxicity. No test item-related irregularities observed in oestrus cyclicity of the females from all tested dose groups during treatment period. No changes were noted for delivery and litter observations in all tested dose groups. No effects were noted for live birth index and pup survival index in all tested dose groups. No test item-related post-implantation losses were noted in all tested dose groups.


There were no treatment-related effects for the developmental toxicity assessment. There were no developmental/external behavioural changes noted and no test item-related mortalities noted during postnatal period in any of the pups from all tested dose group litters. The mean pup weight, mean anogenital distance and its ratio in either sex of pups per litter were unaffected by the test item in all tested dose groups. There were no incidences of retention of nipples in male pups examined on PND 13 from all tested dose and control group litters. The estimated serum T4 levels of PND 13 (from all litters) pups did not reveal any changes in all tested dose group litters. There were no gross pathological changes noted in any of the pups during scheduled sacrifice from all tested dose and control group litters.


Based on these results, the NOAEL of the test substance was determined to be 1000 mg/kg bw/day for maternal and developmental toxicity.

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