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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 June 2019 to 15 September 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Information requirement

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
[As per OECD Guideline No. 443, “Extended One-Generation Reproductive Toxicity Study”, adopted on 25 June 2018]
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS [please address all points below]:

- Premating exposure duration for parental (P0) animals: 10 Weeks
- Basis for dose level selection: Based on data obtained from a 90-day repeated dose-toxicity study conducted according OECD TG 408, the NOAEL of test item Montanol 800 is 750 mg/kg body weight/day, the highest dose tested and data obtained from a pre-natal developmental toxicity study conducted according to OECD TG 414, the NOAEL of test item Montanol 800 is 1000 mg/kg body weight/day. Hence, the doses of 0 (vehicle control), 100 mg/kg body weight/day (low dose), 300 mg/kg body weight/day (mid dose) and 1000 mg/kg body weight/day (high dose) were selected.
- Inclusion/exclusion of extension of Cohort 1B: Excluded extension of Cohort 1B.
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B: Excluded developmental neurotoxicity Cohorts 2A and 2B
- Inclusion/exclusion of developmental immunotoxicity Cohort 3: Excluded developmental immunotoxicity Cohort 3.
- Route of administration: The vehicle and test item formulations were administered through oral (gavage) route as it is the probable route of exposure to human.
- Other considerations, e.g. on choice of species, strain, vehicle and number of animals: Rat-Sprague Dawley (standard laboratory rodent species and strain used for toxicity assessment and also recommended by various regulatory authorities).

The sesame oil was used as vehicle for test item formulations as specified by the sponsor. The test item was clearly miscible with sesame oil at the concentration of 250 mg/mL (the highest dose concentration selected for the study) as per in-house miscibility test results. As sesame oil was used as the vehicle in previous repeated toxicity studies with this test item, sesame oil was selected for the test item formulation preparations.

Total Number of Animals (Parental (P) Generation): 200 (100 Males + 100 Females)
A total of 105 males and 105 females were received. The extra animals were sacrificed after grouping and randomization. Females used were nulliparous and non-pregnant.

Total Number of Animals (First Filial (F1) Generation):
Cohort 1A: 80 (20 Males + 20 Females) - one male and one female/litter/Group
Cohort 1B: 80 (20 Males + 20 Females - one male and one female/litter/Group
A total of 160 males and 160 females were selected and randomly assigned to two cohorts on the day of weaning (PND21).

Test material

Constituent 1
Reference substance name:
1-Hexanol, 2-ethyl-, manuf. of, by-products from, distn. residues
EC Number:
271-832-1
EC Name:
1-Hexanol, 2-ethyl-, manuf. of, by-products from, distn. residues
Cas Number:
68609-68-7
Molecular formula:
UVCB, not applicable, see section 1.2 for information on constituents
IUPAC Name:
1-Hexanol, 2-ethyl-, manuf. of, by-products from, distn. residues
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL

- Source and lot/batch No.of test material: Clariant Produkte (Deutschland) GmbH,
on behalf of BU-OMS, Am Unisys Park 1, D-65843 Sulzbach (Taunus), Germany and Batch No.: 890000038212
- Expiration date of the batch: January 2022
- Purity test date: 21 February 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL

- Storage condition of test material: Ambient (21 to 29oC)
- Stability under test conditions: The stability of dose formulations was established by Analytical Chemistry Department of Bioneeds India Private Limited (Study No.: BIO-ANM 1387). The test item Montanol 800 was stable up to 6 hours at room temperature with concentrations of 25 mg/mL and 250 mg/mL in sesame oil.
However, freshly prepared test item formulations were administered to the animals and homogeneity was achieved by constant and thorough stirring.

- Solubility and stability of the test substance in the vehicle: The sesame oil was used as vehicle for test item formulations as specified by the sponsor. The test item was clearly miscible with sesame oil at the concentration of 250 mg/mL (the highest dose concentration selected for the study) as per in-house miscibility test results. As sesame oil was used as the vehicle in previous repeated toxicity studies with this test item, sesame oil was selected for the test item formulation preparations.

The stability of dose formulations was established by Analytical Chemistry Department of Bioneeds India Private Limited (Study No.: BIO-ANM 1387). The test item Montanol 800 was stable up to 6 hours at room temperature with concentrations of 25 mg/mL and 250 mg/mL in sesame oil.
However, freshly prepared test item formulations were administered to the animals and homogeneity was achieved by constant and thorough stirring.

FORM AS APPLIED IN THE TEST (if different from that of starting material): The test item/vehicle were administered to animals through oral gavage using stainless steel intubation cannula attached to a disposable syringe once daily.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Rat is one of the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: In-house bred animals
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: (P) 5 to 6 weeks ; (F1) 3 weeks (treatment started on PND 21)
- Weight at study initiation: (P) Males: 158.00 g to 189.96 g; Females: 134.25 g to 177.28 g; (F1) - C1A-Males: 35.87 g to 58.64 g; Females: 32.69 g to 59.53 g; C1B-Males: 34.84 g to 59.83 g; Females: 35.04 g to 58.39 g
- Fasting period before study: Animals were not fasted before study.
- Housing: Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted food and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.

P Animals:
i. Pre mating: Maximum of two animals of same sex and group per cage were housed.
ii. Mating: During mating, two animals (one male and one female) of same group were housed.
iii. 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 during gestation and lactation periods. Sterilized paper shreds were provided as a nesting material from gestation day 20 onwards.

F1 Animals:
i. During Lactation period (postnatal period): All F1 pups of both the sex were housed along with the dam.
ii. Post Weaning: Maximum of two animals of same sex and group per cage were housed.

- Diet (e.g. ad libitum): Altromin Maintenance diet for rats and mice 1324 manufactured by Altromin Spezialfutter GmbH & Co. KG was provided ad libitum to the rats throughout the experimental period.

- Water (e.g. ad libitum): Water was provided ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.

- Acclimation period: 5 Days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 to 22.9oC
- Humidity (%): 42 to 67%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle.
IN-LIFE DATES: From: 15 June 2019 To: 10 January 2020

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Sesame Oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item formulations were freshly prepared before dose administration on each treatment day. The required quantity of test item was weighed into a clean beaker and small volume of the vehicle was added into the beaker, mixed well using glass rod and transferred into measuring cylinder. Rinsing procedure was repeated until complete transfer of test item formulation into the measuring cylinder. Finally, the volume was made up to the required quantity with vehicle to get a desired concentration of 25, 75 and 250 mg/mL of test item for low, mid and high dose groups respectively.

VEHICLE
- Justification for use and choice of vehicle: The sesame oil was used as vehicle for test item formulations as specified by the sponsor. The test item was clearly miscible with sesame oil at the concentration of 250 mg/mL (the highest dose concentration selected for the study) as per in-house miscibility test results. As sesame oil was used as the vehicle in previous repeated toxicity studies with this test item, sesame oil was selected for the test item formulation preparations.
- Concentration in vehicle: 0 mg/mL (vehicle control), 25 mg/mL (low dose), 75 mg/mL (mid dose) and 250 mg/mL (high dose)
- Amount of vehicle (if gavage): 4 mL/kg body weight
- Lot/batch no. (if required): Vehicle Lot No. Expiry Date Make
Sesame Oil AG5002 March-2022 MP Biomedicals
AG5003 June-2022
N2190508 June-2022
N2191406 June-2022
O31902003 October-2022
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 Days
- Proof of pregnancy: sperm in vaginal smear, referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: 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 during gestation and lactation periods. Sterilized paper shreds were provided as a nesting material from gestation day 20 onwards.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity and dose formulation analysis for dose concentration verification was done by Analytical Chemistry department of Bioneeds India Private Limited. The analysis was done as per methods detailed in the Study Plan No. BIO-ANM 1387 and the results were presented in the study report. Sampling and analysis of formulations were performed during first and last week of P generation treatment and first and last week of F1 generation treatment. Approximately 5 mL of samples were collected in duplicates from the top, middle and bottom layers from the low, mid and high dose concentrations, and in duplicates from the middle layer for the vehicle control. One set of aliquot of each formulation was analyzed. The second aliquot was stored as a backup at established stability conditions. The second set of samples were discarded, after the analysis results of first set of samples were found within the limits. 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:
parental (P) generation
Males: Pre-mating period (10 weeks) and a two-week mating period.
Females: Pre-mating period (10 weeks), a two-week mating period and during pregnancy and lactation until termination (Lactation Day 21).
F1 Animals:
Cohort 1A - From day of weaning (PND 21) to Post Natal Day (PND) 91 [13 weeks age]
Cohort 1B - From day of weaning (PND 21) to Post Natal Day (PND) 98 [14 weeks age]
Frequency of treatment:
Once Daily
Details on study schedule:
- Age at mating of the mated Parental (P) animals in the study: 15 to 16 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Vehicle Control
Dose / conc.:
100 mg/kg bw/day
Remarks:
Low Dose
Dose / conc.:
300 mg/kg bw/day
Remarks:
Mid Dose
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
High Dose
No. of animals per sex per dose:
Parental Animals: 25 Males + 25 Females per dose
F1 Generation: Cohort 1A and Cohort 1B: 20 Males + 20 Females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on data obtained from a 90-day repeated dose-toxicity study conducted according OECD TG 408, the NOAEL of test item Montanol 800 is 750 mg/kg body weight/day, the highest dose tested and data obtained from a pre-natal developmental toxicity study conducted according to OECD TG 414, the NOAEL of test item Montanol 800 is 1000 mg/kg body weight/day. Hence, the doses of 0 (vehicle control), 100 mg/kg body weight/day (low dose), 300 mg/kg body weight/day (mid dose) and 1000 mg/kg body weight/day (high dose) were selected as recommended by the sponsor.

- Rationale for animal assignment: Parental animals were weighed and arranged in ascending order of their body weights. The body weight stratified animals were distributed to different groups using Microsoft Excel Spreadsheet, such that body weight variation of animals selected for the study did not exceed ±20% (Males: -13.07% to 14.14%; Females: -11.03% to 13.39%) of the mean body weight within each group. The grouping was done one day prior to the initiation of treatment. Body weight of the animals was analyzed statistically for mean body weight to rule out the statistical significant difference between groups.

Allocation of F1 Animals: At weaning (on postnatal day 21), 20 animals per dose and group from all available litters (one male and one female/litter) were selected randomly for further examinations and maintained until sexual maturation.

On postnatal day 21, the selected F1 pups were randomly assigned to following cohorts:
Cohort 1A: 1 male and 1 female/litter/group (20/sex/group)
Cohort 1B: 1 male and 1 female/litter/group (20/sex/group)

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once Daily
- Cage side observations checked in table [No.1] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly Once

BODY WEIGHT: Yes
- Time schedule for examinations:
Pre-mating Period: All the parental generation males and females were weighed on the day of initiation of treatment and once a week thereafter.
Cohabitation (mating) Period: All the parental generation males and females in cohabitation period were weighed once a week.
Gestation Period: All the parental generation females were weighed on gestation day (GD) 0, 7, 14 and 20.
Lactation Period: All the parental generation females were weighed on lactation day (LD) 1, 4, 7, 14 and 21.
The body weight of each animal was taken on the day of termination to calculate the relative organ weight.

Feed Consumption:
Pre-mating Period: Feed consumption was recorded for all the parental generation males and females once a week coinciding with body weight recording.
Cohabitation (mating) Period: Feed consumption was not measured during cohabitation period.
Gestation Period: Feed consumption was recorded for all the parental generation females during gestation day (GD) 0 to 7, 7 to 14 and 14 to 20.
Lactation Period: Feed consumption was recorded for all the parental generation females during lactation day (LD) 1 to 4, 4 to 7, 7 to 14 and 14 to 21.
The feed consumption was calculated as cage wise average feed intake (g/animal/day).

Oestrus Cyclicity:
Oestrus cycles were monitored daily for a period of 2 weeks during pre-mating period before mating and during mating until evidence of mating. When obtaining vaginal/cervical cells, care was taken to avoid disturbance of mucosa, which may induce pseudo-pregnancy. Oestrus cyclicity was also monitored on theday of sacrifice for females.

Mating and Pregnancy
The dates of pairing, mating and parturition were recorded and the precoital interval (pairing to insemination) and the duration of pregnancy (insemination to parturition) was calculated. The P females were examined carefully at the time of expected parturition for any signs of dystocia.
The day on which parturition occurs was considered as lactation day (LD) 1 for the dam and postnatal day (PND) 1 for the offspring.

Terminal Observations
Clinical Pathology
Blood was collected from 10 randomly selected males and 10 randomly selected females from each group at termination and subjected to hematology, clinical chemistry, thyroid hormone (Thyroxine Hormone-T4 and Thyroid Stimulating Hormone-TSH) levels and urinalysis.
One day before scheduled terminal sacrifice, the animals were fasted overnight. Water was provided ad libitum during the fasting period. Blood samples were collected from the animals separately into tubes containing K2-EDTA (2 mg/mL of blood) and sodium heparin (10 IU/mL of blood) for haematology and clinical chemistry analysis, respectively. Sodium citrate (3.2%) tubes were used for Prothrombin time and activated partial thromboplastin time parameters. Blood samples were collected through
retro-orbital plexus puncture method under Isoflurane Anaesthesia with the help of a fine capillary tube.
a. Hematology
Following hematology parameters were estimated using Advia 2120 Hematology System (Siemens Limited).
Parameters Units
Haemoglobin concentration (HGB) g/dL
Haematocrit (HCT) %
Erythrocyte count (RBC) 106 cells/µL
Total Leukocyte Count (WBC) 103 cells/µL
Platelet count (PLT) 103 cells/µL
Differential leucocytes count (DLC) %
Absolute differential leucocytes Count (DLC) 103 cells/µL

Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) were estimated with the help of coagulation analyzer [Tulip Diagnostics (p) Ltd., India]

b. Clinical Chemistry
Following clinical chemistry parameters were analysed using “Rx Daytona+ Clinical Chemistry Analyser” (Randox Laboratories).
Parameters Units
Glucose mg/dL
Blood Urea mg/dL
Creatinine mg/dL
Total cholesterol mg/dL
Total protein g/dL
Albumin g/dL
Aspartate aminotransferase (AST) U/L
Alanine aminotransferase (ALT) U/L
Alkaline phosphatase (ALP) U/L
Plasma was separated by centrifuging the blood samples at 5000 rpm for 10 minutes for determining the clinical chemistry and 1500 rpm for 15 minutes for determining the Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) parameters.

Thyroid Hormone Levels Estimation
Serum T4 and TSH levels were estimated using commercially available ELISA kits from 10 randomly selected males and 10 randomly selected females from each group. The collected blood samples were centrifuged at 5000 rpm for 10 minutes, the serum was separated and stored at frozen conditions (-80ºC).

d. Urinalysis
Urine was collected from 10 randomly selected males and females per dose group at termination and subjected to urinalysis for the following parameters.
The volume of urine collected (mL), appearance and the color was recorded by physical evaluation.

Parameters Units
Blood Ery/µL
Protein mg/dL
Glucose mg/dL

Urine was analyzed using Urine Analyzer DIRUI/H500 (Dirui Industrial Company Ltd.).
In addition, pH and specific gravity was also analyzed. After analysis of the above parameters, the remaining urine was centrifuged at 1500 rpm for 3 minutes and subjected to microscopic examination for urine sediments.
The animals were kept in urine collection cages overnight and did not have access to feed. Water was provided ad libitum during their stay in urine collection cages. Overnight urine volume (mL) collected from these animals was measured.
Oestrous cyclicity (parental animals):
Oestrus cycles were monitored daily for a period of 2 weeks during pre-mating period before mating and during mating until evidence of mating. When obtaining vaginal/cervical cells, care was taken to avoid disturbance of mucosa, which may induce pseudo-pregnancy. Oestrus cyclicity was also monitored on the day of sacrifice for females.
Sperm parameters (parental animals):
Parameters examined in parental males:
Males were randomized throughout all dose groups and restricted to maximum of 20 animals per day for sperm parameters analysis. Sperm parameters were measured in all P generation males.

At termination, testes and epididymis weights were recorded for all P males. One testes and one epididymis was reserved for histopathological examination. Remaining epididymis was used for enumeration of cauda epididymis sperm reserves. In addition, sperm from the cauda epididymis (or vas deferens) was collected for evaluation of sperm motility and morphology.

Sperm motility was evaluated immediately after sacrifice. The percentage of progressively motile sperms was determined subjectively. Samples for evaluating sperm morphology was taken from the suspension used for the sperm motility. Sperm sample was examined as fixed and at least 200 spermatozoa per sample classified as either normal (both head and midpiece/tail appear normal) or abnormal. The other testes were stored in -20˚C and were evaluated for homogenization resistance spermatid head counts.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- For standardizing, the litter size was adjusted to five of each sex by elimination of the additional pups in the litter on PND 4 after birth. The adjustment was made randomly and the procedure was documented.
For those litters with sufficient pups but an unequal number of males and females, the litter was standardized to 10 such that each sex represented as far as possible (e.g. 2 males + 8 females). This variation in procedure was to avoid the unnecessary waste of animals when these were used to generate additional data.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Observations of F1 Pups at Birth: Number of pups born (dead and live) in each litter, sex of pups, sex ratio, live births, and external examination of each pup was recorded at birth. The live birth index was calculated for each dam at birth.
- Daily Clinical Examinations: All the pups were observed for daily clinical examination. The litter was observed daily in order to note the number of alive, dead and cannibalized pups. All the dead and sacrificed pups were examined for malformations and subjected to gross pathological examination.
- Pup Weight: Individual body weight of live pups on lactation day 1, 4, 7, 14 and 21 was recorded.
- Postnatal Developmental Landmarks: The days on which the following postnatal developmental land marks appear in litters were recorded:
a. Pinna Unfolding: Beginning on PND 1, each pup in every litter was observed individually until all pups in the litter met the criterion for pinna unfolding i.e. the point of pinna was detached from the head.
b. Hair coat Development: Beginning on PND 2, each pup in every litter was observed for appearance of fur on the body i.e. first appearance of fur on the body.
c. Incisor Eruption: Beginning on PND 6, each pup in every litter was observed for eruption of all four incisors through the gums until the criterion was met in all the pups.
d. Eye Opening: Beginning on PND 10, all pups in every litter were observed until all pups in the litter met the criterion for eye opening, i.e. the separation of upper eye lid from the lower eye lid in both the eyes.
e. Testes Descent: Beginning on PND 19 till weaning, all male pups in every litter were observed and the days when both testes fully descend into the scrotal sac were recorded.
f. Vaginal Opening: The age of vaginal opening was determined for F1 female weanlings. All selected F1 females were observed daily starting from PND 22 till occurrence for vaginal patency. Sexual maturity of F1 animals was compared to physical development by determining age and body weight at vaginal opening for females.
g. Balano-preputial Separation: The age of balanopreputial separation was determined for F1 male weanlings. All selected F1 males were evaluated daily starting from PND 35 till occurrence for balano-preputial separation. Sexual maturity of F1 animals was compared to physical development by determining age and body weight at balano-preputial separation for males.

- Reflex and Sensory Tests: The following reflex and sensory tests were conducted in F1 litters.
a.Surface Righting Reflex: Surface righting reflex evaluation was initiated on PND 4 and continued till the pups met the passing criterion.
b. Auditory Startle Reflex: Auditory startle reflex was evaluated daily beginning on PND 10 to till the achievement of criterion.
c. Air Righting Reflex: Air righting reflex was evaluated daily beginning on PND 17 to till the achievement of criterion.

- Observations for Surplus Pups on PND 4:
Blood from PND 4 surplus pups was collected by jugular vein incision and were subjected to gross necropsy. The collected blood was separated for serum and was measured for serum thyroid hormone (T4) concentrations. The neonatal (PND 4) blood was pooled by litters for thyroid hormone analysis.

- Ano-genital Distance (AGD) Measurement on PND 4:
Ano-genital distance of each pup was measured on postnatal day 4 and the ratio of AGD to the cube root of pup body weight was calculated.

- Appearance of Nipples/Areolae on PND 13:
All survived male pups were examined for appearance of nipples/areolae on postnatal day 13.

- Observations of F1 Generation surplus pups not allocated to cohorts (at weaning):
Body weights were recorded on PND 21 for all the surplus pups not selected for cohorts.
The blood was collected through retro-orbital plexus puncture method under Isoflurane anaesthesia with the help of a fine capillary tube for T4 and TSH analysis from these weanlings and subjected to gross necropsy on PND 21.
The brain, spleen, thymus (weighed) and mammary tissues were collected and preserved for possible histopathology.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All the survived P males were sacrificed after completion of mating procedure. Males were randomized throughout all dose groups and restricted to maximum of 20 animals per day for necropsy. The animals were fasted overnight, water was provided ad libitum during fasting. The next day, the body weight of all the fasted animals were recorded prior to exsanguination. Gross pathological examination was performed on all the P generation males. A special attention was paid to the organs of the reproductive system. The animals were euthanized using CO2 asphyxiation followed by exsanguination and subjected to gross necropsy, external and internal gross pathological examination.

- Maternal animals: The females confirmed with mating but not littered were sacrificed on gestation day 25. The survived littered females were sacrificed on lactation day 22. The animals were fasted overnight on lactation day 21, water was provided ad libitum during fasting. The next day (lactation day 22), the body weight of all the fasted animals were recorded prior to exsanguination. The animals were euthanized using CO2 asphyxiation followed by exsanguination and subjected to gross necropsy, external and internal gross pathological examination.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera, special attention was paid to the organs of the reproductive system.

HISTOPATHOLOGY / ORGAN WEIGHTS:
Histopathological examination was conducted on all the tissues collected from the vehicle control and high dose group animals (with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure) sacrificed at termination. Additionally, reproductive organs of all animals and all gross lesions, organs and tissue samples were processed, embedded in paraffin, sectioned at a thickness of 3 to 5 micrometers and stained with hematoxylin and eosin. The testes were sectioned at 3 to 4 micrometers and stained with Hematoxylin and eosin stain and also with Periodic Acid-Schiff (PAS) and hematoxylin stain. PAS stain aided in spermatogenesis evaluation.
Quantitative follicle assessment for 10 P females/group in control and high dose groups was conducted.
The investigations were not extended to the lower dose groups as there were no signs of treatment related effects at the high dose level (target organs).
At the time of termination, terminal (fasting) body weights and wet weights of the organs listed below from all P animals were determined as soon as possible after dissection to avoid drying. These organs were preserved under appropriate conditions. Paired organs were weighed combined.
Postmortem examinations (offspring):
SACRIFICE
- For the F1 offsprings not selected for Cohorts, Body weights were recorded on PND 21. The blood was collected through retro-orbital plexus puncture method under Isoflurane anaesthesia with the help of a fine capillary tube for T4 and TSH analysis from these weanlings and subjected to gross necropsy on PND 21.
The brain, spleen, thymus (weighed) and mammary tissues were collected and preserved for possible histopathology.

- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Special attention was paid to the organs of the reproductive system.

ORGAN WEIGTHS
The brain, spleen, thymus were weighed.
Statistics:
The raw data was subjected to computer statistical processing. The computer printout of the data (in the form of appendix) was verified with the raw data. After verification, the data was subjected to various statistical analyses using SPSS software version 22.

All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05), indicated by the aforementioned tests were designated by the superscripts throughout the report as "* Statistically significant (P<0.05) change compared to vehicle control group".
Note: Data of non-pregnant animals excluded for mean calculations and statistical analysis.

The Parametric - One-way ANOVA with Dunnett’s post test was followed to the parameters as mentioned below:
• Body weight (weekly, gestation and lactation)
• Percent change in body weight (weekly, gestation and lactation)
• Feed consumption (weekly, gestation and lactation)
• Copulatory interval
• Gestation length
• Absolute/relative organ weights
• Mean Pup Ano-genital distance
• Mean Pup weight
• Mean postnatal developmental landmarks (F1 pups)
• Mean sensory reflexes (F1 pups)

The Non Parametric - Kruskal-Wallis method was followed to the parameters as mentioned below:

• Implantations/dam
• No. of pups/dam
• Sex ratio (m/f)
• Litter size
• No. of early resorptions
• No. of late resorptions
• Pre implantation loss
• Pre natal loss
• Post natal loss
• Post implantation loss

The Cross Tabs - Chi-square test was followed to the parameters as mentioned below:

• Pregnancy rate
• No. of litters with/without live pups
• No. of litters with/without dead pups
• No. of litters with/without resorptions
Reproductive indices:
Male Mating Index (%) : No. of males with evidence of mating / Total no. of males used for mating X 100
Male Fertility Index (%) : No. of males siring a litter / Total no. of males used for mating X 100
Female Mating Index (%) : No. of females with evidence of mating (or confirmed pregnancy) / Total no. of females used for mating X 100
Female Fertility Index (%) : No. of females with confirmed pregnancy / Total no. of females used for mating X 100
Precoital Interval (Days) : Date of confirmation mating – Date of initiation of cohabitation
Mean Gestational Length : Date of parturition – Date of positive evidence of mating (GD 0)
Gestation Index (%) : No. of females with live born / No. of females with evidence of pregnancy X 100








Offspring viability indices:
Mean No. of Corpora Lutea : Sum of No. of corpora lutea in the group / Total No. of dams in the group X 100
Mean No. of Implantations : Sum of No. of implantations in the group / Total no. of dams in the group X 100
Pre-natal Loss/Post-Implantation Loss (%) : No. of Implantations - No. of Viable pups / No. of Implantations X 100
Pre-natal Loss (No.) : No. of Implantations – No. of Live Births
Postnatal Loss on Lactation Day 4/14/21 (No.) : Live births - pups alive at Lactation Day 4/14/21
Postnatal Loss on Lactation Day 4/14/21 (%) : No. of pups alive on Lactation Day 4/14/21 / No. of live pups at birth X 100
Mean Litter Size per Group : Total No. of pups delivered (live and stillborn) / No. of dams that delivered
Live Birth Index (%) per dam : No. of pups born alive / Total No. of pups born X 100
Pup Survival index (%) on Lactation Day 4 : Total No. of live pups (on Lactation Day 4) / No. of pups born X 100
Sex ratio (m/f) : No. of male offspring / No. of female offspring
Percentage of male/female offspring (%) : No. of male/female offspring / Total No. of offspring X 100
Ano-genital Distance to cube root of the body weight Ratio (AGD ratio) : Ano-genital Distance (mm) / Cube root of the body weight

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs of toxicity noted throughout the experimental period at all the tested dose groups. The detailed clinical examination did not reveal any changes in any of the tested dose groups throughout the experimental period.
Mortality:
no mortality observed
Description (incidence):
There were no mortality/morbidity noted throughout the experimental period at all the tested dose groups.
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 at any of the tested dose groups from both P generation males and females during experimental period when compared with the vehicle control group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no changes noted in mean feed consumption during pre-mating (both males and females) and post-mating (males) treatment period at any of the tested dose groups from both P generation males and females when compared with the vehicle control group.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related changes noted in haematology parameters at any of the tested dose groups from both P generation males and females when compared with the vehicle control group. A statistically significant increase in mean haematocrit levels of group G3 males compared with vehicle control group is considered to be incidental and not test item-related, as this change is lacking dose dependency and all values are still within in-house historical control data range of same species and strain. (In-house Historical Control Range:- Haematocrit levels - Males: 35.7 % to 52.2 %).
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related changes noted in clinical chemistry parameters at any of the tested dose groups from both P generation males and females when compared with the vehicle control group. Statistically significant increases in mean total protein levels of group G4 males as well as in mean albumin levels of groups G3 and G4 males compared with the vehicle control group are considered to be incidental and not test item-related, as these changes are lacking relevant dose dependency and values are still within in-house historical control data range of same species and strain (In-house Historical Control Range:- Total Protein Levels (g/dL) - Males: 35.7 to 52.2; Albumin (g/dL) - Males: 2.3 to 4.0).
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no test item-related changes noted in urinalysis parameters at any of the tested dose groups from both P generation males and females when compared with the vehicle control group.
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related histopathological findings in high dose parental animals of both the sex. Few of the microscopic findings observed in the study were considered incidental as they occurred randomly across the dose groups including controls and/or were expected for laboratory rats.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid Hormone Levels: There were no test item-related changes noted in serum Thyroxine (T4) and Thyroxine Stimulating Hormone (TSH) levels at any of the tested dose groups from both P generation males and females. A statistically significant increase in mean serum TSH levels observed for group G3 males compared with vehicle control group males is considered to be incidental and not test item-related as all values from all dose groups are well within in-house historical control range of same species and strain. In-house Historical Control Range:- TSH Levels (µIU/mL) - Males: 0.172 to 9.398 and Females: 0.385 to 8.806; T4 Levels (ng/mL) - Males: 43.596 to 108.858 and Females: 55.100 to 91.090).

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The oestrus cycles evaluated during pre-mating period did not reveal any irregularities in mean length of oestrus cycle (days) at any of the tested dose groups and vehicle control group.

During cohabitation period, females with at least one complete oestrus cycle are considered for determination and there were no irregularities in mean length of oestrus cycle (days) at any of the tested dose groups and vehicle control group.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
Sperm Motility (%): There were no effects noted in mean sperm motility (%) of any P generation tested dose group males when compared with the vehicle control group.

Sperm Morphology: There were no test item-related sperm morphological changes noted in any P generation tested dose group males when compared with the vehicle control group. The noted sporadic statistically significant differences in occurrence of number and percentage of sperm abnormalities at groups G3 and G4 parental males are considered as toxicologically irrelevant.

Spermatid Head Count and Daily Sperm Production: There were no test item-related changes observed in mean spermatid head count or daily testicular sperm production per animal at any of the tested dose groups from P generation when compared with the vehicle control group. However, sporadic statistically significant differences like, increase in mean spermatid head count at group G2 and increase in mean daily sperm production per testes at groups G2 and G4 are considered as toxicologically irrelevant.

Reproductive performance:
no effects observed
Description (incidence and severity):
There were no effects noted in P generation male reproductive performance/indices [mating index (%) and fertility index (%)] and P generation female reproductive performance/indices [pre-coital interval, gestation length, mating index (%), fertility index (%), fecundity or pregnancy index (%), gestation index (%), post-implantation loss (%) and postnatal loss (%)] at any of the tested dose groups when compared with vehicle control group.

Details on results (P0)

The test item Montanol 800 did not reveal any systemic toxicity, reproductive toxicity and developmental toxicity at all the tested dose levels (100, 300 and 1000 mg/kg body weight/day) when administered for a period of 10 weeks during pre-mating period (both P males and females), 2 weeks during mating period (both P males and P females), throughout gestation and lactation periods up to weaning of their F1 animals.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
urinalysis
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
other: Thyroid hormone (T4 and TSH) Levels

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
Pre Weaning: There were no changes in daily observation of F1 pups noted in any of F1 pups from all the tested dose groups litters noted at birth and during postnatal period till Postnatal Day 21.
Cohort 1A: There were no clinical signs of toxicity noted throughout the treatment period at all the tested dose group animals of either sex.
Cohort 1B : There were no clinical signs of toxicity noted throughout the treatment period at all the tested dose group animals of either sex.
Mortality / viability:
no mortality observed
Description (incidence and severity):
Preweaning: There were no test item related mortalities noted in any of F1 pups from all the tested dose groups litters noted at birth and during postnatal period.
Cohort 1A: There were no mortality/morbidity noted throughout the treatment period at all the tested dose C1A groups animals of both the sex.
Cohort 1B: There were no mortality/morbidity noted throughout the treatment period at all the tested dose C1B groups animals of both the sex.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Pup Weight: There were no test item related changes were noted in mean pup weight of both the sex per litter on postnatal day (PND) 1, 4, 7, 14 and 21 at any of the tested dose group litters when compared with vehicle control group litters. However, statistically significant decrease in mean male pup weight on PND 7 and 14 was noted at group G3 and G4 when compared with vehicle control group. These noted changes could be considered as incidental but not treatment related as there were no changes in daily pup observations and also there were no effects noted in developmental landmarks of F1 pups during postnatal period in both these dose groups.
Cohort 1A: There were no changes noted in mean body weight and percent change in mean body weight gain with respect to postnatal day 21 at any of the tested dose C1A group animals of both the sex during experimental period when compared with vehicle control group.
Cohort 1B: There were no test item-related changes noted in mean body weight and percent change in mean body weight gain with respect to postnatal day (PND) 21 at any of the C1B tested dose group animals of both the sex when compared with the vehicle control group. Statistically significant sporadic decrease in mean body weight on PND 35 at group G3-C1B and G4-C1B females and on PND 42 at group G4-C1B females compared to the vehicle control group are considered as incidental and unrelated to test item due to a lack of dose dependency and also because the percent change in body weight gain and or mean feed consumption were unaffected during these periods at these dose levels.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A: There were no test item-related changes noted in mean feed consumption at any of the tested dose C1A group animals of both the sex during the treatment period when compared with vehicle control group. However, statistically significant reduction in mean feed consumption during PND 70 to 77 was noted at group G3-C1A males when compared with vehicle control group males. This noted reduction is considered as incidental and unrelated to treatment as the change is an isolated occurrence throughout the treatment period and also the body weight and percent change in body weight gain are unaffected during this period at this dose level.
Cohort 1B: There were no test item-related changes noted in mean feed consumption at any of the C1B tested dose group animals of both the sex during the treatment period when compared with the vehicle control group. Sporadically statistically significant reductions in mean feed consumption during PND 35 to 42 at group G2-C1B males and statistically significant increase in mean feed consumption during PND 84 to 91 at group G3-C1B females compared to animals of the vehicle control group are considered as incidental and unrelated to treatment as these changes are sporadic and not consistent throughout the treatment period and also the body weight and percent change in body weight gain remained unaffected during these periods at these dose levels.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A: There were no test item-related changes noted in haematology parameters at any of the tested dose C1A groups of both the sex when compared with concurrent vehicle control group.
Some statistically significant changes were noted in haematology parameters when compared with the vehicle control group:
- decrease in total leucocyte count (WBC) at group G3-C1A males;
- increase in total erythrocyte count (RBC) at groups G2-C1A and G4-C1A males;
- decrease in absolute lymphocytes at group G2-C1A males;
- decrease in absolute monocytes at group G3-C1A males;
- increase in monocyte (%) levels at group G3-C1A females
These observed changes are considered as incidental and not test item-related, as these changes are sporadic, lacking dose dependency and the obtained values are still within the in-house historical control data range of same species and strain.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A: There were no test item-related changes noted in clinical chemistry parameters at any of the tested dose C1A groups of both the sex when compared with vehicle control group. Statistically significant increases in mean total protein and albumin levels of group G4-C1A males; statistically significant increase in alkaline phosphatase (ALP) levels of group G3-C1A were noted when compared with the vehicle control group. These changes are considered as incidental but not test item-related, as these changes are lacking dose dependency and the obtained values are still within in-house historical control data range of same species and strain.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Cohort 1A: There were no changes noted in urinalysis parameters at any of the tested C1A groups of both the sex when compared with the vehicle control group.
Sexual maturation:
no effects observed
Description (incidence and severity):
Cohort 1A:
Balano-Preputial Separation: There were no changes noted in the mean days in occurrence of balano-preputial separation and mean body weight on the day of occurrence of sexual maturation (balano-preputial separation) at any of the tested dose groups when compared with vehicle control group.
Vaginal Patency, Occurrence of First Cornified Cells and Time Interval between Vaginal Patency and Occurrence of First Cornified Cells: There were no changes noted in mean days in occurrence of vaginal patency, mean body weight on the day of occurrence of sexual maturation (vaginal patency), mean occurrence of first cornified cells (days) and time interval between these two events (vaginal patency to occurrence of first cornified cells) at any of the C1A tested dose groups when compared with vehicle control group.

Cohort 1B:
Balano-Preputial Separation (Males)/Vaginal Patency (Females): There were no changes noted in the mean days in occurrence of balano-preputial separation (males), mean days in occurrence of vaginal patency (females) and mean body weight on the day of occurrence of sexual maturation at any of the C1B tested dose groups when compared with the vehicle control group.
Anogenital distance (AGD):
no effects observed
Nipple retention in male pups:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Absolute and Relative Organ Weights (PND 21 Surplus Pups): There were no test item treatment related changes noted in the mean absolute or relative organs weights [thymus, spleen and brain] of PND 21 surplus pups of either sex at any of the tested dose groups when compared with vehicle control group.

Cohort 1A (Absolute and Relative Organ Weight): There were no test item-related changes observed in mean absolute and relative organ weights at any of the tested dose groups from both C1A males and females when compared with the vehicle control group.
Some sporadic statistically significant changes were noted in organ weights (absolute or relative) such as,
- decrease in mean absolute thymus weight at groups G2-C1A, G3-C1A and G4-C1A males;
- decrease in mean relative thymus weight at group G2-C1A males;
- increase in mean relative kidneys weight at group G4-C1A males
These changes are considered as incidental but not test item-related because, neither gross pathological changes (at all the tested dose groups) nor microscopic changes were noted in any of the organs at high dose group animals and the obtained values are still within the in-house historical control data range of same species and strain (In-house Historical Control Range:- Thymus Weight (g) – Males: 0.1698 g to 0.6197 and Females: 0.1379 to 0.5129; Kidneys Weight (g) - Males: 1.8225 to 4.6780 and Females: 1.0006 to 3.4366; Liver Weight (g) - Males: 7.4833 to 16.9590 and Females: 4.7612 to 13.5142 ).

Cohort 1B (Absolute and Relative Organ Weight): There were no test item-related changes observed in mean absolute and relative organ weights at any of the tested dose groups from both C1B males and females when compared with the vehicle control group. Some of the sporadic statistically significant changes such as, decrease in mean pituitary (absolute and relative) weight at group G2-C1B males, in mean mandibular lymph nodes (absolute and relative) weight at all the tested dose group C1B males as well as in mean ovaries (absolute) weight at group G2-C1B females were considered as incidental but not test-item related because, neither gross pathological changes nor microscopic changes were noted in any of the organs at high dose group animals and the obtained values are still within the in-house historical control data range of same species and strain (In-house Historical Control Range:- Pituitary Gland Weight (g) – Males: 0.0120 g to 0.0453 g and Females: 0.0117 g to 0.0341 g; Ovaries Weight (g) – 0.1180 g to 0.2435 g).
Gross pathological findings:
no effects observed
Description (incidence and severity):
PND 21 Surplus Pups: There were no gross pathological changes noted in F1 pups of either sex, whether found dead or sacrificed terminally on PND 4 or 21 at any of the tested dose groups and vehicle control group during.

Cohort 1A: There were no gross pathological changes/findings noted in any of the tested dose group and vehicle control group animals of C1A males and females during conduct of necropsy.

Cohort 1B: There were no gross pathological changes/findings noted in any of the tested dose group and vehicle control group animals of C1B males and females during conduct of necropsy.
Histopathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A: There were no test item-related histopathological findings noted in high dose group parental animals and C1A animals. Few of the microscopic findings observed in the study were considered incidental as they occurred randomly across the dose groups including controls and/or were expected for laboratory rats.
Quantitative ovarian follicular assessment (primordial and primary follicles) in randomly selected parental females and C1A females of control and high dose groups did not reveal any test item related variations.

In addition, quantitative evaluation of corpora luteal count in C1A females of control and high dose group did not reveal any test item related variations.


Other effects:
no effects observed
Description (incidence and severity):
There were no changes noted in serum Thyroxine (T4) and Thyroid Stimulating Hormone (TSH) levels at any of the tested dose C1A group animals of both the sex when compared with vehicle control group. (In-house Historical Control Range:- TSH Levels (µIU/mL) - Males: 0.172 to 9.398 and Females: 0.385 to 8.806; T4 Levels (ng/mL) - Males: 43.596 to 108.858 and Females: 55.100 to 91.09).

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

The test item Montanol 800 did not reveal any systemic and reproduction toxicity at all the tested dose levels (100, 300 and 1000 mg/kg body weight/day) when administered to F1 generation animals from PND 21 to 91 (for C1A animals) and PND 21 to 98 (for C1B animals).

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1 (cohort 1A)
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
sexual maturation
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
urinalysis
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
other: Thyroid Hormone (T4 and TSH) Analysis

Overall reproductive toxicity

Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)

Any other information on results incl. tables

TABLE 25.      SUMMARY OF PUP OBSERVATIONS RECORD DURING POSTNATAL PERIOD - F1 GENERATION

Group & Dose

(mg/kg body weight/day)

At Birth

(PND 1)

PND

1 to 4

Pups Sacrificed for Litter Standardization (No.)*

PND

4 to 7$

PND

7 to 14$

PND

14 to 21$

G1 & 0

No. of Dams#

23

23

21

23

23

23

No. of Live Pups

298

298

68

230

230

230

Pup Observation/

No. of Pups observed

N/298

N/298

-

N/230

N/230

N/230

G2 & 100

No. of Dams#

22

22

18

22

22

22

No. of Live Pups

277

277

60

217

217

217

Pup Observation/

No. of Pups observed

N/277

N/277

-

N/217

N/217

N/217

G3 & 300

No. of Dams#

23

23

16

23

23

23

No. of Live Pups

274

274

51

221

221

221

Pup Observation/

No. of Pups observed

N/274

N/272

D/2

-

N/221

N/221

N/221

G4 & 1000

No. of Dams#

21

21

20

21

21

21

No. of Live Pups

271

271

61

210

210

210

Pup Observation/

No. of Pups observed

N/271

N/271

-

N/210

N/210

N/210

N: Normal; D: Dead; PND: Postnatal Day: #: Dams confirmed with littering; -: Not Applicable

*: Pups selected from dams for litter standardization with litter size more than 10; $: Pups sacrificed on PND 4 were excluded

TABLE 26.      SUMMARY OF MEAN PUP WEIGHT (g) PER LITTER RECORD - F1 GENERATION

Group

& Dose

(mg/kg body

weight/day)

PND 1

PND 4

PND 7

PND 14

PND 21

Pup Weight (g)

Pup Weight (g)

Pup Weight (g)

Pup Weight (g)

Pup Weight (g)

Male

Female

Male

Female

Male

Female

Male

Female

Male

Female

G1 & 0

Mean

6.68

6.26

10.96

10.37

15.73

14.64

30.05

28.43

48.88

47.67

±SD

0.37

0.34

0.37

0.36

0.57

0.31

0.68

0.38

4.50

4.76

n

23

23

23

23

23

23

23

23

23

23

G2 & 100

Mean

6.80

6.25

10.98

10.45

15.67

14.68

30.04

28.53

49.81

48.46

±SD

0.19

0.25

0.24

0.22

0.42

0.32

0.56

0.44

3.09

3.02

n

22

22

22

22

22

22

22

22

22

22

G3 & 300

Mean

6.75

6.39

10.96

10.52

15.34*

14.82

29.30*

28.31

49.22

47.44

±SD

0.35

0.35

0.39

0.27

0.59

0.26

0.82

0.67

5.45

5.13

n

23

22

23

22

23

22

23

22

23

22

G4 & 1000

Mean

6.76

6.22

10.77

10.27

15.11*

14.57

29.34*

28.15

48.33

46.07

±SD

0.21

0.31

0.37

0.36

0.41

0.20

0.47

0.20

4.14

4.04

n

21

21

21

21

21

21

21

21

21

21

 SD: Standard Deviation; n: Number of Animals; PND: Postnatal Day

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

TABLE 27.      SUMMARY OF ANO-GENITAL DISTANCE (mm) AND ANO-GENITAL DISTANCE RATIO OF PUPS ON PND 4 PER LITTER RECORD - F1 GENERATION

Group & Dose

(mg/kg body weight/day)

 

Male Pups AGD (mm)

Female Pups AGD (mm)

Male Pups
AGD Ratio

Female Pups
AGD Ratio

G1 & 0

Mean

5.11

2.68

2.30

1.23

±SD

0.27

0.22

0.11

0.09

n

23

23

23

23

G2 & 100

Mean

5.12

2.68

2.30

1.23

±SD

0.23

0.20

0.10

0.09

n

22

22

22

22

G3 & 300

Mean

5.30

2.61

2.39

1.19

±SD

0.34

0.17

0.15

0.08

n

23

22

23

22

G4 & 1000

Mean

5.34

2.66

2.42*

1.22

±SD

0.30

0.18

0.13

0.08

n

21

21

21

21

SD: Standard Deviation; n: Number of Animals; PND: Postnatal Day; AGD: Anogenital Distance

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

TABLE 28.      SUMMARY OF ASSESSMENT OF MALE PUPS FOR NIPPLE/S RETENTION PER LITTER - F1 GENERATION

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

 

No. of Nipples/Areolae on Postnatal Day 13

G1 & 0

Mean

0.00

±SD

0.00

n

23 (109)

G2 & 100

Mean

0.00

±SD

0.00

n

22 (103)

G3 & 300

Mean

0.00

±SD

0.00

n

23 (113)

G4 & 1000

Mean

0.00

±SD

0.00

n

21 (108)

SD: Standard Deviation; n: Number of Litters (total no. of male pups evaluated)

TABLE 29.      SUMMARY OF ASSESSMENT OF PUPS FOR POSTNATAL DEVELOPMENTAL LANDMARKS RECORD - F1 GENERATION

Group & Dose

(mg/kg body weight/day)

Occurrence of Pinna

Unfolding (PND)

Occurrence of
Hair Coat Development

(PND)

Occurrence of

Incisor Eruption (PND)

Occurrence of Eye

Opening (PND)

Occurrence of

Testes Descent (PND)

G1 & 0

Mean

2.88

5.03

10.32

13.95

19.80

±SD

0.31

0.28

0.24

0.12

0.23

n

23

23

23

23

23

G2 & 100

Mean

2.88

5.11

10.19

13.95

19.77

±SD

0.26

0.11

0.28

0.10

0.24

n

22

22

22

22

22

G3 & 300

Mean

2.73

4.97

10.22

14.15*

19.87

±SD

0.19

0.17

0.23

0.15

0.19

n

23

23

23

23

23

G4 & 1000

Mean

2.71

4.98

10.29

14.15*

19.93

±SD

0.22

0.15

0.22

0.12

0.23

n

21

21

21

21

21

SD: Standard Deviation; n: Number of Animals; PND: Postnatal Day

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

TABLE 30.      SUMMARY OF ASSESSMENT OF PUPS FOR SENSORY REFLEXES RECORD - F1 GENERATION

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

 

Response for Surface

Righting Reflex (PND)

Response for Auditory

Startle Reflex (PND)

Response for

Air Righting Reflex (PND)

G1 & 0

Mean

4.39

13.03

17.26

±SD

0.11

0.13

0.96

n

23

23

23

G2 & 100

Mean

4.35

13.03

17.24

±SD

0.13

0.09

0.95

n

22

22

22

G3 & 300

Mean

4.27*

13.13*

17.22

±SD

0.13

0.14

0.86

n

23

23

23

G4 & 1000

Mean

4.30*

13.10

17.21

±SD

0.10

0.13

0.91

n

21

21

21

SD: Standard Deviation; n: Number of Animals; PND: Postnatal Day

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

TABLE 31.      SUMMARY OF SERUM THYROXINE (T4) LEVELS RECORD - PND 4 PUPS - F1 GENERATION

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

 

Serum T4 Levels

(ng/mL)

G1 & 0

Mean

53.490

±SD

3.764

n

21

G2 & 100

Mean

55.295

±SD

6.747

n

18

G3 & 300

Mean

51.243

±SD

5.098

n

16

G4 & 1000

Mean

51.892

±SD

4.213

n

20

SD: Standard Deviation; n: Number of Litters available with Surplus Pups

TABLE 32.      SUMMARY OF SERUM THYROXINE (T4) LEVELS RECORD - PND 21 PUPS - F1 GENERATION

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

 

Serum T4 Levels

(ng/mL)

G1 & 0

Mean

72.665

±SD

7.124

n

23

G2 & 100

Mean

72.029

±SD

10.333

n

22

G3 & 300

Mean

76.723

±SD

4.038

n

23

G4 & 1000

Mean

77.499

±SD

5.359

n

21

SD: Standard Deviation; n: Number of Litters available with Surplus Pups

TABLE 33.      SUMMARY OF SERUM THYROID STIMULATING HORMONE (TSH) LEVELS RECORD - PND 21 PUPS - F1 GENERATION

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

 

Serum TSH Levels

(µIU/mL)

G1 & 0

Mean

2.576

±SD

5.575

n

23

G2 & 100

Mean

1.602

±SD

4.625

n

22

G3 & 300

Mean

2.378

±SD

4.647

n

23

G4 & 1000

Mean

1.827

±SD

1.919

n

21

SD: Standard Deviation; n: Number of Litters available with Surplus Pups

TABLE 34.      SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) OF PND 21 SURPLUS PUPS RECORD - F1 GENERATION

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

Thymus

Spleen

Brain

Absolute Weight (g)

Absolute Weight (g)

Absolute Weight (g)

Male

Female

Male

Female

Male

Female

G1 & 0

Mean

0.2226

0.2328

0.2357

0.2322

1.5467

1.5240

±SD

0.0384

0.0274

0.0273

0.0332

0.0680

0.0537

n

23

23

23

23

23

23

G2 & 100

Mean

0.2169

0.2165

0.2283

0.2302

1.5418

1.5189

±SD

0.0186

0.0285

0.0249

0.0239

0.0479

0.0404

n

22

22

22

22

22

22

G3 & 300

Mean

0.2082

0.2190

0.2267

0.2363

1.5270

1.5084

±SD

0.0277

0.0262

0.0300

0.0296

0.0592

0.0697

n

23

21#

23

21#

23

21#

G4 & 1000

Mean

0.2140

0.2238

0.2246

0.2250

1.5362

1.5051

±SD

0.0162

0.0299

0.0174

0.0332

0.0547

0.0632

n@

20

20@

20

20@

20

20@

SD: Standard Deviation; n: Number of Animals;

#: Only male surplus pups were available from dam no. Rd7524 and Rd7526 on PND21

@: Only male pups were available from dam no. Rd7548 and only female pups were available from dam no. Rd7544 on PND21

TABLE 35.      SUMMARY OF TERMINAL BODY WEIGHT (g) AND RELATIVE ORGAN WEIGHT (%) OF PND 21 SURPLUS PUPS RECORD - F1 GENERATION

Group

& Dose
(mg/kg body

weight/day)

Thymus

Spleen

Brain

Relative Organ Weight (%)

Relative Organ Weight (%)

Relative Organ Weight (%)

Male

Female

Male

Female

Male

Female

G1 & 0

Mean

0.4610

0.4951

0.4898

0.4918

3.2182

3.2398

±SD

0.0757

0.0665

0.0661

0.0633

0.2852

0.2909

n

23

23

23

23

23

23

G2 & 100

Mean

0.4332

0.4493

0.4556

0.4778

3.0880

3.1519

±SD

0.0348

0.0613

0.0436

0.0552

0.2200

0.2301

n

22

22

22

22

22

22

G3 & 300

Mean

0.4271

0.4688

0.4650

0.5039

3.1555

3.2280

±SD

0.0551

0.0735

0.0575

0.0683

0.3962

0.3431

n

23

21

23

21

23

21

G4 & 1000

Mean

0.4421

0.4916

0.4638

0.4935

3.1713

3.3044

±SD

0.0502

0.0744

0.0521

0.0790

0.2364

0.2928

n

20

20

20

20

20

20

SD: Standard Deviation; n: Number of Animals

TABLE 36.      SUMMARY OF CLINICAL SIGNS OF TOXICITY, DETAILED CLINICAL EXAMINATION AND MORTALITY RECORD - COHORT 1A

Group, Sex & Dose

(mg/kg body weight/day)

Total No. of Animals

Clinical Signs of Toxicitya:

Observation

Detailed Clinical Examinationb:

Observation

Mortalityc:

No. of Mortalities

G1-C1A, M & 0

20

N

NAD

0

G2-C1A, M & 100

20

N

NAD

0

G3-C1A, M & 300

20

N

NAD

0

G4-C1A, M & 1000

20

N

NAD

0

M: Male; N: Normal; NAD: No Abnormality Detected; C1A: Cohort 1A
a: observed daily once; b: observed weekly once; c: observed twice daily

TABLE 36 (Contd…). SUMMARY OF CLINICAL SIGNS OF TOXICITY, DETAILED CLINICAL EXAMINATION AND MORTALITY RECORD - COHORT 1A

 Group, Sex & Dose

(mg/kg body weight/day)

Total No. of Animals

Clinical Signs of Toxicitya:

Observation

Detailed Clinical Examinationb:

Observation

Mortalityc:

No. of Mortalities

G1-C1A, F & 0

20

N

NAD

0

G2-C1A, F & 100

20

N

NAD

0

G3-C1A, F & 300

20

N

NAD

0

G4-C1A, F & 1000

20

N)

NAD

0

F: Female; N: Normal; NAD: No Abnormality Detected;
a: observed daily once; b: observed weekly once; c: observed twice daily

TABLE 37.      SUMMARY OF BODY WEIGHT (g) RECORD - COHORT 1A

Group, Sex

& Dose

(mg/kg body

weight/day)

Body Weight (g) on Postnatal Day

21

25

28

32

35

42

49

56

63

70

77

84

91

G1-C1A,

M & 0

Mean

49.53

68.58

87.11

114.09

141.18

197.77

244.26

275.17

305.22

338.34

369.43

399.15

426.67

±SD

5.01

6.01

6.98

8.40

10.05

12.02

13.35

17.69

23.97

29.38

32.75

34.01

40.74

n

20

20

20

20

20

20

20

20

20

20

20

20

20

G2-C1A,

M & 100

Mean

48.28

66.56

85.02

112.31

137.50

193.54

240.70

270.75

298.13

324.89

355.46

385.27

411.15

±SD

3.75

5.73

6.81

7.60

10.34

16.77

18.47

22.10

27.71

33.99

38.12

39.66

44.57

n

20

20

20

20

20

20

20

20

20

20

20

20

20

G3-C1A,

M & 300

Mean

49.67

69.88

90.37

118.83

145.79

198.64

244.55

272.53

293.54

318.74

352.84

382.39

404.21

±SD

5.78

8.29

14.26

19.03

22.74

22.61

22.23

25.51

30.13

29.46

31.98

36.43

37.82

n

20

20

20

20

20

20

20

20

20

20

20

20

20

G4-C1A,

M & 1000

Mean

48.62

66.43

85.87

112.50

137.22

189.83

240.51

267.09

295.02

326.20

360.27

388.79

410.35

±SD

4.39

6.21

6.40

9.53

12.60

17.02

18.99

18.34

20.81

27.82

29.04

32.46

36.03

n

20

20

20

20

20

20

20

20

20

20

20

20

20

M: Male; C1A: Cohort 1A; SD: Standard Deviation; n: Number of Animals

TABLE 37 (Contd…). SUMMARY OF BODY WEIGHT (g) RECORD - COHORT 1A

Group, Sex

& Dose

(mg/kg body

weight/day)

Body Weight (g) on Postnatal Day

21

25

28

32

35

42

49

56

63

70

77

84

91

G1-C1A,

F & 0

Mean

47.58

65.04

80.82

103.77

121.91

158.37

183.58

203.81

218.68

232.74

246.15

259.66

272.53

±SD

6.62

8.67

10.01

13.21

15.35

17.21

18.60

21.16

22.17

22.07

21.67

21.89

24.14

n

20

20

20

20

20

20

20

20

20

20

20

20

20

G2-C1A,

F & 100

Mean

47.67

65.03

81.61

104.65

124.76

160.58

185.65

203.77

217.82

232.90

247.43

259.92

269.52

±SD

4.52

5.36

6.11

8.81

11.11

12.90

13.62

15.59

16.95

18.48

20.33

21.86

22.62

n

20

20

20

20

20

20

20

20

20

20

20

20

20

G3-C1A,

F & 300

Mean

47.10

65.32

82.07

104.36

124.17

161.60

184.71

204.10

218.03

233.67

248.06

262.59

272.88

±SD

6.37

8.57

9.54

12.03

17.02

15.07

13.45

14.67

15.02

16.60

17.37

18.31

19.19

n

20

20

20

20

20

20

20

20

20

20

20

20

20

G4-C1A,

F & 1000

Mean

46.23

63.31

78.76

101.15

118.85

154.76

178.98

195.69

211.01

225.28

243.27

255.14

264.95

±SD

5.75

6.04

7.59

10.32

11.01

11.64

12.75

12.18

12.40

14.46

13.86

11.67

13.28

n

20

20

20

20

20

20

20

20

20

20

20

20

20

F: Female; C1A: Cohort 1A; SD: Standard Deviation; n: Number of Animals

TABLE 38.      SUMMARY OF PERCENT CHANGE IN BODY WEIGHT (%) WITH RESPECT TO POSTNATAL DAY 21 RECORD - COHORT 1A

Group, Sex

& Dose

(mg/kg body

weight/day)

Percent Change in Body Weight (%) during Postnatal Day

21 to 25

21 to 28

21 to 32

21 to 35

21 to 42

21 to 49

21 to 56

21 to 63

21 to 70

21 to 77

21 to 84

21 to 91

G1-C1A,

M & 0

Mean

38.78

76.37

131.44

186.58

302.33

397.15

460.39

521.04

587.38

650.60

711.06

766.53

±SD

6.38

7.58

15.95

21.17

37.77

47.63

59.85

67.27

68.56

76.54

81.24

90.09

n

20

20

20

20

20

20

20

20

20

20

20

20

G2-C1A,

M & 100

Mean

38.09

76.60

133.53

186.12

303.61

401.79

464.97

522.47

578.79

642.64

704.64

758.20

±SD

9.56

14.05

19.27

27.43

50.42

58.09

72.24

87.55

105.00

115.74

120.52

127.75

n

20

20

20

20

20

20

20

20

20

20

20

20

G3-C1A,

M & 300

Mean

40.78

81.76

139.08

193.24

301.61

395.85

453.56

496.19

547.94

618.35

680.10

724.42

±SD

5.54

15.06

22.14

25.44

35.94

48.65

66.01

74.07

79.34

97.62

120.43

125.48

n

20

20

20

20

20

20

20

20

20

20

20

20

G4-C1A,

M & 1000

Mean

37.09

77.30

132.31

183.27

292.13

397.07

452.81

510.08

573.48

643.77

702.17

746.63

±SD

12.04

13.15

19.83

25.17

37.01

45.18

54.80

55.71

57.02

58.87

59.35

66.55

n

20

20

20

20

20

20

20

20

20

20

20

20

M: Male; C1A: Cohort 1A; SD: Standard Deviation; n: Number of Animals

TABLE 38 (Contd…). SUMMARYOF PERCENT CHANGE IN BODY WEIGHT (%) WITH RESPECT TO POSTNATAL DAY 21 RECORD - COHORT 1A

Group, Sex

& Dose

(mg/kg body

weight/day)

Percent Change in Body Weight (%) duringPostnatal Day

21 to 25

21 to 28

21 to 32

21 to 35

21 to 42

21 to 49

21 to 56

21 to 63

21 to 70

21 to 77

21 to 84

21 to 91

G1-C1A,

F & 0

Mean

37.01

70.59

119.36

158.06

236.89

291.35

335.27

367.02

396.89

425.76

455.22

482.19

±SD

7.03

11.03

19.87

26.82

44.57

56.42

68.93

71.41

73.49

77.77

84.70

86.80

n

20

20

20

20

20

20

20

20

20

20

20

20

G2-C1A,

F & 100

Mean

36.80

71.89

120.46

163.08

238.82

291.83

330.04

359.79

391.64

422.55

449.10

469.16

±SD

7.83

12.04

17.96

25.64

32.97

37.37

41.85

45.84

49.47

56.13

60.97

61.76

n

20

20

20

20

20

20

20

20

20

20

20

20

G3-C1A,

F & 300

Mean

38.97

75.15

122.84

165.05

246.67

297.03

338.86

368.93

402.72

433.26

465.02

486.78

±SD

6.90

12.03

17.28

26.41

37.42

43.97

49.51

52.85

58.85

58.41

66.52

65.53

n

20

20

20

20

20

20

20

20

20

20

20

20

G4-C1A,

F & 1000

Mean

37.53

71.15

119.86

158.67

238.24

291.24

328.30

361.95

393.08

432.67

459.02

480.25

±SD

6.50

9.48

14.62

19.24

36.92

42.73

49.64

53.30

57.31

61.46

65.34

65.94

n

20

20

20

20

20

20

20

20

20

20

20

20

F: Female; C1A: Cohort 1A; SD: Standard Deviation; n: Number of Animals

TABLE 39.      SUMMARY OF AVERAGE FEED CONSUMPTION (g/animal/day) RECORD - COHORT 1A

Group, Sex

& Dose

(mg/kg body

weight/day)

 

Feed Consumption on Postnatal Days

21 to 25

25 to 28

28 to 32

32 to 35

35 to 42

42 to 49

49 to 56

56 to 63

63 to 70

70 to 77

77 to 84

84 to 91

G1-C1A,

M & 0

Mean

7.29

8.91

10.83

14.55

16.08

17.80

21.53

23.45

24.50

26.81

26.93

27.82

±SD

0.74

1.41

1.18

2.76

1.85

1.14

1.53

2.59

2.28

2.05

2.51

2.49

n

20

20

20

20

20

20

20

20

20

20

20

20

G2-C1A,

M & 100

Mean

6.87

8.22

11.09

13.26

15.59

17.11

20.85

22.46

23.93

25.47

26.30

26.57

±SD

0.62

0.96

1.16

1.57

0.99

0.81

1.42

0.91

1.28

1.34

1.41

1.93

n

20

20

20

20

20

20

20

20

20

20

20

20

G3-C1A,

M & 300

Mean

7.25

9.03

10.26

13.73

15.51

16.95

21.15

22.94

23.96

24.64*

27.12

28.00

±SD

0.90

2.25

1.27

1.41

0.83

0.61

1.33

1.29

1.56

1.65

1.60

2.03

n

20

20

20

20

20

20

20

20

20

20

20

20

G4-C1A,

M & 1000

Mean

7.28

8.69

11.58

14.32

17.00

18.08

20.97

23.01

23.09

26.10

26.62

26.67

±SD

0.60

1.02

0.86

1.11

1.13

0.84

1.44

1.61

1.71

1.57

1.02

2.22

n

20

20

20

20

20

20

20

20

20

20

20

20

M: Male; C1A: Cohort 1A; SD: Standard Deviation; n: Number of Animals

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

TABLE 39 (Contd…). SUMMARYOF AVERAGE FEED CONSUMPTION (g/animal/day) RECORD - COHORT 1A

Group, Sex

& Dose

(mg/kg body

weight/day)

 

Feed Consumption (g/animal/day) on Postnatal Day

21 to 25

25 to 28

28 to 32

32 to 35

35 to 42

42 to 49

49 to 56

56 to 63

63 to 70

70 to 77

77 to 84

84 to 91

G1-C1A,

F & 0

Mean

6.69

7.33

10.96

12.76

14.21

16.80

18.89

19.37

19.90

21.22

22.53

24.04

±SD

0.44

0.82

1.89

2.24

0.42

1.14

1.01

1.10

0.91

1.70

1.51

1.51

n

20

20

20

20

20

20

20

20

20

20

20

20

G2-C1A,

F & 100

Mean

6.52

7.56

10.93

12.11

13.73

16.72

18.45

19.46

20.14

21.99

23.17

25.03

±SD

0.80

0.62

1.06

1.20

0.52

0.95

0.97

1.34

1.76

1.61

1.09

1.52

n

20

20

20

20

20

20

20

20

20

20

20

20

G3-C1A,

F & 300

Mean

6.85

7.84

11.21

12.63

13.93

16.43

17.76

18.20

19.13

20.78

22.13

23.71

±SD

1.02

1.89

1.55

1.47

0.68

1.42

1.25

1.14

1.52

1.64

2.01

1.15

n

20

20

20

20

20

20

20

20

20

20

20

20

G4-C1A,

F & 1000

Mean

6.37

7.50

10.92

12.36

14.04

15.92

17.96

19.06

20.42

21.85

23.55

24.87

±SD

0.36

0.74

1.23

1.53

1.21

1.05

1.16

1.18

1.24

2.19

1.59

2.32

n

20

20

20

20

20

20

20

20

20

20

20

20

F: Female; C1A: Cohort 1A; SD: Standard Deviation; n: Number of Animals

TABLE 40.      SUMMARY OF SERUM THYROXINE (T4) HORMONE LEVELS RECORD - COHORT 1A

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

 

Serum T4 Levels

(ng/mL)

G1-C1A, M & 0

Mean

97.397

±SD

10.139

n

10

G2-C1A, M & 100

Mean

90.186

±SD

7.371

n

10

G3-C1A, M & 300

Mean

91.865

±SD

8.089

n

10

G4-C1A, M & 1000

Mean

99.433

±SD

6.816

n

10

M: Male;C1A: Cohort 1A;SD: Standard Deviation; n: Number of Animals

TABLE 40 (Contd…). SUMMARYOF SERUM THYROXINE (T4) HORMONE LEVELS RECORD - COHORT 1A

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

 

Serum T4 Levels

(ng/mL)

G1-C1A, F & 0

Mean

101.189

±SD

22.010

n

10

G1-C1A, F & 100

Mean

87.898

±SD

10.255

n

10

G1-C1A, F & 300

Mean

88.754

±SD

6.467

n

10

G1-C1A, F & 1000

Mean

88.467

±SD

9.804

n

10

F: Female; C1A: Cohort 1A; SD: Standard Deviation; n: Number of Animals

TABLE 41.      SUMMARY OF SERUM THYROID STIMULATING HORMONE (TSH) LEVELS RECORD -
COHORT 1A

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

 

Serum TSH Levels

(µIU/mL)

G1-C1A, M & 0

Mean

2.630

±SD

1.775

n

8

G2-C1A, M & 100

Mean

2.442

±SD

2.158

n

8

G3-C1A, M & 300

Mean

1.982

±SD

2.808

n

7

G4-C1A, M & 1000

Mean

0.695

±SD

0.332

n

10

M: Male;C1A: Cohort 1A;SD: Standard Deviation; n: Number of Animals [The outliered values were excluded for mean calculations];

 

Note: The noted reduced mean TSH levels from group G4 C1A males is considered as incidental and unrelated to test item as the noted values are within in-house historical control range (In-house Historical Control Range:- TSH Levels (µIU/mL) - Males: 0.172 to 9.398).

TABLE 41 (Contd…). SUMMARYOF SERUM THYROID STIMULATING HORMONE (TSH) LEVELS RECORD - COHORT 1A

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

 

Serum TSH Levels

(µIU/mL)

G1-C1A, F & 0

Mean

2.284

±SD

1.762

n

10

G2-C1A, F & 100

Mean

0.640

±SD

0.561

n

9

G3-C1A, F & 300

Mean

2.967

±SD

1.990

n

10

G4-C1A, F & 1000

Mean

0.854

±SD

1.067

n

10

F: Female; C1A: Cohort 1A; SD: Standard Deviation;
n: Number of Animals [
The outliered values were excluded for mean calculations]

Note: The noted reduced mean TSH levels from group G2 and G4 C1A females is considered as incidental and unrelated to test item as the noted values are within in-house historical control range (In-house Historical Control Range:- TSH Levels (µIU/mL) - Females: 0.385 to 8.806).

TABLE 42.      SUMMARY OF HAEMATOLOGY RECORD - COHORT 1A

Group, Sex

& Dose

(mg/kg body

weight/day)

Total Leucocyte

Count

Total Erythrocyte

Count

Hemoglobin

Haematocrit

Platelet Count

Neutrophils

(WBC)

(RBC)

(HGB)

(HCT)

(PLT)

(Neut)

(103cells/µL)

(106cells/µL)

(g/dL)

(%)

(103cells/µL)

(%)

G1-C1A,

M & 0

Mean

13.24

7.28

13.54

43.15

817.10

24.42

±SD

3.09

0.45

0.74

2.01

68.46

4.96

n

10

10

10

10

10

10

G2-C1A,

M & 100

Mean

11.10

8.06*

14.26

45.36

847.70

28.28

±SD

1.57

0.39

0.58

1.41

135.73

4.74

n

10

10

10

10

10

10

G3-C1A,

M & 300

Mean

10.94*

7.56

13.90

43.73

781.40

25.37

±SD

1.33

0.40

0.68

2.51

184.44

7.29

n

10

10

10

10

10

10

G4-C1A,

M & 1000

Mean

11.42

7.88*

13.92

44.75

937.70

24.42

±SD

1.81

0.40

0.80

2.19

73.78

3.94

n

10

10

10

10

10

10

M: Male; C1A: Cohort 1A; SD: Standard Deviation; n: Number of Animals

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

TABLE 42 (Contd…). SUMMARY OF HAEMATOLOGY RECORD - COHORT 1A

Group, Sex

& Dose

(mg/kg body

weight/day)

Lymphocytes

Monocytes

Eosinophils

Basophils

Absolute Neutrophils

Absolute Lymphocytes

(Lymph)

(Mono)

(Eos)

(Baso)

(Neut)

(Lymph)

(%)

(%)

(%)

(%)

(103cells/µL)

(103cells/µL)

G1-C1A,

M & 0

Mean

68.54

4.56

1.17

0.32

3.24

9.08

±SD

4.83

1.30

0.31

0.09

1.05

2.27

n

10

10

10

10

10

10

G2-C1A,

M & 100

Mean

64.14

4.77

1.32

0.30

3.11

7.17*

±SD

6.35

1.51

0.71

0.12

0.59

1.52

n

10

10

10

10

10

10

G3-C1A,

M & 300

Mean

68.02

4.09

1.06

0.28

2.81

7.40

±SD

7.77

0.79

0.25

0.04

1.05

0.99

n

10

10

10

10

10

10

G4-C1A,

M & 1000

Mean

68.28

4.55

1.30

0.30

2.78

7.81

±SD

4.27

0.97

0.58

0.07

0.60

1.42

n

10

10

10

10

10

10

M: Male; C1A: Cohort 1A; SD: Standard Deviation; n: Number of animals

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

TABLE 42 (Contd…). SUMMARY OF HAEMATOLOGY RECORD - COHORT 1A

Group, Sex

& Dose

(mg/kg body

weight/day)

Absolute Monocytes

Absolute Eosinophils

Absolute Basophils

Prothrombin Time

Activated Prothrombin

Time

(Mono)

(Eos)

(Baso)

(PT)

(APTT)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(Seconds)

(Seconds)

G1-C1A,

M & 0

Mean

0.60

0.16

0.04

21.88

28.07

±SD

0.18

0.06

0.02

1.92

4.23

n

10

10

10

10

10

G2-C1A,

M & 100

Mean

0.51

0.14

0.04

20.77

26.82

±SD

0.12

0.06

0.01

4.86

2.50

n

10

10

10

10

10

G3-C1A,

M & 300

Mean

0.45*

0.12

0.03

19.93

27.10

±SD

0.11

0.03

0.01

2.07

2.98

n

10

10

10

10

10

G4-C1A,

M & 1000

Mean

0.51

0.15

0.04

21.23

25.47

±SD

0.12

0.07

0.01

3.31

4.58

n

10

10

10

10

10

M: Male; C1A: Cohort 1A; SD: Standard Deviation; n: Number of Animals

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

TABLE 42 (Contd…). SUMMARY OF HAEMATOLOGY RECORD - COHORT 1A

Group, Sex

& Dose

(mg/kg body

weight/day)

Total Leucocyte

Count

Total Erythrocyte

Count

Hemoglobin

Haematocrit

Platelet Count

Neutrophils

(WBC)

(RBC)

(HGB)

(HCT)

(PLT)

(Neut)

(103cells/µL)

(106cells/µL)

(g/dL)

(%)

(103cells/µL)

(%)

G1-C1A,

F & 0

Mean

9.51

7.18

13.23

41.65

920.70

21.73

±SD

3.30

0.48

0.79

2.23

79.08

5.61

n

10

10

10

10

10

10

G2-C1A,

F & 100

Mean

10.16

7.50

13.60

42.32

957.80

25.04

±SD

4.15

0.57

1.04

3.10

106.06

7.18

n

10

10

10

10

10

10

G3-C1A,

F & 300

Mean

12.20

7.43

13.43

41.71

989.20

28.45

±SD

4.51

0.41

0.56

1.60

140.89

18.84

n

10

10

10

10

10

10

G4-C1A,

F & 1000

Mean

8.80

7.07

12.96

40.37

957.20

24.44

±SD

2.67

0.38

0.78

2.16

154.74

6.44

n

10

10

10

10

10

10

F: Female; C1A: Cohort 1A; SD: Standard Deviation; n: Number of Animals

TABLE 42 (Contd…). SUMMARY OF HAEMATOLOGY RECORD - COHORT 1A

Group, Sex

& Dose

(mg/kg body

weight/day)

Lymphocytes

Monocytes

Eosinophils

Basophils

Absolute Neutrophils

Absolute Lymphocytes

(Lymph)

(Mono)

(Eos)

(Baso)

(Neut)

(Lymph)

(%)

(%)

(%)

(%)

(103cells/µL)

(103cells/µL)

G1-C1A,

F & 0

Mean

72.18

3.42

1.26

0.26

1.96

6.96

±SD

5.76

1.02

0.28

0.07

0.52

2.63

n

10

10

10

10

10

10

G2-C1A,

F & 100

Mean

68.54

3.46

1.20

0.29

2.35

7.19

±SD

7.77

0.90

0.46

0.09

0.60

3.92

n

10

10

10

10

10

10

G3-C1A,

F & 300

Mean

63.22

5.44*

1.20

0.30

4.04

7.09

±SD

20.62

2.67

0.64

0.11

5.02

2.73

n

10

10

10

10

10

10

G4-C1A,

F & 1000

Mean

68.81

3.53

1.54

0.20

2.12

6.07

±SD

6.57

0.95

1.25

0.08

0.78

2.10

n

10

10

10

10

10

10

F: Female; C1A: Cohort 1A; SD: Standard Deviation; n: Number of Animals

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

TABLE 42 (Contd…). SUMMARY OF HAEMATOLOGY RECORD - COHORT 1A

Group, Sex

& Dose

(mg/kg body

weight/day)

Absolute Monocytes

Absolute Eosinophils

Absolute Basophils

Prothrombin Time

Activated Prothrombin

Time

(Mono)

(Eos)

(Baso)

(PT)

(APTT)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(Seconds)

(Seconds)

G1-C1A,

F & 0

Mean

0.33

0.12

0.03

20.86

24.25

±SD

0.16

0.05

0.01

5.30

4.57

n

10

10

10

10

10

G2-C1A,

F & 100

Mean

0.34

0.11

0.03

20.11

26.24

±SD

0.11

0.04

0.02

4.02

5.19

n

10

10

10

10

10

G3-C1A,

F & 300

Mean

0.75

0.12

0.04

24.10

25.98

±SD

0.78

0.05

0.03

7.62

8.04

n

10

10

10

10

10

G4-C1A,

F & 1000

Mean

0.31

0.14

0.02

23.72

27.46

±SD

0.12

0.16

0.01

5.26

3.69

n

10

10

10

10

10

F: Female; C1A: Cohort 1A; SD: Standard Deviation; n: Number of Animals

TABLE 43.      SUMMARY OF CLINICAL CHEMISTRY RECORD - COHORT 1A

Group, Sex

& Dose

(mg/kg body

weight/day)

Glucose

Urea

Creatinine

Total Cholesterol

Total Protein

(GLU)

(CRE)

(CHO)

(TPR)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(g/dL)

G1-C1A,

M & 0

Mean

95.50

25.46

0.48

42.30

5.93

±SD

9.29

5.27

0.05

9.87

0.40

n

10

10

10

10

10

G2-C1A,

M & 100

Mean

92.80

22.90

0.49

44.00

6.28

±SD

6.48

5.55

0.03

8.60

0.30

n

10

10

10

10

10

G3-C1A,

M & 300

Mean

102.10

24.34

0.48

42.90

6.08

±SD

12.41

2.03

0.03

6.31

0.49

n

10

10

10

10

10

G4-C1A,

M & 1000

Mean

96.20

22.51

0.49

47.90

6.40*

±SD

7.51

3.88

0.04

7.82

0.36

n

10

10

10

10

10

M: Male; C1A: Cohort 1A; SD: Standard Deviation; n: Number of Animals

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

TABLE 43 (Contd…). SUMMARY OF CLINICAL CHEMISTRY RECORD - COHORT 1A

Group, Sex

&Dose

(mg/kg body

weight/day)

Albumin

Alanine aminotransferase

Aspartate aminotransferase

Alkaline phosphatase

(ALB)

(ALT)

(AST)

(ALP)

(g/dL)

(U/L)

(U/L)

(U/L)

G1-C1A,

M & 0

Mean

2.93

49.10

86.50

150.40

±SD

0.12

16.95

18.22

66.55

n

10

10

10

10

G2-C1A,

M & 100

Mean

3.00

46.30

88.30

179.10

±SD

0.10

11.30

11.25

79.08

n

10

10

10

10

G3-C1A,

M & 300

Mean

3.02

45.11

81.89

164.60

±SD

0.15

5.21

11.41

28.73

n

10

9@

9@

10

G4-C1A,

M & 1000

Mean

3.15*

41.10

81.70

146.20

±SD

0.10

6.62

8.94

27.08

n

10

10

10

10

M: Male; C1A: Cohort 1A; SD: Standard Deviation; n: Number of Animals; @: The data of one animal was excluded for mean calculation due to outliered value

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

TABLE 43 (Contd…). SUMMARY OF CLINICAL CHEMISTRY RECORD - COHORT 1A

Group, Sex

& Dose

(mg/kg body

weight/day)

Glucose

Urea

Creatinine

Total Cholesterol

Total Protein

(GLU)

-

(CRE)

(CHO)

(TPR)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(g/dL)

G1-C1A,

F & 0

Mean

101.40

26.14

0.53

49.10

6.55

±SD

7.75

4.98

0.05

10.25

0.46

n

10

10

10

10

10

G2-C1A,

F & 100

Mean

113.90

26.08

0.52

46.30

6.37

±SD

13.47

3.68

0.06

11.96

0.35

n

10

10

10

10

10

G3-C1A,

F & 300

Mean

99.40

27.45

0.52

51.30

6.26

±SD

12.86

3.50

0.04

15.92

0.40

n

10

10

10

10

10

G4-C1A,

F & 1000

Mean

118.90

26.07

0.53

51.20

6.52

±SD

26.16

4.30

0.05

13.50

0.38

n

10

10

10

10

10

F: Female; C1A: Cohort 1A; SD: Standard Deviation; n: Number of Animals

TABLE 43 (Contd…). SUMMARY OF CLINICAL CHEMISTRY RECORD - COHORT 1A

Group, Sex

& Dose

(mg/kg body

weight/day)

Albumin

Alanine aminotransferase

Aspartate aminotransferase

Alkaline phosphatase

(ALB)

(ALT)

(AST)

(ALP)

(g/dL)

(U/L)

(U/L)

(U/L)

G1-C1A,

F & 0

Mean

3.18

29.00

70.50

75.50

±SD

0.16

6.16

13.23

19.39

n

10

10

10

10

G2-C1A,

F & 100

Mean

3.12

29.10

68.80

96.10

±SD

0.17

3.81

5.47

22.91

n

10

10

10

10

G3-C1A,

F & 300

Mean

3.17

39.20

79.90

107.80*

±SD

0.27

18.16

12.11

35.86

n

10

10

10

10

G4-C1A,

F & 1000

Mean

3.24

33.50

77.00

91.90

±SD

0.25

8.55

11.40

35.64

n

10

10

10

10

F: Female; C1A: Cohort 1A; SD: Standard Deviation; n: Number of Animals

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

TABLE 44.      SUMMARY OF URINALYSIS RECORD - COHORT 1A

Examination

Group, Sex & Dose

(mg/kg body weight/day)

G1-C1A, M & 0

G2-C1A, M & 100

G3-C1A, M & 300

G4-C1A, M & 1000

Number of Animals

10

10

10

10

Physical Examination

Color

Pale Yellow

9

9

10

8

Yellow

1

1

-

2

Appearance

Clear

9

8

9

9

Turbidity

1

2

1

1

Volume (mL)

Mean

7.5

9.1

9.0

8.8

±SD

2.6

1.2

1.6

1.5

Chemical Examination

pH

Mean

7.8

8.0

8.0

8.3

±SD

0.6

0.5

0.5

0.4

Specific Gravity

Mean

1.013

1.010

1.009

1.009

±SD

0.006

0.004

0.004

0.002

Blood (Ery/µL)

 

Neg

4

3

9

7

Ca10

4

5

-

3

Ca25

2

1

1

-

>=Ca200

-

1

-

-

Protein (mg/dL)

Neg

7

6

6

7

Trace

1

3

4

2

30

2

1

-

1

Glucose (mg/dL)

Neg

10

10

10

10

Microscopic

Examination

Epi Cells

0

6

7

6

7

0-1

3

1

3

2

1-2

1

2

1

1

2-3

-

-

-

-

Casts

Absent

10

10

10

10

Crystals

Present

10

10

10

10

M: Male; C1A: Cohort 1A; SD: Standard Deviation; n: Number of Animals; -: Not Applicable

TABLE 44 (Contd…). SUMMARY OF URINALYSIS RECORD - COHORT 1A

Examination

Group, Sex & Dose

(mg/kg body weight/day)

G1-C1A,

F & 0

G2-C1A,

F & 100

G3-C1A,

F & 300

G4-C1A,

F & 1000

Number of Animals

10

10

10

10

Physical Examination

Color

Pale Yellow

10

10

9

9

Yellow

-

-

1

1

Appearance

Clear

10

5

6

9

Turbidity

-

5

4

1

Volume (mL)

Mean

7.9

8.4

8.1

8.7

±SD

1.7

2.1

2.4

2.9

Chemical Examination

pH

Mean

7.9

8.0

8.2

7.9

±SD

0.5

0.4

0.5

0.6

Specific gravity

Mean

1.010

1.009

1.009

1.011

±SD

0.004

0.002

0.002

0.003

Blood (Ery/µL)

Neg

7

2

7

7

Ca10

1

5

2

1

Ca25

2

3

1

2

Protein (mg/dL)

Neg

9

7

6

8

Trace

1

2

2

1

30

-

1

2

1

Glucose (mg/dL)

Neg

10

10

10

10

Microscopic

Examination

Epi Cells

0

4

3

3

6

0-1

4

4

6

2

1-2

2

1

1

2

2-3

-

2

-

-

Casts

Absent

10

10

10

10

Crystals

Present

10

10

10

10

F: Female; C1A: Cohort 1A; SD: Standard Deviation; n: Number of Animals; -; Not Applicable

TABLE 45.      SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD - COHORT 1A

Group, Sex & Dose

(mg/kg body weight/day)

Adrenals

Thymus

Spleen

Testes

Epididymis

Heart

Kidneys

Brain

Liver

Prostate

G1-C1A, M & 0

Mean

0.0631

0.5320

0.7738

3.5002

1.3252

1.4857

2.8349

2.0758

12.2743

1.1651

±SD

0.0108

0.1024

0.1290

0.2101

0.1635

0.1236

0.2687

0.0962

1.7375

0.2039

n

20

20

20

20

20

20

20

20

20

20

G2-C1A, M & 100

Mean

0.0658

0.4430*

0.7201

3.5110

1.3165

1.4584

2.7818

2.0235

11.4637

1.2598

±SD

0.0147

0.0820

0.1234

0.2890

0.1007

0.2313

0.3881

0.1127

2.0197

0.2047

n

20

20

20

20

20

20

20

20

20

20

G3-C1A, M & 300

Mean

0.0628

0.4583*

0.7520

3.5297

1.3206

1.4376

2.7126

2.0123

11.4183

1.1530

±SD

0.0105

0.0888

0.1248

0.2804

0.1332

0.1968

0.3378

0.1128

1.3926

0.2151

n

20

20

20

20

20

20

20

20

20

20

G4-C1A, M & 1000

Mean

0.0626

0.4485*

0.7145

3.4988

1.2656

1.4512

2.9129

2.0484

11.7978

1.1368

±SD

0.0136

0.0951

0.1124

0.3495

0.1173

0.1597

0.2781

0.0852

1.4795

0.1545

n

20

20

20

20

20

20

20

20

20

20

M: Male; C1A: Cohort 1A; SD: Standard Deviation; n: Number of Animals

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

TABLE 45 (Contd…). SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD - COHORT 1A

Group, Sex & Dose

(mg/kg body weight/day)

Seminal vesicles with

coagulating glands

Pitutary

Mandibular
lymph node

Iliac lymph node

Thyroid along with

parathyroid@

G1-C1A, M & 0

Mean

1.3588

0.0204

0.1537

0.0417

0.0294

±SD

0.2785

0.0076

0.0512

0.0194

0.0032

n

20

20

20

20

20

G2-C1A, M & 100

Mean

1.3743

0.0182

0.1612

0.0419

0.0276

±SD

0.2685

0.0064

0.0536

0.0150

0.0041

n

20

20

20

20

20

G3-C1A, M & 300

Mean

1.2444

0.0165

0.1466

0.0429

0.0278

±SD

0.3055

0.0051

0.0571

0.0158

0.0031

n

20

20

20

20

20

G4-C1A, M & 1000

Mean

1.3673

0.0204

0.1532

0.0351

0.0287

±SD

0.3234

0.0080

0.0518

0.0083

0.0040

n

20

20

20

20

20

M: Male; C1A: Cohort 1A; SD: Standard Deviation; n: Number of Animals; @: weighed post-fixation

TABLE 45 (Contd…). SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD - COHORT 1A

Group, Sex & Dose

(mg/kg body weight/day)

Adrenals

Thymus

Spleen

Ovaries

Uterus

Heart

Kidneys

Brain

G1-C1A, F & 0

Mean

0.0717

0.4731

0.5898

0.1518

0.5271

0.9995

1.7764

1.8994

±SD

0.0094

0.1030

0.0878

0.0356

0.1695

0.0965

0.1786

0.1276

n

20

20

20

20

20

20

20

20

G2-C1A, F & 100

Mean

0.0731

0.4744

0.5999

0.1483

0.5709

1.0207

1.8639

1.9326

±SD

0.0098

0.1295

0.0911

0.0231

0.1802

0.1164

0.2071

0.0949

n

20

20

20

20

20

20

20

20

G3-C1A, F & 300

Mean

0.0742

0.4434

0.5763

0.1368

0.5968

0.9880

1.7760

1.9353

±SD

0.0103

0.1196

0.0972

0.0164

0.1501

0.1205

0.2226

0.0799

n

20

20

20

20

20

20

20

20

G4-C1A, F & 1000

Mean

0.0743

0.4325

0.5550

0.1505

0.5434

1.0072

1.7659

1.9478

±SD

0.0143

0.1289

0.0725

0.0284

0.1609

0.0857

0.2222

0.1008

n

20

20

20

20

20

20

20

20

F: Female; C1A: Cohort 1A; SD: Standard Deviation; n: Number of Animals

TABLE 45 (Contd…). SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD - COHORT 1A

Group, Sex & Dose

(mg/kg body weight/day)

Liver

Pitutary

Mandibular
lymph node

Iliac lymph node

Thyroid along

with parathyroid@

G1-C1A, F & 0

Mean

8.0549

0.0256

0.1445

0.0355

0.0252

±SD

1.1086

0.0103

0.0556

0.0120

0.0029

n

20

20

20

20

20

G2-C1A, F & 100

Mean

8.2016

0.0213

0.1189

0.0366

0.0258

±SD

1.2322

0.0053

0.0448

0.0153

0.0020

n

20

20

20

20

20

G3-C1A, F & 300

Mean

7.8440

0.0217

0.1358

0.0359

0.0253

±SD

0.9848

0.0053

0.0658

0.0118

0.0018

n

20

20

20

20

20

G4-C1A, F & 1000

Mean

7.9738

0.0256

0.1341

0.0372

0.0260

±SD

0.9285

0.0054

0.0334

0.0156

0.0027

n

20

20

20

20

20

F: Female; C1A: Cohort 1A; SD: Standard Deviation; n: Number of Animals; @: Weighed Post-Fixation

TABLE 46.      SUMMARY OF TERMINAL BODY WEIGHT (g) AND RELATIVE ORGAN WEIGHT (%) RECORD -
 COHORT 1A

Group, Sex & Dose

(mg/kg body weight/day)

Terminal Body

Weight (g)

Adrenals

Thymus

Spleen

Testes

Epididymis

Heart

Kidneys

Brain

G1-C1A, M & 0

Mean

401.07

0.0158

0.1334

0.1926

0.8798

0.3320

0.3719

0.7094

0.5222

±SD

38.41

0.0027

0.0245

0.0241

0.0919

0.0417

0.0290

0.0591

0.0567

n

20

20

20

20

20

20

20

20

20

G2-C1A, M & 100

Mean

384.87

0.0171

0.1156*

0.1888

0.9235

0.3457

0.3791

0.7236

0.5306

±SD

44.40

0.0033

0.0199

0.0353

0.1292

0.0418

0.0437

0.0673

0.0497

n

20

20

20

20

20

20

20

20

20

G3-C1A, M & 300

Mean

379.04

0.0166

0.1213

0.1995

0.9361

0.3499

0.3811

0.7159

0.5347

±SD

38.05

0.0024

0.0229

0.0334

0.0836

0.0337

0.0542

0.0601

0.0487

n

20

20

20

20

20

20

20

20

20

G4-C1A, M & 1000

Mean

385.41

0.0163

0.1169

0.1863

0.9145

0.3303

0.3772

0.7584*

0.5350

±SD

34.81

0.0031

0.0259

0.0304

0.1196

0.0367

0.0304

0.0671

0.0472

n

20

20

20

20

20

20

20

20

20

M: Male; C1A: Cohort 1A; SD: Standard Deviation; n: Number of Animals

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

TABLE 46 (Contd…). SUMMARY OF TERMINAL BODY WEIGHT (g) AND RELATIVE ORGAN WEIGHT (%) RECORD -COHORT 1A

Group, Sex & Dose

(mg/kg body weight/day)

Liver

Prostate

Seminal vesicles with coagulating glands

Pitutary

Mandibular lymph node

Iliac lymph node

Thyroid along with parathyroid

G1-C1A, M & 0

Mean

3.0542

0.2925

0.3400

0.0051

0.0384

0.0104

0.0074

±SD

0.2600

0.0553

0.0692

0.0020

0.0120

0.0048

0.0008

n

20

20

20

20

20

20

20

G2-C1A, M & 100

Mean

2.9698

0.3297

0.3604

0.0048

0.0421

0.0111

0.0072

±SD

0.3220

0.0544

0.0747

0.0018

0.0140

0.0043

0.0006

n

20

20

20

20

20

20

20

G3-C1A, M & 300

Mean

3.0137

0.3038

0.3276

0.0043

0.0391

0.0113

0.0074

±SD

0.2308

0.0469

0.0705

0.0012

0.0164

0.0038

0.0007

n

20

20

20

20

20

20

20

G4-C1A, M & 1000

Mean

3.0592

0.2965

0.3548

0.0053

0.0402

0.0092

0.0075

±SD

0.2566

0.0441

0.0773

0.0021

0.0151

0.0024

0.0009

n

20

20

20

20

20

20

20

M: Male; C1A: Cohort 1A; SD: Standard Deviation; n: Number of animals

TABLE 46 (Contd…). SUMMARY OF TERMINAL BODY WEIGHT (g) AND RELATIVE ORGAN WEIGHT (%) RECORD -COHORT 1A

Group, Sex & Dose

(mg/kg body weight/day)

Terminal Body Weight (g)

Adrenals

Thymus

Spleen

Ovaries

Uterus

Heart

G1-C1A, F & 0

Mean

249.77

0.0289

0.1896

0.2367

0.0611

0.2139

0.4021

±SD

22.74

0.0041

0.0385

0.0314

0.0145

0.0761

0.0418

n

20

20

20

20

20

20

20

G2-C1A, F & 100

Mean

245.83

0.0299

0.1926

0.2458

0.0605

0.2328

0.4170

±SD

23.24

0.0044

0.0484

0.0444

0.0094

0.0703

0.0484

n

20

20

20

20

20

20

20

G3-C1A, F & 300

Mean

250.12

0.0298

0.1777

0.2314

0.0549

0.2398

0.3943

±SD

16.74

0.0046

0.0479

0.0422

0.0075

0.0630

0.0324

n

20

20

20

20

20

20

20

G4-C1A, F & 1000

Mean

241.78

0.0307

0.1787

0.2297

0.0622

0.2248

0.4164

±SD

11.36

0.0057

0.0516

0.0291

0.0111

0.0663

0.0273

n

20

20

20

20

20

20

20

F: Female; C1A: Cohort 1A; SD: Standard Deviation; n: Number of Animals

TABLE 46 (Contd…). SUMMARY OF TERMINAL BODY WEIGHT (g) AND RELATIVE ORGAN WEIGHT (%) RECORD -COHORT 1A

Group, Sex & Dose

(mg/kg body weight/day)

Kidneys

Brain

Liver

Pitutary

Mandibular lymph node

Iliac lymph node

Thyroid along with parathyroid

G1-C1A, F & 0

Mean

0.7145

0.7647

3.2301

0.0102

0.0576

0.0143

0.0101

±SD

0.0786

0.0687

0.3793

0.0038

0.0211

0.0047

0.0011

n

20

20

20

20

20

20

20

G2-C1A, F & 100

Mean

0.7617

0.7918

3.3395

0.0088

0.0485

0.0149

0.0105

±SD

0.0892

0.0743

0.3950

0.0026

0.0182

0.0061

0.0009

n

20

20

20

20

20

20

20

G3-C1A, F & 300

Mean

0.7106

0.7767

3.1338

0.0087

0.0544

0.0143

0.0102

±SD

0.0836

0.0574

0.2957

0.0020

0.0259

0.0044

0.0008

n

20

20

20

20

20

20

20

G4-C1A, F & 1000

Mean

0.7299

0.8070

3.2969

0.0106

0.0556

0.0155

0.0108

±SD

0.0815

0.0512

0.3396

0.0024

0.0145

0.0068

0.0013

n

20

20

20

20

20

20

20

F: Female; C1A: Cohort 1A; SD: Standard Deviation; n: Number of Animals

TABLE 47.      SUMMARY OF SPLENIC LYMPHOCYTE SUBPOPULATION ANALYSIS - COHORT 1A


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

 

Percent of Splenic lymphocyte Parental Sub-populations (%)

B Lymphocytes
(B Cells)

T Helper Cells
(Th/CD4+)

T Cytotoxic Cells

(Tc/CD8+)

Natural Killer Cells

(NK Cells)

G1-C1A, M & 0

Mean

28.15

50.49

43.08

7.86

±SD

5.74

7.01

5.89

2.66

n

10

10

10

10

G2-C1A, M & 100

Mean

28.41

55.18

39.93

7.54

±SD

4.56

5.03

6.30

2.37

n

10

10

10

10

G3-C1A, M & 300

Mean

27.45

55.52

40.75

7.68

±SD

3.99

3.42

4.06

3.19

n

10

10

10

10

G4-C1A, M & 1000

Mean

26.45

55.42

41.39

9.64

±SD

5.22

3.11

3.79

3.52

n

10

10

10

10

M: Male; C1A: Cohort 1A; SD: Standard Deviation; n: Number of animals .

TABLE 47 (Contd…). SUMMARY OF SPLENIC LYMPHOCYTE SUBPOPULATION ANALYSIS - COHORT 1A


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

 

Percent of Splenic lymphocyte Parental Sub-populations (%)

B Lymphocytes
(B Cells)

T Helper Cells
(Th/CD4+)

T Cytotoxic Cells

(Tc/CD8+)

Natural Killer Cells

(NK Cells)

G1-C1A, F & 0

Mean

29.99

48.80

46.78

13.12

±SD

5.24

12.28

12.17

8.09

n

10

10

10

10

G2-C1A, F & 100

Mean

28.83

51.02

41.45

10.60

±SD

5.14

7.34

8.67

6.91

n

10

10

10

10

G3-C1A, F & 300

Mean

28.48

51.02

41.16

9.96

±SD

3.28

10.12

11.85

5.73

n

10

10

10

10

G4-C1A, F & 1000

Mean

30.33

49.25

45.84

12.37

±SD

4.54

11.82

12.63

6.08

n

10

10

10

10

F: Female; C1A: Cohort 1A; SD: Standard Deviation; n: Number of Animals

TABLE 48.      SUMMARY OF SEXUAL MATURITY (BALANO-PREPUTIAL SEPERATION) AND BODY WEIGHT (g) RECORD -  COHORT 1A

Group, Sex & Dose

(mg/kg body weight/day)

 

Balano-preputial separation (Day of Occurrence)

Body Weight on

Sexual maturation

G1-C1A, M & 0

Mean

43.30

207.21

±SD

1.45

12.83

n

20

20

G2-C1A, M & 100

Mean

43.55

205.97

±SD

1.32

13.80

n

20

20

G3-C1A, M & 300

Mean

42.85

206.55

±SD

1.81

14.14

n

20

20

G4-C1A, M & 1000

Mean

43.40

201.02

±SD

1.05

11.59

n

20

20

M: Male; C1A: Cohort 1A; SD: Standard Deviation; n: Number of Animals

TABLE 49.      SUMMARY OF SPERM MOTILITY (%) RECORD - COHORT 1A

Group, Sex & Dose

(mg/kg body weight/day)

Sperm Motility (%)

G1-C1A, M & 0

Mean

96.4

±SD

0.5

n

20

G2-C1A, M & 100

Mean

96.4

±SD

0.7

n

20

G3-C1A, M & 300

Mean

96.0

±SD

0.7

n

20

G4-C1A, M & 1000

Mean

96.6

±SD

0.4

n

20

M: Male; C1A: Cohort 1A; SD: Standard Deviation; n: Number of Animals

TABLE 50.      SUMMARY OF SPERM MORPHOLOGY RECORD - COHORT 1A

Group, Sex

& Dose

(mg/kg body weight/day)

Head Abnormalities

Neck Abnormalities

Tail

Abnormalities

Multiple Abnormalities

Sperms with Abnormality

Normal Sperms

Total

%

Total

%

Total

%

Total

%

No.

%

No.

%

G1, M & 0

Mean

4.15

2.08

0.40

0.20

0.75

0.38

0.05

0.03

5.35

2.68

194.65

97.33

±SD

2.06

1.03

0.60

0.30

1.33

0.67

0.22

0.11

2.13

1.07

2.13

1.07

n

20

20

20

20

20

20

20

20

20

20

20

20

G2, M & 100

Mean

4.90

2.45

0.35

0.18

1.20

0.60

0.30

0.15

6.75

3.38

193.25

96.63

±SD

2.07

1.04

0.67

0.34

1.20

0.60

0.57

0.29

3.27

1.64

3.27

1.64

n

20

20

20

20

20

20

20

20

20

20

20

20

G3, M & 300

Mean

4.65

2.33

0.20

0.10

0.80

0.40

0.15

0.08

5.80

2.90

194.20

97.10

±SD

1.81

0.91

0.52

0.26

0.70

0.35

0.49

0.24

2.17

1.08

2.17

1.08

n

20

20

20

20

20

20

20

20

20

20

20

20

G4, M & 1000

Mean

3.85

1.93

0.25

0.13

0.95

0.48

0.25

0.13

5.30

2.65

194.70

97.35

±SD

2.81

1.41

0.44

0.22

1.00

0.50

0.44

0.22

2.41

1.20

2.41

1.20

n

20

20

20

20

20

20

20

20

20

20

20

20

M: Male; C1A: Cohort 1A; SD: Standard Deviation; n: Number of Animals

TABLE 51.      SUMMARY OF SPERMATID HEAD COUNT RECORD - COHORT 1A

Group, Sex & Dose

(mg/kg body weight/day)

Sperm head count (No.)

Daily Sperm Production

(x 106per gram of Testis)

Daily Sperm Production
(x106per Testis)

G1-C1A,

M & 0

Mean

185.00

15.1

52.9

±SD

13.25

1.1

5.9

n

20

20

20

G2-C1A,

M & 100

Mean

173.30*

14.1*

49.6

±SD

6.98

0.6

4.8

n

20

20

20

G3-C1A,

M & 300

Mean

182.66

14.9

52.5

±SD

8.26

0.7

4.6

n

20

20

20

G4-C1A,

M & 1000

Mean

178.36

14.5

50.6

±SD

7.93

0.6

4.3

n

20

20

20

M: Male; C1A: Cohort 1A; SD: Standard Deviation; n: Number of Animals

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

TABLE 52.      SUMMARY OF SEXUAL MATURITY (VAGINAL PATENCY), BODY WEIGHT (g) ON VAGINAL PATENCY AND TIME INTERVAL BETWEEN VAGINAL PATENCY AND OCCURRENCE OF FIRST CORNIFIED CELLS - COHORT 1A

Group, Sex & Dose

(mg/kg body weight/day)

Vaginal Opening

(Day of Occurrence)

Body Weight (g) on

Sexual Maturation

Occurrence of First Cornified Cells
(Postnatal Day)

Time Interval between Vaginal Opening and Occurrence of First Cornified Cells (Days)

G1-C1A, F & 0

Mean

34.75

117.90

36.30

1.55

±SD

2.59

12.65

2.60

1.61

n

20

20

20

20

G2-C1A, F & 100

Mean

34.25

118.02

35.90

1.65

±SD

1.77

8.35

1.62

1.46

n

20

20

20

20

G3-C1A, F & 300

Mean

34.40

117.36

35.55

1.15

±SD

2.06

10.80

2.24

1.31

n

20

20

20

20

G4-C1A, F & 1000

Mean

34.75

116.42

36.25

1.50

±SD

2.10

7.99

1.55

1.54

n

20

20

20

20

F: Female; C1A: Cohort 1A; SD: Standard Deviation; n: Number of Animals

TABLE 53.      SUMMARY OF OESTRUS CYCLE EVALUATION [PND 75 to 88] -COHORT 1A

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

Length of Oestrus Cycle (Days)

G1-C1A, F & 0

Mean

4.98

±SD

0.11

n

20

G2-C1A, F & 100

Mean

4.95

±SD

0.15

n

20

G3-C1A, F & 300

Mean

4.95

±SD

0.15

n

20

G4-C1A, F & 1000

Mean

4.93

±SD

0.18

n

20

F: Female; C1A: Cohort 1A; SD: Standard Deviation; n: Number of Animals

TABLE 54.      SUMMARY OF CLINICAL SIGNS OF TOXICITY, DETAILED CLINICAL EXAMINATION AND MORTALITY RECORD - COHORT 1B

Group, Sex & Dose

(mg/kg body weight/day)

Total No. of Animals

Clinical Signs of Toxicitya:

Observation

Detailed Clinical Examinationb:

Observation

Mortalityc:

No. of Mortalities

G1-C1B, M & 0

20

N

NAD

0

G2-C1B, M & 100

20

N

NAD

0

G3-C1B, M & 300

20

N

NAD

0

G4-C1B, M & 1000

20

N

NAD

0

M: Male; N: Normal; NAD: No Abnormality Detected;
a: observed daily once; b: observed weekly once; c: observed twice daily

TABLE 54 (Contd…). SUMMARYOF CLINICAL SIGNS OF TOXICITY, DETAILED CLINICAL EXAMINATION AND MORTALITY RECORD - COHORT 1B

Group, Sex & Dose

(mg/kg body weight/day)

Total No. of Animals

Clinical Signs of Toxicitya:

Observation

Detailed Clinical Examinationb:

Observation

Mortalityc:

No. of Mortalities

G1-C1B, F & 0

20

N

NAD

0

G2-C1B, F & 100

20

N

NAD

0

G3-C1B, F & 300

20

N

NAD

0

G4-C1B, F & 1000

20

N

NAD

0

F: Female; C1B: Cohort 1B; N: Normal; NAD: No Abnormality Detected;
a: observed daily once; b: observed weekly once; c: observed twice daily

TABLE 55.      SUMMARY OF BODY WEIGHT (g) RECORD - COHORT 1B

Group, Sex

& Dose

(mg/kg body

weight/day)

Body Weight (g) on Postnatal Day

21

25

28

32

35

42

49

56

63

70

77

84

91

98

G1-C1B,

M & 0

Mean

48.39

66.93

86.09

113.79

141.26

193.47

238.58

269.63

303.05

332.26

363.72

391.08

414.45

433.12

±SD

4.93

7.46

8.36

10.58

13.56

17.93

20.15

22.15

24.04

28.11

32.11

39.38

42.67

44.07

n

20

20

20

20

20

20

20

20

20

20

20

20

20

20

G2-C1B,

M & 100

Mean

49.50

67.73

86.66

114.06

139.74

196.22

245.08

274.05

299.60

328.83

362.08

391.75

415.10

438.21

±SD

3.19

4.55

4.69

6.37

9.26

11.89

13.47

16.38

19.25

22.78

27.26

34.42

44.49

47.10

n

20

20

20

20

20

20

20

20

20

20

20

20

20

20

G3-C1B,

M & 300

Mean

49.36

67.72

86.20

113.11

141.30

194.81

242.95

272.22

298.20

327.44

363.29

393.49

411.79

437.00

±SD

5.71

8.36

10.96

13.52

20.70

20.44

19.81

23.24

26.21

28.87

30.49

34.95

39.74

43.67

n

20

20

20

20

20

20

20

20

20

20

20

20

20

20

G4-C1B,

M & 1000

Mean

47.44

64.81

82.21

108.50

131.66

183.43

230.60

256.67

285.62

318.28

353.25

381.10

402.44

424.31

±SD

5.04

5.79

7.29

8.70

13.65

16.72

20.17

23.26

28.51

33.05

35.31

39.47

41.83

44.13

n

20

20

20

20

20

20

20

20

20

20

20

20

20

20

M: Male; C1B: Cohort 1B; SD: Standard Deviation; n: Number of Animals

TABLE 55 (Contd…). SUMMARYOF BODY WEIGHT (g) RECORD - COHORT 1B

Group, Sex

& Dose

(mg/kg body

weight/day)

Body Weight (g) on Postnatal Day

21

25

28

32

35

42

49

56

63

70

77

84

91

98

G1-C1B,

F & 0

Mean

48.06

66.61

82.90

106.16

125.95

162.26

188.47

206.14

220.86

237.70

253.54

267.88

277.59

291.04

±SD

5.61

6.43

6.93

7.94

9.48

9.94

11.84

14.88

17.85

18.55

22.44

25.76

26.69

27.81

n

20

20

20

20

20

20

20

20

20

20

20

20

20

20

G2-C1B,

F & 100

Mean

48.04

65.56

82.67

106.41

126.83

164.46

191.70

209.23

221.78

238.68

252.32

265.66

275.79

288.13

±SD

4.17

4.82

6.36

8.69

10.72

10.80

12.74

15.16

17.82

20.74

20.63

21.82

21.11

20.48

n

20

20

20

20

20

20

20

20

20

20

20

20

20

20

G3-C1B,

F & 300

Mean

47.22

64.66

79.70

100.15

118.06*

154.17

180.27

197.98

212.16

226.85

242.04

256.26

267.70

280.91

±SD

5.27

6.26

7.27

8.00

9.64

11.97

14.14

16.13

17.17

17.26

17.70

18.11

19.11

18.46

n

20

20

20

20

20

20

20

20

20

20

20

20

20

20

G4-C1B,

F & 1000

Mean

45.88

62.16

78.26

100.66

118.34*

152.31*

179.10

197.91

213.45

228.64

246.48

258.07

266.76

280.19

±SD

4.31

6.09

6.75

8.32

9.35

11.96

14.20

16.41

19.11

23.55

24.51

25.42

24.60

25.88

n

20

20

20

20

20

20

20

20

20

20

20

20

20

20

F: Female; C1B: Cohort 1B; SD: Standard Deviation; n: Number of Animals

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

TABLE 56.      SUMMARY OF PERCENT CHANGE IN BODY WEIGHT (%) WITH RESPECT TO POSTNATAL DAY 21 RECORD- COHORT 1B

Group, Sex

& Dose

(mg/kg body weight/day)

Percent Change in Body Weight (%) during Postnatal Day

21 to 25

21 to 28

21 to 32

21 to 35

21 to 42

21 to 49

21 to 56

21 to 63

21 to 70

21 to 77

21 to 84

21 to 91

21 to 98

G1-C1B,

M & 0

Mean

38.38

78.25

135.85

193.05

301.89

395.90

461.02

530.70

590.71

656.15

713.10

761.80

800.31

±SD

7.87

10.67

16.32

25.06

39.14

46.56

57.57

65.23

66.52

76.37

91.97

100.85

101.08

n

20

20

20

20

20

20

20

20

20

20

20

20

20

G2-C1B,

M & 100

Mean

37.07

75.41

131.02

183.00

297.80

396.97

455.85

507.83

566.55

633.68

693.72

740.55

787.44

±SD

8.53

9.25

15.15

20.75

33.04

40.69

49.45

58.49

59.53

65.46

78.64

93.25

99.34

n

20

20

20

20

20

20

20

20

20

20

20

20

20

G3-C1B,

M & 300

Mean

37.22

74.97

129.75

186.62

296.87

396.17

456.52

509.81

570.46

644.63

707.78

744.35

796.69

±SD

5.45

12.90

16.61

27.31

37.23

50.31

64.09

72.59

86.67

100.87

121.36

121.79

136.97

n

20

20

20

20

20

20

20

20

20

20

20

20

20

G4-C1B,

M & 1000

Mean

37.04

73.91

129.86

178.78

289.24

390.20

446.06

506.81

575.73

649.69

709.03

754.32

800.50

±SD

7.74

11.09

17.27

26.70

41.06

58.09

70.21

75.34

82.26

85.96

98.36

102.64

105.90

n

20

20

20

20

20

20

20

20

20

20

20

20

20

M: Male; C1B: Cohort 1B; SD: Standard Deviation; n: Number of Animals

TABLE 56 (Contd…). SUMMARY OF PERCENT CHANGE IN BODY WEIGHT (%) WITH RESPECT TO POSTNATAL DAY 21 RECORD - COHORT 1B

Group, Sex

& Dose

(mg/kg body weight/day)

Percent Change in Body Weight (%) during Postnatal Day

21 to 25

21 to 28

21 to 32

21 to 35

21 to 42

21 to 49

21 to 56

21 to 63

21 to 70

21 to 77

21 to 84

21 to 91

21 to 98

G1-C1B,

F & 0

Mean

39.05

73.42

122.40

164.32

241.37

296.76

333.82

364.24

399.65

432.77

463.21

483.55

511.86

±SD

6.96

11.70

18.04

27.07

38.73

47.13

52.83

54.67

58.03

64.24

72.86

74.92

78.30

n

20

20

20

20

20

20

20

20

20

20

20

20

20

G2-C1B,

F & 100

Mean

36.78

72.49

122.08

164.73

244.07

301.11

337.64

363.86

399.06

427.62

455.49

476.79

502.51

±SD

6.62

9.77

15.47

20.61

29.70

35.23

38.51

43.27

47.52

47.72

49.92

50.03

48.65

n

20

20

20

20

20

20

20

20

20

20

20

20

20

G3-C1B,

F & 300

Mean

37.40

69.62

113.49

151.99

229.25

285.15

323.67

354.20

385.44

417.50

448.30

472.53

500.72

±SD

8.12

12.17

18.19

25.36

34.45

42.37

54.37

59.17

59.27

57.96

64.35

65.57

66.05

n

20

20

20

20

20

20

20

20

20

20

20

20

20

G4-C1B,

F & 1000

Mean

35.51

70.90

119.87

158.72

233.41

292.39

333.62

367.41

400.18

439.15

465.02

484.18

513.31

±SD

5.01

8.39

10.70

15.84

26.72

35.85

41.34

44.99

48.93

49.58

56.93

56.29

56.14

n

20

20

20

20

20

20

20

20

20

20

20

20

20

F: Female; C1B: Cohort 1B; SD: Standard Deviation; n: Number of Animals

TABLE 57.      SUMMARY OF AVERAGE FEED CONSUMPTION (g/animal/day) RECORD - COHORT 1B

Group, Sex

& Dose

(mg/kg body

weight/day)

 

Feed Consumption (g/animal/day) during Postnatal Day

21 to 25

25 to 28

28 to 32

32 to 35

35 to 42

42 to 49

49 to 56

56 to 63

63 to 70

70 to 77

77 to 84

84 to 91

91 to 98

G1-C1B,

M & 0

Mean

7.14

8.54

11.16

13.74

16.73

17.69

21.89

23.77

24.98

26.09

27.27

27.66

29.07

±SD

1.21

0.97

1.41

1.81

1.00

0.87

0.94

1.33

1.42

1.78

1.68

1.73

1.95

n

20

20

20

20

20

20

20

20

20

20

20

20

20

G2-C1B,

M & 100

Mean

6.99

8.70

10.88

12.85

15.64*

17.18

21.17

22.53

24.33

25.49

26.68

27.37

27.84

±SD

1.05

1.30

1.67

1.75

0.66

0.79

1.39

1.70

1.13

1.78

1.94

2.22

1.66

n

20

20

20

20

20

20

20

20

20

20

20

20

20

G3-C1B,

M & 300

Mean

7.10

8.33

10.66

14.23

16.37

17.33

20.95

22.80

23.84

25.65

26.83

28.19

29.43

±SD

1.05

1.92

1.01

0.90

0.79

0.89

1.16

1.59

0.97

1.77

1.71

1.52

0.54

n

20

20

20

20

20

20

20

20

20

20

20

20

20

G4-C1B,

M & 1000

Mean

7.17

9.19

11.76

13.58

15.92

18.20

21.44

23.40

24.21

25.95

27.50

29.04

29.96

±SD

0.54

1.60

1.52

0.97

0.70

1.28

2.07

1.66

1.43

1.30

1.96

1.67

2.70

n

20

20

20

20

20

20

20

20

20

20

20

20

20

M: Male; C1B: Cohort 1B; SD: Standard Deviation; n: Number of Animals

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

TABLE 57 (Contd…). SUMMARYOF AVERAGE FEED CONSUMPTION (g/animal/day) RECORD - COHORT 1B

Group, Sex

& Dose

(mg/kg body

weight/day)

 

Feed Consumption (g/animal/day) on Postnatal Days

21 to 25

25 to 28

28 to 32

32 to 35

35 to 42

42 to 49

49 to 56

56 to 63

63 to 70

70 to 77

77 to 84

84 to 91

91 to 98

G1-C1B,

F & 0

Mean

6.77

7.68

10.06

13.20

14.40

16.96

18.52

19.50

20.83

21.91

22.80

24.11

25.47

±SD

0.56

0.88

1.53

2.22

1.03

0.85

0.74

1.75

2.19

2.02

1.87

2.32

1.89

n

20

20

20

20

20

20

20

20

20

20

20

20

20

G2-C1B,

F & 100

Mean

6.49

7.58

10.52

12.62

14.16

17.18

18.81

20.12

21.85

22.67

23.18

25.10

25.17

±SD

0.74

0.77

1.90

1.40

0.43

0.82

0.58

1.90

1.39

1.11

1.47

1.37

1.95

n

20

20

20

20

20

20

20

20

20

20

20

20

20

G3-C1B,

F & 300

Mean

6.82

7.83

11.01

12.82

14.08

17.00

18.63

20.50

22.02

23.17

23.93

26.06*

26.52

±SD

1.01

2.06

1.44

0.76

0.87

0.64

1.30

1.47

0.85

1.25

1.15

1.79

1.61

n

20

20

20

20

20

20

20

20

20

20

20

20

20

G4-C1B,

F & 1000

Mean

6.41

7.19

11.27

13.21

15.05

16.80

18.43

19.86

21.04

22.06

22.98

24.62

25.14

±SD

0.35

0.43

1.55

1.24

0.53

1.66

1.63

2.05

2.15

2.16

1.31

1.04

1.04

n

20

20

20

20

20

20

20

20

20

20

20

20

20

F: Female; C1B: Cohort 1B; SD: Standard Deviation; n: Number of Animals

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

TABLE 58.      SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD - COHORT 1B

Group, Sex & Dose

(mg/kg body weight/day)

Adrenals

Thymus

Spleen

Testes

Epididymis

G1-C1B, M & 0

Mean

0.0605

0.4214

0.8094

3.4221

1.3108

±SD

0.0107

0.0985

0.1295

0.3335

0.1501

n

20

20

20

20

20

G2-C1B, M & 100

Mean

0.0596

0.4736

0.8431

3.5892

1.3537

±SD

0.0062

0.0868

0.1545

0.2201

0.1101

n

20

20

20

20

20

G3-C1B, M & 300

Mean

0.0625

0.4398

0.8185

3.5764

1.3703

±SD

0.0099

0.0745

0.1017

0.3016

0.1023

n

20

20

20

20

20

G4-C1B, M & 1000

Mean

0.0580

0.4460

0.7735

3.4914

1.2917

±SD

0.0083

0.0863

0.1300

0.1853

0.0882

n

20

20

20

20

20

M: Male; C1B: Cohort 1B; SD: Standard Deviation; n: Number of Animals

TABLE 58 (Contd…). SUMMARYOF ABSOLUTE ORGAN WEIGHT (g) RECORD - COHORT 1B

Group, Sex & Dose

(mg/kg body weight/day)

 

Prostate

Seminal vesicles with

coagulating glands

Pituitary

Mandibular

lymph nodes

Iliac lymph nodes

G1-C1B, M & 0

Mean

1.3519

1.2715

0.0186

0.1730

0.0431

±SD

0.2766

0.2908

0.0083

0.0637

0.0132

n

20

20

20

20

20

G2-C1B, M & 100

Mean

1.4074

1.2399

0.0144*

0.1339*

0.0388

±SD

0.2249

0.2958

0.0029

0.0293

0.0110

n

20

20

20

20

20

G3-C1B, M & 300

Mean

1.3656

1.3833

0.0155

0.1323*

0.0381

±SD

0.2608

0.3204

0.0049

0.0311

0.0088

n

20

20

20

20

20

G4-C1B, M & 1000

Mean

1.3003

1.2616

0.0147

0.1381*

0.0381

±SD

0.1653

0.2482

0.0027

0.0320

0.0108

n

20

20

20

20

20

M: Male; C1B: Cohort 1B; SD: Standard Deviation; n: Number of Animals

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

TABLE 58 (Contd…). SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD - COHORT 1B

Group, Sex & Dose

(mg/kg body weight/day)

Adrenals

Thymus

Spleen

Ovaries

Uterus

Pituitary

Mandibular lymph node

Iliac lymph node

G1-C1B, F & 0

Mean

0.0693

0.4257

0.6051

0.1439

0.5064

0.0171

0.1321

0.0333

±SD

0.0066

0.0650

0.0722

0.0239

0.1114

0.0030

0.0345

0.0061

n

20

20

20

20

20

20

20

20

G2-C1B, F & 100

Mean

0.0673

0.3991

0.6484

0.1251*

0.5034

0.0167

0.1599

0.0306

±SD

0.0066

0.0581

0.1079

0.0156

0.0965

0.0033

0.1986

0.0062

n

20

20

20

20

20

20

20

20

G3-C1B, F & 300

Mean

0.0655

0.3836

0.5638

0.1322

0.4910

0.0158

0.1287

0.0300

±SD

0.0077

0.0653

0.0927

0.0182

0.1010

0.0022

0.0341

0.0066

n

20

20

20

20

20

20

20

20

G4-C1B, F & 1000

Mean

0.0681

0.3933

0.6103

0.1424

0.4691

0.0172

0.1352

0.0322

±SD

0.0077

0.0709

0.0775

0.0309

0.0833

0.0019

0.0273

0.0079

n

20

20

20

20

20

20

20

20

F: Female; C1B: Cohort 1B; SD: Standard Deviation; n: Number of Animals

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

TABLE 59.      INDIVIDUAL ANIMAL TERMINAL BODY WEIGHT (g) AND RELATIVE ORGAN WEIGHT (%) RECORD - COHORT 1B

Group, Sex & Dose

(mg/kg body weight/day)

 

Terminal
 Body Weight (g)

Adrenals

Thymus

Spleen

Testes

Epididymis

G1-C1B, M & 0

Mean

405.98

0.0149

0.1043

0.2021

0.8499

0.3253

±SD

43.79

0.0023

0.0231

0.0422

0.1045

0.0413

n

20

20

20

20

20

20

G2-C1B, M & 100

Mean

411.27

0.0146

0.1165

0.2056

0.8815

0.3317

±SD

48.58

0.0019

0.0230

0.0336

0.0918

0.0306

n

20

20

20

20

20

20

G3-C1B, M & 300

Mean

410.29

0.0153

0.1076

0.2011

0.8799

0.3368

±SD

44.39

0.0026

0.0167

0.0285

0.1073

0.0357

n

20

20

20

20

20

20

G4-C1B, M & 1000

Mean

396.61

0.0148

0.1127

0.1954

0.8911

0.3289

±SD

44.04

0.0028

0.0199

0.0287

0.1117

0.0364

n

20

20

20

20

20

20

M: Male; C1B: Cohort 1B; SD: Standard Deviation; n: Number of Animals

TABLE 59 (Contd…). SUMMARY OF TERMINAL BODY WEIGHT(g) AND RELATIVE ORGAN WEIGHT (%) RECORD - COHORT 1B

Group, Sex & Dose

(mg/kg body weight/day)

Prostate

Seminal vesicles with coagulating glands

Pituitary

Mandibular

lymph node

Iliac lymph node

G1-C1B, M & 0

Mean

0.3334

0.3121

0.0046

0.0433

0.0108

±SD

0.0582

0.0554

0.0022

0.0172

0.0036

n

20

20

20

20

20

G2-C1B, M & 100

Mean

0.3455

0.3061

0.0035*

0.0327*

0.0095

±SD

0.0587

0.0845

0.0007

0.0068

0.0028

n

20

20

20

20

20

G3-C1B, M & 300

Mean

0.3327

0.3382

0.0038

0.0322*

0.0094

±SD

0.0507

0.0781

0.0009

0.0066

0.0025

n

20

20

20

20

20

G4-C1B, M & 1000

Mean

0.3309

0.3217

0.0038

0.0351*

0.0097

±SD

0.0518

0.0701

0.0009

0.0086

0.0027

n

20

20

20

20

20

M: Male; C1B: Cohort 1B; SD: Standard Deviation; n: Number of Animals

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

TABLE 59 (Contd…). SUMMARY OF TERMINAL BODY WEIGHT(g) AND RELATIVE ORGAN WEIGHT (%) RECORD -

COHORT 1B

Group, Sex & Dose

(mg/kg body weight/day)

Terminal Body Weight (g)

Adrenals

Thymus

Spleen

Ovaries

Uterus

Pituitary

Mandibular lymph node

Iliac lymph node

G1-C1B, F & 0

Mean

262.57

0.0265

0.1630

0.2311

0.0551

0.1939

0.0065

0.0505

0.0127

±SD

27.34

0.0024

0.0248

0.0220

0.0093

0.0432

0.0012

0.0127

0.0024

n

20

20

20

20

20

20

20

20

20

G2-C1B, F & 100

Mean

259.00

0.0262

0.1555

0.2516

0.0486

0.1944

0.0065

0.0597

0.0120

±SD

21.42

0.0033

0.0286

0.0452

0.0071

0.0334

0.0015

0.0647

0.0030

n

20

20

20

20

20

20

20

20

20

G3-C1B, F & 300

Mean

252.56

0.0260

0.1523

0.2243

0.0525

0.1957

0.0063

0.0516

0.0119

±SD

17.62

0.0032

0.0273

0.0395

0.0073

0.0440

0.0008

0.0160

0.0027

n

20

20

20

20

20

20

20

20

20

G4-C1B, F & 1000

Mean

252.42

0.0273

0.1568

0.2441

0.0568

0.1880

0.0069

0.0541

0.0128

±SD

28.04

0.0046

0.0289

0.0376

0.0124

0.0387

0.0011

0.0119

0.0028

n

20

20

20

20

20

20

20

20

20

F: Female; C1B: Cohort 1B; SD: Standard Deviation; n: Number of Animals

TABLE 60.      SEXUAL MATURITY (BALANO-PREPUTIAL SEPERATION) AND BODY WEIGHT (g) - COHORT 1B

Group, Sex & Dose

(mg/kg body weight/day)

Balano-preputial separation

(Day of Occurrence)

Body Weight (g) on

Sexual Maturation

G1-C1B, M & 0

Mean

43.55

203.92

±SD

1.19

12.29

n

20

20

G2-C1B, M & 100

Mean

43.30

205.73

±SD

1.22

12.98

n

20

20

G3-C1B, M & 300

Mean

43.60

207.96

±SD

1.39

14.91

n

20

20

G4-C1B, M & 1000

Mean

43.80

197.28

±SD

1.11

11.90

n

20

20

M: Male; C1B: Cohort 1B; SD: Standard Deviation; n: Number of Animals

TABLE 60 (Contd…). SEXUAL MATURITY (VAGINAL PATENCY) AND BODY WEIGHT (g) - COHORT 1B

Group, Sex & Dose

(mg/kg body weight/day)

Vaginal Opening

(Day of Occurrence)

Body Weight (g) on

Sexual maturation

G1-C1B, F & 0

Mean

34.90

123.24

±SD

1.86

11.23

n

20

20

G2-C1B, F & 100

Mean

33.95

119.05

±SD

1.76

9.23

n

20

20

G3-C1B, F & 300

Mean

35.15

117.72

±SD

2.50

10.25

n

20

20

G4-C1B, F & 1000

Mean

35.40

119.37

±SD

1.93

6.07

n

20

20

F: Female; C1B: Cohort 1B; SD: Standard Deviation; n: Number of Animals

Applicant's summary and conclusion

Conclusions:
In conclusion, the test item Montanol 800 did not reveal any systemic toxicity, reproductive toxicity and developmental toxicity at all the tested dose levels (100, 300 and 1000 mg/kg body weight/day) when administered to P generation animals for a period of 10 weeks during pre-mating period (both P males and females), 2 weeks during mating period (both P males and females), throughout gestation and lactation periods up to weaning of F1 animals (for P females); when administered to F1 generation animals from PND 21 to 91 (for Cohort1A animals) and PND 21 to 98 (for Cohort1B animals) under experimental conditions employed.

Therefore, the no-observed-adverse-effect-level (NOAEL) of test item Montanol 800 is considered as 1000 mg/kg body weight/day for systemic, reproduction and developmental toxicity end points.
Executive summary:

The objective of this Extended One-Generation Reproductive Toxicity Study in Sprague Dawley Rats was to evaluate the effects of test item Montanol 800 that may occur as a result of pre and postnatal exposure on development as well as a thorough evaluation of systemic toxicity in pregnant and lactating females, young and adult offspring. The study served as a test for reproductive endpoints that required the interaction of males with females, females with conceptus, females with offspring and the F1 generation before and after sexual maturity.

The study included the evaluation of spermatogenesis for males and oestrous cycles, follicle counts/oocyte maturation and ovarian integrity (histopathology) for females. Specifically, but not exclusively, the following parameters were considered: gonadal function, the oestrus cycle, epididymal sperm maturation, mating behavior, conception, pregnancy, parturition and lactation. Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, was expected to identify specific target organs in the offspring. In addition, the study provided and/or confirmed information about the effects of Montanol 800 on the integrity and performance of the adult male and female reproductive systems.

A total of 200 (100 males + 100 females) Sprague Dawley rats were selected for parental (P) generation and distributed to four main groups. Each P generation group (G1, G2, G3 and G4) consisted of 25 males and 25 females. A total of 160 males and 160 females were selected on the day of weaning (PND21) and randomly assigned to two cohorts [Cohort 1A (80 males + 80 females) and Cohort 1B (80 males + 80 females)]. Each cohort was consisted of four groups and consisted of 20 males and 20 females per group. The animals in G1 (P generation)/G1 -C1A/G1 -C1B (F1 generation) groups were administered with vehicle [Sesame Oil], the animals in G2 (P generation)/G2 -C1A/G2 -C1B (F1 generation), G3 (P generation)/G3 -C1A/G3 -C1B (F1 generation), and G4 (P generation)/G4 -C1A/G4 -C1B (F1 generation) groups were administered with Montanol 800 at the dose levels of 100, 300 and 1000 mg/kg body weight/day respectively. The vehicle and test item formulations were administered orally by gavage at the dose volume of 4 mL/kg body weight.

The stability and homogeneity of test item in dose formulations were established before initiation of the treatment. The test item formulations were stable for 6 hours at room temperature with concentrations of 25 mg/mL and 250 mg/mL in sesame oil.Homogeneity and dose formulation analysis for dose concentration verification was performed during first and lastweek of P generation treatmentand first and lastweek of F1 generation treatmentand the mean results were within the range of 85 to 115% recovery to the nominal concentration and the relative standard deviation (% RSD) was less than 10%.

All the P generation animals were observed once daily for clinical signs, twice daily for mortality/morbidity and weekly once for detailed clinical examination. The body weights (throughout the experimental period) and feed consumption (throughout the experimental period, except during cohabitation period) was recorded once weekly for all the P animals. The assessment for haematology, clinical chemistry, urinalysis and thyroid hormone (Thyroxine Hormone and Thyroid Stimulating Hormone) levels was conducted for randomly selected 10 males and 10 females from each P generation group at termination. Sperm parameters (motility, morphology and daily sperm production) were estimated for all P males. Oestrus cyclicity evaluation was determined during pre-mating and cohabitation period for all P females. The body weights and feed consumption was recorded during gestation (Gestation Day 0, 7, 14 and 20) and during lactation (Lactation Day 1, 4, 7, 14 and 21) for all the P females. The gross pathology and organ weighing was performed on the day of termination for all the P animals. Histopathological examination was conducted on all the tissues collected from the P generation vehicle control and high dose group animals. All the P males and females were evaluated for reproductive performance or indices such as, mating and fertility index (for P males and females) and pre-coital interval, gestation length,fecundity index, gestation index, post-implantation loss and postnatal loss (for P females). All P females were observed for birth parameters (number of live/dead pups born, litter size, sex ratio, and live birth index per litter) and for litter observations (number of live/dead pups during lactation period, sex ratio and pup survival index per litter). All the P males and females did not reveal any test item related changes in systemic and reproductive endpoints at any of the tested dose groups.

The F1 generation pups were observed once daily for external examinations and twice daily for mortalities till termination, weighed individually on postnatal day (PND) 1, 4, 7, 14 and 21, observed for occurrences of postnatal developmental landmarks, observed for responses towards to sensory reflexes during postnatal period, measured for anogenital distance on PND 4, observed for retention of any nipples/areolae in male pups on PND 13, observed for gross pathological observations at termination. The assessment for serum Thyroxine (T4) levels was conducted for PND 4 and 21 pups.

All the F1 pups of both the sex did not reveal any test item related changes in growth parameters, postnatal developmental landmarks, sensory reflexes, thyroid hormone levels and no gross pathological changes noted at any of the tested dose group litters. All the Cohort 1A (F1 generation) animals were observed once daily for clinical signs, twice daily for mortality/morbidity and weekly once for detailed clinical examination. The body weights and feed consumption was recorded once weekly. All the C1A animals were evaluated for occurrence of sexual maturation (for balano-preputial separation in C1A males and for vaginal patency in C1A females). All C1A females were evaluated for mean occurrence of first cornified cells and time interval between vaginal patency and occurrence of first cornified cells. All C1A females were evaluated for oestrus cyclicity during PND 75 to 88. The assessment for haematology, clinical chemistry, urinalysis and thyroid hormone (Thyroxine Hormone and Thyroid Stimulating Hormone) levels was conducted for 10 randomly selected males and 10 randomly selected females from each C1A group at termination. Sperm parameters (motility, morphology and daily sperm production) were estimated for all C1A males. The gross pathology and organ weighing were performed on the day of termination for all the C1A animals. Histopathological examination was conducted on all the tissues collected from the C1A vehicle control and high dose group animals. The flow cytometric analysis of splenic samples for assessment of splenic lymphocyte sub-populations of T "helper" (CD4 +) cells, T cytotoxic (CD8 +) cells, natural killer (NK) cells and B lymphocytes was conducted for 10 randomly selected males and 10 randomly selected females from each C1A group.

All the C1A males and females did not reveal any test item related changes in systemic and reproductive endpoints at any of the tested dose groups when compared with vehicle control group. There were no changes noted insub-population of splenic lymphocytes such as, T "helper" (CD4 +) cells, T cytotoxic (CD8 +) cells, natural killer (NK) cells and B lymphocytes at any of the tested dose groups when compared with vehicle control group.

All the Cohort 1B (F1 generation) animals were observed once daily for clinical signs, twice daily for mortality/morbidity and weekly once for detailed clinical examination. The body weights and feed consumption was recorded once weekly. All the C1B animals were evaluated for occurrence of sexual maturation (for balano-preputial separation in C1B males and for vaginal patency in C1B females). The gross pathology and organ weighing were performed on the day of termination for all the C1B animals. All the C1B males and females did not reveal any test item related changes in systemic and reproductive endpoints at any of the tested dose groups when compared with vehicle control group.