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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Key study: Extended One-Generation Reproductive Toxicity study (EOGRTS). OECD 443, GLP study, performed orally in Sprague Dawley rats. The NOAEL of the test substance was determined to be 1000 mg/kg bw/day for systemic, reproduction and developmental toxicity.


 


Key study: Reproductive/Developmental toxicity screening test. OECD 422, GLP study, 12 female rats administrated orally at doses of 100, 300, 1000 mg/kg/day. The result was as follows: NOAEL maternal toxicity >= 300 mg/kg bw /day; NOAEL reproductive toxicity>= 1000 mg/kg bw/day; NOAEL offspring development >= 1000 mg/kg bw/day.


 


Supporting Study: Dose Range Finding Study in rats for EOGRT study, non GLP. The oral administration of the test item in Sprague Dawley rats did not produce any indication of systemic, reproduction and developmental toxicity at the dose levels of 100, 300 and 1000 mg/kg bw/d under experimental conditions employed and the same are then considered appropriate for the definitive test.

Link to relevant study records

Referenceopen allclose all

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:
11 February 2021 – 19 January 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
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:

- Premating exposure duration for parental (P0) animals: 10 weeks
- Basis for dose level selection: DRF study with no effects up to dose of 1000 mg/kg bw/day.
- Inclusion/exclusion of extension of Cohort 1B: No
- Termination time for F2: F1 generation not mated.
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B: No
- Inclusion/exclusion of developmental immunotoxicity Cohort 3: No
- Route of administration: oral (gavage)
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: In-house bred animals.
- Females (if applicable) nulliparous and non-pregnant: yes
- Weight at study initiation: 188.44-189.75 g (range of average values of each group of males at first day of dosing); 158.26-159.71 g (range of average values of each group of females at first day of dosing)
- Housing: Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with a stainless-steel mesh top grill having facilities for holding pelleted food and drinking water in a water bottle fitted with a stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
Pre mating: max. of 3 animals of the same sex and group per cage.
Mating: 2 animals (one male and one female) of the same group per cage.
Post mating: After confirming presence of sperm in the vaginal smear (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually. Sterilized paper shreds were provided as a nesting material from gestation day 20 onwards.
F1 animals: During Lactation period all F1 pups were housed along with the dam. Post weaning, 2 animals of the same sex and group per cage

- 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 animals throughout the experimental period.
- Water (e.g. ad libitum): Water was provided ad libitum throughout the experimental period. Deep bore-well water passed through a Reverse Osmosis Unit was provided in plastic water bottles with stainless-steel sipper tubes.
- Acclimation period: at least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.2-22.9ºC
- Humidity (%): 48-66 %
- Air changes (per hr): 12-15
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 11 February 2021 To: 07 September 2021
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% w/v Carboxy Methyl Cellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item formulations were prepared and administered to the animals within the established stability conditions. The required quantity of test item was weighed and triturated well in a mortar with a small quantity of vehicle until a homogenous suspension was formed and thereafter the entire quantity of the formulation was transferred into measuring cylinder. A small quantity of vehicle was added to rinse the mortar and this was transferred into the measuring cylinder. The rinsing procedure of mortar and pestle was repeated (many times) to ensure the transfer of the contents to the measuring cylinder. Finally, the volume was made up to required quantity with vehicle to get desired concentration of 10, 30 and 100 mg/mL of test item for low, mid and high dose groups respectively. The test formulations were maintained under stirring conditions using magnetic stirrer to maintain homogeneity of the test item formulations.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item was insoluble in distilled water and uniformly suspended in 0.5% w/v Carboxy Methyl Cellulose at the concentration of 100 mg/mL (the highest dose concentration selected for the study considering the dose volume of 10 mL/kg body weight) as per laboratory solubility/suspendibility test results. Hence, 0.5% w/v Carboxy Methyl Cellulose was used as vehicle for test item formulations.
- Concentration in vehicle: 10 - 100mg/mL
- Amount of vehicle (if gavage): 10mL/kg bw
- Lot/batch no. (if required): BCBN1690V, SLCH1520
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy.
- If mating has not occurred after 2 weeks, the animals will be separated without further continuation of mating. If there are insufficient males (e.g. due to male death before pairing) then male(s) which have already mated will be paired with a second female(s).
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol: No
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test item in dose formulations was established in a preliminary study. The test item formulations were stable at room temperature for 6 hours and up to 48 hours at 2 to 8°C at the concentrations of 10 mg/mL and 500 mg/mL in 0.5% Carboxymethyl Cellulose. Prepared test item formulations were administered to the animals within established stability conditions.
Homogeneity and dose formulation analysis for dose concentration verification was done by a validated ICP-MS method (study nº BIO-ANM 1575). Sampling and analysis of formulations were performed on days 16/02/2021 (Week 1), 15/04/2021 (Week 9), 10/06/2021 (Week 17) and 03/09/2021 (Week 29). Samples were collected in duplicates from top, middle and bottom layers from low, mid and high dose concentrations and in duplicates from middle layer from vehicle control. Formulations were considered acceptable, as the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) was ≤10%.
Duration of treatment / exposure:
Parental males: 10 weeks during pre-mating period + 2 weeks during mating period until sacrifice during post-mating period (a total of 98 to 108 days).
Parental females: 10 weeks during pre-mating period + 2 weeks during mating period + throughout gestation and lactation periods up to weaning of F1 animals (a total of 113 to 127 days).
Cohort 1A - From day of weaning (PND21) to PND 91 (13-weeks age)
Cohort 1B - From day of weaning (PND21) to at PND 98 (14-weeks age)
Frequency of treatment:
Once a day
Details on study schedule:
- F1 animals were not mated.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
G1/ G1-C1A/ G1-C1B
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
G2/ G2-C1A/ G2-C1B
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
G3/ G3-C1A/ G3-C1B
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
G4/ G4-C1A/ G4-C1B
No. of animals per sex per dose:
Parental generation: 25
Cohorts 1A and 1B: 20
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The doses of 100, 300 and 1000 mg/kg/day were selected for test item based on the results obtained from the dose range finding study in which no adverse effects were found up to the dose of 1000 mg/kg/day.
- Fasting period before blood sampling for clinical biochemistry: yes, overnight (water allowed).
Positive control:
None
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily for clinical signs of toxicity and twice daily for mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the treatment on day 1 and weekly thereafter during treatment for all the animals. Signs noted were e.g. changes in skin, fur, eyes and mucous membranes, occurrence of secretions and excretions and autonomic activity such as lacrimation, piloerection, pupil size, and unusual respiratory pattern.

BODY WEIGHT: Yes
- Time schedule for examinations:
P animals were weighed on the first day of dosing, weekly thereafter and at termination. P females were weighed on gestation days 0, 7, 14 and 20 during pregnancy and on days 1, 4, 7, 14 and 21 during lactation period.

FOOD CONSUMPTION: yes
-Feed consumption was recorded for all the P males and P females once a week coinciding with body weight recording. Feed consumption was recorded for all the P females during gestation days 0 to 7, 7 to 14 and 14 to 20, and during lactation days 1 to 4, 4 to 7, 7 to 14 and 14 to 21.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled terminal sacrifice
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 10 males and 10 females randomly selected from each group.
- Parameters checked: Haemoglobin concentration (HGB), Haematocrit (HCT), Erythrocyte count (RBC), Total leukocyte count (WBC), Platelet count (PLT), Differential leucocytes count (DLC), Absolute differential leucocytes count (DLC). Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) were estimated by a coagulation analyzer.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled terminal sacrifice
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 10 males and 10 females randomly selected from each group.
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Total Protein, Albumin, Glucose, Total Cholesterol, Creatinine, Blood urea.

URINALYSIS: Yes
- Time schedule for collection of urine: on the day of scheduled terminal sacrifice.
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 10 males and 10 females randomly selected from each group.
- Parameters checked: Blood, Protein, Glucose. In addition pH and specific gravity were also analyzed. After analysis of the above parameters, the urine was subjected for centrifugation at 1500 rpm for 3 minutes. Then the urine was subjected for microscopic examination for urine sediments.

OTHER:

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.

Oestrous cyclicity (parental animals):
Oestrus cycles were monitored daily during the following occasions:
- for a period of 2 weeks during pre-mating period before initiation of cohabitation;
- during cohabitation period until evidence of mating;
- at termination (on the day of necropsy)
Sperm parameters (parental animals):
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 were reserved for histopathological examination. Remaining epididymis were 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. The other testes were stored in -20 degree and evaluated for homogenization resistance spermatid head counts.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- maximum of 4-5 pups/sex/litter as nearly as possible; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:

In F1 pups:

Number of pups born (dead and live) in each litter, sex of pups, sex ratio, still births, live births, runts and external anomalies like, externally visible abnormalities, including cleft palate; subcutaneous haemorrhages; abnormal skin colour or texture; presence of umbilical cord; lack of milk in stomach; presence of dried secretions (if any) were recorded at birth.
During lactation period all the pups were observed for daily behavioral changes. The litter was observed daily in order to note the number of alive, dead and cannibalized pups.
Individual body weight of live pups on lactation days 1, 4, 7, 14 and 21 was recorded.
Postnatal developmental landmarks: Pinna unfolding, hair coat development, incisor eruption, eye opening and testes descending.
Reflex and Sensory Tests: Surface righting reflex, Auditory Startle reflex and air righting reflex.
Serum Thyroid Hormone (T4) Levels on surplus pups on PND 4.
Anogenital distance (AGD) of each live pup from each litter will be measured on PND 4.
All survived male pups were examined for appearance of nipples/areolae on PND 13.

In F1 surplus pups not allocated to cohorts (at weaning):

Body weight on PND21
Thyroid Hormonal Estimations: T4 and TSH analysis on PND21
Organ Collection, Weights and Preservation: The brain (weighed), spleen (weighed), thymus (weighed) and mammary tissues were collected and preserved for possible histopathology.

In F1 animals after weaning:

Clinical Signs of Toxicity and Mortality/Morbidity: All F1 animals were observed once daily throughout the experiment till sacrifice for treatment related clinical signs and more frequently when signs of toxicity were observed, and twice daily for mortality and morbidity.
Detailed Clinical Examination: All F1 animals were subjected to detailed clinical examinations weekly once after weaning until sacrifice. Signs noted were e.g. changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity such as lacrimation, piloerection, pupil size, and unusual respiratory pattern.
Body weight: All F1 animals were weighed on the day of weaning, any two days for 2 weeks following weaning and at least once a week until sacrifice.
Feed Consumption: The feed consumption was measured for all the animals once a week coinciding with body weight until sacrifice.
Oestrus Cyclicity:
Cohort 1A:
-daily for all F1 females in cohort 1A, after the onset of vaginal patency, until the first cornified smear was recorded, in order to determine the time interval between these two events.
- for a period of two weeks, commencing around PND 75 until sacrifice.
Cohort 1B: on the day of scheduled sacrifice.
Sexual maturation (Cohort 1A and 1B): Vaginal opening and balano-preputial separation.
Clinical Pathology (Cohort 1A): Blood was collected from 10 randomly selected males and 10 randomly selected females from each Cohort 1A group at termination and subjected to hematology, clinical chemistry and urinalysis as described for parental animals.
Thyroid Hormone Levels Estimation (Cohort 1A) Blood was collected from 10 randomly selected males and 10 randomly selected females from each Cohort 1A group at termination and subjected to thyroid hormone levels (T4 and TSH) as described for parental animals.
Sperm Parameters (Cohort 1A): Sperm parameters were measured for all cohort 1A males as described for parental animals.


GROSS EXAMINATION OF DEAD PUPS: Yes, for external and internal abnormalities. Possible cause of death was determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All the survived P males were sacrificed after completion of mating procedure.
- Maternal animals: females not confirmed with mating were sacrificed after 24 to 26 days from the day of termination of cohabitation process. The females confirmed with mating but not littered were sacrificed 24 to 26 days after confirmation of mating. All the surviving littered females were sacrificed on lactation day 22.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.

Additional evaluation:

Ovarian Follicular Assessment: A quantitative ovarian follicle assessment was carried out for all P females from control and high dose groups.
Postmortem examinations (offspring):
SACRIFICE
-The cohort 1A animals were sacrificed on PND 92 and the cohort 1B animals were sacrificed on PND 99.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.
- Vaginal smear were examined on the day of necropsy for all cohort 1A and 1B females to determine the stage of the oestrus cycle and allow correlation with histopathology in reproductive organs.
- Special attention was paid to the organs of the reproductive system. Animals that were humanely sacrificed in a moribund condition and dead pups were examined for possible defects and/or cause of death.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table 2 and 3 were prepared for microscopic examination and weighed, respectively.

Additional evaluations:

Cohort 1A: Histopathological examination of ovaries including quantitative evaluation of primordial and small growing follicles as well as corpora lutea.
Cohort 1B: reproductive and endocrine organs examined for histopathology in case of suspected reproductive or endocrine toxicants or in case of equivocal results from cohort 1A.

Organ-typic Development:
C1A Males: Caput, corpus and cauda of the epididymis and the vas deferens were examined.
C1A Females: Ovary with oviduct, uterus and vagina were examined.

Splenic lymphocyte subpopulation analysis (CD4+ and CD8+ T lymphocytes, B lymphocytes, and natural killer cells) using one half of the spleen, the other half of the spleen preserved for histopathological evaluation. 10 males and 10 females per group from Cohort 1A (1 male or 1 female per litter randomly selected) were subjected to this assessment.
Statistics:
After verification, the data was subjected to statistical analysis using SPSS software, version 22. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05). The statistical analysis was followed to the parameters as mentioned in the table 4 below.
Reproductive indices:
Mating index, fertility index, gestation index, parturation index, pregnancy index, sex ratio, live birth index, post implantation loss (%).
Offspring viability indices:
Pup survival index on lactation day 4/7/14/21, sex ratio on lactation day 4/7/14/21, post natal loss (%), AGD ratio.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs of toxicity noted in any of the tested dose group animals of both sexes throughout the experimental period.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no mortality/morbidity observed at any dose group during the experimental period.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no changes noted in mean body weight and percent change in mean body weight gain with respect to day 1 in all the tested dose group males throughout the experimental period when compared with vehicle control group.

An incidental statistically significant decrease in mean percent change in body weight gain on day 7 with respect to day 1 in group G2 females was noted when compared with vehicle control group. This noted change is unrelated to treatment as there were no changes noted in mean body weight and mean feed consumption during this period. Also, the mean percent change in body weight gain throughout the experimental period in group G2 females was unaffected.

There was no test item related changes noted in mean body weight and percent change in mean body weight gain during gestation and lactation periods in all the tested dose groups when compared with vehicle control group. However, statistically significant increase in mean percent change in body weight gain during lactation day 1 to 4 in group G3 and during lactation day 7 to 14 in group G4 were noted when compared with vehicle control group. These noted differences are considered as incidental and unrelated to treatment as there were no such changes noted in mean body weight and mean feed consumption during these periods from both the dose levels.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There was no test item related changes noted in mean feed consumption in all tested dose groups for both sexes throughout the experimental period (including gestation and lactation periods for females) when compared with vehicle control group.

However, statistically significant decrease in mean feed consumption during week 1 in all tested dose group males was noted when compared with vehicle control group. These noted differences are considered as incidental and unrelated to treatment as there were no changes noted in mean body weight and mean percent change in mean body weight gain with respect to day 1 during this period from any of the dose levels.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant decrease in mean total erythrocyte count in group G4 males, decrease in mean eosinophils (%) in groups G3 and G4 males and decrease in absolute neutrophils in groups G2 and G3 females were noted when compared with vehicle control group. These differences are considered as incidental and unrelated to treatment, as the obtained mean values are within laboratory historical control range of same species and strain and also similar changes were not occurred in other sex of same dose level.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant decrease in mean albumin levels in group G4 females was noted when compared with vehicle control group. This difference is considered as incidental and unrelated to treatment, as the obtained mean value is within laboratory historical control range of same species and strain and also similar changes were not occurred in other sex of same dose level.
Endocrine findings:
no effects observed
Description (incidence and severity):
There were no changes noted in estimated mean serum Thyroxine (T4) and Thyroxine Stimulating Hormone (TSH) levels in any of the tested dose groups of both sexes when compared with vehicle control group.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant increase in mean specific gravity of urine in group G4 females was noted when compared with vehicle control group. This difference is considered as incidental and unrelated to treatment, as the obtained mean value is within laboratory historical control range of same species and strain and also similar changes were not occurred in other sex of same dose level.
Behaviour (functional findings):
not examined
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 was no test item-related histopathological findings in high dose parental animals.

Few of the microscopic findings observed in this study such as ultimobranchial cyst(s) in thyroid gland, epithelial cyst(s)in thymus and all other findings were considered incidental as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats.

Quantitative ovarian follicular assessment (primordial and primary follicles) in all parental females of control and high dose groups did not reveal any test item related variations.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
A total of 3, 3, 2 and 1 female(s) were noted with single irregular oestrus cycle from groups G1, G2, G3 and G4, respectively. These occurrences are considered as incidental and unrelated to treatment exposure as the same animals were proceeded or preceded by normal cycles.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
Sperm motility(%): There were no changes noted in mean sperm motility (%) in all the tested dose groups when compared with vehicle control group.
Sperm Morphology: There were no test item-related changes noted in sperm morphologies in all the tested dose groups when compared with vehicle control group. The noted sporadic statistically significant differences in sperm morphology such as increase in head anomalies in group G3, decrease in tail anomalies in groups G3 and G4, increase in multiple anomalies in group G4 and increase in total sperms with anomalies in group G4 when compared with vehicle control group, are considered as toxicologically irrelevant.
Spermatid Head Count and Daily Sperm Production: There were no test item-related changes noted in mean spermatid head count or daily testicular sperm production per animal in any of the tested dose groups when compared with the vehicle control group. The noted statistically significant increase in mean spermatid head count in group G4 when compared with vehicle control group is considered as toxicologically irrelevant.

Reproductive performance:
no effects observed
Description (incidence and severity):
There were no effects on male and female reproductive performance/indices noted from P generation in any of the tested dose groups when compared with vehicle control group.

Mating index: All the males from all the dose groups i.e. 25 out of 25 males were confirmed as mated (100%). All the females from all the dose groups i.e. 25 out of 25 females were confirmed as sperm positive during vaginal smear examination (100%).
Fertility index: A total of 23 (out of 25), 21 (out of 25), 22 (out of 25) and 21 (out of 25) mated females were confirmed with presence of implantations / presence of live or dead pups / evidence of parturition with a fertility index of 92%, 84%, 88% and 84% from groups G1, G2, G3 and G4 respectively.
Gestation Index: A total of 21 (out of 23), 21 (out of 21), 22 (out of 22) and 21 (out of 21) pregnant females were confirmed with live born pups with a gestation index of 100% for all the tested dose groups and vehicle control group.
Implantation sites and Viable Pups: There were no statistically significant changes noted for both mean implantation sites and viable pups in any of the tested dose groups when compared with the vehicle control group.
Post-Implantation Loss: There were no statistically significant changes noted for mean number and percentage of occurred post-implantation losses in any of the tested dose groups when compared with the vehicle control group.

There were no changes observed in birth parameters such as, total litter size, number of live pups born, sex ratio (m/f) and live birth index in all the tested dose groups when compared with the vehicle control group. Also, the pup survival index per litter during lactation period was unaffected by the test item in all the tested dose groups when compared with the vehicle control group.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects found up to the highest dose tested (1000 mg/kg bw/day)
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
F1 pups: There were no external abnormalities or behavioural changes noted in any of the pups during daily observation from all the tested dose groups and vehicle control group during postnatal period. All the pups had normal behaviour during daily observations.
Cohort 1A /1B: There were no clinical signs of toxicity noted in any of the tested dose group animals of both sexes throughout the experimental period.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
F1 pups. Postnatal Loss: There were no statistically significant changes noted for mean number and percentage of occurred post-natal losses in any of the tested dose groups when compared with the vehicle control group.
Cohort 1A/1B: There were no mortality/morbidity observed at any dose group during the experimental period.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
F1 pups: There was no test item related changes noted in mean pup [both male and female] weight per litter in all the tested dose groups when compared with the vehicle control group, recorded on postnatal day (PND) 1, 4, 7, 14 and 21.
Cohort 1A: 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 in all the tested dose groups of both sexes throughout the experimental period when compared with vehicle control group.
Cohort 1B: There was 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 in all the tested dose groups of both sexes throughout the experimental period when compared with vehicle control group. Some statistically significant changes were noted during the experimental period which were considered due to stress induced by test item administration and not adverse as there were no other systemic toxicity effects noted in any of the tested dose groups.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A: There was no test item related changes noted in mean feed consumption in all the tested dose groups of both sexes throughout the experimental period when compared with vehicle control group. However, an incidental statistically significant decrease in mean feed consumption during week 7 in group G4-C1A was noted when compared with vehicle control group.
Cohort 1B: There was no test item related changes noted in mean feed consumption in all the tested dose groups of both sexes throughout the experimental period when compared with vehicle control group. However, an incidental statistically significant decrease in mean feed consumption during week 7 and 9 in group G2-C1B was noted when compared with vehicle control group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A: A statistically significant increase in mean absolute eosinophils in group G2-C1A males, increase in mean absolute lymphocytes in group G2-C1A females and decrease in mean APTT levels in group G2-C1A were noted when compared with vehicle control group. These differences are considered as incidental and unrelated to treatment, as the obtained mean values are within laboratory historical control range of same species and strain and also similar changes were not occurred in other sex of same dose level.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Cohort 1A: There were no changes noted in the obtained mean clinical chemistry values in all the tested dose groups of both sexes when compared with the vehicle control group.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A: an incidental and toxicologically irrelevant statistically significant decrease in mean volume of urine in group G2-C1A females was noted when compared with vehicle control group. This difference is considered unrelated to treatment and the obtained mean value is within laboratory historical control range of same species.
Sexual maturation:
no effects observed
Description (incidence and severity):
Cohort 1A males: There were no changes noted in mean days of occurrence of balano-preputial separation and mean body weight on the day of occurrence of occurrence of balano-preputial separation in any of the tested dose groups when compared with vehicle control group.
Cohort 1A females: There were no changes noted in mean days of occurrence of vaginal patency/opening, mean body weight on the day of occurrence of vaginal patency, mean occurrence of first cornified cells (days) and mean time interval between vaginal patency to occurrence of first cornified cells in any of the tested dose groups when compared with vehicle control group.
Cohort 1B males and females: There were no changes noted in mean days of occurrence of balano-preputial separation in C1B males and mean days of occurrence of vaginal patency/opening, in C1B females in any of the tested dose groups when compared with vehicle control group.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
F1 pups: There were no changes in mean pup [both male and female] anogenital distance measurement (mm) and its ratio per litter in all the tested dose groups when compared with the vehicle control group, recorded on postnatal day 4.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
F1 pups: There were no occurrences or evidences of retention of nipples in any of the male pups examined on the postnatal day 13 from all the tested dose groups and vehicle control group litters.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
F1 surplus pups: a statistically significant decrease in mean absolute thymus weight was noted in group G4 when compared with vehicle control group. This change is considered as incidental and unrelated to treatment, as the obtained mean value is within laboratory historical control range of PND 21 pups of same species and strain and also no gross pathological changes were noted in any of the pups at this dose level.
Cohort 1A: A statistically significant decrease in mean spleen weight (absolute and relative) in group G3-C1A males, increase in mean relative prostate weight in groups G3-C1A and G4-C1A males, increase in mean relative pituitary weight in all tested dose group males, increase in mean relative thyroid weight in group G4-C1A males, decrease in mean absolute spleen weight in groups G3-C1A and G4-C1A females, decrease in mean absolute ovaries weight in group G3-C1A, increase in mean absolute kidneys weight in group G2-C1A females, decrease in mean relative spleen and ovaries weight in group G3-C1A females and increase in mean relative heart weight in group G4-C1A females were noted when compared with vehicle control group.
These noted differences are considered as incidental and unrelated to treatment, as the obtained mean values are within laboratory historical control range of same species and strain and also neither gross pathological functional changes nor microscopic changes were noted in any of these organs in all the tested dose group animals.
Cohort 1B: There were no test item-related changes noted in mean absolute and relative organ weights in all the tested dose groups of both sexes when compared with vehicle control group. The noted statistically significant differences in some of the organs/tissues from all the tested dose groups of both sexes are not considered as adverse as the obtained mean values are within laboratory historical control range of same species and strain and also gross pathological examination did not reveal any changes in any of organs/tissues in all the tested dose group animals.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross pathological changes/findings noted in any of the tested dose group and vehicle control group of F1 generation animals of both sexes during the necropsy.
Histopathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There was no test item-related histopathological findings in high dose C1A animals.

Few of the microscopic findings observed in this study such as ultimobranchial cyst(s) in thyroid gland, epithelial cyst(s)in thymus and all other findings were considered incidental as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats.

Quantitative ovarian follicular assessment (primordial and primary follicles) in all C1A females of control and high dose groups did not reveal any test item related variations. Similarly, 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:
effects observed, non-treatment-related
Description (incidence and severity):
F1 pups:
Postnatal Developmental Landmarks: There were no changes noted in mean occurrences of postnatal developmental landmarks of F1 pups such as pinna unfold, fur development, incisor eruption, eye opening and testes descent evaluated during postnatal period in all the tested dose groups when compared with vehicle control group.
Sensory Reflexes: There were no changes noted in mean responses of sensory reflexes of F1 pups such as surface righting, auditory startle and air righting performed during postnatal period in all the tested dose groups when compared with vehicle control group.
Thyroid Hormone Levels (T4 and TSH):
There were no changes noted in mean T4 levels of PND 4 pups per litter in all the tested dose groups when compared with the vehicle control group.
There was no test item related changes noted in meanT4 and TSH levels of PND 21 pups per litter in all the tested dose groups when compared with the vehicle control group. The noted statistically significant increase in mean serum T4 levels in all the tested dose groups of PND 21 pups is considered as incidental and also the obtained mean values are within laboratory historical control range of same species and strain.

Cohort 1A:
Thyroid Hormone Levels (T4 and TSH): A statistically significant increase in serum T4 levels in group G4-C1A females and decrease in mean TSH levels were noted in all the tested dose group males when compared with vehicle control group. These differences are considered as incidental and unrelated to treatment, as the obtained mean values are within laboratory historical control range of same species and strain and also similar changes were not occurred in other sex of same dose level.
Splenic Lymphocyte Sub-populations: There was no test item related changes noted in mean splenic sub-populations of T "helper" (CD4+) cells, T cytotoxic (CD8+) cells, natural killer (NK) cells and B lymphocytes in any of the tested dose groups of both sexes when compared with vehicle control group. However, statistically significant increase in mean T "helper" (CD4+) cells populations in group G3-C1A females and statistically significant decrease in mean T cytotoxic (CD8+) cells populations in groups G2-C1A and G3-C1A females were noted when compared with vehicle control group. These differences are considered as incidental and unrelated to treatment, as the obtained mean value is within laboratory historical control range of same species and strain.
Sperm Motility (%): There were no changes noted in mean sperm motility (%)in all the tested dose groups when compared with vehicle control group.
Sperm Morphology: There were no test item-related changes noted in sperm morphology in all the tested dose groups when compared with vehicle control group. The noted sporadic statistically significant differences in sperm morphology such as increase in head anomalies in group G2-C1A, increase in neck anomalies in group G3-C1A and decrease in multiple anomalies in group G3-C1A and increase in total sperms with anomalies in group G3-C1A when compared with vehicle control group are considered as toxicologically irrelevant.
Spermatid Head Count and Daily Sperm Production: There were no changes noted in mean spermatid head count or daily testicular sperm production per animal in any of the tested dose groups when compared with the vehicle control group.
Oestrus Cycle Evaluation: a total of 2, 2, 1 and 1 female(s) were noted with single irregular oestrus cycle from groups G1-C1A, G2-C1A, G3-C1A and G4-C1A, respectively. These occurrences are considered as incidental and unrelated to treatment exposure as the same animals were proceeded or preceded by normal cycles.


Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects found up to the highest dose tested (1000 mg/kg bw/day)
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Table 5. Summary of body weight (g) record - parental (p) generation





































































































































































































































































Group, Sex


& Dose
(mg/kg body


weight/day)



 



Body Weight (g) on Day



1



7



14



21



28



35



42



49



56



63



70



77



84



91



98



104@



G1, M & 0



Mean



189.62



219.90



252.26



276.94



304.05



324.04



353.89



379.50



393.20



407.64



417.21



427.66



439.67



452.70



468.71



489.35



±SD



13.57



16.34



21.85



24.53



31.12



32.92



36.35



38.11



42.19



47.54



48.83



46.82



49.50



50.55



47.97



39.96



n



25



25



25



25



25



25



25



25



25



25



25



25



25



25



25



15



G2, M & 100



Mean



189.75



216.71



248.03



270.02



295.15



318.45



341.70



367.78



382.28



395.43



405.27



415.32



428.25



439.74



457.17



482.50



±SD



13.38



15.87



19.26



23.04



29.27



32.35



36.92



41.69



46.12



48.90



51.42



54.81



55.03



54.72



54.28



61.66



n



25



25



25



25



25



25



25



25



25



25



25



25



25



25



25



15



G3, M & 300



Mean



188.93



217.09



250.78



276.00



300.46



319.54



345.76



366.44



385.21



394.02



407.19



416.29



426.94



437.99



455.09



466.77



±SD



14.56



15.84



23.72



26.26



29.63



32.61



35.61



41.39



42.17



41.81



43.14



44.60



45.67



45.49



44.70



50.72



n



25



25



25



25



25



25



25



25



25



25



25



25



25



25



25



15



G4, M & 1000



Mean



188.44



215.90



251.91



274.96



297.54



317.80



345.10



370.04



382.35



397.21



407.83



418.15



433.05



447.78



467.67



463.80



±SD



12.34



13.21



17.64



23.03



30.44



32.05



32.47



38.89



41.22



41.76



43.16



44.24



44.05



44.48



49.47



41.68



n



25



25



25



25



25



25



25



25



25



25



25



25



25



25



25



15



@: The variation in number of animals is due to termination of males in batches to regulate the number on the day of sacrifice as sperm analysis was performed for each animal.

















































































































































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Body Weight (g) on Day



1



7



14



21



28



35



42



49



56



63



70



77



G1, F & 0



Mean



159.71



175.10



189.42



201.56



214.65



225.46



233.52



245.24



251.38



257.21



262.13



275.76



±SD



10.90



12.87



12.67



13.25



16.87



17.00



16.59



15.27



15.64



16.03



16.76



17.56



n



25



25



25



25



25



25



25



25



25



25



25



9



G2, F & 100



Mean



159.35



171.63



188.78



198.98



212.38



222.98



231.03



241.91



249.85



259.04



262.81



267.61



±SD



9.34



11.18



13.35



14.28



14.40



15.59



16.48



16.67



18.78



20.12



19.13



21.26



n



25



25



25



25



25



25



25



25



25



25



25



12



G3, F & 300



Mean



158.49



172.13



186.58



199.74



211.35



219.40



230.26



239.11



246.07



253.37



255.86



264.00



±SD



10.28



10.00



10.08



8.14



8.67



9.02



9.90



8.57



9.03



12.05



12.93



15.73



n



25



25



25



25



25



25



25



25



25



25



25



11



G4, F & 1000



Mean



158.26



172.69



188.23



202.11



212.14



218.79



231.45



240.51



244.81



252.21



254.19



257.59



±SD



10.15



8.59



9.64



11.66



11.57



11.72



11.03



12.03



12.97



14.19



14.94



14.31



n



25



25



25



25



25



25



25



25



25



25



25



16



 


Table 6. Summary record of gestation body weight (g) - parental (p) generation









































































































Group, Sex


& Dose


(mg/kg body


weight/day)



 



Body Weight (g) on Gestation Day (GD)



0



7



14



20



G1, F & 0



Mean



265.97



280.38



310.98



367.35



±SD



17.21



17.74



18.02



21.17



n



23



23



23



23



G2, F & 100



Mean



265.63



280.02



308.92



365.66



±SD



17.17



17.20



19.61



20.19



n



21



21



21



21



G3, F & 300



Mean



260.48



274.46



304.07



362.34



±SD



14.12



13.66



14.97



20.07



n



22



22



22



22



G4, F & 1000



Mean



260.52



275.17



302.92



363.15



±SD



15.47



16.24



15.94



18.01



n



21



21



21



21



 


Table 7. Summary record of lactation body weight (g) - parental (p) generation






















































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Body Weight (g) on Lactation Day (LD)



1



4



7



14



21



G1, F & 0



Mean



286.50



289.34



298.97



310.30



323.12



±SD



17.81



18.83



18.09



16.57



14.54



n



23



23



23



23



23



G2, F & 100



Mean



285.20



290.07



298.06



310.62



322.55



±SD



16.63



17.12



16.96



15.46



14.40



n



21



21



21



21



21



G3, F & 300



Mean



280.07



284.87



295.11



307.04



318.40



±SD



13.32



13.21



13.98



12.76



11.15



n



22



22



22



22



22



G4, F & 1000



Mean



278.63



282.70



291.07



304.72



316.48



±SD



14.50



14.20



14.05



12.61



12.15



n



21



21



21



21



21



 


Table 8. Summary of haematology record - parental (p) generation












































































































































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Total


Leucocyte


Count



Total


Erythrocyte


Count



Haemoglobin



Haematocrit



Platelet


Count



Neutrophils



Lymphocytes



Monocytes



Eosinophils



Basophils



(WBC)



(RBC)



(HGB)



(HCT)



(PLT)



(Neut)



(Lymph)



(Mono)



(Eos)



(Baso)



(103 cells/µL)



(106 cells/µL)



(g/dL)



(%)



(103 cells/µL)



(%)



(%)



(%)



(%)



(%)



G1, M & 0



Mean



10.63



9.51



15.70



53.63



769.60



29.20



63.58



3.70



2.21



0.35



±SD



3.54



1.94



1.05



14.87



197.51



4.18



4.57



0.46



1.37



0.20



n



10



10



10



10



10



10



10



10



10



10



G2, M & 100



Mean



11.66



8.74



15.52



47.29



758.20



30.97



62.29



3.45



1.67



0.64



±SD



7.12



0.65



1.03



3.99



198.49



6.40



6.39



0.59



0.73



0.57



n



10



10



10



10



10



10



10



10



10



10



G3, M & 300



Mean



10.01



8.25



15.13



46.51



824.30



29.15



65.09



3.15



1.19*



0.39



±SD



1.53



0.77



1.00



2.67



177.39



6.87



7.03



0.86



0.25



0.19



n



10



10



10



10



10



10



10



10



10



10



G4, M & 1000



Mean



10.19



8.09*



14.51



45.07



833.50



31.06



63.33



3.03



1.18*



0.49



±SD



2.93



1.26



1.97



4.91



235.67



7.29



7.14



1.04



0.40



0.54



n



10



10



10



10



10



10



10



10



10



10



 































































































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Absolute


Neutrophils



Absolute


Lymphocytes



Absolute


Monocytes



Absolute


Eosinophils



Absolute


 Basophils



Prothrombin


Time



Activated


Prothrombin


Time



(Neut)



(Lymph)



(Mono)



(Eos)



(Baso)



(PT)



(APTT)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(Seconds)



(Seconds)



G1, M & 0



Mean



3.09



6.75



0.40



0.25



0.03



19.08



29.05



±SD



1.08



2.25



0.14



0.23



0.02



1.55



11.18



n



10



10



10



10



10



10



10



G2, M & 100



Mean



3.68



7.21



0.40



0.19



0.07



19.25



23.76



±SD



2.89



3.99



0.24



0.13



0.06



6.37



6.25



n



10



10



10



10



10



10



10



G3, M & 300



Mean



2.94



6.49



0.32



0.12



0.04



18.44



24.78



±SD



0.88



1.10



0.10



0.04



0.02



1.65



4.65



n



10



10



10



10



10



10



10



G4, M & 1000



Mean



3.33



6.30



0.31



0.13



0.05



18.40



27.99



±SD



1.76



1.17



0.13



0.07



0.05



2.49



10.39



n



10



10



10



10



10



10



10



 












































































































































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Total


Leucocyte


Count



Total


Erythrocyte


Count



Haemoglobin



Haematocrit



Platelet Count



Neutrophils



Lymphocytes



Monocytes



Eosinophils



Basophils



(WBC)



(RBC)



(HGB)



(HCT)



(PLT)



(Neut)



(Lymph)



(Mono)



(Eos)



(Baso)



(103 cells/µL)



(106 cells/µL)



(g/dL)



(%)



(103 cells/µL)



(%)



(%)



(%)



(%)



(%)



G1, F & 0



Mean



11.26



8.42



16.47



49.92



853.20



39.79



52.76



4.44



1.71



0.32



±SD



3.63



0.49



0.66



2.99



333.77



7.02



7.43



1.83



1.30



0.11



n



10



10



10



10



10



10



10



10



10



10



G2, F & 100



Mean



9.20



8.38



16.64



50.35



774.40



32.39



61.00



4.30



1.17



0.31



±SD



2.24



0.98



1.47



5.72



257.15



10.05



9.91



0.97



0.90



0.19



n



10



10



10



10



10



10



10



10



10



10



G3, F & 300



Mean



8.56



7.96



15.74



47.48



946.90



33.55



59.82



4.23



1.33



0.29



±SD



2.79



0.95



1.36



5.19



99.65



9.63



9.57



1.06



0.60



0.16



n



10



10



10



10



10



10



10



10



10



10



G4, F & 1000



Mean



9.37



8.25



16.24



49.85



879.50



34.01



57.58



5.64



1.42



0.31



±SD



2.18



0.74



0.92



1.79



214.37



5.10



4.92



1.59



1.24



0.12



n



10



10



10



10



10



10



10



10



10



10



 































































































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Absolute


Neutrophils



Absolute


Lymphocytes



Absolute


Monocytes



Absolute


Eosinophils



Absolute


Basophils



Prothrombin


Time



Activated


Prothrombin


Time



(Neut)



(Lymph)



(Mono)



(Eos)



(Baso)



(PT)



(APTT)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(Seconds)



(Seconds)



G1, F & 0



Mean



4.51



5.92



0.51



0.17



0.04



16.97



23.81



±SD



1.77



1.95



0.28



0.09



0.02



4.11



15.54



n



10



10



10



10



10



10



10



G2, F & 100



Mean



3.03*



5.56



0.39



0.12



0.03



20.61



20.93



±SD



1.25



1.53



0.11



0.12



0.02



7.28



7.76



n



10



10



10



10



10



10



10



G3, F & 300



Mean



2.96*



5.02



0.38



0.11



0.03



19.04



17.64



±SD



1.33



1.63



0.21



0.05



0.01



3.50



3.12



n



10



10



10



10



10



10



10



G4, F & 1000



Mean



3.19



5.40



0.53



0.14



0.03



20.13



23.48



±SD



0.83



1.36



0.21



0.13



0.02



6.24



5.95



n



10



10



10



10



10



10



10



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


Table 9. Summary of clinical chemistry record - parental (p) generation
















































































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Glucose



Urea



Creatinine



Total Cholesterol



Total Protein



Albumin



(GLU)



-



(CRE)



(CHO)



(TPR)



(ALB)



(mg/dL)



(mg/dL)



(mg/dL)



(mg/dL)



(g/dL)



(g/dL)



G1, M & 0



Mean



89.10



36.80



0.54



53.40



7.49



3.30



±SD



15.46



5.71



0.04



7.73



0.41



0.24



n



10



10



10



10



10



10



G2, M & 100



Mean



92.70



35.98



0.54



45.20



7.48



3.30



±SD



10.33



6.78



0.10



10.03



0.55



0.14



n



10



10



10



10



10



10



G3, M & 300



Mean



109.30



36.04



0.52



54.00



7.58



3.38



±SD



28.58



8.72



0.03



10.27



0.38



0.15



n



10



10



10



10



10



10



G4, M & 1000



Mean



97.40



36.35



0.58



50.10



7.78



3.49



±SD



14.76



5.30



0.09



11.82



0.60



0.19



n



10



10



10



10



10



10



 
















































































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Alanine


aminotransferase



Aspartate


aminotransferase



Alkaline


phosphatase



Globulin



Albumin/


Globulin ratio



Blood Urea


Nitrogen



(ALT)



(AST)



(ALP)



(GLO)



(A/G Ratio)



(BUN)



(U/L)



(U/L)



(U/L)



(g/dL)



-



(mg/dL)



G1, M & 0



Mean



79.30



124.70



132.70



4.19



0.79



17.17



±SD



32.06



45.81



40.28



0.32



0.08



2.66



n



10



10



10



10



10



10



G2, M & 100



Mean



68.50



121.30



144.80



4.18



0.80



16.79



±SD



12.20



17.28



44.86



0.46



0.08



3.16



n



10



10



10



10



10



10



G3, M & 300



Mean



70.90



129.60



153.60



4.20



0.82



16.82



±SD



18.77



22.46



73.38



0.42



0.10



4.07



n



10



10



10



10



10



10



G4, M & 1000



Mean



73.80



118.10



151.50



4.29



0.82



16.96



±SD



14.69



16.64



55.30



0.44



0.06



2.47



n



10



10



10



10



10



10



 
















































































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Glucose



Urea



Creatinine



Total Cholesterol



Total Protein



Albumin



(GLU)



-



(CRE)



(CHO)



(TPR)



(ALB)



(mg/dL)



(mg/dL)



(mg/dL)



(mg/dL)



(g/dL)



(g/dL)



G1, F & 0



Mean



103.00



56.54



0.61



68.70



7.62



3.60



±SD



23.09



14.80



0.06



17.20



0.47



0.27



n



10



10



10



10



10



10



G2, F & 100



Mean



106.60



61.11



0.56



58.60



7.29



3.48



±SD



15.56



14.46



0.06



17.42



0.71



0.22



n



10



10



10



10



10



10



G3, F & 300



Mean



104.50



48.94



0.57



65.80



7.29



3.51



±SD



24.97



12.54



0.06



20.14



0.46



0.22



n



10



10



10



10



10



10



G4, F & 1000



Mean



97.80



71.83



0.55



62.00



7.04



3.32*



±SD



16.12



21.38



0.06



10.75



0.54



0.24



n



10



10



10



10



10



10



 
















































































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Alanine aminotransferase



Aspartate aminotransferase



Alkaline phosphatase



Globulin



Albumin/


Globulin ratio



Blood Urea Nitrogen



(ALT)



(AST)



(ALP)



(GLO)



(A/G Ratio)



(BUN)



(U/L)



(U/L)



(U/L)



(g/dL)



-



(mg/dL)



G1, F & 0



Mean



114.80



143.90



295.60



4.02



0.91



26.38



±SD



36.58



27.64



145.51



0.47



0.15



6.91



n



10



10



10



10



10



10



G2, F & 100



Mean



108.20



158.20



249.90



3.81



0.94



28.52



±SD



46.53



62.94



108.16



0.66



0.18



6.75



n



10



10



10



10



10



10



G3, F & 300



Mean



70.40



114.30



187.60



3.79



0.94



22.84



±SD



28.91



36.62



117.31



0.50



0.16



5.85



n



10



10



10



10



10



10



G4, F & 1000



Mean



117.70



173.30



338.20



3.72



0.92



33.52



±SD



57.63



102.35



246.41



0.67



0.17



9.98



n



10



10



10



10



10



10



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


 


Table 10. Summary of urinalysis record - parental (p) generation















































































































































































































































































































































Examination



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



G1,


M & 0



G2,


M & 100



G3,


M & 300



G4,


M & 1000



Number of Animals



10



10



10



10



Physical


Examination



Color



Pale Yellow



7



7



6



9



Yellow



3



3



4



1



Appearance



Clear



5



8



8



9



Turbid



5



2



2



1



Volume


(mL)



Mean



7.0



7.8



6.9



7.6



±SD



1.7



2.1



1.7



1.5



Chemical


Examination



pH



Mean



7.9



7.8



7.5



7.5



±SD



0.3



0.4



0.8



0.7



Specific gravity



Mean



1.011



1.010



1.011



1.011



±SD



0.004



0.003



0.003



0.005



Urobilinogen


(mg/dL)



Mean



0.3



0.3



0.3



0.3



±SD



0.3



0.3



0.3



0.3



Bilirubin


(mg/dL)



Neg



5



8



8



8



1



5



2



2



2



Ketones


(mg/dL)



Neg



4



4



4



5



5



6



6



6



5



Blood


(Ery/µL)



Neg



1



3



2



-



Ca10



2



-



-



1



Ca25



4



2



2



4



Ca80



1



2



3



3



>=Ca200



2



3



3



2



Protein


(mg/dL)



Neg



3



6



7



7



Trace



2



1



1



-



30



4



2



2



1



100



-



1



-



1



>=300



1



-



-



1



Nitrite



Neg



6



6



5



7



Pos



4



4



5



3



Leucocytes


(Leu/µL)



Neg



-



-



-



2



Ca15



1



5



4



4



Ca70



7



5



5



2



Ca125



2



-



-



2



Glucose


(mg/dL)



Neg



10



10



10



10



Micro Albumin


(mg/dL)



Neg



-



1



-



1



>15



7



4



3



3



15



3



5



7



6



Microscopic


Examination



Epi Cells



0



5



1



5



1



0-1



2



6



2



9



1-2



2



1



3



-



2-3



-



1



-



-



Casts



Absent



10



10



10



10



Crystals



Present



10



10



10



10



 

































































































































































































































































































































Examination



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



G1,


F & 0



G2,


F & 100



G3,


F & 300



G4,


F & 1000



Number of Animals



10



10



10



10



Physical


Examination



Color



Pale Yellow



10



8



8



8



Yellow



-



2



2



2



Appearance



Clear



10



9



9



9



Turbid



-



1



1



1



Volume


(mL)



Mean



6.0



5.6



6.2



6.4



±SD



1.4



1.1



1.0



1.7



Chemical


Examination



pH



Mean



7.9



7.8



7.6



7.4



±SD



0.6



0.5



0.6



0.8



 



Mean



1.008



1.012



1.012



1.014*



±SD



0.005



0.005



0.004



0.004



Urobilinogen


(mg/dL)



Mean



0.4



0.4



0.4



0.3



±SD



0.4



0.6



0.4



0.3



Bilirubin


(mg/dL)



Neg



5



4



5



10



1



5



5



5



-



Ketones


(mg/dL)



Neg



10



9



10



8



5



-



1



-



2



Blood


(Ery/µL)



Neg



8



4



7



9



Ca10



1



2



1



-



Ca25



-



1



1



-



Ca80



1



3



1



1



Protein


(mg/dL)



Neg



2



6



4



9



Trace



4



3



2



-



30



2



-



1



-



100



-



-



1



-



>=300



2



1



2



1



Nitrite



Neg



4



6



4



6



Pos



6



4



6



4



Leucocytes


(Leu/µL)



Neg



1



1



1



3



Ca15



6



4



4



5



Ca70



3



4



5



2



Ca125



-



1



-



-



Glucose


(mg/dL)



Neg



10



10



10



10



Micro Albumin


(mg/dL)



Neg



1



1



4



4



>15



8



4



6



1



15



1



5



-



5



Microscopic


Examination



Epi Cells



0



8



6



6



6



0-1



2



3



3



3



1-2



-



1



1



1



Casts



Absent



10



10



10



10



Crystals



Present



10



10



10



10



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


 


Table 11. Summary of terminal body weight (g) and organ weigtht relative to terminal body weight (%) record - parental (p) generation













































































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Terminal


Body Weight


(g)



Adrenals



Thymus



Spleen



Testes



Epididymides



Heart



G1, M & 0



Mean



458.36



0.0139



0.0660



0.3021



0.7706



0.3619



0.3226



±SD



49.77



0.0020



0.0100



0.0738



0.1159



0.0643



0.0350



n



25



25



25



25



25



25



25



G2, M & 100



Mean



448.24



0.0147



0.0700



0.3046



0.7697



0.3690



0.3324



±SD



56.37



0.0021



0.0122



0.0811



0.1148



0.0574



0.0506



n



25



25



25



25



25



25



25



G3, M & 300



Mean



447.03



0.0143



0.0666



0.3047



0.7814



0.3679



0.3560*



±SD



49.83



0.0023



0.0111



0.0801



0.1153



0.0589



0.0500



n



25



25



25



25



25



25



25



G4, M & 1000



Mean



454.74



0.0136



0.0690



0.3052



0.7701



0.3585



0.3333



±SD



47.89



0.0020



0.0141



0.0606



0.0935



0.0540



0.0403



n



25



25



25



25



25



25



25



 













































































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Kidneys



Brain



Liver



Prostate



Seminal vesicles with coagulating glands



Thyroid along


with parathyroid



Pituitary



G1, M & 0



Mean



0.6130



0.4702



2.4536



0.2976



0.4078



0.0062



0.0035



±SD



0.0817



0.0664



0.3199



0.0666



0.1250



0.0005



0.0003



n



25



25



25



25



25



25



25



G2, M & 100



Mean



0.6116



0.4783



2.4252



0.3059



0.3825



0.0062



0.0038*



±SD



0.0657



0.0538



0.2377



0.0667



0.1117



0.0006



0.0004



n



25



25



25



25



25



25



25



G3, M & 300



Mean



0.6302



0.4865



2.5252



0.2962



0.3941



0.0063



0.0036



±SD



0.0764



0.0606



0.3262



0.0830



0.1306



0.0004



0.0003



n



25



25



25



25



25



25



25



G4, M & 1000



Mean



0.6128



0.4638



2.4626



0.2835



0.3739



0.0061



0.0037



±SD



0.0911



0.0367



0.3435



0.0666



0.1109



0.0005



0.0004



n



25



25



25



25



25



25



25



 
































































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Terminal


Body Weight


(g)



Adrenals



Thymus



Spleen



Ovaries



Uterus with Cervix



G1, F & 0



Mean



307.39



0.0257



0.0720



0.2451



0.0438



0.1860



±SD



14.74



0.0027



0.0133



0.0481



0.0064



0.0377



n



25



25



25



25



25



25



G2, F & 100



Mean



305.35



0.0261



0.0754



0.2429



0.0457



0.1703



±SD



15.18



0.0031



0.0091



0.0421



0.0079



0.0486



n



25



25



25



25



25



25



G3, F & 300



Mean



301.56



0.0275



0.0776



0.2501



0.0443



0.1946



±SD



16.62



0.0030



0.0079



0.0481



0.0074



0.0423



n



25



25



25



25



25



25



G4, F & 1000



Mean



299.24



0.0265



0.0757



0.2522



0.0464



0.1807



±SD



14.69



0.0040



0.0106



0.0333



0.0063



0.0474



n



25



25



25



25



25



25



 
































































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Heart



Kidneys



Brain



Liver



Thyroid along


with parathyroid



Pituitary



G1, F & 0



Mean



0.3899



0.6406



0.6430



3.6558



0.0078



0.0052



±SD



0.0297



0.0522



0.0382



0.3757



0.0006



0.0005



n



25



25



25



25



25



25



G2, F & 100



Mean



0.3621



0.6410



0.6466



3.6413



0.0079



0.0053



±SD



0.1064



0.0609



0.0437



0.3475



0.0005



0.0007



n



25



25



25



25



25



25



G3, F & 300



Mean



0.3852



0.6845



0.6572



3.5934



0.0082



0.0052



±SD



0.0541



0.1793



0.0525



0.3594



0.0008



0.0006



n



25



25



25



25



25



25



G4, F & 1000



Mean



0.3881



0.6652



0.6621



3.6815



0.0080



0.0051



±SD



0.0865



0.0550



0.0552



0.3734



0.0006



0.0005



n



25



25



25



25



25



25



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


 


Table 12. Summary record of sperm motility - parental (p) generation































































Group, Sex & Dose


(mg/kg body weight/day)


 

Mean Percent of Sperm Motility (%)



G1, M & 0



Mean



89.9



±SD



1.9



n



25



G2, M & 100



Mean



90.0



±SD



1.3



n



25



G3, M & 300



Mean



89.7



±SD



1.5



n



25



G4, M & 1000



Mean



89.6



±SD



2.0



n



25



 


Table 13. Summary record of sperm morphology - parental (p) generation






































































































































































































































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



2.68



1.34



1.00



0.50



1.32



0.66



0.12



0.06



5.12



2.56


 

194.88



97.44



±SD



1.68



0.84



1.32



0.66



1.22



0.61



0.33



0.17



2.07



1.03


 

2.07



1.03



n



25



25



25



25



25



25



25



25



25



25


 

25



25



G2, M & 100



Mean



3.08



1.54



1.56



0.78



0.60



0.30



0.44



0.22



5.68



2.84


 

194.32



97.16



±SD



1.55



0.78



1.08



0.54



0.82



0.41



0.58



0.29



1.35



0.67


 

1.35



0.67



n



25



25



25



25



25



25



25



25



25



25


 

25



25



G3, M & 300



Mean



4.16*



2.08*



0.52



0.26



0.36*



0.18*



0.48



0.24



5.52



2.76


 

194.48



97.24



±SD



1.37



0.69



0.65



0.33



0.64



0.32



0.51



0.25



1.08



0.54


 

1.08



0.54



n



25



25



25



25



25



25



25



25



25



25


 

25



25



G4, M & 1000



Mean



3.68



1.84



1.08



0.54



1.12*



0.56*



0.84*



0.42*



6.72*



3.36*


 

193.28*



96.64*



±SD



1.73



0.86



1.00



0.50



0.93



0.46



0.85



0.43



1.40



0.70


 

1.40



0.70



n



25



25



25



25



25



25



25



25



25



25


 

25



25



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


 


Table 14. Summary record of spermatid head count - parental (p) generation

























































































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



 



Average Sperm head count



Daily Sperm Production


(x 106 per gram of Testis)



Daily Sperm Production


(x106 per Animal)



G1, M & 0



Mean



158.88



12.5



47.4



±SD



5.95



0.6



6.0



n



25



25



25



G2, M & 100



Mean



163.43



12.6



49.1



±SD



3.16



0.3



4.6



n



25



25



25



G3, M & 300



Mean



157.05



12.7



50.3



±SD



6.04



0.5



6.4



n



25



25



25



G4, M & 1000



Mean



163.63*



13.0



47.5



±SD



4.36



0.3



7.2



n



25



25



25



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


 


Table 15. Summary record of mean pup weight (g) per litter - F1 generation






























































































































































































































































Group


& Dose


(mg/kg body


weight/day)


 

PND 1


 

PND 4


 

PND 7


 

PND 14


 

PND 21



Mean Pup Weight



 



Mean Pup Weight



 



Mean Pup Weight



 



Mean Pup Weight



 



Mean Pup Weight



Male



Female



 



Male



Female



 



Male



Female



 



Male



Female



 



Male



Female



G1 & 0



Mean



6.60



6.29


 

11.18



10.68


 

16.84



16.13


 

28.05



26.71


 

43.70



40.64



±SD



0.32



0.26


 

0.71



0.70


 

0.68



0.82


 

0.63



0.89


 

2.23



1.50



n



23



23


 

23



23


 

23



23


 

23



23


 

23



23



G2 & 100



Mean



6.54



6.29


 

11.34



10.80


 

16.91



16.22


 

28.17



26.86


 

44.27



41.48



±SD



0.35



0.29


 

0.74



0.80


 

0.75



0.70


 

1.18



0.88


 

2.50



1.93



n



21



21


 

21



21


 

21



21


 

21



21


 

21



21



G3 & 300



Mean



6.63



6.30


 

11.53



10.81


 

17.04



15.99


 

28.32



26.81


 

45.22



42.15*



±SD



0.39



0.24


 

0.68



0.87


 

0.57



0.63


 

1.15



0.87


 

2.81



2.30



n



22



22


 

22



22


 

22



22


 

22



22


 

22



22



G4 & 1000



Mean



6.76



6.53*


 

11.13



10.77


 

16.99



16.47


 

28.18



27.17


 

44.69



42.28*



±SD



0.42



0.39


 

0.88



0.82


 

0.79



0.59


 

0.86



0.70


 

2.73



1.79



n



21



21


 

21



21


 

21



21


 

21



21


 

21



21



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


 


Table 16. Summary of serum T4 hormone levels (ng/ml) record - PND 21 pups - F1 generation















































































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


 

Serum T4 Levels (ng/mL)


 
 

G1 & 0



Mean



55.737


 

±SD



5.117


 

n



23


 

G2 & 100



Mean



61.945*


 

±SD



5.039


 

n



21


 

G3 & 300



Mean



69.303*


 

±SD



5.230


 

n



22


 

G4 & 1000



Mean



71.748*


 

±SD



5.377


 

n



21


 

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


 


Table 17. Summary of serum thyroid stimulating hormone (TSH) levels record - PND 21 pups - F1 generation















































































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


 

Serum TSH Levels (µIU/mL)


 
 

G1, F & 0



Mean



1.667


 

±SD



2.877


 

n



21


 

G2, F & 100



Mean



1.076


 

±SD



1.624


 

n



18


 

G3, F & 300



Mean



2.744


 

±SD



3.830


 

n



22


 

G4, F & 1000



Mean



1.649


 

±SD



2.343


 

n



17


 

n: Number of litters available with surplus pups (Outliers were excluded for mean calcualtions)


 


Table 18. Summary of absolute organ weight (g) record of PND 21 surplus pups - F1 generation








































































































































































Group


& Dose


(mg/kg body


weight/day)


 

Brain


 

Thymus


 

Spleen



Absolute Weight (g)



 



Absolute Weight (g)



 



Absolute Weight (g)



Male



Female


 

Male



Female


 

Male



Female



G1 & 0



Mean



1.5350



1.5319


 

0.2050



0.2067


 

0.2272



0.2307



±SD



0.0948



0.0818


 

0.0239



0.0259


 

0.0200



0.0266



n



23



21


 

23



21


 

23



21



G2 & 100



Mean



1.5300



1.5182


 

0.2053



0.2046


 

0.2265



0.2292



±SD



0.0615



0.0667


 

0.0217



0.0165


 

0.0245



0.0247



n



21



20


 

21



20


 

21



20



G3 & 300



Mean



1.5249



1.4606


 

0.2034



0.1965


 

0.2312



0.2284



±SD



0.1121



0.2169


 

0.0263



0.0265


 

0.0207



0.0197



n



22



21


 

22



21


 

22



21



G4 & 1000



Mean



1.5015



1.5057


 

0.1935



0.1827*


 

0.2239



0.2243



±SD



0.0667



0.0816


 

0.0263



0.0275


 

0.0204



0.0270



n



20



21


 

20



21


 

20



21



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


 


Table 19. Summary of body weight (g) record - Cohort 1A






























































































































































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Body Weight (g) on Day



21



25



28



32



35



42



49



56



63



70



77



84



91



G1-C1A,


M & 0



Mean



43.55



61.67



78.16



101.55



120.35



159.05



198.91



236.59



267.50



296.08



323.80



351.83



383.53



±SD



3.41



6.18



8.33



13.36



18.54



21.93



23.13



28.18



33.32



34.34



29.72



30.54



29.82



n



20



20



20



20



20



20



20



20



20



20



20



20



20



G2-C1A,


M & 100



Mean



44.32



62.36



77.94



99.22



118.42



157.32



196.24



231.03



267.24



295.34



322.64



349.65



379.30



±SD



3.29



5.39



4.79



11.97



18.57



27.24



31.99



37.98



46.04



51.11



51.15



47.30



43.86



n



20



20



20



20



20



20



20



20



20



20



20



20



20



G3-C1A,


M & 300



Mean



46.05*



64.21



78.72



100.74



120.44



157.91



194.27



234.21



271.34



294.85



321.13



346.23



373.63



±SD



3.04



5.19



6.35



11.00



14.07



14.65



18.37



24.65



30.50



37.13



33.57



35.52



34.47



n



20



20



20



20



20



20



20



20



20



20



20



20



20



G4-C1A,


M & 1000



Mean



44.12



63.91



76.90



95.70



110.98



151.03



185.20



220.06



250.81



278.13



304.68



332.81



363.89



±SD



2.79



4.34



3.70



7.91



9.43



18.28



20.21



19.40



24.27



28.43



27.30



25.39



27.93



n



20



20



20



20



20



20



20



20



20



20



20



20



20



 






























































































































































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Body Weight (g) on Day



21



25



28



32



35



42



49



56



63



70



77



84



91



G1-C1A,


F & 0



Mean



40.91



55.03



68.84



85.84



99.86



128.94



155.59



175.76



193.81



208.06



221.69



234.61



245.64



±SD



2.77



4.62



6.10



10.58



13.08



15.37



18.39



19.97



22.61



20.19



17.71



16.60



14.52



n



20



20



20



20



20



20



20



20



20



20



20



20



20



G2-C1A,


F & 100



Mean



41.08



58.27



71.17



91.11



105.23



134.38



158.05



179.32



197.30



212.37



225.84



237.34



247.90



±SD



2.53



4.61



5.44



9.88



12.61



15.28



16.70



19.16



20.00



21.08



18.33



17.68



16.93



n



20



20



20



20



20



20



20



20



20



20



20



20



20



G3-C1A,


F & 300



Mean



41.80



58.54*



70.60



88.32



103.97



128.99



153.59



173.83



190.10



204.41



218.89



232.38



242.33



±SD



2.38



4.18



4.96



9.89



12.35



15.64



18.57



21.65



21.51



21.59



19.26



16.78



14.06



n



20



20



20



20



20



20



20



20



20



20



20



20



20



G4-C1A,


F & 1000



Mean



43.14*



59.77*



71.98



88.96



103.81



128.79



151.32



170.75



189.05



205.99



222.40



232.95



243.62



±SD



2.54



4.44



6.77



10.52



13.35



13.69



16.63



17.71



20.65



18.26



17.86



15.41



15.92



n



20



20



20



20



20



20



20



20



20



20



20



20



20



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


 


Table 20. Summary of feed consumption (g/animal/day) record - Cohort 1A























































































































































































Group, Sex


& Dose


(mg/kg body


weight/day)



 



Feed Consumption on Postnatal Days



Week 1



Week 2



Week 3



Week 4



Week 5



Week 6



Week 7



Week 8



Week 9



Week 10



G1-C1A,


M & 0



Mean



8.10



12.50



16.82



18.40



21.01



23.45



25.96



26.56



27.19



28.45



±SD



0.72



0.69



0.73



0.84



0.87



1.10



0.36



0.43



0.43



1.05



n



10



10



10



10



10



10



10



10



10



10



G2-C1A,


M & 100



Mean



8.15



12.34



16.42



17.95



20.67



23.40



25.33



26.10



27.04



28.14



±SD



0.48



0.40



0.53



0.43



0.58



0.87



0.91



0.85



0.48



1.65



n



10



10



10



10



10



10



10



10



10



10



G3-C1A,


M & 300



Mean



7.99



12.43



16.75



18.13



21.08



23.13



25.62



26.37



27.07



27.93



±SD



0.58



0.62



0.68



0.74



0.68



0.71



0.61



0.57



0.56



1.29



n



10



10



10



10



10



10



10



10



10



10



G4-C1A,


M & 1000



Mean



8.01



12.22



16.40



17.83



20.58



23.32



24.93*



25.87



27.07



28.41



±SD



0.46



0.47



0.50



0.50



0.52



0.66



0.84



0.70



0.51



1.43



n



10



10



10



10



10



10



10



10



10



10



 























































































































































































Group, Sex


& Dose


(mg/kg body weight/day)



 



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



Week 1



Week 2



Week 3



Week 4



Week 5



Week 6



Week 7



Week 8



Week 9



Week 10



G1-C1A,


F & 0



Mean



7.53



11.69



13.71



16.11



18.39



19.45



21.82



22.27



23.08



23.96



±SD



0.55



0.69



0.61



0.61



0.73



0.87



0.47



0.57



0.62



0.65



n



10



10



10



10



10



10



10



10



10



10



G2-C1A,


F & 100



Mean



7.65



11.47



14.00



16.39



18.58



19.25



21.54



22.36



22.75



23.69



±SD



0.55



0.64



0.41



0.40



0.83



0.78



0.56



0.68



0.35



0.97



n



10



10



10



10



10



10



10



10



10



10



G3-C1A,


F & 300



Mean



7.37



11.53



13.67



16.17



18.40



19.25



21.70



22.32



23.18



24.01



±SD



0.46



0.35



0.38



0.45



0.35



0.41



0.47



0.33



0.64



0.82



n



10



10



10



10



10



10



10



10



10



10



G4-C1A,


F & 1000



Mean



7.45



11.67



13.99



16.34



18.49



19.42



21.62



22.46



23.07



25.35



±SD



0.35



0.40



0.61



0.72



0.73



0.98



0.76



0.72



0.65



2.36



n



10



10



10



10



10



10



10



10



10



10



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


 


Table 21. Summary of serum T4 hormone levels (ng/mL) record - Cohort 1A















































































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


 

Serum T4 Levels (ng/mL)


 
 

G1-C1A, M & 0



Mean



59.932


 

±SD



6.028


 

n



10


 

G2-C1A, M & 100



Mean



61.209


 

±SD



4.583


 

n



10


 

G3-C1A, M & 300



Mean



64.605


 

±SD



8.325


 

n



10


 

G4-C1A, M & 1000



Mean



64.589


 

±SD



5.296


 

n



10


 

 















































































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


 

Serum T4 Levels (ng/mL)


 
 

G1-C1A, F & 0



Mean



59.489


 

±SD



5.723


 

n



10


 

G2-C1A, F & 100



Mean



61.644


 

±SD



6.193


 

n



10


 

G3-C1A, F & 300



Mean



60.502


 

±SD



3.310


 

n



10


 

G4-C1A, F & 1000



Mean



65.627*


 

±SD



5.249


 

n



10


 

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


 


Table 22. Summary of serum thyroid stimulating hormone (TSH) levels record - Cohort 1A















































































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


 

Serum TSH Levels_Adj. Result (µIU/mL)


 
 

G1-C1A, M & 0



Mean



4.474


 

±SD



3.683


 

n



9


 

G2-C1A, M & 100



Mean



1.629*


 

±SD



1.460


 

n



10


 

G3-C1A, M & 300



Mean



0.757*


 

±SD



0.595


 

n



8


 

G4-C1A, M & 1000



Mean



1.339*


 

±SD



0.985


 

n



8


 

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















































































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


 

Serum TSH Levels (µIU/mL)


 
 

G1-C1A, F & 0



Mean



0.605


 

±SD



0.549


 

n



10


 

G2-C1A, F & 100



Mean



1.289


 

±SD



1.101


 

n



10


 

G3-C1A, F & 300



Mean



1.891


 

±SD



2.395


 

n



8


 

G4-C1A, F & 1000



Mean



1.414


 

±SD



1.675


 

n



10


 

n: Number of Animals [The outliered values were excluded for mean calculations]


 


Table 23. Summary of haematology record - Cohort 1A












































































































































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Total Leucocyte Count



Total Erythrocyte Count



Haemoglobin



Haematocrit



Platelet Count



Neutrophils



Lymphocytes



Monocytes



Eosinophils



Basophils



(WBC)



(RBC)



(HGB)



(HCT)



(PLT)



(Neut)



(Lymph)



(Mono)



(Eos)



(Baso)



(103 cells/µL)



(106 cells/µL)



(g/dL)



(%)



(103 cells/µL)



(%)



(%)



(%)



(%)



(%)



G1-C1A,


M & 0



Mean



9.16



8.35



15.79



47.52



920.56



19.11



75.73



2.36



1.40



0.53



±SD



1.91



0.48



0.69



2.36



222.23



3.59



4.10



0.40



0.39



0.16



n



9



9



9



9



9



9



9



9



9



9



G2-C1A,


M & 100



Mean



11.43



8.47



15.97



48.17



941.50



21.84



72.42



2.39



2.18



0.36



±SD



3.67



0.63



0.99



2.29



213.75



6.30



7.15



0.51



1.13



0.07



n



10



10



10



10



10



10



10



10



10



10



G3-C1A,


M & 300



Mean



8.25



8.57



16.03



48.52



862.00



21.93



72.01



2.84



1.75



0.48



±SD



2.11



0.63



0.81



1.92



220.27



4.92



4.91



1.33



0.87



0.46



n



10



10



10



10



10



10



10



10



10



10



G4-C1A,


M & 1000



Mean



9.49



8.51



16.19



48.38



804.20



20.14



74.58



2.62



1.44



0.44



±SD



5.24



0.66



1.38



3.84



311.81



3.85



4.23



0.67



0.49



0.24



n



10



10



10



10



10



10



10



10



10



10



 































































































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Absolute Neutrophils



Absolute Lymphocytes



Absolute Monocytes



Absolute Eosinophils



Absolute  Basophils



Prothrombin Time



Activated Prothrombin


Time



(Neut)



(Lymph)



(Mono)



(Eos)



(Baso)



(PT)



(APTT)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(Seconds)



(Seconds)



G1-C1A,


M & 0



Mean



1.75



6.94



0.22



0.13



0.05



16.03



24.60



±SD



0.45



1.52



0.06



0.05



0.01



2.74



5.65



n



9



9



9



9



9



9



9



G2-C1A,


M & 100



Mean



2.53



8.25



0.28



0.24*



0.04



18.21



25.12



±SD



1.18



2.61



0.11



0.10



0.02



2.58



6.03



n



10



10



10



10



10



10



10



G3-C1A,


M & 300



Mean



1.84



5.92



0.22



0.14



0.04



16.46



30.23



±SD



0.69



1.52



0.08



0.06



0.04



3.09



9.49



n



10



10



10



10



10



10



10



G4-C1A,


M & 1000



Mean



1.81



7.18



0.24



0.13



0.04



17.28



23.57



±SD



0.72



4.32



0.12



0.07



0.03



1.86



7.64



n



10



10



10



10



10



10



10



 












































































































































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Total Leucocyte Count



Total Erythrocyte Count



Haemoglobin



Haematocrit



Platelet Count



Neutrophils



Lymphocytes



Monocytes



Eosinophils



Basophils



(WBC)



(RBC)



(HGB)



(HCT)



(PLT)



(Neut)



(Lymph)



(Mono)



(Eos)



(Baso)



(103 cells/µL)



(106 cells/µL)



(g/dL)



(%)



(103 cells/µL)



(%)



(%)



(%)



(%)



(%)



G1-C1A,


F & 0



Mean



6.97



7.88



15.10



44.17



808.40



21.78



72.86



2.52



1.37



0.45



±SD



1.78



0.70



1.35



3.67



346.96



5.34



6.00



0.82



0.43



0.16



n



10



10



10



10



10



10



10



10



10



10



G2-C1A,


F & 100



Mean



10.04



8.43



15.86



46.88



879.60



18.05



76.68



2.32



1.55



0.40



±SD



2.85



0.43



1.10



3.20



184.42



3.39



3.92



0.82



0.86



0.14



n



10



10



10



10



10



10



10



10



10



10



G3-C1A,


F & 300



Mean



9.52



8.27



15.32



45.49



1000.78



20.96



74.51



1.88



1.46



0.29



±SD



3.15



0.39



0.91



2.85



190.62



6.14



6.04



0.38



0.73



0.11



n



9



9



9



9



9



9



9



9



9



9



G4-C1A,


F & 1000



Mean



10.09



7.84



15.48



45.91



847.11



19.74



75.37



2.29



1.47



0.32



±SD



4.22



0.83



1.58



4.56



144.11



3.84



4.00



0.65



0.42



0.19



n



9



9



9



9



9



9



9



9



9



9



 































































































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Absolute Neutrophils



Absolute Lymphocytes



Absolute Monocytes



Absolute Eosinophils



Absolute  Basophils



Prothrombin Time



Activated Prothrombin


Time



(Neut)



(Lymph)



(Mono)



(Eos)



(Baso)



(PT)



(APTT)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(Seconds)



(Seconds)



G1-C1A,


F & 0



Mean



1.50



5.09



0.18



0.10



0.03



17.07



30.47



±SD



0.56



1.41



0.08



0.04



0.01



2.11



12.08



n



10



10



10



10



10



10



10



G2-C1A,


F & 100



Mean



1.77



7.75*



0.23



0.16



0.04



17.41



19.48*



±SD



0.44



2.42



0.09



0.09



0.01



1.68



6.73



n



10



10



10



10



10



10



10



G3-C1A,


F & 300



Mean



1.96



7.12



0.18



0.14



0.03



15.87



25.69



±SD



0.84



2.50



0.09



0.08



0.02



1.83



7.96



n



9



9



9



9



9



9



9



G4-C1A,


F & 1000



Mean



2.07



7.52



0.23



0.15



0.03



16.80



25.77



±SD



1.28



2.81



0.10



0.07



0.02



2.18



5.00



n



9



9



9



9



9



9



9



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


 


Table 24. 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



9



10



10



10



Physical


Examination



Colour



Pale Yellow



4



5



7



8



Yellow



5



5



3



2



Appearance



Clear



3



2



5



4



Turbid



6



8



5



6



Volume


(mL)



Mean



6.0



5.8



7.2



5.9



±SD



2.0



1.8



1.9



1.9



Chemical


Examination



pH



Mean



7.8



7.8



7.8



7.7



±SD



0.6



0.3



0.3



0.4



Specific gravity



Mean



1.008



1.007



1.009



1.009



±SD



0.003



0.003



0.002



0.003



Urobilinogen            


(mg/dL)



Mean



1.0



0.5



0.5



0.3



±SD



1.3



0.4



0.6



0.3



Bilirubin
(mg/dL)



Neg



3



3



5



6



1



4



7



4



4



Ketones


(mg/dL)



Neg



6



6



7



4



5



3



4



3



6



15



-



-



-



-



Blood


(Ery/µL)



Neg



6



7



6



6



Ca10



-



1



2



1



Ca25



2



2



-



2



Ca80



-



-



1



-



>=Ca200



1



-



1



1



Protein 


(mg/dL)



Neg



2



-



3



2



Trace



1



1



2



2



30



1



4



1



2



100



-



1



-



1



>=300



5



4



4



3



Nitrite



Neg



7



7



9



7



Pos



2



3



1



3



Leucocytes
(Leu/µL)



Neg



-



-



1



-



Ca15



3



1



1



3



Ca70



2



6



2



3



Ca125



4



3



6



3



Glucose 


(mg/dL)



Neg



9



10



10



10



Micro Albumin 


(mg/dL)



Neg



1



-



1



1



>15



7



10



7



8



15



1



-



2



1



Microscopic


Examination



Epi Cells



0



8



8



6



7



0-1



-



2



1



2



1-2



1



-



3



-



2-3



-



-



-



1



Casts



Absent



8



10



9



8



Present



1



-



1



2



Crystals



Absent



-



-



-



-



Present



9



10



10



10



 





































































































































































































































































































































































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



9



9



Physical


Examination



Colour



Pale Yellow



7



5



7



5



Yellow



3



5



2



4



Appearance



Clear



2



4



6



1



Turbid



8



6



3



8



Volume


(mL)



Mean



7.0



4.9*



6.2



5.8



±SD



2.2



1.6



1.2



1.5



Chemical


Examination



pH



Mean



8.0



7.7



7.6



7.9



±SD



0.4



0.5



0.2



0.3



Specific gravity



Mean



1.008



1.009



1.008



1.007



±SD



0.003



0.004



0.003



0.003



Urobilinogen
(mg/dL)



Mean



0.6



0.5



0.3



0.5



±SD



0.8



0.6



0.3



0.6



Bilirubin
(mg/dL)



Neg



5



2



6



6



1



2



8



3



3



Ketones


(mg/dL)



Neg



7



8



6



8



5



3



2



3



1



15



-



-



-



-



Blood
(Ery/µL)



Neg



7



9



5



8



Ca10



1



1



2



1



Ca25



1



-



2



-



Ca80



-



-



-



-



>=Ca200



1



-



-



-



Protein


(mg/dL)



Neg



2



2



4



-



Trace



-



2



1



1



30



1



1



1



6



100



1



-



1



-



>=300



6



5



2



2



Nitrite
(NIT)



Neg



7



8



5



8



Pos



3



2



4



1



Leucocytes
(Leu/µL)



Neg



1



-



1



-



Ca15



-



3



-



-



Ca70



6



5



5



6



Ca125



3



2



3



3



Glucose


(mg/dL)



Neg



10



10



9



9



Micro Albumin
(mg/dL)



Neg



-



-



1



-



>15



8



8



5



9



15



2



2



3



-



Microscopic


Examination



Epi Cells



0



7



5



6



6



0-1



-



2



-



2



1-2



2



3



2



1



2-3



1



-



1



-



Casts



Absent



9



10



6



9



Present



1



-



3



-



Crystals



Absent



-



-



-



-



Present



10



10



9



9



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


 


Table 25. Summary of absolute organ weight (g) record - Cohort 1A


























































































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Adrenals



Thymus



Spleen



Testes



Epididymides



Heart



Kidneys



Brain



G1-C1A,


M & 0



Mean



0.0521



0.2671



1.0730



3.3278



1.3713



1.3195



2.3746



1.9340



±SD



0.0066



0.0296



0.1587



0.1459



0.0650



0.2154



0.2800



0.1082



n



20



20



20



20



20



20



20



20



G2-C1A,


M & 100



Mean



0.0545



0.2700



1.0677



3.3446



1.3632



1.3937



2.5491



1.9740



±SD



0.0074



0.0564



0.1221



0.2333



0.0842



0.3014



0.2720



0.1125



n



20



20



20



20



20



20



20



20



G3-C1A,


M & 300



Mean



0.0510



0.2572



0.9012*



3.2638



1.3167



1.3179



2.3903



1.9637



±SD



0.0068



0.0347



0.1886



0.1961



0.0881



0.1934



0.3209



0.0918



n



20



20



20



20



20



20



20



20



G4-C1A,


M & 1000



Mean



0.0537



0.2521



1.0889



3.2345



1.3463



1.3003



2.2548



1.9120



±SD



0.0071



0.0314



0.1480



0.1466



0.0749



0.2759



0.2329



0.0878



n



20



20



20



20



20



20



20



20



 













































































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Liver



Prostate



Seminal vesicles with coagulating glands



Iliac lymph node



Mandibular


lymph node



PituitaryWP



Thyroid along
with parathyroidWP



G1-C1A,


M & 0



Mean



10.6384



0.9206



1.1578



0.0399



0.1361



0.0169



0.0275



±SD



1.4863



0.1193



0.2353



0.0055



0.0147



0.0011



0.0014



n



20



20



20



20



20



20



20



G2-C1A,


M & 100



Mean



10.8144



0.9813



1.2102



0.0423



0.1350



0.0174



0.0278



±SD



1.6681



0.0786



0.2007



0.0102



0.0167



0.0019



0.0020



n



20



20



20



20



20



20



20



G3-C1A,


M & 300



Mean



10.4807



1.0027



1.1563



0.0382



0.1295



0.0176



0.0271



±SD



1.8164



0.1531



0.2624



0.0073



0.0116



0.0019



0.0016



n



20



20



20



20



20



20



20



G4-C1A,


M & 1000



Mean



9.7751



0.9948



1.1467



0.0371



0.1414



0.0174



0.0277



±SD



0.8505



0.1001



0.2035



0.0058



0.0294



0.0014



0.0015



n



20



20



20



20



20



20



20



 













































































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Adrenals



Thymus



Spleen



Ovaries



Uterus with Cervix



Heart



Kidneys



G1-C1A,


F & 0



Mean



0.0600



0.2606



0.9194



0.1322



0.6176



0.9939



1.6052



±SD



0.0051



0.0326



0.1761



0.0166



0.1140



0.1062



0.1619



n



20



20



20



20



20



20



20



G2-C1A,


F & 100



Mean



0.0598



0.2752



0.8742



0.1244



0.6067



1.0463



1.7637*



±SD



0.0083



0.0513



0.2133



0.0124



0.1212



0.0967



0.2534



n



20



20



20



20



20



20



20



G3-C1A,


F & 300



Mean



0.0601



0.2599



0.6836*



0.1105*



0.6075



0.9709



1.6528



±SD



0.0071



0.0401



0.1612



0.0079



0.1078



0.0988



0.2217



n



20



20



20



20



20



20



20



G4-C1A,


F & 1000



Mean



0.0578



0.2517



0.7883*



0.1229



0.5958



1.0611



1.6802



±SD



0.0068



0.0335



0.0921



0.0113



0.1273



0.0822



0.1826



n



20



20



20



20



20



20



20



 
































































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Brain



Liver



Iliac lymph node



Mandibular


lymph node



PituitaryWP



Thyroid along
with parathyroidWP



G1-C1A,


F & 0



Mean



1.8173



8.2701



0.0377



0.1053



0.0144



0.0233



±SD



0.1763



1.3064



0.0087



0.0161



0.0018



0.0027



n



20



20



20



20



20



20



G2-C1A,


F & 100



Mean



1.8609



8.5602



0.0406



0.1089



0.0150



0.0225



±SD



0.1182



1.1930



0.0099



0.0220



0.0019



0.0026



n



20



20



20



20



20



20



G3-C1A,


F & 300



Mean



1.8188



8.0708



0.0395



0.1016



0.0141



0.0227



±SD



0.0801



1.0957



0.0088



0.0127



0.0020



0.0021



n



20



20



20



20



20



20



G4-C1A,


F & 1000



Mean



1.8458



8.8116



0.0420



0.1116



0.0143



0.0236



±SD



0.0604



0.8629



0.0093



0.0187



0.0015



0.0024



n



20



20



20



20



20



20



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


 


Table 26. Summary of terminal body weight (g) and organ weight relative to terminal body weight (%) record - Cohort 1A


























































































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Terminal Body Weight (g)



Adrenals



Thymus



Spleen



Testes



Epididymides



Heart



Kidneys



G1-C1A,


M & 0



Mean



366.84



0.0143



0.0732



0.2945



0.9132



0.3762



0.3590



0.6491



±SD



29.48



0.0019



0.0099



0.0512



0.0905



0.0361



0.0439



0.0756



n



20



20



20



20



20



20



20



20



G2-C1A,


M & 100



Mean



363.59



0.0151



0.0750



0.2980



0.9299



0.3793



0.3851



0.7059



±SD



42.97



0.0024



0.0168



0.0498



0.1078



0.0454



0.0823



0.0791



n



20



20



20



20



20



20



20



20



G3-C1A,


M & 300



Mean



358.18



0.0144



0.0726



0.2537*



0.9202



0.3706



0.3693



0.6711



±SD



34.35



0.0024



0.0134



0.0598



0.1102



0.0407



0.0539



0.0946



n



20



20



20



20



20



20



20



20



G4-C1A,


M & 1000



Mean



348.89



0.0155



0.0726



0.3141



0.9311



0.3878



0.3718



0.6484



±SD



27.55



0.0023



0.0101



0.0509



0.0659



0.0341



0.0705



0.0688



n



20



20



20



20



20



20



20



20



 


























































































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Brain



Liver



Prostate



Seminal vesicles with coagulating glands



Iliac lymph node



Mandibular lymph node



Pituitary



Thyroid


along with parathyroid



G1-C1A,


M & 0



Mean



0.5293



2.9049



0.2516



0.3162



0.0109



0.0373



0.0046



0.0075



±SD



0.0366



0.3814



0.0302



0.0619



0.0017



0.0042



0.0002



0.0004



n



20



20



20



20



20



20



20



20



G2-C1A,


M & 100



Mean



0.5485



2.9919



0.2729



0.3352



0.0118



0.0376



0.0048*



0.0077



±SD



0.0578



0.4341



0.0343



0.0567



0.0033



0.0069



0.0002



0.0005



n



20



20



20



20



20



20



20



20



G3-C1A,


M & 300



Mean



0.5532



2.9364



0.2828*



0.3255



0.0107



0.0366



0.0049*



0.0076



±SD



0.0609



0.4810



0.0539



0.0750



0.0020



0.0052



0.0002



0.0005



n



20



20



20



20



20



20



20



20



G4-C1A,


M & 1000



Mean



0.5502



2.8068



0.2867*



0.3290



0.0107



0.0408



0.0050*



0.0080*



±SD



0.0359



0.2021



0.0366



0.0539



0.0017



0.0091



0.0002



0.0004



n



20



20



20



20



20



20



20



20



 













































































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Terminal


Body Weight


(g)



Adrenals



Thymus



Spleen



Ovaries



Uterus with Cervix



Heart



G1-C1A,


F & 0



Mean



233.44



0.0257



0.1118



0.3944



0.0568



0.2649



0.4261



±SD



14.25



0.0020



0.0136



0.0744



0.0080



0.0474



0.0412



n



20



20



20



20



20



20



20



G2-C1A,


F & 100



Mean



236.08



0.0254



0.1168



0.3707



0.0529



0.2576



0.4440



±SD



16.02



0.0034



0.0216



0.0883



0.0059



0.0521



0.0403



n



20



20



20



20



20



20



20



G3-C1A,


F & 300



Mean



229.79



0.0262



0.1132



0.2989*



0.0482*



0.2641



0.4236



±SD



13.91



0.0034



0.0170



0.0760



0.0044



0.0416



0.0470



n



20



20



20



20



20



20



20



G4-C1A,


F & 1000



Mean



230.71



0.0252



0.1093



0.3424



0.0534



0.2608



0.4616*



±SD



15.78



0.0035



0.0147



0.0406



0.0050



0.0643



0.0437



n



20



20



20



20



20



20



20



 













































































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Kidneys



Brain



Liver



Iliac lymph node



Mandibular


lymph node



Pituitary



Thyroid along
with parathyroid



G1-C1A,


F & 0



Mean



0.6887



0.7803



3.5478



0.0162



0.0454



0.0062



0.0100



±SD



0.0683



0.0810



0.5485



0.0039



0.0079



0.0005



0.0009



n



20



20



20



20



20



20



20



G2-C1A,


F & 100



Mean



0.7502



0.7907



3.6257



0.0173



0.0463



0.0063



0.0095



±SD



0.1217



0.0637



0.4241



0.0043



0.0097



0.0005



0.0008



n



20



20



20



20



20



20



20



G3-C1A,


F & 300



Mean



0.7205



0.7947



3.5154



0.0173



0.0443



0.0061



0.0099



±SD



0.0990



0.0645



0.4427



0.0040



0.0058



0.0006



0.0005



n



20



20



20



20



20



20



20



G4-C1A,


F & 1000



Mean



0.7310



0.8033



3.8322



0.0183



0.0485



0.0062



0.0102



±SD



0.0913



0.0572



0.4196



0.0043



0.0085



0.0003



0.0005



n



20



20



20



20



20



20



20



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


 


Table 27. Summary record of splenic lymphocyte subpopulation analysis - Cohort 1A















































































































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



 



Percent of Splenic lymphocyte Parental Sub-populations (%)



B Lymphocytes



T Helper Cells



T Cytotoxic Cells



Natural Killer Cells



(B Cells)



(Th/CD4+)



(Tc/CD8+)



(NK Cells)



G1-C1A, M & 0



Mean



37.57



51.88



45.37



12.71



±SD



2.29



9.07



8.65



3.51



n



10



10



10



10



G2-C1A, M & 100



Mean



37.85



50.23



45.46



12.12



±SD



4.47



1.30



5.66



3.08



n



10



10



10



10



G3-C1A, M & 300



Mean



37.35



50.44



45.44



13.10



±SD



2.55



3.27



6.36



3.63



n



10



10



10



10



G4-C1A, M & 1000



Mean



37.98



54.99



42.20



12.22



±SD



2.63



15.25



14.93



3.92



n



10



10



10



10



 















































































































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



 



Percent of Splenic lymphocyte Parental Sub-populations (%)



B Lymphocytes



T Helper Cells



T Cytotoxic Cells



Natural Killer Cells



(B Cells)



(Th/CD4+)



(Tc/CD8+)



(NK Cells)



G1-C1A, F & 0



Mean



38.84



52.86



43.73



8.77



±SD



7.60



1.71



1.58



2.28



n



10



10



10



10



G2-C1A, F & 100



Mean



40.64



53.91



41.99*



8.33



±SD



5.02



1.93



1.95



1.08



n



10



10



10



10



G3-C1A, F & 300



Mean



37.76



54.77*



41.00*



9.82



±SD



10.42



1.29



1.04



6.34



n



10



10



10



10



G4-C1A, F & 1000



Mean



40.52



53.68



42.26



9.25



±SD



7.18



1.36



1.38



5.40



n



10



10



10



10



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


 


Table 28. Summary record of sperm motility (%) - Cohort 1A































































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



 



Mean Percent of Sperm Motility



G1-C1A, M & 0



Mean



90.9



±SD



0.9



n



20



G2-C1A, M & 100



Mean



91.0



±SD



1.1



n



20



G3-C1A, M & 300



Mean



90.8



±SD



1.1



n



20



G4-C1A, M & 1000



Mean



91.1



±SD



1.0



n



20



 


Table 29. Summary record of sperm morphology - 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-C1A, M & 0



Mean



2.05



1.03



2.10



1.05



1.40



0.70



1.00



0.50


 

6.55



3.28


 

193.45



96.73



±SD



1.15



0.57



1.62



0.81



1.27



0.64



0.97



0.49


 

1.32



0.66


 

1.32



0.66



n



20



20



20



20



20



20



20



20


 

20



20


 

20



20



G2-C1A, M & 100



Mean



3.20*



1.60*



2.10



1.05



1.15



0.58



0.50



0.25


 

6.95



3.48


 

193.05



96.53



±SD



1.40



0.70



1.25



0.63



1.04



0.52



0.69



0.34


 

1.57



0.79


 

1.57



0.79



n



20



20



20



20



20



20



20



20


 

20



20


 

20



20



G3-C1A, M & 300



Mean



2.95



1.48



3.45*



1.73*



1.55



0.78



0.30*



0.15*


 

8.25*



4.13*


 

191.75*



95.88*



±SD



1.61



0.80



1.28



0.64



1.28



0.64



0.47



0.24


 

1.29



0.65


 

1.29



0.65



n



20



20



20



20



20



20



20



20


 

20



20


 

20



20



G4-C1A, M & 1000



Mean



2.50



1.25



2.75



1.38



1.40



0.70



0.60



0.30


 

7.25



3.63


 

192.75



96.38



±SD



1.24



0.62



1.29



0.65



0.82



0.41



0.68



0.34


 

1.37



0.69


 

1.37



0.69



n



20



20



20



20



20



20



20



20


 

20



20


 

20



20



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


 


Table 30. Summary record of spermatid head count - Cohort 1A

























































































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



 



Average Sperm head count



Daily Sperm Production


(x 106 per gram of Testis)



Daily Sperm Production (x106 per Testis)



G1-C1A, M & 0



Mean



182.00



14.8



51.9



±SD



9.82



0.8



5.0



n



20



20



20



G2-C1A, M & 100



Mean



178.44



14.5



51.0



±SD



5.97



0.5



4.9



n



20



20



20



G3-C1A, M & 300



Mean



182.36



14.9



52.4



±SD



8.01



0.7



4.6



n



20



20



20



G4-C1A, M & 1000



Mean



177.98



14.5



50.5



±SD



7.71



0.6



4.5



n



20



20



20



 


Table 31. Summary of body weight (g) record - Cohort 1B











































































































































































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Body Weight (g) on Day



21



25



28



32



35



42



49



56



63



70



77



84



91



98



G1-C1B,


M & 0



Mean



43.82



62.04



77.25



98.26



118.07



153.98



190.70



225.15



263.80



295.17



321.65



353.98



381.14



407.28



±SD



3.26



4.63



6.11



10.58



15.03



19.02



21.72



28.29



30.25



33.20



30.02



33.60



32.74



32.86



n



20



20



20



20



20



20



20



20



20



20



20



20



20



20



G2-C1B,


M & 100



Mean



44.64



62.80



75.14



91.25



106.32*



142.62



178.68



209.79



245.15



273.64



299.75



332.50



358.60



382.63



±SD



3.47



4.40



6.45



7.97



9.03



12.74



16.33



21.04



25.45



30.48



34.25



29.23



28.02



29.32



n



20



20



20



20



20



20



20



20



20



20



20



20



20



20



G3-C1B,


M & 300



Mean



45.16



65.12



78.09



96.69



114.31



144.64



177.54



213.06



244.97



272.82



298.26



328.94



353.68*



377.12*



±SD



3.82



5.19



6.58



11.15



14.55



19.74



22.81



28.41



35.61



40.66



41.78



44.44



42.05



44.76



n



20



20



20



20



20



20



20



20



20



20



20



20



20



20



G4-C1B,


M & 1000



Mean



45.59



63.03



74.23



91.54



108.43



139.42*



166.95*



191.84*



224.37*



251.57*



279.23*



309.07*



336.26*



356.85*



±SD



3.82



5.58



6.25



9.67



11.57



18.79



21.02



24.77



31.94



37.00



36.29



33.89



32.51



31.50



n



20



20



20



20



20



20



20



20



20



20



20



20



20



20



 











































































































































































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Body Weight (g) on Day



21



25



28



32



35



42



49



56



63



70



77



84



91



98



G1-C1B,


F & 0



Mean



40.06



56.35



70.87



93.49



108.55



138.26



163.53



184.76



203.97



218.34



231.18



242.72



253.25



263.78



±SD



1.89



3.49



5.60



12.26



15.86



18.11



20.70



21.82



22.31



20.61



16.60



14.96



15.67



17.15



n



20



20



20



20



20



20



20



20



20



20



20



20



20



20



G2-C1B,


F & 100



Mean



41.86



58.42



70.50



89.22



103.18



132.10



153.89



174.67



192.28



208.20



220.68



234.02



243.95



254.58



±SD



3.08



3.61



7.05



11.26



12.14



16.72



20.28



21.33



22.10



23.46



21.71



19.89



18.84



18.51



n



20



20



20



20



20



20



20



20



20



20



20



20



20



20



G3-C1B,


F & 300



Mean



42.20*



58.86



72.24



92.63



105.53



132.84



154.49



174.59



191.49



204.96



219.03



232.66



245.03



254.42



±SD



3.09



3.68



3.78



8.36



9.88



13.08



15.79



19.66



24.04



22.53



18.63



18.57



16.95



18.56



n



20



20



20



20



20



20



20



20



20



20



20



20



20



20



G4-C1B,


F & 1000



Mean



42.49*



59.78*



71.19



86.06



98.91*



126.87*



150.04*



170.14*



189.07



204.67



221.26



232.90



243.69



254.08



±SD



2.48



2.92



4.73



6.64



8.93



10.73



11.04



12.94



14.19



14.91



12.41



12.24



10.37



11.79



n



20



20



20



20



20



20



20



20



20



20



20



20



20



20



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


 


Table 32. Summary of percent change in body weight gain (%) with respect to postnatal day 21 record - Cohort 1B






























































































































































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Percent Change in Body Weight (%) during 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



41.84



77.00



125.48



171.03



254.52



338.43



417.53



505.52



577.60



637.90



711.75



773.91



834.07



±SD



9.34



17.44



30.23



41.62



58.95



66.06



82.13



85.56



94.20



87.46



94.05



93.92



99.54



n



20



20



20



20



20



20



20



20



20



20



20



20



20



G2-C1B,


M & 100



Mean



41.24



69.16



105.60



139.66*



221.41



302.45



373.22



453.51



518.40



577.07



650.36



709.42



763.28



±SD



11.86



17.79



23.54



27.71



37.71



46.41



63.84



81.80



98.85



106.33



98.37



102.54



104.98



n



20



20



20



20



20



20



20



20



20



20



20



20



20



G3-C1B,


M & 300



Mean



44.47



73.63



115.74



155.43



223.20



296.83



376.74



448.93



511.87



569.02



637.46



692.53*



744.77*



±SD



8.03



16.22



32.46



43.34



57.76



69.08



87.91



110.51



127.75



134.34



144.15



144.21



150.34



n



20



20



20



20



20



20



20



20



20



20



20



20



20



G4-C1B,


M & 1000



Mean



38.53



63.50*



102.04*



139.15*



206.77*



266.97*



321.15*



392.11*



451.90*



513.36*



579.75*



639.65*



685.47*



±SD



10.30



15.89



26.90



30.50



41.65



42.90



45.88



56.40



67.73



69.65



70.01



67.36



70.22



n



20



20



20



20



20



20



20



20



20



20



20



20



20



 






























































































































































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Percent Change in Body Weight (%) during 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



40.80



77.19



133.92



171.69



246.10



309.39



362.58



410.58



446.55



478.42



507.52



533.81



560.08



±SD



8.72



14.84



32.68



42.13



49.29



57.07



60.98



63.04



60.37



50.66



51.02



52.87



55.65



n



20



20



20



20



20



20



20



20



20



20



20



20



20



G2-C1B,


F & 100



Mean



39.85



68.87



113.88



147.51



217.12



269.63*



319.53*



361.91*



399.87



429.70*



461.95*



485.75*



511.04*



±SD



7.65



16.83



27.61



31.24



44.59



55.01



58.70



62.33



64.68



61.91



62.15



61.63



60.83



n



20



20



20



20



20



20



20



20



20



20



20



20



20



G3-C1B,


F & 300



Mean



39.82



71.89



120.63



151.43



216.60



268.36*



316.55*



357.08*



389.01*



422.02*



454.70*



484.11*



506.45*



±SD



8.65



13.83



26.13



30.89



40.72



49.48



61.25



73.22



71.25



61.93



65.63



64.13



67.75



n



20



20



20



20



20



20



20



20



20



20



20



20



20



G4-C1B,


F & 1000



Mean



40.91



67.88



103.27*



133.54*



199.64*



254.64*



302.07*



346.80*



383.86*



423.14*



450.78*



476.11*



500.66*



±SD



6.48



12.37



20.82



26.00



32.63



38.17



42.67



47.07



51.52



50.80



53.32



51.29



54.55



n



20



20



20



20



20



20



20



20



20



20



20



20



20



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


 


Table 33. Summary of feed consumption (g/animal/day) record - Cohort 1B

































































































































































































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


 

Week 1



Week



Week 3



Week 4



Week 5



Week 6



Week 7



Week 8



Week 9



Week 10



Week 11



G1-C1B, M & 0



Mean



8.16



12.38



16.54



17.93



20.58



23.07



25.55



26.18



27.18



27.56



29.10



±SD



0.55



0.38



0.25



0.44



0.88



0.84



0.69



0.56



0.41



0.49



1.32



n



20



20



20



20



20



20



20



20



20



20



20



G2-C1B, M & 100



Mean



8.27



12.29



16.74



18.14



20.91



23.38



25.30



26.12



26.91



27.68



28.93



±SD



0.72



0.53



0.60



0.57



0.60



0.48



1.05



0.62



0.56



0.78



1.16



n



20



20



20



20



20



20



20



20



20



20



20



G3-C1B, M & 300



Mean



8.16



12.46



16.62



17.86



20.57



22.71



24.86



25.70



26.60



27.24



28.93



±SD



0.66



0.56



0.61



0.72



0.58



0.88



1.36



1.46



0.85



1.01



1.48



n



20



20



20



20



20



20



20



20



20



20



20



G4-C1B, M & 1000



Mean



7.88



11.99



16.17



17.59



20.49



23.13



25.43



26.27



27.17



27.50



29.80



±SD



0.71



0.74



0.60



0.56



0.50



0.86



1.30



1.21



1.02



1.23



1.89



n



20



20



20



20



20



20



20



20



20



20



20



 

































































































































































































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


 

Week 1



Week



Week 3



Week 4



Week 5



Week 6



Week 7



Week 8



Week 9



Week 10



Week 11



G1-C1B, F & 0



Mean



7.54



11.63



13.81



16.22



18.55



19.20



21.93



22.34



23.19



23.10



24.53



±SD



0.66



0.76



0.67



0.90



0.78



0.79



0.60



0.53



0.59



0.75



0.64



n



10



10



10



10



10



10



10



10



10



10



10



G2-C1B, F & 100



Mean



7.03



11.06



13.61



15.76



17.96



18.70



21.06*



21.86



22.24*



23.05



23.95



±SD



0.52



0.58



0.94



0.77



0.84



0.58



0.85



0.71



0.54



0.74



0.75



n



10



10



10



10



10



10



10



10



10



10



10



G3-C1B, F & 300



Mean



7.43



11.47



13.75



15.92



18.22



18.92



21.49



22.04



22.65



23.49



24.37



±SD



0.47



0.66



0.42



0.50



0.56



0.55



0.78



0.63



0.58



0.71



0.99



n



10



10



10



10



10



10



10



10



10



10



10



G4-C1B, F & 1000



Mean



7.33



11.36



13.82



16.18



18.29



19.27



21.82



22.43



23.10



23.51



24.93



±SD



0.43



0.66



0.70



0.75



0.79



0.82



0.59



0.51



0.79



0.78



1.02



n



10



10



10



10



10



10



10



10



10



10



9



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


 


Table 34. Summary of absolute organ weight (g) record - Cohort 1B



















































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Adrenals



Thymus



Spleen



Testes



Epididymides



G1-C1B,


M & 0



Mean



0.0559



0.3412



1.1190



3.2847



1.2667



±SD



0.0063



0.0657



0.1494



0.1994



0.0967



n



20



20



20



20



20



G2-C1B,


M & 100



Mean



0.0506



0.2893*



1.1247



3.2425



1.2746



±SD



0.0096



0.0592



0.1433



0.1452



0.1196



n



20



20



20



20



20



G3-C1B,


M & 300



Mean



0.0513



0.2840*



1.0185



3.1528



1.2425



±SD



0.0069



0.0495



0.1718



0.1810



0.1167



n



20



20



20



20



20



G4-C1B,


M & 1000



Mean



0.0548



0.3333



0.9660*



3.2165



1.2399



±SD



0.0078



0.0687



0.1702



0.2276



0.1311



n



20



20



20



20



20



 
































































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Iliac lymph node



Mandibular


lymph node



Prostate



Seminal vesicles with coagulating glands



PituitaryWP



Thyroid along
with parathyroidWP



G1-C1B,


M & 0



Mean



0.0325



0.2341



1.0201



1.1953



0.0178



0.0282



±SD



0.0069



0.0329



0.1281



0.1446



0.0014



0.0011



n



20



20



20



20



20



20



G2-C1B,


M & 100



Mean



0.0329



0.2278



0.9853



1.2019



0.0172



0.0277



±SD



0.0071



0.0466



0.1098



0.1530



0.0018



0.0014



n



20



20



20



20



20



20



G3-C1B,


M & 300



Mean



0.0340



0.1672*



0.8910*



1.2248



0.0173



0.0280



±SD



0.0088



0.0354



0.1587



0.2050



0.0018



0.0013



n



20



20



20



20



20



20



G4-C1B,


M & 1000



Mean



0.0385



0.1934*



0.9091*



1.1675



0.0176



0.0273



±SD



0.0115



0.0272



0.1314



0.1901



0.0016



0.0011



n



20



20



20



20



20



20



 







































































































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Adrenals



Thymus



Spleen



Ovaries



Uterus


with Cervix



Iliac


lymph node



Mandibular lymph node



PituitaryWP



Thyroid


along with parathyroidWP



G1-C1B,


F & 0



Mean



0.0547



0.2931



0.8804



0.1050



0.5904



0.0280



0.2236



0.0158



0.0244



±SD



0.0103



0.0386



0.1630



0.0168



0.0919



0.0062



0.0236



0.0011



0.0024



n



20



20



20



20



20



20



20



20



20



G2-C1B,


F & 100



Mean



0.0582



0.2235*



0.9592



0.1236*



0.5654



0.0320



0.1652*



0.0161



0.0246



±SD



0.0070



0.0438



0.0692



0.0223



0.1116



0.0057



0.0403



0.0016



0.0025



n



20



20



20



20



20



20



20



20



20



G3-C1B,


F & 300



Mean



0.0624*



0.3224



0.9337



0.1127



0.5303



0.0339*



0.1354*



0.0164



0.0248



±SD



0.0075



0.0410



0.1016



0.0124



0.0558



0.0069



0.0229



0.0017



0.0026



n



20



20



20



20



20



20



20



20



20



G4-C1B,


F & 1000



Mean



0.0618*



0.2673



0.8993



0.1042



0.6006



0.0339*



0.1834*



0.0159



0.0242



±SD



0.0101



0.0519



0.1666



0.0167



0.0857



0.0076



0.0438



0.0012



0.0024



n



20



20



20



20



20



20



20



20



20



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


 


Table 35. Summary of terminal body weight (g) and organ weight relative to terminal body weight (%) record - Cohort 1B
































































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Terminal


Body Weight


(g)



Adrenals



Thymus



Spleen



Testes



Epididymides



G1-C1B,


M & 0



Mean



386.77



0.0145



0.0883



0.2924



0.8563



0.3300



±SD



32.02



0.0021



0.0154



0.0532



0.1008



0.0411



n



20



20



20



20



20



20



G2-C1B,


M & 100



Mean



365.67



0.0139



0.0798



0.3101



0.8926



0.3513



±SD



29.28



0.0028



0.0187



0.0509



0.0856



0.0481



n



20



20



20



20



20



20



G3-C1B,


M & 300



Mean



359.17*



0.0144



0.0799



0.2863



0.8917



0.3498



±SD



45.63



0.0022



0.0147



0.0501



0.1280



0.0443



n



20



20



20



20



20



20



G4-C1B,


M & 1000



Mean



341.18*



0.0162



0.0983



0.2835



0.9495*



0.3655*



±SD



32.23



0.0025



0.0215



0.0450



0.0990



0.0433



n



20



20



20



20



20



20



 
































































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Iliac lymph node



Mandibular


lymph node



Prostate



Seminal vesicles with coagulating glands



Pituitary



Thyroid along
with parathyroid



G1-C1B,


M & 0



Mean



0.0085



0.0609



0.2662



0.3105



0.0046



0.0073



±SD



0.0020



0.0099



0.0459



0.0398



0.0002



0.0004



n



20



20



20



20



20



20



G2-C1B,


M & 100



Mean



0.0091



0.0623



0.2705



0.3309



0.0047



0.0076



±SD



0.0024



0.0115



0.0322



0.0514



0.0002



0.0004



n



20



20



20



20



20



20



G3-C1B,


M & 300



Mean



0.0094



0.0471*



0.2500



0.3494



0.0048*



0.0079*



±SD



0.0020



0.0106



0.0453



0.0897



0.0003



0.0008



n



20



20



20



20



20



20



G4-C1B,


M & 1000



Mean



0.0114*



0.0572



0.2683



0.3449



0.0052*



0.0080*



±SD



0.0036



0.0099



0.0442



0.0628



0.0002



0.0005



n



20



20



20



20



20



20



 




















































































































































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Terminal Body Weight (g)



Adrenals



Thymus



Spleen



Ovaries



Uterus


with Cervix



Iliac


lymph node



Mandibular lymph node



Pituitary



Thyroid along with parathyroid



G1-C1B,


F & 0



Mean



249.90



0.0219



0.1179



0.3550



0.0422



0.2380



0.0112



0.0897



0.0063



0.0098



±SD



18.04



0.0040



0.0174



0.0748



0.0072



0.0446



0.0025



0.0097



0.0003



0.0011



n



20



20



20



20



20



20



20



20



20



20



G2-C1B,


F & 100



Mean



240.88



0.0243



0.0934*



0.4002



0.0516*



0.2363



0.0134



0.0691*



0.0067*



0.0102



±SD



18.94



0.0035



0.0195



0.0400



0.0105



0.0503



0.0027



0.0181



0.0004



0.0010



n



20



20



20



20



20



20



20



20



20



20



G3-C1B,


F & 300



Mean



239.93



0.0261*



0.1352*



0.3917



0.0472



0.2218



0.0142*



0.0568*



0.0068*



0.0103



±SD



19.47



0.0031



0.0203



0.0526



0.0060



0.0239



0.0032



0.0109



0.0003



0.0008



n



20



20



20



20



20



20



20



20



20



20



G4-C1B,


F & 1000



Mean



240.15



0.0258*



0.1117



0.3747



0.0433



0.2508



0.01413



0.0767*



0.0066*



0.0101



±SD



11.09



0.0046



0.0234



0.0671



0.0062



0.0383



0.0033



0.0191



0.0003



0.0010



n



20



20



20



20



20



20



20



20



20



20



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


 


Table 36. Summary record of sexual maturity - Cohort 1B












































































Group, Sex


& Dose
(mg/kg body


weight/day)


 

Occurrence of


Balano-preputial separation


(Postnatal Day)



Body weight (g) on the day of


Occurrence of


Balano-preputial separation



G1-C1B,


M & 0



Mean



43.10



159.69



±SD



1.25



18.08



n



20



20



G2-C1B,


M & 100



Mean



43.25



148.79



±SD



1.33



12.79



n



20



20



G3-C1B,


M & 300



Mean



43.45



152.12



±SD



1.43



17.89



n



20



20



G4-C1B,


M & 1000



Mean



43.30



143.87*



±SD



1.56



14.68



n



20



20



 












































































Group, Sex


& Dose


(mg/kg body


weight/day)


 

Occurrence of Vaginal Opening


(Postnatal day)



Body weight (g) on the day of


Occurrence of Vaginal Opening



G1-C1B,


F & 0



Mean



33.75



99.43



±SD



1.65



10.85



n



20



20



G2-C1B,


F & 100



Mean



34.10



99.00



±SD



1.80



10.99



n



20



20



G3-C1B,


F & 300



Mean



34.35



102.03



±SD



2.11



9.81



n



20



20



G4-C1B,


F & 1000



Mean



34.30



95.84



±SD



2.13



8.81



n



20



20



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


 


 

Conclusions:
In an Extended One-Generation Reproductive Toxicity study (EOGRTS) performed orally in Sprague Dawley rats, the NOAEL of the test substance was determined to be 1000 mg/kg bw/day for systemic, reproduction and developmental toxicity.

Executive summary:

An Extended One-Generation Reproductive Toxicity study (EOGRTS) was conducted in rats by oral administration of the test substance in accordance with OECD test guideline 433, following the GLP principles. 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 and Cohort 1B). Each cohort consisted of four groups 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 (0.5% carboxymethyl cellulose), 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 the test item at the dose levels of 100, 300 and 1000 mg/kg bw/day respectively based on the results of a dose range finding study for which no adverse effects were found up to the dose of 1000 mg/kg/day.. The vehicle and test item formulations were administered orally by gavage at the dose volume of 10 mL/kg bw. Homogeneity and dose formulation analysis for dose concentration verification was performed during weeks 1, 9, 17 and 29 of the treatment period and the mean results were within the range 85-115% recovery to the nominal concentration and RSD was less than 10%.


P generation animals were administered with test item 10 weeks during pre-mating and 2 weeks during mating. Parental males were dosed until sacrifice during post-mating period (a total of 98 to 108 days) and parental females were dosed throughout gestation and lactation periods up to weaning of F1 animals (a total of 113 to 127 days). Cohort 1A animals were dosed from day of weaning (PND21) to PND 91 (13-weeks age) and cohort 1B animals from day of weaning (PND21) to PND 98 (14-weeks age).


For all P generation animals the following observations were performed: clinical signs once daily, mortality/morbidity twice daily and detailed clinical examination weekly; body weights (throughout the experimental period) and feed consumption (throughout the experimental period, except during cohabitation period) once weekly; haematology, clinical chemistry, urinalysis and thyroid hormone (T4 and TSH) levels; Sperm parameters (motility, morphology and daily sperm production) for all P males; Oestrus cyclicity evaluation 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 P females. The gross pathology and organ weighing was performed on the day of termination for all P animals. Histopathological examination was conducted on all the tissues collected from the P generation vehicle control and high dose group animals. All 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 P males and females did not reveal any test item related changes in systemic and reproductive endpoints in 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 and observed for gross pathological observations at termination. Also, assessment for T4 levels was conducted for PND 4 pups and assessment for T4 and TSH levels was conducted for PND 21 pups.


All F1 pups of both sexes did not reveal any test item related changes in growth parameters, postnatal developmental landmarks, sensory reflexes, thyroid hormone levels and no gross pathological changes were noted at any of the tested dose group litters.


For all Cohort 1A (F1 generation) animals the following observations were performed: clinical signs once daily, mortality/morbidity twice daily and detailed clinical examination weekly once; body weights and feed consumption once weekly; occurrence of sexual maturation (for balano-preputial separation in C1A males and for vaginal patency in C1A females); mean occurrence of first cornified cells and time interval between vaginal patency and occurrence of first cornified cells in C1A females; oestrus cyclicity during PND 75 to 88 in C1A females; haematology, clinical chemistry, urinalysis and thyroid hormone (T4 and TSH) levels; Sperm parameters (motility, morphology and daily sperm production) in all C1A males. The gross pathology and organ weighing were performed on the day of termination for all 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 randomly selected males and females from each C1A group.


All C1A males and females did not reveal any test item related changes in systemic and reproductive endpoints in any of the tested dose groups when compared with vehicle control group, except slight reduction in mean body weight and percent change in mean body weight gain in all tested dose group males. There were no changes noted in sub-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.


For all Cohort 1B (F1 generation) animals the following observations were performed: clinical signs once daily, mortality/morbidity twice daily and detailed clinical examination weekly once; body weights and feed consumption once weekly; occurrence of sexual maturation (for balano-preputial separation in C1B males and for vaginal patency in C1B females) and gross pathology and organ weighing for all C1B animals.


All C1B males and females did not reveal any test item related changes in systemic and reproductive endpoints, except reduction in mean body weight and percent change in mean body weight gain in all tested dose group of both sexes.


Therefore, based on the above results, the NOAEL of the test substance is considered to be 1000 mg/kg bw/day for systemic, reproduction and developmental toxicity.

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD guideline 422. GLP study.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
Daiichi Kasei Kogyo (Hyogo), lot No. E0311323
- Composition: SiO2: 39.60%, Al2O3: 23.76%, Fe2O3: 0.45%, S: 12.08%, Na2O: 22.59%, unknown: 1.52%.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
closed container, stored in a cool dark place (2-6 °C).
- Stability under test conditions:
stable (samples were re-analysed at the end of the study to confirm their stability).
- Solubility and stability of the test substance in the solvent/vehicle:
insoluble in water and organic solvents and soluble in aqueous bromine. Stable under the selected administration form. The stability of the test item in the test solution was confirmed to be at least 9 days.
Species:
rat
Strain:
Crj: CD(SD)
Remarks:
[Crj:CD(SD)IGS]
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan Co. Atsugi center (795 Shimokozawa, Atsugi, Kanagawa Prefecture)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 10 wks
- Weight at study initiation: (P) Males: 354-427 g; Females: 208-272 g
- Housing: stainless steel wire mesh cage [260Wx380Dx180H(mm)] / polycarbonate cage [265Wx426Dx200H(mm)] (Rats were housed individually in wire cages in a breeding room. After mating confirmation, females were housed in polycarbonate cages containing nesting material [White flakes, Charles River Japan Co., Ltd.])
- Diet (e.g. ad libitum): standard feed [Lab MR stock, Japan Agro-Industrial Co., Ltd., lot numbers 04046-4, 040576-4, 040861], ad libitum
- Water (e.g. ad libitum): sterile tap water irradiated with UV light after filtration with a cartridge filter with a pore size of 1μm, ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.6 - 24.4ºC
- Humidity (%): 45-59%
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light (7 am to 7 pm)
Route of administration:
oral: gavage
Vehicle:
other: 1.0% (w/v) methylcellulose aqueous solution
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: the solution was prepared as a suspension at a concentration of the specified dose, using purified water as a solvent.

VEHICLE
- Justification for use and choice of vehicle: the test item was insoluble in water and in oils.
- Amount of vehicle (if gavage): 1 mL per 100 g body weight (10 mL/kg)
- Lot/batch no.: methyl cellulose 100 cp, lot No. KLP3708 (Wako Pure Chemical Industries) in purified water (lot No.181382, Kyoei Pharmaceutical Co.,Ltd.)
Details on mating procedure:
- M/F ratio per cage: cohoused 1 to 1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): After mating confirmation, females were housed in polycarbonate cages containing nesting material [White flakes, Charles River Japan Co., Ltd.]) [265Wx426Dx200H(mm)]
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability (at least 9 days) and homogeneity of the test item in the stock solutions had been previously studied. Furthermore, the concentration of the initial preparation was confirmed by analysis.
Duration of treatment / exposure:
The administration period started 14 days prior to mating for both sexes; males were administered for 42 days, females through mating and gestation, and 4 days after parturition, for a minimum of 42 days to a maximum of 46 days, once a day in the morning (9:03 to 12:00). The female recovery group was administered for 42 days.
Frequency of treatment:
Daily
Details on study schedule:
- Age at mating of the mated animals in the study: 12 weeks
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Basis: actual ingested
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Basis: actual ingested
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Basis: actual ingested
No. of animals per sex per dose:
main test: 12 male and 12 female animals per group
satellite groups: 5 male and 5 female animals per group (recovery period)
Control animals:
yes
Details on study design:
- Dose selection rationale: The highest dose to be used in the main study was selected based on the results of range-finding studies. First, 2 male and 2 female rats were administered the test item for 3 days at 2000 mg/kg, and no changes were observed except blue colored stools. Next, the test item was administered to 4 groups of 1 male and 1 female rat at doses of 0, 30, 100, 300 and 1000 mg/kg bw for 14 days. Since no toxic effects resulting from administration were observed, the maximum dose recommended by the test guidelines (1000 mg/kg bw) was selected as the highest dose. The mid and low doses were set at 300 and 100 mg/kg bw, respectively. .
- Rationale for animal assignment: random.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes (appearance, behaviour, mortality)
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The day before starting the administration and once a week after.
- Parameters: ease of removal from cage, ease of handling, body tension (relaxation-tonicity), skin (color), coat, nape, eye secretion, eyelid closure, ocular protrusion, lacrimation, oronasal secretion (dirt), salivation, lower abdominal hair contamination by urine, contamination by stools around the anus, vocalization, breathing, posture, convulsions, tremors, exploratory behavior (degree of alertness), alertness, locomotor activity (activity), walking (stirring), abnormal behavior (self-bite, We observed 29 items of backward walking, etc.), regularity (excessive hairdressing, repeated turning movement, etc.), unconsciousness (mixture, catalepsy, coma), limb muscle tone, urination and feces.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: Weekly. Food intake was measured for 24 hours of food consumption up to the next day according to the weight measurement day. The last day of food consumption measurement was day 41 for males, lactation day 3 for females, and recovery day 13 for animals in the recovery group.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

OTHER:
HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the administration period and at the end of recovery period.
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: all
- Parameters: red blood cell count (electrical resistance detection method), hemoglobin amount (lauryl sulfate) by multi-item automatic hemocytometer (E-4000, Sysmex) Sodium-hemoglobin method), hematocrit value (pulse detection method), average red blood cell volume (MCV), average red blood cell pigment content (MCH), average red blood cell pigment concentration (MCHC, above, calculated value), white blood cell count and platelet count (above, The electric resistance detection method was used, and smears were prepared, and reticulocyte count (Brilliant cresyl blue staining and microscopic examination) and leucocyte percentage (May-Giemsa staining and microscopic examination), prothrombin time (Quick one-step method), blood coagulation automatic measurement device (KC-10A, Amerung Corporation, USA) Thromboplastin time (APTT, eladinic acid activation method).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the administration period and at the end of recovery period.
- Animals fasted: Yes
- How many animals: all
- Parameters: total protein (Biuret method), albumin (BCG method), A / G ratio (calculated value) by a biochemical automatic analyzer (JCA-BM type 8 cleaner, JEOL), blood sugar, total cholesterol, triglyceride (above, enzymatic method), total bilirubin (diazo method), urea nitrogen (Urease-UV method), creatinine (Jaffe), AST, ALT, ALP (above, JSCC method), g − GTP (SSCC method), LDH (SFBC method), cholinesterase (BTC-DNTB method), calcium (OCPC method) and inorganic phosphorus (enzyme method), and with automatic electrolyte analyzer (NAKL-132, Toa Denpa Kogyo) Sodium, potassium and chlorine (above, ion electrode method).

URINALYSIS: Yes (males)
- Time schedule: On day 40 of administration and during second week of recovery.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Not specified
- Parameters: appearance (Martistics, Bayer, Sankyo), qualitative examination of pH, occult blood, protein, sugar, ketone body, bilirubin and urobilinogen and examination of sediment (URI-CELL fluid, stained with Cambridge chemical product and microscopic examination). Urine volume, specific gravity (refractometer, Elma optics), and sodium and potassium (electrolytic analyzer, NAKL-132, Toa Denpa Kogyo) were measured for the urine obtained after 18 hours of storage.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: sensory activity on the last day of administration, for females once during the lactation period, and at the end of the recovery period; motor activity and grip strength on day 41 for males, once during lactation for females, and on the last day of adminstration and on the 13th day of recovery for animals in the recovery group.
- Battery of functions tested: sensory activity / grip strength / motor activity
Oestrous cyclicity (parental animals):
- Estrous cycle
Sperm parameters (parental animals):
- Number of cells in seminiferous epithelia, testis weight, seminal vesicle weight and epididymis weight. Spermatogenic cycle tests (stages II, III, V, VII and XII) were studied in testis.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in offspring:
number of pups born, delivery index (%), number of pups alive on Day 0 of lactation, live birth index (%), sex ratio, number of pups alive on Day 4 of lactation, viability index (%), body weight of live pups on Day 0.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after administration day 42
- Maternal animals: All surviving animals on day 5 of lactation
- Recovery group: on day 14 of recovery

GROSS NECROPSY: Yes
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS: Yes
The tissues indicated were prepared for microscopic examination and weighed, respectively:
- Liver, kidney, adrenal gland, thymus, spleen, brain, heart, pituitary, thyroid and seminal vesicles of five males and females in each group and all male testis and epididymis were weighed (absolute weight). The weight ratio (relative weight) was calculated.
- For females, the number of corpora lutea and the number of implantations of the uterus were examined, and the implantation rate (%) [(number of implantation / number of corpora lutea) × 100] was calculated.
- Brain, pituitary, thyroid, thymus, trachea and lungs (fixed solution injected after immersion), stomach, intestine, heart, liver, spleen, kidney, adrenal, bladder, testis, epididymis, prostate, seminal vesicles, ovary, uterus, spinal cord (neck, chest, and lumbar region), sciatic nerve, bone marrow (femoral bone), lymph node (cervical lymph node, mesenteric lymph node), mammary gland, and other gross abnormal areas were fixed and stored in 10% neutral phosphate buffered formalin.
- Histopathological examination was performed on these preserved organs of 5 males and 5 females in the control group and 1000 mg/kg group.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.

GROSS NECROPSY: Yes
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
Statistics:
- Geometric data: Bartlett's variance test was performed for comparison among many groups. When the variance was uniform, one-way analysis of variance was performed. As a result, when a significant difference was recognized, a Dunnett's method was used to test each group against the control group. When the variance was not uniform, the test method used for non-parametric data was followed.
- Non-parametric data: For comparison among multiple groups, Kruskal-Wallis rank test was conducted, and when there was a significant difference, Dunnett's type test was used to compare each group with the control group. For data on implantation rate, birth rate, parturition rate, sexual cycle, neonatal survival rate etc., Mann-Whitney's U test was performed.
- Category data: Fisher's direct probability method was used.
Reproductive indices:
- Copulation index
- Fertility index
- Implantation index
- Gestation index
Offspring viability indices:
- Delivery index
- Live birth index
- Viability index
Clinical signs:
no effects observed
Description (incidence and severity):
Blue colored stools of the same color as the test substance were observed from day 2 to the final day of administration in all groups of test substance administration, and the degree of blue color tended to increase in a dose-related manner. No treatment-related chages were observed during either the administration period or the recovery period at any dose tested.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no significant changes in body weight and weight gain during the treatment and recovery periods.
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the administration period, the amount of hemoglobin, hematocrit and white blood cell count of males administered the test item (all groups) showed generally lower values ​​compared to the control group. There was a significant difference between the groups. The average red blood cell volume and the average red blood cell blood dye amount in the 100 mg/kg and 1000 mg/kg groups showed significantly lower values, in association with the low values ​​of the hemoglobin amount and the hematocrit value. . However, the average value of each of these groups is within the normal range in the background data of the laboratory [Helopigment: 14.2 to 16.6 (g/dL), Hematocrit: 42.2 to 48.4 (%), White Blood Cell Count: 41 to 120 (102/μL), average red blood cell volume: 49 to 56 (fL), average red blood cell pigment amount: 16.4 to 19.3 (pg)], and no tendency was observed to change in a dose-related manner. In females, no significant change was found.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the administration period, no changes were observed. Male sodium in the 100 mg/kg group showed a significantly lower value, but it was within the normal range [sodium: 142 to 145 (mEq / L)], and there was no dose correlation in the change. At the end of the recovery period, in the examination of the slaughtered animals, female albumin and potassium showed significantly lower values, but these are also within the normal range [Albumin: 2.70 to 3.81 (g/dL), potassium: 4.05 to 5.65 (mEq/L)].
Urinalysis findings:
no effects observed
Description (incidence and severity):
No effects were observed in any male during the treatment period or the recovery period.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
In the recovery group, high value of forelimb grip strength in the forelimbs in males and females was observed. However, this was due to a rather low value in the control group, no other change was observed at the end of the administration period, and the value was within the normal range. It was thought to be an accidental change.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- In animals sacrificed at the end of the administration period: Mild hyperplasia of squamous epithelium near the border of the anterior stomach was observed in 5 males and 4 females in the 1000 mg/kg group, the expression rate was significantly different than in the control group. In one female, edema of the anterior gastric submucosa was also observed. These suggest mild local irritation of the test item. In animals sacrificed at the end of the recovery period: squamous hyperplasia near the border of the anterior stomach was observed in only one female, with a clear tendency toward recovery.
- Other changes, unrelated to the administration of the test item, include arterial wall mineralization and foam cell accumulation in the lung, myocardial degeneration / fibrosis in the heart, focal necrosis in the liver, hepatocellular fatty degeneration (diffuse) and microgranulation. Tumor, cortical lymphocyte infiltration of kidney, solitary cyst, basophilic tubular, hyaline columnar and cortical tubular mineral deposition, interstitial lymphoid infiltrate of prostate were observed in the control group and 1000 mg/kg group only, with a low expression rate. In addition, hyaline droplets of proximal tubular epithelium in male kidney and extramedullary hematopoiesis and brown pigmentation in the spleen of male and female were observed with high expression rate, but there were no significant differences in expression rate and degree of change betweeen the 1000 mg/kg group and the control group. At necropsy, sperm granuloma was found in the epididymal nodular area, which was recognized independently of the administration of the test substance, and seminiferous tubule atrophy and interstitial cell hyperplasia were found in the miniaturized testis.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No significant changes were observed, all cycles were observed in 4-5 days.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
No cases of copulation failure or pregnancy failure were observed, and no significant change was observed in the reproductive indices.
The parental animals exhibited no alterations in reproductive parameters.
A toxic effect was observed due to repeated administration of the test substance: squamous hyperplasia of the anterior stomach, which was observed in males and females in the 1000 mg/kg group by histopathological examination. This gastric change was not observed in the recovery group except for one female, and it was confirmed that this change is a reversible change that recovers in a short period of time.
Key result
Dose descriptor:
NOEL
Remarks:
maternal toxicity
Effect level:
>= 300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction toxicity
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no alterations in reproductive parameters were observed
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
No significant changes were observed in the general condition of newborns.
Mortality / viability:
no mortality observed
Description (incidence and severity):
No significant changes were observed in the total number of offspring per litter, delivery rate, number of newborns on 0 and 4 days of feeding, body weight, birth rate, sex ratio and survival up to day 4. (99.5%)
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant changes were observed.
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no pups with external and visceral abnormalities. Regarding visceral mutations, 3 cases (1.6% expression rate) of thymic cervical residue were found in the control group, and 4 cases (2.4%) in the 1000 mg/kg group; 1 case (0.5%) of the left umbilical artery residue in the control group, 1 in the 1000 mg/kg group (0.6%) and 1 case in the 100 mg/kg group (0.6%). The number of offspring with these visceral mutations is 4 (2.2%) in the control group, 1 (0.6%) in the 100 mg/kg group, 0 (0%) in the 300 mg/kg group, and 5 (3.1%) in the 1000 mg/kg group. There were no significant differences between the control group and treated group in the expression rates of the above visceral mutations by type and in the offspring with visceral mutations, and no dose-related trend was observed.
Histopathological findings:
no effects observed
Other effects:
no effects observed
There were no significant differences in offspring parameters.
Key result
Dose descriptor:
NOAEL
Remarks:
offspring development
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Reproductive effects observed:
no

Table 1. Reproduction results of rats treated orally with the test item in the combined repeated dose toxicity study with the reproductive/developmental toxicity screening test.

Dose (mg/kg) 0 100 300 1000
Estrous cycle (days, mean ± SD) 4.1 ± 0.2 4.0 ± 0.1 4.1 ± 0.4 4.1 ± 0.2
No. of pairs mated 12 12 12 12
No. of successful copulations 12 12 12 12
Copulation index (%) 100 100 100 100
"Pairing days until copulation (days, mean ± SD)" 2.4 ± 1.2 2.8 ± 1.3 2.3 ± 1.0 2.3 ± 1.0
No. pregnant females 12 12 12 12
Fertility index (%) 100 100 100. 100
"No. corpora lutea (mean ± SD)" 16.8 ± 1.7 17.3 ± 1.5 17.0 ± 1.8 15.6 ± 2.2
"No. implantation sites (mean ± SD)" 15.8 ± 1.6 15.9 ± 1.6 16.6 ± 1.8 14.7 ± 2.7
"Implantation index (%, mean ± SD)" 94.8 ± 6.6 92.4 ± 7.4 97.7 ± 5.7 93.8 ± 8.7
No. pregnant females with parturition 12 12 12 12
"Gestation length (days, mean ± SD)" 22.3 ± 0.5 22.3 ± 0.5 22.4 ± 0.5 22.6 ± 0.5
No. pregnant females with live pups 12 12 12 12
Gestation index (%) 100 100 100 100
No. pregnant females with live pups on D4 12 12 12 12

*Copulation index = (No. pairs with successful copulation/No. of pairs mated) x 100

*Fertility index = (No. pregnant females / No. pairs with successful copulation) x 100

*Gestation index = (No. females with live pups / No. pregnant females) x 100

Table 2. Litter results of rats treated orally with the test item in the combined repeated dose toxicity study with the reproductive/developmental toxicity screening test. Each value is expressed as Mean ± SD, except sex ratio.

Dose (mg/kg) 0 100 300 1000
No. pups born 15.0 ± 1.6 14.9 ± 1.2 15.7 ± 1.6 13.6 ± 3.1
Delivery index 94.9 ± 5.0 94.2 ± 7.8 94.9 ± 7.5 91.7 ± 11.0
No. pups alive on D0
Total 14.7 ± 1.7 14.9 ± 1.2 15.3 ± 1.5 13.3 ± 2.9
Male 6.8 ± 2.2 6.8 ± 2.7 7.9 ± 2.2 6.3 ± 2.5
Female 7.9 ± 3.1 8.1 ± 2.2 7.4 ± 2.5 7.0 ± 1.7
Live birth index (%) 97.7 ± 3.5 100 ± 0 98.0 ± 5.1 98.5 ± 3.8
Sex ratio (M/F) 0.88 0.85 1.07 0.87
No. pups alive on D4
Total 14.6 ± 1.6 14.8 ± 1.1 15.3 ± 1.5 13.3 ± 2.9
Male 6.8 ± 2.2 6.8 ± 2.6 7.9 ± 2.2 6.3 ± 2.5
Female 7.8 ± 2.9 8.1 ± 2.2 7.3 ± 2.5 7.0 ± 1.7
Viability index (%) 99.5 ± 1.7 99.5 ± 1.7 99.5 ± 1.8 100 ± 0
Body weight of live pups (g)
on day 0
Male 7.1 ± 0.6 7.0 ± 0.5 7.2 ± 0.7 7.4 ± 0.9
Female 6.7 ± 0.5 6.7 ± 0.4 6.8 ± 0.7 7.1 ± 0.8
on day 4
Male 11.2 ± 1.1 11.3 ± 1.0 11.3 ± 1.2 12.1 ± 1.8
Female 10.9 ± 1.2 10.7 ± 1.0 10.6 ± 1.3 11.6 ± 1.8

*Delivery index = (no. of pups born / No. of implantation sites) x 100

*Live birth index = (no. of live pups on day 0 / no. of pups born) x 100

*Viability index = (no. of live pups on day 4 / no. of live pups on day 0) x 100

* Sex ratio = Total no. of male pups / Total no. of female pups.

Table . External findings of pups from pregnant rats treated orally with the test item in the combined repeated dose toxicity study with the reproduction/developmental screening test.

Dose (mg/kg)

0

100

300

1000

No. pups examined

180

179

188

162

No. pups with external malformations #

0 (0)

0 (0)

0 (0)

0 (0)

External malformations #

0 (0)

0 (0)

0 (0)

0 (0)

#: no. of pups (mean ± SD of individual litter percentages)

Table. Visceral findings of pups from pregnant rats treated orally with the test item in the combined repeated dose toxicity study with the reproduction/developmental screening test.

Dose (mg/kg)

0

100

300

1000

No. pups examined

180

179

188

162

No. pups with visceral malformations

0

(0)

0

(0)

0

(0)

0

(0)

No. pups with visceral variations #

4

(2.2 ± 4.2)

1

(0.6 ± 0.2)

0

(0)

5

(3.1 ± 6.0)

Visceral variations #

- thymic remnant in neck

3

(1.6 ± 3.0)

0

(0)

0

(0)

4

(2.4 ± 4.9)

- persistent left umbilical artery

1

(0.5 ± 1.8)

0

(0)

0

(0)

1

(0.6 ± 2.2)

- convoluted ureters

0

(0)

1

(0.6 ± 2.0)

0

(0)

0

(0)

#: no. of pups (mean ± SD of individual litter percentages)

Table . Number of cells in seminiferous epithelia assessed by the individual examination in male rats treated orally with the test item in the combined repeated dose toxicity study with the reproduction/developmental screening test.

Stage II-III

Stage V

Stage VII

Stage XII

Dose (mg/kg)

No. animals

G

P

T

G

P

T

G

R/P

T

G

Z/P

0

5

Mean

0.72

2.43

7.62

0.77

2.74

7.32

0.11

3.52

7.59

0.10

5.69

SD.

0.08

0.22

0.92

0.07

0.31

0.47

0.02

0.28

0.23

0.01

0.44

1000

5

Mean

0.68

2.23

7.14

0.72

2.50

7.25

0.09

3.73

7.64

0.08

5.26

SD.

0.08

0.23

0.42

0.06

0.29

0.68

0.01

0.38

0.37

0.01

0.22

G: spermatogonia; P: pachytene spermatocyte; R: preleptotene spermacyte; Z: zygotene spermacyte; T: round spermatid.

Conclusions:
The results were:
-NOAEL maternal toxicity >= 300 mg/kg bw /day
-NOAEL reproductive toxicity>= 1000 mg/kg bw/day
-NOAEL offspring development >= 1000 mg/kg bw/day
Executive summary:

The aim of the test was to study the reproductive and developmental toxicity in rats (12 males and 12 females) in a combined repeated dose toxicity study with the reproductive / developmental toxicity screening test at doses of 100, 300 and 1000 mg/kg bw/day.

The test was performed according to OECD Test Guideline 422.

The results were:

-NOAEL maternal toxicity >= 300 mg/kg bw /day

-NOAEL reproductive toxicity>= 1000 mg/kg bw/day

-NOAEL offspring development >= 1000 mg/kg bw/day

Endpoint:
reproductive toxicity, other
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
20 July 2020 to 06 October 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Reason / purpose for cross-reference:
reference to other study
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: Dose Range Finding study for a subsequent definitive Extended One-Generation Reproductive Toxicity Study on the test substance.
- Short description of test conditions: oral administration of test item over a relatively limited period of time i.e. 32 days for male Sprague Dawley rats including pre-mating and mating periods and 49 to 66 days for female Sprague Dawley rats including pre-mating, mating, gestation and lactation (till lactation day 13).
- Parameters analysed / observed: effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition.
GLP compliance:
no
Remarks:
DRF study
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: In-house bred animals
- Age at study initiation: Minimum of 10 weeks
- Weight at study initiation: Males: 254.06 to 298.31 g & Females: 200.23 to 252.96 g
- Fasting period before study: No
- 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.
- During acclimatization, maximum of three animals of same sex were housed.
- Pre mating - Per cage two animals of the same sex and group were housed.
- Cohabitation Period (mating) - Per cage two animals (one male and one female) of the same group were housed.
- Post-mating - After confirming presence of sperm in the vaginal smear (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually. Sterilized paper shreds were provided as a nesting material for main group females from gestation day 20 onwards.
- Diet (e.g. ad libitum): ad libitum - Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG)
- Water (e.g. ad libitum): ad libitum - Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7ºC to 22.9ºC
- Humidity (%): relative humidity 47 to 65%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark

IN-LIFE DATES: From: To: 20 July 2020 to 29 September 2020
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
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 and triturated well in a mortar with a small quantity of vehicle until a homogenous suspension was formed and thereafter the entire quantity of the formulation was transferred into measuring cylinder. A small quantity of vehicle was added to rinse the mortar and this was transferred into the measuring cylinder. The rinsing procedure of mortar and pestle was repeated (many times) to ensure the transfer of the contents to the measuring cylinder. Finally, the volume was made up to required quantity with vehicle to get desired concentration of 10, 30 and 100 mg/mL of test item for low, mid and high dose groups respectively. The test formulations were maintained under stirring conditions using magnetic stirrer to maintain homogeneity of the test item formulations.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item was in-soluble in distilled water and uniformly suspended in 0.5% w/v Carboxy Methyl Cellulose at the concentration of 100 mg/mL (the highest dose concentration selected for the study considering the dose volume of 10 mL/kg body weight) as per in-house solubility/suspendibility test results. Hence, 0.5% w/v Carboxy Methyl Cellulose was used as vehicle for test item formulations and the details were recorded in the raw data and presented in the study report.
- Concentration in vehicle: 10 - 100mg/mL
- Amount of vehicle (if gavage): 10mL/kg bw
- Lot/batch no. (if required): BCBN1690V
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy.
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol: No
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
The stability and homogeneity of the test item in dose formulations was not established under this dose range finding study. However, freshly prepared test item formulations were administered to the animals.
Duration of treatment / exposure:
Males: two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 32 days of treatment).
Females: two weeks pre-mating period, during mating, pregnancy (gestation)
and up to lactation day 13 (total of 49 to 66 days of treatment).
Frequency of treatment:
Once a day
Details on study schedule:
F1 animals were not mated.
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were decided based on the results of a dose range finding study conducted in lines with OECD TG 414 (Bioneeds Study No. BIO-DTX 030) in which no adverse effects were observed for maternal and developmental toxicity at the dose levels of 100, 300 and 1000 mg/kg bw/d.
Positive control:
None
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily for clinical signs of toxicity and twice daily for mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS: No

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

FOOD CONSUMPTION: yes
-Feed consumption was measured for all animals once a week during premating. Feed consumption was not measured during mating period for both males and females. Thereafter, feed consumption for females was recorded during gestation days 0 to 7, 7 to 14 and 14 to 20 and on lactation days 1 to 4, 4 to 7 and 7 to 13.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day before of scheduled terminal sacrifice.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 5 males and 5 females randomly selected from each main group.
- Parameters checked: Haemoglobin concentration (HGB), Haematocrit (HCT), Erythrocyte count (RBC), Total leukocyte count (WBC), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Platelet count (PLT), Differential leucocytes count (DLC), Absolute differential leucocytes count (DLC). Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) were estimated with the help of coagulation analyser [Tulip Diagnostics (p) Ltd., India].

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day before of scheduled terminal sacrifice.
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 5 males and 5 females randomly selected from each main group.
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Total Protein, Albumin, Glucose, Total Cholesterol, Creatinine, Blood Urea.

URINALYSIS: Yes
- Time schedule for collection of urine: on the day before of scheduled terminal sacrifice.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 5 males and 5 females randomly selected from each main group.
- Parameters checked: Blood, Protein, Glucose. In addition pH and specific gravity were also analyzed. After analysis of the above parameters, the urine was subjected for centrifugation at 1500 rpm for 3 minutes. Then the urine was subjected for microscopic examination for urine sediments.
Oestrous cyclicity (parental animals):
Oestrus cycles were monitored daily for a period of 2 weeks during pre-mating period before initiation of cohabitation and during cohabitation until evidence of mating.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Individual pups from each litter were observed for external examination and clinical signs once daily and for mortalities twice daily.
Individual live pup weight from each litter was recorded on PND 1 (within 24 hours of parturition), 4, 7, and 13. The mean pup weight (sex-wise) is reported as per litter basis.
The number of male/female pups born (live/dead/cannibalized) per litter, sex ratio (m/f), live birth index (%), pup survival index (%).

GROSS EXAMINATION OF DEAD PUPS:
The dead pups and pups which were sacrificed on PND 13 were examined for gross abnormalities with particular attention to the external reproductive genitals and the observations were recorded.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after completion of 32 days of treatment.
- Maternal animals: All surviving animals on lactation day 14

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations. Special attention was paid to the organs of the reproductive system.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated below were collected, weighed, and preserved for microscopic examination.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring was sacrificed on postnatal day 13.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations with particular attention to the external reproductive genitals.
Statistics:
After verification, the data was subjected to statistical analysis using SPSS software, version 22. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05). The statistical analysis was followed to the parameters as mentioned in the table below.
Reproductive indices:
See List below.
Offspring viability indices:
See List below.
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
A slight reduction in mean body weight and mean percent change in body
weight gain with respect to day 1 from group G4 males was noted during week 3 and 4. These noted changes are not considered as test item related, as there were no other systemic toxicity effects noted from this dose level in both sexes.

There were no changes noted in mean body weight and mean percent change in body weight gain during gestation or lactation period.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The following statistically significant changes were noted in haematology parameters of tested dose groups from both the sexes when compared with vehicle
control group:
- increase in mean prothrombin time of group G4 males;
- increase in mean activated prothrombin time of groups G2, G3 and G4 males;
- decrease in mean leucocyte (WBC) count of group G2 females;
- decrease in mean absolute lymphocytes of groups G2 and G3 females;
- decrease in mean absolute monocytes of group G2 females.

These noted changes are considered to be incidental and not test item-related, as these changes are lacking relevant dose dependency (except in case of activated prothrombin time) and the obtained values are still within lab historical control data range of same species and strain.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
A statistically significant increase in mean urea levels of group
G3 and G4 males compared with vehicle control group was noted. However, these increases are considered to be incidental and not test item-related, as the obtained values are within lab historical control data range of same species and strain.
Endocrine findings:
not examined
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
A statistically significant increase in mean pH levels of groups G2 and
G4 males compared with vehicle control group was noted. However these changes are considered to be incidental and not test item-related, as the obtained values are within lab historical control data range of same species and strain.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There were no irregularities observed in the oestrus cyclicity of females from all the
tested dose groups and vehicle control group during pre-mating and mating treatment periods. The mean length (days) of oestrus cycle per female during pre-mating and mating treatment period was unaffected by the test item administration at any of the tested dose groups when compared with vehicle control group.
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
A statistically significant increase in mean number of female pups born at birth
and mean survived number of females pups on LD 4, 7 and 13 from group G2 compared with vehicle control group was noted, however, these changes are considered to be incidental with no toxicological relevance.

A slight reduction in male fertility rate of groups G3 and G4 was noted. These noted changes are not considered as test item related, as there were no changes noted in mean organ weight of any of the reproductive organs and also no gross pathological changes noted in any of reproductive organs during conduct of necropsy.

A increase in post implantation losses from group G3 and G4 was noted. However these changes are not considered test item related, as a total of 3 dams were noted with post implantation loss from these dose groups with a maximum of 2 implantation loss and the obtained range of implantation loss is still within the lab historical control data of same species and strain.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
There were no changes noted in daily observation of pups at all the tested and vehicle control groups.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
There were no mortalities of pups during the 13-day lactation period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no changes observed in mean pup [both male and female] weight per litter recorded on postnatal day (PND) 1, 4, 7 and 13 from all the tested dose groups when compared with vehicle control group.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross pathological changes observed in any of the pups found dead or terminally sacrificed from all the tested dose groups and vehicle control group litters during conduct of necropsy.
Histopathological findings:
not examined
Other effects:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

TABLE 1. SUMMARY OF CLINICAL SIGNS OF TOXICITY, DETAILED CLINICAL EXAMINATION AND MORTALITY RECORD




































 


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



 


Total No. of Animals



Clinical Signs of Toxicity (a):


Observation


(No. of Animals revealed)



Mortality (b):


No. of Mortalities (Total No. of Animals)



 


G1, M & 0



 


10



 


N (10)



 


0 (10)



 


G2, M & 100



 


10



 


N (10)



 


0 (10)



 


G3, M & 300



 


10



 


N (10)



 


0 (10)



 


G4, M & 1000



 


10



 


N (10)



 


0 (10)



M: Male; N: Normal;


a: observed daily once; b: observed twice daily


 




































 


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



 


Total No. of Animals



Clinical Signs of Toxicitya:


Observation


(No. of Animals revealed)



Mortalityb:


No. of Mortalities (Total No. of Animals)



 


G1, F & 0



 


10



 


N (10)



 


0 (10)



 


G2, F & 100



 


10



 


N (10)



 


0 (10)



 


G3, F & 300



 


10



 


N (10)



 


0 (10)



 


G4, F & 1000



 


10



 


N (10)



 


0 (10)



F: Female; N: Normal;


a: observed daily once; b: observed twice daily


TABLE 2. SUMMARY OF BODY WEIGHT (g) RECORD

































































































































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



 



 



Body Weight (g) on Day



 



 



1



8



14



21



28



 



Mean



310.66



347.01



367.85



377.61



389.42



G1, M & 0



±SD



19.26



20.33



28.19



30.94



37.34



 



n



10



10



10



10



10



 



Mean



311.95



345.16



361.63



370.24



380.30



G2, M & 100



±SD



16.82



16.67



13.20



17.21



29.36



 



n



10



10



10



10



10



 



Mean



311.55



344.62



362.04



371.47



379.14



G3, M & 300



±SD



17.18



14.85



15.00



21.22



26.93



 



n



10



10



10



10



10



 



Mean



312.67



349.31



365.43



366.94



368.00



G4, M & 1000



±SD



15.21



17.44



25.52



26.55



30.06



 



n



10



10



10



10



10



 

































































































































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



 



 



Body Weight (g) on Day



 



 



1



8



14



21#



28#



 



Mean



235.51



251.81



261.84



-



-



G1, F & 0



±SD



12.71



11.65



14.15



-



-



 



n



10



10



10



-



-



 



Mean



234.68



249.12



256.37



282.83



-



G2, F & 100



±SD



9.43



13.10



15.09



24.95



-



 



n



10



10



10



2



-



 



Mean



235.51



251.56



259.32



267.65



258.65



G3, F & 300



±SD



11.29



16.21



16.70



29.77



23.29



 



n



10



10



10



3



2



 



Mean



233.66



248.77



255.69



261.52



253.16



G4, F & 1000



±SD



12.44



14.29



14.26



13.41



-



 



n



10



10



10



5



1



#: Presented mean values are obtained from the females in cohabitation period. Mated animals data is presented under gestation body weight.


 


TABLE 3. SUMMARY OF PERCENT CHANGE IN BODY WEIGHT (%) WITH RESPECT TO DAY 1


















































































































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



 



Percent Change in Body Weight (%) during Day



 



1 to 8



1 to 14



1 to 21



1 to 28



 



Mean



11.76



18.42



21.56



25.34



G1, M & 0



±SD



2.88



5.70



6.89



9.21



 



n



10



10



10



10



 



Mean



10.71



16.06



18.78



21.97



G2, M & 100



±SD



2.70



3.74



3.74



7.69



 



n



10



10



10



10



 



Mean



10.74



16.49



19.54



22.05



G3, M & 300



±SD



3.93



7.60



9.37



11.38



 



n



10



10



10



10



 



Mean



11.74



16.84



17.36



17.67



G4, M & 1000



±SD



2.54



4.85



6.09



6.95



 



n



10



10



10



10



 


















































































































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



 



Percent Change in Body Weight (%) during Day



 



1 to 8



1 to 14



1 to 21#



1 to 28#



 



Mean



6.97



11.24



-



-



G1, F & 0



±SD



2.22



4.02



-



-



 



n



10



10



-



-



 



Mean



6.14



9.23



18.48



-



G2, F & 100



±SD



3.11



4.38



6.81



-



 



n



10



10



2



-



 



Mean



6.75



10.07



10.13



6.93



G3, F & 300



±SD



2.68



3.84



8.72



0.32



 



n



10



10



3



2



 



Mean



6.47



9.46



12.35



7.61



G4, F & 1000



±SD



2.09



3.23



4.13



-



 



n



10



10



5



1



#: Presented mean values are obtained from the females in cohabitation period. Mated animals data is presented under gestation body weight.


TABLE 4. SUMMARY OF FEED CONSUMPTION (g/animal/day) RECORD























































































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



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



 



Week 1



Week 2



 



Mean



21.76



23.82



G1, M & 0



±SD



0.67



1.52



 



n



10



10



 



Mean



21.35



23.33



G2, M & 100



±SD



1.17



1.62



 



n



10



10



 



Mean



21.87



22.69



G3, M & 300



±SD



0.50



1.26



 



n



10



10



 



Mean



21.52



24.06



G4, M & 1000



±SD



1.82



1.40



 



n



10



10



 


 























































































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



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



 



Week 1



Week 2



 



Mean



16.45



18.14



G1, F & 0



±SD



1.32



1.25



 



n



10



10



 



Mean



16.88



17.73



G2, F & 100



±SD



0.93



1.07



 



n



10



10



 



Mean



17.19



18.59



G3, F & 300



±SD



0.59



1.67



 



n



10



10



 



Mean



17.11



18.01



G4, F & 1000



±SD



1.34



1.30



 



n



10



10



 


TABLE 5. SUMMARY OF HAEMATOLOGY RECORD


























































































































































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



 



Total Leucocyte Count



Total Erythrocyte Count



Haemoglobin



Haematocrit



Platelet Count



Neutrophils



 



(WBC)



(RBC)



(HGB)



(HCT)



(PLT)



(Neut)



 



(103 cells/µL)



(106 cells/µL)



(g/dL)



(%)



(103 cells/µL)



(%)



 



Mean



8.98



7.81



14.70



43.12



853.80



22.54



G1, M & 0



±SD



2.16



0.35



0.50



1.97



255.94



4.12



 



n



5



5



5



5



5



5



 



Mean



7.66



7.37



13.94



40.76



589.20



24.32



G2, M & 100



±SD



2.40



0.83



1.24



3.58



177.01



11.39



 



n



5



5



5



5



5



5



 



Mean



9.51



7.92



14.64



42.66



694.60



20.40



G3, M & 300



±SD



2.99



0.54



1.12



2.71



171.66



5.36



 



n



5



5



5



5



5



5



 



Mean



8.74



7.04



13.06



37.42



742.00



18.22



G4, M & 1000



±SD



1.66



1.38



2.43



6.84



367.30



6.61



 



n



5



5



5



5



5



5



 


























































































































































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



 



Lymphocytes



Monocytes



Eosinophils



Basophils



Absolute Neutrophils



Absolute Lymphocytes



 



(Lymph)



(Mono)



(Eos)



(Baso)



(Neut)



(Lymph)



 



(%)



(%)



(%)



(%)



(103 cells/µL)



(103 cells/µL)



 



Mean



70.50



4.28



1.74



0.34



2.04



6.33



G1, M & 0



±SD



4.59



1.54



0.43



0.11



0.66



1.65



 



n



5



5



5



5



5



5



 



Mean



68.48



3.60



2.44



0.32



1.71



5.39



G2, M & 100



±SD



11.59



2.03



1.20



0.11



0.56



2.45



 



n



5



5



5



5



5



5



 



Mean



73.86



3.12



1.62



0.32



1.86



7.12



G3, M & 300



±SD



5.79



0.77



1.04



0.11



0.46



2.67



 



n



5



5



5



5



5



5



 



Mean



74.78



3.80



1.64



0.46



1.52



6.62



G4, M & 1000



±SD



7.79



0.87



1.71



0.09



0.33



1.81



 



n



5



5



5



5



5



5



 










































































































































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



Absolute Monocytes



Absolute Eosinophils



Absolute Basophils



Prothrombin Time



Activated Prothrombin Time



 



(Mono)



(Eos)



(Baso)



(PT)



(APTT)



 



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(Seconds)



(Seconds)



 



Mean



0.36



0.15



0.03



16.70



19.02



G1, M & 0



±SD



0.09



0.04



0.01



0.82



4.83



 



n



5



5



5



5



5



 



Mean



0.30



0.17



0.03



16.70



25.08*



G2, M & 100



±SD



0.22



0.06



0.02



1.51



1.73



 



n



5



5



5



5



5



 



Mean



0.30



0.15



0.03



16.30



28.36*



G3, M & 300



±SD



0.10



0.06



0.02



1.06



1.47



 



n



5



5



5



5



5



 



Mean



0.34



0.13



0.04



19.00*



31.20*



G4, M & 1000



±SD



0.12



0.11



0.01



1.27



1.89



 



n



5



5



5



5



5



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


























































































































































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



 



Total Leucocyte Count



Total Erythrocyte Count



Haemoglobin



Haematocrit



Platelet Count



Neutrophils



 



(WBC)



(RBC)



(HGB)



(HCT)



(PLT)



(Neut)



 



(103 cells/µL)



(106 cells/µL)



(g/dL)



(%)



(103 cells/µL)



(%)



 



Mean



14.48



6.98



14.08



41.98



697.80



23.32



G1, F & 0



±SD



6.45



0.23



0.28



2.25



99.04



4.34



 



n



5



5



5



5



5



5



 



Mean



6.82*



6.86



13.42



40.56



578.40



28.90



G2, F & 100



±SD



3.71



1.25



2.65



7.73



200.77



6.11



 



n



5



5



5



5



5



5



 



Mean



8.75



7.24



14.04



42.60



639.40



37.32



G3, F & 300



±SD



3.21



0.43



0.77



2.82



250.60



11.79



 



n



5



5



5



5



5



5



 



Mean



11.45



7.39



14.12



43.68



611.80



35.84



G4, F & 1000



±SD



3.12



0.24



0.42



2.07



283.41



12.30



 



n



5



5



5



5



5



5



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


























































































































































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



 



Lymphocytes



Monocytes



Eosinophils



Basophils



Absolute Neutrophils



Absolute Lymphocytes



 



(Lymph)



(Mono)



(Eos)



(Baso)



(Neut)



(Lymph)



 



(%)



(%)



(%)



(%)



(103 cells/µL)



(103 cells/µL)



 



Mean



69.04



4.26



2.08



0.52



3.28



10.14



G1, F & 0



±SD



3.82



1.17



1.09



0.23



1.24



4.92



 



n



5



5



5



5



5



5



 



Mean



61.80



4.58



1.72



0.42



1.91



4.37*



G2, F & 100



±SD



7.16



1.16



0.80



0.22



0.91



2.82



 



n



5



5



5



5



5



5



 



Mean



50.78



4.72



2.42



0.48



3.29



4.53*



G3, F & 300



±SD



17.31



1.71



1.48



0.04



1.55



2.46



 



n



5



5



5



5



5



5



 



Mean



57.34



3.72



1.86



0.32



4.34



6.31



G4, F & 1000



±SD



12.38



0.48



1.26



0.18



2.16



1.11



 



n



5



5



5



5



5



5



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


 











































































































































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



 



Absolute Monocytes



Absolute Eosinophils



Absolute Basophils



Prothrombin Time



Activated Prothrombin Time



 



(Mono)



(Eos)



(Baso)



(PT)



(APTT)



 



(103 cells/µL)



(103 cells/µL)



(103 cells/µL)



(Seconds)



(Seconds)



 



Mean



0.57



0.30



0.08



18.80



24.78



G1, F & 0



±SD



0.21



0.22



0.06



2.49



8.82



 



n



5



5



5



5



5



 



Mean



0.28*



0.11



0.03



21.48



25.06



G2, F & 100



±SD



0.09



0.06



0.03



2.82



9.43



 



n



5



5



5



5



5



 



Mean



0.38



0.20



0.04



18.22



18.54



G3, F & 300



±SD



0.13



0.13



0.02



2.47



6.11



 



n



5



5



5



5



5



 



Mean



0.44



0.21



0.04



19.12



22.80



G4, F & 1000



±SD



0.16



0.15



0.03



3.83



8.89



 



n



5



5



5



5



5



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


 


 


TABLE 6. SUMMARY OF CLINICAL CHEMISTRY RECORD











































































































































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



 



Glucose



Urea



Creatinine



Total Cholesterol



Total Protein



 



(GLU)



-



(CRE)



(CHO)



(TPR)



 



(mg/dL)



(mg/dL)



(mg/dL)



(mg/dL)



(g/dL)



 



Mean



115.60



25.40



0.46



45.40



6.24



G1, M & 0



±SD



9.86



1.95



0.02



5.73



0.40



 



n



5



5



5



5



5



 



Mean



106.60



26.00



0.42



51.80



6.14



G2, M & 100



±SD



8.38



3.44



0.03



9.31



0.28



 



n



5



5



5



5



5



 



Mean



110.00



33.84*



0.45



55.00



5.88



G3, M & 300



±SD



9.43



4.47



0.01



11.02



0.08



 



n



5



5



5



5



5



 



Mean



110.40



36.20*



0.46



47.40



6.12



G4, M & 1000



±SD



15.57



8.17



0.03



9.61



0.27



 



n



5



5



5



5



5



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


 




























































































































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)



 



Mean



3.19



42.80



104.00



107.40



G1, M & 0



±SD



0.11



9.73



14.20



9.40



 



n



5



5



5



5



 



Mean



3.11



35.40



97.00



121.20



G2, M & 100



±SD



0.10



11.26



18.26



39.34



 



n



5



5



5



5



 



Mean



3.15



34.20



99.00



97.40



G3, M & 300



±SD



0.15



4.60



18.59



16.26



 



n



5



5



5



5



 



Mean



2.99



33.80



99.20



106.40



G4, M & 1000



±SD



0.19



5.45



20.90



28.51



 



n



5



5



5



5



 


 











































































































































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)



 



Mean



110.20



46.78



0.60



75.40



6.36



G1, F & 0



±SD



38.71



10.07



0.09



7.16



0.63



 



n



5



5



5



5



5



 



Mean



92.80



57.20



0.66



75.40



6.62



G2, F & 100



±SD



17.31



8.90



0.12



13.07



0.69



 



n



5



5



5



5



5



 



Mean



96.80



50.52



0.59



70.20



6.76



G3, F & 300



±SD



17.98



5.26



0.08



16.42



0.60



 



n



5



5



5



5



5



 



Mean



102.60



53.84



0.60



79.00



6.16



G4, F & 1000



±SD



27.22



13.55



0.06



23.33



0.53



 



n



5



5



5



5



5



 


 




























































































































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)



 



Mean



3.20



66.80



112.80



83.20



G1, F & 0



±SD



0.40



11.58



25.62



22.22



 



n



5



5



5



5



 



Mean



3.48



77.00



165.00



127.20



G2, F & 100



±SD



0.27



32.16



85.40



90.51



 



n



5



5



5



5



 



Mean



3.20



95.00



179.40



195.40



G3, F & 300



±SD



0.38



53.52



144.08



123.33



 



n



5



5



5



5



 



Mean



3.19



93.80



138.00



158.80



G4, F & 1000



±SD



0.19



21.04



50.35



57.61



 



n



5



5



5



5



 


TABLE 7. SUMMARY OF URINALYSIS RECORD




































































































































































































 


Examination



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



G1,


M & 0



G2, M & 100



G3, M & 300



G4,


M & 1000



Number of Animals (randomly selected)



5



5



5



5



 


 


 


Physical Examination



 


Color



Pale Yellow



4



3



4



5



Yellow



1



2



1



-



 


Appearance



Clear



5



5



5



5



Turbidity



-



-



-



-



 


Volume (mL)



Mean



6.8



9.3



9.3



8.4



±SD



3.5



1.4



1.0



1.4



 


 


 


 


 


 


 


 


Chemical Examination



 


pH



Mean



8.2



8.5*



8.4



8.5*



±SD



0.3



0.0



0.2



0.0



 


Specific Gravity



Mean



1.007



1.005



1.008



1.005



±SD



0.003



0.000



0.003



0.000



 


Blood (Ery/µL)



Neg



5



5



4



3



Ca25



-



-



1



2



 


 


 


Protein (mg/dL)



Neg



3



4



2



3



Trace



-



-



2



1



30



1



-



-



-



100



1



-



-



-



>=300



-



1



1



1



Glucose (mg/dL)



Neg



5



5



5



5



 


 


Microscopic Examination



 


Epi Cells



0



3



3



3



4



0-1



1



2



1



1



1-2



1



-



1



-



Casts



Absent



5



5



5



5



Crystals



Present



5



5



5



5



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


 




































































































































































































 


Examination



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



G1,


F & 0



G2,


F & 100



G3,


F & 300



G4,


F & 1000



Number of Animals



5



5



5



5



 


 


 


Physical Examination



 


Color



Pale Yellow



4



3



3



5



Yellow



1



2



2



-



 


Appearance



Clear



5



3



4



2



Turbidity



-



2



1



3



 


Volume (mL)



Mean



5.7



6.4



6.2



5.0



±SD



1.4



2.0



1.8



1.2



 


 


 


 


 


 


 


Chemical Examination



 


pH



Mean



7.3



7.1



6.9



7.4



±SD



0.8



0.4



1.1



0.4



 


Specific gravity



Mean



1.015



1.017



1.019



1.015



±SD



0.005



0.004



0.007



0.004



 


Blood (Ery/µL)



Neg



5



4



3



5



Ca10



-



1



2



-



 


 


Protein (mg/dL)



Neg



4



4



2



5



Trace



1



1



1



-



30



-



-



1



-



>=300



-



-



1



-



Glucose (mg/dL)



Neg



5



5



5



5



 


 


 


Microscopic Examination



 


 


Epi Cells



0



-



3



3



1



0-1



3



2



2



2



1-2



1



-



-



2



2-3



1



-



-



-



Casts



Absent



5



5



5



5



Crystals



Present



5



5



5



5



 


TABLE 8. SUMMARY OF VAGINAL SMEAR EXAMINATION RECORD FOR DETERMINATION OF OESTRUS CYCLICITY





























































































































Determination of Oestrus Cyclicity during Pre-mating Treatment Period



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



Total No. of Females Evaluated



 



Females with Complete Regular Cycles



Females with at least one Irregular Oestrus Cycle



Average Length of Oestrous Cycle (Days)



 



 



n



10



0



Mean



4.95



G1, F & 0



10



%



100.00



0.00



±SD



0.16



 



 



 



 



 



n



10



 



 



n



10



0



Mean



4.95



G2, F & 100



10



%



100.00



0.00



±SD



0.16



 



 



 



 



 



n



10



 



 



n



10



0



Mean



5.00



G3, F & 300



10



%



100.00



0.00



±SD



0.00



 



 



 



 



 



n



10



 



 



n



10



0



Mean



5.00



G4, F & 1000



10



%



100.00



0.00



±SD



0.00



 



 



 



 



 



n



10



 


TABLE 9. SUMMARY RECORD OF GESTATION BODY WEIGHT (g)


 


















































































































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



 



Body Weight (g) on Gestation Day (GD)



 



0



7



14



20



 



Mean



262.66



277.87



309.48



375.80



G1, F & 0



±SD



16.64



16.18



19.11



31.27



 



n



10



10



10



10



 



Mean



257.99



276.92



314.65



396.93



G2, F & 100



±SD



14.34



12.54



14.81



34.46



 



n



9



9



9



9



 



Mean



261.88



277.61



312.40



387.04



G3, F & 300



±SD



17.10



19.10



22.42



31.30



 



n



8



8



8



8



 



Mean



265.30



282.42



317.67



394.73



G4, F & 1000



±SD



11.12



12.76



13.49



27.44



 



n



8



8



8



8



Note: Excluded the data of non-pregnants for mean calculations and statistical analysis.


TABLE 10. SUMMARY RECORD OF PERCENT CHANGE IN BODY WEIGHT GAIN (%) DURING GESTATION PERIOD





































































































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



 



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



 



0 to 7



7 to 14



14 to 20



 



Mean



5.84



11.38



21.43



G1, F & 0



±SD



2.24



2.56



6.88



 



n



10



10



10



 



Mean



7.40



13.63



25.96



G2, F & 100



±SD



1.69



1.55



5.61



 



n



9



9



9



 



Mean



5.99



12.52



23.83



G3, F & 300



±SD



1.41



2.16



2.43



 



n



8



8



8



 



Mean



6.46



12.51



24.23



G4, F & 1000



±SD



1.95



1.90



6.37



 



n



8



8



8



Note: Excluded the data of non-pregnants for mean calculations and statistical analysis


TABLE 11. SUMMARY RECORD OF FEED CONSUMPTION (g/animal/day) DURING GESTATION PERIOD




































































































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



Feed Consumption (g/animal/day) during Gestation Day (GD)



 



0 to 7



7 to 14



14 to 20



 



Mean



18.75



20.30



24.74



G1, F & 0



±SD



0.55



0.54



0.86



 



n



10



10



10



 



Mean



19.15



20.71



25.50



G2, F & 100



±SD



0.61



0.55



0.81



 



n



9



9



9



 



Mean



18.72



20.43



25.32



G3, F & 300



±SD



0.58



0.57



0.94



 



n



8



8



8



 



Mean



18.83



20.61



25.57



G4, F & 1000



±SD



0.60



0.80



1.18



 



n



8



8



8



Note: Excluded the data of non-pregnants for mean calculations and statistical analysis


TABLE 12. SUMMARY RECORD OF LACTATION BODY WEIGHT (g)


















































































































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



 



Body Weight (g) on Lactation Day (LD)



 



1



4



7



13



 



Mean



285.46



293.56



304.10



323.80



G1, F & 0



±SD



19.87



20.53



20.43



19.35



 



n



10



10



10



10



 



Mean



302.20



308.79



318.28



332.30



G2, F & 100



±SD



28.19



25.35



23.94



24.12



 



n



9



9



9



9



 



Mean



293.02



301.01



308.14



324.77



G3, F & 300



±SD



28.36



26.90



26.29



24.30



 



n



8



8



8



8



 



Mean



301.36



310.19



319.60



334.79



G4, F & 1000



±SD



18.35



18.43



17.65



21.71



 



n



8



8



8



8



 


TABLE 13. SUMMARY RECORD OF PERCENT CHANGE IN BODY WEIGHT GAIN (%) DURING LACTATION PERIOD





































































































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



 



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



 



1 to 4



4 to 7



7 to 13



 



Mean



2.84



3.61



6.53



G1, F & 0



±SD



1.32



0.82



1.57



 



n



10



10



10



 



Mean



2.29



3.13



4.44



G2, F & 100



±SD



2.21



1.00



1.97



 



n



9



9



9



 



Mean



2.80



2.41



5.51



G3, F & 300



±SD



1.12



1.36



2.70



 



n



8



8



8



 



Mean



2.94



3.06



4.73



G4, F & 1000



±SD



0.85



1.16



2.59



 



n



8



8



8



TABLE 14. SUMMARY RECORD OF FEED CONSUMTION (g/animal/day) DURING LACTATION PERIOD





































































































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



 



Feed Consumption (g/animal/day) during Lactation Day (LD)



 



1 to 4



4 to 7



7 to 13



 



Mean



28.15



33.73



42.16



G1, F & 0



±SD



1.36



1.40



0.74



 



n



10



10



10



 



Mean



28.29



33.37



41.25



G2, F & 100



±SD



1.39



1.10



1.02



 



n



9



9



9



 



Mean



28.38



33.34



41.59



G3, F & 300



±SD



1.08



0.84



1.43



 



n



8



8



8



 



Mean



28.09



33.87



41.75



G4, F & 1000



±SD



1.34



1.02



1.04



 



n



8



8



8



 


TABLE 15. SUMMARY OF DELIVERY AND LITTER OBSERVATIONS RECORD DURING LACTATION PERIOD


















































































































































































































































































































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



 



Litter Delivered (No.)



 



Total Litter Size (No.)



 



Live Pups (No.)



Dead Pups (No.)



Cannibalized Pups (No.)



 



 



Sex Ratio (m/f) at Birth



 



Live Birth Index(%)



Male



Female



Total



 



Male



Female



Total



 



Undetermined



Male



Female



Total



 



 



 



 



 



Mean



12.80



 



6.40



6.30



12.70



 



0.10



0.00



0.10



 



0.00



0.00



0.00



0.00



 



1.13



 



99.38



G1, F & 0



±SD



2.66



 



2.01



2.31



2.54



 



0.32



0.00



0.32



 



0.00



0.00



0.00



0.00



 



0.50



 



1.98



 



n



10



 



10



10



10



 



10



10



10



 



10



10



10



10



 



10



 



10



 



Mean



14.56



 



6.67



7.89*



14.56



 



0.00



0.00



0.00



 



0.00



0.00



0.00



0.00



 



0.86



 



100.00



G2, F & 100



±SD



1.59



 



1.58



0.78



1.59



 



0.00



0.00



0.00



 



0.00



0.00



0.00



0.00



 



0.23



 



0.00



 



n



9



 



9



9



9



 



9



9



9



 



9



9



9



9



 



9



 



9



 



Mean



13.13



 



7.00



6.00



13.00



 



0.00



0.13



0.13



 



0.00



0.00



0.00



0.00



 



1.23



 



99.17



G3, F & 300



±SD



2.95



 



2.67



1.41



2.88



 



0.00



0.35



0.35



 



0.00



0.00



0.00



0.00



 



0.55



 



2.36



 



n



8



 



8



8



8



 



8



8



8



 



8



8



8



8



 



8



 



8



 



Mean



13.75



 



6.88



6.50



13.38



 



0.13



0.25



0.38



 



0.00



0.00



0.00



0.00



 



1.19



 



97.18



G4, F & 1000



±SD



2.19



 



2.47



2.20



2.39



 



0.35



0.46



0.74



 



0.00



0.00



0.00



0.00



 



0.61



 



5.66



 



n



8



 



8



8



8



 



8



8



8



 



8



8



8



8



 



8



 



8



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


 






















































































































































































































































































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



 



 



During LD 1 to 4



 



 


Sex Ratio (m/f) on LD 4



 



Pup Survival Index (%) during


LD 1 to 4



 



 



Live Pups (No.)



Dead Pups (No.)



Cannibalized Pups (No.)



 



 



Male



Female



Total



 



Male



Female



Total



 



Male



Female



Total



 



Mean



 



6.40



6.30



12.70



 



0.00



0.00



0.00



 



0.00



0.00



0.00



 



1.13



 



100.00



G1, F & 0



±SD



 



2.01



2.31



2.54



 



0.00



0.00



0.00



 



0.00



0.00



0.00



 



0.50



 



0.00



 



n



 



10



10



10



 



10



10



10



 



10



10



10



 



10



 



10



 



Mean



 



6.67



7.89*



14.56



 



0.00



0.00



0.00



 



0.00



0.00



0.00



 



0.86



 



100.00



G2, F & 100



±SD



 



1.58



0.78



1.59



 



0.00



0.00



0.00



 



0.00



0.00



0.00



 



0.23



 



0.00



 



n



 



9



9



9



 



9



9



9



 



9



9



9



 



9



 



9



 



Mean



 



7.00



6.00



13.00



 



0.00



0.00



0.00



 



0.00



0.00



0.00



 



1.23



 



100.00



G3, F & 300



±SD



 



2.67



1.41



2.88



 



0.00



0.00



0.00



 



0.00



0.00



0.00



 



0.55



 



0.00



 



n



 



8



8



8



 



8



8



8



 



8



8



8



 



8



 



8



 



Mean



 



6.88



6.50



13.38



 



0.00



0.00



0.00



 



0.00



0.00



0.00



 



1.19



 



100.00



G4, F & 1000



±SD



 



2.47



2.20



2.39



 



0.00



0.00



0.00



 



0.00



0.00



0.00



 



0.61



 



0.00



 



n



 



8



8



8



 



8



8



8



 



8



8



8



 



8



 



8



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


 








































































































































































































































































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



 



During LD 5 to 7



 



 


Sex Ratio (m/f) on LD 7



 



Pup Survival Index (%) during


LD 5 to 7



 



Live Pups (No.)



 



Dead Pups (No.)



Cannibalized (No.)



 



Male



Female



Total



 



Male



Female



Total



 



Male



Female



Total



 



Mean



6.40



6.30



12.70



 



0.00



0.00



0.00



 



0.00



0.00



0.00



 



1.13



 



100.00



G1, F & 0



±SD



2.01



2.31



2.54



 



0.00



0.00



0.00



 



0.00



0.00



0.00



 



0.50



 



0.00



 



n



10



10



10



 



10



10



10



 



10



10



10



 



10



 



10



 



Mean



6.67



7.89*



14.56



 



0.00



0.00



0.00



 



0.00



0.00



0.00



 



0.86



 



100.00



G2, F & 100



±SD



1.58



0.78



1.59



 



0.00



0.00



0.00



 



0.00



0.00



0.00



 



0.23



 



0.00



 



n



9



9



9



 



9



9



9



 



9



9



9



 



9



 



9



 



Mean



7.00



6.00



13.00



 



0.00



0.00



0.00



 



0.00



0.00



0.00



 



1.23



 



100.00



G3, F & 300



±SD



2.67



1.41



2.88



 



0.00



0.00



0.00



 



0.00



0.00



0.00



 



0.55



 



0.00



 



n



8



8



8



 



8



8



8



 



8



8



8



 



8



 



8



 



Mean



6.88



6.50



13.38



 



0.00



0.00



0.00



 



0.00



0.00



0.00



 



1.19



 



100.00



G4, F & 1000



±SD



2.47



2.20



2.39



 



0.00



0.00



0.00



 



0.00



0.00



0.00



 



0.61



 



0.00



 



n



8



8



8



 



8



8



8



 



8



8



8



 



8



 



8



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


 








































































































































































































































































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



 



During LD 8 to 13



 



 


Sex Ratio (m/f) on LD 13



 



Pup Survival Index (%) during


LD 8 to 13



 



Live Pups (No.)



 



Dead Pups (No.)



Cannibalized (No.)



 



Male



Female



Total



 



Male



Female



Total



 



Male



Female



Total



 



Mean



6.40



6.30



12.70



 



0.00



0.00



0.00



 



0.00



0.00



0.00



 



1.13



 



100.00



G1, F & 0



±SD



2.01



2.31



2.54



 



0.00



0.00



0.00



 



0.00



0.00



0.00



 



0.50



 



0.00



 



n



10



10



10



 



10



10



10



 



10



10



10



 



10



 



10



 



Mean



6.67



7.89*



14.56



 



0.00



0.00



0.00



 



0.00



0.00



0.00



 



0.86



 



100.00



G2, F & 100



±SD



1.58



0.78



1.59



 



0.00



0.00



0.00



 



0.00



0.00



0.00



 



0.23



 



0.00



 



n



9



9



9



 



9



9



9



 



9



9



9



 



9



 



9



 



Mean



7.00



6.00



13.00



 



0.00



0.00



0.00



 



0.00



0.00



0.00



 



1.23



 



100.00



G3, F & 300



±SD



2.67



1.41



2.88



 



0.00



0.00



0.00



 



0.00



0.00



0.00



 



0.55



 



0.00



 



n



8



8



8



 



8



8



8



 



8



8



8



 



8



 



8



 



Mean



6.88



6.50



13.38



 



0.00



0.00



0.00



 



0.00



0.00



0.00



 



1.19



 



100.00



G4, F & 1000



±SD



2.47



2.20



2.39



 



0.00



0.00



0.00



 



0.00



0.00



0.00



 



0.61



 



0.00



 



n



8



8



8



 



8



8



8



 



8



8



8



 



8



 



8



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


TABLE 16. SUMMARY RECORD OF REPRODUCTIVE PERFORMANCE














































 


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



 


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



 


Male Mating Index (%)



 



 


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



 


Male Fertility Index (%)



 


G1, M & 0



 


10 (10)



 


100.0



 



 


10 (10)



 


100.0



 


G2, M & 100



 


10 (10)



 


100.0



 



 


9 (10)



 


90.0



 


G3, M & 300



 


8 (10)



 


80.0



 



 


7 (10)



 


70.0



 


G4, M & 1000



 


9 (10)



 


90.0



 



 


7 (10)



 


70.0



 














































































































































 


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



 



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



 



Gestational Length (duration of pregnancy)



 



Pre-coital Interval


(Days)



 



Conceiving Days


(1 to 5)



Conceiving Days


(More than 5 days)



 



Gestation Length (Days)



 



Mean



2.50



n



9



1



 



22.10



G1, F & 0



±SD



1.58



%



90.00



10.00



 



0.32



 



n



10



 



 



 



 



10



 



Mean



4.10



n



8



2



 



22.00



G2, F & 100



±SD



4.18



%



80.00



20.00



 



0.00



 



n



10



 



 



 



 



9



 



Mean



6.60



n



6



4



 



22.13



G3, F & 300



±SD



6.45



%



60.00



40.00



 



0.35



 



n



10



 



 



 



 



8



 



Mean



7.70



n



5



5



 



22.00



G4, F & 1000



±SD



5.72



%



50.00



50.00



 



0.00



 



n



10



 



 



 



 



8



 














































 


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



 


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



 


Female Mating Index (%)



 



 


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



 


Female Fertility Index (%)



 


G1, F & 0



 


10 (10)



 


100.0



 



 


10 (10)



 


100.0



 


G2, F & 100



 


10 (10)



 


100.0



 



 


9 (10)



 


90.0



 


G3, F & 300



 


10 (10)



 


100.0



 



 


8 (10)



 


80.0



 


G4, F & 1000



 


10 (10)



 


100.0



 



 


8 (10)



 


80.0



 


 




































 


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



 


No. of Pregnant Females



 


No. of Females Confirmed with Mating



 


Female Fecundity or Pregnancy Index (%)



 


G1, F & 0



 


10



 


10



 


100.0



 


G2, F & 100



 


9



 


10



 


90.0



 


G3, F & 300



 


8



 


10



 


80.0



 


G4, F & 1000



 


8



 


10



 


80.0



 
























































 


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



 


With live born pups at Parturition



 


No. of females with evidence of pregnancy



 


Gestation Index (%)



 



 


No. of Females Littered



 


No. of females with evidence of pregnancy



 


Parturition Index (%)



 


G1, F & 0



 


10



 


10



 


100.0



 



 


10



 


10



 


100.0



 


G2, F & 100



 


9



 


9



 


100.0



 



 


9



 


9



 


100.0



 


G3, F & 300



 


8



 


8



 


100.0



 



 


8



 


8



 


100.0



 


G4, F & 1000



 


8



 


8



 


100.0



 



 


8



 


8



 


100.0



 





























































































































































 



 



 



Post-implantation Loss (%)



 



Postnatal Loss (%)



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



 



No. of Implantations



No. of Viable Pups



Post-implantation Loss (No.)



Post-implantation Loss (%)



 



Total No. of Deaths/ Cannibalized during Lactation Period



Postnatal Loss (%)



 



Mean



12.90



12.70



0.20



1.46



 



0.00



0.00



G1, F & 0



±SD



2.60



2.54



0.42



3.11



 



0.00



0.00



 



n



10



10



10



10



 



10



10



 



Mean



14.67



14.56



0.11



0.69



 



0.00



0.00



G2, F & 100



±SD



1.66



1.59



0.33



2.08



 



0.00



0.00



 



n



9



9



9



9



 



9



9



 



Mean



13.50



13.00



0.50



4.47



 



0.00



0.00



G3, F & 300



±SD



2.33



2.88



0.76



7.24



 



0.00



0.00



 



n



8



8



8



8



 



8



8



 



Mean



14.00



13.38



0.63



4.84



 



0.00



0.00



G4, F & 1000



±SD



1.93



2.39



0.92



6.84



 



0.00



0.00



 



n



8



8



8



8



 



8



8



 


TABLE 17. SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD








































































































































Group, Sex & Dose


(mg/kg body weight/day)



 



 


Brain



 


Liver



 


Prostate



Seminal vesicles along with coagulating glands



 


Pituitary



 


Thyroid along with parathyroid#



 



Mean



1.9779



11.2452



1.3347



1.2086



0.0171



0.0248



G1, M & 0



±SD



0.0538



1.3496



0.2966



0.2097



0.0029



0.0033



 



n



10



10



10



10



10



10



 



Mean



1.9943



11.0062



1.2057



1.4115



0.0153



0.0240



G2, M & 100



±SD



0.0948



1.6053



0.2019



0.2512



0.0016



0.0014



 



n



10



10



10



10



10



10



 



Mean



1.9389



10.2393



1.2500



1.4262



0.0181



0.0239



G3, M & 300



±SD



0.1289



0.6409



0.2153



0.3706



0.0030



0.0019



 



n



10



10



10



10



10



10



 



Mean



1.9300



10.4014



1.1476



1.2445



0.0172



0.0243



G4, M & 1000



±SD



0.0858



1.1027



0.2399



0.2545



0.0028



0.0024



 



n



10



10



10



10



10



10



#: Weighed post fixation


 








































































































































Group, Sex & Dose


(mg/kg body weight/day)



 



 


Adrenals



 


Thymus



 


Spleen



 


Ovaries



 


Uterus with Cervix



 


Heart



 



Mean



0.0839



0.2994



0.7358



0.1217



0.4470



1.1678



G1, F & 0



±SD



0.0104



0.0434



0.0621



0.0172



0.0576



0.0877



 



n



10



10



10



10



10



10



 



Mean



0.0912



0.3020



0.6435



0.1174



0.4168



1.1799



G2, F & 100



±SD



0.0117



0.0422



0.0688



0.0156



0.0342



0.1567



 



n



9



9



9



9



9



9



 



Mean



0.0739



0.3114



0.7247



0.1257



0.4685



1.1059



G3, F & 300



±SD



0.0300



0.0442



0.1093



0.0163



0.0745



0.0702



 



n



8



8



8



8



8



8



 



Mean



0.0918



0.3004



0.6786



0.1245



0.4342



1.1530



G4, F & 1000



±SD



0.0082



0.0505



0.0841



0.0126



0.0846



0.0814



 



n



8



8



8



8



8



8



 



























































































































Group, Sex & Dose


(mg/kg body weight/day)



 



 


Kidneys



 


Brain



 


Liver



 


Pituitary



 


Thyroid along with parathyroid#



 



Mean



2.3038



2.1147



13.8717



0.0163



0.0210



G1, F & 0



±SD



0.2673



0.0759



1.3365



0.0031



0.0022



 



n



10



10



10



10



10



 



Mean



2.3605



2.0998



13.7297



0.0159



0.0211



G2, F & 100



±SD



0.1887



0.0940



0.9411



0.0020



0.0020



 



n



9



9



9



9



9



 



Mean



2.4206



2.1097



13.6048



0.0176



0.0213



G3, F & 300



±SD



0.4158



0.1186



1.4003



0.0034



0.0031



 



n



8



8



8



8



8



 



Mean



2.3711



2.0293



13.9262



0.0205*



0.0216



G4, F & 1000



±SD



0.2006



0.1219



1.3631



0.0025



0.0015



 



n



8



8



8



8



8



#: Weighed post fixation


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


TABLE 18. SUMMARY OF TERMINAL BODY WEIGHT (g) AND ORGAN WEIGHT RELATIVE TO TERMINAL BODY WEIGHT (%) RECORD





















































































































































Group, Sex & Dose


(mg/kg body weight/day)



 



 


Terminal Body Weight (g)



 


Adrenals



 


Thymus



 


Spleen



 


Testes



 


Epididymis



 


Heart



 



Mean



385.90



0.0157



0.0854



0.2074



0.8932



0.3658



0.3312



G1, M & 0



±SD



34.53



0.0014



0.0205



0.0346



0.1045



0.0561



0.0128



 



n



10



10



10



10



10



10



10



 



Mean



377.23



0.0174



0.0837



0.2080



0.8844



0.3485



0.3207



G2, M & 100



±SD



26.78



0.0016



0.0094



0.0463



0.1006



0.0259



0.0308



 



n



10



10



10



10



10



10



10



 



Mean



379.22



0.0177



0.0845



0.2192



0.9011



0.3454



0.3264



G3, M & 300



±SD



30.95



0.0023



0.0141



0.0352



0.1057



0.0327



0.0306



 



n



10



10



10



10



10



10



10



 



Mean



369.95



0.0172



0.0845



0.2503



0.8868



0.3411



0.3288



G4, M & 1000



±SD



28.61



0.0021



0.0154



0.0436



0.0875



0.0270



0.0189



 



n



10



10



10



10



10



10



10



 


 





















































































































































Group, Sex & Dose


(mg/kg body weight/day)



 



 


Kidneys



 


Brain



 


Liver



 


Prostate



Seminal vesicles along with coagulating glands



 


Pituitary



 


Thyroid along with parathyroid



 



Mean



0.7219



0.5162



2.9229



0.3438



0.3143



0.0045



0.0064



G1, M & 0



±SD



0.0447



0.0476



0.3293



0.0581



0.0533



0.0007



0.0005



 



n



10



10



10



10



10



10



10



 



Mean



0.7592



0.5305



2.9134



0.3199



0.3739



0.0041



0.0064



G2, M & 100



±SD



0.1150



0.0372



0.3438



0.0524



0.0604



0.0005



0.0007



 



n



10



10



10



10



10



10



10



 



Mean



0.6658



0.5130



2.7132



0.3305



0.3728



0.0048



0.0063



G3, M & 300



±SD



0.0820



0.0379



0.2486



0.0557



0.0724



0.0009



0.0007



 



n



10



10



10



10



10



10



10



 



Mean



0.6970



0.5236



2.8121



0.3089



0.3384



0.0046



0.0066



G4, M & 1000



±SD



0.0655



0.0334



0.1923



0.0506



0.0783



0.0007



0.0004



 



n



10



10



10



10



10



10



10



 


 








































































































































Group, Sex & Dose


(mg/kg body weight/day)



 



 


Terminal Body Weight (g)



 


Adrenals



 


Thymus



 


Spleen



 


Ovaries



 


Uterus with Cervix



 



Mean



309.03



0.0273



0.0973



0.2385



0.0395



0.1449



G1, F & 0



±SD



19.19



0.0039



0.0157



0.0200



0.0062



0.0187



 



n



10



10



10



10



10



10



 



Mean



318.77



0.0288



0.0956



0.2024*



0.0371



0.1321



G2, F & 100



±SD



25.00



0.0047



0.0173



0.0208



0.0065



0.0201



 



n



9



9



9



9



9



9



 



Mean



309.33



0.0242



0.1015



0.2357



0.0407



0.1525



G3, F & 300



±SD



24.53



0.0100



0.0183



0.0426



0.0043



0.0275



 



n



8



8



8



8



8



8



 



Mean



319.83



0.0288



0.0944



0.2124



0.0390



0.1372



G4, F & 1000



±SD



21.30



0.0034



0.0177



0.0251



0.0042



0.0341



 



n



8



8



8



8



8



8



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


 








































































































































Group, Sex & Dose


(mg/kg body weight/day)



 



 


Heart



 


Kidneys



 


Brain



 


Liver



 


Pituitary



 


Thyroid along with parathyroid



 



Mean



0.0395



0.1449



0.6862



4.4899



0.0053



0.0068



G1, F & 0



±SD



0.0062



0.0187



0.0404



0.3469



0.0012



0.0006



 



n



10



10



10



10



10



10



 



Mean



0.0371



0.1321



0.6627



4.3272



0.0050



0.0066



G2, F & 100



±SD



0.0065



0.0201



0.0649



0.4107



0.0009



0.0006



 



n



9



9



9



9



9



9



 



Mean



0.0407



0.1525



0.6856



4.4145



0.0057



0.0069



G3, F & 300



±SD



0.0043



0.0275



0.0643



0.4900



0.0009



0.0008



 



n



8



8



8



8



8



8



 



Mean



0.0390



0.1372



0.6378



4.3741



0.0065



0.0068



G4, F & 1000



±SD



0.0042



0.0341



0.0688



0.5684



0.0010



0.0008



 



n



8



8



8



8



8



8



 


TABLE 19. SUMMARY OF GROSS PATHOLOGY FINDINGS RECORD OF ADULT ANIMALS























































































 


 


 


Parameters



Sex



Male



Female



Group



G1



G2



G3



G4



G1



G2



G3



G4



Dose (mg/kg body weight/day)



0



100



300



1000



0



100



300



1000



No. of Animals



10



10



10



10



10



10



10



10



No. of Terminally Sacrificed Animals



10



10



10



10



10



10



10



10



Gross Pathological Findings



External



No Abnormality Detected



10



10



10



10



10



10



10



10



Internal



No Abnormality Detected



10



10



10



10



10



10



10



10



 


TABLE 20. SUMMARY RECORD OF PUP OBSERVATIONS DURING POSTNATAL PERIOD














































































































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



 



At Birth (PND 1)



PND


1 to 4



PND


5 to 7



PND


8 to 13



 



No. of Dams#



10



10



10



10



G1, F & 0



No. of Live Pups



127



127



127



127



 



Pup Observation/ No. of Pups observed



N/127



N/127



N/127



N/127



 



No. of Dams#



9



9



9



9



G2, F & 100



No. of Live Pups



131



131



131



131



 



Pup Observation/ No. of Pups observed



N/131



N/131



N/131



N/131



 



No. of Dams#



8



8



8



8



G3, F & 300



No. of Live Pups



104



104



104



104



 



Pup Observation/ No. of Pups observed



N/104



N/104



N/104



N/104



 



No. of Dams#



8



8



8



8



G4, F & 1000



No. of Live Pups



107



107



107



107



 



Pup Observation/ No. of Pups observed



N/107



N/107



N/107



N/107



#: Dams confirmed with littering


TABLE 21. SUMMARY RECORD OF MEAN PUP WEIGHT (g) PER LITTER




























































































































































































































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



 



PND 1



 



 



PND 4



 



 



PND 7



 



PND 13



 



Mean Pup Weight (g)



Mean Pup Weight (g)



Mean Pup Weight (g)



Mean Pup Weight (g)



 



Male



Female



 



Male



Female



 



Male



Female



 



Male



Female



 



Mean



6.64



6.37



 



10.97



10.49



 



15.09



14.40



 



28.35



27.58



G1, F & 0



±SD



0.19



0.21



 



0.31



0.23



 



0.28



0.31



 



0.31



0.39



 



n



10



10



 



10



10



 



10



10



 



10



10



 



Mean



6.61



6.28



 



10.86



10.39



 



14.96



14.32



 



28.09



27.41



G2, F & 100



±SD



0.22



0.24



 



0.30



0.22



 



0.35



0.20



 



0.49



0.29



 



n



9



9



 



9



9



 



9



9



 



9



9



 



Mean



6.69



6.36



 



11.18



10.65



 



15.24



14.63



 



28.58



27.94



G3, F & 300



±SD



0.18



0.23



 



0.52



0.41



 



0.56



0.42



 



0.83



0.85



 



n



8



8



 



8



8



 



8



8



 



8



8



 



Mean



6.59



6.17



 



10.97



10.42



 



15.04



14.44



 



28.40



27.65



G4, F & 1000



±SD



0.19



0.20



 



0.29



0.18



 



0.31



0.29



 



0.49



0.53



 



n



8



8



 



8



8



 



8



8



 



8



8



 


TABLE 22. SUMMARY OF GROSS PATHOLOGY FINDINGS RECORD - PUPS












































































 


 


Parameters ↓



Group



G1



G2



G3



G4



Dose


(mg/kg body weight/day)



0



100



300



1000



Sex



M



F



M



F



M



F



M



F



No. of Dead Pups at birth



1



-



-



-



-



1



1



2



No Abnormality Detected



1



-



-



-



-



1



1



2



No. of Pups Sacrificed on PND 13



64



63



60



71



56



48



55



52



No Abnormality Detected



64



63



60



71



56



48



55



52



 


 

Conclusions:
In a dose range finding study for a EOGRT study, the oral administration of the test item in Sprague Dawley rats did not produce any indication of systemic, reproduction and developmental toxicity at the dose levels of 100, 300 and 1000 mg/kg bw/d and the same are then considered appropriate for the definitive test.

Executive summary:

To establish doses for a definitive EOGRT study a dose range finding study non GLP was performed using Sprague Dawley Rats. Doses of 100mg/kg, 300mg/kg and 1000mg/kg were chosen in this study based on available toxicological data in the same substance. 10 male and 10 female rats were allocated to each dose group including a concurrent vehicle control group. Carboxy methyl cellulose was chosen as vehicle and the test item was applied via oral (gavage) once daily. The males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 32 days of treatment). The females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 (total of 49 to 66 days of treatment). For systemic toxicity assessment, animals were observed for clinical signs, mortality and morbidity, body weight and feed consumption (including during gestation and lactation periods), clinical pathological examinations (haematology, clinical chemistry and urinalysis) and organ weighing. For reproductive toxicity assessment, males and dams were evaluated for reproductive performances such as, mating index, fertility index, gestation index, parturition index, pre-coital interval, gestation length, post-implantation loss and prenatal loss. At birth and during lactation, parameters such as, number of live/survived/dead pups, litter size, sex ratio and live birth/survival index per litter were observed. For F1 assessment, survived pups were observed once daily for external examinations and twice daily for mortalities till termination (postnatal day 13). All pups were weighed individually on PND 1, 4, 7 and 13. At termination, all the terminally sacrificed adults and pups were subject to detailed gross pathological examination with special emphasis towards reproductive organs. There were no treatment related effects observed for systemic, reproduction or developmental toxicity at the dose levels of 100, 300 and 1000 mg/kg bw/d. Based on these results, it is concluded that the same doses can be selected as low, mid and high doses for the subsequent Extended One-Generation Reproductive Toxicity Study.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
2 key studies (EOGRTS and OECD 422) available with Klimisch score of 1.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

Key Study: Toxicity to reproduction in rats. OECD 414, GLP study. The "no observed adverse effect level" (NOAEL) for reproduction and developmental toxicity is the highest dose used in this study of 1000mg/kg bw /day for maternal animals and pups in rats.


 


Key Study: Toxicity to reproduction in rabbits. OECD 414, GLP study. The "no observed adverse effect level" (NOAEL) for reproduction and developmental toxicity is the highest dose used in this study of 1000mg/kg bw /day for maternal animals and offspring in rabbits.


 


Supporting Study: Dose Range Finding Study in rats, OECD 414, non GLP. The oral administration of test item by gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 did not produce any indication of maternal and developmental toxicity at the dose levels of 100, 300 and 1000 mg/kg body weight/day under experimental conditions employed and the same are then considered appropriate for the definitive test.


 


Supporting Study: Dose Range Finding Study in rabbits, OECD 414, non GLP. The oral administration of test item by gavage to presumed pregnant female White New Zealand Rabbits from Gestation Day 6 to 28 did not produce any indication of maternal and developmental toxicity at the dose levels of 100, 300 and 1000 mg/kg body weight/day under experimental conditions employed and the same are then considered appropriate for the definitive test.


 


 

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 July 2020 to 04 November 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Remarks:
DRF study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: In-house bred animals
- Age at study initiation: Minimum of 10 weeks
- Weight at study initiation: Males: 251.54 g to 289.11 g & Females: 204.23 g to 250.27 g
- Fasting period before study: No
- Housing: Pre-mating/Acclimatization - Maximum of three animals of same sex per cage were housed in sterilized standard polypropylene cage (Size: L 430 × B 285 × H 150 mm). Steam sterilized clean paddy husk was used as bedding material and was changed along with the cage at least twice a week
- Housing: Mating - During mating, three rats (one male and two females) were housed in standard polypropylene cages.
- Housing: 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 in polypropylene cages.
- Diet (e.g. ad libitum): ad libitum - Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG)
- Water (e.g. ad libitum): ad libitum - Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: for a minimum period of five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1ºC to 23.5 ºC
- Humidity (%): relative humidity 50 to 62%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark

IN-LIFE DATES: From: To: 28 July 2020 to 04 November 2020
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required quantity of test item was weighed and triturated well in a mortar with a small quantity of vehicle until a homogenous suspension was formed and thereafter the entire quantity of the formulation was transferred into measuring cylinder. A small quantity of vehicle was added to rinse the mortar and this was transferred into the measuring cylinder. The rinsing procedure of mortar and pestle was repeated many times to ensure the transfer of the contents to the measuring cylinder. Finally, the volume was made up to required quantity with vehicle to get desired concentration of 10, 30 and 100 mg/mL of test item for low, mid and high dose groups respectively.

VEHICLE
- Justification for use and choice of vehicle (if other than water): 0.5% w/v Carboxy Methyl Cellulose, test item was insoluble in distilled water
- Concentration in vehicle: 100 mg/mL (maximum)
- Amount of vehicle (if gavage): 10mL/kg
- Lot/batch no. (if required): BCBN1690V
- Purity: n/a
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Verification of concentration and homogonetiy of the test item was conduced by BIONEEDS INDIA PRIVATE LIMITED DEVARAHOSAHALLY, SOMPURA HOBLI, NELAMANGALA TALUK, BANGALORE RURAL DISTRICT, PIN - 562 111, KARNATAKA, INDIA which hold the Good Laboratory Practice Certificate (GLP). 0.1 mL of sample was pipetted out into a digestion tube, a volume of 6 mL of concentrated nitric acid was added and placed in microwave sample preparation system and digested by the selected method. After digestion, the digestion tubes were cooled to room temperature and the contents were transferred to suitable volumetric flasks and diluted with Milli-Q water.


TECHNIQUE AND TEST METHOD: ICP-MS


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


ACCEPTANCE CRITERIA: Homogeneity and dose formulation analysis of prepared test formulations for the dose concentration verification were performed during first and last week of the treatment and the obtained mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is ≤10%.
Details on mating procedure:
- Impregnation procedure: Cohoused
- If cohoused:
- M/F ratio per cage: 1:2 ratio (one male and two females)
- Length of cohabitation: until evidence of copulation was observed or for two weeks.
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility. yes
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: In-house bred animals
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: no
Duration of treatment / exposure:
Gestation days 5 to 19
Frequency of treatment:
once daily
Duration of test:
Acclimatization: 28 July 2020 to 20 August 2020
Cohabitation: 04 August 2020 to 03 September 2020
Treatment: 10 August 2020 to 23 September 2020
Necropsy: 25 August 2020 to 24 September 2020
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose range finding study, conducted with the doses of 100, 300 and 1000 mg/kg body weight based on the available literature. Oral administration of test item to pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 did not produce any indication of maternal and prenatal-developmental toxicity at all these tested dose levels in the dose range finding study.
- Rationale for animal assignment (if not random): evenly distributed to all the groups based on their body weights so as to maintain comparable mean body weight for all groups
- Fasting period before blood sampling for (rat) dam thyroid hormones: 0
- Time of day for (rat) dam blood sampling: within 2 hours before necropsy.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily for clinical signs of toxicity and twice daily for mortality and morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on gestation days (GD) 0, 3, 5, 8, 11, 14, 17, 19 and on 20 (day of caesarean section).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): not feeding study
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Recorded as g/animal/day during gestation days 0-3, 3-5, 5-8, 8-11, 11-14, 14-17, 17-19 and 19-20
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No applicable

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not drinking water study


POST-MORTEM EXAMINATIONS: Yes / No / No data
- Sacrifice on gestation day 20
- Organs examined: Ovaries, Uterus with cervix, Thyroid and Parathyroid glands
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: observation of uterine content.
Blood sampling:
- Plasma: No
- Serum: Yes
- Volume collected - n/a
- Other: blood samples were collected as follows:
- From all dams at termination for mandatory assessment of thyroid hormones T4, T3 and thyroid stimulating hormone (TSH) within 2 hours before necropsy.
- Blood samples from non-pregnant females were also collected but were not pooled with the pregnant dams.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No
- Anogenital distance of all live rodent pups: Individual fetus was measured for its anogenital distance on the day of caesarean section
Statistics:
Parametric: One-way ANOVA with Dunnett’s post test: Maternal body weight, Percent change in maternal body weight , Corrected body weight for maternal increase, Gravid uterus weight, Maternal feed consumption, Mean fetal weight per dam, Mean fetal crown rump length per dam, Mean fetal anogenital distance per dam, Serum T3, T4 and TSH levels, Organ weight

Non-Parametric: Kruskal-Wallis: No. of corpora lutea per dam, No. of implantations per dam, Litter size per dam, No. of live/dead fetuses per dam, Percent of live/dead fetuses per dam, No. of early/late resorptions per dam, Percent of early/late resorptions per dam, Sex ratio (m/f) per dam, Pre/Post implantation losses (%) per dam, Fetal external / visceral / skeletal anomalies per dam

Frequencies Comparison: Cross Tabs -Chi-square test: No. of Pregnant / Non-pregnant females (Pregnancy status), No. of dams with / without live fetuses, No. of dams with / without dead fetuses, No. of dams with / without resorptions

Indices:
- Corrected Body weight (g) = (Gestation day 20 body weight - Gestation day 5 body weight) -
Gravid uterus weigh
- Percent of Live Fetuses per dam = (Number of Live Fetuses / Litter Size) x 100
- Percent of Dead Fetuses per dam = (Number of Dead Fetuses/ Litter Size) x 100
- Percent of Early Resorptions (%) per dam = (Number of Early Resorptions/Number of Implantation sites) x 100
- Percent of Late Resorptions (%) per dam = (Number of Late Resorptions / Number of Implantation sites)x 100
- Pre-implantation Loss (%) per dam = [(Number of Corpora lutea - Number of Implantation sites)/ Number of Corpora lutea]x 100
- Post-implantation Loss (%) per Dam = [(Number of Implantation sites - Number of Viable fetuses )/ Number of Implantation sites]x 100
- Sex Ratio (m/f) = Number of live male fetuses / Number of live female fetuses
- Male/Female Fetuses (%) = (Number of live male/female fetuses/ Total number of live fetuses)x 100
- Ano-genital Distance Ratio = Ano-genital distance (mm) / Cube root of Fetal weight (g)
- Fetal incidence (%) = (Number of Fetuses with particular observation per group/Total number of Fetuses examined per group)x 100
- Litter incidence (%) = (Number of Litters/dams with particular observation per group/Total number of Litters per group) x 100










Historical control data:
Historical control data not included in report. Historical control data from in-house of the same species and strain.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs of toxicity noted at all the tested dose groups and vehicle control group animals during the experimental period.
Mortality:
no mortality observed
Description (incidence):
There were no morbidity/mortality noted at all the tested dose groups and vehicle control group animals during the experimental period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no changes noted in mean gestation (maternal) body weight and percent change in mean gestation (maternal) body weight gain at all the tested dose groups 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 gestation (maternal) feed consumption at all the tested dose groups when compared with the vehicle control group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid hormone T3: There were no changes noted for this parameter across the dose groups when compared to the vehicle control group. The statistically significant increase in serum T3 levels (ng/mL) at groups G3 and G4 is considered as incidental and un-related to treatment with test item due to lack of a dose-response relationship and also the obtained values were within the historical control range of same species and strain.

Thyroid hormone T4: There were no changes noted for this parameter across the dose groups when compared to the vehicle control group.

Thyroid stimulating hormone THS: There were no statistically significant differences noted for this parameter across the dose groups when compared to the vehicle control group due to noted larger standard deviations from groups G3 and G4. However, the noted differences in mean value from groups G3 and G4 when compared with vehicle control group are considered as incidental and also the obtained mean values from all the tested dose groups are within in-house historical control range.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The thyroid along with parathyroid was collected, preserved and weighed post fixation from all the dams of each dose group. There were no changes and no significant differences noted for mean absolute and relative weight for this organ in all the tested dose groups when compared to the vehicle control group.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross pathological changes noted in any of the animals from all the tested dose groups and vehicle control group during conduct of the necropsy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related changes noted in thyroid along with the parathyroid subjected to histopathological examination at all the tested dose group animals. The noted ultimobranchial cyst/s in thyroid gland is considered as incidental finding as they occurred randomly across the dose groups including concurrent controls and/or were expected from laboratory rats of this age.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no statistically significant differences noted for percentage of pre- or post-implantation loss per litter at all the tested dose groups when compared to the vehicle control group.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in the number and percentage of early or late resorptions per dam across dose groups when compared to the vehicle control group.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no statistically significant differences noted for number of dead fetuses at all the tested dose groups when compared to the vehicle control group.
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
pregnancy with rates of 88%, 92%, 88% and 92% in groups G1, G2, G3 and G4 respectively
Other effects:
no effects observed
Description (incidence and severity):
The mean number of corpora lutea per litter was 13.00, 13.22, 13.50 and 13.48 for groups G1, G2, G3 and G4 respectively. There were no changes or no statistically significant differences noted at all the tested dose groups when compared with vehicle control group for number of corpora lutea.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: Highest concentration tested, no adverse effects observed
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no changes noted in mean fetal weight of either sex across the dose groups when compared with the vehicle control group.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There were no statistically significant differences noted for number of live fetuses at all the tested dose groups when compared to the vehicle control group.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no statistically significant differences noted for male/female sex ratio across the dose groups when compared to the control group.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The mean litter sizes, assessed as the total number of fetuses in utero (live plus dead) per dam, was 11.64, 11.83, 11.86 and 12.13 for groups G1, G2, G3 and G4, respectively. There were no changes or no statistically significant differences noted at all the tested dose groups when compared with vehicle control group for litter size.
Anogenital distance of all rodent fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
The mean male fetal anogenital distance per litter was 4.25 mm, 4.30 mm, 4.20 mm and 4.28 mm and the mean female anogenital distance per litter was 2.47 mm, 2.51 mm, 2.57 mm and 2.59 mm for groups G1, G2, G3 and G4 respectively. The mean male fetal anogenital distance ratio per litter was 2.61, 2.67, 2.60 and 2.65 and the mean female anogenital distance ratio per litter was 1.55, 1.58, 1.62 and 1.64 for groups G1, G2, G3 and G4 respectively.

Statistically significant increase in mean female fetal anogenital distance and mean fetal anogenital distance ratio per litter was noted at groups G3 and G4 when compared with the vehicle control group. These changes are considered as incidental and un-related to treatment as the obtained range is within in-house historical control range.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The
noted general / developmental variations such as subcutaneous hemorrhagic spot/s beneath the skin on different regions of the body, pale skin colored fetuses, kinked tail and noted external malformations such as unilateral supernumerary digit of hindlimb, unilateral fused digits of hindlimb paw unilateral, hyperextension of forelimb unilateral, misshappened/flat head, short forelimb unilateral across the tested dose group litters are considered incidental as these incidences are comparable with the vehicle control group and also these developmental alterations are common for this species and strain. Also, no remarkable differences or statistically significant changes were noted for these alterations in any of the tested dose groups when compared with vehicle control group.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The below mentioned occasional incidences of skeletal developmental variations were
noted across the tested dose group litters.
- incomplete ossification of skull bones (parietal/interparietal/supraoccipital/ex-occipital/ mandibular/zygoma);
- incomplete or bipartite or unossification of sternabral bones (sternabra 2, 3, 4, 5 and 6);
- wavy ribs (both unilateral and bilateral);
- extra ossification site/s near to first lumbar vertebra i.e. immediately after 13th rib (both unilateral and bilateral);
- dumbbell or semi-bipartite ossification of thoracic verterbral centrum (centrum no. 8, 9, 10, 11, 12 and 13) and lumbar vertebral centrum (centrum no. 2);
- hemicentric ossification of thoracic verterbral centrum (centrum no. 11 and 12);
- incomplete or unossification of metacarpal no. 5 or proximal phalanges of forelimb;
- incomplete ossification of pubis bone unilateral.

The below mentioned occasional incidences of skeletal malformation were noted across
the tested dose group litters.
- fused skull bones (parietal) - single incidence for a fetus from vehicle control group;
- missing rib unilateral (rib no. 12) - single incidence for a fetus from vehicle control group;
- fused rib no. 11 and 12 - single incidence for a fetus from group G3;
- fused rib no. 12 and 13 - single incidence for a fetus from group G1;
- rudimentary rib (14th rib) - single incidences for a fetus from group G2, G3 and G4 each;
- split centrum no. 10 of thoracic vertebra - 1, 2, 0 and 1 fetal and litter incidences from group G1, G2, G3 and G4 respectively.
- split centrum no. 11 of thoracic vertebra - 0, 2, 2 and 3 fetal and litter incidences from group G1, G2, G3 and G4 respectively.
- split centrum no. 12 of thoracic vertebra - 1, 1, 1 and 2 fetal and litter incidences from group G1, G2, G3 and G4 respectively.
- split centrum no. 13 of thoracic vertebra - 0, 0, 1 and 0 fetal and litter incidences from group G1, G2, G3 and G4 respectively.
These skeletal developmental variations and skeletal malformations are considered as incidental and un-related to treatment as these findings occurred infrequently or at a frequency similar to the vehicle control group and did not occur in a dose-dependant manner. Also, the occurred mean litter/fetal proportions were within the in-house historical control range of this species and strain.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related fetal visceral/soft tissue malformations or developmental variations for any of the fetuses examined from all the tested dose group litters. The noted developmental variations such as pale or discolored adrenals/liver lobes/lung lobes, fused liver lobes, dilatation of renal pelvis and dilatation of ureters across the tested dose group litters are considered incidental as these incidences are comparable with the vehicle control group and also these developmental variations are common for this species and strain. There were no remarkable differences or statistically significant changes noted for these variations in any of the tested dose groups when compared with vehicle control group.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
The mean male fetal crown rump length per litter was 37.28 mm, 37.40 mm, 37.56 mm and 37.67 mm and the female mean crown rump length per litter was 36.61 mm, 36.96 mm, 36.79 mm and 36.92 mm for groups G1, G2, G3 and G4 respectively. Statistically significant increase in mean male fetal crown rump length per litter was noted at group G4 when compared with the vehicle control group. This change is considered as incidental and un-related to treatment as the obtained range is within in-house historical control range.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Highest dose tested, no adverse effects observed
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
TABLE 1. SUMMARY OF PREGNANCY STATUS, CLINICAL SIGNS OF TOXICITY AND MORTALITY RECORD                                                                                                                     
















































Group
& Dose
(mg/kg body
weight/day)
Pregnancy StatusClinical Signs and Mortality Record
No. of Presumed
Pregnant Animals / Group
No. of Presumed
Pregnant Animals / Group
Rate of Pregnancy (%)Clinical Signs of
Toxicity revealed
(No. of Animals)
No. of Mortalities /
Total No. of animals
G1 & 0252288.0N (25)0/25
G2 & 100252392.0N (25)0/25
G3 & 300252288.0N (25)0/25
G4 & 1000252392.0N (25)0/25

TABLE 2. SUMMARY OF GESTATION BODY WEIGHT (g) RECORD










































































































































































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



 



Body Weight (g) on Gestation Day (GD)



0



3



5



8



11



14



17



19



20



G1  & 0



Mean ±SD



251.79



258.46



265.3



274.34



285.93



300.86



323.93



343.93



358.5



13.58



12.74



12.08



12.31



15.43



15.91



19.21



24.16



28.5



 



n



22



22



22



22



22



22



22



22



22



G2 & 100



Mean ±SD



257.16



264.34



271.41



281.48



292.19



305.77



328.69



350.58



366.59



12.26



10.56



10.52



12.39



13.49



14.67



17.94



21.18



27.52



 



n



23



23



23



23



23



23



23



23



23



G3 & 300



Mean ±SD



252.48



259.66



266.37



275.52



287.86



301.9



326.49



349.47



365.92



16.2



16.12



16.59



16.32



16.93



19.56



21.46



24.03



27.12



 



n



22



22



22



22



22



22



22



22



22



G4 & 1000



Mean ±SD



257.53



265.91



273.18



283.86



295.36



309.37



336.11



359.99



372.98



17.79



16.62



15.73



15.79



15.18



16.49



19.04



24.34



25.84



 



n



23



23



23



23



23



23



23



23



23



Table 3 SUMMARY OF PERCENT CHANGE IN GESTATION BODY WEIGHT (%) RECORD
































































































































































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



 



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



0 to 3



3 to 5



5 to 8



8 to 11



11 to 14



14 to 17



17 to 19



19 to 20



G1 & 0



Mean ±SD



2.68



2.67



3.42



4.2



5.24



7.7



6.14



4.19



1.05



1.03



1.34



1.77



1.37



4.19



2.74



2.26



 



n



22



22



22



22



22



22



22



22



G2 & 100



Mean ±SD



2.83



2.68



3.7



3.8



4.65



7.5



6.65



4.49



1.38



1.01



1.19



0.77



1.46



3.16



2.19



1.96



 



n



23



23



23



23



23



23



23



23



G3 & 300



Mean ±SD



2.86



2.59



3.46



4.49



4.85



8.18



7.04



4.68



1.1



0.78



1.03



1.45



1.32



2.85



2.33



1.62



 



n



22



22



22



22



22



22



22



22



G4 & 1000



Mean ±SD



3.31



2.77



3.93



4.08



4.75



8.66



7.08



3.61



1.58



1.25



1.39



1.19



1.45



2.92



2.95



1.85



 



n



23



23



23



23



23



23



23



23



SD: Standard Deviation; n: Number of Dams.



TABLE 4. SUMMARY OF GRAVID UTERUS WEIGHT (g) AND MATERNAL BODY WEIGHT CHANGE CORRECTED FOR GRAVID UTERINE WEIGHT (g)





















































































































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



 



Body Weight Change (g) from GD 5 to 20



Gravid Uterus Weight (g)



 Corrected Body Weight


 

g



%



G1 & 0



Mean ±SD



93.21



70.87



22.34



8.43



21.52



19.66



10.17



3.81



 



n



22



22



22



22



G2 & 100



Mean ±SD



95.21



71.6



23.61



8.67



21.61



20.26



9.42



3.37



 



n



23



23



23



23



G3 & 300



Mean ±SD



99.55



74



25.55



9.62



18.69



15.96



11.52



4.35



 



n



22



22



22



22



G4 & 1000



Mean ±SD



99.8



73.06



26.74



9.89



23.15



21.85



10.02



3.9



 



n



23



23



23



23


      

SD: Standard Deviation; n: Number of Dams; GD: Gestation Day. *: Statistically significant (P<0.05) change than the control group.



TABLE 5. SUMMARY OF AVERAGE GESTATION FEED CONSUMPTION (g/animal/day) RECORD





























































































































































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



 



Feed Consumption (g/animal/day) during Gestation Day (GD)



0 to 3



3 to 5



5 to 8



8 to 11



11 to 14



14 to 17



17 to 19



19 to 20



G1 & 0



Mean ±SD



14.98



18.97



15.96



19.76



22.45



24.77



26.64



28.78



1.31



1.13



1.05



1.57



1.52



1.69



2.22



2.01



 



n



22



22



22



22



22



22



22



22



G2 & 100



Mean ±SD



14.91



19.19



16.26



19.53



22.39



24.6



26.8



29.19



1.23



1.17



0.97



1.89



1.41



2.27



2.45



2.38



 



n



23



23



23



23



23



23



23



23



G3 & 300



Mean ±SD



14.71



19.52



16.83



19.62



22.28



24.85



25.79



28.62



1.59



1.76



1.27



1.55



1.23



1.33



2.65



2.37



 



n



22



22



22



22



22



22



22



22



G4 & 1000



Mean ±SD



15.51



19.75



16.94



19.9



22.88



25.5



26.02



28.06



1.69



1.62



2.17



1.54



0.89



0.89



2.17



2.63



 



n



23



23



23



23



23



23



23



23



 


TABLE 6. SUMMARY OF UTERI OBSERVATIONS PER LITTER RECORD











































































































































































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


 

No. of Corpora lutea



No. of Implantations



Litter size



No. of Live Fetuses



No. of Dead Fetuses



No. of Early Resorptions



No. of Late Resorptions


 

Total



Male



Female



G1 & 0



Mean ±SD



13



12.27



11.64



11.64



5.5



6.14



0



0.64



0



2.94



2.88



3.39



3.39



2.3



2.59



0



1.05



0



 



n



22



22



22



22



22



22



22



22



22



G2 & 100



Mean ±SD



13.22



12.57



11.83



11.83



6.57



5.26



0



0.74



0



3.48



3.76



4.05



4.05



2.89



2.68



0



1.05



0



 



n



23



23



23



23



23



23



23



23



23



G3 & 300



Mean ±SD



13.5



12.73



11.86



11.77



5.59



6.18



0.09



0.86



0



2.86



2.6



2.95



2.91



1.76



2.17



0.43



1.25



0



 



n



22



22



22



22



22



22



22



22



22



G4 & 1000



Mean ±SD



13.48



12.96



12.13



12.09



6.3



5.78



0.04



0.78



0.04



3.57



3.36



3.81



3.8



2.55



2.5



0.21



0.9



0.21



 



n



23



23



23



23



23



23



23



23



23



TABLE 7. SUMMARY OF MATERNAL DATA PER LITTER RECORD


















































































































































































































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



 



Pre-Implantation Loss (%)



Post-Implantation Loss (%)



Percent of Dead Fetus (%)



Percent of Early Resorptions (%)



Percent of Late Resorptions (%)



Male/Female Sex Ratio



Male  Fetuses (%)



Female Fetuses (%)



Percent of Live Fetuses



 


 
  
  
  

G1 & 0



Mean ±SD



5.57



6.23



0



6.23



0



1.06



47.55



52.45



100


  

6.11



10.88



0



10.88



0



0.61



14.03



14.03



0


  

 



n



22



22



22



22



22



22



22



22



22


  

G2 & 100



Mean ±SD



6.13



7.32



0



7.32



0



1.69



56.19



43.81



100


  

8.5



11.88



0



11.88



0



1.28



15.94



15.94



0


  

 



n



23



23



23



23



23



23



23



23



23


  

G3 & 300



Mean ±SD



5.51



8.04



0.65



7.4



0



1.02



48



52



99.35


  

4.82



11.95



3.05



11.98



0



0.46



11.41



11.41



3.05


  

 



n



22



22



22



22



22



22



22



22



22


  

G4 & 1000



Mean ±SD



3.71



8.28



0.33



7.61



0.33



1.14



53.02



46.98



99.67


  

5.74



12.48



1.6



12.5



1.6



0.65



16.95



16.95



1.6


  

 



n



23



23



23



23



23



23



23



23



23


  

SD: Standard Deviation; n: Number of Dams.


  

TABLE 8. SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

































































Group & Dose (mg/kg body weight/day)  Absolute Thyroid along with Parathyroid Weight (g)# 
G1 & 0 Mean ±SD 0.0183
0.0027
 22
G2 & 100 Mean ±SD 0.0177
0.0025
 23
G3 & 300 Mean ±SD 0.0185
0.0025
 22
G4 & 1000 Mean ±SD 0.019
0.0019
 23
SD: Standard Deviation; n: Number of Dams; #: Weighed post fixation. 

TABLE 9. SUMMARY OF TERMINAL BODY WEIGHT (g) AND ORGAN
WEIGHT (%) RELATIVE TO TERMINAL BODY WEIGHT RECORD














































































Group & Dose (mg/kg body weight/day)  Terminal Body Weight (g) Relative Thyroid along with Parathyroid weight (%) 
G1 & 0 Mean ±SD 358.50.0051
28.50.0008
 2222
G2 & 100 Mean ±SD 366.590.0049
27.520.0007
 2323
G3 & 300 Mean ±SD 365.920.0051
27.120.0006
 2222
G4 & 1000 Mean ±SD 372.980.0051
25.840.0006
 2323
SD: Standard Deviation; n: Number of Dams. 

TABLE 10. SUMMARY OF SERUM TRIIODOTHYRONINE (T3) LEVELS
(ng/mL) RECORD


































































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



 



Serum T3 Levels (ng/mL)



G1 & 0



Mean ±SD



2.587



0.249



 



n



22



G2 & 100



Mean ±SD



2.659



0.273



 



n



23



G3 & 300



Mean ±SD



3.082*



0.225



 



n



22



G4 & 1000



Mean ±SD



2.849*



0.535



 



n



23



SD: Standard Deviation;   n: Number of Dams considered for mean calculations.



TABLE 11. SUMMARY OF SERUM THYROXINE (T4) LEVELS (ng/mL)
RECORD































































Group & Dose  (mg/kg body weight/day)



 



Serum T4 Levels (ng/mL)



G1 & 0



Mean ±SD



72.608



16.419



 



n



22



G2 & 100



Mean ±SD



71.998



13.244



 



n



23



G3 & 300



Mean ±SD



67.222



15.074



 



n



22



G4 & 1000



Mean ±SD



62.908



11.753



 



n



23



TABLE 12. SUMMARY OF SERUM THYROID STIMULATING HORMONE
(TSH) LEVELS (µIU/mL) RECORD































































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



 



Serum TSH Levels (µIU/mL)



G1 & 0



Mean ±SD



3.853



4.033



 



n



20



G2 & 100



Mean ±SD



3.441



3.896



 



n



20



G3 & 300



Mean ±SD



7.357



6.235



 



n



21



G4 & 1000



Mean ±SD



6.445



5.991



 



n



21



TABLE 13. SUMMARY OF MEAN FETAL WEIGHT, MEAN FETAL CROWN RUMP LENGTH, MEAN FETAL ANOGENITAL DISTANCE AND RATIO RECORD


























































































































































































































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


 

Fetal Weight (g)



Crown Rump Length (mm)



Anogenital Distance
Measurement (mm)



Anogenital Distance Ratio



 


 
 

Male



Female



Male



Female



Male



Female



Male



Female



 


 
  
  

G1 & 0



Mean ±SD



4.24



4.06



37.28



36.61



4.25



2.47



2.61



1.55


  

0.33



0.31



0.57



0.67



0.18



0.12



0.18



0.07


  
 

n



22



22



22



22



22



22



22



22


  
  
  

G2 & 100



Mean ±SD



4.2



4.01



37.4



36.96



4.3



2.51



2.67



1.58


  

0.38



0.4



0.56



0.59



0.23



0.1



0.14



0.08


  
 

n



23



23



23



23



23



23



23



23


  
  
  

G3 & 300



Mean ±SD



4.25



4.02



37.56



36.79



4.2



2.57*



2.6



1.62*


  

0.33



0.21



0.5



0.65



0.27



0.12



0.18



0.07


  
 

n



22



22



22



22



22



22



22



22


  
  
  

G4 & 1000



Mean ±SD



4.22 0.30



3.93 0.28



37.67*



36.92



4.28



2.59*



2.65 0.17



1.64*


  

0.49



0.6



0.24



0.1



0.07


  

 



n



23



22



23



22



23



22



23



22


  

Table 14 SUMMARY RECORD OF FETAL EXTERNAL EXAMINATION PER LITTER











































































































































































































































































































































Group



G1



G2



G3



G4



Dose (mg/kg body weight/dry)



0



100



300



1000


            

Litters Evaluated for External Examination



No



22



23



22



23



Fetuses Evaluated for External Examination



No



256



272



259



278



Variations



Region/Tissue



Alteration


         

General



Subcutaneous hemorrhagic spot/s beneach the skin on different regions of the body


 

Litter Incidences



No (%)



7



(31.8)



10



(43.5)



6



(27.3)



8



(34.8)


 

Fetal Incidences



No (%)



9



(3.5)



14



(5.1)



8



(3.1)



10



(3.6)



General



Pale colored skin


 

Litter Incidences



No (%)



2



(9.1)



1



(4.3)



1



(4.5)



3



(13.0)


 

Fetal Incidences



No (%)



2



(0.8)



1



(0.4)



1



(0.4)



3



(1.1)



Tail



Kinked


 

Litter Incidences



No (%)



0



(0.0)



0



(0.0)



0



(0.0)



1



(4.3)


 

Fetal Incidences



No (%)



0



(0.0)



0



(0.0)



0



(0.0)



1



(0.4)



Malformations



Region/Tissue



Alteration


         

Hindlimb



Supernumerary digit (unilateral)


 

Litter Incidences



No (%)



0



(0.0)



1



(4.3)



0



(0.0)



0



(0.0)


 

Fetal Incidences



No (%)



0



(0.0)



1



(0.4)



0



(0.0)



0



(0.0)



Hindlimb



Fused digits (unilateral)


 

Litter Incidences



No (%)



0



(0.0)



1



(4.3)



0



(0.0)



1



(4.3)


 

Fetal Incidences



No (%)



0



(0.0)



1



(0.4)



0



(0.0)



1



(0.4)



Forelimb



Hyperextension (unilateral)


 

Litter Incidences



No (%)



0



(0.0)



0



(0.0)



1



(4.5)



0



(0.0)


 

Fetal Incidences



No (%)



0



(0.0)



0



(0.0)



2



(0.8)



0



(0.0)



Head



Misshappen (flat shaped)


 

Litter Incidences



No (%)



0



(0.0)



0



(0.0)



0



(0.0)



1



(4.3)


 

Fetal Incidences



No (%)



0



(0.0)



0



(0.0)



0



(0.0)



1



(0.4)



Forelimb



Short (unilateral)


         
 

Litter Incidences



No (%)



0



(0.0)



0



(0.0)



0



(0.0)



1



(4.3)


 

Fetal Incidences



No (%)



0



(0.0)



0



(0.0)



0



(0.0)



1



(0.4)



TABLE 15. SUMMARY OF FETAL VISCERAL EXAMINATION PER LITTER
RECORD
































































































































































































































































Group



G1



G2



G3



G4



Dose (mg/kg body weight/dry)



0



100



300



1000


            

Litters Evaluated for External Examination



No



22



23



22



23



Fetuses Evaluated for External Examination



No



123



129



124



133



Variations



Region/Tissue



Alteration


         

Adrenals



Pale/Discoloration


         
 

Litter Incidences



No (%)



1



4.5



0



0


 

0



0



0


 

Fetal Incidences



No (%)



1



0.8



0



0


 

0



0



0



Kidneys



Dilatation of renal pelvis
(bilateral)


 

Litter Incidences



No (%)



4



18.2



2



8.7



0



13.6



3



13


 

Fetal Incidences



No (%)



6



4.8



4



3.1



0



4



5



3.8



Liver



Pale/Discoloration


 

Litter Incidences



No (%)



4



18.2



3



13



3



4.5



2



8.7


 

Fetal Incidences



No (%)



5



4



3



2.3



5



0.8



3



2.3



Liver



Fused Lobes


 
 

Litter Incidences



No (%)



1



4.5



0



0



1



0



0



0


 

Fetal Incidences



No (%)



1



0.8



0



0



1



0



0



0



Lungs



Pale/Discoloration


 

Litter Incidences



No (%)



0



0



1



4.3



0



0



1



4.3


 

Fetal Incidences



No (%)



0



0



1



0.8



0



0



2



1.5



Ureters



Dilatation


 

Litter Incidences



No (%)



2



9.1



4



17.4



4



18.2



3



13


 

Fetal Incidences



No (%)



3



2.4



5



3.9



4



3.2



5



3.8



TABLE 16. SUMMARY OF SKELETAL EXAMINATION OF FETUSES PER LITTER RECORD









































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































Group



G1



G2



G3



G4



Dose (mg/kg body weight/dry)



0



100



300



1000


            

Litters Evaluated for External Examination



No



22



23



22



23



Fetuses Evaluated for External Examination



No



132



143



136



146



Variations



Region/Tissue



Alteration


         

Skull



Parietal bones  Incomplete ossification


       

Litter Incidences



No (%)



2



(9.1)



1



(4.3)



1



(4.5)



0



(0.0)



Fetal Incidences



No (%)



2



(1.5)



1



(0.7)



1



(0.7)



0



(0.0)



Interparietal bones  Incomplete ossification
(bilateral)


  

 



 



 


 

 



 



 



 



Litter Incidences



No (%)



1



(4.5)



4



(17.4)



2



(9.1)



1



(4.3)



Fetal Incidences



No (%)



1



(0.8)



5



(3.5)



2



(1.5)



1



(0.7)



Supraoccipital bones  Incomplete ossification


          

Litter Incidences



No (%)



0



(0.0)



1



(4.3)



2



(9.1)



2



(8.7)



Fetal Incidences



No (%)



0



(0.0)



1



(0.7)



2



(1.5)



2



(1.4)



Exoccipital bones  Incomplete ossification


      

Litter Incidences



No (%)



0



(0.0)



2



(8.7)



1



(4.5)



1



(4.3)



Fetal Incidences



No (%)



0



(0.0)



2



(1.4)



1



(0.7)



1



(0.7)



Mandibular bones  Incomplete ossification


      

Litter Incidences



No (%)



0



(0.0)



0



(0.0)



0



(0.0)



1



(4.3)



Fetal Incidences



No (%)



0



(0.0)



0



(0.0)



0



(0.0)



1



(0.7)



Zygomatic Arch  Incomplete ossification


       

Litter Incidences



No (%)



0



(0.0)



1



(4.3)



0



(0.0)



2



(8.7)



Fetal Incidences



No (%)



0



(0.0)



1



(0.7)



0



(0.0)



2



(1.4)



Sternum



Sternebra No. 3  Bipartite ossification


       

Litter Incidences



No (%)



0



(0.0)



1



(4.3)



0



(0.0)



0



(0.0)



Fetal Incidences



No (%)



0



(0.0)



1



(0.7)



0



(0.0)



0



(0.0)



Sternebra No. 4  Bipartite ossification


       

Litter Incidences



No (%)



0



(0.0)



1



(4.3)



0



(0.0)



1



(4.3)



Fetal Incidences



No (%)



0



(0.0)



1



(0.7)



0



(0.0)



1



(0.7)



Sternebra No. 5  Bipartite ossification


       

Litter Incidences



No (%)



1



(4.5)



1



(4.3)



2



(9.1)



1



(4.3)



Fetal Incidences



No (%)



1



(0.8)



1



(0.7)



2



(1.5)



1



(0.7)



Sternebra No. 6  Bipartite ossification



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



1



(4.3)



1



(4.5)



0



(0.0)



Fetal Incidences



No (%)



0



(0.0)



1



(0.7)



1



(0.7)



0



(0.0)



Sternebra No. 2  Bipartite ossification



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



0



(0.0)



0



(0.0)



1



(4.3)



Fetal Incidences



No (%)



0



(0.0)



0



(0.0)



0



(0.0)



1



(0.7)



Sternebra No. 2  Incomplete ossification


       

Litter Incidences



No (%)



0



(0.0)



1



(4.3)



0



(0.0)



0



(0.0)



Fetal Incidences



No (%)



0



(0.0)



1



(0.7)



0



(0.0)



0



(0.0)



Sternebra No. 5  Incomplete ossification



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



10



(45.5)



12



(52.2)



8



(36.4)



4



(17.4)



Fetal Incidences



No (%)



14



(10.6)



15



(10.5)



11



(8.1)



5



(3.4)



Sternebra No. 6  Incomplete ossification



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



8



(36.4)



15



(65.2)



12



(54.5)



14



(60.9)



Fetal Incidences



No (%)



14



(10.6)



25



(17.5)



18



(13.2)



22



(15.1)



Sternebra No. 5  Unossified



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



2



(9.1)



6



(26.1)



6



(27.3)



6



(26.1)



Fetal Incidences



No (%)



2



(1.5)



8



(5.6)



7



(5.1)



6



(4.1)



Sternebra No. 6  Unossified



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



2



(9.1)



5



(21.7)



4



(18.2)



5



(21.7)



Fetal Incidences



No (%)



2



(1.5)



10



(7.0)



4



(2.9)



5



(3.4)



Ribs



Rib No. 9 Wavy (Unilateral)



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



0



(0.0)



1



(4.5)



0



(0.0)



Fetal Incidences



No (%)



0



(0.0)



0



(0.0)



1



(0.7)



0



(0.0)



Rib No. 10 Wavy (Unilateral)



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



0



(0.0)



1



(4.5)



0



(0.0)



Fetal Incidences



No (%)



0



(0.0)



0



(0.0)



1



(0.7)



0



(0.0)



Rib No. 5 Wavy (bilateral)



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



0



(0.0)



1



(4.5)



0



(0.0)



Fetal Incidences



No (%)



0



(0.0)



0



(0.0)



1



(0.7)



0



(0.0)



Rib No. 6 Wavy (bilateral)



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



0



(0.0)



1



(4.5)



0



(0.0)



Fetal Incidences



No (%)



0



(0.0)



0



(0.0)



1



(0.7)



0



(0.0)



Rib No. 7 Wavy (bilateral)



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



0



(0.0)



1



(4.5)



0



(0.0)



Fetal Incidences



No (%)



0



(0.0)



0



(0.0)



1



(0.7)



0



(0.0)



Rib No. 8 Wavy (bilateral)



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



0



(0.0)



1



(4.5)



0



(0.0)



Fetal Incidences



No (%)



0



(0.0)



0



(0.0)



1



(0.7)



0



(0.0)



Rib No. 9 Wavy (bilateral)


        

Litter Incidences



No (%)



0



(0.0)



1



(4.3)



1



(4.5)



0



(0.0)



Fetal Incidences



No (%)



0



(0.0)



1



(0.7)



1



(0.7)



0



(0.0)



Rib No. 10 Wavy (bilateral)



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



1



(4.3)



1



(4.5)



0



(0.0)



Fetal Incidences



No (%)



0



(0.0)



1



(0.7)



1



(0.7)



0



(0.0)



Rib No. 11 Wavy (bilateral)



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



1



(4.3)



1



(4.5)



0



(0.0)



Fetal Incidences



No (%)



0



(0.0)



1



(0.7)



1



(0.7)



0



(0.0)



Rib No. 12 Wavy (bilateral)



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



1



(4.3)



1



(4.5)



0



(0.0)



Fetal Incidences



No (%)



0



(0.0)



1



(0.7)



1



(0.7)



0



(0.0)



Rib No. 13 Wavy (bilateral)



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



1



(4.5)



2



(8.7)



3



(13.6)



0



(0.0)



Fetal Incidences



No (%)



1



(0.8)



3



(2.1)



3



(2.2)



0



(0.0)



Ossification Site at First Lumbar Vertebra (Unilateral)



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



2



(8.7)



3



(13.6)



4



(17.4)



4



(18.2)



Fetal Incidences



No (%)



2



(1.5)



4



(2.8)



5



(3.7)



5



(3.4)



Ossification Site at First Lumbar Vertebra (Bilateral)



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



2



(9.1)



2



(8.7)



2



(9.1)



Fetal Incidences



No (%)



0



(0.0)



2



(1.4)



3



(2.2)



3



(2.1)



Thoracic Vertebra



Centrum No. 8  Dumbbellshaped



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



1



(4.3)



0



(0.0)



0



(0.0)



0



(0.0)



Fetal Incidences



No (%)



1



(0.8)



0



(0.0)



0



(0.0)



0



(0.0)



Centrum No. 9  Dumbbellshaped



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



2



(9.1)



3



(13.0)



3



(13.6)



Fetal Incidences



No (%)



0



(0.0)



2



(1.4)



3



(2.2)



3



(2.1)



Centrum No. 10  Dumbbellshaped



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



6



(26.1)



7



(31.8)



9



(39.1)



9



(40.9)



Fetal Incidences



No (%)



6



(4.5)



11



(7.7)



9



(6.6)



9



(6.2)



Centrum No. 11  Dumbbellshaped



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



7



(30.4)



2



(9.1)



8



(34.8)



8



(36.4)



Fetal Incidences



No (%)



10



(7.6)



3



(2.1)



10



(7.4)



12



(8.2)



Centrum No. 12  Dumbbellshaped



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



7



(30.4)



8



(36.4)



9



(39.1)



10



(45.5)



Fetal Incidences



No (%)



10



(7.6)



10



(7.0)



9



(6.6)



14



(9.6)



Centrum No. 13  Dumbbellshaped



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



4



(17.4)



4



(18.2)



2



(8.7)



6



(27.3)



Fetal Incidences



No (%)



4



(3.0)



4



(2.8)



2



(1.5)



8



(5.5)



Centrum No. 11  Hemicentric



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.00



0



(0.0)



1



(4.3)



0



(0.0)



Fetal Incidences



No (%)



0



(0.0)



0



(0.0)



1



(0.7)



0



(0.0)



Centrum No. 12  Hemicentric



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



0



(0.0)



1



(4.3)



0



(0.0)



Fetal Incidences



No (%)



0



(0.0)



0



(0.0)



1



(0.7)



0



(0.0)



Sacral Vertebra



Arch No. 1  Ossification Site



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



0



(0.0)



0



(0.0)



1



(4.5)



Fetal Incidences



No (%)



0



(0.0)



0



(0.0)



0



(0.0)



1



(0.7)



Lumbar Vertebra



Centrum No. 2  Dumbbellshaped



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.00



1



(4.5)



0



(0.0)



0



(0.0)



Fetal Incidences



No (%)



0



(0.0)



1



(0.7)



0



(0.0)



0



(0.0)



Forelimb



Metacarpal No. 5  Unossified (Bilateral)



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



3



(13.6)



2



(8.70



3



(13.6)



Fetal Incidences



No (%)



0



(0.0)



4



(2.8)



2



(1.5)



3



(2.1)



Metacarpal No. 5  Incomplete
ossification (Bilateral) Litter



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



1



(4.3)



2



(9.1)



0



(0.0)



4



(18.2)



Fetal Incidences



No (%)



1



(0.8)



2



(1.4)



0



(0.0)



4



(2.7)



Proximal Phalanges  Unossified
(Bilateral)



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



18



(78.3)



12



(54.5)



10



(43.5)



15



(68.2)



Fetal Incidences



No (%)



20



(15.2)



18



(12.6)



14



(10.3)



18



(12.3)



Proximal Phalanges  Incomplete
ossification (Bilateral) Litter



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



1



(4.3)



3



(13.6)



3



(13.0)



1



(4.5)



Fetal Incidences



No (%)



1



(0.8)



3



92.1)



4



(2.9)



1



(0.7)



Pelvic Girdle



Pubis  Incomplete ossification
(Unilateral)



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



0



(0.0)



0



(0.0)



1



(4.5)



Fetal Incidences



No (%)



0



(0.0)



0



(0.0)



0



(0.0)



1



(0.7)



Malformations



Skull



Parietal bones - fused



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



1



(4.3)



0



(0.0)



0



(0.0)



0



(0.0)



Fetal Incidences



No (%)



1



(0.8)



0



(0.0)



0



(0.0)



0



(0.0)



Ribs



Rib No. 12 - Absent (unilateral)



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



1



(4.3)



0



(0.0)



0



(0.0)



0



(0.0)



Fetal Incidences



No (%)



1



(0.8)



0



(0.0)



0



(0.0)



0



(0.0)



Rib No. 11 and 12 - Fused



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



0



(0.0)



1



(4.3)



0



(0.0)



Fetal Incidences



No (%)



0



(0.0)



0



(0.0)



1



(0.7)



0



(0.0)



Rib No. 12 and 13 - Fused



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



1



(4.3)



0



(0.0)



0



(0.0)



0



(0.0)



Fetal Incidences



No (%)



1



(0.8)



0



(0.0)



0



(0.0)



0



(0.0)



Rudimentary (bilateral)



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



1



(4.5)



1



(4.3)



1



(4.5)



Fetal Incidences



No (%)



0



(0.0)



1



(0.7)



1



(0.7)



1



(0.7)



Thoracic Vertebra



Centrum No. 10 - Split



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



1



(4.3)



2



(9.1)



0



(0.0)



1



(4.5)



Fetal Incidences



No (%)



1



(0.8)



2



(1.4)



0



(0.0)



1



(0.7)



Centrum No. 11 - Split



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



2



(9.1)



2



(8.7)



3



(13.6)



Fetal Incidences



No (%)



0



(0.0)



2



(1.4)



2



(1.5)



3



(2.1)



Centrum No. 12 - Split



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



1



(4.3)



1



(4.5)



1



(4.3)



2



(9.1)



Fetal Incidences



No (%)



1



(0.8)



1



(0.7)



1



(0.7)



2



(1.4)



Centrum No. 13 - Split



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



0



(0.0)



1



(4.3)



0



(0.0)



Fetal Incidences



No (%)



0



(0.0)



0



(0.0)



1



(0.7)



0



(0.0)



 

Conclusions:
In a pre-natal developmental toxicity study conducted in Sprague Dawley Rats, the NOAEL of the test substance for both maternal and fetal toxicity was determined to be 1000 mg/kg body weight/day under the experimental conditions employed.
Executive summary:

The developmental toxicity of the test item was determined using OECD Guideline 414 under GLP conditions. The study used spraque dawley rats from an in-house line to test the daily oral administration via gavage of the test item using Carboxymethyl cellulose as vehicle. Based on a preliminary dose range study doses of 100, 300 and 1000 mg/kg bw/day were set with a concurrent vehicle control. On day 5 until day 19 of gestation, the test item was administered orally once daily to 25 pregnant females per group. During this time weight, food consumption, clinical signs of toxicity, mortality were observed. On day 20 all animals were euthanized, and tissue, organs and fetuses extracted and examined. There were no clinical signs of toxicity, mortality, morbidity, changes in body weight, food consumption changes, gross pathological or visceral changes in any group of maternal animals. Pregnancy rate, mean gravid uterus weight, live fetuses, mean litter size, mean sex ratio, mean number of implantation sites, mean number of resorptions, mean number of corpora lutae were similarly not affected by the treatment. Also unaffected were pre-and post- implantation losses, mean absolute and relative thyroid along with parathyropid weight, histopathology of the thryoid along with the parathyroid. The fetal toxicity assessment concluded no effect on mean fetal weight or crown-rump length in either sex. No test item related effects on developmental or structural alterations could be observed. Some visceral and skeletal alterations are considered incidental and not related to the test item as they occur in all groups, including the control and are not dose dependent. During the study no test item related effects in maternal or offspring animals could be observed. Therefore the "no observed adverse effect level (NOAEL) for reproduction and developmental toxicity is the highest dose used in this study of 1000mg/kg bw /day for maternal animals and offspring in rats.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 September 2020 - 20 December 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Remarks:
DRF study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: approved breeder, Adita Biosys Private Limited, Plot No. SPL 26, 2nd Stage, KSSIDC, Industrial Estate, Antharasanahalli, Tumakuru, Karnataka 572106. CPCSEA Registration No. 1868/PO/RcBt/S/16/CPCSEA
- Age at study initiation: 4 to 5 Months
- Weight at study initiation: Males: 2.26298 kg to 2.86128 kg; Females: 2.01942 kg to 2.51023 kg
- Fasting period before study: n/a
- Housing: Pre-mating/Acclimatization - The animals were housed individually in a stainless steel wire mesh cage (Size: L 24 x B 18 x H 18 inches) having facilities for holding pelleted food and drinking water.
- Housing: Mating - During mating, two rabbits (one male and one female) were housed together.
- Housing: Post mating - After confirmation of mating by visual observation, (Day 0 of pregnancy), the mated female was allotted with their permanent number and housed individually. The male was housed in its original cage.
- Diet (e.g. ad libitum): ad libitum, Altromin maintenance diet for rabbits (manufactured by Altromin Spezialfutter GmbH & Co. KG)
- Water (e.g. ad libitum): ad libitum Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes
- Acclimation period: minimum period of five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.6 to 20.9°C
- Humidity (%): relative humidity 47 to 65%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle

IN-LIFE DATES: From: 09 September 2020 To: 20 December 2020
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item formulations were prepared and administered to the animals within the established stability conditions. The required quantity of test item was weighed and triturated well in a mortar with a small quantity of vehicle until a homogenous suspension is formed and thereafter the entire quantity of the formulation were transferred into measuring cylinder. A small quantity of vehicle was added to rinse the mortar and this was transferred into the measuring cylinder. The rinsing procedure of mortar and pestle was repeated for many times to ensure the transfer of the contents to the measuring cylinder. Finally, the volume was made up to required quantity with vehicle to get desired concentration of 50, 150 and 500 mg/mL of test item for low, mid, and high dose groups, respectively.

VEHICLE
- Justification for use and choice of vehicle (if other than water): test item was insoluble in distilled water at the concentration of 100 mg/mL and uniformly suspended in 0.5% w/v Carboxymethyl cellulose at the concentration of 500 mg/mL (the highest dose concentration selected for the study considering the dose volume of 2 mL/kg body weight) as per in-house solubility/suspendibility test results. Hence, 0.5% w/v Carboxymethyl cellulose was used as vehicle for test item formulations
- Concentration in vehicle: 50, 150 and 500 mg/mL
- Amount of vehicle (if gavage): 2mL/kg
- Lot/batch no. (if required): BCBN1690V
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Verification of concentration and homogonetiy of the test item was conduced by BIONEEDS INDIA PRIVATE LIMITED DEVARAHOSAHALLY, SOMPURA HOBLI, NELAMANGALA TALUK, BANGALORE RURAL DISTRICT, PIN - 562 111, KARNATAKA, INDIA which hold the Good Laboratory Practice Certificate (GLP). 0.1 mL of sample was pipetted out into a digestion tube, a volume of 6 mL of concentrated nitric acid was added and placed in microwave sample preparation system and digested by the selected method. After digestion, the digestion tubes were cooled to room temperature and the contents were transferred to suitable volumetric flasks and diluted with Milli-Q water.


TECHNIQUE AND TEST METHOD: ICP-MS


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


ACCEPTANCE CRITERIA: The dose formulation samples of the test substance were analyzed for homogeneity and dose concentration analysis by ICP-MS and the results are found within the range 85-115% recovery to the nominal concentration with ˂10% RSD.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: until evidence of copulation was observed visually
- After an unspecified amount of days of unsuccessful pairing female returned to its original cage and was given another opportunity to mate on later occasions
- Further matings after two unsuccessful attempts: not detailed.
- Verification of same strain and source of both sexes: yes, same source
- Proof of pregnancy: day of confirmation of mating referred to as day 0 of pregnancy
Duration of treatment / exposure:
Gestation days 6 to 28
Frequency of treatment:
once daily
Duration of test:
29 days
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
25 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose range finding study for Prenatal Developmental Toxicity Study with New Zealand White Rabbits [Bioneeds Study No. BIO-DTX 039] was conducted with the doses of 100, 300 and 1000 mg/kg body weight based on the available literature. Based on the results obtained from the dose range finding study, the same doses of 0, 100, 300 and 1000 mg/kg body weight were selected as control, low, mid and high dose groups.
- Rationale for animal assignment (if not random): evenly distributed to all the groups based on their body weights so as to maintain comparable mean body weights for all groups
- Fasting period before blood sampling for (rat) dam thyroid hormones: not performed
- Time of day for (rat) dam blood sampling: not performed
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily for clinical signs of toxicity and twice daily for mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily for clinical signs of toxicity and twice daily for mortality and morbidity.

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 3, 6, 9, 12, 15, 18, 21, 24, 26, 28 and on 29

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Not feeding study

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not drinking study

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29
- Organs examined: Ovaries, Uterus with cervix, Thryoid and Parathyroid glands
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Blood sampling:
not collected
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter, half per litter without a head
- Head examinations: Yes: half per litter
- Anogenital distance of all live rodent pups: rabbits used in study
Statistics:
Parametric: One-way ANOVA with Dunnett’s post test - Maternal body weight, Percent change in maternal body weight, Corrected body weight for maternal increase, Gravid uterus weight, Maternal Feed consumption, Absolute / relative thyroid weights, Mean fetal weight per doe, Mean fetal crown rump length per doe

Non-Parametric: Kruskal-Wallis - No. of corpora lutea per doe, No. of implantations per doe,
Litter size per doe, No. of live fetuses per doe, Percent of live fetuses per doe, No. of early/late resorptions per doe, Percent of early/late resorptions per doe, Sex ratio (m/f) per doe, Pre/Post implantation losses (%) per doe, Fetal external / visceral / skeletal anomalies per doe

Frequencies Comparison: Cross Tabs - Chi-square test - No. of Pregnant / Non-pregnant females
(Pregnancy status), No. of does with / without live fetuses, No. of does with / without dead fetuses
No. of does with / without resorptions
Historical control data:
in-house historical control data, not included in report
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs of toxicity noted at all the tested dose groups and vehicle control group animals during the experimental period.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no morbidity/mortality noted at all the tested dose groups and vehicle control group animals during the experimental period.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related changes noted in mean gestation (maternal) body weight and percent change in mean gestation (maternal) body weight gain at all the tested dose groups when compared with the vehicle control group.
Statistically significant increase in percent change in body weight gain during GD 6 to 9 at group G3 were noted compared with vehicle control group; statistically significant decrease in percent change in body weight gain during GD 26 to 28 at groups G2 and G3 were noted compared with vehicle control group. However, these changes are considered as incidental and toxicologically insignificant.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related changes noted in mean gestation (maternal) feed consumption at all the tested dose groups when compared with the vehicle control group.
Statistically significant increase in mean feed consumption during different gestation periods were noted at all the tested dose groups when compared with vehicle control group but they are considered as incidental and toxicologically insignificant.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The thyroid along with parathyroid was collected, preserved and weighed post fixation from all the does of each dose group. There were no test item-related changes noted for mean absolute and relative weight for this organ in all the tested dose groups when compared to the vehicle control group.
The noted statistically significant increase in mean relative thyroid along with parathyroid at group G3 is considered as incidental as there were no changes in mean absolute weight of this organ and also no gross pathological changes noted during necropsy.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross pathological changes noted in any of the animals from all the tested dose groups and vehicle control group during conduct of the necropsy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related histopathological findings in thyroid and parathyroid glands of all treated animals. However, single incidence of ultimobranchial cyst/s in thyroid gland were noted in groups G1 and G3 each. These changes are considered as incidental findings as they occurred randomly across the dose groups including concurrent controls.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no statistically significant differences noted for percentage of pre-or post-implantation loss per litter at all the tested dose groups when compared to the vehicle control group.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in the number and percentage of early or late resorptions per doe across dose groups when compared to the vehicle control group.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no dead fetuses noted in any of the litters from all the tested dose and vehicle control groups during caesarean section.
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
A total of 21, 20, 21 and 22 females from groups G1, G2, G3 and G4 were found with implantations yielding to pregnancy with rates of 84%, 80%, 84% and 88% respectively.
Other effects:
no effects observed
Description (incidence and severity):
The mean number of corpora lutea per litter was 4.95, 5.15, 4.90 and 5.18 for groups G1, G2, G3 and G4 respectively. There were no changes or no statistically significant differences noted at all the tested dose groups when compared with vehicle control group for number of corpora lutea.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: highest dose tested, no adverse effects observed
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no changes noted in mean fetal weight of either sex across the dose groups when compared with the vehicle control group.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There were no statistically significant differences noted for number of live fetuses at all the tested dose groups when compared to the vehicle control group.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no statistically significant differences noted for male/female sex ratio across the dose groups when compared to the control group.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The mean litter sizes, assessed as the total number of fetuses in utero [live plus dead] per doe, was 4.05, 4.30, 3.95 and 4.23 for groups G1, G2, G3 and G4, respectively. There were no changes or no statistically significant differences noted at all the tested dose groups when compared with vehicle control group for litter size.
Anogenital distance of all rodent fetuses:
not examined
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related fetal external malformations or developmental variations for any of the fetuses examined from all the tested dose group litters. The noted general / developmental variations such as subcutaneous hemorrhagic spot/s beneath the skin on different regions of the body and noted external malformations such as hyperextension of forelimb unilateral and hyperextension of hindlimb unilateral across the tested dose group litters are considered incidental as these incidences are comparable with the vehicle control group and also these developmental alterations are common for this species and strain. Also, no remarkable differences or statistically significant changes noted for these alterations in any of the tested dose groups when compared with vehicle control group.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The below mentioned occasional incidences of skeletal developmental variations were
noted across the tested dose group litters.
- incomplete ossification of skull bones (hyoid bones, nasal bones, frontal, parietal);
- incomplete or bipartite or unossification of sternabral bones (sternabra 2, 4, 5 and
6);
- dumbbell or semi-bipartite ossification of thoracic verterbral centrum (centrum no.
11 and 12);
- incomplete ossification of pubis bones;
- incomplete /poor or unossification of metacarpal no. 1, 2, 4 and 5;
- incomplete /poor or unossification of middle/proximal phalanges of forelimb;
The below mentioned occasional incidences of skeletal malformation were noted across
the tested dose group litters.
- rudimentary rib (13th rib)
- supplementary rib (13th rib)
These skeletal developmental variations and skeletal malformations are considered as
incidental and un-related to treatment as these findings occurred infrequently or at a
frequency similar to the vehicle control group and did not occur in a dose-dependent
manner. Also, the occurred mean litter/fetal proportions were within the in-house
historical control range of this species and strain.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The noted developmental variations such as discoloured liver/thymus/adrenals/kidneys,
discoloured contents of gall bladder, dilatation of renal pelvis and dilatation of ureters
across the tested dose group litters are considered incidental as these incidences are
comparable with the vehicle control group and also these developmental variations are
common for this species and strain.
The noted developmental malformations lateral ventricular dilatation of brain, malpositioned kidneys, mis-shappened liver lobes, cyst in liver lobes, retrocaval ureters,
retinal fold across the tested dose group litters are considered incidental as these
incidences are comparable with the vehicle control group and also these developmental
alterations are common for this species and strain.
There were no remarkable differences or statistically significant changes noted for these
variations in any of the tested dose groups when compared with vehicle control group.
Other effects:
no effects observed
Description (incidence and severity):
There were no changes noted in mean fetal crown rump length per litter of either across the dose groups when compared with the vehicle control group.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Highest dose tested, no adverse effect observed
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

TABLE 1. SUMMARY OF PREGNANCY STATUS, CLINICAL SIGNS OF TOXICITY AND MORTALITY RECORD



















































Group


& Dose


(mg/kg body weight/day)



Pregnancy Status



 



Clinical Signs and Mortality Record



No. of Presumed


Pregnant Animals / Group



No. of Females


confirmed with


Pregnancy



Rate of Pregnancy (%)



Clinical Signs of


Toxicity revealed


(No. of Animals)



No. of Mortalities /


Total No. of animals



G1 & 0



25



21



84.0



N (25)



0/25



G2 & 100



25



20



80.0



N (25)



0/25



G3 & 300



25



21



84.0



N (25)



0/25



G4 & 1000



25



22



88.0



N (25)



0/25



TABLE 2. SUMMARY OF GESTATION BODY WEIGHT (kg) RECORD

















































































































































































































Group & Dose


(mg/kg body weight/day)


 

Body Weight (kg) on Gestation Day (GD)



0



3



6



9



12



15



18



21



24



26



28



29



G1 & 0



Mean



2.29566



2.36140



2.41082



2.46109



2.54458



2.61633



2.68157



2.74805



2.81948



2.88697



3.00956



3.10470



±SD



0.10872



0.10239



0.11277



0.13066



0.14922



0.14056



0.12835



0.13476



0.13750



0.14154



0.18962



0.23137



n



21



21



21



21



21



21



21



21



21



21



21



21



G2 & 100



Mean



2.29969



2.35634



2.41640



2.47141



2.54248



2.61253



2.68693



2.75395



2.83006



2.90218



2.97094



3.03621



±SD



0.09843



0.08784



0.08707



0.08016



0.07846



0.07785



0.07318



0.07094



0.07635



0.07760



0.08256



0.08249



n



20



20



20



20



20



20



20



20



20



20



20



20



G3 & 300



Mean



2.28398



2.32251



2.36867



2.43613



2.49699



2.56718



2.63950



2.70757



2.77324



2.84513



2.91838



2.98939



±SD



0.11322



0.12476



0.12555



0.12973



0.13174



0.13235



0.12764



0.13320



0.13918



0.14768



0.14961



0.15151



n



21



21



21



21



21



21



21



21



21



21



21



21



G4 & 1000



Mean



2.28688



2.31976



2.36018



2.41750



2.48551



2.55193



2.62423



2.68620



2.75992



2.83492



2.92486



3.00433



±SD



0.11850



0.10486



0.10024



0.09892



0.10160



0.10163



0.10804



0.12053



0.13392



0.15129



0.18653



0.23117



n



22



22



22



22



22



22



22



22



22



22



22



22



TABLE 3. SUMMARY OF PERCENT CHANGE IN GESTATION BODY WEIGHT GAIN (%) RECORD




































































































































































































Group & Dose


(mg/kg body weight/day)



 



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



0 to 3



3 to 6



6 to 9



9 to 12



12 to 15



15 to 18



18 to 21



21 to 24



24 to 26



26 to 28



28 to 29



G1 & 0



Mean



2.90529



2.09096



2.06280



3.39241



2.85267



2.52499



2.47635



2.60284



2.39595



4.22709



3.10025



±SD



2.30791



1.72348



1.41075



2.43775



1.19645



0.87592



0.85997



0.77788



0.90407



3.63466



1.73788



n



21



21



21



21



21



21



21



21



21



21



21



G2 & 100



Mean



2.50848



2.56634



2.29188



2.88226



2.76043



2.85628



2.49979



2.76714



2.55241



2.36805*



2.20173



±SD



2.31506



1.69914



0.81976



0.69112



0.62844



0.54524



0.63368



1.20868



0.86601



0.62377



0.83784



n



20



20



20



20



20



20



20



20



20



20



20



G3 & 300



Mean



1.67990



1.99793



2.84753*



2.50282



2.82240



2.83757



2.57778



2.42230



2.58691



2.57892*



2.43773



±SD



1.69009



1.19451



0.50875



0.76321



1.12178



0.87666



0.66839



0.48652



0.66254



0.61394



0.68864



n



21



21



21



21



21



21



21



21



21



21



21



G4 & 1000



Mean



1.48842



1.76394



2.44024



2.81513



2.67967



2.83121



2.34878



2.73045



2.69688



3.13420



2.66727



±SD



2.02579



1.45651



1.05621



0.78365



0.87497



0.89481



0.75474



0.70278



0.81172



2.08907



2.79445



n



22



22



22



22



22



22



22



22



22



22



22



TABLE 4. SUMMARY OF GRAVID UTERUS WEIGHT AND MATERNAL
BODY WEIGHT CHANGE CORRECTED FOR GRAVID UTERINE
WEIGHT




































































































































Group


& Dose


(mg/kg body weight/day)


 

Body Weight Change (kg) from GD 6 to 29



Gravid Uterus Weight (g)



Gravid Uterus Weight (kg)



Corrected Body weight


 

kg



%


 

G1 & 0



Mean



0.69388



218.28



0.21828



0.47560



19.66


 

±SD



0.18334



56.84



0.05684



0.17176



6.82


 

n



21



21



21



21



21


 

G2 & 100



Mean



0.61981



221.00



0.22100



0.39881



16.58


 

±SD



0.08184



49.43



0.04943



0.07286



3.38


 

n



20



20



20



20



20


 

G3 & 300



Mean



0.62072



211.96



0.21196



0.40876



17.32


 

±SD



0.09168



67.55



0.06755



0.05879



2.73


 

n



21



21



21



21



21


 

G4 & 1000



Mean



0.64415



223.49



0.22349



0.42066



17.76


 

±SD



0.17995



78.71



0.07871



0.15570



6.24


 

n



22



22



22



22



22


 

TABLE 5. SUMMARY OF AVERAGE GESTATION FEED CONSUMPTION (g/animal/day) RECORD




































































































































































































Group


& Dose


(mg/kg body weight/day)



 



Feed Consumption (g/animal/day) during Gestation Day (GD)



0 to 3



3 to 6



6 to 9



9 to 12



12 to 15



15 to 18



18 to 21



21 to 24



24 to 26



26 to 28



28 to 29



G1 & 0



Mean



64.83



63.51



64.41



66.36



66.70



67.53



69.30



72.29



76.66



81.10



84.01



±SD



5.85



5.66



5.72



4.83



3.23



2.45



3.27



3.60



3.59



2.56



4.17



n



21



21



21



21



21



21



21



21



21



21



21



G2 & 100



Mean



65.66



66.71*



68.87*



70.93*



74.47*



76.09*



78.19*



79.82*



82.71*



86.10*



88.25*



±SD



3.53



4.92



4.61



4.99



4.73



4.90



4.18



4.51



4.92



4.25



6.61



n



20



20



20



20



20



20



20



20



20



20



20



G3 & 300



Mean



65.78



66.65*



68.46*



69.56



71.95*



72.80*



74.92*



77.33*



79.47



80.76



83.24



±SD



4.41



2.23



3.77



3.54



4.42



4.70



3.94



3.61



3.46



3.06



3.43



n



21



21



21



21



21



21



21



21



21



21



21



G4 & 1000



Mean



63.63



64.49



65.72



67.32



69.44



71.89*



73.93*



76.22*



82.36*



82.81



86.60



±SD



3.63



2.76



3.51



4.26



3.49



4.19



3.45



3.77



9.88



4.92



5.56



n



22



22



22



22



22



22



22



22



22



22



22



TABLE 6. SUMMARY OF UTERI OBSERVATIONS PER LITTER RECORD










































































































































































Group


& Dose


(mg/kg body weight/day)


 

No. of


Corpora lutea



No. of Implantations



Litter size



No. of Live Fetuses



No. of Dead Fetuses



No. of Early Resorptions



No. of Late Resorptions



Total



Male



Female



G1 & 0



Mean



4.95



4.38



4.05



4.05



2.05



2.00



0.00



0.14



0.19



±SD



1.20



1.12



1.28



1.28



0.86



1.00



0.00



0.36



0.51



n



21



21



21



21



21



21



21



21



21



G2 & 100



Mean



5.15



4.60



4.30



4.30



2.10



2.20



0.00



0.15



0.15



±SD



0.93



1.10



1.13



1.13



0.64



1.01



0.00



0.37



0.37



n



20



20



20



20



20



20



20



20



20



G3 & 300



Mean



4.90



4.38



3.95



3.95



2.14



1.81



0.00



0.24



0.19



±SD



1.04



1.36



1.47



1.47



0.85



1.12



0.00



0.54



0.51



n



21



21



21



21



21



21



21



21



21



G4 & 1000



Mean



5.18



4.64



4.23



4.23



1.95



2.27



0.00



0.23



0.18



±SD



1.01



1.40



1.63



1.63



1.36



1.24



0.00



0.43



0.50



n



22



22



22



22



22



22



22



22



22



TABLE 7. SUMMARY OF MATERNAL DATA PER LITTER RECORD




















































































































































































Group


& Dose


(mg/kg body weight/day)



 



Pre-Implantation Loss (%)



Post-Implantation Loss (%)



Percent of Dead Fetus (%)



Percent of Early Resorptions (%)



Percent of Late Resorptions (%)



Male/Female Sex Ratio



Male


Fetuses (%)



Female Fetuses (%)



Percent of Live Fetuses


 

G1 & 0



Mean



11.09



8.10



0.00



3.73



4.37



1.19



52.01



47.99



100.00


 

±SD



11.34



14.43



0.00



9.60



12.25



0.86



21.01



21.01



0.00


 

n



21



21



21



21



21



21



21



21



21


 

G2 & 100



Mean



10.63



6.42



0.00



3.75



2.67



1.24



50.39



49.61



100.00


 

±SD



13.14



10.28



0.00



9.16



6.54



0.81



15.41



15.41



0.00


 

n



20



20



20



20



20



20



20



20



20


 

G3 & 300



Mean



11.27



9.52



0.00



5.24



4.29



1.37



56.64



43.36



100.00


 

±SD



17.04



14.48



0.00



11.56



11.10



0.84



17.76



17.76



0.00


 

n



21



21



21



21



21



21



21



21



21


 

G4 & 1000



Mean



11.52



10.23



0.00



4.77



5.45



0.95



44.65



55.35



100.00


 

±SD



15.33



16.73



0.00



9.06



15.89



0.83



24.20



24.20



0.00


 

n



22



22



22



22



22



22



22



22



22


 

TABLE 8. SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD































































Group & Dose


(mg/kg body weight/day)


 

Absolute Thyroid along with Parathyroid Weight (g)#



G1 & 0



Mean



0.2274



±SD



0.0410



n



21



G2 & 100



Mean



0.2138



±SD



0.0260



n



20



G3 & 300



Mean



0.2526



±SD



0.0483



n



21



G4 & 1000



Mean



0.2175



±SD



0.0410



n



22



TABLE 9. SUMMARY OF TERMINAL BODY WEIGHT (kg) AND ORGAN
WEIGHT (%) RELATIVE TO TERMINAL BODY WEIGHT RECORD












































































Group & Dose


(mg/kg body weight/day)


 

Terminal Body Weight (kg)



Relative Thyroid along


with Parathyroid weight (%)



G1 & 0



Mean



3104.71



0.0073



±SD



231.36



0.0010



n



21



21



G2 & 100



Mean



3036.21



0.0070



±SD



82.49



0.0007



n



20



20



G3 & 300



Mean



2989.39



0.0084*



±SD



151.51



0.0014



n



21



21



G4 & 1000



Mean



3004.33



0.0072



±SD



231.17



0.0009



n



22



22



TABLE 10. SUMMARY OF MEAN FETAL WEIGHT AND
MEAN CROWN RUMP LENGTH RECORD











































































































Group & Dose


(mg/kg body weight/day)


 

Fetal Weight (g)


 

Crown Rump Length (mm)



Male



Female



Male



Female



G1, & 0



Mean



41.30



39.27



90.64



87.88



±SD



4.28



4.18



2.34



2.10



n



20



20



20



20



G2, & 100



Mean



40.55



38.41



90.51



87.71



±SD



2.05



2.57



1.75



2.23



n



20



20



20



20



G3, & 300



Mean



41.19



39.97



90.94



89.02



±SD



2.51



2.34



2.61



2.83



n



21



20



21



20



G4, & 1000



Mean



41.19



39.23



90.27



88.49



±SD



2.15



3.12



2.17



2.95



n



20



21



20



21



TABLE 11. SUMMARY RECORD OF FETAL EXTERNAL EXAMINATION PER
LITTER
















































































Group


 

G1



G2



G3



G4



Dose (mg/kg body weight/day)


 

0



100



300



1000


 

Litters Evaluated for External Examination



No.



21



20



21



22



Fetuses Evaluated for External Examination



No.



85



86



83



93


 

Region/Tissue↓



Alteration↓


 

General



Subcutaneous Hemorrhagic spot


on different regions of the body


 
 

Litter Incidences



No. (%)



2



9.5



2



10.0



1



4.8



1



4.5


 

Fetal Incidences



No. (%)



2



2.4



2



2.3



1



1.2



1



1.1



TABLE 11 (Contd…). SUMMARY RECORD OF FETAL EXTERNAL
EXAMINATION PER LITTER















































































































Group


 

G1



G2



G3



G4



Dose (mg/kg body weight/day)


 

0



100



300



1000



 



Litters Evaluated for External Examination



No.



21



20



21



22



Fetuses Evaluated for External Examination



No.



85



86



83



93


 

Region/Tissue↓



Alteration↓



 



Forelimb



Hyperextension - Unilateral



 


 

Litter Incidences



No. (%)



1



4.8



0



0.0



1



4.8



1



4.5


 

Fetal Incidences



No. (%)



1



1.2



0



0.0



1



1.2



1



1.1



Hindlimb



Hyperextension - Unilateral


 
 

Litter Incidences



No. (%)



1



4.8



1



5.0



0



0.0



1



4.5


 

Fetal Incidences



No. (%)



1



1.2



1



1.2



0



0.0



1



1.1



TABLE 12. SUMMARY OF FETAL VISCERAL EXAMINATION PER LITTER
RECORD










































































































































































































































































Group


 

G1



G2



G3



G4



Dose (mg/kg body weight/day)


 

0



100



300



1000


 

Litters Evaluated for  Visceral Examination



No.



21



20



21



22



Fetuses Evaluated for  Visceral Examination



No.



85



86



83



93


 

Region/Tissue↓



Alteration↓


 

Adrenals



Discoloured


 
 

Litter Incidences



No. (%)



2



9.5



1



5.0



0



0.0



2



9.1


 

Fetal Incidences



No. (%)



2



2.4



1



1.2



0



0.0



2



2.2



Gall bladder



Discoloured contents


 
 

Litter Incidences



No. (%)



1



4.8



0



0.0



1



4.8



1



4.5


 

Fetal Incidences



No. (%)



1



1.2



0



0.0



1



1.2



1



1.1



Kidneys



Dilatation of renal pelvis  - bilateral


 
 

Litter Incidences



No. (%)



3



14.3



4



20.0



3



14.3



2



9.1


 

Fetal Incidences



No. (%)



3



3.5



4



4.7



3



3.6



3



3.2



Kidneys



Discoloured


 
 

Litter Incidences



No. (%)



2



9.5



1



5.0



0



0.0



2



9.1


 

Fetal Incidences



No. (%)



2



2.4



1



1.2



0



0.0



2



2.2



Liver



Discoloured


 
 

Litter Incidences



No. (%)



3



14.3



2



10.0



2



9.5



1



4.5


 

Fetal Incidences



No. (%)



3



3.5



2



2.3



2



2.4



1



1.1



Thymus



Discoloured


 
 

Litter Incidences



No. (%)



1



4.8



0



0.0



2



9.5



1



4.5


 

Fetal Incidences



No. (%)



1



1.2



0



0.0



2



2.4



1



1.1



Ureters



Dilatation


 
 

Litter Incidences



No. (%)



3



14.3



2



10.0



6



28.6



4



18.2


 

Fetal Incidences



No. (%)



4



4.7



3



3.5



6



7.2



5



5.4



 


TABLE 12 (Contd…). SUMMARY RECORD OF FETAL
VISCERAL EXAMINATION PER LITTER RECORD











































































































































































































































Group


 

G1



G2



G3



G4



Dose (mg/kg body weight/day)


 

0



100



300



1000


 

Litters Evaluated for Visceral Examination



No.



21



20



21



22



Fetuses Evaluated for Visceral Examination 



No.



85



86



83



93


 

Region/Tissue↓



Alteration↓


 

Brain



Lateral ventricular dilatation


 
 

Litter Incidences



No. (%)



1



4.8



0



0.0



0



0.0



1



4.5


 

Fetal Incidences



No. (%)



1



1.2



0



0.0



0



0.0



1



1.1



Kidneys



Malpositioned


 
 

Litter Incidences



No. (%)



2



9.5



0



0.0



1



4.8



1



4.5


 

Fetal Incidences



No. (%)



2



2.4



0



0.0



1



1.2



1



1.1



Liver



Misshappen


 
 

Litter Incidences



No. (%)



1



4.8



1



5.0



0



0.0



1



4.5


 

Fetal Incidences



No. (%)



1



1.2



1



1.2



0



0.0



1



1.1



Liver



Cyst


 
 

Litter Incidences



No. (%)



0



0.0



1



5.0



1



4.8



0



0.0


 

Fetal Incidences



No. (%)



0



0.0



1



1.2



1



1.2



0



0.0



Ureters



Retrocaval


 
 

Litter Incidences



No. (%)



1



4.8



0



0.0



1



4.8



1



4.5


 

Fetal Incidences



No. (%)



1



1.2



0



0.0



1



1.2



1



1.1



Eye (retina)



Fold


 
 

Litter Incidences



No. (%)



0



0.0



0



0.0



1



4.8



0



0.0


 

Fetal Incidences



No. (%)



0



0.0



0



0.0



1



1.2



0



0.0



TABLE 13. SUMMARY OF SKELETAL EXAMINATION OF FETUSES PER
LITTER RECORD























































































































































































































































































































































































































































Group



 



G1



G2



G3



G4



Dose (mg/kg body weight/day)



 



0



100



300



1000



 



Litters Evaluated for Skeletal Examination



No.



21



20



21



22



Fetuses Evaluated for Skeletal Examination



No.



85



86



83



93



 



Region/Tissue↓



Alteration↓



 



Skull



Hyoid bone - Incomplete ossification


 

Litter Incidences



No. (%)



2



9.5



1



5.0



2



9.5



2



9.1



 



Fetal Incidences



No. (%)



2



2.4



1



1.2



2



2.4



2



2.2



Skull



Nasal bone - Incomplete ossification 


 

Litter Incidences



No. (%)



2



9.5



0



0.0



0



0.0



1



4.5



 



Fetal Incidences



No. (%)



2



2.4



0



0.0



0



0.0



1



1.1



Skull



Frontal bone - Incomplete ossification 


 

Litter Incidences



No. (%)



1



4.8



0



0.0



1



4.8



0



0.0



 



Fetal Incidences



No. (%)



1



1.2



0



0.0



1



1.2



0



0.0



Skull



Parietal bone - Incomplete ossification 


 

Litter Incidences



No. (%)



0



0.0



1



5.0



0



0.0



1



4.5



 



Fetal Incidences



No. (%)



0



0.0



1



1.2



0



0.0



1



1.1



Sternum



Sternebra No. 5 - Unossified 


 

Litter Incidences



No. (%)



2



9.5



1



5.0



1



4.8



0



0.0



 



Fetal Incidences



No. (%)



2



2.4



1



1.2



1



1.2



0



0.0



Sternum



Sternebra No. 6 - Unossified 


 

Litter Incidences



No. (%)



1



4.8



0



0.0



0



0.0



0



0.0



 



Fetal Incidences



No. (%)



1



1.2



0



0.0



0



0.0



0



0.0



Sternum



Sternebra No. 2 - Poor ossification 


 

Litter Incidences



No. (%)



0



0.0



1



5.0



1



4.8



0



0.0



 



Fetal Incidences



No. (%)



0



0.0



2



2.3



1



1.2



0



0.0



Sternum



Sternebra No. 4 - Poor ossification 


 

Litter Incidences



No. (%)



0



0.0



1



5.0



0



0.0



0



0.0



 



Fetal Incidences



No. (%)



0



0.0



1



1.2



0



0.0



0



0.0



Sternum



Sternebra No. 5 - Poor ossification 


 

Litter Incidences



No. (%)



4



19.0



4



20.0



2



9.5



5



22.7



 



Fetal Incidences



No. (%)



5



5.9



4



4.7



4



4.8



7



7.5



Sternum



Sternebra No. 6 - Poor ossification 


 

Litter Incidences



No. (%)



3



14.3



3



15.0



3



14.3



5



22.7



 



Fetal Incidences



No. (%)



3



3.5



3



3.5



3



3.6



5



5.4



Sternum



Sternebra No. 4 - Incomplete ossification 


 

Litter Incidences



No. (%)



0



0.0



0



0.0



1



4.8



0



0.0



 



Fetal Incidences



No. (%)



0



0.0



0



0.0



1



1.2



0



0.0



Sternum



Sternebra No. 5 - Incomplete ossification 


 

Litter Incidences



No. (%)



2



9.5



2



10.0



1



4.8



4



18.2



 



Fetal Incidences



No. (%)



2



2.4



2



2.3



1



1.2



4



4.3



Sternum



Sternebra No. 6 - Incomplete ossification 


 

Litter Incidences



No. (%)



3



14.3



4



20.0



2



9.5



4



18.2



 



Fetal Incidences



No. (%)



3



3.5



4



4.7



2



2.4



4



4.3



TABLE 13 (Contd…). SUMMARY OF SKELETAL EXAMINATION OF FETUSES
PER LITTER RECORD



































































































































































































































































Group



 



G1



G2



G3



G4



Dose (mg/kg body weight/day)



 



0



100



300



1000



 



Litters Evaluated for Skeletal Examination



No.



21



20



21



22



Fetuses Evaluated for Skeletal Examination



No.



85



86



83



93



 



Region/Tissue↓



Alteration↓



 



Thoracic Vertebra



Centrum No. 11 - Dumbbell-shaped ossification 


 

Litter Incidences



No. (%)



2



9.5



3



15.0



1



4.8



3



13.6



 



Fetal Incidences



No. (%)



2



2.4



3



3.5



2



2.4



3



3.2



Thoracic Vertebra



Centrum No. 12 - Dumbbell-shaped ossification 


 

Litter Incidences



No. (%)



2



9.5



3



15.0



2



9.5



2



9.1



 



Fetal Incidences



No. (%)



2



2.4



3



3.5



2



2.4



2



2.2



Pelvic girdle



Pubis bone - Incomplete ossification (Bilateral) 


 

Litter Incidences



No. (%)



3



14.3



0



0.0



4



19.0



2



9.1



 



Fetal Incidences



No. (%)



3



3.5



0



0.0



4



4.8



2



2.2



Forelimb



Metacarpal No. 1 - Unossified (Bilateral) 


 

Litter Incidences



No. (%)



3



14.3



2



10.0



5



23.8



3



13.6



 



Fetal Incidences



No. (%)



3



3.5



2



2.3



5



6.0



3



3.2



Forelimb



Metacarpal No. 2 - Poor ossification (Bilateral) 


 

Litter Incidences



No. (%)



0



0.0



1



5.0



0



0.0



0



0.0



 



Fetal Incidences



No. (%)



0



0.0



1



1.2



0



0.0



0



0.0



Forelimb



Metacarpal No. 4 - Poor ossification (Bilateral)  


 

Litter Incidences



No. (%)



0



0.0



1



5.0



1



4.8



0



0.0



 



Fetal Incidences



No. (%)



0



0.0



1



1.2



1



1.2



0



0.0



Forelimb



Metacarpal No. 5 - Poor ossification (Bilateral) 


 

Litter Incidences



No. (%)



0



0.0



0



0.0



1



4.8



0



0.0



 



Fetal Incidences



No. (%)



0



0.0



0



0.0



1



1.2



0



0.0



TABLE 13 (Contd…). SUMMARY OF SKELETAL EXAMINATION OF FETUSES
PER LITTER RECORD



































































































































































































































































Group



 



G1



G2



G3



G4



Dose (mg/kg body weight/day)



 



0



100



300



1000



 



Litters Evaluated for Skeletal Examination



No.



21



20



21



22



Fetuses Evaluated for Skeletal Examination



No.



85



86



83



93



 



Region/Tissue↓



Alteration↓



 



Forelimb



Middle phalanges - Poor ossification (Bilateral) 


 

Litter Incidences



No. (%)



2



9.5



1



5.0



2



9.5



3



13.6



 



Fetal Incidences



No. (%)



2



2.4



1



1.2



2



2.4



3



3.2



Forelimb



Proximal phalanx No. 1 - Poor ossification (Bilateral) 


 

Litter Incidences



No. (%)



1



4.8



2



10.0



0



0.0



0



0.0



 



Fetal Incidences



No. (%)



1



1.2



3



3.5



0



0.0



0



0.0



Forelimb



Proximal phalanx No. 2 - Poor ossification (Bilateral)  


 

Litter Incidences



No. (%)



0



0.0



1



5.0



0



0.0



0



0.0



 



Fetal Incidences



No. (%)



0



0.0



1



1.2



0



0.0



0



0.0



Forelimb



Proximal phalanx No. 3 - Poor ossification (Bilateral) 


 

Litter Incidences



No. (%)



0



0.0



2



10.0



0



0.0



0



0.0



 



Fetal Incidences



No. (%)



0



0.0



2



2.3



0



0.0



0



0.0



Forelimb



Proximal phalanx No. 4 - Poor ossification (Bilateral)  


 

Litter Incidences



No. (%)



0



0.0



2



10.0



1



4.8



0



0.0



 



Fetal Incidences



No. (%)



0



0.0



2



2.3



1



1.2



0



0.0



Forelimb



Proximal phalanx No. 5 - Poor ossification (Bilateral) 


 

Litter Incidences



No. (%)



0



0.0



1



5.0



1



4.8



0



0.0



 



Fetal Incidences



No. (%)



0



0.0



1



1.2



1



1.2



0



0.0



Forelimb



Proximal phalanx No. 1 - Incomplete ossification (Bilateral) 


 

Litter Incidences



No. (%)



0



0.0



0



0.0



1



4.8



0



0.0



 



Fetal Incidences



No. (%)



0



0.0



0



0.0



1



1.2



0



0.0



TABLE 13 (Contd…). SUMMARY OF SKELETAL EXAMINATION OF FETUSES
PER LITTER RECORD













































































































Group



 



G1



G2



G3



G4



Dose (mg/kg body weight/day)



 



0



100



300



1000



 



Litters Evaluated for Skeletal Examination



No.



21



20



21



22



Fetuses Evaluated for Skeletal Examination



No.



85



86



83



93



 



Region/Tissue↓



Alteration↓



 



Ribs



Rib No. 13 - Supplementary rib (Bilateral)


 

Litter Incidences



No. (%)



2



9.5



1



5.0



2



9.5



4



18.2



 



Fetal Incidences



No. (%)



2



2.4



1



1.2



2



2.4



4



4.3



Ribs



Rib No. 13 - Rudimentary rib (Bilateral)


 

Litter Incidences



No. (%)



0



0.0



1



5.0



0



0.0



2



9.1



 



Fetal Incidences



No. (%)



0



0.0



1



1.2



0



0.0



2



2.2


Conclusions:
In a pre-natal developmental toxicity study conducted in New Zealand White rabbits, the NOAEL of the test substance for both maternal and fetal toxicity was determined to be 1000 mg/kg body weight/day under the experimental conditions employed.
Executive summary:

The developmental toxicity of the test item was determined using OECD Guideline 414 under GLP conditions. The study used New Zealand White rabbits from a certified breeder to test the daily oral administration via gavage of the test item using Carboxymethyl cellulose as vehicle. Based on a preliminary dose range study doses of 100, 300 and 1000 mg/kg bw/day were set with a concurrent vehicle control. On day 6 until day 28 of gestation, the test item was administered orally once daily to 25 pregnant females per group. During this time weight, food consumption, clinical signs of toxicity, mortality were observed. On day 29 all animals were euthanized, and tissue, organs and fetuses extracted and examined. There were no clinical signs of toxicity, mortality, morbidity, changes in body weight, food consumption changes, gross pathological or visceral changes in any group of maternal animals. Pregnancy rate, mean gravid uterus weight, live fetuses, mean litter size, mean sex ratio, mean number of implantation sites, mean number of resorptions, mean number of corpora lutae were similarly not affected by the treatment. Also unaffected were pre-and post implantation losses, mean absolute and relative thyroid along with parathyropid weight, histopathology of the thryoid along with the parathyroid. The fetal toxicity assessment concluded no effect on mean fetal weight or crown-rump length in either sex. No test item related effects on developmental or structural alterations could be observed. Some visceral and skeletal alterations are considered incidental and not related to the test item as they occur in all groups, including the control and are not dose dependent. During the study no test item related effects in maternal or offspring animals could be observed. Therefore the "no observed adverse effect level (NOAEL) for reproduction and developmental toxicity is the highest dose used in this study of 1000mg/kg bw /day for maternal animals and offspring in rabbits.


 

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
2 key studies (in rat and rabbit) are available with a Klimisch score of 1. Also, the respective dose range finding studies as supporting studies are available with Klimisch score of 1.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available information, the substance is not classified for toxicity to reproduction in accordance with CLP Regulation (EC) no 1272/2008.

Additional information