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Toxicological information

Toxicity to reproduction

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Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 Apr 2018 to 14 Jun 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 Apr 2018 to 14 Jun 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
HanTac: WH
Details on species / strain selection:
Rat is the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Vivo Bio Tech Ltd.; Sy. #349/A, Pregnapur-502311, Gajwel Mandal, Medak District, Telangana
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 11-12 weeks
- Weight at study initiation: Males: 239.32 to 282.57 g; Females: 173.83 to 204.99 g
- Housing:
Pre-mating: Two rats of the same sex were housed per cage in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with a stainless-steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles with a stainless-steel sipper.
Mating and post-mating: During mating, two rats (one male and one female) were housed in standard polysulfone cages with a stainless-steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles. After confirming the presence of sperm in the vaginal smear or vaginal plugs (Day ‘0’ pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually in polysulfone cages. The sterilised nesting material (paper shreds) was provided near-term. Steam sterilized clean corn cob was used as bedding and changed along with the cage twice a week.
- Diet: Teklad Certified (2014C) Global 14% Protein Rodent Maintenance Diet – Pellet (Certified) was provided ad libitum to the animals.
- Water: Water was provided ad libitum to rats.
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY: Food and water was checked for contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25
- Humidity (%): 59-68
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 20 Apr 2018 To: 14 Jun 2018
Route of administration:
oral: gavage
Details on route of administration:
The route of test item administration was through oral gavage. The oral route was chosen because it provides an exaggerated model of the normal exposure in humans.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Required quantities of the test item were weighed in a beaker (previously calibrated to a desired volume) for each dose level separately and a small volume of vehicle [corn oil] was added and stirred using a glass rod till a uniform suspension was obtained. The volume was made up to the mark using the vehicle to get the final desired concentration of 20, 60 and 200 mg/mL for the G2, G3 and G4 groups, respectively. The suspensions were mixed well by stirring using a magnetic stirrer. The formulations were constantly stirred using a magnetic stirrer during test item administration and during sampling.

VEHICLE
- Concentration in vehicle: 20, 60 and 200 mg/L
- Amount of vehicle: 5 mL/kg/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For homogeneity and active ingredient (a.i.) concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 of the treatment period and during the 2nd month (day 33) of treatment and was analysed inhouse. For each set, duplicate samples were drawn from the top, middle and bottom layers of each preparation and in case of the control duplicate samples from the middle layer were drawn.
Dose formulations were considered acceptable as the overall mean results were within ± 15.0% of the claimed concentration and the overall relative standard deviation (RSD) was less than 10.0%.
Duration of treatment / exposure:
Males: Male animals were dosed during a 2-week premating period, during mating and post-mating until sacrifice (42 days of treatment).
Females: The female animals were dosed during a 2-week premating period, and during mating, pregnancy and up to LD 13.
Frequency of treatment:
Once daily
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Group 4
No. of animals per sex per dose:
10 rats per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels of 100 (G2), 300 (G3) and 1000 (G4) mg/kg bw/day were selected for this study based on the results of the 14-Day Repeated Dose Oral Toxicity Study in Wistar Rats (Study No. N3629) and in consultation with the Sponsor.
- Fasting period before blood sampling for clinical biochemistry: yes, overnight fasting (water allowed)
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
- Time schedule: All rats were observed for morbidity and mortality twice daily i.e., once in the morning and once in the afternoon. As the observed clinical signs did not warrant twice daily observations, the observation for morbidity and mortality was carried out once in the morning during holidays. All rats were observed for clinical signs twice (pre and post dose) daily during the treatment period.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: prior to the treatment on Day 1 (± 2 day) and at weekly intervals thereafter during the treatment period.
- Parameters checked: During detailed clinical examination, all rats were observed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g., excessive grooming, repetitive circling or bizarre behaviour like self-mutilation, walking backwards).

BODY WEIGHT:
- Time schedule for examinations: Individual body weights of males were recorded on Day 1 and at weekly intervals thereafter. Individual body weights of females were recorded on Day 1 and at weekly intervals thereafter till cohabitation (till mating confirmation) with males. All dams were weighed on Gestation Day (GD) 0, 7, 14 and 20 and on Lactation Day (LD) 0, 4 and 13.

FOOD CONSUMPTION:
- Cagewise average food consumption was calculated by using the food consumed at each interval per cage and dividing by the number of rats per cage and the number of days in the intervening period to determine the food consumption/rat/day. Food consumption was not measured during the cohabitation period. Food consumption of pregnant dams was recorded on GD 7, 14 and 20 and on Day 4 and 13 of the lactation period.

HAEMATOLOGY:
- Time schedule for collection of blood: At the end of the pre-mating period (Day 16)
- Anaesthetic used for blood collection: Yes, Isoflurane anaesthesia
- Animals fasted: Yes, overnight (water allowed)
- How many animals: randomly selected 5 males and 5 females of each group
- Parameters checked in Tables 1 and 2 in ‘Any other information on materials and methods incl. tables’ were examined.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: At the end of the pre-mating period (Day 16)
- Animals fasted: Yes, overnight (water allowed)
- How many animals: randomly selected 5 males and 5 females of each group
- Parameters checked in Table 3 in ‘Any other information on materials and methods incl. tables’ were examined.

URINALYSIS:
- Time schedule for collection of urine: At the end of the pre-mating period
- Metabolism cages used for collection of urine: Yes, each rat was placed overnight in a specially fabricated cage
- Animals fasted: Yes, overnight (water allowed)
- Parameters checked in Table 4 in ‘Any other information on materials and methods incl. tables’ were examined.

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule for examinations: towards the end of the dosing period for males (shortly prior to their scheduled kill) and during the lactation period for females (shortly before the scheduled kill).
- Dose groups that were examined: All groups
- Battery of functions tested:
Home cage observations: Each rat was observed in the home cage for posture and for presence or absence of abnormal vocalizations, tremors and convulsions.
Observations during removal of animal from home cage and handling: Each rat was observed for the following examinations: ease of removal from home cage, handling reactivity, palpebral closure, eye examination, piloerection, lacrimation, salivation, skin/fur examination, perineum wetness, respiration, muscle tone and extensor thrust response. The observations were recorded using scores/ranks.
Open Field Observation: A rat was placed in an open arena, on a flat surface with a clean absorbent pa-per and observed for at least 2 minutes. Absorbent paper was replaced for each group. During this observation period, the rat was evaluated as it moves about freely/unperturbed and the following observations were made and recorded using score/ranks: gait, posture, tremors, mobility score, arousal level, clonic or tonic movements, stereotypic behaviour, bizarre behaviour, urination, defecation, rearing, abnormal vocalizations,
Functional tests: Functional testing included motor activity, sensory evaluation, landing hindlimbs foot splay and measurement of grip performance.
Physiological observations: Body temperature (rectal temperature) was measured in degree Celsius (°C) using a digital thermometer. At the end of the functional test, body weight of each rat was measured.

HORMONE ANALYSIS
Blood samples were collected and serum was separated as per the following schedule for the determination of total T4 and TSH:
- All dams prior to sacrifice
- All adult males, prior to sacrifice
Blood samples were collected in plain labelled tubes and kept on the bench top for 20 minutes before centrifugation. Serum was separated by centrifuging the whole blood samples at 5000 rpm for 5 minutes at 4°C. The serum samples were placed in labelled plastic tubes and stored at ~-70°C until they were analyzed.
Blood samples were not collected from the non-littered females and the dams in which all pups were dead/cannibalized during lactation.

THYROID PROFILE HORMONES
The serum samples were analysed for Rodent Thyroid Stimulating Hormone (TSH; ng/mL) and Rodent Thyroxin (T4; ng/mL).
Sacrifice and pathology:
GROSS NECROPSY/ ORGAN WEIGHTS: Table 5 in ‘Any other information on materials and methods incl. tables’.
All adult animals were subjected for detailed necropsy and findings were recorded. The adult animals killed at term were fasted overnight (water allowed), weighed and exsanguinated under isoflurane anaesthesia.
On completion of the gross pathology examination, the tissues/organs listed in table 5 in ‘Any other information on results, incl. tables’ were collected, weighed and preserved in 10% neutral buffered formalin (NBF) from all adult (parents) animals. In addition, the tissues shown in table 6 in ‘Any other information on results, incl. tables’ were collected, weighed and preserved from randomly selected 5 males and 5 females from each group. The organ weights as percentage of body weights and brain weights were determined and presented in the report.

HISTOPATHOLOGY: Table 6 in ‘Any other information on materials and methods incl. tables’.
Tissues/organs collected from randomly selected 5 males and 5 females in the control and high dose groups (including reproductive organs) were examined microscopically for histopathological changes. Further, liver and kidneys in randomly selected parental male and female rats and lungs from all randomly selected females of lower dose groups were also subjected for microscopic examination. The thyroid gland from all adult males and females, from all the groups was also evaluated. All gross lesions were examined from all the groups. The reproductive organs were examined from the non-pregnant females. The tissues were processed for routine paraffin embedding and 4 - 5 micron thickness sections were stained with Mayer’s Haematoxylin and Eosin stain. In addition, testes were sectioned at 3-4 µm thickness and stained with PAS reagent and haematoxylin to aid in the qualitative assessment of spermatogenesis. Unused tissues were archived.
Statistics:
Data captured using ProvantisTM : Parameters of body weight, organ weights and organ weight ratios body weight, laboratory investigations - Haematology (Coagulation tests PT and APTT data were directly captured in ProvantisTM ) and Clinical Chemistry data was analysed using Provantis built-in statistical tests. The statistical analysis of the experimental data was carried out using the validated package in Excel and also using licensed copies of SYSTAT Statistical package ver.12.0. All quantitative variables like neurological observations (neuromuscular observation/body temperature/body weights) were tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene’s test) within the group before performing a one-factor ANOVA modelling by treatment groups. Non-optimal (non-normal or heteroschedastic) data was transformed, before ANOVA is performed. Comparison of means between treatment groups and control group was done using Dunnett’s test when the overall treatment, ‘F’ test found significant. Derived data like net body weight change and food consumption were analysed using above mentioned methods. All analyses and comparisons were evaluated at the 5% (p<0.05) level. Statistically significant differences (p<0.05), indicated by the aforementioned tests were designated throughout the report as stated below:
*: Significantly different from the vehicle control group
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were no clinical signs observed in both sexes in the 100 and 300 mg/kg bw/day dose groups. The following transient clinical signs were observed in the high dose (1000 mg/kg bw/day) group animals for a few hours after dose formulation administration and the animals recovered within 1-2 hours. (see tables 2 and 3 in ‘Any other information on results incl. tables’). The ataxia and recumbence were observed for a short period of time post-dose administration and the animals recovered within hours, indicating an adaptation to treatment.
Mortality:
no mortality observed
Description (incidence):
There were no mortalities observed.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weights and net weight gains were unaffected by the treatment at the low and mid doses in both sexes when compared to vehicle control. At the 1000 mg/kg bw/day dose, the mean body weights on Day 42 and absolute weight gain (Day 1-42) were significantly lower in males. On days 22, 29 and 36, the mean body weights were apparently lower without statistical significance. In females, the mean body weight and absolute weight gains were comparable with the vehicle control. Hence, the treatment decreased the body weight in males towards the end of treatment at 1000 mg/kg bw/day in males.

DURING GESTATION
Treatment had no effect on the maternal body weights measured during different intervals of the gestation period at all the doses tested. Incidence of significantly lower absolute weight gain during gestation days (GD) 0-7 was observed at 300 mg/kg bw/day. This statistically significant difference was toxicologically not significant as there was no dose-dependency observed.

DURING LACTATION
Treatment had no effects on the maternal body weights measured during different intervals of the lactation period at all the doses tested when compared to the vehicle control. Incidences of significantly higher absolute weight gain was observed during lactation day (LD) 0-4 at 100 mg/kg bw/day. This statistically significant difference was toxicologically not significant as there was no dose-dependency observed.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The food consumption was not altered throughout the treatment period when compared to the vehicle control. Incidences of significantly lower food consumption during days 1-8 in the 1000 mg/kg bw/day dose group in males and during days 1-8 in the 300 and 1000 mg/kg bw/day dose groups in females. These statistically significant differences observed in food consumption were toxicologically not significant as the mean body weights were not altered by the treatment.

DURING GESTATION
Significantly lower total and per day food consumption during GD 0-7 was observed at 1000 mg/kg bw/day. This statistically significant decrease in food consumption was toxicologically not significant as the mean maternal body weights were not altered during the same period.

DURING LACTATION
Significantly lower total and per day food consumption during LD 0-4 and 0-13 was observed at 1000 mg/kg bw/day. This statistically significant decrease in food consumption was toxicologically not significant as the mean maternal body weights were not altered during the same period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no test item-related changes in haematology parameters in males and females. All the minimal differences and the statistical significances obtained were considered as incidental changes in the absence of dose relation. The prothrombin time and APTT values were not affected by test item administration.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, increased plasma glucose concentration was noted in both males (65%) and females (45%) and was considered related to test item related change. Lower BUN value was noted in 1000 mg/kg bw/day males and ≥300 mg/kg bw/day females. This finding was not associated with any morphological correlates in kidneys and liver. Further, there were no changes in urinalysis parameters and hence considered as toxicologically insignificant. All the other changes including the statistical significances obtained were considered as toxicologically insignificant in the absence of dose relation/correlating morphological alterations.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no significant intergroup differences in the urinalysis parameters.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related abnormalities were observed for home cage and handling observations, open field observations, sensory observations, grip strength, landing hind limb foot-splay, body temperature and body weights recorded at the end of functional tests, in all the tested dose groups in both sexes.
For motor activity, higher ambulatory time (sec) at interval 1 was observed at 300 mg/kg bw/day in males. This change was considered incidental as there was no dose-dependency observed.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
An increase in liver weight was noted in 1000 mg/kg bw/day males and females and considered as test item related. This weight increase was microscopic correlated with hepatocellular hypertrophy. At 1000 mg/kg bw/day, increased seminal vesicle and coagulating gland weight without any associated microscopic changes was considered as toxicologically insignificant and not related to test item administration. All the other minor variations in weights were also considered as incidental findings as the values were within the range of normal biological variation.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Grossly, thickening of non-glandular stomach noted in males at 1000 mg/kg bw/day (2/10) was considered related to test item administration. Microscopically, non-glandular epithelial hyperplasia was noted in these rats. All the other gross lesions were considered as spontaneous and or associated with physiological changes (uterus-dilatation). The cause of infertility could not be determined for the two non-pregnant females. Dilatation of uterus was noted in these animals on gross and microscopic examination and ovaries were normal.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item related microscopic changes were present in liver and stomach. In the liver, hepatocellular hypertrophy was noted at 1000 mg/kg bw/day in males and females. This finding was of minimal and mild degree in severity of centrilobular distribution without any significant alteration in biochemical parameters. This was considered as an adaptive response to the test item administration. The non-glandular epithelial hyperplasia of stomach in 1000 mg/kg/day males and females and a single incidence of focal non-glandular ulceration in 1000 mg/kg bw/day females were considered as local responses to the presence of test item. All the other microscopic findings in test item treated groups were considered either as incidental/spontaneous changes common to this age group rats.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
clinical biochemistry
Key result
Critical effects observed:
no

Dose fromulation homogeneity and stability

The test item, was observed to be stable in the vehicle at dose concentrations of 1.15 mg/mL and 250.19 mg/mL up to 6 days when stored at room temperature. The test item was found to be homogeneous in all the dose formulation samples and they met the acceptance limits of variation (85 to 115%) from the claimed concentrations and had a %RSD of less than 10 %.

TABLE 1. Summary of Detailed Clinical Examination, Clinical Signs and Mortality – Males

 
Observation Type: All Types Male
From Day 1 (Start Date) to 43 (Start Date) G1 G2 G3 G4
  0 100 300 1000
  mg/kg/day mg/kg/day mg/kg/day mg/kg/day
Normal 10 10 10 10
Gait Characteristics, Ataxia (uncoordinated, staggering and wobbling gait) 0 0 0 7
Piloerection, Slight 0 0 0 10
Salivation, Slight 0 0 0 10
Posture, Recumbent (limbs spread out or lying on one side) 0 0 0 2
Values = Number of Animals Affected

TABLE 2. Summary of Detailed Clinical Examination, Clinical Signs and Mortality – Females
Observation Type: All Types Female

From Day 1 (Start Date) to 57 (Start Date)
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
Normal 10 10 10 10
Gait Characteristics, Ataxia (uncoordinated, staggering and wobbling gait) 0 0 0 5
Piloerection, Slight 0 0 0 10
Salivation, Slight 0 0 0 10
Posture, Recumbent (limbs spread out or lying on one side) 0 0 0 2
Total litter loss 1 2 1 5
Not littered 0 0 1 1
Values = Number of Animals Affected

TABLE 3. Summary of Functional Observation Battery – Males
         
Parameters Group
Dose
G1
0
G2
100
G3
300
G4
1000
(mg/kg/ day)
  No. of rats 5 5 5 5
Neurobehavioral Observations          
Home Cage Observations
Posture
Sitting or Standing alert, watching 4 4 4 4
Rearing 1 1 1 1
Abnormal Vocalizations
Absent 5 5 5 5
Tremors
Absent 5 5 5 5
Convulsions - Clonic Movement
Absent 5 5 5 5
Convulsions - Tonic Movement
Absent 5 5 5 5
Handling Observations
Ease of Removing from Cage
Easy (minimally avoids capture but is not aggressive) 5 5 5 5
Handling Reactivity
Moderately low (slight resistance) 5 5 5 5
Palpebral Closure
Eyelids wide open i.e., Normal 5 5 5 5
Eye Examination
Normal 5 5 5 5
Piloerection
Absent 5 5 5 5
Lacrimation
Absent 5 5 5 5
Salivation
None (no external salivation observed) 5 5 5 5
Skin/Fur Examination
Normal   5 5 5 5
Perineum Wetness          
Absent 5 5 5 5
Respiration
Normal 5 5 5 5
Muscle Tone
Moderate (animal is neither very relaxed nor tense; Normal muscle tone) 5 5 5 5
Extensor Thrust Response
Moderate (clearly detectable; Normal) 5 5 5 5
Open Field Observations
Gait
Normal gait 5 5 5 5
Posture
Normal posture 5 5 5 5
Tremors
Absent 5 5 5 5
Mobility Score
No impairment 5 5 5 5
Arousal Level
Alert (some exploratory movements; normal) 5 5 5 5
Clonic Movement
Absent 5 5 5 5
Tonic Movement
Absent 5 5 5 5
Stereotypic Behavior
Absent 5 5 5 5
Bizarre Behavior
Absent   5 5 5 5

TABLE 3 contd. Summary of Functional Observation Battery – Males
           
Parameters Group
Dose
G1
0
G2
100
G3
300
G4
1000
(mg/kg/ day)
  No. of rats 5 5 5 5
Urination          
Absent 4 3 4 5
Normal 1 2 1 0
Defecation
Absent 3 4 4 3
Normal 2 1 1 2
Abnormal Vocalization
Absent 5 5 5 5
Rears 32 32 29 22
Sensory Reactivity Measurements
Approach Response
Normal response (approaches towards the object) 5 5 5 5
Touch Response
Normal Response (spontaneously lifts tail when touched) 5 5 5 5
Click Response
Normal Response (clear movements of the head, neck and ears) 5 5 5 5
Tail-Pinch Response
Normal Response (Flinches, moves rapidly and/or vocalizes) 5 5 5 5
Pupil Response
Normal 5 5 5 5
Aerial Righting Reflex
Normal 5 5 5 5
Physiological Observation
Body Temperature (°C) Mean 37.42 37.80 37.56 37.40
Body Weight (g) Mean 353.47 328.68 356.86 329.51
Hindlimbs Footsplay (mm)
Average Mean 69.60 71.27 75.00 71.20
  SD 14.37 11.49 8.16 13.12
Grip Strength (gf)          
Forelimbs Grip Strength
Average Mean 1024.13 1028.67 1045.93 1022.33
SD 4.79 17.00 27.63 25.74
Hindlimbs Grip Strength
Average Mean 858.67 872.33 878.07 887.07
SD 39.37 68.27 38.27 35.36
Motor Activity Score
Stereotypic Time (sec)
Interval 1 Mean 139.00 114.80 81.20 97.20
SD 53.63 20.35 23.68 30.13
Interval 2 Mean 150.20 117.40 97.20 90.80
SD 75.04 52.28 33.80 39.23
Interval 3 Mean 55.40 78.20 76.20 81.00
SD 52.36 37.31 25.30 44.84
Total Mean 344.60 310.40 254.60 269.00
SD 130.34 80.75 24.52 93.55
Ambulatory Time (sec)
Interval 1 Mean 319.40 310.00 436.40* 360.80
SD 51.39 84.77 32.12 52.37
Interval 2 Mean 110.00 189.00 237.20 186.40
SD 58.61 89.63 56.45 89.72
Interval 3 Mean 56.80 104.60 169.20 132.00
SD 55.18 119.06 85.20 98.15
Total Mean 486.20 603.60 842.80 679.20
  SD 135.07 282.05 157.23 202.71
Horizontal Counts          
Interval 1 Mean 3180.80 3032.80 4242.80 3495.00
SD 764.67 739.47 1091.17 929.10
Interval 2 Mean 1109.00 1514.80 2071.00 1577.80
SD 530.31 664.95 731.59 914.18
Interval 3 Mean 495.00 808.80 1109.60 1184.80
SD 423.03 872.51 455.35 922.02
Total Mean 4784.80 5356.40 7423.40 6257.60
SD 1493.72 2201.95 1702.21 2417.27
Ambulatory Counts
Interval 1 Mean 2413.80 2425.00 3463.00 2822.40
SD 528.34 703.56 889.04 743.44
Interval 2 Mean 708.00 1131.40 1611.60 1228.00
SD 346.57 562.91 574.00 719.13
Interval 3 Mean 336.20 576.20 831.00 920.60
SD 290.90 720.17 357.96 740.89
Total Mean 3458.00 4132.60 5905.60 4971.00
SD 940.51 1913.18 1348.61 1892.36
*: Significantly different from vehicle control group

TABLE 4. Summary of Functional Observation Battery – Females
           
Parameters Group
Dose
G1
0
G2
100
G3
300
G4
1000
(mg/kg/ day)
  No. of rats 5 5 5 5
Neurobehavioral Observations          
Home Cage Observations
Posture
Sitting or Standing alert, watching 3 2 4 4
Rearing 2 3 1 1
Abnormal Vocalizations
Absent 5 5 5 5
Tremors
Absent 5 5 5 5
Convulsions - Clonic Movement
Absent 5 5 5 5
Convulsions - Tonic Movement
Absent 5 5 5 5
Handling Observations
Ease of Removing from Cage
Easy (minimally avoids capture but is not aggressive) 5 5 5 5
Handling Reactivity
Moderately low (slight resistance) 5 5 5 5
Palpebral Closure
Eyelids wide open i.e., Normal 5 5 5 5
Eye Examination
Normal 5 5 5 5
Piloerection
Absent 5 5 5 5
Lacrimation
Absent 5 5 5 5
Salivation
None (no external salivation observed) 5 5 5 5
Skin/Fur Examination
Normal   5 5 5 5
Perineum Wetness          
Absent 5 5 5 5
Respiration
Normal 5 5 5 5
Muscle Tone
Moderate (animal is neither very relaxed nor tense; Normal muscle tone) 5 5 5 5
Extensor Thrust Response
Moderate (clearly detectable; Normal) 5 5 5 5
Open Field Observations
Gait
Normal gait 5 5 5 5
Posture
Normal posture 5 5 5 5
Tremors
Absent 5 5 5 5
Mobility Score
No impairment 5 5 5 5
Arousal Level
Alert (some exploratory movements; normal) 5 5 5 5
Clonic Movement
Absent 5 5 5 5
Tonic Movement
Absent 5 5 5 5
Stereotypic Behavior
Absent 5 5 5 5
Bizarre Behavior
Absent   5 5 5 5
Urination          
Absent 4 5 4 5
Normal 1 0 1 0
Defecation
Absent 3 5 3 2
Normal 2 0 2 3
Abnormal Vocalization
Absent 5 5 5 5
Rears 41 41 41 50
Sensory Reactivity Measurements
Approach Response
Normal response (approaches towards the object) 5 5 5 5
Touch Response
Normal Response (spontaneously lifts tail when touched) 5 5 5 5
Click Response
Normal Response (clear movements of the head, neck and ears) 5 5 5 5
Tail-Pinch Response
Normal Response (Flinches, moves rapidly and/or vocalizes) 5 5 5 5
Pupil Response
Normal 5 5 5 5
Aerial Righting Reflex
Normal 5 5 5 5
Physiological Observation
Body Temperature (°C) Mean 37.92 37.80 38.02 37.86
Body Weight (g) Mean 239.31 257.24 243.86 235.89
Hindlimbs Footsplay (mm)
Average Mean 884.00 908.07 908.60 890.53
  SD 25.43 26.02 17.54 23.37
Grip Strength (gf)          
Forelimbs Grip Strength
Average Mean 800.20 788.40 783.00 753.47
SD 45.29 44.45 53.84 39.45
Hindlimbs Grip Strength
Average Mean 63.20 61.00 65.80 69.93
SD 5.54 6.70 7.14 16.58
Motor Activity Score
Stereotypic Time (sec)
Interval 1 Mean 116.00 120.40 87.60 108.20
SD 45.37 11.28 25.53 29.91
Interval 2 Mean 122.20 156.80 105.20 124.60
SD 57.82 27.13 42.61 61.52
Interval 3 Mean 91.40 96.20 115.00 53.40
SD 28.28 10.76 79.97 42.56
Total Mean 329.60 373.40 307.80 286.20
SD 98.79 25.45 137.95 89.63
Ambulatory Time (sec)
Interval 1 Mean 268.00 278.00 292.60 292.40
SD 11.81 56.71 73.10 66.87
Interval 2 Mean 150.80 168.60 150.60 137.80
SD 43.56 34.24 92.83 61.72
Interval 3 Mean 90.00 124.20 90.60 51.40
SD 55.76 42.36 44.17 51.44
Total Mean 508.80 570.80 533.80 481.60
  SD 92.12 100.19 190.90 119.57
Horizontal Counts          
Interval 1 Mean 2411.40 2629.40 2607.00 2389.20
SD 466.09 877.16 1184.84 616.36
Interval 2 Mean 1179.00 1467.00 1237.40 1011.00
SD 219.15 293.61 992.66 406.65
Interval 3 Mean 647.20 1053.20 776.40 354.20
SD 356.14 399.70 536.37 340.60
Total Mean 4237.60 5149.60 4620.80 3754.40
SD 449.07 1287.83 2641.45 763.89
Ambulatory Counts
Interval 1 Mean 1803.60 1957.60 1963.00 1834.00
SD 355.27 688.91 839.55 499.35
Interval 2 Mean 789.20 972.80 846.00 660.60
SD 189.71 164.04 680.37 296.68
Interval 3 Mean 413.80 747.40 408.00 233.20
SD 324.88 339.72 310.98 260.09
Total Mean 3006.60 3677.80 3217.00 2727.80
  SD 501.96 915.81 1695.82 503.65

TABLE 5. Summary of Body Weights (g) and Net Body Weight Gains (g) – Males
Sex: Male


Day(s) Relative to Start Date
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
Body 1 [a] Mean 261.42 259.62 261.92 259.62
Weight   SD 12.96 13.56 11.68 13.85
(g)   N 10 10 10 10
  8 [a] Mean 277.40 274.38 276.47 265.19
    SD 15.55 18.14 18.18 19.59
    N 10 10 10 10
  15 [a] Mean 292.53 285.69 286.97 277.31
    SD 16.27 18.38 20.51 21.37
    N 10 10 10 10
  22 [a] Mean 306.18 295.85 297.29 285.54
    SD 22.05 20.92 20.53 23.10
    N 10 10 10 10
  29 [a] Mean 327.01 313.51 314.76 300.67
    SD 22.10 23.92 25.39 23.05
    N 10 10 10 10
  36 [a] Mean 344.56 329.04 332.37 311.72
    SD 22.60 27.19 28.46 28.52
    N 10 10 10 10
  42 [a] Mean 355.65 340.81 342.11 317.78 *
    SD 22.93 27.44 27.40 25.76
    N 10 10 10 10
Absolute 1 → 42 [a] Mean 94.23 81.19 80.19 58.15 *
Weight Gain   SD 13.63 18.29 17.96 16.62
(g)   N 10 10 10 10
[a] - Anova & Dunnett(Log): * = p < 0.05
*: Significantly different from vehicle control group

TABLE 6. Summary of Body Weights (g) and Net Body Weight Gains (g) – Females
Sex: Female


Day(s) Relative to Start Date
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
Body 1 [a] Mean 192.21 192.52 190.77 189.57
Weight   SD 9.39 7.63 6.45 8.47
(g)   N 10 10 10 10
  8 [a] Mean 195.36 195.53 196.83 187.08
    SD 7.86 8.74 10.01 11.64
    N 10 10 10 10
  15 [a] Mean 199.94 203.49 202.79 197.43
    SD 9.20 8.17 12.22 13.23
    N 10 10 10 10
Absolute 1 → 15 [a] Mean 7.74 10.97 12.02 7.87
Weight Gain   SD 4.26 4.26 7.62 9.40
(g)   N 10 10 10 10
[a] - Anova & Dunnett(Log)

TABLE 7. Summary of Food Consumption (g/rat/day) – Males
Food Mean Daily Consumption (g/animal/day)
Sex: Male

Day(s) Relative to
Start Date
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
1 → 8

Mean
SD

N

17.57
1.48
10
17.65
1.63
10
17.23
1.31
10
14.75 *
2.29
10
8 → 15 Mean 19.47 18.48 18.32 18.50
  SD 0.73 1.12 1.47 1.10
  N 10 10 10 10
29 → 36 Mean 17.93 17.17 17.46 18.26
  SD 1.14 1.14 1.11 1.44
  N 10 10 10 10
36 → 42 Mean 18.41 18.20 18.32 18.15
  SD 0.81 1.37 0.95 0.58
  N 10 10 10 10
Anova & Dunnett(Log): * = p < 0.05 
*: Significantly different from vehicle control group 

TABLE 8. Summary of Food Consumption (g/rat/day) – Females
Food Mean Daily Consumption (g/animal/day)
Sex: Female

Day(s) Relative to
Start Date
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
1 → 8

Mean
SD

N

14.19
1.44
10
13.37
0.56
10
12.91 *
1.43
10
8.50 *
0.30
10
8 → 15 Mean 15.33 14.82 14.88 15.32
  SD 1.18 0.66 1.58 1.98
  N 10 10 10 10
Anova & Dunnett(Log): * = p < 0.05; 
*: Significantly different from vehicle control group 

TABLE 9. Summary of Maternal Body Weights (g) and Weight Change During Gestation Period
Sex: Female


Day(s) Relative to Mating
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
Body 0 [a] Mean 199.76 202.25 206.24 199.48
Weight   SD 11.78 8.15 12.36 10.86
(g)   N 10 10 9 9
  7 [a] Mean 229.51 230.15 227.63 224.62
    SD 10.73 8.46 11.20 13.43
    N 10 10 9 9
  14 [a] Mean 259.36 256.98 251.33 249.05
    SD 12.16 14.64 13.74 17.22
    N 10 10 9 9
  20 [a] Mean 313.00 309.19 303.38 301.82
    SD 19.79 21.18 16.01 25.45
    N 10 10 9 9
Absolute 0→7 [a] Mean 29.75 27.90 21.39 * 25.14
Weight Gain   SD 4.56 4.65 4.80 6.68
(g)   N 10 10 9 9
  7 →14 [a] Mean 29.85 26.83 23.70 24.43
    SD 5.62 10.82 3.84 5.51
    N 10 10 9 9
  14 →20 [a] Mean 53.63 52.21 52.05 52.77
    SD 9.55 17.24 5.45 9.09
    N 10 10 9 9
  0 →20 [a] Mean 113.24 106.93 97.14 102.34
    SD 15.19 18.56 8.04 18.20
    N 10 10 9 9
[a] - Anova & Dunnett(Log): * = p < 0.05 
*: Significantly different from vehicle control group 

TABLE 10. Summary of Maternal Food Intake (g) During Gestation Period
Sex: Female


Day(s) Relative to Mating
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
Total Food 0-7 [a] Mean 117.52 115.49 106.15 103.08 *
Consumption   SD 14.96 7.40 13.05 10.53
(g)   N 10 10 9 9
  7-14 [a] Mean 135.45 132.60 122.83 125.14
    SD 13.09 9.24 9.35 14.06
    N 10 10 9 9
  14-20 [a] Mean 128.27 133.48 122.88 123.19
    SD 17.22 18.65 16.81 10.70
    N 10 10 9 9
  0-20 [a] Mean 381.24 381.57 351.85 351.41
    SD 41.73 22.70 35.26 29.87
    N 10 10 9 9
Food Mean 0-7 [a] Mean 16.79 16.50 15.16 14.73 *
Consumption   SD 2.14 1.06 1.86 1.50
(g/day)   N 10 10 9 9
  7-14 [a] Mean 19.35 18.94 17.55 17.88
    SD 1.87 1.32 1.34 2.01
    N 10 10 9 9
  14-20 [a] Mean 21.38 22.25 20.48 20.53
    SD 2.87 3.11 2.80 1.78
    N 10 10 9 9
  0-20 [a] Mean 19.06 19.08 17.59 17.57
    SD 2.09 1.14 1.76 1.49
    N 10 10 9 9
[a] - Anova & Dunnett(Log): * = p < 0.05
*: Significantly different from vehicle control group

TABLE 11. Summary of Maternal Body Weights (g) and Weight Change During Lactation Period
Sex: Female


Day(s) Relative to Littering
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
Body 0 [a] Mean 230.70 219.91 230.13 218.08
Weight   SD 19.33 20.57 15.38 21.43
(g)   N 10 10 9 9
  4 [a] Mean 237.33 240.56 238.61 224.86
    SD 16.34 20.24 15.50 17.36
    N 9 9 9 5
  13 [a] Mean 252.65 259.32 250.96 241.29
    SD 17.54 17.97 13.73 25.58
    N 9 8 8 4
Absolute 0 - 4 [a] Mean 5.25 18.55 * 8.48 7.27
Weight Gain   SD 7.97 9.91 10.57 10.26
(g)   N 9 9 9 5
  4 - 13 [a] Mean 15.32 18.49 11.85 15.19
    SD 12.68 10.65 10.03 5.92
    N 9 8 8 4
  0 - 13 [a] Mean 20.57 36.54 19.93 26.83
    SD 11.20 15.57 15.37 3.10
    N 9 8 8 4
[a] - Anova & Dunnett(Log): * = p < 0.05  
*: Significantly different from vehicle control group

TABLE 12. Summary of Maternal Food Intake (g) During Lactation Period
Sex: Female


Day(s) Relative to Littering
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
 
Total Food 0-4 [a] Mean 102.48 101.23 98.40 75.24 *
Consumption   SD 15.03 16.06 15.40 16.54  
(g)   N 9 9 9 4  
  4-13 [a] Mean 383.85 399.82 392.05 354.49  
    SD 23.25 34.09 30.66 63.59  
    N 9 8 8 4  
  0-13 [a] Mean 486.33 504.12 493.88 429.73 *
    SD 19.86 41.92 36.10 59.11  
    N 9 8 8 4  
Food Mean 0-4 [a] Mean 25.62 25.31 24.60 18.81 *
Consumption   SD 3.76 4.02 3.85 4.14  
(g/day)   N 9 9 9 4  
  4-13 [a] Mean 42.65 44.42 43.56 39.39  
    SD 2.58 3.79 3.41 7.07  
    N 9 8 8 4  
  0-13 [a] Mean 37.41 38.78 37.99 33.06 *
    SD 1.53 3.22 2.78 4.55  
    N 9 8 8 4  
[a] - Anova & Dunnett(Log): * = p < 0.05
*: Significantly different from vehicle control group

TABLE 13. Summary of Haematological and Coagulation Parameters – Males
Sex: Male


Day(s) Relative to Start Date
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
RBC Day 16 Mean 9.05 9.05 8.60 8.93
(10^12/L)   SD 0.16 0.39 0.22 0.38
    N 5 5 5 5
    %Diff   -- -- --
HGB Day 16 Mean 167 166 161 164
(g/L)   SD 5 3 5 3
    N 5 5 5 5
    %Diff   -- -- --
HCT Day 16 Mean 0.499 0.504 0.481 0.495
(L/L)   SD 0.019 0.010 0.015 0.010
    N 5 5 5 5
    %Diff   -- -- --
MCV Day 16 Mean 55.2 55.8 55.9 55.5
(fL)   SD 2.2 1.5 0.4 1.3
    N 5 5 5 5
    %Diff   -- -- --
MCH Day 16 Mean 18.5 18.4 18.8 18.4
(pg)   SD 0.6 0.5 0.2 0.6
    N 5 5 5 5
    %Diff   -- -- --
MCHC Day 16 Mean 335 330 * 336 332
(g/L)   SD 3 1 1 4
    N 5 5 5 5
    %Diff   2 -- --
Retic A Day 16 Mean 0.244 0.217 0.225 0.264
(10^12/L)   SD 0.043 0.025 0.019 0.058
    N 5 5 5 5
    %Diff   -- -- --
PLT Day 16 Mean 1100 968 1072 1088
(10^9/L)   SD 56 97 86 124
    N 5 5 5 5
    %Diff   -- -- --
WBC Day 16 Mean 6.36 5.85 6.43 7.61
(10^9/L)   SD 0.99 1.00 1.68 1.17
    N 5 5 5 5
    %Diff   -- -- --
Neut A Day 16 Mean 1.02 0.96 1.00 1.02
(10^9/L)   SD 0.22 0.20 0.28 0.22
    N 5 5 5 5
    %Diff   -- -- --
Lymp A Day 16 Mean 5.11 4.66 5.20 6.26
(10^9/L)   SD 0.88 0.97 1.50 0.96
    N 5 5 5 5
    %Diff   -- -- --
Mono A Day 16 Mean 0.15 0.16 0.14 0.21
(10^9/L)   SD 0.05 0.05 0.08 0.07
    N 5 5 5 5
    %Diff   -- -- --
Baso A Day 16 Mean 0.01 0.01 0.01 0.01
(10^9/L)   SD 0.00 0.00 0.01 0.00
    N 5 5 5 5
    %Diff   -- -- --
Eosi A Day 16 Mean 0.06 0.06 0.05 0.09
(10^9/L)   SD 0.04 0.03 0.01 0.07
    N 5 5 5 5
    %Diff   -- -- --
PT Day 16 Mean 16.6 15.3 14.7 * 16.4
    SD 1.3 1.1 0.7 0.5
(seconds)   N 5 5 5 5
    %Diff   -- 11 --
APTT Day 16 Mean 15.5 15.2 18.3 14.8
(seconds)   SD 1.0 1.5 2.2 2.0
    N 5 5 5 5
    %Diff   -- -- --
--: Not calculated as difference is not statistically significant
*: Significantly different from the vehicle control group at p < 0.05

TABLE 14. Summary of Haematological and Coagulation Parameters – Females
Sex: Female


Day(s) Relative to Start Date
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
RBC Day 16 Mean 8.34 8.36 8.28 7.95
(10^12/L)   SD 0.17 0.47 0.60 0.36
    N 5 5 5 5
    %Diff   -- -- --
HGB Day 16 Mean 157 159 153 154
(g/L)   SD 1 3 6 10
    N 5 5 5 5
    %Diff   -- -- --
HCT Day 16 Mean 0.458 0.475 0.453 0.463
(L/L)   SD 0.009 0.016 0.015 0.020
    N 5 5 5 5
    %Diff   -- -- --
MCV Day 16 Mean 54.9 56.9 54.9 58.3 *
(fL)   SD 0.4 2.5 2.4 1.6
    N 5 5 5 5
    %Diff   -- -- 6
MCH Day 16 Mean 18.9 19.0 18.5 19.4
(pg)   SD 0.3 1.0 0.9 0.6
    N 5 5 5 5
    %Diff   -- -- --
MCHC Day 16 Mean 343 334 338 333
(g/L)   SD 5 6 5 10
    N 5 5 5 5
    %Diff   -- -- --
Retic A Day 16 Mean 0.268 0.261 0.207 0.249
(10^12/L)   SD 0.055 0.042 0.041 0.088
    N 5 5 5 5
    %Diff   -- -- --
PLT Day 16 Mean 1132 1017 1093 1177
(10^9/L)   SD 143 91 68 272
    N 5 5 5 5
    %Diff   -- -- --
WBC Day 16 Mean 5.38 4.40 4.89 5.28
(10^9/L)   SD 0.43 0.81 1.47 1.71
    N 5 5 5 5
    %Diff   -- -- --
Neut A Day 16 Mean 0.81 0.58 0.74 0.63
(10^9/L)   SD 0.04 0.14 0.14 0.16
    N 5 5 5 5
    %Diff   -- -- --
Lymp A Day 16 Mean 4.34 3.67 3.99 4.49
(10^9/L)   SD 0.41 0.73 1.35 1.57
    N 5 5 5 5
    %Diff   -- -- --
Mono A Day 16 Mean 0.12 0.09 0.10 0.09
(10^9/L)   SD 0.01 0.04 0.01 0.01
    N 5 5 5 5
    %Diff   -- -- --
Baso A Day 16 Mean 0.01 0.01 0.01 0.00
(10^9/L)   SD 0.01 0.01 0.01 0.01
    N 5 5 5 5
    %Diff   -- -- --
Eosi A Day 16 Mean 0.08 0.04 0.05 0.05
(10^9/L)   SD 0.04 0.02 0.02 0.03
    N 5 5 5 5
    %Diff   -- -- --
PT Day 16 Mean 15.9 14.8 17.5 16.2
    SD 1.3 0.4 1.1 1.2
(seconds)   N 5 5 5 5
    %Diff   -- -- --
APTT Day 16 Mean 14.7 13.9 16.9 13.1
(seconds)   SD 1.6 1.5 2.3 1.9
    N 5 5 5 5
    %Diff   -- -- --
--: Not calculated as difference is not statistically significant
*: Significantly different from the vehicle control group at p < 0.05

TABLE 15. Summary of Clinical Chemistry Parameters – Males
Sex: Male


Day(s) Relative to Start Date
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
Glu Day 16 Mean 3.90 3.86 3.91 6.45 *
(mmol/L)   SD 0.19 0.29 0.44 0.82
    N 5 5 5 5
    %Diff   -- -- 65
BUN Day 16 Mean 5.05 4.31 4.14 3.12 *
(mmol/L)   SD 0.81 0.59 0.51 0.97
    N 5 5 5 5
    %Diff   -- -- 38
Creat Day 16 Mean 40 40 39 45 *
(µmol/L)   SD 2 4 2 4
    N 5 5 5 5
    %Diff   -- -- 14
AST Day 16 Mean 81 83 70 85
(U/L)   SD 9 18 11 8
    N 5 5 5 5
    %Diff   -- -- --
ALT Day 16 Mean 38 34 34 41
(U/L)   SD 4 8 9 6
    N 5 5 5 5
    %Diff   -- -- --
GGT Day 16 Mean 6 5 5 6
(U/L)   SD 1 0 1 1
    N 5 5 5 5
    %Diff   -- -- --
Alp Day 16 Mean 114 113 114 133
(U/L)   SD 9 9 24 33
    N 5 5 5 5
    %Diff   -- -- --
CK Day 16 Mean 354 315 280 356
(U/L)   SD 53 88 57 105
    N 5 5 5 5
    %Diff   -- -- --
T.Bil Day 16 Mean 1.62 2.19 1.76 1.66
(µmol/L)   SD 0.53 0.66 0.18 0.76
    N 5 3a 5 5
    %Diff   -- -- --
T.Chol Day 16 Mean 2.19 2.07 2.03 1.94
(mmol/L)   SD 0.24 0.23 0.24 0.20
    N 5 5 5 5
    %Diff   -- -- --
BA Day 16 Mean 31.8 9.6 * 15.8 34.0
(µmol/L)   SD 18.8 4.3 5.3 24.9
    N 5 5 5 5
    %Diff   70 -- --
Trig Day 16 Mean 1.04 0.89 0.70 0.65
(mmol/L)   SD 0.25 0.32 0.13 0.21
    N 5 5 5 5
    %Diff   -- -- --
T.Pro Day 16 Mean 69.4 70.7 69.9 68.9
(g/L)   SD 2.1 2.8 1.8 2.6
    N 5 5 5 5
    %Diff   -- -- --
ALB Day 16 Mean 31.6 32.5 30.8 31.0
(g/L)   SD 1.2 0.7 1.0 1.1
    N 5 5 5 5
    %Diff   -- -- --
GLOB Day 16 Mean 37.8 38.2 39.1 38.0
(g/L)   SD 1.0 2.3 0.9 1.8
    N 5 5 5 5
    %Diff   -- -- --
A/G Day 16 Mean 0.84 0.85 0.79 * 0.82
(ratio)   SD 0.02 0.04 0.01 0.03
    N 5 5 5 5
    %Diff   -- 6 --
Pi Day 16 Mean 2.34 2.11 * 2.31 2.43
(mmol/L)   SD 0.04 0.06 0.17 0.16
    N 5 5 5 5
    %Diff   10 -- --
Ca Day 16 Mean 2.63 2.59 2.62 2.41 *
(mmol/L)   SD 0.07 0.05 0.09 0.11
    N 5 5 5 5
    %Diff   -- -- 8
Na Day 16 Mean 146.8 147.6 148.3 149.0
(mEq/L)   SD 0.7 2.3 1.9 1.4
    N 5 5 5 5
    %Diff   -- -- --
K Day 16 Mean 3.53 3.89 3.71 3.93
(mEq/L)   SD 0.16 0.43 0.13 0.43
    N 5 5 5 5
    %Diff   -- -- --
Cl Day 16 Mean 102.7 102.3 102.7 104.1
(mEq/L)   SD 1.3 1.3 1.5 1.1
    N 5 5 5 5
    %Diff   -- -- --
a: Values below LLOQ (Lower Limit of Quantification for T.bil is below 0.40 µmol/L) were not considered for analysis.
--: Not calculated as difference is not statistically significant
*: Significantly different from the vehicle control group at p < 0.05

TABLE 16. Summary of Clinical Chemistry Parameters – Females
Sex: Female


Day(s) Relative to Start Date
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
Glu Day 16 Mean 4.70 4.85 5.70 6.83 *
(mmol/L)   SD 0.73 0.26 1.01 0.99
    N 5 5 5 5
    %Diff   -- -- 45
BUN Day 16 Mean 7.10 5.26 4.90 * 2.13 *
(mmol/L)   SD 1.35 1.50 1.46 0.17
    N 5 5 5 5
    %Diff   -- 31 70
Creat Day 16 Mean 52 47 52 50
(µmol/L)   SD 7 2 11 11
    N 5 5 5 5
    %Diff   -- -- --
AST Day 16 Mean 92 78 77 86
(U/L)   SD 6 5 8 15
    N 5 5 5 5
    %Diff   -- -- --
ALT Day 16 Mean 26 28 32 39
(U/L)   SD 4 8 12 7
    N 5 5 5 5
    %Diff   -- -- --
GGT Day 16 Mean 6 5 5 6
(U/L)   SD 1 1 1 1
    N 5 5 5 5
    %Diff   -- -- --
Alp Day 16 Mean 59 48 67 93 *
(U/L)   SD 15 6 21 27
    N 5 5 5 5
    %Diff   -- -- 59
CK Day 16 Mean 363 247 279 468
(U/L)   SD 117 67 29 191
    N 5 5 5 5
    %Diff   -- -- --
T.Bil Day 16 Mean 1.61 2.12 1.25 1.30
(µmol/L)   SD 0.92 0.60 0.64 0.32
    N 4a 5 5 4a
    %Diff   -- -- --
T.Chol Day 16 Mean 1.74 1.53 1.66 n 1.92
(mmol/L)   SD 0.35 0.16 0.17 0.52
    N 5 5 2a 5
    %Diff   -- -- --
BA Day 16 Mean 24.6 10.2 9.2 23.4
(µmol/L)   SD 31.3 5.1 3.4 6.4
    N 5 5 5 5
    %Diff   -- -- --
Trig Day 16 Mean 0.34 0.43 0.37 0.48
(mmol/L)   SD 0.12 0.15 0.04 0.12
    N 5 5 5 5
    %Diff   -- -- --
T.Pro Day 16 Mean 70.8 71.7 70.7 71.9
(g/L)   SD 1.7 4.9 2.7 4.4
    N 5 5 5 5
    %Diff   -- -- --
ALB Day 16 Mean 33.2 34.0 32.6 33.1
(g/L)   SD 1.1 2.4 1.4 2.1
    N 5 5 5 5
    %Diff   -- -- --
GLOB Day 16 Mean 37.5 37.7 38.1 38.8
(g/L)   SD 0.7 2.6 1.4 2.3
    N 5 5 5 5
    %Diff   -- -- --
A/G Day 16 Mean 0.88 0.90 0.85 0.85
(ratio)   SD 0.02 0.02 0.02 0.02
    N 5 5 5 5
    %Diff   -- -- --
Pi Day 16 Mean 1.86 1.76 1.75 2.16
(mmol/L)   SD 0.20 0.15 0.23 0.25
    N 5 5 5 5
    %Diff   -- -- --
Ca Day 16 Mean 2.56 2.59 2.52 2.44
(mmol/L)   SD 0.06 0.08 0.08 0.10
    N 5 5 5 5
    %Diff   -- -- --
Na Day 16 Mean 144.7 146.6 147.2 146.7
(mEq/L)   SD 1.9 1.1 0.6 1.4
    N 5 5 5 5
    %Diff   -- -- --
K Day 16 Mean 3.46 3.18 3.45 4.09
(mEq/L)   SD 0.28 0.22 0.61 0.51
    N 5 5 5 5
    %Diff   -- -- --
Cl Day 16 Mean 101.0 103.0 102.7 104.5 *
(mEq/L)   SD 1.0 1.2 0.9 1.8
    N 5 5 5 5
    %Diff   -- -- 3
a: Values below LLOQ (Lower Limit of Quantification for T.bil is below 0.40 µmol/L and for T.chol is below 1.30 mmol/L) were not considered for analysis.
n: Inappropriate for statistics 
--: Not calculated as difference is not statistically significant
*: Significantly different from the vehicle control group at p < 0.05

TABLE 17. Summary of Clinical Analysis of Urine – Males 
Group No. G1 G2 G3 G4
Dose (mg/kg Bwt/day) 0 100 300 1000
No. of rats 5 5 5 5
  Day 16      
Volume (mL) $ 13.0 15.0 13.0 14.0
Glucose* 0 0 0 0
Bilirubin* 2 0 0 0
Ketone Bodies* 5 3 3 2
Specific gravity $ 1.021 1.013 1.017 1.025
pH $ 7.9 7.6 7.4 7.5
Proteins* 5 2 3 3
Urobilinogen* # 0 0 0 0
Nitrite* 1 4 4 2
$: Mean value *: Incidences of findings
#: values less than or equal to 3.2 are normal and hence not considered as positive findings/incidence.

TABLE 18. Summary of Clinical Analysis of Urine – Females
Group No. G1 G2 G3 G4
Dose (mg/kg Bwt/day) 0 100 300 1000
No. of rats 5 5 5 5
  Day 16      
Volume (mL) $ 13.5 13.0 12.5 19.0
Glucose* 0 0 0 0
Bilirubin* 1 1 0 0
Ketone Bodies* 0 1 0 0
Specific gravity $ 1.020 1.019 1.017 1.015
pH $ 8.0 7.8 7.4 7.6
Proteins* 3 2 2 1
Urobilinogen* # 1 0 0 0
Nitrite* 4 5 3 5
$: Mean value *: Incidences of findings
#: values less than or equal to 3.2 are normal and hence not considered as positive findings/incidence.

TABLE 19. Summary of Thyroid Hormones Profile – Males
Group No.
Dose
(mg/kg/day)
Day No.   T4
ng/mL
TSH
ng/mL
G1 Day 42 Mean 23.00 0.69
0 SD 4.24 0.61
    N 10 10
G2 Day 42 Mean 18.06* 0.86
100 SD 1.96 0.40
  N 10 10
    % Diff 21 --
G3 Day 42 Mean 19.42* 0.82
300 SD 2.35 0.33
  N 10 10
    % Diff 16 --
G4 Day 42 Mean 16.52* 0.73
1000 SD 2.98 0.26
  N 10 10
    % Diff 28 --
--: Not calculated as difference is not statistically significant.
*: Significantly different from the vehicle control group at p < 0.05

TABLE 20. Summary of Thyroid Hormones Profile – Females
Group No.
Dose
(mg/kg/day)
Day No.   T4
ng/mL
TSH
ng/mL
G1 LD 13 Mean 14.84 0.48
0 SD 2.27 0.20
    N 9 9
G2 LD 13 Mean 15.72 0.50
100 SD 2.36 0.12
  N 8 8
    % Diff -- --
G3 LD 13 Mean 16.80 0.37
300 SD 2.67 0.18
  N 8 8
    % Diff -- --
G4 LD 13 Mean 14.72 0.47
1000 SD 2.23 0.20
  N 4 4
    % Diff -- --
--: Not calculated as difference is not statistically significant.

TABLE 21. Summary of Thyroid Hormones Profile – Pup Lactation Day 4
Group No.
Dose
(mg/kg/day)
Day No.   T4
ng/mL
TSH
ng/mL
G1 LD 4 Mean 12.22 0.28
0 SD 2.18 0.12
    N 9 9
G2 LD 4 Mean 12.16 0.33
100 SD 2.12 0.12
  N 7 7
    % Diff -- --
G3 LD 4 Mean 10.96 0.29
300 SD 1.67 0.12
  N 7 7
    % Diff -- --
G4 LD 4 Mean 9.75 0.41
1000 SD 2.23 0.33
  N 3 3
    % Diff -- --
--: Not calculated as difference is not statistically significant.

TABLE 22. Summary of Thyroid Hormones Profile – Pup Lactation Day 13
Group No.
Dose
(mg/kg/day)
Day No.   T4
ng/mL
TSH
ng/mL
G1 LD 13 Mean 18.50 0.36
0 SD 7.83 0.35
    N 9 9
G2 LD 13 Mean 16.77 0.38
100 SD 8.89 0.25
  N 8 8
    % Diff -- --
G3 LD 13 Mean 20.03 0.32
300 SD 7.00 0.29
  N 8 8
    % Diff -- --
G4 LD 13 Mean 14.12 0.31
1000 SD 2.42 0.17
  N 4 4
    % Diff -- --
--: Not calculated as difference is not statistically significant.

TABLE 23. Summary of Terminal Fasting Body Weights and Organ Weights – 
Reproductive Organs - Males
Sex: Male


Day(s) Relative to Start Date
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
Terminal Day 43 Mean 335.66 322.04 324.67 296.71 *
Fasting BW   SD 22.69 28.65 26.89 24.59
(g)   N 10 10 10 10
    %Diff   -- -- 12
Epididymides Day 43 Mean 1.4935 1.4450 1.5744 1.3504
(g)   SD 0.1522 0.1863 0.1145 0.1438
    N 10 10 10 10
    %Diff   -- -- --
Prostate Day 43 Mean 0.9308 1.0514 1.0659 0.8227
(g)   SD 0.1370 0.1978 0.2331 0.1389
    N 10 10 10 10
    %Diff   -- -- --
Seminal vesicles Day 43 Mean 1.5197 1.5618 1.3673 1.1843 *
& coagulating   SD 0.1933 0.2708 0.2279 0.1804
glands   N 10 10 10 10
(g)   %Diff   -- -- 22
Testes Day 43 Mean 3.9138 3.8008 3.6773 3.7619
(g)   SD 0.3619 0.1849 0.3904 0.4404
    N 10 10 10 10
    %Diff   -- -- --
Thyroid with Day 48~ Mean 0.0288 0.0286 0.0302 0.0285
parathyroids   SD 0.0060 0.0043 0.0055 0.0075
(g)   N 10 10 10 10
    %Diff   -- -- --
Levator ani Day 43 Mean 1.1521 1.1792 1.0599 1.0376
bulbocavernosus   SD 0.1222 0.1262 0.1607 0.1470
muscle complex   N 10 10 10 10
(g)   %Diff   -- -- --
Cowper’s Day 43 Mean 0.1222 0.1315 0.1154 0.1096
glands   SD 0.0386 0.0248 0.0248 0.0282
(g)   N 10 10 10 10
    %Diff   -- -- --
Glans penis Day 43 Mean 0.0891 0.0934 0.0924 0.0857
(g)   SD 0.0077 0.0099 0.0063 0.0062
    N 10 10 10 10
    %Diff   -- -- --
~: Weighed after fixation
--: Not calculated as difference is not statistically significant
*: Significantly different from the vehicle control group at p < 0.05

TABLE 24. Summary of Terminal Fasting Body Weights and Organ to Body
Weight Ratios – Reproductive Organs - Males 
Sex: Male


Day(s) Relative to Start Date
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
Terminal Day 43 Mean 335.66 322.04 324.67 296.71 *
Fasting BW   SD 22.69 28.65 26.89 24.59
(g)   N 10 10 10 10
    %Diff   -- -- 12
Epididymides Day 43 Mean 0.4454 0.4527 0.4871 0.4563
(%)   SD 0.0395 0.0769 0.0460 0.0445
    N 10 10 10 10
    %Diff   -- -- --
Prostate Day 43 Mean 0.2779 0.3284 0.3307 0.2759
(%)   SD 0.0408 0.0628 0.0814 0.0291
    N 10 10 10 10
    %Diff   -- -- --
Seminal vesicles Day 43 Mean 0.4553 0.4859 0.4218 0.4037
& coagulating   SD 0.0702 0.0774 0.0709 0.0803
glands   N 10 10 10 10
(%)   %Diff   -- -- --
Testes Day 43 Mean 1.1658 1.1895 1.1344 1.2652 *
(%)   SD 0.0701 0.1273 0.1006 0.0654
    N 10 10 10 10
    %Diff   -- -- 9
Thyroid with Day 48~ Mean 0.0086 0.0089 0.0093 0.0097
parathyroids   SD 0.0020 0.0014 0.0015 0.0027
(%)   N 10 10 10 10
    %Diff   -- -- --
Levator ani Day 43 Mean 0.3431 0.3674 0.3283 0.3512
bulbocavernosus   SD 0.0271 0.0400 0.0538 0.0537
muscle complex   N 10 10 10 10
(%)   %Diff   -- -- --
Cowper’s Day 43 Mean 0.0365 0.0411 0.0358 0.0370
glands   SD 0.0111 0.0086 0.0085 0.0093
(%)   N 10 10 10 10
    %Diff   -- -- --
Glans penis Day 43 Mean 0.0266 0.0291 0.0286 0.0290
(%)   SD 0.0026 0.0033 0.0027 0.0020
    N 10 10 10 10
    %Diff   -- -- --
~: Weighed after fixation
--: Not calculated as difference is not statistically significant
*: Significantly different from the vehicle control group at p < 0.05

TABLE 25. Summary of Terminal Fasting Body Weights and Organ Weights –Males
Sex: Male


Day(s) Relative to Start Date
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
Terminal Day 43 Mean 331.50 309.94 342.62 308.99
Fasting BW   SD 26.70 29.97 24.95 18.12
(g)   N 5 5 5 5
    %Diff   -- -- --
Adrenals Day 43 Mean 0.0615 0.0637 0.0649 0.0607
(g)   SD 0.0096 0.0036 0.0091 0.0074
    N 5 5 5 5
    %Diff   -- -- --
Brain Day 43 Mean 2.0570 2.1944 2.0849 2.0442
(g)   SD 0.0882 0.1426 0.1043 0.1201
    N 5 5 5 5
    %Diff   -- -- --
Heart Day 43 Mean 0.8950 0.8748 0.9125 0.8914
(g)   SD 0.0993 0.0679 0.0945 0.0548
    N 5 5 5 5
    %Diff   -- -- --
Kidneys Day 43 Mean 2.0016 1.9522 2.1446 2.1872
(g)   SD 0.1368 0.1582 0.3248 0.1758
    N 5 5 5 5
    %Diff   -- -- --
Liver Day 43 Mean 7.5659 7.5518 8.5659 10.0726 *
(g)   SD 0.7353 0.9654 0.8578 0.4292
    N 5 5 5 5
    %Diff   -- -- 33
Pituitary Day 48~ Mean 0.0105 0.0115 0.0094 0.0097
(g)   SD 0.0021 0.0011 0.0028 0.0007
    N 5 5 5 5
    %Diff   -- -- --
Spleen Day 43 Mean 0.5844 0.6548 0.6598 0.5527
(g)   SD 0.0568 0.0711 0.0227 0.0501
    N 5 5 5 5
    %Diff   -- -- --
Thymus Day 43 Mean 0.3367 0.3497 0.4012 0.2827
(g)   SD 0.0670 0.0960 0.0523 0.0157
    N 5 5 5 5
    %Diff   -- -- --
~: Weighed after fixation
--: Not calculated as difference is not statistically significant.
*: Significantly different from the vehicle control group at p < 0.05

TABLE 26. Summary of Terminal Fasting Body Weights and Organ to Body  
 Weight Ratios – Males
Sex: Male


Day(s) Relative to Start Date
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
Terminal Day 43 Mean 331.50 309.94 342.62 308.99
Fasting BW   SD 26.70 29.97 24.95 18.12
(g)   N 5 5 5 5
    %Diff   -- -- --
Adrenals Day 43 Mean 0.0188 0.0208 0.0189 0.0197
(%)   SD 0.0042 0.0031 0.0018 0.0026
    N 5 5 5 5
    %Diff   -- -- --
Brain Day 43 Mean 0.6228 0.7106 * 0.6097 0.6619
(%)   SD 0.0419 0.0464 0.0251 0.0230
    N 5 5 5 5
    %Diff   14 -- --
Heart Day 43 Mean 0.2698 0.2832 0.2659 0.2895
(%)   SD 0.0173 0.0213 0.0104 0.0279
    N 5 5 5 5
    %Diff   -- -- --
Kidneys Day 43 Mean 0.6066 0.6309 0.6233 0.7074 *
(%)   SD 0.0609 0.0246 0.0577 0.0271
    N 5 5 5 5
    %Diff   -- -- 17
Liver Day 43 Mean 2.2815 2.4330 2.4977 * 3.2634 *
(%)   SD 0.0970 0.1454 0.1260 0.1096
    N 5 5 5 5
    %Diff   -- 9 43
Pituitary Day 48~ Mean 0.0032 0.0037 0.0027 0.0032
(%)   SD 0.0007 0.0005 0.0007 0.0004
    N 5 5 5 5
    %Diff   -- -- --
Spleen Day 43 Mean 0.1770 0.2117 * 0.1932 0.1789
(%)   SD 0.0209 0.0187 0.0126 0.0137
    N 5 5 5 5
    %Diff   20 -- --
Thymus Day 43 Mean 0.1012 0.1130 0.1171 0.0918
(%)   SD 0.0160 0.0315 0.0129 0.0087
    N 5 5 5 5
    %Diff   -- -- --
~: Weighed after fixation
--: Not calculated as difference is not statistically significant.
*: Significantly different from the vehicle control group at p < 0.05

TABLE 27. Summary of Terminal Fasting Body Weights and Organ to Brain  
Weight Ratios – Males 
Sex: Male


Day(s) Relative to Start Date
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
Brain Day 43 Mean 2.0570 2.1944 2.0849 2.0442
(g)   SD 0.0882 0.1426 0.1043 0.1201
    N 5 5 5 5
    %Diff   -- -- --
Adrenals Day 43 Mean 3.0064 2.9183 3.1095 2.9733
(%)   SD 0.5752 0.3096 0.3658 0.3432
    N 5 5 5 5
    %Diff   -- -- --
Heart Day 43 Mean 43.5037 39.9956 43.7234 43.6970
(%)   SD 4.2980 4.0483 3.4082 3.2119
    N 5 5 5 5
    %Diff   -- -- --
Kidneys Day 43 Mean 97.6321 88.9290 102.6609 106.8997
(%)   SD 10.3871 3.4501 13.2362 3.1588
    N 5 5 5 5
    %Diff   -- -- --
Liver Day 43 Mean 368.0663 343.6177 410.2766 493.4064 *
(%)   SD 35.1248 31.8014 27.2170 20.2049
    N 5 5 5 5
    %Diff   -- -- 34
Pituitary Day 48~ Mean 0.5080 0.5264 0.4474 0.4775
(%)   SD 0.0981 0.0540 0.1153 0.0647
    N 5 5 5 5
    %Diff   -- -- --
Spleen Day 43 Mean 28.3901 29.9244 31.7085 27.0248
(%)   SD 2.1312 3.7537 1.9274 1.7011
    N 5 5 5 5
    %Diff   -- -- --
Thymus Day 43 Mean 16.3026 15.9098 19.2489 13.8826
(%)   SD 2.7957 4.2537 2.4729 1.3234
    N 5 5 5 5
    %Diff   -- -- --
~: Weighed after fixation
--: Not calculated as difference is not statistically significant.
*: Significantly different from the vehicle control group at p < 0.05

TABLE 28. Summary of Terminal Fasting Body Weights and Organ Weights – 
Reproductive Organs - Females
Sex: Female


Day(s) Relative to Start Date
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
Terminal LD 14 Mean 226.41 225.78 230.28 216.94
Fasting BW   SD 16.01 16.90 14.45 20.11
(g)   N 10 10 10 10
    %Diff   -- -- --
Ovaries LD 14 Mean 0.0940 0.0947 0.0894 0.0980
(g)   SD 0.0190 0.0265 0.0173 0.0208
    N 10 10 10 10
    %Diff   -- -- --
Uterus with LD 14 Mean 0.6030 0.5587 0.5209 0.9089
cervix   SD 0.3646 0.1725 0.1023 0.5455
(g)   N 10 10 10 10
    %Diff   -- -- --
Thyroid with Day 69~ Mean 0.0250 0.0244 0.0245 0.0273
parathyroids   SD 0.0061 0.0041 0.0040 0.0061
(g)   N 10 10 10 10
    %Diff   -- -- --
~: Weighed after fixation
--: Not calculated as difference is not statistically significant.

TABLE 29. Summary of Terminal Fasting Body Weights and Organ to Body  
 WeightR atios – Reproductive Organs - Females
Sex: Female


Day(s) Relative to Start Date
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
Terminal LD 14 Mean 226.41 225.78 230.28 216.94
Fasting BW   SD 16.01 16.90 14.45 20.11
(g)   N 10 10 10 10
    %Diff   -- -- --
Ovaries LD 14 Mean 0.0418 0.0418 0.0388 0.0455
(%)   SD 0.0095 0.0110 0.0068 0.0103
    N 10 10 10 10
    %Diff   -- -- --
Uterus with LD 14 Mean 0.2702 0.2495 0.2262 0.4247
cervix   SD 0.1726 0.0839 0.0422 0.2693
(%)   N 10 10 10 10
    %Diff   -- -- --
Thyroid with Day 69~ Mean 0.0111 0.0108 0.0107 0.0128
parathyroids   SD 0.0030 0.0017 0.0020 0.0035
(%)   N 10 10 10 10
    %Diff   -- -- --
~: Weighed after fixation
--: Not calculated as difference is not statistically significant.

TABLE 30. Summary of Terminal Fasting Body Weights and Organ Weights – Females
Sex: Female


Day(s) Relative to Start Date
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
Terminal LD 14 Mean 224.74 234.46 229.51 222.82
Fasting BW   SD 8.51 7.15 19.84 16.65
(g)   N 5 5 5 5
    %Diff   -- -- --
Adrenals LD 14 Mean 0.0765 0.0738 0.0797 0.0825
(g)   SD 0.0164 0.0147 0.0084 0.0109
    N 5 5 5 5
    %Diff   -- -- --
Brain LD 14 Mean 1.9611 1.9305 1.9730 1.9011
(g)   SD 0.0653 0.1237 0.0450 0.0577
    N 5 5 5 5
    %Diff   -- -- --
Heart LD 14 Mean 0.7479 0.8056 0.7670 0.7944
(g)   SD 0.0477 0.0646 0.0460 0.0826
    N 5 5 5 5
    %Diff   -- -- --
Kidneys LD 14 Mean 1.5412 1.5733 1.6233 1.7405
(g)   SD 0.0789 0.1408 0.1145 0.2873
    N 5 5 5 5
    %Diff   -- -- --
Liver LD 14 Mean 7.8764 8.4014 8.4129 9.1161
(g)   SD 1.3563 0.8210 1.1036 1.1622
    N 5 5 5 5
    %Diff   -- -- --
Pituitary Day 69~ Mean 0.0116 0.0132 0.0119 0.0122
(g)   SD 0.0008 0.0016 0.0016 0.0016
    N 5 5 5 5
    %Diff   -- -- --
Spleen LD 14 Mean 0.5291 0.5314 0.4949 0.5864
(g)   SD 0.0374 0.0397 0.0584 0.1351
    N 5 5 5 5
    %Diff   -- -- --
Thymus LD 14 Mean 0.1908 0.2493 0.2702 0.2149
(g)   SD 0.0535 0.0255 0.1765 0.0589
    N 5 5 5 5
    %Diff   -- -- --
~: Weighed after fixation
--: Not calculated as difference is not statistically significant.

TABLE 31. Summary of Terminal Fasting Body Weights and Organ to Body
 Weight Ratios – Females
Sex: Female


Day(s) Relative to Start Date
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
Terminal LD 14 Mean 224.74 234.46 229.51 222.82
Fasting BW   SD 8.51 7.15 19.84 16.65
(g)   N 5 5 5 5
    %Diff   -- -- --
Adrenals LD 14 Mean 0.0342 0.0314 0.0349 0.0369
(%)   SD 0.0081 0.0058 0.0047 0.0025
    N 5 5 5 5
    %Diff   -- -- --
Brain LD 14 Mean 0.8730 0.8229 0.8639 0.8561
(%)   SD 0.0241 0.0353 0.0623 0.0511
    N 5 5 5 5
    %Diff   -- -- --
Heart LD 14 Mean 0.3327 0.3433 0.3355 0.3565
(%)   SD 0.0146 0.0208 0.0249 0.0263
    N 5 5 5 5
    %Diff   -- -- --
Kidneys LD 14 Mean 0.6865 0.6702 0.7086 0.7770 *
(%)   SD 0.0429 0.0421 0.0327 0.0709
    N 5 5 5 5
    %Diff   -- -- 13
Liver LD 14 Mean 3.5108 3.5798 3.6709 4.0856
(%)   SD 0.6492 0.2846 0.4112 0.3334
    N 5 5 5 5
    %Diff   -- -- --
Pituitary Day 69~ Mean 0.0052 0.0056 0.0052 0.0055
(%)   SD 0.0003 0.0006 0.0007 0.0005
    N 5 5 5 5
    %Diff   -- -- --
Spleen LD 14 Mean 0.2360 0.2267 0.2162 0.2627
(%)   SD 0.0239 0.0173 0.0243 0.0551
    N 5 5 5 5
    %Diff   -- -- --
Thymus LD 14 Mean 0.0848 0.1066 0.1177 0.0961
(%)   SD 0.0233 0.0132 0.0762 0.0222
    N 5 5 5 5
    %Diff   -- -- --
~: Weighed after fixation
--: Not calculated as difference is not statistically significant.
*: Significantly different from the vehicle control group at p < 0.05

TABLE 32. Summary of Terminal Fasting Body Weights and Organ to Brain Weight Ratios – Females
Sex: Female


Day(s) Relative to Start Date
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
Brain LD 14 Mean 1.9611 1.9305 1.9730 1.9011
(g)   SD 0.0653 0.1237 0.0450 0.0577
    N 5 5 5 5
    %Diff   -- -- --
Adrenals LD 14 Mean 3.9224 3.8103 4.0456 4.3387
(%)   SD 0.9445 0.6738 0.4557 0.5323
    N 5 5 5 5
    %Diff   -- -- --
Heart LD 14 Mean 38.1285 41.7096 38.8579 41.7563
(%)   SD 1.8042 1.4609 1.7615 3.8290
    N 5 5 5 5
    %Diff   -- -- --
Kidneys LD 14 Mean 78.6417 81.4567 82.2241 91.4354
(%)   SD 4.5457 4.0953 4.5254 14.1637
    N 5 5 5 5
    %Diff   -- -- --
Liver LD 14 Mean 402.9605 435.4030 426.2677 479.3454
(%)   SD 78.9801 35.5620 52.7760 57.5572
    N 5 5 5 5
    %Diff   -- -- --
Pituitary Day 69~ Mean 0.5938 0.6866 0.6032 0.6408
(%)   SD 0.0432 0.0775 0.0698 0.0713
    N 5 5 5 5
    %Diff   -- -- --
Spleen LD 14 Mean 27.0445 27.5910 25.0444 30.7498
(%)   SD 2.7281 2.3456 2.4713 6.5454
    N 5 5 5 5
    %Diff   -- -- --
Thymus LD 14 Mean 9.7305 12.9871 13.6167 11.3142
(%)   SD 2.8090 1.7937 8.6694 3.1267
    N 5 5 5 5
    %Diff   -- -- --
~: Weighed after fixation
--: Not calculated as difference is not statistically significant.

TABLE 33. Summary of Terminal Body Weights, Organ Weights and Organ to  
 Body Weight Ratios – Pups
Sex: Female


Day(s) Relative to Littering (Litter: A)
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
Pup TerminalBwt-Males 13 Mean
SD
N
%Diff
22.73
3.57
9
22.96
1.95
8
--
25.72
4.66
8
--
20.81
3.77
4
--
Pup TerminalBwt-Females 13 Mean
SD
N
%Diff
22.44
4.54
9
22.33
2.19
8
--
25.82
4.29
8
--
21.34
2.53
4
--
Pup Thyroid
weights (g)
Males
14 Mean
SD
N
%Diff
0.0060
0.0010
9
0.0067
0.0008
8
--
0.0074 *
0.0010
8
23
0.0062
0.0012
4
--
Pup Thyroid
weights (g)
Females
14 Mean
SD
N
%Diff
0.0067
0.0012
9
0.0069
0.0015
8
--
0.0066
0.0015
8
--
0.0059
0.0009
4
--
Pup Thyroid
weights (%)
Males
14 Mean
SD
N
%Diff
0.0276
0.0083
9
0.0294
0.0032
8
--
0.0297
0.0070
8
--
0.0302
0.0070
4
--
Pup Thyroid
weights (%)
Females
14 Mean
SD
N
%Diff
0.0321
0.0127
9
0.0307
0.0056
8
--
0.0258
0.0048
8
--
0.0277
0.0016
4
--
--: Not calculated as difference is not statistically significant.
*: Significantly different from the vehicle control group at p < 0.05
Reason / purpose:
reference to same study
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 Apr 2018 to 14 Jun 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
HanTac: WH
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Vivo Bio Tech Ltd.; Sy. #349/A, Pregnapur-502311, Gajwel Mandal, Medak District, Telangana
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 11-12 weeks
- Weight at study initiation: Males: 239.32 to 282.57 g; Females: 173.83 to 204.99 g
- Housing:
Pre-mating: Two rats of the same sex were housed per cage in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with a stainless-steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles with a stainless-steel sipper.
Mating and post-mating: During mating, two rats (one male and one female) were housed in standard polysulfone cages with a stainless-steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles. After confirming the presence of sperm in the vaginal smear or vaginal plugs (Day ‘0’ pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually in polysulfone cages. The sterilised nesting material (paper shreds) was provided near-term. Steam sterilized clean corn cob was used as bedding and changed along with the cage twice a week.
- Diet: Teklad Certified (2014C) Global 14% Protein Rodent Maintenance Diet – Pellet (Certified) was provided ad libitum to the animals.
- Water: Water was provided ad libitum to rats.
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY: Food and water was checked for contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25
- Humidity (%): 59-68
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 20 Apr 2018 To: 14 Jun 2018
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Required quantities of the test item were weighed in a beaker (previously calibrated to a desired volume) for each dose level separately and a small volume of vehicle [corn oil] was added and stirred using a glass rod till a uniform suspension was obtained. The volume was made up to the mark using the vehicle to get the final desired concentration of 20, 60 and 200 mg/mL for the G2, G3 and G4 groups, respectively. The suspensions were mixed well by stirring using a magnetic stirrer. The formulations were constantly stirred using a magnetic stirrer during test item administration and during sampling.

VEHICLE
- Concentration in vehicle: 20, 60 and 200 mg/L
- Amount of vehicle: 5 mL/kg/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For homogeneity and active ingredient (a.i.) concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 of the treatment period and during the 2nd month (day 33) of treatment and was analysed inhouse. For each set, duplicate samples were drawn from the top, middle and bottom layers of each preparation and in case of the control duplicate samples from the middle layer were drawn.
Dose formulations were considered acceptable as the overall mean results were within ± 15.0% of the claimed concentration and the overall relative standard deviation (RSD) was less than 10.0%.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: Cohabitation was continued until there was evidence of sperm in the vaginal smear and/or vaginal plug.
- Proof of pregnancy: sperm in vaginal smear and/or presence of vaginal plug referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually
Duration of treatment / exposure:
Males: Male animals were dosed during a 2-week premating period, during mating and post-mating until sacrifice (42 days of treatment).
Females: The female animals were dosed during a 2-week premating period, and during mating, pregnancy and up to LD 13.
Frequency of treatment:
Once daily
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Group 4
No. of animals per sex per dose:
All groups: 10 rats per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels of 100 (G2), 300 (G3) and 1000 (G4) mg/kg bw/day were selected for this study based on the results of the 14-Day Repeated Dose Oral Toxicity Study in Wistar Rats (Study No. N3629) and in consultation with the Sponsor.
- Fasting period before blood sampling for clinical biochemistry: yes, overnight fasting (water allowed)
Maternal examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: All rats were observed for morbidity and mortality twice daily i.e., once in the morning and once in the afternoon. As the observed clinical signs did not warrant twice daily observations, the observation for morbidity and mortality was carried out once in the morning during holidays. All rats were observed for clinical signs twice (pre and post dose) daily during the treatment period.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: prior to the treatment on Day 1 (± 2 day) and at weekly intervals thereafter during the treatment period.
- Parameters checked: During detailed clinical examination, all rats were observed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g., excessive grooming, repetitive circling or bizarre behaviour like self-mutilation, walking backwards).

BODY WEIGHT:
- Time schedule for examinations: Individual body weights of males were recorded on Day 1 and at weekly intervals thereafter. Individual body weights of females were recorded on Day 1 and at weekly intervals thereafter till cohabitation (till mating confirmation) with males. All dams were weighed on Gestation Day (GD) 0, 7, 14 and 20 and on Lactation Day (LD) 0, 4 and 13.

FOOD CONSUMPTION:
- Cagewise average food consumption was calculated by using the food consumed at each interval per cage and dividing by the number of rats per cage and the number of days in the intervening period to determine the food consumption/rat/day. Food consumption was not measured during the cohabitation period. Food consumption of pregnant dams was recorded on GD 7, 14 and 20 and on Day 4 and 13 of the lactation period.

HAEMATOLOGY:
- Time schedule for collection of blood: At the end of the pre-mating period (Day 16)
- Anaesthetic used for blood collection: Yes, Isoflurane anaesthesia
- Animals fasted: Yes, overnight (water allowed)
- How many animals: randomly selected 5 males and 5 females of each group
- Parameters checked in Tables 1 and 2 in ‘Any other information on materials and methods incl. tables’ were examined.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: At the end of the pre-mating period (Day 16)
- Animals fasted: Yes, overnight (water allowed)
- How many animals: randomly selected 5 males and 5 females of each group
- Parameters checked in Table 3 in ‘Any other information on materials and methods incl. tables’ were examined.

URINALYSIS:
- Time schedule for collection of urine: At the end of the pre-mating period
- Metabolism cages used for collection of urine: Yes, each rat was placed overnight in a specially fabricated cage
- Animals fasted: Yes, overnight (water allowed)
- Parameters checked in Table 4 in ‘Any other information on materials and methods incl. tables’ were examined.

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule for examinations: during the lactation period for females (shortly before the scheduled kill).
- Dose groups that were examined: All groups
- Battery of functions tested:
Home cage observations: Each rat was observed in the home cage for posture and for presence or absence of abnormal vocalizations, tremors and convulsions.
Observations during removal of animal from home cage and handling: Each rat was observed for the following examinations: ease of removal from home cage, handling reactivity, palpebral closure, eye examination, piloerection, lacrimation, salivation, skin/fur examination, perineum wetness, respiration, muscle tone and extensor thrust response. The observations were recorded using scores/ranks.
Open Field Observation: A rat was placed in an open arena, on a flat surface with a clean absorbent pa-per and observed for at least 2 minutes. Absorbent paper was replaced for each group. During this observation period, the rat was evaluated as it moves about freely/unperturbed and the following observations were made and recorded using score/ranks: gait, posture, tremors, mobility score, arousal level, clonic or tonic movements, stereotypic behaviour, bizarre behaviour, urination, defecation, rearing, abnormal vocalizations,
Functional tests: Functional testing included motor activity, sensory evaluation, landing hindlimbs foot splay and measurement of grip performance.
Physiological observations: Body temperature (rectal temperature) was measured in degree Celsius (°C) using a digital thermometer. At the end of the functional test, body weight of each rat was measured.

OTHER:
PARTURITION
The duration of gestation was calculated from Day ‘0’ of pregnancy to the day of parturition (Gestation Length).
Females were observed for signs of difficult or prolonged parturition.

HORMONE ANALYSIS
Blood samples were collected and serum was separated as per the following schedule for the determination of total T4 and TSH:
- Available surplus pups on Day 4 after birth.
- All dams prior to sacrifice and at least two available pups per litter on Day 13.
Blood collected in pup was pooled for thyroid hormone analyses. Pups were lightly anaesthetized with isoflurane and incised at the jugular vein in the neck region. The collected samples were pooled together for each litter. Blood samples were collected in plain labelled tubes and kept on the bench top for 20 minutes before centrifugation. Serum was separated by centrifuging the whole blood samples at 5000 rpm for 5 minutes at 4°C. The serum samples were placed in labelled plastic tubes and stored at ~-70°C until they were analyzed.
Blood samples were not collected from the non-littered females and the dams in which all pups were dead/cannibalized during lactation.

THYROID PROFILE HORMONES
The serum samples were analysed for Rodent Thyroid Stimulating Hormone (TSH; ng/mL) and Rodent Thyroxin (T4; ng/mL).
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
Statistics:
Data captured using ProvantisTM : Parameters of body weight, oestrous cycle, ano-genital distance, organ weights and organ weight ratios body weight, laboratory investigations - Haematology (Coagulation tests PT and APTT data were directly captured in ProvantisTM ) and Clinical Chemistry data was analysed using Provantis built-in statistical tests. The statistical analysis of the experimental data was carried out using the validated package in Excel and also using licensed copies of SYSTAT Statistical package ver.12.0. All quantitative variables like neurological observations (neuromuscular observation/body temperature/body weights) were tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene’s test) within the group before performing a one-factor ANOVA modelling by treatment groups. Non-optimal (non-normal or heteroschedastic) data was transformed, before ANOVA is performed. Comparison of means between treatment groups and control group was done using Dunnett’s test when the overall treatment, ‘F’ test found significant. Derived data like net body weight change, food consumption, ano-genital ratio, oestrous cycle length, organ weight ratios, post implantation loss (%), number of nipples/areolae in male pups, no. of implantations, pre-coital interval, mean litter size, sex ratio and gestation length (days), mating, fertility and survival indices were analysed using above mentioned methods. All analyses and comparisons were evaluated at the 5% (p<0.05) level. Statistically significant differences (p<0.05), indicated by the aforementioned tests were designated throughout the report as stated below:
*: Significantly different from the vehicle control group
Indices:
Male mating index (%) = [Number of males with evidence of mating / Number of males cohabited] x 100
Male fertility index (%) = [Number of males siring a litter/impregnated a female / Number of males cohabited] x 100
Female mating index (%) = [Number of females mated / Number of females cohabited] x 100
Female fertility index (%) = [Number of pregnant females / Number of females used for mating] x 100
Mean number of implantations/group = Total number of implantations / Total number of pregnant animals
Post implantation loss (%) = [(Number of implantations - Number of live pups) / Number of implantations] x 100
Mean litter size per group = Total Number of pups born / Total Number of littered animals
Mean viable litter size = No. of viable pups / No. of females littered
Live birth index (%) = [No. of viable pups born (at first observation) / Total no. of pups born (at first observation)] x 100
Day 4 survival index (%) = [Number of viable pups on lactation Day 4 / Number of viable pups born] x 100
Sex Ratio (%) = [No. of male pups born / Total no. of pups born] x 100
Ano-genital Distance Ratio (mm/g^1/3 ) = Ano-genital distance / Cube root of body weight
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were no clinical signs observed in both sexes in the 100 and 300 mg/kg bw/day dose groups. The following transient clinical signs were observed in the high dose (1000 mg/kg bw/day) group animals for a few hours after dose formulation administration and the animals recovered within 1-2 hours. (see tables 2 and 3 in ‘Any other information on results incl. tables’). The ataxia and recumbence were observed for a short period of time post-dose administration and the animals recovered within hours, indicating an adaptation to treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no mortalities observed.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
In females, the mean body weights and net weight gains were unaffected by the treatment at all doses tested when compared to vehicle control.

DURING GESTATION
Treatment had no effect on the maternal body weights measured during different intervals of the gestation period at all the doses tested. Incidence of significantly lower absolute weight gain during gestation days (GD) 0-7 was observed at 300 mg/kg bw/day. This statistically significant difference was toxicologically not significant as there was no dose-dependency observed.

DURING LACTATION
Treatment had no effects on the maternal body weights measured during different intervals of the lactation period at all the doses tested when compared to the vehicle control. Incidences of significantly higher absolute weight gain was observed during lactation day (LD) 0-4 at 100 mg/kg bw/day. This statistically significant difference was toxicologically not significant as there was no dose-dependency observed.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The food consumption was not altered throughout the treatment period when compared to the vehicle control. Incidences of significantly lower food consumption during days 1-8 in the 300 and 1000 mg/kg bw/day dose groups in females. These statistically significant differences observed in food consumption were toxicologically not significant as the mean body weights were not altered by the treatment.

DURING GESTATION
Significantly lower total and per day food consumption during GD 0-7 was observed at 1000 mg/kg bw/day. This statistically significant decrease in food consumption was toxicologically not significant as the mean maternal body weights were not altered during the same period.

DURING LACTATION
Significantly lower total and per day food consumption during LD 0-4 and 0-13 was observed at 1000 mg/kg bw/day. This statistically significant decrease in food consumption was toxicologically not significant as the mean maternal body weights were not altered during the same period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no test item-related changes in haematology parameters in females. All the minimal differences and the statistical significances obtained were considered as incidental changes in the absence of dose relation. The prothrombin time and APTT values were not affected by test item administration.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, increased plasma glucose concentration was noted in females (45%) and was considered related to test item related change. Lower BUN value was noted in ≥300 mg/kg bw/day females. This finding was not associated with any morphological correlates in kidneys and liver. Further, there were no changes in urinalysis parameters and hence considered as toxicologically insignificant. All the other changes including the statistical significances obtained were considered as toxicologically insignificant in the absence of dose relation/correlating morphological alterations.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no significant intergroup differences in the urinalysis parameters.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No treatment-related abnormalities were observed in all the tested dose groups in both sexes
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
An increase in liver weight was noted in 1000 mg/kg bw/day females and considered as test item related. This weight increase was microscopic correlated with hepatocellular hypertrophy. All the other minor variations in weights were also considered as incidental findings as the values were within the range of normal biological variation.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
All the gross lesions were considered as spontaneous and or associated with physiological changes (uterus-dilatation). The cause of infertility could not be determined for the two non-pregnant females. Dilatation of uterus was noted in these animals on gross and microscopic examination and ovaries were normal.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item related microscopic changes were present in liver and stomach. In the liver, hepatocellular hypertrophy was noted at 1000 mg/kg bw/day in females. This finding was of minimal and mild degree in severity of centrilobular distribution without any significant alteration in biochemical parameters. This was considered as an adaptive response to the test item administration. The non-glandular epithelial hyperplasia of stomach in 1000 mg/kg/day females and a single incidence of focal non-glandular ulceration in 1000 mg/kg bw/day females were considered as local responses to the presence of test item. All the other microscopic findings in test item treated groups were considered either as incidental/spontaneous changes common to this age group rats.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No treatment-related effects were observed with respect to implantations, and percentage of post-implantation loss when compared to the control group means.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
The test item had no treatment-related effects on the number littered.

Early or late resorptions:
not examined
Dead fetuses:
no effects observed
Description (incidence and severity):
The test item had no treatment-related effects on the number of dead pups at first observation.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on the mean gestation length at all the tested doses.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The test item had no treatment-related effects on the number of pregnancies.
Other effects:
not examined
Description (incidence and severity):
ANO-GENITAL DISTANCE (AGT)
No changes attributable to the test item were detected in the AGD, body weight and ratio of AGD to the cube root of body weight of either sex.

NIPPLE RETENTION
The male pups did not exhibit areola/nipple retention on PND 13.

HORMONE LEVELS
There were no significant intergroup differences in the thyroid profile parameters in females and in pups on lactation Day 4 and 13.
Key result
Dose descriptor:
NOAEL
Remarks:
Systemic toxicity
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical signs
body weight and weight gain
clinical biochemistry
Key result
Dose descriptor:
NOAEL
Remarks:
Reproductive toxicity
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean number and body weight of male and female (and total number) pups per litter were not affected by the treatment at all the doses tested.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The test item had no treatment-related effects on the number of dead pups at first observation.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The test item had no treatment-related effects on the mean litter size.
Changes in postnatal survival:
effects observed, treatment-related
Description (incidence and severity):
The Day 4 survival index was significantly lower at 1000 mg/kg bw/day. This significant difference observed is due to the total litter loss (dead/cannibalism) observed in five dams. The lower Day survival index could be due to poor lactation ability of the dam, maternal neglect, and acute intoxication during the treatment period.
External malformations:
no effects observed
Description (incidence and severity):
There were no gross lesions/developmental malformations in pups.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
Developmental toxicity
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
changes in postnatal survival
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
yes

Dose fromulation homogeneity and stability

The test item, was observed to be stable in the vehicle at dose concentrations of 1.15 mg/mL and 250.19 mg/mL up to 6 days when stored at room temperature. The test item was found to be homogeneous in all the dose formulation samples and they met the acceptance limits of variation (85 to 115%) from the claimed concentrations and had a %RSD of less than 10 %.

TABLE 1. Summary of Detailed Clinical Examination, Clinical Signs and Mortality – Males
 
Observation Type: All Types Male
From Day 1 (Start Date) to 43 (Start Date) G1 G2 G3 G4
  0 100 300 1000
  mg/kg/day mg/kg/day mg/kg/day mg/kg/day
Normal 10 10 10 10
Gait Characteristics, Ataxia (uncoordinated, staggering and wobbling gait) 0 0 0 7
Piloerection, Slight 0 0 0 10
Salivation, Slight 0 0 0 10
Posture, Recumbent (limbs spread out or lying on one side) 0 0 0 2
Values = Number of Animals Affected

TABLE 2. Summary of Detailed Clinical Examination, Clinical Signs and Mortality – Females
Observation Type: All Types Female

From Day 1 (Start Date) to 57 (Start Date)
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
Normal 10 10 10 10
Gait Characteristics, Ataxia (uncoordinated, staggering and wobbling gait) 0 0 0 5
Piloerection, Slight 0 0 0 10
Salivation, Slight 0 0 0 10
Posture, Recumbent (limbs spread out or lying on one side) 0 0 0 2
Total litter loss 1 2 1 5
Not littered 0 0 1 1
Values = Number of Animals Affected

TABLE 3. Summary of Functional Observation Battery – Males
         
Parameters Group
Dose
G1
0
G2
100
G3
300
G4
1000
(mg/kg/ day)
  No. of rats 5 5 5 5
Neurobehavioral Observations          
Home Cage Observations
Posture
Sitting or Standing alert, watching 4 4 4 4
Rearing 1 1 1 1
Abnormal Vocalizations
Absent 5 5 5 5
Tremors
Absent 5 5 5 5
Convulsions - Clonic Movement
Absent 5 5 5 5
Convulsions - Tonic Movement
Absent 5 5 5 5
Handling Observations
Ease of Removing from Cage
Easy (minimally avoids capture but is not aggressive) 5 5 5 5
Handling Reactivity
Moderately low (slight resistance) 5 5 5 5
Palpebral Closure
Eyelids wide open i.e., Normal 5 5 5 5
Eye Examination
Normal 5 5 5 5
Piloerection
Absent 5 5 5 5
Lacrimation
Absent 5 5 5 5
Salivation
None (no external salivation observed) 5 5 5 5
Skin/Fur Examination
Normal   5 5 5 5
Perineum Wetness          
Absent 5 5 5 5
Respiration
Normal 5 5 5 5
Muscle Tone
Moderate (animal is neither very relaxed nor tense; Normal muscle tone) 5 5 5 5
Extensor Thrust Response
Moderate (clearly detectable; Normal) 5 5 5 5
Open Field Observations
Gait
Normal gait 5 5 5 5
Posture
Normal posture 5 5 5 5
Tremors
Absent 5 5 5 5
Mobility Score
No impairment 5 5 5 5
Arousal Level
Alert (some exploratory movements; normal) 5 5 5 5
Clonic Movement
Absent 5 5 5 5
Tonic Movement
Absent 5 5 5 5
Stereotypic Behavior
Absent 5 5 5 5
Bizarre Behavior
Absent   5 5 5 5

TABLE 3 contd. Summary of Functional Observation Battery – Males
           
Parameters Group
Dose
G1
0
G2
100
G3
300
G4
1000
(mg/kg/ day)
  No. of rats 5 5 5 5
Urination          
Absent 4 3 4 5
Normal 1 2 1 0
Defecation
Absent 3 4 4 3
Normal 2 1 1 2
Abnormal Vocalization
Absent 5 5 5 5
Rears 32 32 29 22
Sensory Reactivity Measurements
Approach Response
Normal response (approaches towards the object) 5 5 5 5
Touch Response
Normal Response (spontaneously lifts tail when touched) 5 5 5 5
Click Response
Normal Response (clear movements of the head, neck and ears) 5 5 5 5
Tail-Pinch Response
Normal Response (Flinches, moves rapidly and/or vocalizes) 5 5 5 5
Pupil Response
Normal 5 5 5 5
Aerial Righting Reflex
Normal 5 5 5 5
Physiological Observation
Body Temperature (°C) Mean 37.42 37.80 37.56 37.40
Body Weight (g) Mean 353.47 328.68 356.86 329.51
Hindlimbs Footsplay (mm)
Average Mean 69.60 71.27 75.00 71.20
  SD 14.37 11.49 8.16 13.12
Grip Strength (gf)          
Forelimbs Grip Strength
Average Mean 1024.13 1028.67 1045.93 1022.33
SD 4.79 17.00 27.63 25.74
Hindlimbs Grip Strength
Average Mean 858.67 872.33 878.07 887.07
SD 39.37 68.27 38.27 35.36
Motor Activity Score
Stereotypic Time (sec)
Interval 1 Mean 139.00 114.80 81.20 97.20
SD 53.63 20.35 23.68 30.13
Interval 2 Mean 150.20 117.40 97.20 90.80
SD 75.04 52.28 33.80 39.23
Interval 3 Mean 55.40 78.20 76.20 81.00
SD 52.36 37.31 25.30 44.84
Total Mean 344.60 310.40 254.60 269.00
SD 130.34 80.75 24.52 93.55
Ambulatory Time (sec)
Interval 1 Mean 319.40 310.00 436.40* 360.80
SD 51.39 84.77 32.12 52.37
Interval 2 Mean 110.00 189.00 237.20 186.40
SD 58.61 89.63 56.45 89.72
Interval 3 Mean 56.80 104.60 169.20 132.00
SD 55.18 119.06 85.20 98.15
Total Mean 486.20 603.60 842.80 679.20
  SD 135.07 282.05 157.23 202.71
Horizontal Counts          
Interval 1 Mean 3180.80 3032.80 4242.80 3495.00
SD 764.67 739.47 1091.17 929.10
Interval 2 Mean 1109.00 1514.80 2071.00 1577.80
SD 530.31 664.95 731.59 914.18
Interval 3 Mean 495.00 808.80 1109.60 1184.80
SD 423.03 872.51 455.35 922.02
Total Mean 4784.80 5356.40 7423.40 6257.60
SD 1493.72 2201.95 1702.21 2417.27
Ambulatory Counts
Interval 1 Mean 2413.80 2425.00 3463.00 2822.40
SD 528.34 703.56 889.04 743.44
Interval 2 Mean 708.00 1131.40 1611.60 1228.00
SD 346.57 562.91 574.00 719.13
Interval 3 Mean 336.20 576.20 831.00 920.60
SD 290.90 720.17 357.96 740.89
Total Mean 3458.00 4132.60 5905.60 4971.00
SD 940.51 1913.18 1348.61 1892.36
*: Significantly different from vehicle control group

TABLE 4. Summary of Functional Observation Battery – Females
           
Parameters Group
Dose
G1
0
G2
100
G3
300
G4
1000
(mg/kg/ day)
  No. of rats 5 5 5 5
Neurobehavioral Observations          
Home Cage Observations
Posture
Sitting or Standing alert, watching 3 2 4 4
Rearing 2 3 1 1
Abnormal Vocalizations
Absent 5 5 5 5
Tremors
Absent 5 5 5 5
Convulsions - Clonic Movement
Absent 5 5 5 5
Convulsions - Tonic Movement
Absent 5 5 5 5
Handling Observations
Ease of Removing from Cage
Easy (minimally avoids capture but is not aggressive) 5 5 5 5
Handling Reactivity
Moderately low (slight resistance) 5 5 5 5
Palpebral Closure
Eyelids wide open i.e., Normal 5 5 5 5
Eye Examination
Normal 5 5 5 5
Piloerection
Absent 5 5 5 5
Lacrimation
Absent 5 5 5 5
Salivation
None (no external salivation observed) 5 5 5 5
Skin/Fur Examination
Normal   5 5 5 5
Perineum Wetness          
Absent 5 5 5 5
Respiration
Normal 5 5 5 5
Muscle Tone
Moderate (animal is neither very relaxed nor tense; Normal muscle tone) 5 5 5 5
Extensor Thrust Response
Moderate (clearly detectable; Normal) 5 5 5 5
Open Field Observations
Gait
Normal gait 5 5 5 5
Posture
Normal posture 5 5 5 5
Tremors
Absent 5 5 5 5
Mobility Score
No impairment 5 5 5 5
Arousal Level
Alert (some exploratory movements; normal) 5 5 5 5
Clonic Movement
Absent 5 5 5 5
Tonic Movement
Absent 5 5 5 5
Stereotypic Behavior
Absent 5 5 5 5
Bizarre Behavior
Absent   5 5 5 5
Urination          
Absent 4 5 4 5
Normal 1 0 1 0
Defecation
Absent 3 5 3 2
Normal 2 0 2 3
Abnormal Vocalization
Absent 5 5 5 5
Rears 41 41 41 50
Sensory Reactivity Measurements
Approach Response
Normal response (approaches towards the object) 5 5 5 5
Touch Response
Normal Response (spontaneously lifts tail when touched) 5 5 5 5
Click Response
Normal Response (clear movements of the head, neck and ears) 5 5 5 5
Tail-Pinch Response
Normal Response (Flinches, moves rapidly and/or vocalizes) 5 5 5 5
Pupil Response
Normal 5 5 5 5
Aerial Righting Reflex
Normal 5 5 5 5
Physiological Observation
Body Temperature (°C) Mean 37.92 37.80 38.02 37.86
Body Weight (g) Mean 239.31 257.24 243.86 235.89
Hindlimbs Footsplay (mm)
Average Mean 884.00 908.07 908.60 890.53
  SD 25.43 26.02 17.54 23.37
Grip Strength (gf)          
Forelimbs Grip Strength
Average Mean 800.20 788.40 783.00 753.47
SD 45.29 44.45 53.84 39.45
Hindlimbs Grip Strength
Average Mean 63.20 61.00 65.80 69.93
SD 5.54 6.70 7.14 16.58
Motor Activity Score
Stereotypic Time (sec)
Interval 1 Mean 116.00 120.40 87.60 108.20
SD 45.37 11.28 25.53 29.91
Interval 2 Mean 122.20 156.80 105.20 124.60
SD 57.82 27.13 42.61 61.52
Interval 3 Mean 91.40 96.20 115.00 53.40
SD 28.28 10.76 79.97 42.56
Total Mean 329.60 373.40 307.80 286.20
SD 98.79 25.45 137.95 89.63
Ambulatory Time (sec)
Interval 1 Mean 268.00 278.00 292.60 292.40
SD 11.81 56.71 73.10 66.87
Interval 2 Mean 150.80 168.60 150.60 137.80
SD 43.56 34.24 92.83 61.72
Interval 3 Mean 90.00 124.20 90.60 51.40
SD 55.76 42.36 44.17 51.44
Total Mean 508.80 570.80 533.80 481.60
  SD 92.12 100.19 190.90 119.57
Horizontal Counts          
Interval 1 Mean 2411.40 2629.40 2607.00 2389.20
SD 466.09 877.16 1184.84 616.36
Interval 2 Mean 1179.00 1467.00 1237.40 1011.00
SD 219.15 293.61 992.66 406.65
Interval 3 Mean 647.20 1053.20 776.40 354.20
SD 356.14 399.70 536.37 340.60
Total Mean 4237.60 5149.60 4620.80 3754.40
SD 449.07 1287.83 2641.45 763.89
Ambulatory Counts
Interval 1 Mean 1803.60 1957.60 1963.00 1834.00
SD 355.27 688.91 839.55 499.35
Interval 2 Mean 789.20 972.80 846.00 660.60
SD 189.71 164.04 680.37 296.68
Interval 3 Mean 413.80 747.40 408.00 233.20
SD 324.88 339.72 310.98 260.09
Total Mean 3006.60 3677.80 3217.00 2727.80
  SD 501.96 915.81 1695.82 503.65

TABLE 5. Summary of Body Weights (g) and Net Body Weight Gains (g) – Males
Sex: Male


Day(s) Relative to Start Date
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
Body 1 [a] Mean 261.42 259.62 261.92 259.62
Weight   SD 12.96 13.56 11.68 13.85
(g)   N 10 10 10 10
  8 [a] Mean 277.40 274.38 276.47 265.19
    SD 15.55 18.14 18.18 19.59
    N 10 10 10 10
  15 [a] Mean 292.53 285.69 286.97 277.31
    SD 16.27 18.38 20.51 21.37
    N 10 10 10 10
  22 [a] Mean 306.18 295.85 297.29 285.54
    SD 22.05 20.92 20.53 23.10
    N 10 10 10 10
  29 [a] Mean 327.01 313.51 314.76 300.67
    SD 22.10 23.92 25.39 23.05
    N 10 10 10 10
  36 [a] Mean 344.56 329.04 332.37 311.72
    SD 22.60 27.19 28.46 28.52
    N 10 10 10 10
  42 [a] Mean 355.65 340.81 342.11 317.78 *
    SD 22.93 27.44 27.40 25.76
    N 10 10 10 10
Absolute 1 → 42 [a] Mean 94.23 81.19 80.19 58.15 *
Weight Gain   SD 13.63 18.29 17.96 16.62
(g)   N 10 10 10 10
[a] - Anova & Dunnett(Log): * = p < 0.05
*: Significantly different from vehicle control group

TABLE 6. Summary of Body Weights (g) and Net Body Weight Gains (g) – Females
Sex: Female


Day(s) Relative to Start Date
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
Body 1 [a] Mean 192.21 192.52 190.77 189.57
Weight   SD 9.39 7.63 6.45 8.47
(g)   N 10 10 10 10
  8 [a] Mean 195.36 195.53 196.83 187.08
    SD 7.86 8.74 10.01 11.64
    N 10 10 10 10
  15 [a] Mean 199.94 203.49 202.79 197.43
    SD 9.20 8.17 12.22 13.23
    N 10 10 10 10
Absolute 1 → 15 [a] Mean 7.74 10.97 12.02 7.87
Weight Gain   SD 4.26 4.26 7.62 9.40
(g)   N 10 10 10 10
[a] - Anova & Dunnett(Log)

TABLE 7. Summary of Food Consumption (g/rat/day) – Males
Food Mean Daily Consumption (g/animal/day)
Sex: Male

Day(s) Relative to
Start Date
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
1 → 8

Mean
SD

N

17.57
1.48
10
17.65
1.63
10
17.23
1.31
10
14.75 *
2.29
10
8 → 15 Mean 19.47 18.48 18.32 18.50
  SD 0.73 1.12 1.47 1.10
  N 10 10 10 10
29 → 36 Mean 17.93 17.17 17.46 18.26
  SD 1.14 1.14 1.11 1.44
  N 10 10 10 10
36 → 42 Mean 18.41 18.20 18.32 18.15
  SD 0.81 1.37 0.95 0.58
  N 10 10 10 10
Anova & Dunnett(Log): * = p < 0.05 
*: Significantly different from vehicle control group 

TABLE 8. Summary of Food Consumption (g/rat/day) – Females
Food Mean Daily Consumption (g/animal/day)
Sex: Female

Day(s) Relative to
Start Date
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
1 → 8

Mean
SD

N

14.19
1.44
10
13.37
0.56
10
12.91 *
1.43
10
8.50 *
0.30
10
8 → 15 Mean 15.33 14.82 14.88 15.32
  SD 1.18 0.66 1.58 1.98
  N 10 10 10 10
Anova & Dunnett(Log): * = p < 0.05; 
*: Significantly different from vehicle control group 

TABLE 9. Summary of of Oestrous Cycle Length Prior to Cohabitation Period
Sex: Female


Day(s) Relative to Start Date
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
Mean 1 → 16 [a] Mean 4.3 4.1 4.2 4.2
Cycle   SD 0.3 0.2 0.2 0.3
(days)   N 10 10 10 10
[a] - Anova & Dunnett(Log)

TABLE 10. Summary of Maternal Body Weights (g) and Weight Change During Gestation Period
Sex: Female


Day(s) Relative to Mating
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
Body 0 [a] Mean 199.76 202.25 206.24 199.48
Weight   SD 11.78 8.15 12.36 10.86
(g)   N 10 10 9 9
  7 [a] Mean 229.51 230.15 227.63 224.62
    SD 10.73 8.46 11.20 13.43
    N 10 10 9 9
  14 [a] Mean 259.36 256.98 251.33 249.05
    SD 12.16 14.64 13.74 17.22
    N 10 10 9 9
  20 [a] Mean 313.00 309.19 303.38 301.82
    SD 19.79 21.18 16.01 25.45
    N 10 10 9 9
Absolute 0→7 [a] Mean 29.75 27.90 21.39 * 25.14
Weight Gain   SD 4.56 4.65 4.80 6.68
(g)   N 10 10 9 9
  7 →14 [a] Mean 29.85 26.83 23.70 24.43
    SD 5.62 10.82 3.84 5.51
    N 10 10 9 9
  14 →20 [a] Mean 53.63 52.21 52.05 52.77
    SD 9.55 17.24 5.45 9.09
    N 10 10 9 9
  0 →20 [a] Mean 113.24 106.93 97.14 102.34
    SD 15.19 18.56 8.04 18.20
    N 10 10 9 9
[a] - Anova & Dunnett(Log): * = p < 0.05 
*: Significantly different from vehicle control group 

TABLE 11. Summary of Maternal Food Intake (g) During Gestation Period
Sex: Female


Day(s) Relative to Mating
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
Total Food 0-7 [a] Mean 117.52 115.49 106.15 103.08 *
Consumption   SD 14.96 7.40 13.05 10.53
(g)   N 10 10 9 9
  7-14 [a] Mean 135.45 132.60 122.83 125.14
    SD 13.09 9.24 9.35 14.06
    N 10 10 9 9
  14-20 [a] Mean 128.27 133.48 122.88 123.19
    SD 17.22 18.65 16.81 10.70
    N 10 10 9 9
  0-20 [a] Mean 381.24 381.57 351.85 351.41
    SD 41.73 22.70 35.26 29.87
    N 10 10 9 9
Food Mean 0-7 [a] Mean 16.79 16.50 15.16 14.73 *
Consumption   SD 2.14 1.06 1.86 1.50
(g/day)   N 10 10 9 9
  7-14 [a] Mean 19.35 18.94 17.55 17.88
    SD 1.87 1.32 1.34 2.01
    N 10 10 9 9
  14-20 [a] Mean 21.38 22.25 20.48 20.53
    SD 2.87 3.11 2.80 1.78
    N 10 10 9 9
  0-20 [a] Mean 19.06 19.08 17.59 17.57
    SD 2.09 1.14 1.76 1.49
    N 10 10 9 9
[a] - Anova & Dunnett(Log): * = p < 0.05
*: Significantly different from vehicle control group

TABLE 12. Summary of Maternal Body Weights (g) and Weight Change During Lactation Period
Sex: Female


Day(s) Relative to Littering
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
Body 0 [a] Mean 230.70 219.91 230.13 218.08
Weight   SD 19.33 20.57 15.38 21.43
(g)   N 10 10 9 9
  4 [a] Mean 237.33 240.56 238.61 224.86
    SD 16.34 20.24 15.50 17.36
    N 9 9 9 5
  13 [a] Mean 252.65 259.32 250.96 241.29
    SD 17.54 17.97 13.73 25.58
    N 9 8 8 4
Absolute 0 - 4 [a] Mean 5.25 18.55 * 8.48 7.27
Weight Gain   SD 7.97 9.91 10.57 10.26
(g)   N 9 9 9 5
  4 - 13 [a] Mean 15.32 18.49 11.85 15.19
    SD 12.68 10.65 10.03 5.92
    N 9 8 8 4
  0 - 13 [a] Mean 20.57 36.54 19.93 26.83
    SD 11.20 15.57 15.37 3.10
    N 9 8 8 4
[a] - Anova & Dunnett(Log): * = p < 0.05  
*: Significantly different from vehicle control group

TABLE 13. Summary of Maternal Food Intake (g) During Lactation Period
Sex: Female


Day(s) Relative to Littering
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
 
Total Food 0-4 [a] Mean 102.48 101.23 98.40 75.24 *
Consumption   SD 15.03 16.06 15.40 16.54  
(g)   N 9 9 9 4  
  4-13 [a] Mean 383.85 399.82 392.05 354.49  
    SD 23.25 34.09 30.66 63.59  
    N 9 8 8 4  
  0-13 [a] Mean 486.33 504.12 493.88 429.73 *
    SD 19.86 41.92 36.10 59.11  
    N 9 8 8 4  
Food Mean 0-4 [a] Mean 25.62 25.31 24.60 18.81 *
Consumption   SD 3.76 4.02 3.85 4.14  
(g/day)   N 9 9 9 4  
  4-13 [a] Mean 42.65 44.42 43.56 39.39  
    SD 2.58 3.79 3.41 7.07  
    N 9 8 8 4  
  0-13 [a] Mean 37.41 38.78 37.99 33.06 *
    SD 1.53 3.22 2.78 4.55  
    N 9 8 8 4  
[a] - Anova & Dunnett(Log): * = p < 0.05
*: Significantly different from vehicle control group

TABLE 14. Summary of Mean Pup Body Weights (g), Ano-genital Distance (AGD), Ano-genital Ratio (AGR) and Ano-genital Index (AGI)
Sex: Female


Day(s) Relative to Littering
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
Litter Mean 0 [a] Mean 5.98 5.42 6.13 5.70
Pup BW - M   SD 0.86 0.64 0.61 0.37
    N 10 10 9 8
  4 [a] Mean 9.32 8.02 9.45 8.41
    SD 1.53 1.47 1.49 0.72
    N 9 9 9 4
  13 [a] Mean 22.63 23.35 25.53 21.58
    SD 3.46 1.86 3.45 3.15
    N 9 8 8 4
Litter Mean 0 [a] Mean 5.70 5.25 5.99 5.50
Pup BW - F   SD 0.93 0.67 0.86 0.38
    N 10 10 9 8
  4 [a] Mean 9.14 7.95 9.52 7.93
    SD 1.58 1.36 2.48 0.88
    N 9 9 9 4
  13 [a] Mean 22.67 22.95 26.27 21.17
    SD 4.34 2.09 4.40 3.00
    N 9 8 8 4
Litter Mean 0 [a] Mean 5.85 5.34 6.07 5.59
Pup BW   SD 0.88 0.63 0.68 0.38
    N 10 10 9 8
  4 [a] Mean 9.24 7.99 9.47 8.17
    SD 1.56 1.39 1.77 0.81
    N 9 9 9 4
  13 [a] Mean 22.66 23.15 25.85 21.34
    SD 3.79 1.95 3.78 3.08
    N 9 8 8 4
Mean of pup 0 [a] Mean 3.69 3.64 3.74 3.70
AGD males   SD 0.05 0.09 0.08 0.06
    N 10 10 9 8
Mean of pup 0 [a] Mean 1.64 1.67 1.72 1.65
AGD females   SD 0.07 0.11 0.08 0.03
    N 10 10 9 8
Mean of pup 0 [a] Mean 2.04 2.08 2.05 2.07
AGR males   SD 0.08 0.06 0.05 0.04
    N 10 10 9 8
Mean of pup 0 [a] Mean 0.92 0.96 0.95 0.94
AGR females   SD 0.05 0.05 0.02 0.03
    N 10 10 9 8
Mean of pup 0 [a] Mean 2.0 2.1 2.0 2.1
AGI males   SD 0.1 0.1 0.0 0.0
    N 10 10 9 8
Mean of pup 0 [a] Mean 0.9 1.0 0.9 0.9
AGI females   SD 0.1 0.1 0.0 0.0
    N 10 10 9 8
[a] - Anova & Dunnett(Log)

TABLE 15. Summary of Fertility Data
Sex: Both


Day(s) Relative to Mating
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
Paired Males - N 10 10 10 10
Paired Females - N 10 10 10 10
Mated - N+ve 10 10 10 10
Fertile Male? - N+ve 10 10 9 9
Fertile Female? - N+ve 10 10 9 9
M Mating Index - [a] % 100.0 100.0 100.0 100.0
Female Mating Index - [a] % 100.0 100.0 100.0 100.0
Pregnant? - N+ve 10 10 9 9
Littered - N+ve 10 10 9 9
Pre-coital
Interval (f)
(days)
- [a] Mean
SD
N
3.0
1.2
10
2.6
1.5
10
3.4
1.7
10
2.4
1.1
10
Female Fertility Index - [a] % 100.0 100.0 90.0 90.0
Male Impregd a Female - [a] % 100.0 100.0 90.0 90.0
[a] - Anova & Dunnett(Log)

TABLE 16. Summary of Litter Data
Sex: Female


Day(s) Relative to Littering (Litter: A)
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
Littered LD 0 N+ve 10   10   9   9  
Gest. GD 0 - LD 0 [a] Mean 22.2   22.1   22.1   22.8  
Length   SD 0.6   0.6   0.6   0.8  
(days)   N 10   10   9   9  
Total No of Pups Born LD 0 Sum 106   118   89   95  
Litter Size LD 0 [a1] Mean 10.6   11.8   9.9   10.6  
on D0   N 10   10   9   9  
Live Male Pups D0 LD 0 Sum 55   62   52   44  
Live Female Pups D0 LD 0 Sum 51   56   37   51  
Live Pups LD 0 [a] Mean 11   12   10   11  
Day 0   SD 2   2   2   1  
    N 10   10   9   9  
    Sum 106   118   89   95  
Dead/Cani Pups on D0 LD 0 Sum   .   .   . 12  
Live Birth Index (%) D0 LD 0 [a1] Mean 100   100   100   100  
Sex Ratio D0 LD 0 [a1] Mean 53.7   51.8   58.9   46.9  
Dead/Canni Pups on D1 LD 1 Sum 8   11     . 26  
Live Pups LD 1 [a] Mean 10   11   10   6  
Day 1   SD 4   4   2   4  
    N 10   10   9   9  
    Sum 98   107   89   57  
Dead/Canni Pups D2-4 LD 2-4 Sum   . 12   2   19  
Live Pups Day 4 LD 4 Sum 98   95   87   38  
Survival Idx LD 4 [a2] Mean 89.2   83.3   98.1   42.0 *
on LD4   SD 31.4   36.0   5.6   50.2  
    N 10   10   9   9  
No. of Pups Culled D4 4 Sum 26   26   17   7  
No.of LivPup Post Cul LD 4 Sum 72   69   70   31  
Dead/Canni Pups D5-12 LD 5-12 Sum 3   5   9     .
Live Pups Day 13 LD 13 Sum 69   64   61   31  
Dead/Can/TS Pups on D13 LD 13 Sum 69   64   61   31  
No. of Implantation LD 14 [a] Mean 12.2   13.4   12.1   11.7  
    SD 1.9   1.8   1.4   1.0  
    N 10   10   9   9  
    Sum 122.0   134.0   109.0   105.0  
%Post-impt' Loss (%) LD 14 [a] Mean 13.66 12.03 18.69 9.31
SD 9.93 9.77 13.23 9.68
N 10 10 9 9
[a] - Anova & Dunnett(Log)
[a1]- Anova & Dunnett(Arc SineSQRT)
[a2]- Anova & Dunnett(Square Root): * = p < 0.05

TABLE 17. Summary of Haematological and Coagulation Parameters – Males
Sex: Male


Day(s) Relative to Start Date
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
RBC Day 16 Mean 9.05 9.05 8.60 8.93
(10^12/L)   SD 0.16 0.39 0.22 0.38
    N 5 5 5 5
    %Diff   -- -- --
HGB Day 16 Mean 167 166 161 164
(g/L)   SD 5 3 5 3
    N 5 5 5 5
    %Diff   -- -- --
HCT Day 16 Mean 0.499 0.504 0.481 0.495
(L/L)   SD 0.019 0.010 0.015 0.010
    N 5 5 5 5
    %Diff   -- -- --
MCV Day 16 Mean 55.2 55.8 55.9 55.5
(fL)   SD 2.2 1.5 0.4 1.3
    N 5 5 5 5
    %Diff   -- -- --
MCH Day 16 Mean 18.5 18.4 18.8 18.4
(pg)   SD 0.6 0.5 0.2 0.6
    N 5 5 5 5
    %Diff   -- -- --
MCHC Day 16 Mean 335 330 * 336 332
(g/L)   SD 3 1 1 4
    N 5 5 5 5
    %Diff   2 -- --
Retic A Day 16 Mean 0.244 0.217 0.225 0.264
(10^12/L)   SD 0.043 0.025 0.019 0.058
    N 5 5 5 5
    %Diff   -- -- --
PLT Day 16 Mean 1100 968 1072 1088
(10^9/L)   SD 56 97 86 124
    N 5 5 5 5
    %Diff   -- -- --
WBC Day 16 Mean 6.36 5.85 6.43 7.61
(10^9/L)   SD 0.99 1.00 1.68 1.17
    N 5 5 5 5
    %Diff   -- -- --
Neut A Day 16 Mean 1.02 0.96 1.00 1.02
(10^9/L)   SD 0.22 0.20 0.28 0.22
    N 5 5 5 5
    %Diff   -- -- --
Lymp A Day 16 Mean 5.11 4.66 5.20 6.26
(10^9/L)   SD 0.88 0.97 1.50 0.96
    N 5 5 5 5
    %Diff   -- -- --
Mono A Day 16 Mean 0.15 0.16 0.14 0.21
(10^9/L)   SD 0.05 0.05 0.08 0.07
    N 5 5 5 5
    %Diff   -- -- --
Baso A Day 16 Mean 0.01 0.01 0.01 0.01
(10^9/L)   SD 0.00 0.00 0.01 0.00
    N 5 5 5 5
    %Diff   -- -- --
Eosi A Day 16 Mean 0.06 0.06 0.05 0.09
(10^9/L)   SD 0.04 0.03 0.01 0.07
    N 5 5 5 5
    %Diff   -- -- --
PT Day 16 Mean 16.6 15.3 14.7 * 16.4
    SD 1.3 1.1 0.7 0.5
(seconds)   N 5 5 5 5
    %Diff   -- 11 --
APTT Day 16 Mean 15.5 15.2 18.3 14.8
(seconds)   SD 1.0 1.5 2.2 2.0
    N 5 5 5 5
    %Diff   -- -- --
--: Not calculated as difference is not statistically significant
*: Significantly different from the vehicle control group at p < 0.05

TABLE 18. Summary of Haematological and Coagulation Parameters – Females
Sex: Female


Day(s) Relative to Start Date
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
RBC Day 16 Mean 8.34 8.36 8.28 7.95
(10^12/L)   SD 0.17 0.47 0.60 0.36
    N 5 5 5 5
    %Diff   -- -- --
HGB Day 16 Mean 157 159 153 154
(g/L)   SD 1 3 6 10
    N 5 5 5 5
    %Diff   -- -- --
HCT Day 16 Mean 0.458 0.475 0.453 0.463
(L/L)   SD 0.009 0.016 0.015 0.020
    N 5 5 5 5
    %Diff   -- -- --
MCV Day 16 Mean 54.9 56.9 54.9 58.3 *
(fL)   SD 0.4 2.5 2.4 1.6
    N 5 5 5 5
    %Diff   -- -- 6
MCH Day 16 Mean 18.9 19.0 18.5 19.4
(pg)   SD 0.3 1.0 0.9 0.6
    N 5 5 5 5
    %Diff   -- -- --
MCHC Day 16 Mean 343 334 338 333
(g/L)   SD 5 6 5 10
    N 5 5 5 5
    %Diff   -- -- --
Retic A Day 16 Mean 0.268 0.261 0.207 0.249
(10^12/L)   SD 0.055 0.042 0.041 0.088
    N 5 5 5 5
    %Diff   -- -- --
PLT Day 16 Mean 1132 1017 1093 1177
(10^9/L)   SD 143 91 68 272
    N 5 5 5 5
    %Diff   -- -- --
WBC Day 16 Mean 5.38 4.40 4.89 5.28
(10^9/L)   SD 0.43 0.81 1.47 1.71
    N 5 5 5 5
    %Diff   -- -- --
Neut A Day 16 Mean 0.81 0.58 0.74 0.63
(10^9/L)   SD 0.04 0.14 0.14 0.16
    N 5 5 5 5
    %Diff   -- -- --
Lymp A Day 16 Mean 4.34 3.67 3.99 4.49
(10^9/L)   SD 0.41 0.73 1.35 1.57
    N 5 5 5 5
    %Diff   -- -- --
Mono A Day 16 Mean 0.12 0.09 0.10 0.09
(10^9/L)   SD 0.01 0.04 0.01 0.01
    N 5 5 5 5
    %Diff   -- -- --
Baso A Day 16 Mean 0.01 0.01 0.01 0.00
(10^9/L)   SD 0.01 0.01 0.01 0.01
    N 5 5 5 5
    %Diff   -- -- --
Eosi A Day 16 Mean 0.08 0.04 0.05 0.05
(10^9/L)   SD 0.04 0.02 0.02 0.03
    N 5 5 5 5
    %Diff   -- -- --
PT Day 16 Mean 15.9 14.8 17.5 16.2
    SD 1.3 0.4 1.1 1.2
(seconds)   N 5 5 5 5
    %Diff   -- -- --
APTT Day 16 Mean 14.7 13.9 16.9 13.1
(seconds)   SD 1.6 1.5 2.3 1.9
    N 5 5 5 5
    %Diff   -- -- --
--: Not calculated as difference is not statistically significant
*: Significantly different from the vehicle control group at p < 0.05

TABLE 19. Summary of Clinical Chemistry Parameters – Males
Sex: Male


Day(s) Relative to Start Date
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
Glu Day 16 Mean 3.90 3.86 3.91 6.45 *
(mmol/L)   SD 0.19 0.29 0.44 0.82
    N 5 5 5 5
    %Diff   -- -- 65
BUN Day 16 Mean 5.05 4.31 4.14 3.12 *
(mmol/L)   SD 0.81 0.59 0.51 0.97
    N 5 5 5 5
    %Diff   -- -- 38
Creat Day 16 Mean 40 40 39 45 *
(µmol/L)   SD 2 4 2 4
    N 5 5 5 5
    %Diff   -- -- 14
AST Day 16 Mean 81 83 70 85
(U/L)   SD 9 18 11 8
    N 5 5 5 5
    %Diff   -- -- --
ALT Day 16 Mean 38 34 34 41
(U/L)   SD 4 8 9 6
    N 5 5 5 5
    %Diff   -- -- --
GGT Day 16 Mean 6 5 5 6
(U/L)   SD 1 0 1 1
    N 5 5 5 5
    %Diff   -- -- --
Alp Day 16 Mean 114 113 114 133
(U/L)   SD 9 9 24 33
    N 5 5 5 5
    %Diff   -- -- --
CK Day 16 Mean 354 315 280 356
(U/L)   SD 53 88 57 105
    N 5 5 5 5
    %Diff   -- -- --
T.Bil Day 16 Mean 1.62 2.19 1.76 1.66
(µmol/L)   SD 0.53 0.66 0.18 0.76
    N 5 3a 5 5
    %Diff   -- -- --
T.Chol Day 16 Mean 2.19 2.07 2.03 1.94
(mmol/L)   SD 0.24 0.23 0.24 0.20
    N 5 5 5 5
    %Diff   -- -- --
BA Day 16 Mean 31.8 9.6 * 15.8 34.0
(µmol/L)   SD 18.8 4.3 5.3 24.9
    N 5 5 5 5
    %Diff   70 -- --
Trig Day 16 Mean 1.04 0.89 0.70 0.65
(mmol/L)   SD 0.25 0.32 0.13 0.21
    N 5 5 5 5
    %Diff   -- -- --
T.Pro Day 16 Mean 69.4 70.7 69.9 68.9
(g/L)   SD 2.1 2.8 1.8 2.6
    N 5 5 5 5
    %Diff   -- -- --
ALB Day 16 Mean 31.6 32.5 30.8 31.0
(g/L)   SD 1.2 0.7 1.0 1.1
    N 5 5 5 5
    %Diff   -- -- --
GLOB Day 16 Mean 37.8 38.2 39.1 38.0
(g/L)   SD 1.0 2.3 0.9 1.8
    N 5 5 5 5
    %Diff   -- -- --
A/G Day 16 Mean 0.84 0.85 0.79 * 0.82
(ratio)   SD 0.02 0.04 0.01 0.03
    N 5 5 5 5
    %Diff   -- 6 --
Pi Day 16 Mean 2.34 2.11 * 2.31 2.43
(mmol/L)   SD 0.04 0.06 0.17 0.16
    N 5 5 5 5
    %Diff   10 -- --
Ca Day 16 Mean 2.63 2.59 2.62 2.41 *
(mmol/L)   SD 0.07 0.05 0.09 0.11
    N 5 5 5 5
    %Diff   -- -- 8
Na Day 16 Mean 146.8 147.6 148.3 149.0
(mEq/L)   SD 0.7 2.3 1.9 1.4
    N 5 5 5 5
    %Diff   -- -- --
K Day 16 Mean 3.53 3.89 3.71 3.93
(mEq/L)   SD 0.16 0.43 0.13 0.43
    N 5 5 5 5
    %Diff   -- -- --
Cl Day 16 Mean 102.7 102.3 102.7 104.1
(mEq/L)   SD 1.3 1.3 1.5 1.1
    N 5 5 5 5
    %Diff   -- -- --
a: Values below LLOQ (Lower Limit of Quantification for T.bil is below 0.40 µmol/L) were not considered for analysis.
--: Not calculated as difference is not statistically significant
*: Significantly different from the vehicle control group at p < 0.05

TABLE 20. Summary of Clinical Chemistry Parameters – Females
Sex: Female


Day(s) Relative to Start Date
G1
0
mg/kg/day
G2
100
mg/kg/day
G3
300
mg/kg/day
G4
1000
mg/kg/day
Glu Day 16 Mean 4.70 4.85 5.70 6.83 *
(mmol/L)   SD 0.73 0.26 1.01 0.99
    N 5 5 5 5
    %Diff   -- -- 45
BUN Day 16 Mean 7.10 5.26 4.90 * 2.13 *
(mmol/L)   SD 1.35 1.50 1.46 0.17
    N 5 5 5 5
    %Diff   -- 31 70
Creat Day 16 Mean 52 47 52 50
(µmol/L)   SD 7 2 11 11
    N 5 5 5 5
    %Diff   -- -- --
AST Day 16 Mean 92 78 77 86
(U/L)   SD 6 5 8 15
    N 5 5 5 5
    %Diff   -- -- --
ALT Day 16 Mean 26 28 32 39
(U/L)   SD 4 8 12 7
    N 5 5 5 5
    %Diff   -- -- --
GGT Day 16 Mean 6 5 5 6
(U/L)   SD 1 1 1 1
    N 5 5 5 5
    %Diff   -- -- --
Alp Day 16 Mean 59 48 67 93 *
(U/L)   SD 15 6 21 27
    N 5 5 5 5
    %Diff   -- -- 59
CK Day 16 Mean 363 247 279 468
(U/L)   SD 117 67 29 191
    N 5 5 5 5
    %Diff   -- -- --
T.Bil Day 16 Mean 1.61 2.12 1.25 1.30
(µmol/L)   SD 0.92 0.60 0.64 0.32
    N 4a 5 5 4a
    %Diff   -- -- --
T.Chol Day 16 Mean 1.74 1.53 1.66 n 1.92
(mmol/L)   SD 0.35 0.16 0.17 0.52
    N 5 5 2a 5
    %Diff   -- -- --
BA Day 16 Mean 24.6 10.2 9.2 23.4
(µmol/L)   SD 31.3 5.1 3.4 6.4
    N 5 5 5 5
    %Diff   -- -- --
Trig Day 16 Mean 0.34 0.43 0.37 0.48
(mmol/L)   SD 0.12 0.15 0.04 0.12
    N 5 5 5 5
    %Diff   -- -- --
T.Pro Day 16 Mean 70.8 71.7 70.7 71.9
(g/L)   SD 1.7 4.9 2.7 4.4
    N 5 5 5 5
    %Diff   -- -- --
ALB Day 16 Mean 33.2 34.0 32.6 33.1
(g/L)   SD 1.1 2.4 1.4 2.1
    N 5 5 5 5
    %Diff   -- -- --
GLOB Day 16 Mean 37.5 37.7 38.1 38.8
(g/L)   SD 0.7 2.6 1.4 2.3
    N 5 5 5 5
    %Diff   -- -- --
A/G Day 16 Mean 0.88 0.90 0.85 0.85
(ratio)   SD 0.02 0.02 0.02 0.02
    N 5 5 5 5
    %Diff   -- -- --
Pi Day 16 Mean 1.86 1.76 1.75 2.16
(mmol/L)   SD 0.20 0.15 0.23 0.25
    N 5 5 5 5
    %Diff   -- -- --
Ca Day 16 Mean 2.56 2.59 2.52 2.44
(mmol/L)   SD 0.06 0.08 0.08 0.10
    N 5 5 5 5
    %Diff   -- -- --
Na Day 16 Mean 144.7 146.6 147.2 146.7
(mEq/L)   SD 1.9 1.1 0.6 1.4
    N 5 5 5 5
    %Diff   -- -- --
K Day 16 Mean 3.46 3.18 3.45 4.09
(mEq/L)   SD