(2-methoxyethyl)benzene

Toxicity to reproduction

Administrative data

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-reference

Cross-reference 1

Reason / purpose for cross-reference:

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 for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

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 or test system 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:

body weight and weight gain

clinical biochemistry

clinical signs

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	4^a
		%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

Cross-reference 2

Reason / purpose for cross-reference:

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 for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

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 or test system 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:

body weight and weight gain

clinical biochemistry

clinical signs

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	&