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REACH

2,9-dichloro-5,12-dihydroquino[2,3-b]acridine-7,14-dione

EC number: 221-424-4 | CAS number: 3089-17-6

Life Cycle description
Uses advised against

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:: repeated dose toxicity: oral, other
Remarks:: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test by Oral Gavage in Sprague Dawley Rats
Type of information:: experimental study
Adequacy of study:: key study
Study period:: 15 June 2021 to 22 December 2021
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study

Cross-reference

Reason / purpose for cross-reference:: reference to same study

Reference

Endpoint:: screening for reproductive / developmental toxicity
Type of information:: experimental study
Adequacy of study:: key study
Study period:: 15 June 2021 to 22 December 2021
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Qualifier:: according to guideline
Guideline:: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:: As per OECD Guideline for Testing of Chemicals, Number 422, adopted on 29 July 2016
Deviations:: no
GLP compliance:: yes (incl. QA statement)
Justification for study design:: - Basis for dose level selection: There was no information or data available from repeated dose/reproduction toxicity and teratology studies for the test item.
The doses of 0, 111, 333 and 1000 mg/kg body weight/day were selected as control, low, mid and high dose groups for the dose range finding study (BIO-DTX 056) in consultation with the sponsor.
There were no test item-related related changes noted in any of the systemic, reproduction and developmental parameters up to the dose level of 1000 mg/kg body weight/day in the Dose Range Finding Study (BIO-DTX 056).
Hence, the same doses of 0, 111, 333 and 1000 mg/kg body weight/day were selected as control, low, mid and high dose groups respectively, for the present study in consultation with the sponsor.
Specific details on test material used for the study:: STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 21 to 29ºC
- Stability under test conditions: The test item was stable for 6 hours and 48 hours at room temperature in 0.5% w/v Carboxymethyl cellulose with the concentrations of 5 mg/mL and 100 mg/mL.
- Solubility of the test substance: The test item was insoluble in distilled water and uniformly suspended in 0.5% w/v Carboxy Methyl Cellulose at the concentration of 100 mg/mL (the highest dose concentration selected for the study considering the dose volume of 10 mL/kg body weight) as per in-house solubility/suspendibility test results.
Hence, 0.5% w/v Carboxymethyl Cellulose was used as vehicle for test item formulations in consultation with sponsor.
0.5% w/v Carboxymethyl Cellulose is a routinely used and universally accepted vehicle of choice for the oral toxicity studies.
Species:: rat
Strain:: Sprague-Dawley
Details on species / strain selection:: Rat is one of the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.
Sex:: male/female
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: Hylasco Biotechnology India Pvt. Ltd, Charles River Technology Licensee,
CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) Registration No.: 1808/PO/RcBt/S/15/CPCSEA.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8 to 10 weeks
- Weight at study initiation: Males: 254.36 to 291.64 g; Females: 200.54 to 211.92 g
- Housing: Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted food and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
During acclimatization, two animals of same sex were housed.
i. Main Group Animals:
- Pre mating: Per cage two animals of same sex and group were housed.
- Cohabitation Period (mating): Per cage two animals (one male and one female) of same group were housed.
- Post-mating: After confirmation on presence of sperm in the vaginal smear (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually. Sterilized paper shreds were provided as a nesting material for main group females from gestation day 20 onwards.
ii. Recovery Group Animals:
- Maximum of two animals per cage per group of same sex were housed, tunnels were provided as enrichment for the animals which were housed individually.
- Diet (e.g. ad libitum): Altromin maintenance diet
- Water (e.g. ad libitum): Water was provided ad libitum throughout the experimental period. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.9 to 22.9 oC
- Humidity (%): 48 to 63%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle
IN-LIFE DATES: From: 24 June 2021 To: 16 September 2021
Route of administration:: oral: gavage
Details on mating procedure:: - M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy.
- After successful mating each pregnant female was caged (how): After confirmation on presence of sperm in the vaginal smear (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually. Sterilized paper shreds were provided as a nesting material for main group females from gestation day 20 onwards.
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: Homogeneity and dose formulation analysis for dose concentration verification was performed during week 1 and 5 of the treatment. The results of dose concentration verifications for the prepared test item formulations and the mean results of analysis were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) was less than 15%.
Duration of treatment / exposure:: The test item or vehicle were administered to the animals through the oral (gavage) route once daily.
The males were treated for two weeks pre-mating, during mating period and up to the day before sacrifice (total of completion of 31 days of administration).
The females were treated for a two-week pre-mating period (14-days), during mating, pregnancy (gestation) and up to lactation day 13 (ranging from a total period of 50 to 62 days).
The females with positive mating signal, but no-evidence of pregnancy / littering were treated during two-week pre-mating period, during mating and further 24 days from the day of positive mating signal (group G1 - one female until experimental day 39; group G2 - one female until experimental day 52, one female until experimental day 46; G3 - two females until experimental day 49; group G4 - one female until experimental day 39).
The recovery group animals were treated until the first scheduled sacrifice of dams (total of 52 days of administration) and kept without treatment for further 18-days of observation period.
The test item/vehicle was administered by oral (gavage) route using a stainless-steel intubation cannula attached to a disposable syringe. All the doses were administered in an equivolume of 10 mL/kg body weight/day with the concentration of 11.1, 33.3 and 100 mg/mL for low, mid, and high dose groups, respectively. Vehicle was administered to the vehicle control group at an equivolume of 10 mL/kg body weight/day.
The actual dose volume for each animal was calculated based on the most recent body weight. The test item formulations were administered as soon as possible after preparation.
The prepared formulations were stirred using magnetic stirrer during administration to maintain homogeneity.
Frequency of treatment:: Once Daily
Details on study schedule:: - Age at mating of the mated animals in the study: 13 to 14 weeks
Dose / conc.:: 0 mg/kg bw/day
Remarks:: Vehicle Control
Dose / conc.:: 111 mg/kg bw/day
Remarks:: Low Dose
Dose / conc.:: 333 mg/kg bw/day
Remarks:: Mid Dose
Dose / conc.:: 1 000 mg/kg bw/day
Remarks:: High Dose
No. of animals per sex per dose:: Main Group: 24 (12 Males + 12 Females)
Recovery Group: 10 (5 Males + 5 Females)
Control animals:: yes, concurrent vehicle
Details on study design:: - Dose selection rationale: There was no information or data available from repeated dose/reproduction toxicity and teratology studies for the test item.
Based on the above available information, the doses of 0, 111, 333 and 1000 mg/kg body weight/day were selected as control, low, mid and high dose groups for the dose range finding study (BIO-DTX 056) in consultation with the sponsor.
There were no test item-related related changes noted in any of the systemic, reproduction and developmental parameters up to the dose level of 1000 mg/kg body weight/day in the Dose Range Finding Study (BIO-DTX 056).
Hence, the same doses of 0, 111, 333 and 1000 mg/kg body weight/day were selected as control, low, mid and high dose groups respectively, for the present study in consultation with the sponsor.

- Rationale for animal assignment: The animals were weighed and arranged in ascending order of their body weights. These body weight stratified animals were distributed to all the main and recovery groups using Microsoft Excel Spreadsheet, such that body weight variation of animals selected for the study does not exceed ±20% (Males: -6.80 to 11.14%; Females: -12.58 to 8.29%) of the mean body weight of each sex. The grouping was done one day prior to initiation of treatment. Body weight of the animals were analyzed statistically for mean body weight to rule out statistical significant differences between groups within each sex.
Parental animals: Observations and examinations:: CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily for general clinical signs of toxicity.
- Cage side observations checked in table [No.1] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Day 1 before treatment and weekly thereafter during treatment.

BODY WEIGHT: Yes
- Time schedule for examinations: The main group males were weighed at receipt, on the first day of dosing, weekly thereafter and at termination.
The main group females were weighed at receipt, on the first day of dosing, weekly thereafter during pre-mating and until confirmation of mating. All the pregnant animals were weighed on gestation days (GD) 0, 7, 14 and 20 and on lactation days (LD) 1, 4, 7, 13 and 14 (terminal body weight). The non-pregnant animals were weighed weekly once until termination and the terminal body weights were recorded before scheduled sacrifice.

The recovery group animals were weighed at receipt, on the first day of dosing, weekly thereafter and at termination.
Oestrous cyclicity (parental animals):: Oestrus cyclicity was monitored for two weeks after five days of acclimatization to evaluate the normal oestrus cycle (4 to 5 days). Females with normal oestrus cyclicity only were selected for the treatment. Vaginal smears were monitored daily from beginning of the treatment period until evidence of mating. When obtaining vaginal/cervical cells, care was taken to avoid disturbance of mucosa, which may induce pseudopregnancy. The status of oestrus cyclicity of females was determined on termination day.

Recovery group females were not evaluated for oestrus cyclicity
Sperm parameters (parental animals):: Parameters examined in all males: testis weight, epididymis weight.
Litter observations:: STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):: SACRIFICE
- Male animals: All animals were sacrificed after completion of 31 days of treatment. The males were necropsied in randomized manner.
- Maternal animals: All pregnant females were sacrificed on lactation day 14 and the non-pregnant females were sacrificed after 24 days from the last day of confirmation of mating.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [22] were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):: SACRIFICE
- Dead pups and pups which were sacrificed on PND 4/13, examined for gross abnormalities with particular attention to the external reproductive genitals and the findings were recorded.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
Statistics:: The raw data was subjected to computer statistical processing. The computer printout of the data (in the form of an appendix) was verified with the raw data. After verification, the data was subjected to various statistical analyses using SPSS software version 22.
All analysis and comparisons were evaluated at 95% with the level of confidence of P<0.05 respectively, indicated by the aforementioned tests and were designated by the superscripts throughout the report as stated below:
* Statistically significant (P<0.05) change than the vehicle control group.
Note: Data of non-pregnant females was excluded for mean calculations and statistical analysis for reproductive end points but considered for systemic parameters. However, the individual animal data is presented in appendices.
The statistical analysis tests followed for the interpretation of data are as mentioned below:

Parametric - One-way ANOVA
• Body weight (weekly/gestation/lactation)
• Percent Change in body weight (weekly/gestation/lactation)
• Feed consumption (weekly/gestation/lactation)
• Copulatory interval
• Gestation length
• Hematology
• Clinical chemistry
• Urinalysis
• Absolute/relative organ weights
• Functional Observation parameters
• Mean pup weight per litter
• Mean pup anogenital distance ratio per litter
• Serum T4 values (adults/pups).

Non Parametric - Kruskal-Wallis
• Implantations/litter
• No. of pups/litter
• Sex ratio/litter
• Litter size
• No. of early resorptions/litter
• No. of late resorptions/litter
• Prenatal loss/litter
• Postnatal loss/litter

Cross Tabs - Chi-square test
• Reproductive performances (frequency occurrences)
- mated/non-mated;
- fertile/infertile;
- pregnant/non-pregnant;
- with/without live pups;
- with/without dead pups
Reproductive indices:: The following reproductive performance indices were calculated as mentioned below and presented in the study report,

Male Mating Index (%) = No. of males with confirmed mating / Total No. of males cohabited x 100
Female Mating Index (%) = No. of sperm-positive females / Total No. of females cohabited x 100
Male Fertility Index (%) = No. of males impregnating a female / Total No. of males mated x 100
Female Fertility Index (%) = No. of pregnant females / No. of sperm-positive females x 100
Pre-coital Interval (Days) = Date of confirmation of mating – Date of initiation of cohabitation
Gestation Length (days) = Date of parturition – Date of positive evidence of mating (GD 0)
Gestation Index (%) = No. of females with live born / No. of females with evidence of pregnancy x 100
Parturition Index (%) = No. of females littered / No. of females with evidence of pregnancy x 100
Pregnancy Index (%) = No. of pregnant females / No. of females with confirmed mating x 100
Offspring viability indices:: Sex ratio (m/f) = No. of male offspring / Number of female offspring
Live Birth Index (%) per litter = No. of pups born alive / Total No. of pups born x 100
Pup Survival index (%) on LD 4/7/13 = Total No. of live pups on LD 4/7/13 / No. of pups born/4/7/13 x 100
Sex ratio (m/f) on LD 4/7/13 = No. of male offspring on LD 4/7/13 / No. of female offspring on LD 4/7/13
Post-Implantation Loss (%) = No. of implantations - No. of viable pups / No. of implantations x 100
Post-implantation Loss (No.) = No. of implantations - No. of live births
Postnatal loss on LD 13 (%) = No. of pups alive on postnatal day 13 /No. of live pups at birth x 100
Postnatal Loss on LD 13 (No.) = Live births - pups alive at lactation day 13
Anogenital Distance (AGD) Ratio = Anogenital distance (mm) / Cube root of PND 4 pup weight x 100
Clinical signs:: no effects observed
Mortality:: no mortality observed
Body weight and weight changes:: no effects observed
Food efficiency:: no effects observed
Ophthalmological findings:: no effects observed
Haematological findings:: no effects observed
Clinical biochemistry findings:: no effects observed
Urinalysis findings:: no effects observed
Behaviour (functional findings):: no effects observed
Organ weight findings including organ / body weight ratios:: no effects observed
Histopathological findings: non-neoplastic:: no effects observed
Other effects:: no effects observed
Reproductive function: oestrous cycle:: no effects observed
Reproductive performance:: no effects observed
: Key result
Dose descriptor:: NOAEL
Effect level:: ca. 1 000 mg/kg bw/day
Based on:: test mat.
Sex:: male/female
Basis for effect level:: clinical signs; mortality; body weight and weight gain; food efficiency; ophthalmological examination; haematology; clinical biochemistry; urinalysis; organ weights and organ / body weight ratios; gross pathology; histopathology: non-neoplastic; reproductive function (oestrous cycle); reproductive function (sperm measures); reproductive performance; other: Thyroid hormonal levels
: Key result
Critical effects observed:: no
Clinical signs:: no effects observed
Mortality / viability:: no mortality observed
Body weight and weight changes:: no effects observed
Gross pathological findings:: no effects observed
Other effects:: effects observed, non-treatment-related
Description (incidence and severity):: There were no changes in mean serum T4 hormone levels of pups at postnatal day 13 in any of the tested dose group litters when compared with vehicle control group litters. However, an incidental statistically significant decrease in mean T4 levels in pups was noted in groups G2, G3 and G4 when compared with vehicle control group.
Description (incidence and severity):: The oral administration of test item Hostaperm Red Violet ER 02 over the time of the study, did not produce any indication of developmental toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day.
: Key result
Dose descriptor:: NOAEL
Generation:: F1
Effect level:: ca. 1 000 mg/kg bw/day
Based on:: test mat.
Sex:: male/female
Basis for effect level:: viability; clinical signs; mortality; body weight and weight gain; gross pathology; other: Serum Thyroid hormonal levels (T4)
: Key result
Critical effects observed:: no
: Key result
Reproductive effects observed:: no

Group, Sex & Dose (mg/kg body weight/day)	Total No. of Animals	Clinical Signs of Toxicity^a: Observation (No. of Animals revealed)	Detailed Clinical Examination^b: Observation (No. of Animals revealed)	Mortality^c: No. of Mortalities (Total No. of Animals)
G1, M & 0	12	N (12)	NAD (12)	0 (12)
G2, M & 111	12	N (12)	NAD (12)	0 (12)
G3, M & 333	12	N (12)	NAD (12)	0 (12)
G4, M & 1000	12	N (12)	NAD (12)	0 (12)

Group, Sex & Dose (mg/kg body weight/day)	Total No. of Animals	Clinical Signs of Toxicity^a: Observation (No. of Animals revealed)	Detailed Clinical Examination^b: Observation (No. of Animals revealed)	Mortality^c: No. of Mortalities (Total No. of Animals)
G1, F & 0	12	N (12)	NAD (12)	0 (12)
G2, F & 111	12	N (12)	NAD (12)	0 (12)
G3, F & 333	12	N (12)	NAD (12)	0 (12)
G4, F & 1000	12	N (12)	NAD (12)	0 (12)

Group, Sex & Dose (mg/kg body weight/day)	Total No. of Animals	Clinical Signs of Toxicity^a: Observation (No. of Animals revealed)	Detailed Clinical Examination^b: Observation (No. of Animals revealed)	Mortality^c: No. of Mortalities (Total No. of Animals)
G1R, M & 0	5	N (5)	NAD (5)	0 (5)
G4R, M & 1000	5	N (5)	NAD (5)	0 (5)
G1R, F & 0	5	N (5)	NAD (5)	0 (5)
G4R, F & 1000	5	N (5)	NAD (5)	0 (5)

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Day
		Body Weight (g) on Day
		1	7	14	21	28
G1, M & 0	Mean	400.75	421.20	438.19	446.01	458.77
	±SD	22.07	32.34	36.49	37.08	38.53
	n	12	12	12	12	12
G2, M & 111	Mean	401.74	417.26	433.41	442.75	452.56
	±SD	20.39	24.35	30.70	31.29	30.16
	n	12	12	12	12	12
G3, M & 333	Mean	402.72	419.51	438.54	444.84	456.36
	±SD	19.35	23.54	30.11	27.23	24.95
	n	12	12	12	12	12
G4, M & 1000	Mean	401.99	422.76	443.74	453.94	463.38
	±SD	21.50	27.21	34.32	36.83	39.17
	n	12	12	12	12	12

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Day
		Body Weight (g) on Day
		1	7	14	21^#
G1, F & 0	Mean	250.88	257.35	267.91	280.34
	±SD	14.86	17.17	18.25	26.26
	n	12	12	12	3
G2, F & 111	Mean	251.05	256.43	266.53	272.56
	±SD	12.78	15.62	15.30	25.98
	n	12	12	12	3
G3, F & 333	Mean	251.13	256.32	261.08	260.46
	±SD	13.59	14.00	15.94	17.45
	n	12	12	12	4
G4, F & 1000	Mean	251.24	261.46	265.51	269.05
	±SD	15.56	18.91	20.71	24.37
	n	12	12	12	4

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Gestation Day (GD)
Group, Sex & Dose (mg/kg body weight/day)		0	7	14	20
G1, F & 0	Mean	272.42	287.38	319.92	375.62
	±SD	18.33	21.41	28.89	35.87
	n	11	11	11	11
G2, F & 111	Mean	269.81	286.87	328.45	391.71
	±SD	11.98	12.37	15.11	22.46
	n	10	10	10	10
G3, F & 333	Mean	264.91	282.42	317.51	369.85
	±SD	15.69	14.58	20.68	32.90
	n	10	10	10	10
G4, F & 1000	Mean	273.49	291.35	327.07	382.89
	±SD	17.37	17.37	23.26	39.30
	n	11	11	11	11

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Lactation Day (LD)
Group, Sex & Dose (mg/kg body weight/day)		1	4	7	13
G1, F & 0	Mean	294.60	300.48	308.18	323.25
	±SD	22.07	22.28	22.06	20.39
	n	11	11	11	11
G2, F & 111	Mean	306.24	309.48	314.78	330.57
	±SD	22.46	21.39	20.72	19.26
	n	10	10	10	10
G3, F & 333	Mean	293.35	297.31	304.48	319.65
	±SD	19.47	20.23	20.46	22.22
	n	10	10	10	10
G4, F & 1000	Mean	303.31	306.82	313.01	325.64
	±SD	22.77	24.10	23.73	23.27
	n	11	11	11	11

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight (%) during Day
		Percent Change in Body Weight (%) during Day
		1 to 7	1 to 14	1 to 21	1 to 28
G1, M & 0	Mean	5.01	9.25	11.19	14.36
	±SD	3.10	4.96	4.86	4.98
	n	12	12	12	12
G2, M & 111	Mean	3.84	7.80	10.13	12.59
	±SD	2.04	3.09	3.48	3.20
	n	12	12	12	12
G3, M & 333	Mean	4.15	8.89	10.50	13.42
	±SD	2.15	5.14	5.14	5.61
	n	12	12	12	12
G4, M & 1000	Mean	5.12	10.29	12.82	15.16
	±SD	1.91	3.83	4.55	5.27
	n	12	12	12	12

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight Gain (%) during Gestation Day (GD)
Group, Sex & Dose (mg/kg body weight/day)		0 to 7	7 to 14	14 to 20
G1, F & 0	Mean	5.46	11.22	17.44
	±SD	1.77	2.82	4.18
	n	11	11	11
G2, F & 111	Mean	6.35	14.52	19.23
	±SD	2.12	3.20	3.16
	n	10	10	10
G3, F & 333	Mean	6.67	12.40	16.35
	±SD	1.92	3.55	3.43
	n	10	10	10
G4, F & 1000	Mean	6.56	12.23	16.88
	±SD	1.60	3.35	4.81
	n	11	11	11

Group, Sex & Dose (mg/kg body weight/day)		Feed Consumption (g/animal/day) during Pre-mating Period
Group, Sex & Dose (mg/kg body weight/day)		Week 1	Week 2
G1, M & 0	Mean	21.31	24.01
	±SD	2.38	1.81
	n	12 (6)	12 (6)
G2, M & 111	Mean	21.39	23.12
	±SD	1.76	0.75
	n	12 (6)	12 (6)
G3, M & 333	Mean	22.25	23.93
	±SD	2.39	1.85
	n	12 (6)	12 (6)
G4, M & 1000	Mean	22.28	24.17
	±SD	1.23	0.67
	n	12 (6)	12 (6)

Group, Sex & Dose (mg/kg body weight/day)		Feed Consumption (g/animal/day)
Group, Sex & Dose (mg/kg body weight/day)		Week 1	Week 2	Week 3	Week 4	Week 5	Week 6	Week 7	Week 8	Week 9
G1R, M & 0	Mean	23.52	24.34	25.11	26.35	27.86	29.05	30.75	31.83	32.47
	±SD	3.71	1.67	1.55	1.27	0.97	1.11	0.47	3.66	3.01
	n	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)
G4R, M & 1000	Mean	22.84	23.75	24.26	27.17	28.86	30.05	31.07	32.19	33.03
	±SD	0.77	1.04	1.34	2.09	2.61	2.18	2.12	1.59	1.67
	n	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)
G1R, F & 0	Mean	17.44	18.36	19.45	10.21	23.17	24.20	25.35	26.19	27.04
	±SD	1.79	0.40	0.40	9.72	0.99	0.82	0.62	0.41	0.99
	n	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)
G4R, F & 1000	Mean	17.21	19.05	20.42	11.29	24.18	25.37	26.19	27.14	27.89
	±SD	0.69	1.77	0.91	10.38	0.03	0.80	0.84	0.69	0.68
	n	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)

Day -1 (Grouping and randomization)
Group, Sex & Dose (mg/kg body weight/day) →	G1, M & 0		G2, M & 111		G3, M & 333		G4, M & 1000
Number of Animals→	12		12		12		12
Observations↓
Eye→ Region↓	LE	RE	LE	RE	LE	RE	LE	RE
Eye Lids	N	N	N	N	N	N	N	N
Cornea	N	N	N	N	N	N	N	N
Iris	N	N	N	N	N	N	N	N
Aqueous Humour	N	N	N	N	N	N	N	N
Lens	N	N	N	N	N	N	N	N
Vitreous Humour	N	N	N	N	N	N	N	N
Retina/Optic disc	N	N	N	N	N	N	N	N

Day 30
Group, Sex & Dose (mg/kg body weight/day) →	G1, M & 0		G2, M & 111		G3, M & 333		G4, M & 1000
Number of Animals→	12		12		12		12
Observations↓
Eye→ Region↓	LE	RE	LE	RE	LE	RE	LE	RE
Eye Lids	N	N	N	N	N	N	N	N
Cornea	N	N	N	N	N	N	N	N
Iris	N	N	N	N	N	N	N	N
Aqueous Humour	N	N	N	N	N	N	N	N
Lens	N	N	N	N	N	N	N	N
Vitreous Humour	N	N	N	N	N	N	N	N
Retina/Optic disc	N	N	N	N	N	N	N	N
Other findings:	N	N	N	N	N	N	N	N

Pregnant/Littered females: Lactation day 13; Non-pregnant females: (Experimental day 39 to 52)
Group, Sex & Dose (mg/kg body weight/day) →	G1, F & 0		G2, F & 111		G3, F & 333		G4, F & 1000
Number of Animals→	12		12		12		12
Observations↓
Eye→ Region↓	LE	RE	LE	RE	LE	RE	LE	RE
Eye Lids	N	N	N	N	N	N	N	N
Cornea	N	N	N	N	N	N	N	N
Iris	N	N	N	N	N	N	N	N
Aqueous Humour	N	N	N	N	N	N	N	N
Lens	N	N	N	N	N	N	N	N
Vitreous Humour	N	N	N	N	N	N	N	N
Retina/Optic disc	N	N	N	N	N	N	N	N
Other findings:	N	N	N	N	N	N	N	N

Day 65
Group, Sex & Dose (mg/kg body weight/day) →	G1R, M & 0		G4R, M & 1000
Number of Animals→	5		5
Observations↓
Eye→ Region↓	LE	RE	LE	RE
Eye Lids	N	N	N	N
Cornea	N	N	N	N
Iris	N	N	N	N
Aqueous Humour	N	N	N	N
Lens	N	N	N	N
Vitreous Humour	N	N	N	N
Retina/Optic disc	N	N	N	N
Other findings:	N	N	N	N

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Day
		Body Weight (g) on Day
		1	7	14	21	28	35	42	49	56	63
G1R, M & 0	Mean	395.45	407.17	422.63	436.11	448.75	458.35	473.23	489.33	502.87	514.37
	±SD	21.72	21.10	31.29	32.45	32.78	34.62	33.67	33.58	34.92	37.99
	n	5	5	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	396.36	412.35	430.15	444.31	454.98	464.82	475.04	493.68	508.20	519.69
	±SD	20.86	20.17	12.53	16.96	17.27	14.72	15.50	9.28	11.65	11.69
	n	5	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	245.24	250.97	262.44	268.72	277.56	284.92	295.96	306.72	315.86	324.75
	±SD	10.97	10.11	13.68	15.80	15.60	15.72	19.40	19.59	17.41	15.21
	n	5	5	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	245.66	248.99	264.41	270.43	278.43	286.33	298.35	308.32	318.51	327.87
	±SD	12.82	12.43	13.84	12.16	11.19	12.10	12.79	13.72	10.97	12.44
	n	5	5	5	5	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight (%) during Day
		Percent Change in Body Weight (%) during Day
		1 to 7	1 to 14	1 to 21	1 to 28	1 to 35	1 to 42	1 to 49	1 to 56	1 to 63
G1R, M & 0	Mean	2.98	6.83	10.25	13.47	15.92	19.69	23.73	27.13	30.00
	±SD	0.92	4.09	4.67	5.29	6.27	5.81	4.88	4.25	4.30
	n	5	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	4.06	8.66	12.20	14.91	17.44	20.09	24.77	28.45	31.32
	±SD	1.73	3.60	3.33	3.84	4.90	6.78	5.51	6.10	5.14
	n	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	2.35	7.00	9.55	13.20	16.19	20.62	25.02	28.77	32.44
	±SD	0.72	2.36	3.15	4.61	4.45	4.14	4.54	3.55	3.44
	n	5	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	1.37	7.66	10.20	13.45	16.65	21.52	25.60	29.78	33.57
	±SD	1.07	2.71	4.80	4.07	3.73	3.02	4.10	3.75	3.31
	n	5	5	5	5	5	5	5	5	5

Day 30
Parameters↓	Group & Sex		G1 & M	G2 & M	G3 & M	G4 & M
	Dose (mg/kg body weight/day)		0	111	333	1000
	Number of Animals (randomly selected)		5	5	5	5
Home Cage Observations
Home cage posture			1	1	1	1
Respiratory pattern			1	1	1	1
Clonic involuntary movements			1	1	1	1
Tonic involuntary movements			1	1	1	1
Vocalization			1	1	1	1
Palpebral closure			1	1	1	1
Handling Observations
Ease of removal from the cage			2	2	2	2
Ease of handling animal in hand			2	2	2	2
Red or crusty deposits		Eyes	1	1	1	1
		Nose	1	1	1	1
		Mouth	1	1	1	1
Lacrimation			1	1	1	1
Salivation			1	1	1	1
Fur appearance			1	1	1	1
Piloerection			1	1	1	1
Eye prominence			1	1	1	1
Muscle tone			1	1	1	1
Home Cage Observations:Home cage posture - 1=Normal; Respiratory pattern - 1=Normal; Clonic involuntary movements - 1=None; Tonic involuntary movements - 1=None; Vocalization - 1=None; Palpebral closure - 1=Normal;Handling Observations:Ease of removal from the cage - 2=Normal; Ease of handling animal in hand - 2=Normal; Red or crusty deposits - 1=Absent; Lacrimation - 1=None; Salivation- 1=None; Fur appearance - 1=Normal; Piloerection - 1=None; Eye Prominence - 1=Normal; Muscle tone - 1=Normal

Day 30
Parameters↓	Group & Sex		G1 & M	G2 & M	G3 & M	G4 & M
	Dose (mg/kg body weight/day)		0	111	333	1000
	Number of Animals (randomly selected)		5	5	5	5
Open Field Observations
Mobility			1	1	1	1
Gait			1	1	1	1
Arousal			3	3	3	3
Number of rearing		Mean	4.4	4.8	4.8	4.8
Number of rearing		±SD	1.1	0.8	0.8	0.8
Number of urine pools		Mean	3.4	3.8	3.8	3.8
Number of urine pools		±SD	0.5	0.8	0.8	0.8
Number of defecations		Mean	3.4	3.2	3.4	3.2
Number of defecations		±SD	0.5	0.8	0.5	0.4
Clonic involuntary movement			1	1	1	1
Tonic involuntary movement			1	1	1	1
Stereotype behaviour			1	1	1	1
Number of grooming		Mean	3.4	3.2	3.4	3.2
Number of grooming		±SD	0.5	0.8	0.5	0.8
Sensory Observations
Approach response			2	2	2	2
Auditory response			2	2	2	2
Touch response			2	2	2	2
Pupil reflex			2	2	2	2
Tail pinch response			2	2	2	2
Righting reflex			1	1	1	1
Physiological Observation
Body temperature (°F)		Mean	98.4	98.4	98.1	98.4
Body temperature (°F)		±SD	0.5	0.4	0.3	0.5
Open Field Observations: Mobility- 1=Normal;Gait- 1=Normal;Arousal- 3=Normal;Clonic involuntary movement- 1=None;Tonic involuntary movement -1=None;Stereotype behaviour -1=Absent;Sensory Observations:Approach response- 2=Normal;Auditory response- 2=Normal;Touch response -2=Normal;Pupil reflex- 2=Normal;Tail pinch response- 2=Normal;Righting reflex- 1=Present

Lactation Day 13
Parameters↓	Group & Sex		G1 & F	G2 & F	G3 & F	G4 & F
	Dose (mg/kg body weight/day)		0	111	333	1000
	Number of Animals (randomly selected)		5	5	5	5
Home Cage Observations
Home cage posture			1	1	1	1
Respiratory pattern			1	1	1	1
Clonic involuntary movements			1	1	1	1
Tonic involuntary movements			1	1	1	1
Vocalization			1	1	1	1
Palpebral closure			1	1	1	1
Handling Observations
Ease of removal from the cage			2	2	2	2
Ease of handling animal in hand			2	2	2	2
Red or crusty deposits		Eyes	1	1	1	1
		Nose	1	1	1	1
		Mouth	1	1	1	1
Lacrimation			1	1	1	1
Salivation			1	1	1	1
Fur appearance			1	1	1	1
Piloerection			1	1	1	1
Eye prominence			1	1	1	1
Muscle tone			1	1	1	1
Home Cage Observations:Home cage posture - 1=Normal; Respiratory pattern - 1=Normal; Clonic involuntary movements - 1=None; Tonic involuntary movements - 1=None; Vocalization - 1=None; Palpebral closure - 1=Normal;Handling Observations:Ease of removal from the cage - 2=Normal; Ease of handling animal in hand - 2=Normal; Red or crusty deposits - 1=Absent; Lacrimation - 1=None; Salivation- 1=None; Fur appearance - 1=Normal; Piloerection - 1=None; Eye Prominence - 1=Normal; Muscle tone - 1=Normal

Day 30
Group, Sex & Dose (mg/kg body weight/day)		Movement Counts (no.)
G1, M & 0	Mean	1911.4
	±SD	108.9
	n	5
G2, M & 111	Mean	1961.8
	±SD	138.9
	n	5
G3, M & 333	Mean	1954.2
	±SD	126.1
	n	5
G4, M & 1000	Mean	1931.4
	±SD	104.0
	n	5

Day 30
Group, Sex & Dose (mg/kg body weight/day)		Grip Strength (kgf)
Group, Sex & Dose (mg/kg body weight/day)		Forelimb	Hindlimb
G1, M & 0	Mean	1.529	0.510
	±SD	0.023	0.025
	n	5	5
G2, M & 111	Mean	1.501	0.539
	±SD	0.020	0.021
	n	5	5
G3, M & 333	Mean	1.521	0.552*
	±SD	0.048	0.008
	n	5	5
G4, M & 1000	Mean	1.539	0.544*
	±SD	0.040	0.015
	n	5	5

Day 30
Group, Sex & Dose (mg/kg body weight/day)		Hindlimb Foot Splay (cm)
G1, M & 0	Mean	9.0
	±SD	1.0
	n	5
G2, M & 111	Mean	8.8
	±SD	1.3
	n	5
G3, M & 333	Mean	9.3
	±SD	0.6
	n	5
G4, M & 1000	Mean	8.6
	±SD	0.6
	n	5

Group, Sex & Dose (mg/kg body weight/day)		Total Leucocyte Count	Total Erythrocyte Count	Haemoglobin	Haematocrit	Mean Corpuscular Volume	Mean Corpuscular Haemoglobin	Mean Corpuscular Haemoglobin Concentration	Platelet Count	Mean Platelet Volume
		(WBC)	(RBC)	(HGB)	(HCT)	(MCV)	(MCH)	(MCHC)	(PLT)	(MPV)
		(10³cells/µL)	(10⁶cells/µL)	(g/dL)	(%)	(fL)	(pg)	(g/dL)	(10³cells/µL)	(fL)
G1, M & 0	Mean	8.26	9.64	17.56	52.80	55.00	18.30	33.26	776.40	7.72
	±SD	1.95	1.09	1.34	4.49	3.09	1.14	0.53	330.86	0.52
	n	5	5	5	5	5	5	5	5	5
G2, M & 111	Mean	9.68	9.37	16.82	51.32	54.72	17.94	32.80	1135.20	7.46
	±SD	2.59	0.15	0.33	0.89	1.12	0.29	0.58	184.30	0.19
	n	5	5	5	5	5	5	5	5	5
G3, M & 333	Mean	8.67	9.40	16.58	50.78	54.12	17.64	32.62	962.60	7.72
	±SD	3.67	0.62	0.87	1.91	1.72	0.59	0.70	256.00	0.52
	n	5	5	5	5	5	5	5	5	5
G4, M & 1000	Mean	8.14	9.35	16.88	52.02	55.74	18.06	32.46	1017.60	7.50
	±SD	2.42	0.54	0.55	1.58	2.71	0.74	0.53	93.02	0.38
	n	5	5	5	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Reticulocyte Count	Neutrophils	Lymphocytes	Monocytes	Eosinophils	Basophils
		(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)
		(%)	(%)	(%)	(%)	(%)	(%)
G1, M & 0	Mean	2.25	23.48	72.02	2.38	0.44	0.80
	±SD	1.24	9.09	9.80	1.15	0.17	0.49
	n	5	5	5	5	5	5
G2, M & 111	Mean	1.87	19.66	75.74	2.28	1.04	0.54
	±SD	0.64	3.44	3.55	0.74	0.59	0.23
	n	5	5	5	5	5	5
G3, M & 333	Mean	1.87	21.98	70.58	2.40	1.04	0.62
	±SD	0.60	5.35	7.57	2.37	0.34	0.61
	n	5	5	5	5	5	5
G4, M & 1000	Mean	2.01	20.86	75.78	1.42	0.72	0.54
	±SD	0.79	3.34	2.92	0.23	0.25	0.15
	n	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Absolute Reticulocyte Count	Absolute Neutrophils	Absolute Lymphocytes	Absolute Monocytes	Absolute Eosinophils	Absolute Basophils	Prothrombin Time	Activated Prothrombin Time
		(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)	(PT)	(APTT)
		(10⁹cells/L)	(10³cells/µL)	(10³cells/µL)	(10³cells/µL)	(10³cells/µL)	(10³cells/µL)	(Seconds)	(Seconds)
G1, M & 0	Mean	212.96	1.83	6.07	0.19	0.04	0.07	15.54	22.26
	±SD	101.87	0.42	2.01	0.08	0.02	0.06	0.78	6.12
	n	5	5	5	5	5	5	5	5
G2, M & 111	Mean	174.50	1.91	7.33	0.23	0.10*	0.05	15.72	25.28
	±SD	56.93	0.64	1.96	0.10	0.04	0.02	1.03	1.56
	n	5	5	5	5	5	5	5	5
G3, M & 333	Mean	175.20	1.80	5.98	0.27	0.09*	0.07	16.72	24.30
	±SD	59.25	0.52	1.97	0.39	0.03	0.11	1.49	1.59
	n	5	5	5	5	5	5	5	5
G4, M & 1000	Mean	184.78	1.70	6.17	0.11	0.06	0.04	15.98	26.78
	±SD	67.09	0.55	1.89	0.04	0.02	0.02	0.89	1.29
	n	5	5	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Absolute Reticulocyte Count	Absolute Neutrophils	Absolute Lymphocytes	Absolute Monocytes	Absolute Eosinophils	Absolute Basophils	Prothrombin Time	Activated Prothrombin Time
		(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)	(PT)	(APTT)
		(10⁹cells/L)	(10³cells/µL)	(10³cells/µL)	(10³cells/µL)	(10³cells/µL)	(10³cells/µL)	(Seconds)	(Seconds)
G1, F & 0	Mean	177.36	3.64	6.21	0.40	0.15	0.06	17.60	23.34
	±SD	60.94	2.11	2.30	0.19	0.09	0.03	1.02	2.05
	n	5	5	5	5	5	5	5	5
G2, F & 111	Mean	313.86	3.59	6.91	0.56	0.12	0.05	22.06	27.48
	±SD	276.22	2.17	1.72	0.29	0.04	0.02	8.41	10.47
	n	5	5	5	5	5	5	5	5
G3, F & 333	Mean	117.60	2.64	6.29	0.39	0.12	0.06	16.88	22.08
	±SD	50.69	2.06	2.26	0.31	0.07	0.08	3.84	7.81
	n	5	5	5	5	5	5	5	5
G4, F & 1000	Mean	291.70	4.81	6.94	0.38	0.10	0.04	15.66	19.92
	±SD	145.98	3.66	3.06	0.17	0.03	0.02	3.85	5.41
	n	5	5	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Absolute Reticulocyte Count	Absolute Neutrophils	Absolute Lymphocytes	Absolute Monocytes	Absolute Eosinophils	Absolute Basophils	Prothrombin Time	Activated Prothrombin Time
		(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)	(PT)	(APTT)
		(10⁹cells/L)	(10³cells/µL)	(10³cells/µL)	(10³cells/µL)	(10³cells/µL)	(10³cells/µL)	(Seconds)	(Seconds)
G1R, M & 0	Mean	150.40	2.27	6.35	0.16	0.21	0.08	16.00	21.56
	±SD	23.36	0.40	1.23	0.04	0.07	0.02	2.04	2.05
	n	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	186.92	2.06	7.02	0.14	0.19	0.05	15.50	22.48
	±SD	52.68	0.53	1.09	0.04	0.04	0.03	1.17	3.12
	n	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	140.08	1.78	4.83	0.15	0.09	0.05	14.98	24.56
	±SD	23.05	0.73	1.73	0.05	0.02	0.01	0.53	1.98
	n	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	192.10*	2.06	4.98	0.17	0.25	0.05	15.92	19.34*
	±SD	30.17	0.60	2.77	0.07	0.24	0.02	1.17	2.34
	n	5	5	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Glucose	Urea	Creatinine	Total Cholesterol	Triglycerides	Total Protein	Albumin
		(GLU)	-	(CRE)	(CHO)	(TRI)	(TPR)	(ALB)
		(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(g/dL)	(g/dL)
G1, M & 0	Mean	126.60	30.52	0.58	51.60	25.60	7.74	3.47
	±SD	15.47	2.56	0.03	3.36	2.30	0.26	0.19
	n	5	5	5	5	5	5	5
G2, M & 111	Mean	123.60	33.30	0.57	60.60	30.40	7.24*	3.55
	±SD	5.55	3.91	0.04	4.83	6.07	0.33	0.15
	n	5	5	5	5	5	5	5
G3, M & 333	Mean	122.60	33.78	0.60	58.00	29.40	7.44	3.65
	±SD	12.92	2.83	0.03	6.71	7.73	0.22	0.29
	n	5	5	5	5	5	5	5
G4, M & 1000	Mean	131.00	36.04	0.59	56.00	27.00	7.58	3.68
	±SD	19.79	4.26	0.02	9.57	6.16	0.29	0.24
	n	5	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Alanine aminotransferase	Aspartate aminotransferase	Alkaline phosphatase	Total Bilirubin	Calcium	Phosphorous
		(ALT)	(AST)	(ALP)	(BIT)	(CAL)	(PHO)
		(U/L)	(U/L)	(U/L)	(mg/dL)	(mg/dL)	(mg/dL)
G1, M & 0	Mean	61.20	111.20	125.80	0.05	9.36	6.48
	±SD	16.93	12.34	21.11	0.01	0.11	0.58
	n	5	5	5	5	5	5
G2, M & 111	Mean	47.40	93.20	120.00	0.06	9.64	6.54
	±SD	8.96	13.46	31.92	0.03	0.47	0.62
	n	5	5	5	5	5	5
G3, M & 333	Mean	40.00	81.60*	127.20	0.06	10.40*	6.82
	±SD	5.96	12.30	38.01	0.03	1.05	0.62
	n	5	5	5	5	5	5
G4, M & 1000	Mean	52.20	91.80	114.80	0.06	9.94	6.62
	±SD	24.52	18.69	28.70	0.02	0.30	0.49
	n	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Globulin	Albumin/ Globulin ratio	Blood Urea Nitrogen	Sodium	Potassium	Chloride
		(GLO)	(A/G Ratio)	(BUN)	(Na)	(K)	(CLO)
		(g/dL)	-	(mg/dL)	(mmol/L)	(mmol/L)	(mmol/L)
G1, M & 0	Mean	4.27	0.82	14.24	141.64	3.96	104.10
	±SD	0.30	0.09	1.19	0.94	0.25	2.51
	n	5	5	5	5	5	5
G2, M & 111	Mean	3.69*	0.97	15.54	142.16	3.71	105.38
	±SD	0.38	0.12	1.83	0.53	0.15	1.40
	n	5	5	5	5	5	5
G3, M & 333	Mean	3.79*	0.97	15.77	142.44	3.86	105.78
	±SD	0.12	0.10	1.32	0.93	0.36	1.73
	n	5	5	5	5	5	5
G4, M & 1000	Mean	3.90	0.95	16.82	142.76	3.85	106.40
	±SD	0.29	0.11	1.99	0.66	0.09	0.86
	n	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Globulin	Albumin/Globulin ratio	Blood Urea Nitrogen	Sodium	Potassium	Chloride
		(GLO)	(A/G Ratio)	(BUN)	(Na)	(K)	(CLO)
		(g/dL)	-	(mg/dL)	(mmol/L)	(mmol/L)	(mmol/L)
G1, F & 0	Mean	3.69	0.98	22.72	143.04	4.02	106.18
	±SD	0.50	0.11	3.79	1.34	0.81	2.96
	n	5	5	5	5	5	5
G2, F & 111	Mean	3.93	0.88	23.87	142.18	3.57	107.22
	±SD	0.66	0.14	6.20	1.47	0.28	3.01
	n	5	5	5	5	5	5
G3, F & 333	Mean	3.98	0.91	20.90	142.36	3.60	107.94
	±SD	0.40	0.16	9.25	1.61	0.33	2.63
	n	5	5	5	5	5	5
G4, F & 1000	Mean	4.06	0.92	24.11	142.62	3.86	107.06
	±SD	0.50	0.09	7.55	1.62	0.40	3.20
	n	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Alanine aminotransferase	Aspartate aminotransferase	Alkaline phosphatase	Total Bilirubin	Calcium	Phosphorous
		(ALT)	(AST)	(ALP)	(BIT)	(CAL)	(PHO)
		(U/L)	(U/L)	(U/L)	(mg/dL)	(mg/dL)	(mg/dL)
G1R, M & 0	Mean	52.00	101.80	99.00	0.02	10.42	5.72
	±SD	40.34	53.17	23.40	0.02	0.37	0.34
	n	5	5	5	5	5	5
G4R, M & 1000	Mean	42.60	98.40	105.20	0.02	10.02	5.26
	±SD	9.86	18.49	29.84	0.01	0.15	0.50
	n	5	5	5	5	5	5
G1R, F & 0	Mean	54.00	94.00	70.80	0.02	11.20	4.20
	±SD	28.82	36.89	40.92	0.02	0.45	0.38
	n	5	5	5	5	5	5
G4R, F & 1000	Mean	53.80	103.20	69.00	0.04	10.90	4.42
	±SD	21.53	39.30	60.10	0.02	0.37	0.18
	n	5	5	5	5	5	5

Examination	Group, Sex & Dose (mg/kg body weight/day)		G1, M & 0	G2, M & 111	G3, M & 333	G4, M & 1000
Examination	Number of Animals (randomly selected)		5	5	5	5
Physical Examination	Color	Pale Yellow	5	5	5	5
	Appearance	Clear	5	5	5	5
	Volume (mL)	Mean	10.6	10.2	11.0	11.4
	Volume (mL)	±SD	0.7	1.4	0.8	0.4
Chemical Examination	pH	Mean	7.5	7.4	7.4	7.5
	pH	±SD	0.0	0.4	0.5	0.0
	Specific Gravity	Mean	1.008	1.011	1.009	1.009
	Specific Gravity	±SD	0.003	0.002	0.002	0.002
	Urobilinogen (mg/dL)	Mean	0.2	0.2	0.2	0.2
	Urobilinogen (mg/dL)	±SD	0.0	0.0	0.0	0.0
	Bilirubin (mg/dL)	Neg	5	5	5	5
	Ketones (mg/dL)	Neg	2	-	2	1
	Ketones (mg/dL)	5	3	5	3	4
	Blood (Ery/µL)	Neg	5	4	5	5
	Blood (Ery/µL)	Ca80	-	1	-	-
	Protein (mg/dL)	Neg	3	-	3	1
		Trace	2	4	1	4
		30	-	1	1	-
	Nitrite	Neg	4	4	5	1
	Nitrite	Pos	1	1	-	4
	Leucocytes	Neg	3	-	3	1
		Ca15	1	1	1	2
		Ca70	1	3	1	2
		Ca125	-	1	-	-
	Glucose (mg/dL)	Neg	5	5	5	5
	Microalbumin (mg/dL)	Neg	-	-	2	1
		>15	2	5	2	4
		15	3	-	1	-
Microscopic Examination	Epi Cells	0	3	4	2	4
		0-1	1	1	2	1
		1-2	1	-	1	-
	Casts	Absent	4	4	4	5
	Casts	Present	1	1	1	-
	Crystals	Present	5	5	5	5

Examination	Group, Sex & Dose (mg/kg body weight/day)		G1R, M & 0	G4R, M &1000	G1R, F & 0	G4R, F & 1000
Examination	Number of Animals (randomly selected)		5	5	5	5
Physical Examination	Color	Pale Yellow	2	5	3	3
	Color	Yellow	3	-	2	2
	Appearance	Clear	3	2	4	4
	Appearance	Turbid	2	3	1	1
	Volume (mL)	Mean	6.4	8.6	7.9	7.8
	Volume (mL)	±SD	1.7	3.1	2.2	0.8
Chemical Examination	pH	Mean	6.7	7.3	6.8	7.6
	pH	±SD	0.9	0.4	1.0	1.0
	Specific Gravity	Mean	1.020	1.014	1.014	1.012
	Specific Gravity	±SD	0.007	0.002	0.007	0.008
	Urobilinogen (mg/dL)	Mean	0.2	0.2	0.2	0.6
	Urobilinogen (mg/dL)	±SD	0.0	0.0	0.0	0.8
	Bilirubin (mg/dL)	Neg	2	5	5	2
	Bilirubin (mg/dL)	1	3	-	-	3
	Ketones (mg/dL)	Neg	1	2	5	5
	Ketones (mg/dL)	5	4	3	-	-
	Blood (Ery/µL)	Neg	5	5	5	4
	Blood (Ery/µL)	Ca10	-	-	-	1
	Protein (mg/dL)	Neg	2	5	4	2
		Trace	2	-	1	-
		30	1	-	-	-
		100	-	-	-	1
		>=300	-	-	-	2
	Nitrite	Neg	3	5	5	5
	Nitrite	Pos	2	-	-	-
	Leucocytes	Neg	-	1	3	2
		Ca15	1	4	2	-
		Ca70	4	-	-	3
	Glucose (mg/dL)	Neg	5	5	5	5
	Microalbumin (mg/dL)	Neg	-	1	3	1
		>15	3	-	1	3
		15	2	4	1	1
Microscopic Examination\	Epi Cells	0	3	3	2	2
		0-1	1	2	2	1
		1-2	1	-	1	-
		2-3	-	-	-	2
	Casts	Absent	5	2	5	4
	Casts	Present	-	3	-	1
	Crystals	Absent	-	2	1	2
	Crystals	Present	5	3	4	3

Group, Sex & Dose (mg/kg body weight/day)	Total No. of Females Evaluated		Females with Complete Regular Cycles	Females with at least one Irregular Oestrus Cycle	Average Length of Oestrus Cycle (Days)
G1, F & 0	12	n	11	1	Mean	4.65
		%	91.7	8.3	±SD	0.41
					n	12
G2, F & 111	12	n	11	0	Mean	4.43
		%	91.7	0.0	±SD	0.42
					n	12
G3, F & 333	12	n	12	0	Mean	4.68
		%	100.0	0.0	±SD	0.27
					n	12
G4, F & 1000	12	n	11	1	Mean	4.53
		%	91.7	8.3	±SD	0.34
					n	12

Group, Sex & Dose (mg/kg body weight/day)		Delivery Record At birth
		Litter Size (No.)	Live Pups (No.)			Dead Pups (No.)			Cannibalized Pups (No.)				Sex Ratio (m/f) at Birth	Live Birth Index (%)
		Litter Size (No.)	Male	Female	Total	Male	Female	Total	Undetermined	Male	Female	Total	Sex Ratio (m/f) at Birth	Live Birth Index (%)
G1, F & 0	Mean	12.18	6.27	5.73	12.00	0.18	0.00	0.18	0.00	0.00	0.00	0.00	1.14	98.86
	±SD	2.68	1.90	1.35	2.41	0.40	0.00	0.40	0.00	0.00	0.00	0.00	0.42	2.54
	n	11	11	11	11	11	11	11	11	11	11	11	11	11
G2, F & 111	Mean	12.20	6.00	6.00	12.00	0.10	0.10	0.20	0.00	0.00	0.00	0.00	1.12	98.70
	±SD	3.29	1.94	1.89	3.09	0.32	0.32	0.42	0.00	0.00	0.00	0.00	0.57	2.76
	n	10	10	10	10	10	10	10	10	10	10	10	10	10
G3, F & 333	Mean	10.90	5.30	5.50	10.80	0.10	0.00	0.10	0.00	0.00	0.00	0.00	1.00	99.41
	±SD	2.77	2.00	1.27	2.53	0.32	0.00	0.32	0.00	0.00	0.00	0.00	0.39	1.86
	n	10	10	10	10	10	10	10	10	10	10	10	10	10
G4, F & 1000	Mean	12.82	6.36	6.09	12.45	0.18	0.18	0.36	0.00	0.00	0.00	0.00	1.16	96.57
	±SD	1.99	2.11	1.64	2.54	0.40	0.60	0.92	0.00	0.00	0.00	0.00	0.71	9.11
	n	11	11	11	11	11	11	11	11	11	11	11	11	11

Group, Sex & Dose (mg/kg body weight/day)		LD 1 to 4									Sex Ratio (m/f) on LD 4	Pup Survival Index (%) [LD 1 to 4]
		Live Pups (No.)			Dead Pups (No.)			Cannibalized Pups (No.)
		Male	Female	Total	Male	Female	Total	Male	Female	Total
G1, F & 0	Mean	6.27	5.73	12.00	0.00	0.00	0.00	0.00	0.00	0.00	1.14	100.00
	±SD	1.90	1.35	2.41	0.00	0.00	0.00	0.00	0.00	0.00	0.42	0.00
	n	11	11	11	11	11	11	11	11	11	11	11
G2, F & 111	Mean	6.00	6.00	12.00	0.00	0.00	0.00	0.00	0.00	0.00	1.12	100.00
	±SD	1.94	1.89	3.09	0.00	0.00	0.00	0.00	0.00	0.00	0.57	0.00
	n	10	10	10	10	10	10	10	10	10	10	10
G3, F & 333	Mean	5.30	5.50	10.80	0.00	0.00	0.00	0.00	0.00	0.00	1.00	100.00
	±SD	2.00	1.27	2.53	0.00	0.00	0.00	0.00	0.00	0.00	0.39	0.00
	n	10	10	10	10	10	10	10	10	10	10	10
G4, F & 1000	Mean	6.36	6.09	12.45	0.00	0.00	0.00	0.00	0.00	0.00	1.16	100.00
	±SD	2.11	1.64	2.54	0.00	0.00	0.00	0.00	0.00	0.00	0.71	0.00
	n	11	11	11	11	11	11	11	11	11	11	11

Group, Sex & Dose (mg/kg body weight/day)		Pups Sacrificed for Litter Standardization / Hormonal analysis (No.)			Live Pups (No.) on LD 4 after Litter Standardization / Hormonal analysis (No.)			LD 5 to 7									Sex Ratio (m/f) on LD 7	Pup Survival Index (%) [LD 5 to 7]
								Live Pups (No.)			Dead Pups (No.)			Cannibalized (No.)
		Male	Female	Total	Male	Female	Total	Male	Female	Total	Male	Female	Total	Male	Female	Total
G1, F & 0	Mean	0.00	1.09	1.09	6.27	4.64	10.91	6.27	4.64	10.91	0.00	0.00	0.00	0.00	0.00	0.00	1.55	100.00
	±SD	0.00	1.04	1.04	1.90	1.36	1.58	1.90	1.36	1.58	0.00	0.00	0.00	0.00	0.00	0.00	0.86	0.00
	n	11	11	11	11	11	11	11	11	11	11	11	11	11	11	11	11	11
G2, F & 111	Mean	0.00	1.20	1.20	6.00	4.80	10.80	6.00	4.80	10.80	0.00	0.00	0.00	0.00	0.00	0.00	1.37	100.00
	±SD	0.00	1.03	1.03	1.94	1.23	2.20	1.94	1.23	2.20	0.00	0.00	0.00	0.00	0.00	0.00	0.62	0.00
	n	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
G3, F & 333	Mean	0.00	0.70	0.70	5.30	4.80	10.10	5.30	4.80	10.10	0.00	0.00	0.00	0.00	0.00	0.00	1.21	100.00
	±SD	0.00	0.95	0.95	2.00	1.03	1.73	2.00	1.03	1.73	0.00	0.00	0.00	0.00	0.00	0.00	0.67	0.00
	n	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
G4, F & 1000	Mean	0.00	1.64	1.64	6.36	4.45	10.82	6.36	4.45	10.82	0.00	0.00	0.00	0.00	0.00	0.00	2.07	100.00
	±SD	0.00	0.81	0.81	2.11	1.57	1.99	2.11	1.57	1.99	0.00	0.00	0.00	0.00	0.00	0.00	2.39	0.00
	n	11	11	11	11	11	11	11	11	11	11	11	11	11	11	11	11	11