Sodium polysulfide aluminosilicate with a SOD-type framework structure

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 February 2021 – 19 January 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

reproductive toxicity, other

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

20 July 2020 to 06 October 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Dose Range Finding study for a subsequent definitive Extended One-Generation Reproductive Toxicity Study on the test substance.
- Short description of test conditions: oral administration of test item over a relatively limited period of time i.e. 32 days for male Sprague Dawley rats including pre-mating and mating periods and 49 to 66 days for female Sprague Dawley rats including pre-mating, mating, gestation and lactation (till lactation day 13).
- Parameters analysed / observed: effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

GLP compliance:

no

Remarks:

DRF study

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-house bred animals
- Age at study initiation: Minimum of 10 weeks
- Weight at study initiation: Males: 254.06 to 298.31 g & Females: 200.23 to 252.96 g
- Fasting period before study: No
- Housing: Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding
pelleted food and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
- During acclimatization, maximum of three animals of same sex were housed.
- Pre mating - Per cage two animals of the same sex and group were housed.
- Cohabitation Period (mating) - Per cage two animals (one male and one female) of the same group were housed.
- Post-mating - After confirming presence of sperm in the vaginal smear (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually. Sterilized paper shreds were provided as a nesting material for main group females from gestation day 20 onwards.
- Diet (e.g. ad libitum): ad libitum - Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG)
- Water (e.g. ad libitum): ad libitum - Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: five days

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

IN-LIFE DATES: From: To: 20 July 2020 to 29 September 2020

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item formulations were freshly prepared before dose administration on each treatment day. The required quantity of test item was weighed and triturated well in a mortar with a small quantity of vehicle until a homogenous suspension was formed and thereafter the entire quantity of the formulation was transferred into measuring cylinder. A small quantity of vehicle was added to rinse the mortar and this was transferred into the measuring cylinder. The rinsing procedure of mortar and pestle was repeated (many times) to ensure the transfer of the contents to the measuring cylinder. Finally, the volume was made up to required quantity with vehicle to get desired concentration of 10, 30 and 100 mg/mL of test item for low, mid and high dose groups respectively. The test formulations were maintained under stirring conditions using magnetic stirrer to maintain homogeneity of the test item formulations.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item was in-soluble in distilled water and uniformly suspended in 0.5% w/v Carboxy Methyl Cellulose at the concentration of 100 mg/mL (the highest dose concentration selected for the study considering the dose volume of 10 mL/kg body weight) as per in-house solubility/suspendibility test results. Hence, 0.5% w/v Carboxy Methyl Cellulose was used as vehicle for test item formulations and the details were recorded in the raw data and presented in the study report.
- Concentration in vehicle: 10 - 100mg/mL
- Amount of vehicle (if gavage): 10mL/kg bw
- Lot/batch no. (if required): BCBN1690V

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy.
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol: No

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the test item in dose formulations was not established under this dose range finding study. However, freshly prepared test item formulations were administered to the animals.

Duration of treatment / exposure:

Males: two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 32 days of treatment).
Females: two weeks pre-mating period, during mating, pregnancy (gestation)
and up to lactation day 13 (total of 49 to 66 days of treatment).

Frequency of treatment:

Once a day

Details on study schedule:

F1 animals were not mated.

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were decided based on the results of a dose range finding study conducted in lines with OECD TG 414 (Bioneeds Study No. BIO-DTX 030) in which no adverse effects were observed for maternal and developmental toxicity at the dose levels of 100, 300 and 1000 mg/kg bw/d.

Positive control:

None

Parental animals: Observations and examinations:

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

DETAILED CLINICAL OBSERVATIONS: No

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

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

OPHTHALMOSCOPIC EXAMINATION: No

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

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

URINALYSIS: Yes
- Time schedule for collection of urine: on the day before of scheduled terminal sacrifice.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 5 males and 5 females randomly selected from each main group.
- Parameters checked: Blood, Protein, Glucose. In addition pH and specific gravity were also analyzed. After analysis of the above parameters, the urine was subjected for centrifugation at 1500 rpm for 3 minutes. Then the urine was subjected for microscopic examination for urine sediments.

Oestrous cyclicity (parental animals):

Oestrus cycles were monitored daily for a period of 2 weeks during pre-mating period before initiation of cohabitation and during cohabitation until evidence of mating.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Individual pups from each litter were observed for external examination and clinical signs once daily and for mortalities twice daily.
Individual live pup weight from each litter was recorded on PND 1 (within 24 hours of parturition), 4, 7, and 13. The mean pup weight (sex-wise) is reported as per litter basis.
The number of male/female pups born (live/dead/cannibalized) per litter, sex ratio (m/f), live birth index (%), pup survival index (%).

GROSS EXAMINATION OF DEAD PUPS:
The dead pups and pups which were sacrificed on PND 13 were examined for gross abnormalities with particular attention to the external reproductive genitals and the observations were recorded.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after completion of 32 days of treatment.
- Maternal animals: All surviving animals on lactation day 14

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated below were collected, weighed, and preserved for microscopic examination.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring was sacrificed on postnatal day 13.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations with particular attention to the external reproductive genitals.

Statistics:

After verification, the data was subjected to statistical analysis using SPSS software, version 22. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05). The statistical analysis was followed to the parameters as mentioned in the table below.

Reproductive indices:

See List below.

Offspring viability indices:

See List below.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

A slight reduction in mean body weight and mean percent change in body
weight gain with respect to day 1 from group G4 males was noted during week 3 and 4. These noted changes are not considered as test item related, as there were no other systemic toxicity effects noted from this dose level in both sexes.

There were no changes noted in mean body weight and mean percent change in body weight gain during gestation or lactation period.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The following statistically significant changes were noted in haematology parameters of tested dose groups from both the sexes when compared with vehicle
control group:
- increase in mean prothrombin time of group G4 males;
- increase in mean activated prothrombin time of groups G2, G3 and G4 males;
- decrease in mean leucocyte (WBC) count of group G2 females;
- decrease in mean absolute lymphocytes of groups G2 and G3 females;
- decrease in mean absolute monocytes of group G2 females.

These noted changes are considered to be incidental and not test item-related, as these changes are lacking relevant dose dependency (except in case of activated prothrombin time) and the obtained values are still within lab historical control data range of same species and strain.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant increase in mean urea levels of group
G3 and G4 males compared with vehicle control group was noted. However, these increases are considered to be incidental and not test item-related, as the obtained values are within lab historical control data range of same species and strain.

Endocrine findings:

not examined

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant increase in mean pH levels of groups G2 and
G4 males compared with vehicle control group was noted. However these changes are considered to be incidental and not test item-related, as the obtained values are within lab historical control data range of same species and strain.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no irregularities observed in the oestrus cyclicity of females from all the
tested dose groups and vehicle control group during pre-mating and mating treatment periods. The mean length (days) of oestrus cycle per female during pre-mating and mating treatment period was unaffected by the test item administration at any of the tested dose groups when compared with vehicle control group.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant increase in mean number of female pups born at birth
and mean survived number of females pups on LD 4, 7 and 13 from group G2 compared with vehicle control group was noted, however, these changes are considered to be incidental with no toxicological relevance.

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

A increase in post implantation losses from group G3 and G4 was noted. However these changes are not considered test item related, as a total of 3 dams were noted with post implantation loss from these dose groups with a maximum of 2 implantation loss and the obtained range of implantation loss is still within the lab historical control data of same species and strain.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no changes noted in daily observation of pups at all the tested and vehicle control groups.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no mortalities of pups during the 13-day lactation period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no changes observed in mean pup [both male and female] weight per litter recorded on postnatal day (PND) 1, 4, 7 and 13 from all the tested dose groups when compared with vehicle control group.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological changes observed in any of the pups found dead or terminally sacrificed from all the tested dose groups and vehicle control group litters during conduct of necropsy.

Histopathological findings:

not examined

Other effects:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

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

Group, Sex & Dose (mg/kg body weight/day)	Total No. of Animals	Clinical Signs of Toxicity (a): Observation (No. of Animals revealed)	Mortality (b): No. of Mortalities (Total No. of Animals)
G1, M & 0	10	N (10)	0 (10)
G2, M & 100	10	N (10)	0 (10)
G3, M & 300	10	N (10)	0 (10)
G4, M & 1000	10	N (10)	0 (10)

M: Male; N: Normal;

a: observed daily once; b: observed twice daily

 

Group, Sex & Dose (mg/kg body weight/day)	Total No. of Animals	Clinical Signs of Toxicity^a: Observation (No. of Animals revealed)	Mortality^b: No. of Mortalities (Total No. of Animals)
G1, F & 0	10	N (10)	0 (10)
G2, F & 100	10	N (10)	0 (10)
G3, F & 300	10	N (10)	0 (10)
G4, F & 1000	10	N (10)	0 (10)

F: Female; N: Normal;

a: observed daily once; b: observed twice daily

TABLE 2. SUMMARY OF BODY WEIGHT (g) RECORD

Group, Sex & Dose (mg/kg body weight/day)			Body Weight (g) on Day
		1	8	14	21	28
	Mean	310.66	347.01	367.85	377.61	389.42
G1, M & 0	±SD	19.26	20.33	28.19	30.94	37.34
	n	10	10	10	10	10
	Mean	311.95	345.16	361.63	370.24	380.30
G2, M & 100	±SD	16.82	16.67	13.20	17.21	29.36
	n	10	10	10	10	10
	Mean	311.55	344.62	362.04	371.47	379.14
G3, M & 300	±SD	17.18	14.85	15.00	21.22	26.93
	n	10	10	10	10	10
	Mean	312.67	349.31	365.43	366.94	368.00
G4, M & 1000	±SD	15.21	17.44	25.52	26.55	30.06
	n	10	10	10	10	10

 

Group, Sex & Dose (mg/kg body weight/day)			Body Weight (g) on Day
		1	8	14	21^#	28^#
	Mean	235.51	251.81	261.84	-	-
G1, F & 0	±SD	12.71	11.65	14.15	-	-
	n	10	10	10	-	-
	Mean	234.68	249.12	256.37	282.83	-
G2, F & 100	±SD	9.43	13.10	15.09	24.95	-
	n	10	10	10	2	-
	Mean	235.51	251.56	259.32	267.65	258.65
G3, F & 300	±SD	11.29	16.21	16.70	29.77	23.29
	n	10	10	10	3	2
	Mean	233.66	248.77	255.69	261.52	253.16
G4, F & 1000	±SD	12.44	14.29	14.26	13.41	-
	n	10	10	10	5	1

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

 

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

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight (%) during Day
		1 to 8	1 to 14	1 to 21	1 to 28
	Mean	11.76	18.42	21.56	25.34
G1, M & 0	±SD	2.88	5.70	6.89	9.21
	n	10	10	10	10
	Mean	10.71	16.06	18.78	21.97
G2, M & 100	±SD	2.70	3.74	3.74	7.69
	n	10	10	10	10
	Mean	10.74	16.49	19.54	22.05
G3, M & 300	±SD	3.93	7.60	9.37	11.38
	n	10	10	10	10
	Mean	11.74	16.84	17.36	17.67
G4, M & 1000	±SD	2.54	4.85	6.09	6.95
	n	10	10	10	10

 

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight (%) during Day
		1 to 8	1 to 14	1 to 21^#	1 to 28^#
	Mean	6.97	11.24	-	-
G1, F & 0	±SD	2.22	4.02	-	-
	n	10	10	-	-
	Mean	6.14	9.23	18.48	-
G2, F & 100	±SD	3.11	4.38	6.81	-
	n	10	10	2	-
	Mean	6.75	10.07	10.13	6.93
G3, F & 300	±SD	2.68	3.84	8.72	0.32
	n	10	10	3	2
	Mean	6.47	9.46	12.35	7.61
G4, F & 1000	±SD	2.09	3.23	4.13	-
	n	10	10	5	1

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

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

Group, Sex & Dose (mg/kg body weight/day)	Feed Consumption (g/animal/day) during Pre-mating Period
		Week 1	Week 2
	Mean	21.76	23.82
G1, M & 0	±SD	0.67	1.52
	n	10	10
	Mean	21.35	23.33
G2, M & 100	±SD	1.17	1.62
	n	10	10
	Mean	21.87	22.69
G3, M & 300	±SD	0.50	1.26
	n	10	10
	Mean	21.52	24.06
G4, M & 1000	±SD	1.82	1.40
	n	10	10

 

 

Group, Sex & Dose (mg/kg body weight/day)	Feed Consumption (g/animal/day) during Pre-mating Period
		Week 1	Week 2
	Mean	16.45	18.14
G1, F & 0	±SD	1.32	1.25
	n	10	10
	Mean	16.88	17.73
G2, F & 100	±SD	0.93	1.07
	n	10	10
	Mean	17.19	18.59
G3, F & 300	±SD	0.59	1.67
	n	10	10
	Mean	17.11	18.01
G4, F & 1000	±SD	1.34	1.30
	n	10	10

 

TABLE 5. SUMMARY OF HAEMATOLOGY RECORD

Group, Sex & Dose (mg/kg body weight/day)		Total Leucocyte Count	Total Erythrocyte Count	Haemoglobin	Haematocrit	Platelet Count	Neutrophils
		(WBC)	(RBC)	(HGB)	(HCT)	(PLT)	(Neut)
		(10^3 cells/µL)	(10^6 cells/µL)	(g/dL)	(%)	(10^3 cells/µL)	(%)
	Mean	8.98	7.81	14.70	43.12	853.80	22.54
G1, M & 0	±SD	2.16	0.35	0.50	1.97	255.94	4.12
	n	5	5	5	5	5	5
	Mean	7.66	7.37	13.94	40.76	589.20	24.32
G2, M & 100	±SD	2.40	0.83	1.24	3.58	177.01	11.39
	n	5	5	5	5	5	5
	Mean	9.51	7.92	14.64	42.66	694.60	20.40
G3, M & 300	±SD	2.99	0.54	1.12	2.71	171.66	5.36
	n	5	5	5	5	5	5
	Mean	8.74	7.04	13.06	37.42	742.00	18.22
G4, M & 1000	±SD	1.66	1.38	2.43	6.84	367.30	6.61
	n	5	5	5	5	5	5

 

Group, Sex & Dose (mg/kg body weight/day)		Lymphocytes	Monocytes	Eosinophils	Basophils	Absolute Neutrophils	Absolute Lymphocytes
		(Lymph)	(Mono)	(Eos)	(Baso)	(Neut)	(Lymph)
		(%)	(%)	(%)	(%)	(10^3 cells/µL)	(10^3 cells/µL)
	Mean	70.50	4.28	1.74	0.34	2.04	6.33
G1, M & 0	±SD	4.59	1.54	0.43	0.11	0.66	1.65
	n	5	5	5	5	5	5
	Mean	68.48	3.60	2.44	0.32	1.71	5.39
G2, M & 100	±SD	11.59	2.03	1.20	0.11	0.56	2.45
	n	5	5	5	5	5	5
	Mean	73.86	3.12	1.62	0.32	1.86	7.12
G3, M & 300	±SD	5.79	0.77	1.04	0.11	0.46	2.67
	n	5	5	5	5	5	5
	Mean	74.78	3.80	1.64	0.46	1.52	6.62
G4, M & 1000	±SD	7.79	0.87	1.71	0.09	0.33	1.81
	n	5	5	5	5	5	5

 

Group, Sex & Dose (mg/kg body weight/day)	Absolute Monocytes		Absolute Eosinophils	Absolute Basophils	Prothrombin Time	Activated Prothrombin Time
		(Mono)	(Eos)	(Baso)	(PT)	(APTT)
		(10^3 cells/µL)	(10^3 cells/µL)	(10^3 cells/µL)	(Seconds)	(Seconds)
	Mean	0.36	0.15	0.03	16.70	19.02
G1, M & 0	±SD	0.09	0.04	0.01	0.82	4.83
	n	5	5	5	5	5
	Mean	0.30	0.17	0.03	16.70	25.08*
G2, M & 100	±SD	0.22	0.06	0.02	1.51	1.73
	n	5	5	5	5	5
	Mean	0.30	0.15	0.03	16.30	28.36*
G3, M & 300	±SD	0.10	0.06	0.02	1.06	1.47
	n	5	5	5	5	5
	Mean	0.34	0.13	0.04	19.00*	31.20*
G4, M & 1000	±SD	0.12	0.11	0.01	1.27	1.89
	n	5	5	5	5	5

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

Group, Sex & Dose (mg/kg body weight/day)		Total Leucocyte Count	Total Erythrocyte Count	Haemoglobin	Haematocrit	Platelet Count	Neutrophils
		(WBC)	(RBC)	(HGB)	(HCT)	(PLT)	(Neut)
		(10^3 cells/µL)	(10^6 cells/µL)	(g/dL)	(%)	(10^3 cells/µL)	(%)
	Mean	14.48	6.98	14.08	41.98	697.80	23.32
G1, F & 0	±SD	6.45	0.23	0.28	2.25	99.04	4.34
	n	5	5	5	5	5	5
	Mean	6.82*	6.86	13.42	40.56	578.40	28.90
G2, F & 100	±SD	3.71	1.25	2.65	7.73	200.77	6.11
	n	5	5	5	5	5	5
	Mean	8.75	7.24	14.04	42.60	639.40	37.32
G3, F & 300	±SD	3.21	0.43	0.77	2.82	250.60	11.79
	n	5	5	5	5	5	5
	Mean	11.45	7.39	14.12	43.68	611.80	35.84
G4, F & 1000	±SD	3.12	0.24	0.42	2.07	283.41	12.30
	n	5	5	5	5	5	5

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

Group, Sex & Dose (mg/kg body weight/day)		Lymphocytes	Monocytes	Eosinophils	Basophils	Absolute Neutrophils	Absolute Lymphocytes
		(Lymph)	(Mono)	(Eos)	(Baso)	(Neut)	(Lymph)
		(%)	(%)	(%)	(%)	(10^3 cells/µL)	(10^3 cells/µL)
	Mean	69.04	4.26	2.08	0.52	3.28	10.14
G1, F & 0	±SD	3.82	1.17	1.09	0.23	1.24	4.92
	n	5	5	5	5	5	5
	Mean	61.80	4.58	1.72	0.42	1.91	4.37*
G2, F & 100	±SD	7.16	1.16	0.80	0.22	0.91	2.82
	n	5	5	5	5	5	5
	Mean	50.78	4.72	2.42	0.48	3.29	4.53*
G3, F & 300	±SD	17.31	1.71	1.48	0.04	1.55	2.46
	n	5	5	5	5	5	5
	Mean	57.34	3.72	1.86	0.32	4.34	6.31
G4, F & 1000	±SD	12.38	0.48	1.26	0.18	2.16	1.11
	n	5	5	5	5	5	5

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

 

Group, Sex & Dose (mg/kg body weight/day)		Absolute Monocytes	Absolute Eosinophils	Absolute Basophils	Prothrombin Time	Activated Prothrombin Time
		(Mono)	(Eos)	(Baso)	(PT)	(APTT)
		(10^3 cells/µL)	(10^3 cells/µL)	(10^3 cells/µL)	(Seconds)	(Seconds)
	Mean	0.57	0.30	0.08	18.80	24.78
G1, F & 0	±SD	0.21	0.22	0.06	2.49	8.82
	n	5	5	5	5	5
	Mean	0.28*	0.11	0.03	21.48	25.06
G2, F & 100	±SD	0.09	0.06	0.03	2.82	9.43
	n	5	5	5	5	5
	Mean	0.38	0.20	0.04	18.22	18.54
G3, F & 300	±SD	0.13	0.13	0.02	2.47	6.11
	n	5	5	5	5	5
	Mean	0.44	0.21	0.04	19.12	22.80
G4, F & 1000	±SD	0.16	0.15	0.03	3.83	8.89
	n	5	5	5	5	5

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

 

 

TABLE 6. SUMMARY OF CLINICAL CHEMISTRY RECORD

Group, Sex & Dose (mg/kg body weight/day)		Glucose	Urea	Creatinine	Total Cholesterol	Total Protein
		(GLU)	-	(CRE)	(CHO)	(TPR)
		(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(g/dL)
	Mean	115.60	25.40	0.46	45.40	6.24
G1, M & 0	±SD	9.86	1.95	0.02	5.73	0.40
	n	5	5	5	5	5
	Mean	106.60	26.00	0.42	51.80	6.14
G2, M & 100	±SD	8.38	3.44	0.03	9.31	0.28
	n	5	5	5	5	5
	Mean	110.00	33.84*	0.45	55.00	5.88
G3, M & 300	±SD	9.43	4.47	0.01	11.02	0.08
	n	5	5	5	5	5
	Mean	110.40	36.20*	0.46	47.40	6.12
G4, M & 1000	±SD	15.57	8.17	0.03	9.61	0.27
	n	5	5	5	5	5

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

 

Group, Sex & Dose (mg/kg body weight/day)		Albumin	Alanine aminotransferase	Aspartate aminotransferase	Alkaline phosphatase
		(ALB)	(ALT)	(AST)	(ALP)
		(g/dL)	(U/L)	(U/L)	(U/L)
	Mean	3.19	42.80	104.00	107.40
G1, M & 0	±SD	0.11	9.73	14.20	9.40
	n	5	5	5	5
	Mean	3.11	35.40	97.00	121.20
G2, M & 100	±SD	0.10	11.26	18.26	39.34
	n	5	5	5	5
	Mean	3.15	34.20	99.00	97.40
G3, M & 300	±SD	0.15	4.60	18.59	16.26
	n	5	5	5	5
	Mean	2.99	33.80	99.20	106.40
G4, M & 1000	±SD	0.19	5.45	20.90	28.51
	n	5	5	5	5

 

 

Group, Sex & Dose (mg/kg body weight/day)		Glucose	Urea	Creatinine	Total Cholesterol	Total Protein
		(GLU)	-	(CRE)	(CHO)	(TPR)
		(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(g/dL)
	Mean	110.20	46.78	0.60	75.40	6.36
G1, F & 0	±SD	38.71	10.07	0.09	7.16	0.63
	n	5	5	5	5	5
	Mean	92.80	57.20	0.66	75.40	6.62
G2, F & 100	±SD	17.31	8.90	0.12	13.07	0.69
	n	5	5	5	5	5
	Mean	96.80	50.52	0.59	70.20	6.76
G3, F & 300	±SD	17.98	5.26	0.08	16.42	0.60
	n	5	5	5	5	5
	Mean	102.60	53.84	0.60	79.00	6.16
G4, F & 1000	±SD	27.22	13.55	0.06	23.33	0.53
	n	5	5	5	5	5

 

 

Group, Sex & Dose (mg/kg body weight/day)		Albumin	Alanine aminotransferase	Aspartate aminotransferase	Alkaline phosphatase
		(ALB)	(ALT)	(AST)	(ALP)
		(g/dL)	(U/L)	(U/L)	(U/L)
	Mean	3.20	66.80	112.80	83.20
G1, F & 0	±SD	0.40	11.58	25.62	22.22
	n	5	5	5	5
	Mean	3.48	77.00	165.00	127.20
G2, F & 100	±SD	0.27	32.16	85.40	90.51
	n	5	5	5	5
	Mean	3.20	95.00	179.40	195.40
G3, F & 300	±SD	0.38	53.52	144.08	123.33
	n	5	5	5	5
	Mean	3.19	93.80	138.00	158.80
G4, F & 1000	±SD	0.19	21.04	50.35	57.61
	n	5	5	5	5

 

TABLE 7. SUMMARY OF URINALYSIS RECORD

Examination	Group, Sex & Dose (mg/kg body weight/day)		G1, M & 0	G2, M & 100	G3, M & 300	G4, M & 1000
	Number of Animals (randomly selected)		5	5	5	5
Physical Examination	Color	Pale Yellow	4	3	4	5
		Yellow	1	2	1	-
	Appearance	Clear	5	5	5	5
		Turbidity	-	-	-	-
	Volume (mL)	Mean	6.8	9.3	9.3	8.4
		±SD	3.5	1.4	1.0	1.4
Chemical Examination	pH	Mean	8.2	8.5*	8.4	8.5*
		±SD	0.3	0.0	0.2	0.0
	Specific Gravity	Mean	1.007	1.005	1.008	1.005
		±SD	0.003	0.000	0.003	0.000
	Blood (Ery/µL)	Neg	5	5	4	3
		Ca25	-	-	1	2
	Protein (mg/dL)	Neg	3	4	2	3
		Trace	-	-	2	1
		30	1	-	-	-
		100	1	-	-	-
		>=300	-	1	1	1
	Glucose (mg/dL)	Neg	5	5	5	5
Microscopic Examination	Epi Cells	0	3	3	3	4
		0-1	1	2	1	1
		1-2	1	-	1	-
	Casts	Absent	5	5	5	5
	Crystals	Present	5	5	5	5

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

 

Examination	Group, Sex & Dose (mg/kg body weight/day)		G1, F & 0	G2, F & 100	G3, F & 300	G4, F & 1000
	Number of Animals		5	5	5	5
Physical Examination	Color	Pale Yellow	4	3	3	5
		Yellow	1	2	2	-
	Appearance	Clear	5	3	4	2
		Turbidity	-	2	1	3
	Volume (mL)	Mean	5.7	6.4	6.2	5.0
		±SD	1.4	2.0	1.8	1.2
Chemical Examination	pH	Mean	7.3	7.1	6.9	7.4
		±SD	0.8	0.4	1.1	0.4
	Specific gravity	Mean	1.015	1.017	1.019	1.015
		±SD	0.005	0.004	0.007	0.004
	Blood (Ery/µL)	Neg	5	4	3	5
		Ca10	-	1	2	-
	Protein (mg/dL)	Neg	4	4	2	5
		Trace	1	1	1	-
		30	-	-	1	-
		>=300	-	-	1	-
	Glucose (mg/dL)	Neg	5	5	5	5
Microscopic Examination	Epi Cells	0	-	3	3	1
		0-1	3	2	2	2
		1-2	1	-	-	2
		2-3	1	-	-	-
	Casts	Absent	5	5	5	5
	Crystals	Present	5	5	5	5

 

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

Determination of Oestrus Cyclicity during Pre-mating Treatment Period
Group, Sex & Dose (mg/kg body weight/day)	Total No. of Females Evaluated		Females with Complete Regular Cycles	Females with at least one Irregular Oestrus Cycle	Average Length of Oestrous Cycle (Days)
		n	10	0	Mean	4.95
G1, F & 0	10	%	100.00	0.00	±SD	0.16
					n	10
		n	10	0	Mean	4.95
G2, F & 100	10	%	100.00	0.00	±SD	0.16
					n	10
		n	10	0	Mean	5.00
G3, F & 300	10	%	100.00	0.00	±SD	0.00
					n	10
		n	10	0	Mean	5.00
G4, F & 1000	10	%	100.00	0.00	±SD	0.00
					n	10

 

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

 

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Gestation Day (GD)
		0	7	14	20
	Mean	262.66	277.87	309.48	375.80
G1, F & 0	±SD	16.64	16.18	19.11	31.27
	n	10	10	10	10
	Mean	257.99	276.92	314.65	396.93
G2, F & 100	±SD	14.34	12.54	14.81	34.46
	n	9	9	9	9
	Mean	261.88	277.61	312.40	387.04
G3, F & 300	±SD	17.10	19.10	22.42	31.30
	n	8	8	8	8
	Mean	265.30	282.42	317.67	394.73
G4, F & 1000	±SD	11.12	12.76	13.49	27.44
	n	8	8	8	8

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

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

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight Gain (%) during Gestation Day GD)
		0 to 7	7 to 14	14 to 20
	Mean	5.84	11.38	21.43
G1, F & 0	±SD	2.24	2.56	6.88
	n	10	10	10
	Mean	7.40	13.63	25.96
G2, F & 100	±SD	1.69	1.55	5.61
	n	9	9	9
	Mean	5.99	12.52	23.83
G3, F & 300	±SD	1.41	2.16	2.43
	n	8	8	8
	Mean	6.46	12.51	24.23
G4, F & 1000	±SD	1.95	1.90	6.37
	n	8	8	8

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

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

Group, Sex & Dose (mg/kg body weight/day)	Feed Consumption (g/animal/day) during Gestation Day (GD)
		0 to 7	7 to 14	14 to 20
	Mean	18.75	20.30	24.74
G1, F & 0	±SD	0.55	0.54	0.86
	n	10	10	10
	Mean	19.15	20.71	25.50
G2, F & 100	±SD	0.61	0.55	0.81
	n	9	9	9
	Mean	18.72	20.43	25.32
G3, F & 300	±SD	0.58	0.57	0.94
	n	8	8	8
	Mean	18.83	20.61	25.57
G4, F & 1000	±SD	0.60	0.80	1.18
	n	8	8	8

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

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

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Lactation Day (LD)
		1	4	7	13
	Mean	285.46	293.56	304.10	323.80
G1, F & 0	±SD	19.87	20.53	20.43	19.35
	n	10	10	10	10
	Mean	302.20	308.79	318.28	332.30
G2, F & 100	±SD	28.19	25.35	23.94	24.12
	n	9	9	9	9
	Mean	293.02	301.01	308.14	324.77
G3, F & 300	±SD	28.36	26.90	26.29	24.30
	n	8	8	8	8
	Mean	301.36	310.19	319.60	334.79
G4, F & 1000	±SD	18.35	18.43	17.65	21.71
	n	8	8	8	8

 

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

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight Gain (%) during Lactation Day (LD)
		1 to 4	4 to 7	7 to 13
	Mean	2.84	3.61	6.53
G1, F & 0	±SD	1.32	0.82	1.57
	n	10	10	10
	Mean	2.29	3.13	4.44
G2, F & 100	±SD	2.21	1.00	1.97
	n	9	9	9
	Mean	2.80	2.41	5.51
G3, F & 300	±SD	1.12	1.36	2.70
	n	8	8	8
	Mean	2.94	3.06	4.73
G4, F & 1000	±SD	0.85	1.16	2.59
	n	8	8	8

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

Group, Sex & Dose (mg/kg body weight/day)		Feed Consumption (g/animal/day) during Lactation Day (LD)
		1 to 4	4 to 7	7 to 13
	Mean	28.15	33.73	42.16
G1, F & 0	±SD	1.36	1.40	0.74
	n	10	10	10
	Mean	28.29	33.37	41.25
G2, F & 100	±SD	1.39	1.10	1.02
	n	9	9	9
	Mean	28.38	33.34	41.59
G3, F & 300	±SD	1.08	0.84	1.43
	n	8	8	8
	Mean	28.09	33.87	41.75
G4, F & 1000	±SD	1.34	1.02	1.04
	n	8	8	8

 

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

Group, Sex & Dose (mg/kg body weight/day)		Litter Delivered (No.)
		Total Litter Size (No.)		Live Pups (No.)			Dead Pups (No.)				Cannibalized Pups (No.)						Sex Ratio (m/f) at Birth		Live Birth Index(%)
				Male	Female	Total		Male	Female	Total		Undetermined	Male	Female	Total
	Mean	12.80		6.40	6.30	12.70		0.10	0.00	0.10		0.00	0.00	0.00	0.00		1.13		99.38
G1, F & 0	±SD	2.66		2.01	2.31	2.54		0.32	0.00	0.32		0.00	0.00	0.00	0.00		0.50		1.98
	n	10		10	10	10		10	10	10		10	10	10	10		10		10
	Mean	14.56		6.67	7.89*	14.56		0.00	0.00	0.00		0.00	0.00	0.00	0.00		0.86		100.00
G2, F & 100	±SD	1.59		1.58	0.78	1.59		0.00	0.00	0.00		0.00	0.00	0.00	0.00		0.23		0.00
	n	9		9	9	9		9	9	9		9	9	9	9		9		9
	Mean	13.13		7.00	6.00	13.00		0.00	0.13	0.13		0.00	0.00	0.00	0.00		1.23		99.17
G3, F & 300	±SD	2.95		2.67	1.41	2.88		0.00	0.35	0.35		0.00	0.00	0.00	0.00		0.55		2.36
	n	8		8	8	8		8	8	8		8	8	8	8		8		8
	Mean	13.75		6.88	6.50	13.38		0.13	0.25	0.38		0.00	0.00	0.00	0.00		1.19		97.18
G4, F & 1000	±SD	2.19		2.47	2.20	2.39		0.35	0.46	0.74		0.00	0.00	0.00	0.00		0.61		5.66
	n	8		8	8	8		8	8	8		8	8	8	8		8		8

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

 

Group, Sex & Dose (mg/kg body weight/day)			During LD 1 to 4												Sex Ratio (m/f) on LD 4		Pup Survival Index (%) during LD 1 to 4
			Live Pups (No.)			Dead Pups (No.)				Cannibalized Pups (No.)
			Male	Female	Total		Male	Female	Total		Male	Female	Total
	Mean		6.40	6.30	12.70		0.00	0.00	0.00		0.00	0.00	0.00		1.13		100.00
G1, F & 0	±SD		2.01	2.31	2.54		0.00	0.00	0.00		0.00	0.00	0.00		0.50		0.00
	n		10	10	10		10	10	10		10	10	10		10		10
	Mean		6.67	7.89*	14.56		0.00	0.00	0.00		0.00	0.00	0.00		0.86		100.00
G2, F & 100	±SD		1.58	0.78	1.59		0.00	0.00	0.00		0.00	0.00	0.00		0.23		0.00
	n		9	9	9		9	9	9		9	9	9		9		9
	Mean		7.00	6.00	13.00		0.00	0.00	0.00		0.00	0.00	0.00		1.23		100.00
G3, F & 300	±SD		2.67	1.41	2.88		0.00	0.00	0.00		0.00	0.00	0.00		0.55		0.00
	n		8	8	8		8	8	8		8	8	8		8		8
	Mean		6.88	6.50	13.38		0.00	0.00	0.00		0.00	0.00	0.00		1.19		100.00
G4, F & 1000	±SD		2.47	2.20	2.39		0.00	0.00	0.00		0.00	0.00	0.00		0.61		0.00
	n		8	8	8		8	8	8		8	8	8		8		8

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

 

Group, Sex & Dose (mg/kg body weight/day)		During LD 5 to 7												Sex Ratio (m/f) on LD 7		Pup Survival Index (%) during LD 5 to 7
		Live Pups (No.)			Dead Pups (No.)				Cannibalized (No.)
		Male	Female	Total		Male	Female	Total		Male	Female	Total
	Mean	6.40	6.30	12.70		0.00	0.00	0.00		0.00	0.00	0.00		1.13		100.00
G1, F & 0	±SD	2.01	2.31	2.54		0.00	0.00	0.00		0.00	0.00	0.00		0.50		0.00
	n	10	10	10		10	10	10		10	10	10		10		10
	Mean	6.67	7.89*	14.56		0.00	0.00	0.00		0.00	0.00	0.00		0.86		100.00
G2, F & 100	±SD	1.58	0.78	1.59		0.00	0.00	0.00		0.00	0.00	0.00		0.23		0.00
	n	9	9	9		9	9	9		9	9	9		9		9
	Mean	7.00	6.00	13.00		0.00	0.00	0.00		0.00	0.00	0.00		1.23		100.00
G3, F & 300	±SD	2.67	1.41	2.88		0.00	0.00	0.00		0.00	0.00	0.00		0.55		0.00
	n	8	8	8		8	8	8		8	8	8		8		8
	Mean	6.88	6.50	13.38		0.00	0.00	0.00		0.00	0.00	0.00		1.19		100.00
G4, F & 1000	±SD	2.47	2.20	2.39		0.00	0.00	0.00		0.00	0.00	0.00		0.61		0.00
	n	8	8	8		8	8	8		8	8	8		8		8

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

 

Group, Sex & Dose (mg/kg body weight/day)		During LD 8 to 13												Sex Ratio (m/f) on LD 13		Pup Survival Index (%) during LD 8 to 13
		Live Pups (No.)			Dead Pups (No.)				Cannibalized (No.)
		Male	Female	Total		Male	Female	Total		Male	Female	Total
	Mean	6.40	6.30	12.70		0.00	0.00	0.00		0.00	0.00	0.00		1.13		100.00
G1, F & 0	±SD	2.01	2.31	2.54		0.00	0.00	0.00		0.00	0.00	0.00		0.50		0.00
	n	10	10	10		10	10	10		10	10	10		10		10
	Mean	6.67	7.89*	14.56		0.00	0.00	0.00		0.00	0.00	0.00		0.86		100.00
G2, F & 100	±SD	1.58	0.78	1.59		0.00	0.00	0.00		0.00	0.00	0.00		0.23		0.00
	n	9	9	9		9	9	9		9	9	9		9		9
	Mean	7.00	6.00	13.00		0.00	0.00	0.00		0.00	0.00	0.00		1.23		100.00
G3, F & 300	±SD	2.67	1.41	2.88		0.00	0.00	0.00		0.00	0.00	0.00		0.55		0.00
	n	8	8	8		8	8	8		8	8	8		8		8
	Mean	6.88	6.50	13.38		0.00	0.00	0.00		0.00	0.00	0.00		1.19		100.00
G4, F & 1000	±SD	2.47	2.20	2.39		0.00	0.00	0.00		0.00	0.00	0.00		0.61		0.00
	n	8	8	8		8	8	8		8	8	8		8		8

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

TABLE 16. SUMMARY RECORD OF REPRODUCTIVE PERFORMANCE

Group, Sex & Dose (mg/kg body weight/day)	No. of Males with Evidence of Mating (No. of Males used for Mating)	Male Mating Index (%)		No. of Males Capable of Impregnating a Female (No. of males used for Mating)	Male Fertility Index (%)
G1, M & 0	10 (10)	100.0		10 (10)	100.0
G2, M & 100	10 (10)	100.0		9 (10)	90.0
G3, M & 300	8 (10)	80.0		7 (10)	70.0
G4, M & 1000	9 (10)	90.0		7 (10)	70.0

 

Group, Sex & Dose (mg/kg body weight/day)		Cohabitation Record and Pre-coital Interval (Mean Time to Mating)					Gestational Length (duration of pregnancy)
		Pre-coital Interval (Days)		Conceiving Days (1 to 5)	Conceiving Days (More than 5 days)		Gestation Length (Days)
	Mean	2.50	n	9	1		22.10
G1, F & 0	±SD	1.58	%	90.00	10.00		0.32
	n	10					10
	Mean	4.10	n	8	2		22.00
G2, F & 100	±SD	4.18	%	80.00	20.00		0.00
	n	10					9
	Mean	6.60	n	6	4		22.13
G3, F & 300	±SD	6.45	%	60.00	40.00		0.35
	n	10					8
	Mean	7.70	n	5	5		22.00
G4, F & 1000	±SD	5.72	%	50.00	50.00		0.00
	n	10					8

 

Group, Sex & Dose (mg/kg body weight/day)	No. of Females with Evidence of Mating (No. of Females used for Mating)	Female Mating Index (%)		No. of Females Confirmed as Fertile (No. of Females used for Mating)	Female Fertility Index (%)
G1, F & 0	10 (10)	100.0		10 (10)	100.0
G2, F & 100	10 (10)	100.0		9 (10)	90.0
G3, F & 300	10 (10)	100.0		8 (10)	80.0
G4, F & 1000	10 (10)	100.0		8 (10)	80.0

 

 

Group, Sex & Dose (mg/kg body weight/day)	No. of Pregnant Females	No. of Females Confirmed with Mating	Female Fecundity or Pregnancy Index (%)
G1, F & 0	10	10	100.0
G2, F & 100	9	10	90.0
G3, F & 300	8	10	80.0
G4, F & 1000	8	10	80.0

 

Group, Sex & Dose (mg/kg body weight/day)	With live born pups at Parturition	No. of females with evidence of pregnancy	Gestation Index (%)		No. of Females Littered	No. of females with evidence of pregnancy	Parturition Index (%)
G1, F & 0	10	10	100.0		10	10	100.0
G2, F & 100	9	9	100.0		9	9	100.0
G3, F & 300	8	8	100.0		8	8	100.0
G4, F & 1000	8	8	100.0		8	8	100.0

 

			Post-implantation Loss (%)			Postnatal Loss (%)
Group, Sex & Dose (mg/kg body weight/day)		No. of Implantations	No. of Viable Pups	Post-implantation Loss (No.)	Post-implantation Loss (%)		Total No. of Deaths/ Cannibalized during Lactation Period	Postnatal Loss (%)
	Mean	12.90	12.70	0.20	1.46		0.00	0.00
G1, F & 0	±SD	2.60	2.54	0.42	3.11		0.00	0.00
	n	10	10	10	10		10	10
	Mean	14.67	14.56	0.11	0.69		0.00	0.00
G2, F & 100	±SD	1.66	1.59	0.33	2.08		0.00	0.00
	n	9	9	9	9		9	9
	Mean	13.50	13.00	0.50	4.47		0.00	0.00
G3, F & 300	±SD	2.33	2.88	0.76	7.24		0.00	0.00
	n	8	8	8	8		8	8
	Mean	14.00	13.38	0.63	4.84		0.00	0.00
G4, F & 1000	±SD	1.93	2.39	0.92	6.84		0.00	0.00
	n	8	8	8	8		8	8

 

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

Group, Sex & Dose (mg/kg body weight/day)		Brain	Liver	Prostate	Seminal vesicles along with coagulating glands	Pituitary	Thyroid along with parathyroid^#
	Mean	1.9779	11.2452	1.3347	1.2086	0.0171	0.0248
G1, M & 0	±SD	0.0538	1.3496	0.2966	0.2097	0.0029	0.0033
	n	10	10	10	10	10	10
	Mean	1.9943	11.0062	1.2057	1.4115	0.0153	0.0240
G2, M & 100	±SD	0.0948	1.6053	0.2019	0.2512	0.0016	0.0014
	n	10	10	10	10	10	10
	Mean	1.9389	10.2393	1.2500	1.4262	0.0181	0.0239
G3, M & 300	±SD	0.1289	0.6409	0.2153	0.3706	0.0030	0.0019
	n	10	10	10	10	10	10
	Mean	1.9300	10.4014	1.1476	1.2445	0.0172	0.0243
G4, M & 1000	±SD	0.0858	1.1027	0.2399	0.2545	0.0028	0.0024
	n	10	10	10	10	10	10

#: Weighed post fixation

 

Group, Sex & Dose (mg/kg body weight/day)		Adrenals	Thymus	Spleen	Ovaries	Uterus with Cervix	Heart
	Mean	0.0839	0.2994	0.7358	0.1217	0.4470	1.1678
G1, F & 0	±SD	0.0104	0.0434	0.0621	0.0172	0.0576	0.0877
	n	10	10	10	10	10	10
	Mean	0.0912	0.3020	0.6435	0.1174	0.4168	1.1799
G2, F & 100	±SD	0.0117	0.0422	0.0688	0.0156	0.0342	0.1567
	n	9	9	9	9	9	9
	Mean	0.0739	0.3114	0.7247	0.1257	0.4685	1.1059
G3, F & 300	±SD	0.0300	0.0442	0.1093	0.0163	0.0745	0.0702
	n	8	8	8	8	8	8
	Mean	0.0918	0.3004	0.6786	0.1245	0.4342	1.1530
G4, F & 1000	±SD	0.0082	0.0505	0.0841	0.0126	0.0846	0.0814
	n	8	8	8	8	8	8

 

Group, Sex & Dose (mg/kg body weight/day)		Kidneys	Brain	Liver	Pituitary	Thyroid along with parathyroid^#
	Mean	2.3038	2.1147	13.8717	0.0163	0.0210
G1, F & 0	±SD	0.2673	0.0759	1.3365	0.0031	0.0022
	n	10	10	10	10	10
	Mean	2.3605	2.0998	13.7297	0.0159	0.0211
G2, F & 100	±SD	0.1887	0.0940	0.9411	0.0020	0.0020
	n	9	9	9	9	9
	Mean	2.4206	2.1097	13.6048	0.0176	0.0213
G3, F & 300	±SD	0.4158	0.1186	1.4003	0.0034	0.0031
	n	8	8	8	8	8
	Mean	2.3711	2.0293	13.9262	0.0205*	0.0216
G4, F & 1000	±SD	0.2006	0.1219	1.3631	0.0025	0.0015
	n	8	8	8	8	8

#: Weighed post fixation

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

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

Group, Sex & Dose (mg/kg body weight/day)		Terminal Body Weight (g)	Adrenals	Thymus	Spleen	Testes	Epididymis	Heart
	Mean	385.90	0.0157	0.0854	0.2074	0.8932	0.3658	0.3312
G1, M & 0	±SD	34.53	0.0014	0.0205	0.0346	0.1045	0.0561	0.0128
	n	10	10	10	10	10	10	10
	Mean	377.23	0.0174	0.0837	0.2080	0.8844	0.3485	0.3207
G2, M & 100	±SD	26.78	0.0016	0.0094	0.0463	0.1006	0.0259	0.0308
	n	10	10	10	10	10	10	10
	Mean	379.22	0.0177	0.0845	0.2192	0.9011	0.3454	0.3264
G3, M & 300	±SD	30.95	0.0023	0.0141	0.0352	0.1057	0.0327	0.0306
	n	10	10	10	10	10	10	10
	Mean	369.95	0.0172	0.0845	0.2503	0.8868	0.3411	0.3288
G4, M & 1000	±SD	28.61	0.0021	0.0154	0.0436	0.0875	0.0270	0.0189
	n	10	10	10	10	10	10	10

 

 

Group, Sex & Dose (mg/kg body weight/day)		Kidneys	Brain	Liver	Prostate	Seminal vesicles along with coagulating glands	Pituitary	Thyroid along with parathyroid
	Mean	0.7219	0.5162	2.9229	0.3438	0.3143	0.0045	0.0064
G1, M & 0	±SD	0.0447	0.0476	0.3293	0.0581	0.0533	0.0007	0.0005
	n	10	10	10	10	10	10	10
	Mean	0.7592	0.5305	2.9134	0.3199	0.3739	0.0041	0.0064
G2, M & 100	±SD	0.1150	0.0372	0.3438	0.0524	0.0604	0.0005	0.0007
	n	10	10	10	10	10	10	10
	Mean	0.6658	0.5130	2.7132	0.3305	0.3728	0.0048	0.0063
G3, M & 300	±SD	0.0820	0.0379	0.2486	0.0557	0.0724	0.0009	0.0007
	n	10	10	10	10	10	10	10
	Mean	0.6970	0.5236	2.8121	0.3089	0.3384	0.0046	0.0066
G4, M & 1000	±SD	0.0655	0.0334	0.1923	0.0506	0.0783	0.0007	0.0004
	n	10	10	10	10	10	10	10

 

 

Group, Sex & Dose (mg/kg body weight/day)		Terminal Body Weight (g)	Adrenals	Thymus	Spleen	Ovaries	Uterus with Cervix
	Mean	309.03	0.0273	0.0973	0.2385	0.0395	0.1449
G1, F & 0	±SD	19.19	0.0039	0.0157	0.0200	0.0062	0.0187
	n	10	10	10	10	10	10
	Mean	318.77	0.0288	0.0956	0.2024*	0.0371	0.1321
G2, F & 100	±SD	25.00	0.0047	0.0173	0.0208	0.0065	0.0201
	n	9	9	9	9	9	9
	Mean	309.33	0.0242	0.1015	0.2357	0.0407	0.1525
G3, F & 300	±SD	24.53	0.0100	0.0183	0.0426	0.0043	0.0275
	n	8	8	8	8	8	8
	Mean	319.83	0.0288	0.0944	0.2124	0.0390	0.1372
G4, F & 1000	±SD	21.30	0.0034	0.0177	0.0251	0.0042	0.0341
	n	8	8	8	8	8	8

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

 

Group, Sex & Dose (mg/kg body weight/day)		Heart	Kidneys	Brain	Liver	Pituitary	Thyroid along with parathyroid
	Mean	0.0395	0.1449	0.6862	4.4899	0.0053	0.0068
G1, F & 0	±SD	0.0062	0.0187	0.0404	0.3469	0.0012	0.0006
	n	10	10	10	10	10	10
	Mean	0.0371	0.1321	0.6627	4.3272	0.0050	0.0066
G2, F & 100	±SD	0.0065	0.0201	0.0649	0.4107	0.0009	0.0006
	n	9	9	9	9	9	9
	Mean	0.0407	0.1525	0.6856	4.4145	0.0057	0.0069
G3, F & 300	±SD	0.0043	0.0275	0.0643	0.4900	0.0009	0.0008
	n	8	8	8	8	8	8
	Mean	0.0390	0.1372	0.6378	4.3741	0.0065	0.0068
G4, F & 1000	±SD	0.0042	0.0341	0.0688	0.5684	0.0010	0.0008
	n	8	8	8	8	8	8

 

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

Parameters	Sex	Male				Female
	Group	G1	G2	G3	G4	G1	G2	G3	G4
	Dose (mg/kg body weight/day)	0	100	300	1000	0	100	300	1000
	No. of Animals	10	10	10	10	10	10	10	10
No. of Terminally Sacrificed Animals		10	10	10	10	10	10	10	10
Gross Pathological Findings
External
No Abnormality Detected		10	10	10	10	10	10	10	10
Internal
No Abnormality Detected		10	10	10	10	10	10	10	10

 

TABLE 20. SUMMARY RECORD OF PUP OBSERVATIONS DURING POSTNATAL PERIOD

Group, Sex & Dose (mg/kg body weight/day)		At Birth (PND 1)	PND 1 to 4	PND 5 to 7	PND 8 to 13
	No. of Dams^#	10	10	10	10
G1, F & 0	No. of Live Pups	127	127	127	127
	Pup Observation/ No. of Pups observed	N/127	N/127	N/127	N/127
	No. of Dams^#	9	9	9	9
G2, F & 100	No. of Live Pups	131	131	131	131
	Pup Observation/ No. of Pups observed	N/131	N/131	N/131	N/131
	No. of Dams^#	8	8	8	8
G3, F & 300	No. of Live Pups	104	104	104	104
	Pup Observation/ No. of Pups observed	N/104	N/104	N/104	N/104
	No. of Dams^#	8	8	8	8
G4, F & 1000	No. of Live Pups	107	107	107	107
	Pup Observation/ No. of Pups observed	N/107	N/107	N/107	N/107

#: Dams confirmed with littering

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

Group, Sex & Dose (mg/kg body weight/day)		PND 1			PND 4			PND 7		PND 13
		Mean Pup Weight (g)		Mean Pup Weight (g)			Mean Pup Weight (g)			Mean Pup Weight (g)
		Male	Female		Male	Female		Male	Female		Male	Female
	Mean	6.64	6.37		10.97	10.49		15.09	14.40		28.35	27.58
G1, F & 0	±SD	0.19	0.21		0.31	0.23		0.28	0.31		0.31	0.39
	n	10	10		10	10		10	10		10	10
	Mean	6.61	6.28		10.86	10.39		14.96	14.32		28.09	27.41
G2, F & 100	±SD	0.22	0.24		0.30	0.22		0.35	0.20		0.49	0.29
	n	9	9		9	9		9	9		9	9
	Mean	6.69	6.36		11.18	10.65		15.24	14.63		28.58	27.94
G3, F & 300	±SD	0.18	0.23		0.52	0.41		0.56	0.42		0.83	0.85
	n	8	8		8	8		8	8		8	8
	Mean	6.59	6.17		10.97	10.42		15.04	14.44		28.40	27.65
G4, F & 1000	±SD	0.19	0.20		0.29	0.18		0.31	0.29		0.49	0.53
	n	8	8		8	8		8	8		8	8

 

TABLE 22. SUMMARY OF GROSS PATHOLOGY FINDINGS RECORD - PUPS

Parameters ↓	Group	G1		G2		G3		G4
	Dose (mg/kg body weight/day)	0		100		300		1000
	Sex	M	F	M	F	M	F	M	F
No. of Dead Pups at birth		1	-	-	-	-	1	1	2
No Abnormality Detected		1	-	-	-	-	1	1	2
No. of Pups Sacrificed on PND 13		64	63	60	71	56	48	55	52
No Abnormality Detected		64	63	60	71	56	48	55	52

 

 

Conclusions:

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

Executive summary:

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

- Premating exposure duration for parental (P0) animals: 10 weeks
- Basis for dose level selection: DRF study with no effects up to dose of 1000 mg/kg bw/day.
- Inclusion/exclusion of extension of Cohort 1B: No
- Termination time for F2: F1 generation not mated.
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B: No
- Inclusion/exclusion of developmental immunotoxicity Cohort 3: No
- Route of administration: oral (gavage)

Test material

Test material information

Constituent 1

Reference substance name:

Sodium polysulfide aluminosilicate with a SOD-type framework structure

EC Number:

701-340-9

Molecular formula:

|Na+6-x+y+z (S2•-)y(S3•-)z|[Al6-x Si6+x O24] - SOD Where: 6 ≤ 6-x+y+z ≤ 8 0 ≤ x ≤ 1.2 1 ≤ y+z ≤ 2

IUPAC Name:

Sodium polysulfide aluminosilicate with a SOD-type framework structure

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-house bred animals.
- Females (if applicable) nulliparous and non-pregnant: yes
- Weight at study initiation: 188.44-189.75 g (range of average values of each group of males at first day of dosing); 158.26-159.71 g (range of average values of each group of females at first day of dosing)
- Housing: Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with a stainless-steel mesh top grill having facilities for holding pelleted food and drinking water in a water bottle fitted with a stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
Pre mating: max. of 3 animals of the same sex and group per cage.
Mating: 2 animals (one male and one female) of the same group per cage.
Post mating: After confirming presence of sperm in the vaginal smear (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually. Sterilized paper shreds were provided as a nesting material from gestation day 20 onwards.
F1 animals: During Lactation period all F1 pups were housed along with the dam. Post weaning, 2 animals of the same sex and group per cage

- Diet (e.g. ad libitum): Altromin maintenance diet for rats and mice 1324 (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to the animals throughout the experimental period.
- Water (e.g. ad libitum): Water was provided ad libitum throughout the experimental period. Deep bore-well water passed through a Reverse Osmosis Unit was provided in plastic water bottles with stainless-steel sipper tubes.
- Acclimation period: at least 5 days.

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

IN-LIFE DATES: From: 11 February 2021 To: 07 September 2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% w/v Carboxy Methyl Cellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item formulations were prepared and administered to the animals within the established stability conditions. The required quantity of test item was weighed and triturated well in a mortar with a small quantity of vehicle until a homogenous suspension was formed and thereafter the entire quantity of the formulation was transferred into measuring cylinder. A small quantity of vehicle was added to rinse the mortar and this was transferred into the measuring cylinder. The rinsing procedure of mortar and pestle was repeated (many times) to ensure the transfer of the contents to the measuring cylinder. Finally, the volume was made up to required quantity with vehicle to get desired concentration of 10, 30 and 100 mg/mL of test item for low, mid and high dose groups respectively. The test formulations were maintained under stirring conditions using magnetic stirrer to maintain homogeneity of the test item formulations.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item was insoluble in distilled water and uniformly suspended in 0.5% w/v Carboxy Methyl Cellulose at the concentration of 100 mg/mL (the highest dose concentration selected for the study considering the dose volume of 10 mL/kg body weight) as per laboratory solubility/suspendibility test results. Hence, 0.5% w/v Carboxy Methyl Cellulose was used as vehicle for test item formulations.
- Concentration in vehicle: 10 - 100mg/mL
- Amount of vehicle (if gavage): 10mL/kg bw
- Lot/batch no. (if required): BCBN1690V, SLCH1520

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy.
- If mating has not occurred after 2 weeks, the animals will be separated without further continuation of mating. If there are insufficient males (e.g. due to male death before pairing) then male(s) which have already mated will be paired with a second female(s).
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol: No

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test item in dose formulations was established in a preliminary study. The test item formulations were stable at room temperature for 6 hours and up to 48 hours at 2 to 8°C at the concentrations of 10 mg/mL and 500 mg/mL in 0.5% Carboxymethyl Cellulose. Prepared test item formulations were administered to the animals within established stability conditions.
Homogeneity and dose formulation analysis for dose concentration verification was done by a validated ICP-MS method (study nº BIO-ANM 1575). Sampling and analysis of formulations were performed on days 16/02/2021 (Week 1), 15/04/2021 (Week 9), 10/06/2021 (Week 17) and 03/09/2021 (Week 29). Samples were collected in duplicates from top, middle and bottom layers from low, mid and high dose concentrations and in duplicates from middle layer from vehicle control. Formulations were considered acceptable, as the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) was ≤10%.

Duration of treatment / exposure:

Parental males: 10 weeks during pre-mating period + 2 weeks during mating period until sacrifice during post-mating period (a total of 98 to 108 days).
Parental females: 10 weeks during pre-mating period + 2 weeks during mating period + throughout gestation and lactation periods up to weaning of F1 animals (a total of 113 to 127 days).
Cohort 1A - From day of weaning (PND21) to PND 91 (13-weeks age)
Cohort 1B - From day of weaning (PND21) to at PND 98 (14-weeks age)

Frequency of treatment:

Once a day

Details on study schedule:

- F1 animals were not mated.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

G1/ G1-C1A/ G1-C1B

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

G2/ G2-C1A/ G2-C1B

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

G3/ G3-C1A/ G3-C1B

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

G4/ G4-C1A/ G4-C1B

No. of animals per sex per dose:

Parental generation: 25
Cohorts 1A and 1B: 20

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The doses of 100, 300 and 1000 mg/kg/day were selected for test item based on the results obtained from the dose range finding study in which no adverse effects were found up to the dose of 1000 mg/kg/day.
- Fasting period before blood sampling for clinical biochemistry: yes, overnight (water allowed).

Positive control:

None

Examinations

Parental animals: Observations and examinations:

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

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

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

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

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

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

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

OTHER:

Serum T4 and TSH levels were estimated using commercially available ELISA kits from 10 randomly selected males and 10 randomly selected females from each group.

Oestrous cyclicity (parental animals):

Oestrus cycles were monitored daily during the following occasions:
- for a period of 2 weeks during pre-mating period before initiation of cohabitation;
- during cohabitation period until evidence of mating;
- at termination (on the day of necropsy)

Sperm parameters (parental animals):

Sperm parameters were measured in all P generation males.
At termination, testes and epididymis weights were recorded for all P males. One testes and one epididymis were reserved for histopathological examination. Remaining epididymis were used for enumeration of cauda epididymis sperm reserves. In addition, sperm from the cauda epididymis (or vas deferens) was collected for evaluation of sperm motility and morphology. The other testes were stored in -20 degree and evaluated for homogenization resistance spermatid head counts.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- maximum of 4-5 pups/sex/litter as nearly as possible; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:

In F1 pups:

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

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

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

In F1 animals after weaning:

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


GROSS EXAMINATION OF DEAD PUPS: Yes, for external and internal abnormalities. Possible cause of death was determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All the survived P males were sacrificed after completion of mating procedure.
- Maternal animals: females not confirmed with mating were sacrificed after 24 to 26 days from the day of termination of cohabitation process. The females confirmed with mating but not littered were sacrificed 24 to 26 days after confirmation of mating. All the surviving littered females were sacrificed on lactation day 22.

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

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

Additional evaluation:

Ovarian Follicular Assessment: A quantitative ovarian follicle assessment was carried out for all P females from control and high dose groups.

Postmortem examinations (offspring):

SACRIFICE
-The cohort 1A animals were sacrificed on PND 92 and the cohort 1B animals were sacrificed on PND 99.

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

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

Additional evaluations:

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

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

Splenic lymphocyte subpopulation analysis (CD4+ and CD8+ T lymphocytes, B lymphocytes, and natural killer cells) using one half of the spleen, the other half of the spleen preserved for histopathological evaluation. 10 males and 10 females per group from Cohort 1A (1 male or 1 female per litter randomly selected) were subjected to this assessment.

Statistics:

After verification, the data was subjected to statistical analysis using SPSS software, version 22. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05). The statistical analysis was followed to the parameters as mentioned in the table 4 below.

Reproductive indices:

Mating index, fertility index, gestation index, parturation index, pregnancy index, sex ratio, live birth index, post implantation loss (%).

Offspring viability indices:

Pup survival index on lactation day 4/7/14/21, sex ratio on lactation day 4/7/14/21, post natal loss (%), AGD ratio.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs of toxicity noted in any of the tested dose group animals of both sexes throughout the experimental period.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no mortality/morbidity observed at any dose group during the experimental period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no changes noted in mean body weight and percent change in mean body weight gain with respect to day 1 in all the tested dose group males throughout the experimental period when compared with vehicle control group.

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

There was no test item related changes noted in mean body weight and percent change in mean body weight gain during gestation and lactation periods in all the tested dose groups when compared with vehicle control group. However, statistically significant increase in mean percent change in body weight gain during lactation day 1 to 4 in group G3 and during lactation day 7 to 14 in group G4 were noted when compared with vehicle control group. These noted differences are considered as incidental and unrelated to treatment as there were no such changes noted in mean body weight and mean feed consumption during these periods from both the dose levels.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There was no test item related changes noted in mean feed consumption in all tested dose groups for both sexes throughout the experimental period (including gestation and lactation periods for females) when compared with vehicle control group.

However, statistically significant decrease in mean feed consumption during week 1 in all tested dose group males was noted when compared with vehicle control group. These noted differences are considered as incidental and unrelated to treatment as there were no changes noted in mean body weight and mean percent change in mean body weight gain with respect to day 1 during this period from any of the dose levels.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant decrease in mean total erythrocyte count in group G4 males, decrease in mean eosinophils (%) in groups G3 and G4 males and decrease in absolute neutrophils in groups G2 and G3 females were noted when compared with vehicle control group. These differences are considered as incidental and unrelated to treatment, as the obtained mean values are within laboratory historical control range of same species and strain and also similar changes were not occurred in other sex of same dose level.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant decrease in mean albumin levels in group G4 females was noted when compared with vehicle control group. This difference is considered as incidental and unrelated to treatment, as the obtained mean value is within laboratory historical control range of same species and strain and also similar changes were not occurred in other sex of same dose level.

Endocrine findings:

no effects observed

Description (incidence and severity):

There were no changes noted in estimated mean serum Thyroxine (T4) and Thyroxine Stimulating Hormone (TSH) levels in any of the tested dose groups of both sexes when compared with vehicle control group.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant increase in mean specific gravity of urine in group G4 females was noted when compared with vehicle control group. This difference is considered as incidental and unrelated to treatment, as the obtained mean value is within laboratory historical control range of same species and strain and also similar changes were not occurred in other sex of same dose level.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test item-related histopathological findings in high dose parental animals.

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

Quantitative ovarian follicular assessment (primordial and primary follicles) in all parental females of control and high dose groups did not reveal any test item related variations.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

A total of 3, 3, 2 and 1 female(s) were noted with single irregular oestrus cycle from groups G1, G2, G3 and G4, respectively. These occurrences are considered as incidental and unrelated to treatment exposure as the same animals were proceeded or preceded by normal cycles.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

Sperm motility(%): There were no changes noted in mean sperm motility (%) in all the tested dose groups when compared with vehicle control group.
Sperm Morphology: There were no test item-related changes noted in sperm morphologies in all the tested dose groups when compared with vehicle control group. The noted sporadic statistically significant differences in sperm morphology such as increase in head anomalies in group G3, decrease in tail anomalies in groups G3 and G4, increase in multiple anomalies in group G4 and increase in total sperms with anomalies in group G4 when compared with vehicle control group, are considered as toxicologically irrelevant.
Spermatid Head Count and Daily Sperm Production: There were no test item-related changes noted in mean spermatid head count or daily testicular sperm production per animal in any of the tested dose groups when compared with the vehicle control group. The noted statistically significant increase in mean spermatid head count in group G4 when compared with vehicle control group is considered as toxicologically irrelevant.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no effects on male and female reproductive performance/indices noted from P generation in any of the tested dose groups when compared with vehicle control group.

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

There were no changes observed in birth parameters such as, total litter size, number of live pups born, sex ratio (m/f) and live birth index in all the tested dose groups when compared with the vehicle control group. Also, the pup survival index per litter during lactation period was unaffected by the test item in all the tested dose groups when compared with the vehicle control group.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects found up to the highest dose tested (1000 mg/kg bw/day)

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

F1 pups: There were no external abnormalities or behavioural changes noted in any of the pups during daily observation from all the tested dose groups and vehicle control group during postnatal period. All the pups had normal behaviour during daily observations.
Cohort 1A /1B: There were no clinical signs of toxicity noted in any of the tested dose group animals of both sexes throughout the experimental period.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

F1 pups. Postnatal Loss: There were no statistically significant changes noted for mean number and percentage of occurred post-natal losses in any of the tested dose groups when compared with the vehicle control group.
Cohort 1A/1B: There were no mortality/morbidity observed at any dose group during the experimental period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

F1 pups: There was no test item related changes noted in mean pup [both male and female] weight per litter in all the tested dose groups when compared with the vehicle control group, recorded on postnatal day (PND) 1, 4, 7, 14 and 21.
Cohort 1A: There were no test item related changes noted in mean body weight and percent change in mean body weight gain with respect to postnatal day (PND) 21 in all the tested dose groups of both sexes throughout the experimental period when compared with vehicle control group.
Cohort 1B: There was no test item related changes noted in mean body weight and percent change in mean body weight gain with respect to postnatal day (PND) 21 in all the tested dose groups of both sexes throughout the experimental period when compared with vehicle control group. Some statistically significant changes were noted during the experimental period which were considered due to stress induced by test item administration and not adverse as there were no other systemic toxicity effects noted in any of the tested dose groups.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1A: There was no test item related changes noted in mean feed consumption in all the tested dose groups of both sexes throughout the experimental period when compared with vehicle control group. However, an incidental statistically significant decrease in mean feed consumption during week 7 in group G4-C1A was noted when compared with vehicle control group.
Cohort 1B: There was no test item related changes noted in mean feed consumption in all the tested dose groups of both sexes throughout the experimental period when compared with vehicle control group. However, an incidental statistically significant decrease in mean feed consumption during week 7 and 9 in group G2-C1B was noted when compared with vehicle control group.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1A: A statistically significant increase in mean absolute eosinophils in group G2-C1A males, increase in mean absolute lymphocytes in group G2-C1A females and decrease in mean APTT levels in group G2-C1A were noted when compared with vehicle control group. These differences are considered as incidental and unrelated to treatment, as the obtained mean values are within laboratory historical control range of same species and strain and also similar changes were not occurred in other sex of same dose level.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Cohort 1A: There were no changes noted in the obtained mean clinical chemistry values in all the tested dose groups of both sexes when compared with the vehicle control group.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1A: an incidental and toxicologically irrelevant statistically significant decrease in mean volume of urine in group G2-C1A females was noted when compared with vehicle control group. This difference is considered unrelated to treatment and the obtained mean value is within laboratory historical control range of same species.

Sexual maturation:

no effects observed

Description (incidence and severity):

Cohort 1A males: There were no changes noted in mean days of occurrence of balano-preputial separation and mean body weight on the day of occurrence of occurrence of balano-preputial separation in any of the tested dose groups when compared with vehicle control group.
Cohort 1A females: There were no changes noted in mean days of occurrence of vaginal patency/opening, mean body weight on the day of occurrence of vaginal patency, mean occurrence of first cornified cells (days) and mean time interval between vaginal patency to occurrence of first cornified cells in any of the tested dose groups when compared with vehicle control group.
Cohort 1B males and females: There were no changes noted in mean days of occurrence of balano-preputial separation in C1B males and mean days of occurrence of vaginal patency/opening, in C1B females in any of the tested dose groups when compared with vehicle control group.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

F1 pups: There were no changes in mean pup [both male and female] anogenital distance measurement (mm) and its ratio per litter in all the tested dose groups when compared with the vehicle control group, recorded on postnatal day 4.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

F1 pups: There were no occurrences or evidences of retention of nipples in any of the male pups examined on the postnatal day 13 from all the tested dose groups and vehicle control group litters.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

F1 surplus pups: a statistically significant decrease in mean absolute thymus weight was noted in group G4 when compared with vehicle control group. This change is considered as incidental and unrelated to treatment, as the obtained mean value is within laboratory historical control range of PND 21 pups of same species and strain and also no gross pathological changes were noted in any of the pups at this dose level.
Cohort 1A: A statistically significant decrease in mean spleen weight (absolute and relative) in group G3-C1A males, increase in mean relative prostate weight in groups G3-C1A and G4-C1A males, increase in mean relative pituitary weight in all tested dose group males, increase in mean relative thyroid weight in group G4-C1A males, decrease in mean absolute spleen weight in groups G3-C1A and G4-C1A females, decrease in mean absolute ovaries weight in group G3-C1A, increase in mean absolute kidneys weight in group G2-C1A females, decrease in mean relative spleen and ovaries weight in group G3-C1A females and increase in mean relative heart weight in group G4-C1A females were noted when compared with vehicle control group.
These noted differences are considered as incidental and unrelated to treatment, as the obtained mean values are within laboratory historical control range of same species and strain and also neither gross pathological functional changes nor microscopic changes were noted in any of these organs in all the tested dose group animals.
Cohort 1B: There were no test item-related changes noted in mean absolute and relative organ weights in all the tested dose groups of both sexes when compared with vehicle control group. The noted statistically significant differences in some of the organs/tissues from all the tested dose groups of both sexes are not considered as adverse as the obtained mean values are within laboratory historical control range of same species and strain and also gross pathological examination did not reveal any changes in any of organs/tissues in all the tested dose group animals.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological changes/findings noted in any of the tested dose group and vehicle control group of F1 generation animals of both sexes during the necropsy.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test item-related histopathological findings in high dose C1A animals.

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

Quantitative ovarian follicular assessment (primordial and primary follicles) in all C1A females of control and high dose groups did not reveal any test item related variations. Similarly, quantitative evaluation of corpora luteal count in C1A females of control and high dose group did not reveal any test item related variations.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

F1 pups:
Postnatal Developmental Landmarks: There were no changes noted in mean occurrences of postnatal developmental landmarks of F1 pups such as pinna unfold, fur development, incisor eruption, eye opening and testes descent evaluated during postnatal period in all the tested dose groups when compared with vehicle control group.
Sensory Reflexes: There were no changes noted in mean responses of sensory reflexes of F1 pups such as surface righting, auditory startle and air righting performed during postnatal period in all the tested dose groups when compared with vehicle control group.
Thyroid Hormone Levels (T4 and TSH):
There were no changes noted in mean T4 levels of PND 4 pups per litter in all the tested dose groups when compared with the vehicle control group.
There was no test item related changes noted in meanT4 and TSH levels of PND 21 pups per litter in all the tested dose groups when compared with the vehicle control group. The noted statistically significant increase in mean serum T4 levels in all the tested dose groups of PND 21 pups is considered as incidental and also the obtained mean values are within laboratory historical control range of same species and strain.

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects found up to the highest dose tested (1000 mg/kg bw/day)

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

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

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Day
		1	7	14	21	28	35	42	49	56	63	70	77	84	91	98	104^@
G1, M & 0	Mean	189.62	219.90	252.26	276.94	304.05	324.04	353.89	379.50	393.20	407.64	417.21	427.66	439.67	452.70	468.71	489.35
	±SD	13.57	16.34	21.85	24.53	31.12	32.92	36.35	38.11	42.19	47.54	48.83	46.82	49.50	50.55	47.97	39.96
	n	25	25	25	25	25	25	25	25	25	25	25	25	25	25	25	15
G2, M & 100	Mean	189.75	216.71	248.03	270.02	295.15	318.45	341.70	367.78	382.28	395.43	405.27	415.32	428.25	439.74	457.17	482.50
	±SD	13.38	15.87	19.26	23.04	29.27	32.35	36.92	41.69	46.12	48.90	51.42	54.81	55.03	54.72	54.28	61.66
	n	25	25	25	25	25	25	25	25	25	25	25	25	25	25	25	15
G3, M & 300	Mean	188.93	217.09	250.78	276.00	300.46	319.54	345.76	366.44	385.21	394.02	407.19	416.29	426.94	437.99	455.09	466.77
	±SD	14.56	15.84	23.72	26.26	29.63	32.61	35.61	41.39	42.17	41.81	43.14	44.60	45.67	45.49	44.70	50.72
	n	25	25	25	25	25	25	25	25	25	25	25	25	25	25	25	15
G4, M & 1000	Mean	188.44	215.90	251.91	274.96	297.54	317.80	345.10	370.04	382.35	397.21	407.83	418.15	433.05	447.78	467.67	463.80
	±SD	12.34	13.21	17.64	23.03	30.44	32.05	32.47	38.89	41.22	41.76	43.16	44.24	44.05	44.48	49.47	41.68
	n	25	25	25	25	25	25	25	25	25	25	25	25	25	25	25	15

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

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Day
		1	7	14	21	28	35	42	49	56	63	70	77
G1, F & 0	Mean	159.71	175.10	189.42	201.56	214.65	225.46	233.52	245.24	251.38	257.21	262.13	275.76
	±SD	10.90	12.87	12.67	13.25	16.87	17.00	16.59	15.27	15.64	16.03	16.76	17.56
	n	25	25	25	25	25	25	25	25	25	25	25	9
G2, F & 100	Mean	159.35	171.63	188.78	198.98	212.38	222.98	231.03	241.91	249.85	259.04	262.81	267.61
	±SD	9.34	11.18	13.35	14.28	14.40	15.59	16.48	16.67	18.78	20.12	19.13	21.26
	n	25	25	25	25	25	25	25	25	25	25	25	12
G3, F & 300	Mean	158.49	172.13	186.58	199.74	211.35	219.40	230.26	239.11	246.07	253.37	255.86	264.00
	±SD	10.28	10.00	10.08	8.14	8.67	9.02	9.90	8.57	9.03	12.05	12.93	15.73
	n	25	25	25	25	25	25	25	25	25	25	25	11
G4, F & 1000	Mean	158.26	172.69	188.23	202.11	212.14	218.79	231.45	240.51	244.81	252.21	254.19	257.59
	±SD	10.15	8.59	9.64	11.66	11.57	11.72	11.03	12.03	12.97	14.19	14.94	14.31
	n	25	25	25	25	25	25	25	25	25	25	25	16

 

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

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Gestation Day (GD)
		0	7	14	20
G1, F & 0	Mean	265.97	280.38	310.98	367.35
	±SD	17.21	17.74	18.02	21.17
	n	23	23	23	23
G2, F & 100	Mean	265.63	280.02	308.92	365.66
	±SD	17.17	17.20	19.61	20.19
	n	21	21	21	21
G3, F & 300	Mean	260.48	274.46	304.07	362.34
	±SD	14.12	13.66	14.97	20.07
	n	22	22	22	22
G4, F & 1000	Mean	260.52	275.17	302.92	363.15
	±SD	15.47	16.24	15.94	18.01
	n	21	21	21	21

 

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

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Lactation Day (LD)
		1	4	7	14	21
G1, F & 0	Mean	286.50	289.34	298.97	310.30	323.12
	±SD	17.81	18.83	18.09	16.57	14.54
	n	23	23	23	23	23
G2, F & 100	Mean	285.20	290.07	298.06	310.62	322.55
	±SD	16.63	17.12	16.96	15.46	14.40
	n	21	21	21	21	21
G3, F & 300	Mean	280.07	284.87	295.11	307.04	318.40
	±SD	13.32	13.21	13.98	12.76	11.15
	n	22	22	22	22	22
G4, F & 1000	Mean	278.63	282.70	291.07	304.72	316.48
	±SD	14.50	14.20	14.05	12.61	12.15
	n	21	21	21	21	21

 

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

Group, Sex & Dose (mg/kg body weight/day)		Total Leucocyte Count	Total Erythrocyte Count	Haemoglobin	Haematocrit	Platelet Count	Neutrophils	Lymphocytes	Monocytes	Eosinophils	Basophils
		(WBC)	(RBC)	(HGB)	(HCT)	(PLT)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)
		(10^3 cells/µL)	(10^6 cells/µL)	(g/dL)	(%)	(10^3 cells/µL)	(%)	(%)	(%)	(%)	(%)
G1, M & 0	Mean	10.63	9.51	15.70	53.63	769.60	29.20	63.58	3.70	2.21	0.35
	±SD	3.54	1.94	1.05	14.87	197.51	4.18	4.57	0.46	1.37	0.20
	n	10	10	10	10	10	10	10	10	10	10
G2, M & 100	Mean	11.66	8.74	15.52	47.29	758.20	30.97	62.29	3.45	1.67	0.64
	±SD	7.12	0.65	1.03	3.99	198.49	6.40	6.39	0.59	0.73	0.57
	n	10	10	10	10	10	10	10	10	10	10
G3, M & 300	Mean	10.01	8.25	15.13	46.51	824.30	29.15	65.09	3.15	1.19*	0.39
	±SD	1.53	0.77	1.00	2.67	177.39	6.87	7.03	0.86	0.25	0.19
	n	10	10	10	10	10	10	10	10	10	10
G4, M & 1000	Mean	10.19	8.09*	14.51	45.07	833.50	31.06	63.33	3.03	1.18*	0.49
	±SD	2.93	1.26	1.97	4.91	235.67	7.29	7.14	1.04	0.40	0.54
	n	10	10	10	10	10	10	10	10	10	10

 

Group, Sex & Dose (mg/kg body weight/day)		Absolute Neutrophils	Absolute Lymphocytes	Absolute Monocytes	Absolute Eosinophils	Absolute  Basophils	Prothrombin Time	Activated Prothrombin Time
		(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)	(PT)	(APTT)
		(10^3 cells/µL)	(10^3 cells/µL)	(10^3 cells/µL)	(10^3 cells/µL)	(10^3 cells/µL)	(Seconds)	(Seconds)
G1, M & 0	Mean	3.09	6.75	0.40	0.25	0.03	19.08	29.05
	±SD	1.08	2.25	0.14	0.23	0.02	1.55	11.18
	n	10	10	10	10	10	10	10
G2, M & 100	Mean	3.68	7.21	0.40	0.19	0.07	19.25	23.76
	±SD	2.89	3.99	0.24	0.13	0.06	6.37	6.25
	n	10	10	10	10	10	10	10
G3, M & 300	Mean	2.94	6.49	0.32	0.12	0.04	18.44	24.78
	±SD	0.88	1.10	0.10	0.04	0.02	1.65	4.65
	n	10	10	10	10	10	10	10
G4, M & 1000	Mean	3.33	6.30	0.31	0.13	0.05	18.40	27.99
	±SD	1.76	1.17	0.13	0.07	0.05	2.49	10.39
	n	10	10	10	10	10	10	10

 

Group, Sex & Dose (mg/kg body weight/day)		Total Leucocyte Count	Total Erythrocyte Count	Haemoglobin	Haematocrit	Platelet Count	Neutrophils	Lymphocytes	Monocytes	Eosinophils	Basophils
		(WBC)	(RBC)	(HGB)	(HCT)	(PLT)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)
		(103 cells/µL)	(106 cells/µL)	(g/dL)	(%)	(103 cells/µL)	(%)	(%)	(%)	(%)	(%)
G1, F & 0	Mean	11.26	8.42	16.47	49.92	853.20	39.79	52.76	4.44	1.71	0.32
	±SD	3.63	0.49	0.66	2.99	333.77	7.02	7.43	1.83	1.30	0.11
	n	10	10	10	10	10	10	10	10	10	10
G2, F & 100	Mean	9.20	8.38	16.64	50.35	774.40	32.39	61.00	4.30	1.17	0.31
	±SD	2.24	0.98	1.47	5.72	257.15	10.05	9.91	0.97	0.90	0.19
	n	10	10	10	10	10	10	10	10	10	10
G3, F & 300	Mean	8.56	7.96	15.74	47.48	946.90	33.55	59.82	4.23	1.33	0.29
	±SD	2.79	0.95	1.36	5.19	99.65	9.63	9.57	1.06	0.60	0.16
	n	10	10	10	10	10	10	10	10	10	10
G4, F & 1000	Mean	9.37	8.25	16.24	49.85	879.50	34.01	57.58	5.64	1.42	0.31
	±SD	2.18	0.74	0.92	1.79	214.37	5.10	4.92	1.59	1.24	0.12
	n	10	10	10	10	10	10	10	10	10	10

 

Group, Sex & Dose (mg/kg body weight/day)		Absolute Neutrophils	Absolute Lymphocytes	Absolute Monocytes	Absolute Eosinophils	Absolute Basophils	Prothrombin Time	Activated Prothrombin Time
		(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)	(PT)	(APTT)
		(103 cells/µL)	(103 cells/µL)	(103 cells/µL)	(103 cells/µL)	(103 cells/µL)	(Seconds)	(Seconds)
G1, F & 0	Mean	4.51	5.92	0.51	0.17	0.04	16.97	23.81
	±SD	1.77	1.95	0.28	0.09	0.02	4.11	15.54
	n	10	10	10	10	10	10	10
G2, F & 100	Mean	3.03*	5.56	0.39	0.12	0.03	20.61	20.93
	±SD	1.25	1.53	0.11	0.12	0.02	7.28	7.76
	n	10	10	10	10	10	10	10
G3, F & 300	Mean	2.96*	5.02	0.38	0.11	0.03	19.04	17.64
	±SD	1.33	1.63	0.21	0.05	0.01	3.50	3.12
	n	10	10	10	10	10	10	10
G4, F & 1000	Mean	3.19	5.40	0.53	0.14	0.03	20.13	23.48
	±SD	0.83	1.36	0.21	0.13	0.02	6.24	5.95
	n	10	10	10	10	10	10	10

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

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

Group, Sex & Dose (mg/kg body weight/day)		Glucose	Urea	Creatinine	Total Cholesterol	Total Protein	Albumin
		(GLU)	-	(CRE)	(CHO)	(TPR)	(ALB)
		(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(g/dL)	(g/dL)
G1, M & 0	Mean	89.10	36.80	0.54	53.40	7.49	3.30
	±SD	15.46	5.71	0.04	7.73	0.41	0.24
	n	10	10	10	10	10	10
G2, M & 100	Mean	92.70	35.98	0.54	45.20	7.48	3.30
	±SD	10.33	6.78	0.10	10.03	0.55	0.14
	n	10	10	10	10	10	10
G3, M & 300	Mean	109.30	36.04	0.52	54.00	7.58	3.38
	±SD	28.58	8.72	0.03	10.27	0.38	0.15
	n	10	10	10	10	10	10
G4, M & 1000	Mean	97.40	36.35	0.58	50.10	7.78	3.49
	±SD	14.76	5.30	0.09	11.82	0.60	0.19
	n	10	10	10	10	10	10

 

Group, Sex & Dose (mg/kg body weight/day)		Alanine aminotransferase	Aspartate aminotransferase	Alkaline phosphatase	Globulin	Albumin/ Globulin ratio	Blood Urea Nitrogen
		(ALT)	(AST)	(ALP)	(GLO)	(A/G Ratio)	(BUN)
		(U/L)	(U/L)	(U/L)	(g/dL)	-	(mg/dL)
G1, M & 0	Mean	79.30	124.70	132.70	4.19	0.79	17.17
	±SD	32.06	45.81	40.28	0.32	0.08	2.66
	n	10	10	10	10	10	10
G2, M & 100	Mean	68.50	121.30	144.80	4.18	0.80	16.79
	±SD	12.20	17.28	44.86	0.46	0.08	3.16
	n	10	10	10	10	10	10
G3, M & 300	Mean	70.90	129.60	153.60	4.20	0.82	16.82
	±SD	18.77	22.46	73.38	0.42	0.10	4.07
	n	10	10	10	10	10	10
G4, M & 1000	Mean	73.80	118.10	151.50	4.29	0.82	16.96
	±SD	14.69	16.64	55.30	0.44	0.06	2.47
	n	10	10	10	10	10	10

 

Group, Sex & Dose (mg/kg body weight/day)		Glucose	Urea	Creatinine	Total Cholesterol	Total Protein	Albumin
		(GLU)	-	(CRE)	(CHO)	(TPR)	(ALB)
		(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(g/dL)	(g/dL)
G1, F & 0	Mean	103.00	56.54	0.61	68.70	7.62	3.60
	±SD	23.09	14.80	0.06	17.20	0.47	0.27
	n	10	10	10	10	10	10
G2, F & 100	Mean	106.60	61.11	0.56	58.60	7.29	3.48
	±SD	15.56	14.46	0.06	17.42	0.71	0.22
	n	10	10	10	10	10	10
G3, F & 300	Mean	104.50	48.94	0.57	65.80	7.29	3.51
	±SD	24.97	12.54	0.06	20.14	0.46	0.22
	n	10	10	10	10	10	10
G4, F & 1000	Mean	97.80	71.83	0.55	62.00	7.04	3.32*
	±SD	16.12	21.38	0.06	10.75	0.54	0.24
	n	10	10	10	10	10	10

 

Group, Sex & Dose (mg/kg body weight/day)		Alanine aminotransferase	Aspartate aminotransferase	Alkaline phosphatase	Globulin	Albumin/ Globulin ratio	Blood Urea Nitrogen
		(ALT)	(AST)	(ALP)	(GLO)	(A/G Ratio)	(BUN)
		(U/L)	(U/L)	(U/L)	(g/dL)	-	(mg/dL)
G1, F & 0	Mean	114.80	143.90	295.60	4.02	0.91	26.38
	±SD	36.58	27.64	145.51	0.47	0.15	6.91
	n	10	10	10	10	10	10
G2, F & 100	Mean	108.20	158.20	249.90	3.81	0.94	28.52
	±SD	46.53	62.94	108.16	0.66	0.18	6.75
	n	10	10	10	10	10	10
G3, F & 300	Mean	70.40	114.30	187.60	3.79	0.94	22.84
	±SD	28.91	36.62	117.31	0.50	0.16	5.85
	n	10	10	10	10	10	10
G4, F & 1000	Mean	117.70	173.30	338.20	3.72	0.92	33.52
	±SD	57.63	102.35	246.41	0.67	0.17	9.98
	n	10	10	10	10	10	10

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

 

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

Examination	Group, Sex & Dose (mg/kg body weight/day)		G1, M & 0	G2, M & 100	G3, M & 300	G4, M & 1000
	Number of Animals		10	10	10	10
Physical Examination	Color	Pale Yellow	7	7	6	9
		Yellow	3	3	4	1
	Appearance	Clear	5	8	8	9
		Turbid	5	2	2	1
	Volume (mL)	Mean	7.0	7.8	6.9	7.6
		±SD	1.7	2.1	1.7	1.5
Chemical Examination	pH	Mean	7.9	7.8	7.5	7.5
		±SD	0.3	0.4	0.8	0.7
	Specific gravity	Mean	1.011	1.010	1.011	1.011
		±SD	0.004	0.003	0.003	0.005
	Urobilinogen (mg/dL)	Mean	0.3	0.3	0.3	0.3
		±SD	0.3	0.3	0.3	0.3
	Bilirubin (mg/dL)	Neg	5	8	8	8
		1	5	2	2	2
	Ketones (mg/dL)	Neg	4	4	4	5
		5	6	6	6	5
	Blood (Ery/µL)	Neg	1	3	2	-
		Ca10	2	-	-	1
		Ca25	4	2	2	4
		Ca80	1	2	3	3
		>=Ca200	2	3	3	2
	Protein (mg/dL)	Neg	3	6	7	7
		Trace	2	1	1	-
		30	4	2	2	1
		100	-	1	-	1
		>=300	1	-	-	1
	Nitrite	Neg	6	6	5	7
		Pos	4	4	5	3
	Leucocytes (Leu/µL)	Neg	-	-	-	2
		Ca15	1	5	4	4
		Ca70	7	5	5	2
		Ca125	2	-	-	2
	Glucose (mg/dL)	Neg	10	10	10	10
	Micro Albumin (mg/dL)	Neg	-	1	-	1
		>15	7	4	3	3
		15	3	5	7	6
Microscopic Examination	Epi Cells	0	5	1	5	1
		0-1	2	6	2	9
		1-2	2	1	3	-
		2-3	-	1	-	-
	Casts	Absent	10	10	10	10
	Crystals	Present	10	10	10	10

 

Examination	Group, Sex & Dose (mg/kg body weight/day)		G1, F & 0	G2, F & 100	G3, F & 300	G4, F & 1000
	Number of Animals		10	10	10	10
Physical Examination	Color	Pale Yellow	10	8	8	8
		Yellow	-	2	2	2
	Appearance	Clear	10	9	9	9
		Turbid	-	1	1	1
	Volume (mL)	Mean	6.0	5.6	6.2	6.4
		±SD	1.4	1.1	1.0	1.7
Chemical Examination	pH	Mean	7.9	7.8	7.6	7.4
		±SD	0.6	0.5	0.6	0.8
		Mean	1.008	1.012	1.012	1.014*
		±SD	0.005	0.005	0.004	0.004
	Urobilinogen (mg/dL)	Mean	0.4	0.4	0.4	0.3
		±SD	0.4	0.6	0.4	0.3
	Bilirubin (mg/dL)	Neg	5	4	5	10
		1	5	5	5	-
	Ketones (mg/dL)	Neg	10	9	10	8
		5	-	1	-	2
	Blood (Ery/µL)	Neg	8	4	7	9
		Ca10	1	2	1	-
		Ca25	-	1	1	-
		Ca80	1	3	1	1
	Protein (mg/dL)	Neg	2	6	4	9
		Trace	4	3	2	-
		30	2	-	1	-
		100	-	-	1	-
		>=300	2	1	2	1
	Nitrite	Neg	4	6	4	6
		Pos	6	4	6	4
	Leucocytes (Leu/µL)	Neg	1	1	1	3
		Ca15	6	4	4	5
		Ca70	3	4	5	2
		Ca125	-	1	-	-
	Glucose (mg/dL)	Neg	10	10	10	10
	Micro Albumin (mg/dL)	Neg	1	1	4	4
		>15	8	4	6	1
		15	1	5	-	5
Microscopic Examination	Epi Cells	0	8	6	6	6
		0-1	2	3	3	3
		1-2	-	1	1	1
	Casts	Absent	10	10	10	10
	Crystals	Present	10	10	10	10

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

 

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

Group, Sex & Dose (mg/kg body weight/day)		Terminal Body Weight (g)	Adrenals	Thymus	Spleen	Testes	Epididymides	Heart
G1, M & 0	Mean	458.36	0.0139	0.0660	0.3021	0.7706	0.3619	0.3226
	±SD	49.77	0.0020	0.0100	0.0738	0.1159	0.0643	0.0350
	n	25	25	25	25	25	25	25
G2, M & 100	Mean	448.24	0.0147	0.0700	0.3046	0.7697	0.3690	0.3324
	±SD	56.37	0.0021	0.0122	0.0811	0.1148	0.0574	0.0506
	n	25	25	25	25	25	25	25
G3, M & 300	Mean	447.03	0.0143	0.0666	0.3047	0.7814	0.3679	0.3560*
	±SD	49.83	0.0023	0.0111	0.0801	0.1153	0.0589	0.0500
	n	25	25	25	25	25	25	25
G4, M & 1000	Mean	454.74	0.0136	0.0690	0.3052	0.7701	0.3585	0.3333
	±SD	47.89	0.0020	0.0141	0.0606	0.0935	0.0540	0.0403
	n	25	25	25	25	25	25	25

 

Group, Sex & Dose (mg/kg body weight/day)		Kidneys	Brain	Liver	Prostate	Seminal vesicles with coagulating glands	Thyroid along with parathyroid	Pituitary
G1, M & 0	Mean	0.6130	0.4702	2.4536	0.2976	0.4078	0.0062	0.0035
	±SD	0.0817	0.0664	0.3199	0.0666	0.1250	0.0005	0.0003
	n	25	25	25	25	25	25	25
G2, M & 100	Mean	0.6116	0.4783	2.4252	0.3059	0.3825	0.0062	0.0038*
	±SD	0.0657	0.0538	0.2377	0.0667	0.1117	0.0006	0.0004
	n	25	25	25	25	25	25	25
G3, M & 300	Mean	0.6302	0.4865	2.5252	0.2962	0.3941	0.0063	0.0036
	±SD	0.0764	0.0606	0.3262	0.0830	0.1306	0.0004	0.0003
	n	25	25	25	25	25	25	25
G4, M & 1000	Mean	0.6128	0.4638	2.4626	0.2835	0.3739	0.0061	0.0037
	±SD	0.0911	0.0367	0.3435	0.0666	0.1109	0.0005	0.0004
	n	25	25	25	25	25	25	25

 

Group, Sex & Dose (mg/kg body weight/day)		Terminal Body Weight (g)	Adrenals	Thymus	Spleen	Ovaries	Uterus with Cervix
G1, F & 0	Mean	307.39	0.0257	0.0720	0.2451	0.0438	0.1860
	±SD	14.74	0.0027	0.0133	0.0481	0.0064	0.0377
	n	25	25	25	25	25	25
G2, F & 100	Mean	305.35	0.0261	0.0754	0.2429	0.0457	0.1703
	±SD	15.18	0.0031	0.0091	0.0421	0.0079	0.0486
	n	25	25	25	25	25	25
G3, F & 300	Mean	301.56	0.0275	0.0776	0.2501	0.0443	0.1946
	±SD	16.62	0.0030	0.0079	0.0481	0.0074	0.0423
	n	25	25	25	25	25	25
G4, F & 1000	Mean	299.24	0.0265	0.0757	0.2522	0.0464	0.1807
	±SD	14.69	0.0040	0.0106	0.0333	0.0063	0.0474
	n	25	25	25	25	25	25

 

Group, Sex & Dose (mg/kg body weight/day)		Heart	Kidneys	Brain	Liver	Thyroid along with parathyroid	Pituitary
G1, F & 0	Mean	0.3899	0.6406	0.6430	3.6558	0.0078	0.0052
	±SD	0.0297	0.0522	0.0382	0.3757	0.0006	0.0005
	n	25	25	25	25	25	25
G2, F & 100	Mean	0.3621	0.6410	0.6466	3.6413	0.0079	0.0053
	±SD	0.1064	0.0609	0.0437	0.3475	0.0005	0.0007
	n	25	25	25	25	25	25
G3, F & 300	Mean	0.3852	0.6845	0.6572	3.5934	0.0082	0.0052
	±SD	0.0541	0.1793	0.0525	0.3594	0.0008	0.0006
	n	25	25	25	25	25	25
G4, F & 1000	Mean	0.3881	0.6652	0.6621	3.6815	0.0080	0.0051
	±SD	0.0865	0.0550	0.0552	0.3734	0.0006	0.0005
	n	25	25	25	25	25	25

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

 

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

Group, Sex & Dose (mg/kg body weight/day)		Mean Percent of Sperm Motility (%)
G1, M & 0	Mean	89.9
	±SD	1.9
	n	25
G2, M & 100	Mean	90.0
	±SD	1.3
	n	25
G3, M & 300	Mean	89.7
	±SD	1.5
	n	25
G4, M & 1000	Mean	89.6
	±SD	2.0
	n	25

 

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

Group, Sex & Dose (mg/kg body weight/day)		Head Abnormalities		Neck Abnormalities		Tail Abnormalities		Multiple Abnormalities			Sperms with Abnormality			Normal Sperms
		Total	%	Total	%	Total	%	Total	%		No.	%		No.	%
G1, M & 0	Mean	2.68	1.34	1.00	0.50	1.32	0.66	0.12	0.06		5.12	2.56		194.88	97.44
	±SD	1.68	0.84	1.32	0.66	1.22	0.61	0.33	0.17		2.07	1.03		2.07	1.03
	n	25	25	25	25	25	25	25	25		25	25		25	25
G2, M & 100	Mean	3.08	1.54	1.56	0.78	0.60	0.30	0.44	0.22		5.68	2.84		194.32	97.16
	±SD	1.55	0.78	1.08	0.54	0.82	0.41	0.58	0.29		1.35	0.67		1.35	0.67
	n	25	25	25	25	25	25	25	25		25	25		25	25
G3, M & 300	Mean	4.16*	2.08*	0.52	0.26	0.36*	0.18*	0.48	0.24		5.52	2.76		194.48	97.24
	±SD	1.37	0.69	0.65	0.33	0.64	0.32	0.51	0.25		1.08	0.54		1.08	0.54
	n	25	25	25	25	25	25	25	25		25	25		25	25
G4, M & 1000	Mean	3.68	1.84	1.08	0.54	1.12*	0.56*	0.84*	0.42*		6.72*	3.36*		193.28*	96.64*
	±SD	1.73	0.86	1.00	0.50	0.93	0.46	0.85	0.43		1.40	0.70		1.40	0.70
	n	25	25	25	25	25	25	25	25		25	25		25	25

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

 

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

Group, Sex & Dose (mg/kg body weight/day)		Average Sperm head count	Daily Sperm Production (x 10^6 per gram of Testis)	Daily Sperm Production (x10^6 per Animal)
G1, M & 0	Mean	158.88	12.5	47.4
	±SD	5.95	0.6	6.0
	n	25	25	25
G2, M & 100	Mean	163.43	12.6	49.1
	±SD	3.16	0.3	4.6
	n	25	25	25
G3, M & 300	Mean	157.05	12.7	50.3
	±SD	6.04	0.5	6.4
	n	25	25	25
G4, M & 1000	Mean	163.63*	13.0	47.5
	±SD	4.36	0.3	7.2
	n	25	25	25

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

 

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

Group & Dose (mg/kg body weight/day)		PND 1			PND 4			PND 7			PND 14			PND 21
		Mean Pup Weight			Mean Pup Weight			Mean Pup Weight			Mean Pup Weight			Mean Pup Weight
		Male	Female		Male	Female		Male	Female		Male	Female		Male	Female
G1 & 0	Mean	6.60	6.29		11.18	10.68		16.84	16.13		28.05	26.71		43.70	40.64
	±SD	0.32	0.26		0.71	0.70		0.68	0.82		0.63	0.89		2.23	1.50
	n	23	23		23	23		23	23		23	23		23	23
G2 & 100	Mean	6.54	6.29		11.34	10.80		16.91	16.22		28.17	26.86		44.27	41.48
	±SD	0.35	0.29		0.74	0.80		0.75	0.70		1.18	0.88		2.50	1.93
	n	21	21		21	21		21	21		21	21		21	21
G3 & 300	Mean	6.63	6.30		11.53	10.81		17.04	15.99		28.32	26.81		45.22	42.15*
	±SD	0.39	0.24		0.68	0.87		0.57	0.63		1.15	0.87		2.81	2.30
	n	22	22		22	22		22	22		22	22		22	22
G4 & 1000	Mean	6.76	6.53*		11.13	10.77		16.99	16.47		28.18	27.17		44.69	42.28*
	±SD	0.42	0.39		0.88	0.82		0.79	0.59		0.86	0.70		2.73	1.79
	n	21	21		21	21		21	21		21	21		21	21

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

 

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

Group & Dose (mg/kg body weight/day)		Serum T4 Levels (ng/mL)
G1 & 0	Mean	55.737
	±SD	5.117
	n	23
G2 & 100	Mean	61.945*
	±SD	5.039
	n	21
G3 & 300	Mean	69.303*
	±SD	5.230
	n	22
G4 & 1000	Mean	71.748*
	±SD	5.377
	n	21

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

 

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

Group, Sex & Dose (mg/kg body weight/day)		Serum TSH Levels (µIU/mL)
G1, F & 0	Mean	1.667
	±SD	2.877
	n	21
G2, F & 100	Mean	1.076
	±SD	1.624
	n	18
G3, F & 300	Mean	2.744
	±SD	3.830
	n	22
G4, F & 1000	Mean	1.649
	±SD	2.343
	n	17

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

 

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

Group & Dose (mg/kg body weight/day)		Brain			Thymus			Spleen
		Absolute Weight (g)			Absolute Weight (g)			Absolute Weight (g)
		Male	Female		Male	Female		Male	Female
G1 & 0	Mean	1.5350	1.5319		0.2050	0.2067		0.2272	0.2307
	±SD	0.0948	0.0818		0.0239	0.0259		0.0200	0.0266
	n	23	21		23	21		23	21
G2 & 100	Mean	1.5300	1.5182		0.2053	0.2046		0.2265	0.2292
	±SD	0.0615	0.0667		0.0217	0.0165		0.0245	0.0247
	n	21	20		21	20		21	20
G3 & 300	Mean	1.5249	1.4606		0.2034	0.1965		0.2312	0.2284
	±SD	0.1121	0.2169		0.0263	0.0265		0.0207	0.0197
	n	22	21		22	21		22	21
G4 & 1000	Mean	1.5015	1.5057		0.1935	0.1827*		0.2239	0.2243
	±SD	0.0667	0.0816		0.0263	0.0275		0.0204	0.0270
	n	20	21		20	21		20	21

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

 

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

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Day
		21	25	28	32	35	42	49	56	63	70	77	84	91
G1-C1A, M & 0	Mean	43.55	61.67	78.16	101.55	120.35	159.05	198.91	236.59	267.50	296.08	323.80	351.83	383.53
	±SD	3.41	6.18	8.33	13.36	18.54	21.93	23.13	28.18	33.32	34.34	29.72	30.54	29.82
	n	20	20	20	20	20	20	20	20	20	20	20	20	20
G2-C1A, M & 100	Mean	44.32	62.36	77.94	99.22	118.42	157.32	196.24	231.03	267.24	295.34	322.64	349.65	379.30
	±SD	3.29	5.39	4.79	11.97	18.57	27.24	31.99	37.98	46.04	51.11	51.15	47.30	43.86
	n	20	20	20	20	20	20	20	20	20	20	20	20	20
G3-C1A, M & 300	Mean	46.05*	64.21	78.72	100.74	120.44	157.91	194.27	234.21	271.34	294.85	321.13	346.23	373.63
	±SD	3.04	5.19	6.35	11.00	14.07	14.65	18.37	24.65	30.50	37.13	33.57	35.52	34.47
	n	20	20	20	20	20	20	20	20	20	20	20	20	20
G4-C1A, M & 1000	Mean	44.12	63.91	76.90	95.70	110.98	151.03	185.20	220.06	250.81	278.13	304.68	332.81	363.89
	±SD	2.79	4.34	3.70	7.91	9.43	18.28	20.21	19.40	24.27	28.43	27.30	25.39	27.93
	n	20	20	20	20	20	20	20	20	20	20	20	20	20

 

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Day
		21	25	28	32	35	42	49	56	63	70	77	84	91
G1-C1A, F & 0	Mean	40.91	55.03	68.84	85.84	99.86	128.94	155.59	175.76	193.81	208.06	221.69	234.61	245.64
	±SD	2.77	4.62	6.10	10.58	13.08	15.37	18.39	19.97	22.61	20.19	17.71	16.60	14.52
	n	20	20	20	20	20	20	20	20	20	20	20	20	20
G2-C1A, F & 100	Mean	41.08	58.27	71.17	91.11	105.23	134.38	158.05	179.32	197.30	212.37	225.84	237.34	247.90
	±SD	2.53	4.61	5.44	9.88	12.61	15.28	16.70	19.16	20.00	21.08	18.33	17.68	16.93
	n	20	20	20	20	20	20	20	20	20	20	20	20	20
G3-C1A, F & 300	Mean	41.80	58.54*	70.60	88.32	103.97	128.99	153.59	173.83	190.10	204.41	218.89	232.38	242.33
	±SD	2.38	4.18	4.96	9.89	12.35	15.64	18.57	21.65	21.51	21.59	19.26	16.78	14.06
	n	20	20	20	20	20	20	20	20	20	20	20	20	20
G4-C1A, F & 1000	Mean	43.14*	59.77*	71.98	88.96	103.81	128.79	151.32	170.75	189.05	205.99	222.40	232.95	243.62
	±SD	2.54	4.44	6.77	10.52	13.35	13.69	16.63	17.71	20.65	18.26	17.86	15.41	15.92
	n	20	20	20	20	20	20	20	20	20	20	20	20	20

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

 

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

Group, Sex & Dose (mg/kg body weight/day)		Feed Consumption on Postnatal Days
		Week 1	Week 2	Week 3	Week 4	Week 5	Week 6	Week 7	Week 8	Week 9	Week 10
G1-C1A, M & 0	Mean	8.10	12.50	16.82	18.40	21.01	23.45	25.96	26.56	27.19	28.45
	±SD	0.72	0.69	0.73	0.84	0.87	1.10	0.36	0.43	0.43	1.05
	n	10	10	10	10	10	10	10	10	10	10
G2-C1A, M & 100	Mean	8.15	12.34	16.42	17.95	20.67	23.40	25.33	26.10	27.04	28.14
	±SD	0.48	0.40	0.53	0.43	0.58	0.87	0.91	0.85	0.48	1.65
	n	10	10	10	10	10	10	10	10	10	10
G3-C1A, M & 300	Mean	7.99	12.43	16.75	18.13	21.08	23.13	25.62	26.37	27.07	27.93
	±SD	0.58	0.62	0.68	0.74	0.68	0.71	0.61	0.57	0.56	1.29
	n	10	10	10	10	10	10	10	10	10	10
G4-C1A, M & 1000	Mean	8.01	12.22	16.40	17.83	20.58	23.32	24.93*	25.87	27.07	28.41
	±SD	0.46	0.47	0.50	0.50	0.52	0.66	0.84	0.70	0.51	1.43
	n	10	10	10	10	10	10	10	10	10	10

 

Group, Sex & Dose (mg/kg body weight/day)		Feed Consumption (g/animal/day) on Postnatal Day
		Week 1	Week 2	Week 3	Week 4	Week 5	Week 6	Week 7	Week 8	Week 9	Week 10
G1-C1A, F & 0	Mean	7.53	11.69	13.71	16.11	18.39	19.45	21.82	22.27	23.08	23.96
	±SD	0.55	0.69	0.61	0.61	0.73	0.87	0.47	0.57	0.62	0.65
	n	10	10	10	10	10	10	10	10	10	10
G2-C1A, F & 100	Mean	7.65	11.47	14.00	16.39	18.58	19.25	21.54	22.36	22.75	23.69
	±SD	0.55	0.64	0.41	0.40	0.83	0.78	0.56	0.68	0.35	0.97
	n	10	10	10	10	10	10	10	10	10	10
G3-C1A, F & 300	Mean	7.37	11.53	13.67	16.17	18.40	19.25	21.70	22.32	23.18	24.01
	±SD	0.46	0.35	0.38	0.45	0.35	0.41	0.47	0.33	0.64	0.82
	n	10	10	10	10	10	10	10	10	10	10
G4-C1A, F & 1000	Mean	7.45	11.67	13.99	16.34	18.49	19.42	21.62	22.46	23.07	25.35
	±SD	0.35	0.40	0.61	0.72	0.73	0.98	0.76	0.72	0.65	2.36
	n	10	10	10	10	10	10	10	10	10	10

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

 

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

Group, Sex & Dose (mg/kg body weight/day)		Serum T4 Levels (ng/mL)
G1-C1A, M & 0	Mean	59.932
	±SD	6.028
	n	10
G2-C1A, M & 100	Mean	61.209
	±SD	4.583
	n	10
G3-C1A, M & 300	Mean	64.605
	±SD	8.325
	n	10
G4-C1A, M & 1000	Mean	64.589
	±SD	5.296
	n	10

 

Group, Sex & Dose (mg/kg body weight/day)		Serum T4 Levels (ng/mL)
G1-C1A, F & 0	Mean	59.489
	±SD	5.723
	n	10
G2-C1A, F & 100	Mean	61.644
	±SD	6.193
	n	10
G3-C1A, F & 300	Mean	60.502
	±SD	3.310
	n	10
G4-C1A, F & 1000	Mean	65.627*
	±SD	5.249
	n	10

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

 

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

Group, Sex & Dose (mg/kg body weight/day)		Serum TSH Levels_Adj. Result (µIU/mL)
G1-C1A, M & 0	Mean	4.474
	±SD	3.683
	n	9
G2-C1A, M & 100	Mean	1.629*
	±SD	1.460
	n	10
G3-C1A, M & 300	Mean	0.757*
	±SD	0.595
	n	8
G4-C1A, M & 1000	Mean	1.339*
	±SD	0.985
	n	8

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

Group, Sex & Dose (mg/kg body weight/day)		Serum TSH Levels (µIU/mL)
G1-C1A, F & 0	Mean	0.605
	±SD	0.549
	n	10
G2-C1A, F & 100	Mean	1.289
	±SD	1.101
	n	10
G3-C1A, F & 300	Mean	1.891
	±SD	2.395
	n	8
G4-C1A, F & 1000	Mean	1.414
	±SD	1.675
	n	10

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

 

Table 23. Summary of haematology record - Cohort 1A

Group, Sex & Dose (mg/kg body weight/day)		Total Leucocyte Count	Total Erythrocyte Count	Haemoglobin	Haematocrit	Platelet Count	Neutrophils	Lymphocytes	Monocytes	Eosinophils	Basophils
		(WBC)	(RBC)	(HGB)	(HCT)	(PLT)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)
		(103 cells/µL)	(106 cells/µL)	(g/dL)	(%)	(103 cells/µL)	(%)	(%)	(%)	(%)	(%)
G1-C1A, M & 0	Mean	9.16	8.35	15.79	47.52	920.56	19.11	75.73	2.36	1.40	0.53
	±SD	1.91	0.48	0.69	2.36	222.23	3.59	4.10	0.40	0.39	0.16
	n	9	9	9	9	9	9	9	9	9	9
G2-C1A, M & 100	Mean	11.43	8.47	15.97	48.17	941.50	21.84	72.42	2.39	2.18	0.36
	±SD	3.67	0.63	0.99	2.29	213.75	6.30	7.15	0.51	1.13	0.07
	n	10	10	10	10	10	10	10	10	10	10
G3-C1A, M & 300	Mean	8.25	8.57	16.03	48.52	862.00	21.93	72.01	2.84	1.75	0.48
	±SD	2.11	0.63	0.81	1.92	220.27	4.92	4.91	1.33	0.87	0.46
	n	10	10	10	10	10	10	10	10	10	10
G4-C1A, M & 1000	Mean	9.49	8.51	16.19	48.38	804.20	20.14	74.58	2.62	1.44	0.44
	±SD	5.24	0.66	1.38	3.84	311.81	3.85	4.23	0.67	0.49	0.24
	n	10	10	10	10	10	10	10	10	10	10

 

Group, Sex & Dose (mg/kg body weight/day)		Absolute Neutrophils	Absolute Lymphocytes	Absolute Monocytes	Absolute Eosinophils	Absolute  Basophils	Prothrombin Time	Activated Prothrombin Time
		(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)	(PT)	(APTT)
		(103 cells/µL)	(103 cells/µL)	(103 cells/µL)	(103 cells/µL)	(103 cells/µL)	(Seconds)	(Seconds)
G1-C1A, M & 0	Mean	1.75	6.94	0.22	0.13	0.05	16.03	24.60
	±SD	0.45	1.52	0.06	0.05	0.01	2.74	5.65
	n	9	9	9	9	9	9	9
G2-C1A, M & 100	Mean	2.53	8.25	0.28	0.24*	0.04	18.21	25.12
	±SD	1.18	2.61	0.11	0.10	0.02	2.58	6.03
	n	10	10	10	10	10	10	10
G3-C1A, M & 300	Mean	1.84	5.92	0.22	0.14	0.04	16.46	30.23
	±SD	0.69	1.52	0.08	0.06	0.04	3.09	9.49
	n	10	10	10	10	10	10	10
G4-C1A, M & 1000	Mean	1.81	7.18	0.24	0.13	0.04	17.28	23.57
	±SD	0.72	4.32	0.12	0.07	0.03	1.86	7.64
	n	10	10	10	10	10	10	10

 

Group, Sex & Dose (mg/kg body weight/day)		Total Leucocyte Count	Total Erythrocyte Count	Haemoglobin	Haematocrit	Platelet Count	Neutrophils	Lymphocytes	Monocytes	Eosinophils	Basophils
		(WBC)	(RBC)	(HGB)	(HCT)	(PLT)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)
		(103 cells/µL)	(106 cells/µL)	(g/dL)	(%)	(103 cells/µL)	(%)	(%)	(%)	(%)	(%)
G1-C1A, F & 0	Mean	6.97	7.88	15.10	44.17	808.40	21.78	72.86	2.52	1.37	0.45
	±SD	1.78	0.70	1.35	3.67	346.96	5.34	6.00	0.82	0.43	0.16
	n	10	10	10	10	10	10	10	10	10	10
G2-C1A, F & 100	Mean	10.04	8.43	15.86	46.88	879.60	18.05	76.68	2.32	1.55	0.40
	±SD	2.85	0.43	1.10	3.20	184.42	3.39	3.92	0.82	0.86	0.14
	n	10	10	10	10	10	10	10	10	10	10
G3-C1A, F & 300	Mean	9.52	8.27	15.32	45.49	1000.78	20.96	74.51	1.88	1.46	0.29
	±SD	3.15	0.39	0.91	2.85	190.62	6.14	6.04	0.38	0.73	0.11
	n	9	9	9	9	9	9	9	9	9	9
G4-C1A, F & 1000	Mean	10.09	7.84	15.48	45.91	847.11	19.74	75.37	2.29	1.47	0.32
	±SD	4.22	0.83	1.58	4.56	144.11	3.84	4.00	0.65	0.42	0.19
	n	9	9	9	9	9	9	9	9	9	9

 

Group, Sex & Dose (mg/kg body weight/day)		Absolute Neutrophils	Absolute Lymphocytes	Absolute Monocytes	Absolute Eosinophils	Absolute  Basophils	Prothrombin Time	Activated Prothrombin Time
		(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)	(PT)	(APTT)
		(103 cells/µL)	(103 cells/µL)	(103 cells/µL)	(103 cells/µL)	(103 cells/µL)	(Seconds)	(Seconds)
G1-C1A, F & 0	Mean	1.50	5.09	0.18	0.10	0.03	17.07	30.47
	±SD	0.56	1.41	0.08	0.04	0.01	2.11	12.08
	n	10	10	10	10	10	10	10
G2-C1A, F & 100	Mean	1.77	7.75*	0.23	0.16	0.04	17.41	19.48*
	±SD	0.44	2.42	0.09	0.09	0.01	1.68	6.73
	n	10	10	10	10	10	10	10
G3-C1A, F & 300	Mean	1.96	7.12	0.18	0.14	0.03	15.87	25.69
	±SD	0.84	2.50	0.09	0.08	0.02	1.83	7.96
	n	9	9	9	9	9	9	9
G4-C1A, F & 1000	Mean	2.07	7.52	0.23	0.15	0.03	16.80	25.77
	±SD	1.28	2.81	0.10	0.07	0.02	2.18	5.00
	n	9	9	9	9	9	9	9

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

 

Table 24. Summary of urinalysis record - Cohort 1A

Examination	Group, Sex & Dose (mg/kg body weight/day)		G1-C1A, M & 0	G2-C1A, M & 100	G3-C1A, M & 300	G4-C1A, M & 1000
	Number of Animals		9	10	10	10
Physical Examination	Colour	Pale Yellow	4	5	7	8
		Yellow	5	5	3	2
	Appearance	Clear	3	2	5	4
		Turbid	6	8	5	6
	Volume (mL)	Mean	6.0	5.8	7.2	5.9
		±SD	2.0	1.8	1.9	1.9
Chemical Examination	pH	Mean	7.8	7.8	7.8	7.7
		±SD	0.6	0.3	0.3	0.4
	Specific gravity	Mean	1.008	1.007	1.009	1.009
		±SD	0.003	0.003	0.002	0.003
	Urobilinogen             (mg/dL)	Mean	1.0	0.5	0.5	0.3
		±SD	1.3	0.4	0.6	0.3
	Bilirubin (mg/dL)	Neg	3	3	5	6
		1	4	7	4	4
	Ketones (mg/dL)	Neg	6	6	7	4
		5	3	4	3	6
		15	-	-	-	-
	Blood (Ery/µL)	Neg	6	7	6	6
		Ca10	-	1	2	1
		Ca25	2	2	-	2
		Ca80	-	-	1	-
		>=Ca200	1	-	1	1
	Protein  (mg/dL)	Neg	2	-	3	2
		Trace	1	1	2	2
		30	1	4	1	2
		100	-	1	-	1
		>=300	5	4	4	3
	Nitrite	Neg	7	7	9	7
		Pos	2	3	1	3
	Leucocytes (Leu/µL)	Neg	-	-	1	-
		Ca15	3	1	1	3
		Ca70	2	6	2	3
		Ca125	4	3	6	3
	Glucose  (mg/dL)	Neg	9	10	10	10
	Micro Albumin  (mg/dL)	Neg	1	-	1	1
		>15	7	10	7	8
		15	1	-	2	1
Microscopic Examination	Epi Cells	0	8	8	6	7
		0-1	-	2	1	2
		1-2	1	-	3	-
		2-3	-	-	-	1
	Casts	Absent	8	10	9	8
		Present	1	-	1	2
	Crystals	Absent	-	-	-	-
		Present	9	10	10	10

 

Examination	Group, Sex & Dose (mg/kg body weight/day)		G1-C1A, F & 0	G2-C1A, F & 100	G3-C1A, F & 300	G4-C1A, F & 1000
	Number of Animals		10	10	9	9
Physical Examination	Colour	Pale Yellow	7	5	7	5
		Yellow	3	5	2	4
	Appearance	Clear	2	4	6	1
		Turbid	8	6	3	8
	Volume (mL)	Mean	7.0	4.9*	6.2	5.8
		±SD	2.2	1.6	1.2	1.5
Chemical Examination	pH	Mean	8.0	7.7	7.6	7.9
		±SD	0.4	0.5	0.2	0.3
	Specific gravity	Mean	1.008	1.009	1.008	1.007
		±SD	0.003	0.004	0.003	0.003
	Urobilinogen (mg/dL)	Mean	0.6	0.5	0.3	0.5
		±SD	0.8	0.6	0.3	0.6
	Bilirubin (mg/dL)	Neg	5	2	6	6
		1	2	8	3	3
	Ketones (mg/dL)	Neg	7	8	6	8
		5	3	2	3	1
		15	-	-	-	-
	Blood (Ery/µL)	Neg	7	9	5	8
		Ca10	1	1	2	1
		Ca25	1	-	2	-
		Ca80	-	-	-	-
		>=Ca200	1	-	-	-
	Protein (mg/dL)	Neg	2	2	4	-
		Trace	-	2	1	1
		30	1	1	1	6
		100	1	-	1	-
		>=300	6	5	2	2
	Nitrite (NIT)	Neg	7	8	5	8
		Pos	3	2	4	1
	Leucocytes (Leu/µL)	Neg	1	-	1	-
		Ca15	-	3	-	-
		Ca70	6	5	5	6
		Ca125	3	2	3	3
	Glucose (mg/dL)	Neg	10	10	9	9
	Micro Albumin (mg/dL)	Neg	-	-	1	-
		>15	8	8	5	9
		15	2	2	3	-
Microscopic Examination	Epi Cells	0	7	5	6	6
		0-1	-	2	-	2
		1-2	2	3	2	1
		2-3	1	-	1	-
	Casts	Absent	9	10	6	9
		Present	1	-	3	-
	Crystals	Absent	-	-	-	-
		Present	10	10	9	9

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

 

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

Group, Sex & Dose (mg/kg body weight/day)		Adrenals	Thymus	Spleen	Testes	Epididymides	Heart	Kidneys	Brain
G1-C1A, M & 0	Mean	0.0521	0.2671	1.0730	3.3278	1.3713	1.3195	2.3746	1.9340
	±SD	0.0066	0.0296	0.1587	0.1459	0.0650	0.2154	0.2800	0.1082
	n	20	20	20	20	20	20	20	20
G2-C1A, M & 100	Mean	0.0545	0.2700	1.0677	3.3446	1.3632	1.3937	2.5491	1.9740
	±SD	0.0074	0.0564	0.1221	0.2333	0.0842	0.3014	0.2720	0.1125
	n	20	20	20	20	20	20	20	20
G3-C1A, M & 300	Mean	0.0510	0.2572	0.9012*	3.2638	1.3167	1.3179	2.3903	1.9637
	±SD	0.0068	0.0347	0.1886	0.1961	0.0881	0.1934	0.3209	0.0918
	n	20	20	20	20	20	20	20	20
G4-C1A, M & 1000	Mean	0.0537	0.2521	1.0889	3.2345	1.3463	1.3003	2.2548	1.9120
	±SD	0.0071	0.0314	0.1480	0.1466	0.0749	0.2759	0.2329	0.0878
	n	20	20	20	20	20	20	20	20

 

Group, Sex & Dose (mg/kg body weight/day)		Liver	Prostate	Seminal vesicles with coagulating glands	Iliac lymph node	Mandibular lymph node	Pituitary^WP	Thyroid along with parathyroid^WP
G1-C1A, M & 0	Mean	10.6384	0.9206	1.1578	0.0399	0.1361	0.0169	0.0275
	±SD	1.4863	0.1193	0.2353	0.0055	0.0147	0.0011	0.0014
	n	20	20	20	20	20	20	20
G2-C1A, M & 100	Mean	10.8144	0.9813	1.2102	0.0423	0.1350	0.0174	0.0278
	±SD	1.6681	0.0786	0.2007	0.0102	0.0167	0.0019	0.0020
	n	20	20	20	20	20	20	20
G3-C1A, M & 300	Mean	10.4807	1.0027	1.1563	0.0382	0.1295	0.0176	0.0271
	±SD	1.8164	0.1531	0.2624	0.0073	0.0116	0.0019	0.0016
	n	20	20	20	20	20	20	20
G4-C1A, M & 1000	Mean	9.7751	0.9948	1.1467	0.0371	0.1414	0.0174	0.0277
	±SD	0.8505	0.1001	0.2035	0.0058	0.0294	0.0014	0.0015
	n	20	20	20	20	20	20	20

 

Group, Sex & Dose (mg/kg body weight/day)		Adrenals	Thymus	Spleen	Ovaries	Uterus with Cervix	Heart	Kidneys
G1-C1A, F & 0	Mean	0.0600	0.2606	0.9194	0.1322	0.6176	0.9939	1.6052
	±SD	0.0051	0.0326	0.1761	0.0166	0.1140	0.1062	0.1619
	n	20	20	20	20	20	20	20
G2-C1A, F & 100	Mean	0.0598	0.2752	0.8742	0.1244	0.6067	1.0463	1.7637*
	±SD	0.0083	0.0513	0.2133	0.0124	0.1212	0.0967	0.2534
	n	20	20	20	20	20	20	20
G3-C1A, F & 300	Mean	0.0601	0.2599	0.6836*	0.1105*	0.6075	0.9709	1.6528
	±SD	0.0071	0.0401	0.1612	0.0079	0.1078	0.0988	0.2217
	n	20	20	20	20	20	20	20
G4-C1A, F & 1000	Mean	0.0578	0.2517	0.7883*	0.1229	0.5958	1.0611	1.6802
	±SD	0.0068	0.0335	0.0921	0.0113	0.1273	0.0822	0.1826
	n	20	20	20	20	20	20	20

 

Group, Sex & Dose (mg/kg body weight/day)		Brain	Liver	Iliac lymph node	Mandibular lymph node	Pituitary^WP	Thyroid along with parathyroid^WP
G1-C1A, F & 0	Mean	1.8173	8.2701	0.0377	0.1053	0.0144	0.0233
	±SD	0.1763	1.3064	0.0087	0.0161	0.0018	0.0027
	n	20	20	20	20	20	20
G2-C1A, F & 100	Mean	1.8609	8.5602	0.0406	0.1089	0.0150	0.0225
	±SD	0.1182	1.1930	0.0099	0.0220	0.0019	0.0026
	n	20	20	20	20	20	20
G3-C1A, F & 300	Mean	1.8188	8.0708	0.0395	0.1016	0.0141	0.0227
	±SD	0.0801	1.0957	0.0088	0.0127	0.0020	0.0021
	n	20	20	20	20	20	20
G4-C1A, F & 1000	Mean	1.8458	8.8116	0.0420	0.1116	0.0143	0.0236
	±SD	0.0604	0.8629	0.0093	0.0187	0.0015	0.0024
	n	20	20	20	20	20	20

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

 

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

Group, Sex & Dose (mg/kg body weight/day)		Terminal Body Weight (g)	Adrenals	Thymus	Spleen	Testes	Epididymides	Heart	Kidneys
G1-C1A, M & 0	Mean	366.84	0.0143	0.0732	0.2945	0.9132	0.3762	0.3590	0.6491
	±SD	29.48	0.0019	0.0099	0.0512	0.0905	0.0361	0.0439	0.0756
	n	20	20	20	20	20	20	20	20
G2-C1A, M & 100	Mean	363.59	0.0151	0.0750	0.2980	0.9299	0.3793	0.3851	0.7059
	±SD	42.97	0.0024	0.0168	0.0498	0.1078	0.0454	0.0823	0.0791
	n	20	20	20	20	20	20	20	20
G3-C1A, M & 300	Mean	358.18	0.0144	0.0726	0.2537*	0.9202	0.3706	0.3693	0.6711
	±SD	34.35	0.0024	0.0134	0.0598	0.1102	0.0407	0.0539	0.0946
	n	20	20	20	20	20	20	20	20
G4-C1A, M & 1000	Mean	348.89	0.0155	0.0726	0.3141	0.9311	0.3878	0.3718	0.6484
	±SD	27.55	0.0023	0.0101	0.0509	0.0659	0.0341	0.0705	0.0688
	n	20	20	20	20	20	20	20	20

 

Group, Sex & Dose (mg/kg body weight/day)		Brain	Liver	Prostate	Seminal vesicles with coagulating glands	Iliac lymph node	Mandibular lymph node	Pituitary	Thyroid along with parathyroid
G1-C1A, M & 0	Mean	0.5293	2.9049	0.2516	0.3162	0.0109	0.0373	0.0046	0.0075
	±SD	0.0366	0.3814	0.0302	0.0619	0.0017	0.0042	0.0002	0.0004
	n	20	20	20	20	20	20	20	20
G2-C1A, M & 100	Mean	0.5485	2.9919	0.2729	0.3352	0.0118	0.0376	0.0048*	0.0077
	±SD	0.0578	0.4341	0.0343	0.0567	0.0033	0.0069	0.0002	0.0005
	n	20	20	20	20	20	20	20	20
G3-C1A, M & 300	Mean	0.5532	2.9364	0.2828*	0.3255	0.0107	0.0366	0.0049*	0.0076
	±SD	0.0609	0.4810	0.0539	0.0750	0.0020	0.0052	0.0002	0.0005
	n	20	20	20	20	20	20	20	20
G4-C1A, M & 1000	Mean	0.5502	2.8068	0.2867*	0.3290	0.0107	0.0408	0.0050*	0.0080*
	±SD	0.0359	0.2021	0.0366	0.0539	0.0017	0.0091	0.0002	0.0004
	n	20	20	20	20	20	20	20	20

 

Group, Sex & Dose (mg/kg body weight/day)		Terminal Body Weight (g)	Adrenals	Thymus	Spleen	Ovaries	Uterus with Cervix	Heart
G1-C1A, F & 0	Mean	233.44	0.0257	0.1118	0.3944	0.0568	0.2649	0.4261
	±SD	14.25	0.0020	0.0136	0.0744	0.0080	0.0474	0.0412
	n	20	20	20	20	20	20	20
G2-C1A, F & 100	Mean	236.08	0.0254	0.1168	0.3707	0.0529	0.2576	0.4440
	±SD	16.02	0.0034	0.0216	0.0883	0.0059	0.0521	0.0403
	n	20	20	20	20	20	20	20
G3-C1A, F & 300	Mean	229.79	0.0262	0.1132	0.2989*	0.0482*	0.2641	0.4236
	±SD	13.91	0.0034	0.0170	0.0760	0.0044	0.0416	0.0470
	n	20	20	20	20	20	20	20
G4-C1A, F & 1000	Mean	230.71	0.0252	0.1093	0.3424	0.0534	0.2608	0.4616*
	±SD	15.78	0.0035	0.0147	0.0406	0.0050	0.0643	0.0437
	n	20	20	20	20	20	20	20

 

Group, Sex & Dose (mg/kg body weight/day)		Kidneys	Brain	Liver	Iliac lymph node	Mandibular lymph node	Pituitary	Thyroid along with parathyroid
G1-C1A, F & 0	Mean	0.6887	0.7803	3.5478	0.0162	0.0454	0.0062	0.0100
	±SD	0.0683	0.0810	0.5485	0.0039	0.0079	0.0005	0.0009
	n	20	20	20	20	20	20	20
G2-C1A, F & 100	Mean	0.7502	0.7907	3.6257	0.0173	0.0463	0.0063	0.0095
	±SD	0.1217	0.0637	0.4241	0.0043	0.0097	0.0005	0.0008
	n	20	20	20	20	20	20	20
G3-C1A, F & 300	Mean	0.7205	0.7947	3.5154	0.0173	0.0443	0.0061	0.0099
	±SD	0.0990	0.0645	0.4427	0.0040	0.0058	0.0006	0.0005
	n	20	20	20	20	20	20	20
G4-C1A, F & 1000	Mean	0.7310	0.8033	3.8322	0.0183	0.0485	0.0062	0.0102
	±SD	0.0913	0.0572	0.4196	0.0043	0.0085	0.0003	0.0005
	n	20	20	20	20	20	20	20

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

 

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

Group, Sex & Dose (mg/kg body weight/day)		Percent of Splenic lymphocyte Parental Sub-populations (%)
		B Lymphocytes	T Helper Cells	T Cytotoxic Cells	Natural Killer Cells
		(B Cells)	(Th/CD4+)	(Tc/CD8+)	(NK Cells)
G1-C1A, M & 0	Mean	37.57	51.88	45.37	12.71
	±SD	2.29	9.07	8.65	3.51
	n	10	10	10	10
G2-C1A, M & 100	Mean	37.85	50.23	45.46	12.12
	±SD	4.47	1.30	5.66	3.08
	n	10	10	10	10
G3-C1A, M & 300	Mean	37.35	50.44	45.44	13.10
	±SD	2.55	3.27	6.36	3.63
	n	10	10	10	10
G4-C1A, M & 1000	Mean	37.98	54.99	42.20	12.22
	±SD	2.63	15.25	14.93	3.92
	n	10	10	10	10

 

Group, Sex & Dose (mg/kg body weight/day)		Percent of Splenic lymphocyte Parental Sub-populations (%)
		B Lymphocytes	T Helper Cells	T Cytotoxic Cells	Natural Killer Cells
		(B Cells)	(Th/CD4+)	(Tc/CD8+)	(NK Cells)
G1-C1A, F & 0	Mean	38.84	52.86	43.73	8.77
	±SD	7.60	1.71	1.58	2.28
	n	10	10	10	10
G2-C1A, F & 100	Mean	40.64	53.91	41.99*	8.33
	±SD	5.02	1.93	1.95	1.08
	n	10	10	10	10
G3-C1A, F & 300	Mean	37.76	54.77*	41.00*	9.82
	±SD	10.42	1.29	1.04	6.34
	n	10	10	10	10
G4-C1A, F & 1000	Mean	40.52	53.68	42.26	9.25
	±SD	7.18	1.36	1.38	5.40
	n	10	10	10	10

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

 

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

Group, Sex  & Dose (mg/kg body weight/day)		Mean Percent of Sperm Motility
G1-C1A, M & 0	Mean	90.9
	±SD	0.9
	n	20
G2-C1A, M & 100	Mean	91.0
	±SD	1.1
	n	20
G3-C1A, M & 300	Mean	90.8
	±SD	1.1
	n	20
G4-C1A, M & 1000	Mean	91.1
	±SD	1.0
	n	20

 

Table 29. Summary record of sperm morphology - Cohort 1A

Group, Sex & Dose (mg/kg body weight/day)		Head Abnormalities		Neck Abnormalities		Tail Abnormalities		Multiple Abnormalities			Sperms with Abnormality			Normal Sperms
		Total	%	Total	%	Total	%	Total	%		No.	%		No.	%
G1-C1A, M & 0	Mean	2.05	1.03	2.10	1.05	1.40	0.70	1.00	0.50		6.55	3.28		193.45	96.73
	±SD	1.15	0.57	1.62	0.81	1.27	0.64	0.97	0.49		1.32	0.66		1.32	0.66
	n	20	20	20	20	20	20	20	20		20	20		20	20
G2-C1A, M & 100	Mean	3.20*	1.60*	2.10	1.05	1.15	0.58	0.50	0.25		6.95	3.48		193.05	96.53
	±SD	1.40	0.70	1.25	0.63	1.04	0.52	0.69	0.34		1.57	0.79		1.57	0.79
	n	20	20	20	20	20	20	20	20		20	20		20	20
G3-C1A, M & 300	Mean	2.95	1.48	3.45*	1.73*	1.55	0.78	0.30*	0.15*		8.25*	4.13*		191.75*	95.88*
	±SD	1.61	0.80	1.28	0.64	1.28	0.64	0.47	0.24		1.29	0.65		1.29	0.65
	n	20	20	20	20	20	20	20	20		20	20		20	20
G4-C1A, M & 1000	Mean	2.50	1.25	2.75	1.38	1.40	0.70	0.60	0.30		7.25	3.63		192.75	96.38
	±SD	1.24	0.62	1.29	0.65	0.82	0.41	0.68	0.34		1.37	0.69		1.37	0.69
	n	20	20	20	20	20	20	20	20		20	20		20	20

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

 

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

Group, Sex & Dose (mg/kg body weight/day)		Average Sperm head count	Daily Sperm Production (x 10^6 per gram of Testis)	Daily Sperm Production (x10^6 per Testis)
G1-C1A, M & 0	Mean	182.00	14.8	51.9
	±SD	9.82	0.8	5.0
	n	20	20	20
G2-C1A, M & 100	Mean	178.44	14.5	51.0
	±SD	5.97	0.5	4.9
	n	20	20	20
G3-C1A, M & 300	Mean	182.36	14.9	52.4
	±SD	8.01	0.7	4.6
	n	20	20	20
G4-C1A, M & 1000	Mean	177.98	14.5	50.5
	±SD	7.71	0.6	4.5
	n	20	20	20

 

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

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Day
		21	25	28	32	35	42	49	56	63	70	77	84	91	98
G1-C1B, M & 0	Mean	43.82	62.04	77.25	98.26	118.07	153.98	190.70	225.15	263.80	295.17	321.65	353.98	381.14	407.28
	±SD	3.26	4.63	6.11	10.58	15.03	19.02	21.72	28.29	30.25	33.20	30.02	33.60	32.74	32.86
	n	20	20	20	20	20	20	20	20	20	20	20	20	20	20
G2-C1B, M & 100	Mean	44.64	62.80	75.14	91.25	106.32*	142.62	178.68	209.79	245.15	273.64	299.75	332.50	358.60	382.63
	±SD	3.47	4.40	6.45	7.97	9.03	12.74	16.33	21.04	25.45	30.48	34.25	29.23	28.02	29.32
	n	20	20	20	20	20	20	20	20	20	20	20	20	20	20
G3-C1B, M & 300	Mean	45.16	65.12	78.09	96.69	114.31	144.64	177.54	213.06	244.97	272.82	298.26	328.94	353.68*	377.12*
	±SD	3.82	5.19	6.58	11.15	14.55	19.74	22.81	28.41	35.61	40.66	41.78	44.44	42.05	44.76
	n	20	20	20	20	20	20	20	20	20	20	20	20	20	20
G4-C1B, M & 1000	Mean	45.59	63.03	74.23	91.54	108.43	139.42*	166.95*	191.84*	224.37*	251.57*	279.23*	309.07*	336.26*	356.85*
	±SD	3.82	5.58	6.25	9.67	11.57	18.79	21.02	24.77	31.94	37.00	36.29	33.89	32.51	31.50
	n	20	20	20	20	20	20	20	20	20	20	20	20	20	20

 

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Day
		21	25	28	32	35	42	49	56	63	70	77	84	91	98
G1-C1B, F & 0	Mean	40.06	56.35	70.87	93.49	108.55	138.26	163.53	184.76	203.97	218.34	231.18	242.72	253.25	263.78
	±SD	1.89	3.49	5.60	12.26	15.86	18.11	20.70	21.82	22.31	20.61	16.60	14.96	15.67	17.15
	n	20	20	20	20	20	20	20	20	20	20	20	20	20	20
G2-C1B, F & 100	Mean	41.86	58.42	70.50	89.22	103.18	132.10	153.89	174.67	192.28	208.20	220.68	234.02	243.95	254.58
	±SD	3.08	3.61	7.05	11.26	12.14	16.72	20.28	21.33	22.10	23.46	21.71	19.89	18.84	18.51
	n	20	20	20	20	20	20	20	20	20	20	20	20	20	20
G3-C1B, F & 300	Mean	42.20*	58.86	72.24	92.63	105.53	132.84	154.49	174.59	191.49	204.96	219.03	232.66	245.03	254.42
	±SD	3.09	3.68	3.78	8.36	9.88	13.08	15.79	19.66	24.04	22.53	18.63	18.57	16.95	18.56
	n	20	20	20	20	20	20	20	20	20	20	20	20	20	20
G4-C1B, F & 1000	Mean	42.49*	59.78*	71.19	86.06	98.91*	126.87*	150.04*	170.14*	189.07	204.67	221.26	232.90	243.69	254.08
	±SD	2.48	2.92	4.73	6.64	8.93	10.73	11.04	12.94	14.19	14.91	12.41	12.24	10.37	11.79
	n	20	20	20	20	20	20	20	20	20	20	20	20	20	20

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

 

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

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight (%) during Day
		21 to 25	21 to 28	21 to 32	21 to 35	21 to 42	21 to 49	21 to 56	21 to 63	21 to 70	21 to 77	21 to 84	21 to 91	21 to 98
G1-C1B, M & 0	Mean	41.84	77.00	125.48	171.03	254.52	338.43	417.53	505.52	577.60	637.90	711.75	773.91	834.07
	±SD	9.34	17.44	30.23	41.62	58.95	66.06	82.13	85.56	94.20	87.46	94.05	93.92	99.54
	n	20	20	20	20	20	20	20	20	20	20	20	20	20
G2-C1B, M & 100	Mean	41.24	69.16	105.60	139.66*	221.41	302.45	373.22	453.51	518.40	577.07	650.36	709.42	763.28
	±SD	11.86	17.79	23.54	27.71	37.71	46.41	63.84	81.80	98.85	106.33	98.37	102.54	104.98
	n	20	20	20	20	20	20	20	20	20	20	20	20	20
G3-C1B, M & 300	Mean	44.47	73.63	115.74	155.43	223.20	296.83	376.74	448.93	511.87	569.02	637.46	692.53*	744.77*
	±SD	8.03	16.22	32.46	43.34	57.76	69.08	87.91	110.51	127.75	134.34	144.15	144.21	150.34
	n	20	20	20	20	20	20	20	20	20	20	20	20	20
G4-C1B, M & 1000	Mean	38.53	63.50*	102.04*	139.15*	206.77*	266.97*	321.15*	392.11*	451.90*	513.36*	579.75*	639.65*	685.47*
	±SD	10.30	15.89	26.90	30.50	41.65	42.90	45.88	56.40	67.73	69.65	70.01	67.36	70.22
	n	20	20	20	20	20	20	20	20	20	20	20	20	20

 

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight (%) during Day
		21 to 25	21 to 28	21 to 32	21 to 35	21 to 42	21 to 49	21 to 56	21 to 63	21 to 70	21 to 77	21 to 84	21 to 91	21 to 98
G1-C1B, F & 0	Mean	40.80	77.19	133.92	171.69	246.10	309.39	362.58	410.58	446.55	478.42	507.52	533.81	560.08
	±SD	8.72	14.84	32.68	42.13	49.29	57.07	60.98	63.04	60.37	50.66	51.02	52.87	55.65
	n	20	20	20	20	20	20	20	20	20	20	20	20	20
G2-C1B, F & 100	Mean	39.85	68.87	113.88	147.51	217.12	269.63*	319.53*	361.91*	399.87	429.70*	461.95*	485.75*	511.04*
	±SD	7.65	16.83	27.61	31.24	44.59	55.01	58.70	62.33	64.68	61.91	62.15	61.63	60.83
	n	20	20	20	20	20	20	20	20	20	20	20	20	20
G3-C1B, F & 300	Mean	39.82	71.89	120.63	151.43	216.60	268.36*	316.55*	357.08*	389.01*	422.02*	454.70*	484.11*	506.45*
	±SD	8.65	13.83	26.13	30.89	40.72	49.48	61.25	73.22	71.25	61.93	65.63	64.13	67.75
	n	20	20	20	20	20	20	20	20	20	20	20	20	20
G4-C1B, F & 1000	Mean	40.91	67.88	103.27*	133.54*	199.64*	254.64*	302.07*	346.80*	383.86*	423.14*	450.78*	476.11*	500.66*
	±SD	6.48	12.37	20.82	26.00	32.63	38.17	42.67	47.07	51.52	50.80	53.32	51.29	54.55
	n	20	20	20	20	20	20	20	20	20	20	20	20	20

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

 

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

Group, Sex & Dose (mg/kg body weight/day)		Week 1	Week	Week 3	Week 4	Week 5	Week 6	Week 7	Week 8	Week 9	Week 10	Week 11
G1-C1B, M & 0	Mean	8.16	12.38	16.54	17.93	20.58	23.07	25.55	26.18	27.18	27.56	29.10
	±SD	0.55	0.38	0.25	0.44	0.88	0.84	0.69	0.56	0.41	0.49	1.32
	n	20	20	20	20	20	20	20	20	20	20	20
G2-C1B, M & 100	Mean	8.27	12.29	16.74	18.14	20.91	23.38	25.30	26.12	26.91	27.68	28.93
	±SD	0.72	0.53	0.60	0.57	0.60	0.48	1.05	0.62	0.56	0.78	1.16
	n	20	20	20	20	20	20	20	20	20	20	20
G3-C1B, M & 300	Mean	8.16	12.46	16.62	17.86	20.57	22.71	24.86	25.70	26.60	27.24	28.93
	±SD	0.66	0.56	0.61	0.72	0.58	0.88	1.36	1.46	0.85	1.01	1.48
	n	20	20	20	20	20	20	20	20	20	20	20
G4-C1B, M & 1000	Mean	7.88	11.99	16.17	17.59	20.49	23.13	25.43	26.27	27.17	27.50	29.80
	±SD	0.71	0.74	0.60	0.56	0.50	0.86	1.30	1.21	1.02	1.23	1.89
	n	20	20	20	20	20	20	20	20	20	20	20

 

Group, Sex & Dose (mg/kg body weight/day)		Week 1	Week	Week 3	Week 4	Week 5	Week 6	Week 7	Week 8	Week 9	Week 10	Week 11
G1-C1B, F & 0	Mean	7.54	11.63	13.81	16.22	18.55	19.20	21.93	22.34	23.19	23.10	24.53
	±SD	0.66	0.76	0.67	0.90	0.78	0.79	0.60	0.53	0.59	0.75	0.64
	n	10	10	10	10	10	10	10	10	10	10	10
G2-C1B, F & 100	Mean	7.03	11.06	13.61	15.76	17.96	18.70	21.06*	21.86	22.24*	23.05	23.95
	±SD	0.52	0.58	0.94	0.77	0.84	0.58	0.85	0.71	0.54	0.74	0.75
	n	10	10	10	10	10	10	10	10	10	10	10
G3-C1B, F & 300	Mean	7.43	11.47	13.75	15.92	18.22	18.92	21.49	22.04	22.65	23.49	24.37
	±SD	0.47	0.66	0.42	0.50	0.56	0.55	0.78	0.63	0.58	0.71	0.99
	n	10	10	10	10	10	10	10	10	10	10	10
G4-C1B, F & 1000	Mean	7.33	11.36	13.82	16.18	18.29	19.27	21.82	22.43	23.10	23.51	24.93
	±SD	0.43	0.66	0.70	0.75	0.79	0.82	0.59	0.51	0.79	0.78	1.02
	n	10	10	10	10	10	10	10	10	10	10	9

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

 

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

Group, Sex & Dose (mg/kg body weight/day)		Adrenals	Thymus	Spleen	Testes	Epididymides
G1-C1B, M & 0	Mean	0.0559	0.3412	1.1190	3.2847	1.2667
	±SD	0.0063	0.0657	0.1494	0.1994	0.0967
	n	20	20	20	20	20
G2-C1B, M & 100	Mean	0.0506	0.2893*	1.1247	3.2425	1.2746
	±SD	0.0096	0.0592	0.1433	0.1452	0.1196
	n	20	20	20	20	20
G3-C1B, M & 300	Mean	0.0513	0.2840*	1.0185	3.1528	1.2425
	±SD	0.0069	0.0495	0.1718	0.1810	0.1167
	n	20	20	20	20	20
G4-C1B, M & 1000	Mean	0.0548	0.3333	0.9660*	3.2165	1.2399
	±SD	0.0078	0.0687	0.1702	0.2276	0.1311
	n	20	20	20	20	20

 

Group, Sex & Dose (mg/kg body weight/day)		Iliac lymph node	Mandibular lymph node	Prostate	Seminal vesicles with coagulating glands	Pituitary^WP	Thyroid along with parathyroid^WP
G1-C1B, M & 0	Mean	0.0325	0.2341	1.0201	1.1953	0.0178	0.0282
	±SD	0.0069	0.0329	0.1281	0.1446	0.0014	0.0011
	n	20	20	20	20	20	20
G2-C1B, M & 100	Mean	0.0329	0.2278	0.9853	1.2019	0.0172	0.0277
	±SD	0.0071	0.0466	0.1098	0.1530	0.0018	0.0014
	n	20	20	20	20	20	20
G3-C1B, M & 300	Mean	0.0340	0.1672*	0.8910*	1.2248	0.0173	0.0280
	±SD	0.0088	0.0354	0.1587	0.2050	0.0018	0.0013
	n	20	20	20	20	20	20
G4-C1B, M & 1000	Mean	0.0385	0.1934*	0.9091*	1.1675	0.0176	0.0273
	±SD	0.0115	0.0272	0.1314	0.1901	0.0016	0.0011
	n	20	20	20	20	20	20

 

Group, Sex & Dose (mg/kg body weight/day)		Adrenals	Thymus	Spleen	Ovaries	Uterus with Cervix	Iliac lymph node	Mandibular lymph node	Pituitary^WP	Thyroid along with parathyroid^WP
G1-C1B, F & 0	Mean	0.0547	0.2931	0.8804	0.1050	0.5904	0.0280	0.2236	0.0158	0.0244
	±SD	0.0103	0.0386	0.1630	0.0168	0.0919	0.0062	0.0236	0.0011	0.0024
	n	20	20	20	20	20	20	20	20	20
G2-C1B, F & 100	Mean	0.0582	0.2235*	0.9592	0.1236*	0.5654	0.0320	0.1652*	0.0161	0.0246
	±SD	0.0070	0.0438	0.0692	0.0223	0.1116	0.0057	0.0403	0.0016	0.0025
	n	20	20	20	20	20	20	20	20	20
G3-C1B, F & 300	Mean	0.0624*	0.3224	0.9337	0.1127	0.5303	0.0339*	0.1354*	0.0164	0.0248
	±SD	0.0075	0.0410	0.1016	0.0124	0.0558	0.0069	0.0229	0.0017	0.0026
	n	20	20	20	20	20	20	20	20	20
G4-C1B, F & 1000	Mean	0.0618*	0.2673	0.8993	0.1042	0.6006	0.0339*	0.1834*	0.0159	0.0242
	±SD	0.0101	0.0519	0.1666	0.0167	0.0857	0.0076	0.0438	0.0012	0.0024
	n	20	20	20	20	20	20	20	20	20

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

 

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

Group, Sex & Dose (mg/kg body weight/day)		Terminal Body Weight (g)	Adrenals	Thymus	Spleen	Testes	Epididymides
G1-C1B, M & 0	Mean	386.77	0.0145	0.0883	0.2924	0.8563	0.3300
	±SD	32.02	0.0021	0.0154	0.0532	0.1008	0.0411
	n	20	20	20	20	20	20
G2-C1B, M & 100	Mean	365.67	0.0139	0.0798	0.3101	0.8926	0.3513
	±SD	29.28	0.0028	0.0187	0.0509	0.0856	0.0481
	n	20	20	20	20	20	20
G3-C1B, M & 300	Mean	359.17*	0.0144	0.0799	0.2863	0.8917	0.3498
	±SD	45.63	0.0022	0.0147	0.0501	0.1280	0.0443
	n	20	20	20	20	20	20
G4-C1B, M & 1000	Mean	341.18*	0.0162	0.0983	0.2835	0.9495*	0.3655*
	±SD	32.23	0.0025	0.0215	0.0450	0.0990	0.0433
	n	20	20	20	20	20	20

 

Group, Sex & Dose (mg/kg body weight/day)		Iliac lymph node	Mandibular lymph node	Prostate	Seminal vesicles with coagulating glands	Pituitary	Thyroid along with parathyroid
G1-C1B, M & 0	Mean	0.0085	0.0609	0.2662	0.3105	0.0046	0.0073
	±SD	0.0020	0.0099	0.0459	0.0398	0.0002	0.0004
	n	20	20	20	20	20	20
G2-C1B, M & 100	Mean	0.0091	0.0623	0.2705	0.3309	0.0047	0.0076
	±SD	0.0024	0.0115	0.0322	0.0514	0.0002	0.0004
	n	20	20	20	20	20	20
G3-C1B, M & 300	Mean	0.0094	0.0471*	0.2500	0.3494	0.0048*	0.0079*
	±SD	0.0020	0.0106	0.0453	0.0897	0.0003	0.0008
	n	20	20	20	20	20	20
G4-C1B, M & 1000	Mean	0.0114*	0.0572	0.2683	0.3449	0.0052*	0.0080*
	±SD	0.0036	0.0099	0.0442	0.0628	0.0002	0.0005
	n	20	20	20	20	20	20

 

Group, Sex & Dose (mg/kg body weight/day)		Terminal Body Weight (g)	Adrenals	Thymus	Spleen	Ovaries	Uterus with Cervix	Iliac lymph node	Mandibular lymph node	Pituitary	Thyroid along with parathyroid
G1-C1B, F & 0	Mean	249.90	0.0219	0.1179	0.3550	0.0422	0.2380	0.0112	0.0897	0.0063	0.0098
	±SD	18.04	0.0040	0.0174	0.0748	0.0072	0.0446	0.0025	0.0097	0.0003	0.0011
	n	20	20	20	20	20	20	20	20	20	20
G2-C1B, F & 100	Mean	240.88	0.0243	0.0934*	0.4002	0.0516*	0.2363	0.0134	0.0691*	0.0067*	0.0102
	±SD	18.94	0.0035	0.0195	0.0400	0.0105	0.0503	0.0027	0.0181	0.0004	0.0010
	n	20	20	20	20	20	20	20	20	20	20
G3-C1B, F & 300	Mean	239.93	0.0261*	0.1352*	0.3917	0.0472	0.2218	0.0142*	0.0568*	0.0068*	0.0103
	±SD	19.47	0.0031	0.0203	0.0526	0.0060	0.0239	0.0032	0.0109	0.0003	0.0008
	n	20	20	20	20	20	20	20	20	20	20
G4-C1B, F & 1000	Mean	240.15	0.0258*	0.1117	0.3747	0.0433	0.2508	0.01413	0.0767*	0.0066*	0.0101
	±SD	11.09	0.0046	0.0234	0.0671	0.0062	0.0383	0.0033	0.0191	0.0003	0.0010
	n	20	20	20	20	20	20	20	20	20	20

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

 

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

Group, Sex & Dose (mg/kg body weight/day)		Occurrence of Balano-preputial separation (Postnatal Day)	Body weight (g) on the day of Occurrence of Balano-preputial separation
G1-C1B, M & 0	Mean	43.10	159.69
	±SD	1.25	18.08
	n	20	20
G2-C1B, M & 100	Mean	43.25	148.79
	±SD	1.33	12.79
	n	20	20
G3-C1B, M & 300	Mean	43.45	152.12
	±SD	1.43	17.89
	n	20	20
G4-C1B, M & 1000	Mean	43.30	143.87*
	±SD	1.56	14.68
	n	20	20

 

Group, Sex & Dose (mg/kg body weight/day)		Occurrence of Vaginal Opening (Postnatal day)	Body weight (g) on the day of Occurrence of Vaginal Opening
G1-C1B, F & 0	Mean	33.75	99.43
	±SD	1.65	10.85
	n	20	20
G2-C1B, F & 100	Mean	34.10	99.00
	±SD	1.80	10.99
	n	20	20
G3-C1B, F & 300	Mean	34.35	102.03
	±SD	2.11	9.81
	n	20	20
G4-C1B, F & 1000	Mean	34.30	95.84
	±SD	2.13	8.81
	n	20	20

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

 

 

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

An Extended One-Generation Reproductive Toxicity study (EOGRTS) was conducted in rats by oral administration of the test substance in accordance with OECD test guideline 433, following the GLP principles. A total of 200 (100 males + 100 females) Sprague Dawley rats were selected for parental (P) generation and distributed to four main groups. Each P generation group (G1, G2, G3 and G4) consisted of 25 males and 25 females. A total of 160 males and 160 females were selected on the day of weaning (PND21) and randomly assigned to two cohorts (Cohort 1A and Cohort 1B). Each cohort consisted of four groups of 20 males and 20 females per group. The animals in G1 (P generation)/G1-C1A/G1-C1B (F1 generation) groups were administered with vehicle (0.5% carboxymethyl cellulose), the animals in G2 (P generation)/G2-C1A/G2-C1B (F1 generation), G3 (P generation)/G3-C1A/G3-C1B (F1 generation), and G4 (P generation)/G4-C1A/G4-C1B (F1 generation) groups were administered with the test item at the dose levels of 100, 300 and 1000 mg/kg bw/day respectively based on the results of a dose range finding study for which no adverse effects were found up to the dose of 1000 mg/kg/day.. The vehicle and test item formulations were administered orally by gavage at the dose volume of 10 mL/kg bw. Homogeneity and dose formulation analysis for dose concentration verification was performed during weeks 1, 9, 17 and 29 of the treatment period and the mean results were within the range 85-115% recovery to the nominal concentration and RSD was less than 10%.

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

For all P generation animals the following observations were performed: clinical signs once daily, mortality/morbidity twice daily and detailed clinical examination weekly; body weights (throughout the experimental period) and feed consumption (throughout the experimental period, except during cohabitation period) once weekly; haematology, clinical chemistry, urinalysis and thyroid hormone (T4 and TSH) levels; Sperm parameters (motility, morphology and daily sperm production) for all P males; Oestrus cyclicity evaluation pre-mating and cohabitation period for all P females. The body weights and feed consumption was recorded during gestation (Gestation Day 0, 7, 14 and 20) and during lactation (Lactation Day 1, 4, 7, 14 and 21) for all P females. The gross pathology and organ weighing was performed on the day of termination for all P animals. Histopathological examination was conducted on all the tissues collected from the P generation vehicle control and high dose group animals. All P males and females were evaluated for reproductive performance or indices such as, mating and fertility index (for P males and females) and pre-coital interval, gestation length, fecundity index, gestation index, post-implantation loss and postnatal loss (for P females). All P females were observed for birth parameters (number of live/dead pups born, litter size, sex ratio, and live birth index per litter) and for litter observations (number of live/dead pups during lactation period, sex ratio and pup survival index per litter).

All P males and females did not reveal any test item related changes in systemic and reproductive endpoints in any of the tested dose groups.

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

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

For all Cohort 1A (F1 generation) animals the following observations were performed: clinical signs once daily, mortality/morbidity twice daily and detailed clinical examination weekly once; body weights and feed consumption once weekly; occurrence of sexual maturation (for balano-preputial separation in C1A males and for vaginal patency in C1A females); mean occurrence of first cornified cells and time interval between vaginal patency and occurrence of first cornified cells in C1A females; oestrus cyclicity during PND 75 to 88 in C1A females; haematology, clinical chemistry, urinalysis and thyroid hormone (T4 and TSH) levels; Sperm parameters (motility, morphology and daily sperm production) in all C1A males. The gross pathology and organ weighing were performed on the day of termination for all C1A animals. Histopathological examination was conducted on all the tissues collected from the C1A vehicle control and high dose group animals. The flow cytometric analysis of splenic samples for assessment of splenic lymphocyte sub-populations of T "helper" (CD4+) cells, T cytotoxic (CD8+) cells, natural killer (NK) cells and B lymphocytes was conducted for randomly selected males and females from each C1A group.

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

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

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

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