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REACH

N,N'-(methylenedi-p-phenylene)bis(aziridine-1-carboxamide)

EC number: 231-034-6 | CAS number: 7417-99-4

Life Cycle description
Uses advised against

Toxicological information

Developmental toxicity / teratogenicity

Administrative data

Endpoint:: developmental toxicity
Type of information:: experimental study
Adequacy of study:: key study
Study period:: 27 February 2020 – 22 July 2020
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study

Cross-referenceopen all close all

Cross-reference 1

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

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 October 2019 – 19 November 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

(The study period was only 14 days as it was conducted as a dose range finding study for a subsequent more extensive toxicity study)

GLP compliance:

Remarks:

DRF study performed without GLP

Limit test:

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
- Age at study initiation: 10 weeks (at receipt)
- Weight at study initiation: 253.87-255.78 g (range of average values of each group of males); 237.34-238.38 g (range of average values of each group of females)
- Fasting period before study: No
- Housing: Maximum of 3 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.
- Diet (e.g. ad libitum): Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to the animals throughout the acclimatization and experimental period.
- Water (e.g. ad libitum): Water was provided ad libitum throughout the acclimatization and 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: 5 days

DETAILS OF FOOD AND WATER QUALITY:
Analyses of feed and water were performed before starting the study. The corresponding certificates are annexed to the test report.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6-22.9 ºC
- Humidity (%): 46-68 %
- Air changes (per hr): 12-15
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 31 October 2019 To: 19 November 2019

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral 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. Rinsing procedure was repeated until complete transfer of test item formulation into the measuring cylinder. Finally, the volume was made up to the required quantity with vehicle to get the desired concentration. 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 substance was tested in a solubility test with distilled water and also in a suspendibility test with 0.5% w/v carboxy methyl cellulose. Finally, the test item was found to form uniform suspension with corn oil at the concentration of 60 mg/mL (the highest dose concentration selected for the study considering the dose volume of 5 mL/kg body weight). Hence, corn oil was selected as vehicle for test item formulation preparations. Corn oil is universally accepted and routinely used vehicle in oral toxicity studies.
- Concentration in vehicle: 10, 20 and 60 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): N2192016
- Purity: N/A

Analytical verification of doses or concentrations:

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 and homogeneity was achieved by thorough stirring using magnetic stirrer. The actual dose volume for each animal was calculated based on the most recent body weight.

Duration of treatment / exposure:

14 days

Frequency of treatment:

Once a day

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

G1 (vehicle only)

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Remarks:

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

No. of animals per sex per dose:

Control animals:

yes, concurrent vehicle

Details on study design:

Positive control:

None

Observations and examinations performed and frequency:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All the surviving animals and animals found dead were subjected to necropsy and detailed gross pathological examination of external surfaces, external orifices, abdominal, thoracic and cranial cavities, as well as organs and tissues of each animal with special emphasis on reproductive organs.
The following organs from all animals at the scheduled sacrifices were weighed wet as soon as possible to avoid drying: Kidneys, Adrenals, Spleen, Heart, Liver, Thymus, Brain, Lungs Testes/Ovaries, Epididymides/Uterus, Prostate along with seminal vesicles with coagulating gland. Relative organ weights were calculated against fasting body weight.

-HISTOPATHOLOGY: No

Statistics:

After verification, the data was subjected to statistical analysis using SPSS software, version 22. Body weight, percent change in body weight with respect to day 1, hematological, clinical chemistry estimations, urinalysis parameters (whichever applicable), absolute and relative organ weights were subjected to statistical analysis. One way ANOVA followed by Dunnett’s post test was done for different treatment groups comparing with the control group data. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Group G2 (50 mg/kg bw/d) males and females were noted with treatment related clinical signs like lethargy, soft stools/diarrhoea, blood stains around anal region, ataxia/changes in gait, abdominal breathing/unusual respiratory pattern, chromodacryorrhea, wet perineum/urine staining, rough hair coat and piloerection from day 3 and continued till termination.
Group G3 (100 mg/kg bw/d) males and females were noted with treatment related clinical signs like lethargy, salivation, soft stools/diarrhoea, ataxia, abdominal breathing/unusual respiratory pattern, chromodacryorrhea, wet perineum/urine staining, rough hair coat and piloerection from day 2 and continued till termination/death.
Group G4 (300 mg/kg bw/d) males and females were noted with test item related clinical signs like lethargy, salivation, chromodacryorrhea, wet perineum, rough hair coat and piloerection from day 1 and continued till termination/death.

Mortality:

mortality observed, treatment-related

Description (incidence):

A total of 3/5 males and 3/5 females were found dead in group G2, and all the males and females were found dead in groups G3 and G4.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In group G2 males and females, statistically significant reductions in body weight were found on day 7 & 14 (mean body weight) and during day 1 to 7 & 7 to 14 (percent change in mean body weight).
In group G3 males and females, statistically significant reductions in body weight were found on day 7 (mean body weight) and during day 1 to 7 (percent change in mean body weight) in both sexes. The mean body weight on day 14 and percent change in mean body weight on day 14 with respect to day 1 was not available from both sexes due to noted mortalities.
In group G4 males, the mean body weight and percent change in mean body weight with respect to day 1 were not available due to noted mortalities. For females statistically significant reductions in body weight were found on day 7 (mean body weight) and during day 1 to 7 (percent change in mean body weight) in both the sex. The mean body weight on day 14 and percent change in mean body weight on day 14 with respect to day 1 was not available from both sexes due to noted mortalities.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In group G2 males and females, statistically significant reductions in mean feed consumption were found during week 1 and 2.
In group G3 males and females, statistically significant reductions in mean feed consumption were found during week 1 in both sexes. The mean feed consumption was not available from both sexes during week 2 due to noted mortalities.
In group G4 males, the mean feed consumption was not available due to noted mortalities. For females statistically significant reductions in mean feed consumption were found during week 1 and the mean feed consumption during week 2 was not available due to noted mortalities.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ocular changes observed during the opthalmoscopic examination carried out during week 2 (day 14) for all group G1 males and females, and for all surviving group G2 males and females. A total of 2 males and 2 females from group G2 was not subjected to opthalmoscopic examination due to mortalities.
The group G3 and G4 males and females were either dead or moribund sacrificed before scheduled sacrifice. Hence, there was no data on opthalmoscopic examination available from these dose groups.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In group G2 test item related changes in haematology parameters were noticed which were analysed on the day of termination (day 15) for all surviving animals.
The group G3 and G4 males and females were either dead or moribund sacrificed before scheduled sacrifice. Hence, there was no data available on heamatolgy parameters from these groups.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In group G2 test item related changes in clinical chemistry parameters were noticed which were analysed on the day of termination (day 15) for all surviving animals.
The group G3 and G4 males and females were either dead or moribund sacrificed before scheduled sacrifice. Hence, there was no data available on chemistry parameters from these dose groups.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

There were no changes observed in urinalysis parameters analysed on the day of termination (day 15) in group G2 males and females when compared with concurrent control group.
The group G3 and G4 males and females were either dead or moribund sacrificed before scheduled sacrifice. Hence, there was no data available on urinalysis parameters from these groups.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In group G2, test item related changes in all of the organs weighed in both the sex were noted when compared with concurrent control group.
The group G3 and G4 males and females were either dead or moribund sacrificed before scheduled sacrifice. Hence, there was no data available on absolute / relative organ weights from these groups.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In group G2, test item related gross pathological changes like wet perineum (all males and females) externally and reduced thymus size (4 males and 2 females), reduced thymus along with spleen and testes size (1 male) and reduced thymus along with spleen size (3 females) were noted in both sexes.
In group G3 all 5 males and 5 females were found dead during treatment period and subjected to gross pathology. The external gross pathological observation revealed with wet perineum (all males and females) and internal gross pathological observation noted with reduced thymus along with spleen size (all males and females).
In group G4 all 5 males and 5 females were found dead during treatment period and subjected to gross pathology. The external gross pathological observations revealed with wet perineum (all males and 2 females), wet perineum along with wet mouth (3 females). The internal gross pathological observation noted with reduced thymus along with spleen size (all males and females).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

< 50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Treatment related effects observed at the lowest dose tested (50 mg/kg bw).

Key result

Critical effects observed:

Table 1.1. Summary of clinical signs of toxicity, detailed clinical examination and mortality record. Males

Group, Sex & Dose (mg/kg body weight/day)	No. of Animals	Clinical Signs of Toxicity: Observation (No. of Animals noted)	Detailed Clinical Examination: Observation (No. of Animals noted)	Mortality (No. of incidence/ No. of animals)
G1, M & 0	5	N (5)	No Abnormality Detected (5)	0/5
G2, M & 50	5	1 (5); 37 (5); 38 (1); 47(3); 71 (2); 82 (5); 110 (5); 116 (5); 117(1)	Urine staining (5); Diarrhoea (3); Piloerection (3); Unusual respiratory pattern (1); Changes in gait (3); Chromodacryorrhea (5); Piloerectiion (3); Lethargy (1); Rough hair coat (1)	3/5
G3, M & 100	5	1 (5); 6 (2); 37 (5); 71 (4); 82 (5);110 (5); 116 (5); 117 (1);	Urine staining (5); Diarrhoea (2); Unusual respiratory pattern (1); Chromodacryorrhea (5); Lethargy (5); Rough hair coat (3)	5/5
G4, M & 300	5	1 (5); 6 (5); 82 (5); 110 (5); 116 (5); 117 (1)	-	5/5

M: Male; N: Normal; 1: Lethargy; 6: Salivation; 37: Soft stool; 38: Blood stains around anal region; 47: Ataxia; 71: Abdominal breathing; 82: Chromodacryorrhea; 110: Wet perineum; 116: Rough hair coat; 117: Piloerection.

Note: G2 males did not reveal any clinical signs till Day 3 and started to reveal treatment related clinical signs from Day 4 onwards.

G3 males did not reveal any clinical signs till Day 2 and started to reveal treatment related clinical signs from Day 3 onwards.

G4 males did not reveal any clinical signs after first day of dose administration, started to reveal treatment related clinical signs from Day 2 onwards.

Table 1.2. Summary of clinical signs of toxicity, detailed clinical examination and mortality record. Females

Group, Sex & Dose (mg/kg body weight/day)	No. of Animals	Clinical Signs of Toxicity: Observation (No. of Animals noted)	Detailed Clinical Examination: Observation (No. of Animals noted)	Mortality (No. of incidence/ No. of animals)
G1, F & 0	5	N (5)	No Abnormality Detected (5)	0/5
G2, F & 50	5	1 (5); 37 (4); 47 (1); 71 (3); 82 (5); 110 (5); 116 (5); 117 (1)	Urine Staining (5); Diarrhoea (4); Piloerection (1); Unusual repiratory pattern (2); Changes in gait (2); Chromodacryorrhea (4); Lethargy (5); Piloerection (1)	3/5
G3, F & 100	5	1 (5); 6 (1); 37 (5); 47 (1); 71 (3); 82 (5); 110 (5); 116 (3)	Urine Staining (5); Diarrhoea (1); Chromodacryorrhea (4); Lethargy (5); Rough hair coat (2)	5/5
G4, F & 300	5	1 (5); 6 (3); 37 (5); 71 (2); 82 (5); 110 (5); 116 (5); 117 (1)	Salivation (2); Urine staining (4); Diarrhoea (4); Piloerection (1); Unusual repiratory pattern (1); Chromodacryorrhea (4); Rough hair coat (3)	5/5

F: Female; N: Normal; 1: Lethargy; 6: Salivation; 37: Soft stool; 47: Ataxia; 71: Abdominal breathing; 82: Chromodacryorrhea; 110: Wet perineum; 116: Rough hair coat; 117: Piloerection.

Note: G2 females did not reveal any clinical signs till Day 4 and started to reveal treatment related clinical signs from Day 5 onwards.

G3 females did not reveal any clinical signs till Day 2 and started to reveal treatment related clinical signs from Day 3 onwards.

G4 females did not reveal any clinical signs after first day of dose administration, started to reveal treatment related clinical signs from Day 2 onwards.

Table 2.1. Summary of body weight (g) record. Males

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Day
Group, Sex & Dose (mg/kg body weight/day)		1	7	14
G1, M & 0	Mean	255.49	293.79	314.10
	±SD	13.01	19.26	28.70
	n	5	5	5
G2, M & 50	Mean	253.87	232.28*	186.34*
	±SD	17.37	13.95	2.88
	n	5	5	3
G3, M & 100	Mean	254.79	206.73*	-
	±SD	17.47	17.37	-
	n	5	5	-
G4, M & 300	Mean	255.78	-	-
	±SD	13.49	-	-
	n	5	-	-

M: Male; SD: Standard deviation; n: Number of animals; -: Not applicable

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

Table 2.2. Summary of body weight (g) record. Females

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Day
Group, Sex & Dose (mg/kg body weight/day)		1	7	14
G1, F & 0	Mean	237.34	245.39	253.88
	±SD	14.08	13.78	12.54
	n	5	5	5
G2, F & 50	Mean	238.25	220.84*	177.09*
	±SD	13.67	11.41	20.29
	n	5	5	3
G3, F & 100	Mean	238.38	216.09*	-
	±SD	13.57	8.98	-
	n	5	5	-
G4, F & 300	Mean	238.09	182.91*	-
	±SD	11.91	14.31	-
	n	5	4	-

F: Female; SD: Standard deviation; n: Number of animals; -: Not applicable

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

Table 3.1. Summary of percent change in body weight (%) with respect to day 1. Males

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight (%) during Day
Group, Sex & Dose (mg/kg body weight/day)		1 to 7	1 to 14
G1, M & 0	Mean	14.92	22.75
	±SD	2.21	5.61
	n	5	5
G2, M & 50	Mean	-8.43*	-56.86*
	±SD	2.80	39.50
	n	5	5
G3, M & 100	Mean	-18.74*	-
	±SD	6.60	-
	n	5	-

M: Male; SD: Standard deviation; n: Number of animals; -: Not applicable

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

Table 3.2. Summary of percent change in body weight (%) with respect to day 1. Females

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight (%) during Days
Group, Sex & Dose (mg/kg body weight/day)		1 to 7	1 to 14
G1, F & 0	Mean	3.44	7.04
	±SD	2.55	2.50
	n	5	5
G2, F & 50	Mean	-7.17*	-54.44*
	±SD	5.05	42.25
	n	5	5
G3, F & 100	Mean	-9.19*	-
	±SD	4.91	-
	n	5	-
G4, F & 300	Mean	-38.69*	-
	±SD	34.36	-
	n	5	-

F: Female; SD: Standard deviation; n: Number of animals; -: Not applicable

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

Table 4.1. Summary of average feed consumption (g/animal/day) record. Males

Group, Sex & Dose (mg/kg body weight/day)		Week 1 Feed Consumption	Week 2 Feed Consumption
G1, M & 0	Mean	20.64	21.53
	±SD	3.47	4.88
	n	5	5
G2, M & 50	Mean	10.68*	4.65*
	±SD	1.17	1.21
	n	5	5
G3, M & 100	Mean	7.31*	-
	±SD	1.36	-
	n	5	-

M: Male; SD: Standard deviation; n: Number of animals; -: Not applicable

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

Table 4.2. Summary of average feed consumption (g/animal/day) record. Females

Group, Sex & Dose (mg/kg body weight/day)		Week 1 Feed Consumption	Week 2 Feed Consumption


G1, F & 0	Mean	14.19	16.13
	±SD	1.19	0.89
	n	5	5
G2, F & 50	Mean	8.13*	3.39*
	±SD	2.01	1.85
	n	5	5
G3, F & 100	Mean	8.22*	-
	±SD	1.58	-
	n	5	-
G4, F & 300	Mean	2.51*	-
	±SD	0.19	-
	n	5	-

F: Female; SD: Standard deviation; n: Number of animals; -: Not applicable

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

Table 5. Summary of haematology record.

Group, Sex & Dose (mg/kg body weight/day)		Total Leucocyte Count	Total Erythrocyte Count	Hemoglobin	Haematocrit	Mean Corpuscular Volume	Mean Corpuscular Hemoglobin	Mean Corpuscular Hemoglobin Concentration	Platelet Count
		(WBC)	(RBC)	(HGB)	(HCT)	(MCV)	(MCH)	(MCHC)	(PLT)
		(10³cells/µL)	(10⁶cells/µL)	(g/dL)	(%)	(fL)	(pg)	(g/dL)	(10³cells/µL)
G1, M & 0	Mean	9.45	7.48	13.98	45.02	60.18	18.66	31.06	703.00
	±SD	1.38	0.16	0.52	1.53	1.40	0.63	0.40	192.08
	n	5	5	5	5	5	5	5	5
G2, M & 50	Mean	1.45*	5.72	10.75	33.25	57.85	18.70	32.40*	6.00*
	±SD	0.78	1.67	3.46	10.96	2.33	0.57	0.28	2.83
	n	2	2	2	2	2	2	2	2
G1, F & 0	Mean	10.60	7.11	12.76	40.68	57.28	17.98	31.38	828.60
	±SD	3.91	0.41	0.44	1.58	1.20	0.50	0.33	130.66
	n	5	5	5	5	5	5	5	5
G2, F & 50	Mean	4.59*	6.65	11.80	36.00	54.10*	17.80	32.90	71.50*
	±SD	1.11	0.92	1.41	5.09	0.14	0.42	0.85	30.41
	n	2	2	2	2	2	2	2	2

Group, Sex & Dose (mg/kg body weight/day)		Mean Platelet Volume	Reticulocyte Count	Neutrophils	Lymphocytes	Monocytes	Eosinophils	Basophils
		(MPV)	(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)
		(fL)	(%)	(%)	(%)	(%)	(%)	(%)
G1, M & 0	Mean	6.56	2.20	21.30	72.80	3.40	1.04	0.30
	±SD	0.11	0.31	5.76	5.57	0.79	0.43	0.10
	n	5	5	5	5	5	5	5
G2, M & 50	Mean	10.35	0.02*	4.95*	93.15*	1.35*	0.05*	0.25
	±SD	3.04	0.01	1.91	3.04	1.20	0.07	0.07
	n	2	2	2	2	2	2	2
G1, F & 0	Mean	6.60	1.98	21.14	73.66	2.70	0.90	0.26
	±SD	0.19	0.45	7.31	7.70	0.69	0.37	0.11
	n	5	5	5	5	5	5	5
G2, F & 50	Mean	8.25	0.03*	51.05	48.15	0.05*	0.05*	0.25
	±SD	1.63	0.00	68.94	68.09	0.07	0.07	0.07
	n	2	2	2	2	2	2	2

Group, Sex & Dose (mg/kg body weight/day)		Absolute Reticulocyte Count	Absolute Neutrophils	Absolute Lymphocytes	Absolute Monocytes	Absolute Eosinophils	Absolute Basophils	Prothrombin Time	Activated Prothrombin Time
		(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)	(PT)	(APTT)
		(10⁹cells/L)	(10³cells/µL)	(10³cells/µL)	(10³cells/µL)	(10³cells/µL)	(10³cells/µL)	(Seconds)	(Seconds)
G1, M & 0	Mean	164.88	2.00	6.90	0.32	0.10	0.03	21.70	19.74
	±SD	25.87	0.56	1.29	0.08	0.05	0.01	5.44	4.54
	n	5	5	5	5	5	5	5	5
G2, M & 50	Mean	0.75*	0.07*	1.36*	0.02*	0.00*	0.00*	17.30	29.55
	±SD	0.07	0.01	0.78	0.01	0.00	0.00	1.98	7.14
	n	2	2	2	2	2	2	2	2
G1, F & 0	Mean	140.72	2.07	8.00	0.27	0.09	0.03	17.30	31.62
	±SD	33.65	0.47	3.67	0.05	0.03	0.02	2.09	9.25
	n	5	5	5	5	5	5	5	5
G2, F & 50	Mean	2.00*	1.97	2.59	0.01*	0.00*	0.02	16.85	29.00
	±SD	0.00	2.60	3.66	0.01	0.00	0.01	1.06	2.83
	n	2	2	2	2	2	2	2	2

*: 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	Triglycerides	Total Protein	Albumin
		(GLU)		(CRE)	(CHO)	(TRI)	(TPR)	(ALB)
		(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(g/dL)	(g/dL)
G1, M & 0	Mean	97.20	33.60	0.52	45.80	38.00	6.20	3.15
	±SD	21.32	6.72	0.04	8.47	10.39	0.16	0.14
	n	5	5	5	5	5	5	5
G2, M & 50	Mean	129.50	46.75	0.48	27.00*	35.50*	5.15	2.64*
	±SD	27.58	11.53	0.00	1.41	4.95	0.21	0.05
	n	2	2	2	2	2	2	2
G1, F & 0	Mean	90.60	38.06	0.63	57.40	26.40	7.26	3.57
	±SD	11.48	4.31	0.05	6.27	5.90	0.26	0.27
	n	5	5	5	5	5	5	5
G2, F & 50	Mean	65.50*	167.20	1.82	48.50	45.00	6.70	3.34
	±SD	9.19	192.05	1.80	14.85	26.87	0.42	0.15
	n	2	2	2	2	2	2	2

Group, Sex & Dose (mg/kg body weight/day)		Alanine aminotransferase	Aspartate aminotransferase	Alkaline phosphatase	Total Bilirubin	Calcium	Phosphorous
		(ALT)	(AST)	(ALP)	(BIT)	(CAL)	(PHO)
		(U/L)	(U/L)	(U/L)	(mg/dL)	(mg/dL)	(mg/dL)
G1, M & 0	Mean	55.00	98.00	235.20	0.11	9.28	6.12
	±SD	14.20	9.30	70.69	0.00	0.40	0.47
	n	5	5	5	5	5	5
G2, M & 50	Mean	37.50	96.50	241.00	0.25	8.45	5.35
	±SD	14.85	19.09	291.33	0.05	0.49	0.64
	n	2	2	2	2	2	2
G1, F & 0	Mean	41.00	103.60	116.80	0.09	9.46	4.90
	±SD	7.78	41.69	63.35	0.04	0.30	0.32
	n	5	5	5	5	5	5
G2, F & 50	Mean	37.50	82.50	84.00	0.20*	10.65*	9.05
	±SD	7.78	3.54	53.74	0.00	0.49	7.14
	n	2	2	2	2	2	2

Group, Sex & Dose (mg/kg body weight/day)		Cholinesterase	Globulin	Albumin/ Globulin ratio	Blood Urea Nitrogen	Sodium	Potassium	Chloride
		(CHE)	(GLO)	(A/G Ratio)	(BUN)	(Na)	(K)	(CLO)
		(U/L)	(g/dL)		(mg/dL)	(mmol/L)	(mmol/L)	(mmol/L)
G1, M & 0	Mean	155.00	3.05	1.03	15.68	146.56	3.52	115.18
	±SD	55.51	0.13	0.08	3.14	1.42	0.69	1.76
	n	5	5	5	5	5	5	5
G2, M & 50	Mean	136.00	2.52*	1.06	21.82	142.25*	4.19	112.25
	±SD	25.46	0.26	0.13	5.38	1.06	0.22	3.61
	n	2	2	2	2	2	2	2
G1, F & 0	Mean	830.00	3.69	0.97	17.76	146.06	3.09	115.82
	±SD	313.93	0.20	0.11	2.01	1.38	0.16	1.45
	n	5	5	5	5	5	5	5
G2, F & 50	Mean	521.50	3.37	1.00	78.03	151.20	5.50	120.75
	±SD	169.00	0.28	0.04	89.63	6.93	2.44	7.28
	n	2	2	2	2	2	2	2

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

Table 7.1. Summary of urinalysis record. Males

Examination	Group & Sex		G1 & M	G2 & M
	Dose (mg/kg body weight/day)		0	50
	Number of animals		5	2
Physical	Colour	Pale yellow	5	2
	Appearance	Clear	4	2
	Appearance	Turbid	1	-
	Volume (mL)	Mean	4.9	4.3
	Volume (mL)	±SD	1.0	0.4
Chemical	pH	Mean	7.9	8.5
	pH	±SD	0.9	0.0
	Specific Gravity	Mean	1.013	1.005
	Specific Gravity	±SD	0.010	0.000
	Urobilinogen (mg/dL)	Mean	0.6	0.2
	Urobilinogen (mg/dL)	±SD	0.8	0.0
	Bilirubin (mg/dL)	Neg	4	1
		3	1	-
		1	-	1
	Ketones (mg/dL)	Neg	5	2
	Blood (Ery/µL)	Neg	5	-
	Blood (Ery/µL)	>=Ca200	-	2
	Proteins (mg/dL)	Neg	2	-
		Trace	1	-
		100	-	1
		30	1	-
		>=300	1	1
	Nitrite	Pos	5	2
	Leucocytes (Leu/µL)	Neg	4	1
	Leucocytes (Leu/µL)	Ca70	1	1
	Glucose (mg/dL)	Neg	5	2
Microscopic	Epithelial cells	0	2	1
		0-1	1	1
		1-2	1	-
		2-3	1	-
	Casts	Absent	5	2
	Crystals	Present	5	2

Table 7.2. Summary of urinalysis record. Females

Examination	Group & Sex		G1 & F	G2 & F
	Dose (mg/kg body weight/day)		0	50
	Number of animals		5	2
Physical	Colour	Pale yellow	3	2
	Colour	Yellow	2	-
	Appearance	Clear	3	2
	Appearance	Turbid	2	-
	Volume (mL)	Mean	4.6	5.8
	Volume (mL)	±SD	1.0	1.1
Chemical	pH	Mean	8.0	8.5
	pH	±SD	0.5	0.0
	Specific Gravity	Mean	1.012	1.008
	Specific Gravity	±SD	0.004	0.004
	Urobilinogen (mg/dL)	Mean	0.2	0.2
	Urobilinogen (mg/dL)	±SD	0.0	0.0
	Bilirubin (mg/dL)	Neg	5	2
	Ketones (mg/dL)	Neg	5	2
	Blood (Ery/µL)	Neg	3	2
	Blood (Ery/µL)	Ca10	2	-
	Proteins (mg/dL)	Neg	3	-
		Trace	1	1
		>=300	1	1
	Nitrite	Neg	2	-
	Nitrite	Pos	3	2
	Leucocytes (Leu/µL)	Neg	5	2
	Glucose (mg/dL)	Neg	5	2
Microscopic	Epithelial cells	0	3	1
	Epithelial cells	0-1	2	1
	Casts	Absent	5	2
	Crystals	Present	5	2

Table 8.1. Summary of absolute organ weights (g) record. Males

Group, Sex & Dose (mg/kg body weight/day)		Adrenals	Thymus	Spleen	Testes	Epididymes	Heart	Kidneys	Brain	Liver	Prostate+Seminal vesicles with coagulating glands (PSC)	Lungs
G1, M & 0	Mean	0.0666	0.4265	0.6070	3.1496	1.9348	1.1320	2.2874	2.0027	9.8748	2.3939	2.0915
	±SD	0.0225	0.0675	0.0719	0.4489	1.5506	0.0205	0.2215	0.1075	1.2209	0.2261	0.2787
	n	5	5	5	5	5	5	5	5	5	5	5
G2, M & 50	Mean	0.0904	0.1271*	0.3547*	2.6744	0.8543	0.8612*	1.8948	1.9436	8.3810	1.0995*	1.4195*
	±SD	0.0115	0.0011	0.0154	0.0822	0.0748	0.0507	0.2379	0.0167	0.1577	0.0587	0.0660
	n	2	2	2	2	2	2	2	2	2	2	2

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

Table 8.2. Summary of absolute organ weights (g) record. Females

Group, Sex & Dose (mg/kg body weight/day)		Adrenals	Thymus	Spleen	Ovaries	Uterus	Heart	Kidneys	Brain	Liver	Lungs
G1, F & 0	Mean	0.0767	0.3629	0.5325	0.1519	0.8539	0.8522	1.6618	1.8790	7.1617	1.7757
	±SD	0.0115	0.0255	0.0640	0.0121	0.3953	0.1163	0.3867	0.0718	0.7306	0.2630
	n	5	5	5	5	5	5	5	5	5	5
G2, F & 50	Mean	0.1050	0.2220*	0.3407	0.1148	0.4232	0.7189	1.6481	1.9438	6.5787	1.5437
	±SD	0.0339	0.0347	0.2169	0.0431	0.2209	0.1127	0.2593	0.3224	0.7404	0.3969
	n	2	2	2	2	2	2	2	2	2	2

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

Table 9.1. Summary of fasting body weight (g) and organ weight relative to fasting body weight (%) record. Males

Group, Sex & Dose (mg/kg body weight/day)		Fasting Body Weight (g)	Adrenals	Thymus	Spleen	Testes	Epididymes	Heart	Kidneys	Brain	Liver	Prostate+Seminal vesicles with coagulating glands (PSC)	Lungs
G1, M & 0	Mean	283.58	0.0236	0.1501	0.2154	1.1150	0.6753	0.4017	0.8092	0.7116	3.4832	0.8477	0.7384
	±SD	26.08	0.0079	0.0187	0.0312	0.1608	0.5107	0.0346	0.0759	0.0799	0.3013	0.0879	0.0834
	n	5	5	5	5	5	5	5	5	5	5	5	5
G2, M & 50	Mean	176.09*	0.0514*	0.0722*	0.2015	1.5190*	0.4853	0.4892*	1.0756*	1.1038*	4.7594*	0.6246*	0.8063
	±SD	1.31	0.0069	0.0011	0.0103	0.0579	0.0461	0.0324	0.1271	0.0013	0.0542	0.0380	0.0435
	n	2	2	2	2	2	2	2	2	2	2	2	2

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

Table 9.2. Summary of fasting body weight (g) and organ weight relative to fasting body weight (%) record. Females

Group, Sex & Dose (mg/kg body weight/day)		Fasting Body Weight (g)	Adrenals	Thymus	Spleen	Ovaries	Uterus	Heart	Kidneys	Brain	Liver	Lungs
G1, F & 0	Mean	233.10	0.0331	0.1556	0.2282	0.0655	0.3656	0.3645	0.7121	0.8070	3.0738	0.7636
	±SD	12.51	0.0059	0.0047	0.0229	0.0075	0.1616	0.0332	0.1547	0.0297	0.2826	0.1191
	n	5	5	5	5	5	5	5	5	5	5	5
G2, F & 50	Mean	155.40*	0.0674	0.1430	0.2200	0.0737	0.2731	0.4630	1.0615*	1.2520	4.2361*	0.9947
	±SD	1.58	0.0211	0.0238	0.1418	0.0270	0.1449	0.0772	0.1776	0.2202	0.5195	0.2655
	n	2	2	2	2	2	2	2	2	2	2	2

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

Table 10.1. Summary of gross pathology record. Males

Group, Sex and Dose(mg/kg body weight/day)	G1, M & 0	G2, M & 50	G3, M & 100	G4, M & 300
Total No. of Animals	5	5	5	5
No. of Animals Euthanized Terminally	5	2	0	0
No. of Animals Found Dead	0	3	5	5
External Gross pathological Observation
No Abnormality Detected	5	0	0	0
Wet perineum	0	5	5	0
Wet perineum and wet mouth	0	0	0	5
Internal Gross pathological Observation
No Abnormality Detected	5	0	0	0
Thymus: Small in size	0	4	0	0
Thymus: Small in size; Spleen: Small in size; Testes: Small in size,unilateral	0	1	0	0
Thymus: Small in size; Spleen: Small in size	0	0	5	5

Table 10.2. Summary of gross pathology record. Females

Group, Sex and Dose(mg/kg body weight/day)	G1, F & 0	G2, F & 50	G3, F & 100	G4, F & 300
Total No. of Animals	5	5	5	5
No. of Animals Euthanized Terminally	5	2	0	0
No. of Animals Euthanized for Moribund	0	0	0	1
No. of Animals Found Dead	0	3	5	4
External Gross pathological Observation
No Abnormality Detected	5	0	0	0
Wet perineum	0	5	5	2
Wet perineum and wet mouth	0	0	0	3
Internal Gross pathological Observation
No Abnormality Detected	5	0	0	0
Thymus: Small in size	0	2	0	0
Thymus: Small in size; Spleen: Small in size	0	3	5	5

Conclusions:

In this dose range finding study, after an oral exposure of the test substance for 14 days in males and females rats, treatment related effects including mortalities were found at the lowest level tested (50 mg/kg bw /day). Thus, it is not possible to properly decide on doses for the main study and another dose range finding study is considered to be performed.

Executive summary:

A dose range finding study was performed for the test substance in accordance with OECD guideline 407 in order to evaluate its toxic potential after repeated exposure and select the appropriate doses for a subsequent toxicity study. The test item was diluted in corn oil (vehicle) and added to Sprague-Dawley rats (5 per sex per dose) at doses of 0 (vehicle only), 50, 100 and 300 mg/kg bw/day for a period of 14 days. The vehicle and test item formulations were administered orally (gavage) at a dose volume of 5 mL/kg body weight. Freshly prepared test item formulations were administered to the animals as soon as possible after preparation.

All animals for each group were observed for clinical signs of toxicity once daily, mortality and morbidity twice daily, and detailed clinical examination, body weight and feed consumption weekly. Ophthalmological examination was carried out during week 2 (day 14) for all group animals. Clinical pathology (haematology, clinical chemistry analysis and urinalysis) was conducted for all the animals sacrificed at termination. All the animals were subjected to gross pathological examination and the organs were weighed, collected and preserved.

The dose of 50 mg/kg body weight revealed test item related clinical signs of toxicity and mortalities (60%). The doses of 100 and 300 mg/kg body weight revealed test item related clinical signs of toxicity and mortalities (100%). At all dose levels, test item related reduced body weight and feed consumption and test item related gross pathological changes in both sexes were noted. At dose of 50

mg/kg body weight, test item related effects in clinical pathological parameters, absolute / relative organ weights were noted. At doses of 100 and 300 mg/kg body weight, data on clinical pathological parameters and absolute / relative organ weights were not available because all animals were either dead or moribund sacrificed before scheduled sacrifice. In the dose of 50 mg/kg body weight, gross pathological findings were wet perineum (all males and females), reduced thymus size (4 males and 2 females), reduced thymus along with spleen and testes size (1 male) and reduced thymus along with spleen size (3 females). In the dose of 100 mg/kg body weight, gross pathological findings were wet perineum (all males and females) and reduced thymus along with spleen size (all males and females). In the dose of 300 mg/kg body weight, gross pathological findings were wet perineum (all males and 2 females), wet perineum along with wet mouth (3 females) and reduced thymus along with spleen size (all males and females). Based on these results, it is not possible to properly decide on doses for the main study and another dose range finding study is considered to be performed.

Cross-reference 2

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

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 December 2019 – 08 January 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

(The study period was only 14 days as it was conducted as a dose range finding study for a subsequent more extensive toxicity study)

GLP compliance:

Remarks:

DRF study performed without GLP

Limit test:

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
- Age at study initiation: 10 weeks (at receipt)
- Weight at study initiation: 283.88-289.46 g (range of average values of each group of males); 245.80-247.72 g (range of average values of each group of females)
- Fasting period before study: No
- Housing: Maximum of 3 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.
- Diet (e.g. ad libitum): Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to the animals throughout the acclimatization and experimental period.
- Water (e.g. ad libitum): Water was provided ad libitum throughout the acclimatization and 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: 5 days

DETAILS OF FOOD AND WATER QUALITY:
Analyses of feed and water were performed before starting the study. The corresponding certificates are annexed to the test report.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.4-22.8 ºC
- Humidity (%): 47-66 %
- Air changes (per hr): 12-15
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 20 December 2019 To: 08 January 2020

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral 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. Rinsing procedure was repeated until complete transfer of test item formulation into the measuring cylinder. Finally, the volume was made up to the required quantity with vehicle to get the desired concentration. 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 solubility/suspendibility test for the test item was assessed in a previous dose range finding study with the following results: the test substance was tested in a solubility test with distilled water and also in a suspendibility test with 0.5% w/v carboxy methyl cellulose. Finally, the test item was found to form uniform suspension with corn oil at the concentration of 60 mg/mL (the highest dose concentration selected for the study considering the dose volume of 5 mL/kg body weight). Hence, corn oil was selected as vehicle for test item formulation preparations. Corn oil is universally accepted and routinely used vehicle in oral toxicity studies.
- Concentration in vehicle: 0.54, 1.66 and 5 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): N2192016
- Purity: N/A

Analytical verification of doses or concentrations:

Details on analytical verification of doses or concentrations:

Duration of treatment / exposure:

14 days

Frequency of treatment:

Once a day

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

G1 (vehicle only)

Dose / conc.:

2.7 mg/kg bw/day (actual dose received)

Remarks:

Dose / conc.:

8.3 mg/kg bw/day (actual dose received)

Remarks:

Dose / conc.:

25 mg/kg bw/day (actual dose received)

Remarks:

No. of animals per sex per dose:

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
According to ECHA registration dossier, in an acute oral toxicity study (OECD guideline 423), Wistar female rats were treated at a dose of 300 mg/kg bw and 2000 mg/kg bw at single dose. There were no mortalities or clinical signs noted in female rats at 300 mg/kg bw and noted 100% mortalities with treatment related clinical signs at 2000 mg/kg bw. Based on these results it was concluded that the LD50 of the test item was lower than 2000 mg/kg bw and the LD50 cut-off value could be considered around 500 mg/kg bw. Hence, the doses of 0, 50, 100 and 300 mg/kg bw/day were selected as control, low, mid and high dose groups for a first dose range finding study.
The dose of 50 mg/kg bw/d revealed test item related clinical signs of toxicity and mortalities (60%) and test item related effects in clinical pathology parameters and organ weights. Doses of 100 and 300 mg/kg bw/d revealed test item related clinical signs of toxicity and mortalities (100%). All doses revealed test-item related reduced body weight and feed consumption and test item related gross pathological changes in both sexes. In view of these results, it was not possible to properly decide on doses for the main study, thus the present dose range finding study was considered to be performed with doses of 0, 2.7, 8.3 and 25 mg/kg bw/day.

- Fasting period before blood sampling for clinical biochemistry: The animals were fasted overnight before blood collection. Water was provided ad libitum during fasting period.

Positive control:

None

Observations and examinations performed and frequency:

Sacrifice and pathology:

Statistics:

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In group G2 (2.7 mg/kg bw/day), there were no clinical signs of toxicity noted and no changes were noted in detailed clinical examination.
In group G3 (8.3 mg/kg bw/day), there were no adverse test item-related effects noted in any of the parameters of both sexes, except noted wet perineum in one female (recovered later).
In group G4 (25 mg/kg bw/day), the animals of both sexes were noted with test item-related clinical signs like, lethargy, chromodacryorrhea, wet perineum, rough hair coat during the treatment period.

Mortality:

no mortality observed

Description (incidence):

No mortalities were found in any dose level.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In group G2 there were no changes noted in mean body weight and percent change in mean body weight gain.
In group G3 and G4 test item-related slight reduction in mean body weight/body weight gain was noted in both sexes.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In group G2 there were no changes noted in mean feed consumption.
In group G3 and G4 test item-related slight reduction in mean feed consumption was noted in both sexes.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The opthalmoscopic examination did not reveal any changes in any dose group.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In group G2 the haematology assessment did not reveal any test item-related changes in both sexes.
In group G3 the haematological examination revealed test item-related reduced platelet count in males and reduced absolute/percent eosinophils in both males and females.
In group G4 test item-related effects were noted in haematology parameters of either sex.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In group G2 the clinical chemistry assessment did not reveal any test item-related changes in both sexes.
In group G3 the clinical chemistry did not reveal any test item-related changes in both sexes.
In group G4 test item-related effects were noted in clinical chemistry parameters of either sex.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Urinalysis assessment did not reveal any test item-related changes in any dose group. However, statistically significant increase in mean pH of urine at group G3 and G4 females was noted when compared with vehicle control group. This change is considered as incidental and unrelated to treatment as the change was not occurred in dose dependent manner and also no effects were noted on other urinalysis parameters at these dose levels.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There were no test item-related changes observed in mean absolute and relative organ weights at group G2 and G3 in both sexes when compared with the control. However, statistically significant decrease in mean absolute and relative thymus weight at group G3 female was noted when compared with the control.
In group G4 statistically significant reduction in mean absolute thymus, relative thymus, absolute PSC, relative adrenals, relative testes, relative brain weights were noted from males; statistically significant reduction in mean absolute thymus, relative thymus, absolute spleen, relative spleen, absolute uterus, relative uterus, absolute liver, relative brain weights were noted from females. These noted changes are considered as test item-related due to noted clinical signs, reduced body weight and noted gross pathological changes at this dose level.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

There were no external and internal gross pathological changes observed in group G1, G2 and G3 animals of both sexes during necropsy.
In group G4 there were no external gross pathological changes noted in either sex. Internal gross pathological observations found were reduced thymus size (2 males and 4 females), reduced thymus size along with spleen and PCS size (1 male), reduced thymus along with spleen size (1 male and 1 female) and reduced thymus along with PCS size (1 male). These changes are considered as test item-related due to noted significant reduction in absolute or relative weights of the same organs at this dose level.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

8.3 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

organ weights and organ / body weight ratios

Remarks on result:

other: Treatment related adverse effects observed at the dose level of 25 mg/kg bw /d.

Key result

Critical effects observed:

Table 1.1. Summary of clinical signs of toxicity, detailed clinical examination and mortality record. Males

Group, Sex & Dose (mg/kg body weight/day)	No. of Animals	Clinical Signs of Toxicity: Observation (No. of Animals noted)	Detailed Clinical Examination: Observation (No. of Animals noted)	Mortality (No. of incidence/ No. of animals)
G1, M & 0	5	Day 1 to 15: N (5)	No Abnormality Detected (5)	0/5
G2, M & 2.7	5	Day 1 to 15: N (5)	No Abnormality Detected (5)	0/5
G3, M & 8.3	5	Day 1 to 15: N (5)	No Abnormality Detected (5)	0/5
G4, M & 25	5	- Day 1 to 5:N (5) - Day 6:N (1); 82+ (4) - Day 8:N (1); 82+ (3); 1,82+,116 (1) - Day 9:N (1); 82+ (2);1,82+(1); 1,82+,116, 110+(1) - Day 10 & 11:N (1); 82+ (1); - 1,82+ (2); 1,82+,110+,116(1) - Day 12:N (1);82+ (1);1,82+ (1); 1,82+,110+ (1); 1,82+,110+,116(1) - Day 13 & 15:1,82+,110+,116(2); 116(1);82+(1);1,82+(1)	Day 7:No Abnormality Detected (1); Chromodacryorrhea (4) Day 14:Chromodacryorrhea, Urine staining/Wet perineum, Rough hair coat (2); Rough hair coat (1); Chromodacryorrhea (2),	0/5

M: Male; N: Normal; 1: Lethargy; 82: Chromodacryorrhea; 110: Wet perineum; 116: Rough hair coat; +: Slight

Table 1.2. Summary of clinical signs of toxicity, detailed clinical examination and mortality record. Females

Group, Sex & Dose (mg/kg body weight/day)	No. of Animals	Clinical Signs of Toxicity: Observation (No. of Animals noted)	Detailed Clinical Examination: Observation (No. of Animals noted)	Mortality (No. of incidence/ No. of animals)
G1, F & 0	5	Day 1 to 15: N (5)	No Abnormality Detected (5)	0/5
G2, F & 2.7	5	Day 1 to 15: N (5)	No Abnormality Detected (5)	0/5
G3, F & 8.3	5	- Day 1 to 5:N (5) - Day 6 to 8:N (4); 110+ (1) - Day 9 to 15:N (5)	Day 7:No Abnormality Detected (4); Urine staining/Wet perineum(1) Day 14:No Abnormality Detected (5)	0/5
G4, F & 25	5	- Day 1 to 5:N (5) - Day 6:N (4); 82+ (1) - Day 8:N (2); 1,116(2);1,82+(1) - Day 9 to 11:N (2); 1,110+,116(1); 1,110++,116(1);1,82+,110+,116(1) - Day 12:N (2); 1,110++,116(1); 1,110+,116(1);1,82+,116(1) - Day 13 & 15:N (2); 1,110++,116(1); 1,116(1);1,82+,116(1)	Day 7:No Abnormality Detected (4); Chromodacryorrhea (1) Day 14:No Abnormality Detected (2); Urine staining/Wet perineum, Rough Hair Coat (1); Chromodacryorrhea, Rough Hair Coat (1); Rough Hair Coat (1)	0/5

F: Female; N: Normal; 1: Lethargy; 82: Chromodacryorrhea; 110: Wet perineum; 116: Rough hair coat; +: Slight; ++: Moderate

Table 2.1. Summary of body weight (g) record. Males

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Day
Group, Sex & Dose (mg/kg body weight/day)		1	7	14
G1, M & 0	Mean	285.46	300.43	325.69
	±SD	18.62	24.79	32.43
	n	5	5	5
G2, M & 2.7	Mean	283.88	295.31	321.63
	±SD	21.02	27.37	33.83
	n	5	5	5
G3, M & 8.3	Mean	289.46	295.90	318.17
	±SD	20.50	28.36	39.12
	n	5	5	5
G4, M & 25	Mean	286.90	265.40	234.77*
	±SD	19.93	16.83	28.67
	n	5	5	5

M: Male; SD: Standard deviation; n: Number of animals;

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

Table 2.2. Summary of body weight (g) record. Females

Group, Sex & Dose(mg/kg body weight/day)		Body Weight (g) on Day
Group, Sex & Dose(mg/kg body weight/day)		1	7	14
G1, F & 0	Mean	247.72	259.41	268.45
	±SD	13.16	18.07	18.21
	n	5	5	5
G2, F & 2.7	Mean	245.80	253.42	262.64
	±SD	16.67	14.87	14.77
	n	5	5	5
G3, F & 8.3	Mean	246.32	248.25	252.76
	±SD	20.18	17.48	20.72
	n	5	5	5
G4, F & 25	Mean	246.86	218.96*	214.85*
	±SD	19.67	26.97	51.46
	n	5	5	5

F: Female; SD: Standard deviation; n: Number of animals;

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

Table 3.1. Summary of percent change in body weight (%) with respect to day 1. Males

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight (%) during Day
		Percent Change in Body Weight (%) during Day
		1 to 7	1 to 14
G1, M & 0	Mean	5.15	13.93
	±SD	2.58	5.84
	n	5	5
G2, M & 2.7	Mean	3.94	13.20
	±SD	2.91	5.87
	n	5	5
G3, M & 8.3	Mean	2.13	9.67
	±SD	4.22	7.46
	n	5	5
G4, M & 25	Mean	-7.45*	-18.12*
	±SD	1.74	8.72
	n	5	5

M: Male; SD: Standard deviation; n: Number of animals;

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

Table 3.2. Summary of percent change in body weight (%) with respect to day 1. Females

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight (%) during Day
		Percent Change in Body Weight (%) during Day
		1 to 7	1 to 14
G1, F & 0	Mean	4.68	8.33
	±SD	3.51	3.41
	n	5	5
G2, F & 2.7	Mean	3.16	6.94
	±SD	2.02	2.88
	n	5	5
G3, F & 8.3	Mean	0.89	2.67
	±SD	3.10	3.84
	n	5	5
G4, F & 25	Mean	-11.52*	-13.76*
	±SD	4.70	14.61
	n	5	5

F: Female; SD: Standard deviation; n: Number of animals;

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

Table 4.1. Summary of average feed consumption (g/animal/day) record. Males

Group, Sex & Dose (mg/kg body weight/day)		Average Feed consumption(g/rat/day)
		Average Feed consumption(g/rat/day)
		Week 1	Week 2
G1, M & 0	Mean	21.41	21.91
	±SD	3.71	3.70
	n	5	5
G2, M & 2.7	Mean	19.89	20.70
	±SD	2.81	2.36
	n	5	5
G3, M & 8.3	Mean	19.09	20.46
	±SD	3.58	1.24
	n	5	5
G4, M & 25	Mean	13.31	7.62*
	±SD	0.31	2.48
	n	5	5

M: Male; SD: Standard deviation; n: Number of animals;

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

Table 4.2. Summary of average feed consumption (g/animal/day) record. Females

Group, Sex & Dose (mg/kg body weight/day)		Average Feed consumption(g/rat/day)
		Average Feed consumption(g/rat/day)
		Week 1	Week 2
G1, F & 0	Mean	18.06	19.39
	±SD	3.54	4.93
	n	5	5
G2, F & 2.7	Mean	16.66	17.19
	±SD	0.18	0.35
	n	5	5
G3, F & 8.3	Mean	15.71	16.18
	±SD	1.56	1.57
	n	5	5
G4, F & 25	Mean	8.07*	9.76
	±SD	3.17	6.63
	n	5	5

F: Female; SD: Standard deviation; n: Number of animals;

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

Table 5. Summary of haematology record.

Group, Sex & Dose (mg/kg body weight/day)		Total Leucocyte Count	Total Erythrocyte Count	Hemoglobin	Haematocrit	Mean Corpuscular Volume	Mean Corpuscular Hemoglobin	Mean Corpuscular Hemoglobin Concentration	Platelet Count	Mean Platelet Volume
		(WBC)	(RBC)	(HGB)	(HCT)	(MCV)	(MCH)	(MCHC)	(PLT)	(MPV)
		(10³cells/µL)	(10⁶cells/µL)	(g/dL)	(%)	(fL)	(pg)	(g/dL)	(10³cells/µL)	(fL)
G1, M & 0	Mean	10.56	7.41	13.50	44.98	61.06	18.32	30.06	797.20	8.32
	±SD	2.80	0.56	0.45	1.19	5.94	1.46	1.74	109.08	2.34
	n	5	5	5	5	5	5	5	5	5
G2, M & 2.7	Mean	8.56	7.67	14.12	46.90	61.36	18.46	30.16	772.80	8.04
	±SD	1.40	0.62	0.66	2.39	4.95	1.09	1.99	182.93	2.24
	n	5	5	5	5	5	5	5	5	5
G3, M & 8.3	Mean	8.82	7.19	13.46	44.88	63.82	18.82	30.08	564.40*	8.16
	±SD	2.59	1.03	1.32	1.26	12.59	1.20	3.54	174.14	1.39
	n	5	5	5	5	5	5	5	5	5
G4, M & 25	Mean	3.79*	4.81*	9.26*	29.76*	64.70	19.58	30.50	43.40*	18.54
	±SD	2.16	2.13	3.84	9.73	8.14	1.57	2.82	27.86	14.25
	n	5	5	5	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Reticulocyte Count	Neutrophils	Lymphocytes	Monocytes	Eosinophils	Basophils
		(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)
		(%)	(%)	(%)	(%)	(%)	(%)
G1, M & 0	Mean	2.88	20.46	71.36	6.38	1.08	0.26
	±SD	0.79	2.19	1.37	3.12	0.41	0.11
	n	5	5	5	5	5	5
G2, M & 2.7	Mean	3.07	24.74	66.88	6.42	1.12	0.32
	±SD	0.79	2.24	5.61	3.45	0.34	0.08
	n	5	5	5	5	5	5
G3, M & 8.3	Mean	3.17	16.34	76.32	5.82	0.26*	0.34
	±SD	1.89	3.86	4.14	1.10	0.11	0.09
	n	5	5	5	5	5	5
G4, M & 25	Mean	0.23*	5.50*	93.18*	0.52*	0.30*	0.14
	±SD	0.34	3.66	4.16	0.35	0.42	0.13
	n	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Absolute Reticulocyte Count	Absolute Neutrophils	Absolute Lymphocytes	Absolute Monocytes	Absolute Eosinophils	Absolute Basophils	Prothrombin Time	Activated Prothrombin Time
		(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)	(PT)	(APTT)
		(10⁹cells/L)	(10³cells/µL)	(10³cells/µL)	(10³cells/µL)	(10³cells/µL)	(10³cells/µL)	(Seconds)	(Seconds)
G1, M & 0	Mean	211.22	2.19	7.55	0.63	0.10	0.03	19.22	30.30
	±SD	50.39	0.74	2.10	0.32	0.03	0.02	3.49	7.63
	n	5	5	5	5	5	5	5	5
G2, M & 2.7	Mean	232.20	2.14	5.67	0.58	0.10	0.03	19.30	30.82
	±SD	45.06	0.55	0.53	0.38	0.05	0.01	1.15	6.69
	n	5	5	5	5	5	5	5	5
G3, M & 8.3	Mean	221.56	1.46	6.74	0.50	0.02*	0.03	17.38	26.80
	±SD	128.95	0.62	2.05	0.11	0.01	0.01	2.46	7.05
	n	5	5	5	5	5	5	5	5
G4, M & 25	Mean	8.02*	0.18*	3.57*	0.01*	0.00*	0.01*	20.10	24.32
	±SD	11.19	0.17	2.14	0.01	0.01	0.01	4.60	2.50
	n	5	5	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Total Leucocyte Count	Total Erythrocyte Count	Hemoglobin	Haematocrit	Mean Corpuscular Volume	Mean Corpuscular Hemoglobin	Mean Corpuscular Hemoglobin Concentration	Platelet Count	Mean Platelet Volume
		(WBC)	(RBC)	(HGB)	(HCT)	(MCV)	(MCH)	(MCHC)	(PLT)	(MPV)
		(10³cells/µL)	(10⁶cells/µL)	(g/dL)	(%)	(fL)	(pg)	(g/dL)	(10³cells/µL)	(fL)
G1, F & 0	Mean	10.14	7.09	13.50	42.28	59.62	19.04	31.90	847.20	6.82
	±SD	2.55	0.28	1.16	2.90	2.35	1.07	0.63	82.33	0.28
	n	5	5	5	5	5	5	5	5	5
G2, F & 2.7	Mean	10.69	7.26	13.94	42.58	58.66	19.24	32.78	836.00	6.74
	±SD	4.77	0.46	0.75	3.10	1.60	0.43	0.80	97.22	0.22
	n	5	5	5	5	5	5	5	5	5
G3, F & 8.3	Mean	9.46	7.35	14.02	43.46	59.04	19.04	32.22	837.40	6.82
	±SD	2.67	0.40	0.88	3.00	1.36	0.30	0.38	106.09	0.28
	n	5	5	5	5	5	5	5	5	5
G4, F & 25	Mean	5.37	6.30*	11.64*	35.34*	56.20*	18.52	32.94*	106.60*	8.18*
	±SD	1.67	0.53	0.88	2.27	2.07	0.45	0.57	85.69	0.97
	n	5	5	5	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Reticulocyte Count	Neutrophils	Lymphocytes	Monocytes	Eosinophils	Basophils
		(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)
		(%)	(%)	(%)	(%)	(%)	(%)
G1, F & 0	Mean	1.91	20.50	73.62	3.72	1.20	0.30
	±SD	0.18	5.04	4.17	1.33	0.59	0.00
	n	5	5	5	5	5	5
G2, F & 2.7	Mean	2.04	18.08	76.72	3.46	1.00	0.28
	±SD	0.52	2.70	2.88	0.23	0.46	0.11
	n	5	5	5	5	5	5
G3, F & 8.3	Mean	1.93	14.22	80.62	3.68	0.28*	0.32
	±SD	0.56	4.76	5.86	1.08	0.19	0.08
	n	5	5	5	5	5	5
G4, F & 25	Mean	0.11*	3.72*	94.28*	0.64*	0.12*	0.14*
	±SD	0.13	2.50	4.46	0.71	0.08	0.05
	n	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Absolute Reticulocyte Count	Absolute Neutrophils	Absolute Lymphocytes	Absolute Monocytes	Absolute Eosinophils	Absolute Basophils	Prothrombin Time	Activated Prothrombin Time
		(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)	(PT)	(APTT)
		(10⁹cells/L)	(10³cells/µL)	(10³cells/µL)	(10³cells/µL)	(10³cells/µL)	(10³cells/µL)	(Seconds)	(Seconds)
G1, F & 0	Mean	135.36	2.07	7.48	0.36	0.13	0.03	20.80	28.84
	±SD	15.95	0.72	1.95	0.13	0.09	0.01	4.42	5.02
	n	5	5	5	5	5	5	5	5
G2, F & 2.7	Mean	148.76	1.85	8.28	0.36	0.10	0.03	18.18	30.84
	±SD	42.97	0.61	3.94	0.16	0.06	0.02	1.29	12.02
	n	5	5	5	5	5	5	5	5
G3, F & 8.3	Mean	141.26	1.29	7.70	0.33	0.03*	0.03	18.16	24.54
	±SD	38.31	0.44	2.60	0.08	0.02	0.01	2.05	3.25
	n	5	5	5	5	5	5	5	5
G4, F & 25	Mean	6.68*	0.19*	5.07	0.03*	0.00*	0.01*	19.28	24.34
	±SD	7.70	0.15	1.66	0.04	0.01	0.00	3.29	4.53
	n	5	5	5	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	Triglycerides	Total Protein	Albumin
		(GLU)		(CRE)	(CHO)	(TRI)	(TPR)	(ALB)
		(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(g/dL)	(g/dL)
G1, M & 0	Mean	84.20	32.72	0.47	42.80	38.00	5.98	2.89
	±SD	13.59	8.79	0.02	5.54	17.48	0.15	0.06
	n	5	5	5	5	5	5	5
G2, M & 2.7	Mean	83.00	34.44	0.51	50.80	30.40	6.24	3.00
	±SD	10.42	6.49	0.05	7.05	6.80	0.33	0.15
	n	5	5	5	5	5	5	5
G3, M & 8.3	Mean	98.00	32.24	0.49	46.00	46.00	6.18	3.03
	±SD	23.95	6.69	0.05	10.84	10.34	0.31	0.13
	n	5	5	5	5	5	5	5
G4, M & 25	Mean	100.40	43.08	0.45	26.40*	27.20	5.48	2.55
	±SD	7.20	23.31	0.05	2.97	5.89	0.63	0.41
	n	5	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Alanine aminotransferase	Aspartate aminotransferase	Alkaline phosphatase	Total Bilirubin	Calcium	Phosphorous
		(ALT)	(AST)	(ALP)	(BIT)	(CAL)	(PHO)
		(U/L)	(U/L)	(U/L)	(mg/dL)	(mg/dL)	(mg/dL)
G1, M & 0	Mean	48.80	92.20	164.60	0.05	9.22	8.92
	±SD	12.93	13.18	39.37	0.03	0.29	1.45
	n	5	5	5	5	5	5
G2, M & 2.7	Mean	52.00	96.40	194.60	0.08	9.38	7.22*
	±SD	4.95	5.03	24.00	0.04	0.29	0.73
	n	5	5	5	5	5	5
G3, M & 8.3	Mean	41.40	94.80	238.20	0.12	9.24	7.54
	±SD	6.43	28.03	89.83	0.16	0.27	0.46
	n	5	5	5	5	5	5
G4, M & 25	Mean	36.20	73.20	95.00	0.15	8.70*	7.22*
	±SD	12.76	24.68	37.24	0.09	0.24	1.00
	n	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Cholinesterase	Globulin	Albumin/Globulin ratio	Blood Urea Nitrogen	Sodium	Potassium	Chloride
		(CHE)	(GLO)	(A/G Ratio)	(BUN)	(Na)	(K)	(CLO)
		(U/L)	(g/dL)		(mg/dL)	(mmol/L)	(mmol/L)	(mmol/L)
G1, M & 0	Mean	113.00	3.09	0.94	15.27	146.54	3.54	109.44
	±SD	20.72	0.15	0.05	4.11	0.88	0.27	0.71
	n	5	5	5	5	5	5	5
G2, M & 2.7	Mean	135.40	3.24	0.92	16.07	146.60	3.84	109.56
	±SD	47.95	0.20	0.03	3.03	1.22	0.21	1.59
	n	5	5	5	5	5	5	5
G3, M & 8.3	Mean	123.80	3.15	0.97	15.05	146.30	4.15	110.46
	±SD	38.58	0.32	0.11	3.12	1.73	1.07	1.97
	n	5	5	5	5	5	5	5
G4, M & 25	Mean	79.80	2.93	0.88	19.98	146.20	3.97	112.40*
	±SD	16.48	0.41	0.16	10.62	0.86	0.32	2.11
	n	5	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Glucose	Urea	Creatinine	Total Cholesterol	Triglycerides	Total Protein	Albumin
		(GLU)		(CRE)	(CHO)	(TRI)	(TPR)	(ALB)
		(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(g/dL)	(g/dL)
G1, F & 0	Mean	96.20	35.78	0.54	57.80	22.80	6.62	3.17
	±SD	16.81	9.49	0.02	10.80	4.38	0.42	0.06
	n	5	5	5	5	5	5	5
G2, F & 2.7	Mean	87.00	30.58	0.51	50.00	27.20	6.48	3.26
	±SD	16.61	5.09	0.04	9.03	5.07	0.16	0.11
	n	5	5	5	5	5	5	5
G3, F & 8.3	Mean	101.20	30.90	0.53	52.60	28.80	6.64	3.40
	±SD	19.66	2.65	0.04	7.06	10.47	0.44	0.30
	n	5	5	5	5	5	5	5
G4, F & 25	Mean	120.40	30.20	0.50	36.20*	29.20	5.82*	2.95
	±SD	29.14	6.01	0.06	15.51	15.99	0.61	0.28
	n	5	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Alanine aminotransferase	Aspartate aminotransferase	Alkaline phosphatase	Total Bilirubin	Calcium	Phosphorous
		(ALT)	(AST)	(ALP)	(BIT)	(CAL)	(PHO)
		(U/L)	(U/L)	(U/L)	(mg/dL)	(mg/dL)	(mg/dL)
G1, F & 0	Mean	45.20	87.00	145.00	0.04	9.32	6.94
	±SD	8.01	15.78	76.97	0.01	0.25	0.64
	n	5	5	5	5	5	5
G2, F & 2.7	Mean	45.20	84.40	136.80	0.04	9.32	7.24
	±SD	4.27	10.06	48.87	0.02	0.23	1.34
	n	5	5	5	5	5	5
G3, F & 8.3	Mean	39.40	82.20	100.60	0.06	9.44	9.02
	±SD	6.27	4.97	14.98	0.01	0.19	4.07
	n	5	5	5	5	5	5
G4, F & 25	Mean	36.60	86.60	113.60	0.09*	8.90*	5.84
	±SD	3.91	12.54	59.95	0.02	0.27	1.07
	n	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Cholinesterase	Globulin	Albumin/Globulin ratio	Blood Urea Nitrogen	Sodium	Potassium	Chloride
		(CHE)	(GLO)	(A/G Ratio)	(BUN)	(Na)	(K)	(CLO)
		(U/L)	(g/dL)		(mg/dL)	(mmol/L)	(mmol/L)	(mmol/L)
G1, F & 0	Mean	483.80	3.45	0.93	16.70	145.16	3.76	110.48
	±SD	120.58	0.39	0.09	4.42	4.34	1.07	3.60
	n	5	5	5	5	5	5	5
G2, F & 2.7	Mean	488.40	3.22	1.02	14.27	147.20	3.27	110.60
	±SD	99.60	0.23	0.11	2.37	0.73	0.18	1.60
	n	5	5	5	5	5	5	5
G3, F & 8.3	Mean	443.20	3.24	1.05	14.42	147.34	3.15	111.32
	±SD	127.83	0.25	0.11	1.23	1.63	0.28	1.47
	n	5	5	5	5	5	5	5
G4, F & 25	Mean	347.20	2.87*	1.04	14.09	146.06	3.73	111.58
	±SD	254.50	0.38	0.10	2.80	2.74	0.61	2.28
	n	5	5	5	5	5	5	5

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

Table 7.1. Summary of urinalysis record. Males

Examination	Group, Sex		G1, M	G2, M	G3, M	G4, M
	Dose (mg/kg body weight/day)		0	2.7	8.3	25
	Number of Animals		5	5	5	5
Physical Examination	Color	Pale Yellow	5	5	4	5
	Color	Yellow	0	0	1	0
	Apperance	Clear	4	5	4	4
	Apperance	Turbidity	1	0	1	1
	Volume (mL)	Mean	4.8	6.2	6.0	4.4
	Volume (mL)	±SD	0.7	0.8	0.9	1.1
Chemical Examination	pH	Mean	7.3	7.4	7.5	7.9
	pH	±SD	0.4	0.2	0.4	0.7
	Specific gravity (SG)	Mean	1.020	1.015	1.016	1.014
	Specific gravity (SG)	±SD	0.008	0.004	0.009	0.007
	Urobilinogen (UBG) (mg/dL)	Mean	0.2	0.2	0.2	0.2
	Urobilinogen (UBG) (mg/dL)	±SD	0.0	0.0	0.0	0.0
	Bilirubin (BIL) (mg/dL)	Neg	5	5	5	5
	Bilirubin (BIL) (mg/dL)	1	0	0	0	0
	Ketones (KET) (mg/dL)	5	5	3	2	3
		15	0	0	1	0
		Neg	0	2	2	2
	Blood (BLD) (Ery/µL)	Ca10	0	0	0	1
		Ca80	4	1	3	3
		Ca25	0	0	1	0
		>=Ca200	1	4	1	1
	Protein (PRO) (mg/dL)	Trace	4	1	2	1
		Neg	0	4	2	1
		30	1	0	1	2
		100	0	0	0	1
	Nitrite (NIT)	Neg	2	5	2	4
	Nitrite (NIT)	Pos	3	0	3	1
	Leucocytes (LEU) (Leu/µL)	Neg	5	5	2	4
	Leucocytes (LEU) (Leu/µL)	Ca15	0	0	3	1
	Glucose(GLU) (mg/dL)	Neg	5	5	5	5
Microscopic Examination	Epi Cells	0	4	3	4	3
		0-1	0	2	0	1
		1-2	1	0	1	0
		2-3	0	0	0	1
	Casts	Absent	5	5	5	5
	Crystals	Present	5	5	5	5

M: Male; Neg: Negative; Pos: Positive; Ca: Calculated approximately; SD: Standard deviation; >= more than equal to

Table 7.2. Summary of urinalysis record. Females

Examination	Group, Sex Dose (mg/kg body weight/day)		G1, F	G2, F	G3, F	G4, F
	Dose (mg/kg body weight/day)		0	2.7	8.3	25
	Number of Animals		5	5	5	5
Physical Examination	Color	Pale Yellow	5	5	5	5
	Apperance	Clear	5	5	5	5
	Volume (mL)	Mean	5.3	5.6	6.1	5.8
	Volume (mL)	±SD	1.5	1.1	2.0	1.0
Chemical Examination	pH	Mean	6.8	7.3	7.8*	7.6*
	pH	±SD	0.3	0.4	0.4	0.2
	Specific gravity (SG)	Mean	1.015	1.016	1.014	1.013
	Specific gravity (SG)	±SD	0.004	0.004	0.004	0.003
	Urobilinogen (UBG) (mg/dL)	Mean	0.2	0.2	0.2	0.2
	Urobilinogen (UBG) (mg/dL)	±SD	0.0	0.0	0.0	0.0
	Bilirubin (BIL) (mg/dL)	Neg	5	5	5	5
	Ketones (KET) (mg/dL)	5	0	0	1	0
	Ketones (KET) (mg/dL)	Neg	5	5	4	5
	Blood (BLD) (Ery/µL)	Ca10	2	1	3	1
		Ca25	0	0	0	1
		Ca80	0	0	0	2
		>=Ca200	0	0	0	1
		Neg	3	4	2	0
	Protein (PRO) (mg/dL)	Trace	1	0	0	0
		Neg	4	5	4	5
		100	0	0	1	0
	Nitrite (NIT)	Neg	5	5	3	1
	Nitrite (NIT)	Pos	0	0	2	4
	Leucocytes (LEU) (Leu/µL)	Neg	5	5	5	5
	Glucose(GLU)(mg/dL)	Neg	5	5	5	5
Microscopic Examination	Epi Cells	0	4	4	3	3
		0-1	1	1	2	1
		1-2	0	0	0	1
	Casts	Absent	5	5	5	5
	Crystals	Present	5	5	5	5

F: Female; Neg: Negative; Pos: Positive; Ca: Calculated approximately; >= more than equal to; SD: Standard deviation;

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

Table 8.1. Summary of absolute organ weights (g) record. Males

Group, Sex & Dose (mg/kg body weight/day)		Adrenals	Thymus	Spleen	Testes	Epididymis	Heart	Kidneys	Brain	Liver	Prostate+Seminal vesicles with coagulating glands	Lungs
G1, M & 0	Mean	0.0518	0.3537	1.0897	3.2889	1.0565	1.1689	2.4151	1.9058	10.0892	2.2410	2.2864
	±SD	0.0089	0.0873	0.3362	0.3593	0.1060	0.2408	0.4920	0.1369	1.9523	0.3177	0.6103
	n	5	5	5	5	5	5	5	5	5	5	5
G2, M & 2.7	Mean	0.0518	0.3044	1.2695	3.2207	0.9811	1.0864	2.1333	1.9364	9.2042	2.2023	2.4908
	±SD	0.0034	0.1268	0.3010	0.6065	0.1643	0.3076	0.4350	0.1494	1.8123	0.2864	0.9321
	n	5	5	5	5	5	5	5	5	5	5	5
G3, M & 8.3	Mean	0.0483	0.2635	1.0093	3.2752	1.0884	1.1165	2.2240	1.9264	9.3631	2.3537	2.4398
	±SD	0.0084	0.0552	0.5907	0.3214	0.0988	0.1110	0.3576	0.1069	1.3706	0.2593	0.2491
	n	5	5	5	5	5	5	5	5	5	5	5
G4, M & 25	Mean	0.0564	0.0994*	0.6824	3.0020	0.9072	0.9152	1.8692	1.8565	7.9519	1.2656*	1.8941
	±SD	0.0122	0.0324	0.3706	0.2183	0.0792	0.1738	0.1842	0.0640	1.1102	0.4555	0.3959
	n	5	5	5	5	5	5	5	5	5	5	5

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

Table 8.2. Summary of absolute organ weights (g) record. Females

Group, Sex & Dose (mg/kg body weight/day)		Adrenals	Thymus	Spleen	Ovaries	Uterus	Heart	Kidneys	Brain	Liver	Lungs
G1, F & 0	Mean	0.0662	0.3965	0.6406	0.1213	0.7294	0.9054	1.6172	1.9606	7.6942	2.1865
	±SD	0.0057	0.1017	0.1435	0.0295	0.2004	0.1340	0.0606	0.0663	0.5108	0.2787
	n	5	5	5	5	5	5	5	5	5	5
G2, F & 2.7	Mean	0.0671	0.3437	0.5401	0.1200	0.5062	0.9308	1.6759	1.9225	7.6816	2.1255
	±SD	0.0043	0.0650	0.0689	0.0209	0.1498	0.0711	0.2399	0.0820	0.5841	0.4252
	n	5	5	5	5	5	5	5	5	5	5
G3, F & 8.3	Mean	0.0689	0.2658*	0.4884	0.1133	0.4804	0.9128	1.5927	1.8693	7.7629	1.9623
	±SD	0.0023	0.0386	0.0602	0.0085	0.1262	0.0749	0.0321	0.0587	0.3641	0.3719
	n	5	5	5	5	5	5	5	5	5	5
G4, F & 25	Mean	0.0616	0.1210*	0.3617*	0.0991	0.3365*	0.8106	1.4502	1.9254	6.3781*	1.7117
	±SD	0.0098	0.0593	0.1010	0.0279	0.2478	0.1277	0.2116	0.1213	1.3336	0.1206
	n	5	5	5	5	5	5	5	5	5	5

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

Table 9.1. Summary of fasting body weight (g) and organ weight relative to fasting body weight (%) record. Males

Group, Sex & Dose (mg/kg body weight/day)		Fasting (Terminal) Body Weight (g)	Adrenals	Thymus	Spleen	Testes	Epididymes	Heart	Kidneys	Brain	Liver	Prostate+Seminal vesicles with coagulating glands	Lungs
G1, M & 0	Mean	298.76	0.0175	0.1168	0.3745	1.1068	0.3549	0.3884	0.8026	0.6409	3.3658	0.7526	0.7647
	±SD	31.45	0.0036	0.0199	0.1454	0.1269	0.0283	0.0508	0.0942	0.0464	0.4342	0.0929	0.1740
	n	5	5	5	5	5	5	5	5	5	5	5	5
G2, M & 2.7	Mean	296.54	0.0177	0.1031	0.4353	1.0866	0.3328	0.3630	0.7172	0.6570	3.0872	0.7538	0.8221
	±SD	39.24	0.0025	0.0443	0.1302	0.1705	0.0581	0.0609	0.0782	0.0441	0.2126	0.1522	0.1828
	n	5	5	5	5	5	5	5	5	5	5	5	5
G3, M & 8.3	Mean	292.42	0.0165	0.0895	0.3370	1.1285	0.3764	0.3841	0.7591	0.6638	3.2043	0.8226	0.8430
	±SD	36.73	0.0017	0.0104	0.1673	0.1278	0.0546	0.0343	0.0509	0.0519	0.2836	0.1871	0.1264
	n	5	5	5	5	5	5	5	5	5	5	5	5
G4, M & 25	Mean	217.93*	0.0263*	0.0465*	0.3217	1.3941*	0.4182	0.4184	0.8599	0.8592*	3.6619	0.5764	0.8629
	±SD	23.72	0.0074	0.0182	0.1877	0.2039	0.0370	0.0524	0.0620	0.0880	0.5003	0.1844	0.0900
	n	5	5	5	5	5	5	5	5	5	5	5	5

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

Table 9.2. Summary of fasting body weight (g) and organ weight relative to fasting body weight (%) record. Females

Group, Sex & Dose (mg/kg body weight/day)		Fasting (Terminal) Body Weight (g)	Adrenals	Thymus	Spleen	Ovaries	Uterus	Heart	Kidneys	Brain	Liver	Lungs
G1, F & 0	Mean	247.71	0.0267	0.1590	0.2594	0.0487	0.2933	0.3662	0.6549	0.7942	3.1091	0.8899
	±SD	16.90	0.0015	0.0344	0.0589	0.0103	0.0712	0.0515	0.0444	0.0564	0.1455	0.1652
	n	5	5	5	5	5	5	5	5	5	5	5
G2, F & 2.7	Mean	242.24	0.0278	0.1414	0.2226	0.0494	0.2111	0.3848	0.6918	0.7952	3.1742	0.8775
	±SD	15.94	0.0021	0.0215	0.0189	0.0067	0.0705	0.0262	0.0875	0.0390	0.2013	0.1657
	n	5	5	5	5	5	5	5	5	5	5	5
G3, F & 8.3	Mean	232.28	0.0298	0.1145*	0.2103	0.0489	0.2074	0.3958	0.6913	0.8119	3.3683	0.8452
	±SD	22.96	0.0027	0.0117	0.0153	0.0033	0.0522	0.0470	0.0720	0.0930	0.3709	0.1298
	n	5	5	5	5	5	5	5	5	5	5	5
G4, F & 25	Mean	197.16*	0.0320	0.0587*	0.1815*	0.0509	0.1603*	0.4167	0.7509	1.0096*	3.2569	0.8914
	±SD	42.13	0.0071	0.0184	0.0135	0.0120	0.0827	0.0432	0.1288	0.2107	0.3711	0.1371
	n	5	5	5	5	5	5	5	5	5	5	5

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

Table 10.1. Summary of gross pathology record. Males

Group, Sex and Dose (mg/kg body weight/day)	G1, M & 0	G2, M & 2.7	G3, M & 8.3	G4, M & 25
Total No. of Animals	5	5	5	5
No. of Animals Euthanized Terminally	5	5	5	5
External Gross pathological Observation
No Abnormality Detected	5	5	5	5
Internal Gross pathological Observation
No Abnormality Detected	5	5	5	0
Thymus: Small in size	0	0	0	2
Thymus: Small in size; Spleen: Small in size; PSC: Small in size	0	0	0	1
Thymus: Small in size; Spleen: Small in size	0	0	0	1
Thymus: Small in size; PSC: Small in size	0	0	0	1

PSC: Prostate+Seminal vesicles with coagulating glands

Table 10.2. Summary of gross pathology record. Females

Group, Sex and Dose(mg/kg body weight/day)	G1, F & 0	G2, F & 2.7	G3, F & 8.3	G4, F & 25
Total No. of Animals	5	5	5	5
No. of Animals Euthanized Terminally	5	5	5	5
External Gross pathological Observation
No Abnormality Detected	5	5	5	5
Internal Gross pathological Observation
No Abnormality Detected	5	5	5	0
Thymus: Small in size	0	0	0	4
Thymus-small, Spleen-small	0	0	0	1

Conclusions:

In this dose range finding study, after an oral exposure of the test substance for 14 days in males and females rats, treatment related adverse effects were found at the top tested dose level of 25 mg/kg bw /d.

Executive summary:

A second dose range finding study was performed for the test substance in accordance with OECD guideline 407 in order to select the appropriate doses for a subsequent toxicity study. The test item was diluted in corn oil (vehicle) and added to Sprague-Dawley rats (5 per sex per dose) at doses of 0 (vehicle only), 2.7, 8.3 and 25 mg/kg bw/day for a period of 14 days. The vehicle and test item formulations were administered orally (gavage) at a dose volume of 5 mL/kg bw. Freshly prepared test item formulations were administered to the animals as soon as possible after preparation.

At dose of 2.7 mg/kg bw/d there were no test item-related effects noted in any parameter of both sexes throughout the treatment period and no gross pathological changes noted during necropsy. At dose of 8.3 mg/kg bw/d there were no adverse test item-related effects noted in any parameter of both sexes throughout the treatment period, except noted wet perineum in one female (recovered later), slight reduction in mean body weight/body weight gain, mean feed consumption and reduced platelet counts and eosinophils. There were no gross pathological changes noted during necropsy. At dose of 25 mg/kg bw/d, animals of both sexes were noted with test item-related clinical signs, significant reduction in body weight/body weight gain, feed consumption, significant effects in haematology / clinical chemistry parameters and reduced absolute / relative organ weights. The gross pathological examination revealed test item-related significant reduction in size of thymus, spleen and prostate along with seminal vesicles with coagulating gland. No ophthalmological effects were found in any dose group.

Based on these results, it can be concluded that the tested dose of 25 mg/kg bw /d was the lowest dose level at which treatment related adverse effects were found.

Cross-reference 3

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

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 February 2020 – 22 July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

GLP compliance:

yes (incl. QA statement)

Limit test:

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: 343.19-343.99 g (range of average values of each group of males at first day of dosing); 254.10-255.22 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.
Acclimatization: maximum of 2 animals of same sex per cage.
Pre mating: 2 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.
Recovery animals: 2 animals of same sex per cage.
- Diet (e.g. ad libitum): Altromin maintenance diet for rats and mice (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. Females were screened for normal oestrous cycles (4 to 5 days) in a two weeks pre-treatment period before initiation of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6-23.2ºC
- Humidity (%): 46-65 %
- Air changes (per hr): 12-15
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The required quantity of test item was weighed into a clean beaker and a little volume of vehicle was added into the beaker and mixed well with glass rod. The formulation was transferred into a measuring cylinder, the beaker was rinsed with some more amount of vehicle also transferred into the measuring cylinder. This procedure was repeated until the entire quantity of the test item formulation was transferred into the measuring cylinder. Finally, the volume was made up to the required quantity with vehicle to get the desired concentration for the different dose levels.
Test item formulations were prepared daily before administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item was found insoluble in distilled water but formed uniform suspension with corn oil at the concentration of 1.9 mg/mL (considering highest dose of 9.5 mg/kg bw with a dose volume of 5 mL/kg bw) based on the in-house suspendibility test results. Hence, corn oil was selected as vehicle for test item formulation preparations. Corn oil is universally accepted and routinely used vehicle in oral toxicity studies.
- Concentration in vehicle: 0.4, 0.86 and 1.9 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): L12017004, L32011001.

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 at the concentrations of 0.4 mg/mL and 1.9 mg/mL in corn oil. 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 HPLC method (study nº BIO-ANM 1513). Sampling and analysis of formulations were performed during week 1 (16-03-2020) and week 5 (14-04-2020) of the treatment. Samples were collected in duplicates from top, middle and bottom layers from low, mid and high dose concentrations and in duplicates from single 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:

Main group males: two weeks pre-mating, during mating and up to the day before sacrifice during the post-mating period (total of 37 days of treatment).
Main group females: two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13.
Recovery animals: same period as for the main group females until the first scheduled sacrifice of dams and kept without treatment for a further 14 days observation.

Frequency of treatment:

Once a day

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

G1 - Vehicle control + G1R - Vehicle Control Recovery Group

Dose / conc.:

2 mg/kg bw/day (actual dose received)

Remarks:

G2 - Low dose

Dose / conc.:

4.3 mg/kg bw/day (actual dose received)

Remarks:

G3 - Mid dose

Dose / conc.:

9.5 mg/kg bw/day (actual dose received)

Remarks:

G4 - High dose + G4R - High dose Recovery Group

No. of animals per sex per dose:

Main Group: 12
Recovery Group: 5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Doses of 0, 50, 100 and 300 mg/kg bw /day were selected in a first DRF study (BIO-CTX 139) based on the available results for an acute oral toxicity study conducted as per OECD 423 guideline. Treatment related effects including mortalities were found at the lowest level tested. Thus, it was not possible to properly decide on doses for the main study and a second DRF study (BIO-CTX 171) was performed at doses of 0, 2.7, 8.3 and 25 mg/kg bw /day. The dose of 25 mg/kg bw /d was the lowest dose level at which treatment related adverse effects were found and based on these results the doses 2, 4.3 and 9.5 mg/kg bw /day were considered for present main study.
- Fasting period before blood sampling for clinical biochemistry: yes, overnight (water allowed).
- Post-exposure recovery period in satellite groups: 14 days

Positive control:

None

Observations and examinations performed and frequency:

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 included, but not be limited to, 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: The main group animals were weighed at receipt, on the first day of dosing, weekly thereafter and at termination. The main group females were weighed on gestation days 0, 7, 14 and 20 during pregnancy and on days 1, 4, 7 and 13 during lactation period. The recovery group animals (both males and females) were weighed at receipt, on the first day of dosing, weekly thereafter and at termination.

FOOD CONSUMPTION: yes
-feed consumption was measured for main group animals (both males and females) once a week during premating and weekly once for main group males during the post mating period. Feed consumption was not measured during the mating period for both males and females. Thereafter, feed consumption for main group 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. Feed consumption was measured for recovery group animals (both males and females) once a week throughout the experimental period. Average feed intake per rat (g/rat/day) was calculated using the amount of feed given and left over in each cage and the number of rats surviving in each cage.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once before treatment for all animals, at the end of the dosing period for males (shortly prior to scheduled sacrifice) and during the lactation period for females (shortly prior to scheduled sacrifice) of vehicle control and high dose main group animals and during the last week for the recovery group animals.
- Dose groups that were examined: vehicle control and high dose main group.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled terminal sacrifice (for main group males on day 38, for main group females on lactation day 14 and for recovery group animals on the day 66).
- 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 and all recovery group animals.
- Parameters checked: Haemoglobin concentration (HGB), Haematocrit (HCT), Erythrocyte count (RBC), Total leukocyte count (WBC), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet count (PLT), Mean platelet volume (MPV), Reticulocyte count (Retic), Absolute reticulocyte count, 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 (for main group males on day 38, for main group females on lactation day 14 and for recovery group animals on the day 66).
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 5 males and 5 females randomly selected from each main group and all recovery group animals.
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Cholinesterase, Total Protein, Albumin, Total bilirubin, Glucose, Total Cholesterol, Creatinine, Urea, Blood urea nitrogen (BUN), Triglycerides, Phosphorous, Calcium, Globulin. Sodium (mmol/L), Potassium (mmol/L) and Chloride (mmol/L) were estimated using a Na/K/Cl analyzer.

URINALYSIS: Yes
- Time schedule for collection of urine: on the day of scheduled terminal sacrifice.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 5 randomly selected males of each main group and all recovery animals.
- Parameters checked: Blood, Bilirubin, Urobilinogen, Ketones, Protein, Glucose, Microalbumin, Leucocytes. In addition pH, nitrite 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.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Towards the end of the dosing period for males (on day 37) and during lactation period for females (on lactation day 13). Towards the end of the recovery period (shortly prior to scheduled sacrifice on day 65) for the recovery group.
- Dose groups that were examined: all main and recovery groups.
- Battery of functions tested: Home Cage Observations (convulsions, tremors and palpebral closure), Handling Observations, Open Field Observations, Sensory Observations, Neuromuscular Observations, Physiological Observation (Rectal temperature), Grip strength assessment and Motor activity assessment.

OTHER:

Oestrous Cyclicity: Estrous cycles were monitored during the acclimatization to evaluate its normal oestrous cyclicity (4 to 5 days). Only females with normal oestrous cyclicity were selected for the treatment. For the main group females, the vaginal smears were monitored daily from the beginning of the treatment period until evidence of mating. Oestrous cyclicity was also monitored on the day of sacrifice for main group females.

Litter Observation: The number of pups born (dead and live) in a litter, sex and external observations were recorded at birth. Individual body weight of live pups on lactation day 1 (within 24 hours of parturition), 4, 7 and 13 was recorded. The anogenital distance of each pup was measured on postnatal day 4 (lactation day 4). The number of nipples/areolae in male pups was counted on postnatal day 13 (lactation day 13). Fertility index for dams and sires, pup survival index and sex ratio at birth were calculated.

Thyroxine Hormone (T4) Level estimation: Blood samples were collected for measurement of serum T4 levels on the following schedule:
a. Two pups per litter on lactation day 4 based on the following conditions:
- Two female pups in order to retain more male pups for nipple retention on PND 13.
- No pups were eliminated when litter size dropped below 10 pups/litter.
- Only one pup was eliminated and used for blood collection in case of only one pup was available above ten.
b. All main group females (dams) at termination (lactation day 14).
c. Two pups per litter (same sex) at termination (lactation day 13).
d. All adult main group males, at termination (after completion of 37 days of treatment).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see tables 1 and 2)

HISTOPATHOLOGY: Yes (see tables 1 and 2)

Other examinations:

Indices calculation (see table 3)

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.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinical signs of toxicity observed at groups G1/G1R, G2 and G3 animals of either sex throughout the experimental period.

Animals in groups G4/G4R were noted with test item-related clinical signs of toxicity such as, lethargic, rough hair coat, wet perineum, chromodacryorrhea, hair thinning or blood stains around vagina. However, all the animals from group G4R started to recover from the above mentioned clinical signs during recovery period and found normal at termination. In group G4 all the animals did not reveal any clinical signs till day 23 of treatment period. However, from day 24 onwards the animals started to reveal clinical signs. In group G4R all the animals did not reveal any clinical signs till day 25 of treatment period. However, from day 26 onwards the animals started to reveal clinical signs.

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, 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 at groups G2 and G3 in both sexes throughout the experimental period including gestation and lactation periods.

In group G4, there were no changes noted in mean body weight and percent change in mean body weight gain with respect to day 1 till week 3 in both sexes. However, reduction in mean body weight and percent change in mean body weight gain was noted from week 4 onwards and continued till termination. Even though these reductions were not statistically significant, these changes are considered as test item-related due to noted test item-related clinical signs and test item-related effects in neurological observations during these periods.

In group G4R, there were no changes noted in mean body weight and percent change in mean body weight gain with respect to day 1 till week 3 in both sexes. However, reduction in mean body weight and percent change in mean body weight gain was noted from week 4 onwards and continued till end of dosing period. Furthermore, some of these reductions were found to be statistically significant compared to the control group. These changes are considered as test item-related due to noted test item-related clinical signs and test item-related decreased mean feed consumption during these periods. However, the body weight and body weight gains were started to recover during recovery period in both sexes.

In group G4, reduction in mean body weight and percent change in mean body weight gain was noted throughout the gestation period which is statitistically significant on gestation day (GD) 14 and 20 for mean body weight and statitistically significant during GD 7 to 14 and 14 to 20 for percent change in mean body weight gain when compared with vehicle control group. These reductions are considered as test item-related due to noted test item-related clinical signs and test item-related reduction in feed consumption during these periods.

In group G4, reduction in mean body weight and percent change in mean body weight gain was noted throughout the lactation period which is statitistically significant on lactation day (LD) 4, 7 and 13 for mean body weight and statitistically significant during LD 4 to 7 and 7 to 13 for percent change in mean body weight gain when compared with vehicle control group. These reductions are considered as test item-related due to noted test item-related clinical signs and test item-related reduction in feed consumption during these periods.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There were no changes noted in mean feed consumption at groups G2 and G3 in both sexes during pre-mating, gestation and lactation periods when compared with vehicle control group.

In group G4, there were no changes noted in mean feed consumption in both sexes during pre-mating period when compared with vehicle control group. However, reductions in mean feed consumption were noted during gestation and lactation periods which are statitistically significant during GD 14 to 20 and during LD 1 to 4, 4 to 7 and 7 to 13, when compared with vehicle control group. These reductions are considered as test item-related due to noted test item-related clinical signs and test item-related reduction in body weights during these periods.

In group G4R, there were no changes noted in mean feed cosnumption till week 3 in both sexes. However, reduction in mean feed consumption was noted from week 4 onwards and continued till end of dosing period. These reductions are statistically significant during week 5 to 9 in G4R males; statistically significant during week 6 in G4R females when compared with respective vehicle control group. These changes are considered as test item-related due to noted test item-related clinical signs, decreased mean body weight and or percent body weight gain during these periods in both sexes. However, the mean feed consumption was started to recover during recovery period in both sexes.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ocular changes observed in any of the animals from all the tested dose and vehicle control main groups in both sexes during ophthalmological examination conducted towards end of the dosing period. Likewise, there were no ocular changes observed in any of the animals from both recovery groups in either sex during ophthalmological examination conducted towards end of the recovery period.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

There were no changes noted in mean haematology parameters in groups G2 and G3 of both sexes when compared with vehicle control group. In group G4/G4R, the following statistically significant changes were noted: decrease in mean absolute differential leucocyte count (nuetrophills, lymphocytes, monocytes, eosinophils, basophils), eosinophils (%), WBC count of group G4 males; decrease in mean RBC count, HGB, HCT, monocytes (absolute and %), prothrombin time of group G4 females; increase in mean reticulocyte count (absolute and %), eosinophils (absolute and %) and monocytes (%), decrease in mean lymphocytes (%) and prothrombin time of group G4R males; and decrease in RBC count and Activated Prothrombin Time, increase in MCV, MCH, reticulocyte count (absolute and %) of group G4R females.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

There were no test item-related changes noted in mean clinical chemistry parameters of groups G2 and G3 in both sexes when compared with vehicle control group. In group G4/G4R, the following statistically significant changes were noted: decrease in mean alanine aminotransferase (ALT), aspartate aminotransferase (AST) levels of group G4 males; decrease in mean urea and blood urea nitrogen levels of group G4 females; decrease in mean creatinine levels and increase in mean calcium and phosphorous levels of group G4R males; increase in mean creatinine, sodium and total bilirubin levels and decrease in mean total cholesterol levels of group G4R females.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes observed in urinalysis parameters in any of the tested dose levels of both main and recovery groups when compared with vehicle control group. Some of the sporadically occurring statistically significant differences like, increase in mean urine pH levels and decrease in urine specific gravity levels in group G4R females were considered incidental and therefore of no toxicological relevance.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

There were no changes noted in neurological/functional examinations like home cage/ handling/open-field/sensory/neuromuscular (hind limb foot splay)/physiological observations and grip strength or motor activity assessments in any of the animals from groups G2 and G3 when compared with vehicle control groups.

In group G4, there were no changes noted in home cage/handling/open-field/sensory/ neuromuscular (hind limb foot splay)/physiological observations when compared with vehicle control group in both sexes. However, a slight (non-statistically significant) reduction in mean movements count during motor activity assessment in G4 males, statistically significant reduction in mean fore/hind limb grip strengths in G4 males were noted when compared with vehicle control group. These changes can be considered as secondary effects to the test item exposure, noted due to test item-related clinical signs, decreased mean body weight, percent body weight gain and mean feed consumption during this period.

There were no changes noted in neurological/functional examinations like home cage/ handling/open-field/sensory/neuromuscular (hind limb foot splay)/physiological observations and grip strength or motor activity assessments in any of the animals from group G4R in both sexes when compared with vehicle control group.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There were no changes noted in both mean absolute and relative organ weights of groups G2 and G3 in either sex when compared with vehicle control group.

In group G4, statistically significant decrease in mean thymus weight (absolute and relative) of males, statistically significant decrease in mean spleen weight (absolute), mean liver weight (absolute) and thymus weight (absolute and relative) of females was noted when compared with vehicle control group. These observed changes can be considered as test item-related due to noted mild decrease in lymphocytes in white pulp of spleen and cortical area of thymus during microscopic examination of these organs at this dose level. These changes can also correlate with test item-related and statistically significant reduction in mean terminal body weight at this dose level.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

There were no gross pathological changes observed in any of the adult animals from groups G1/G1R, G2 and G3.

The animals of group G4 were noted with test item related external gross pathological changes like wet pernineum (4 males and 7 females), wet pernineum along with blood stains around vagina (2 females) and test item related internal gross pathological changes like uterus with resorptions/dead fetuses during necropsy. However, the group G4R animals of both sexes treated with high dose did not reveal any external or internal gross pathological changes during necropsy. These observations are because of recovery of animals from clinical signs during recovery period and hence all the animals were found normal at termination.

At necropsy, the dead pups which were found dead by birth and during lactation period were noted with pale discoloration and red discoloration at nasal region from group G4 litters. No gross pathological changes were noted in any of the survived pups from group G4 and all pups from other dose group litters.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related changes were observed in spleen, thymus and bone marrow of femur in both sexes at group G4. The microscopic changes were characterized by mild decrease in lymphocytes in white pulp of spleen, cortical area of thymus and also mild decrease in marrow cellularity of femoral bone. Similar changes were not observed in lower and recovery group of animals. All these changes in thymus, spleen and femoral bone marrow were considered to be reversible, secondary effects of reduced food consumption and decreased body weight gain (Everds et.al., 2013). Few of the other microscopic findings observed in the study such as accessory cortical tissue in adrenal glands, ultimobranchial cysts(s) in thyroid gland 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.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Oestrus cycle: There were no irregularities observed in the oestrus cyclicity of main group females during pre-mating period at any of the tested dose groups. The mean length of oestrus cycle (days) per dam during pre-mating period was unaffected at all the tested dose groups when compared with vehicle control group.

Litter observations: In group G4, test item-related reduced gestation index, increased post-implantation loss, increased postnatal loss, reduced litter size, reduced live birth index per litter, increased number of dead pups at birth / during postnatal period and reduced pup survival index were noted. These noted changes are considered as test item-related due to noted test item-related clinical signs, test item-related reduction in maternal body weights and feed consumption during gestation and lactation periods.

Serum Thyroxine (T4) Levels: There were no changes noted in T4 levels of males and dams of main groups and PND 13 pups when compared with vehicle control group. The obtained T4 levels are within the normal range of this species and strain. Also, there were no changes noted in T4 levels of PND 4 pups from groups G2 and G3 when compared with vehicle control group. In group G4, T4 levels of PND 4 pups was not conducted as there were no surplus pups remained from this dose level on PND 4. However, the obtained T4 levels of PND 4 pups from groups G2 and G3 are within the normal range of this species and strain.

Key result

Dose descriptor:

LOAEL

Effect level:

9.5 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Effect level:

4.3 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Remarks on result:

other: Effects observed at the highest tested dose of 9.5 mg/kg bw/day.

Key result

Critical effects observed:

Lowest effective dose / conc.:

9.5 mg/kg bw/day (actual dose received)

System:

immune system

Organ:

bone marrow

spleen

thymus

Table 5.1. Summary of clinical signs of toxicity, detailed clinical examination and mortality record. Males

Group, Sex & Dose (mg/kg body weight/day)	Total No. of Animals	Clinical Signs of Toxicity^a: Observation (No. of Animals revealed)	Detailed Clinical Examination^b: Observation (No. of Animals revealed)	Mortality^c: No. of Mortalities (Total No. of Animals)
G1, M & 0	12	N (12)	NAD (12)	0 (12)
G2, M & 2	12	N (12)	NAD (12)	0 (12)
G3, M & 4.3	12	N (12)	NAD (12)	0 (12)
G4, M & 9.5	12	116 (7); 121 (1); 110+ (7); 82+ (5)	Rough hair coat (4); Hair thinning (1); Wet perineum (6); Chromodacryorrhea (5)	0 (12)

M: Male; N: Normal; NAD: No Abnormality Detected; 82: Chromodacryorrhea; 110: Wet perineum; 116: Rough hair coat; 121: Hair thinning; +: Slight

a: observed daily once; b: observed weekly once; c: observed twice daily

Table 5.2. Summary of clinical signs of toxicity, detailed clinical examination and mortality record. Females

Group, Sex & Dose (mg/kg body weight/day)	Total No. of Animals	Clinical Signs of Toxicity^a: Observation (No. of Animals revealed)	Detailed Clinical Examination^b: Observation (No. of Animals revealed)	Mortality^c: No. of Mortalities (Total No. of Animals)
G1, F & 0	12	N (12)	NAD (12)	0 (12)
G2, F & 2	12	N (12)	NAD (12)	0 (12)
G3, F & 4.3	12	N (12)	NAD (12)	0 (12)
G4, F & 9.5	12	116 (4); 121 (1); 110+ (9); 82+ (3); 145 (1); 1 (1)	Rough hair coat (3); Wet perineum (8); Chromodacryorrhea (3); Hair thinning (1)	0 (12)

F: Female; N: Normal; NAD: No Abnormality Detected; 1: Lethargy; 82: Chromodacryorrhea; 110: Wet perineum; 116: Rough hair coat; 121: Hair thinning; 145: Blood stains around vaginal region +: Slight

a: observed daily once; b: observed weekly once; c: observed twice daily

Table 5.3. Summary of clinical signs of toxicity, detailed clinical examination and mortality record. Recovery groups

Group, Sex & Dose (mg/kg body weight/day)	Total No. of Animals	Clinical Signs of Toxicity^a: Observation (No. of Animals revealed)	Detailed Clinical Examination^b: Observation (No. of Animals revealed)	Mortality^c: No. of Mortalities (Total No. of Animals)
G1R, M & 0	5	N (5)	NAD (5)	0 (5)
G4R, M & 9.5	5	1 (4); 82+ (4); 110+ (5); 116 (4)	Rough hair coat (4); Chromodacryorrhea (4); Wet perineum (5); Lethargy (4)	0 (5)
G1R, F & 0	5	N (5)	NAD (5)	0 (5)
G4R, F & 9.5	5	1 (1); 2 (1); 82+ (2); 110+ (3); 116 (2)	Rough hair coat (2); Chromodacryorrhea (2); Wet perineum (3); Lethargy (1); Dehydration (1)	0 (5)

M: Male; F: Female; R: Recovery; N: Normal; 1: Lethargy; 2: Dehydration; 82: Chromodacryorrhea; 110: Wet perineum; 116: Rough hair coat; +: Slight

a: observed daily once; b: observed weekly once; c: observed twice daily

Table 6.1. Summary of body weight (g) record. Males

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Day
Group, Sex & Dose (mg/kg body weight/day)		1	8	15	22	28	35
G1, M & 0	Mean	343.80	354.30	372.89	381.94	392.62	399.38
	±SD	28.76	30.14	27.43	28.36	29.44	33.00
	n	12	12	12	12	12	12
G2, M & 2	Mean	343.19	355.48	375.06	382.19	395.24	404.80
	±SD	27.66	28.50	31.44	30.40	32.57	34.95
	n	12	12	12	12	12	12
G3, M & 4.3	Mean	343.22	352.18	369.94	377.53	393.85	401.39
	±SD	28.59	31.35	34.05	37.72	41.80	46.65
	n	12	12	12	12	12	12
G4, M & 9.5	Mean	343.99	356.69	372.18	376.97	374.98	376.93
	±SD	29.28	27.87	30.98	33.95	37.26	41.44
	n	12	12	12	12	12	12

Table 6.2. Summary of body weight (g) record. Females

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Day
Group, Sex & Dose (mg/kg body weight/day)		1	8	15	22^#	28^#
G1, F & 0	Mean	254.53	259.57	269.83	275.95	256.16
	±SD	20.28	18.04	19.70	19.89	-
	n	12	12	12	7	1
G2, F & 2	Mean	254.62	262.12	271.75	263.95	268.77
	±SD	19.45	18.11	17.30	20.71	-
	n	12	12	12	4	1
G3, F & 4.3	Mean	255.22	260.53	267.92	267.32	326.14
	±SD	20.49	21.14	21.57	27.80	-
	n	12	12	12	6	1
G4, F & 9.5	Mean	254.10	259.09	269.40	274.25	253.66
	±SD	18.53	19.62	19.05	19.13	-
	n	12	12	12	4	1

#: Presented mean values are obtained from the females in cohabitation period. Data of females confirmed with mating considered for gestation body weight

Table 6.3. Summary of body weight (g) record. Recovery groups

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Days
Group, Sex & Dose (mg/kg body weight/day)		1	8	15	22	28	35	42	49	56	63
G1R, M & 0	Mean	338.71	351.28	367.49	378.53	397.05	408.94	415.18	422.50	436.74	442.08
	±SD	23.47	21.70	23.01	23.71	28.55	27.70	28.66	28.44	35.50	33.17
	n	5	5	5	5	5	5	5	5	5	5
G4R, M & 9.5	Mean	337.91	342.76	361.70	368.88	370.56	362.34*	357.64*	366.45*	383.76*	394.64*
	±SD	25.62	25.46	28.70	28.16	24.16	21.46	24.58	23.15	28.63	27.68
	n	5	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	251.59	257.19	267.20	273.45	278.38	282.59	284.20	287.64	296.38	301.07
	±SD	18.16	21.21	23.27	23.59	22.25	23.55	24.39	23.98	25.02	26.17
	n	5	5	5	5	5	5	5	5	5	5
G4R, F & 9.5	Mean	252.36	258.10	264.50	268.36	259.94	255.07	255.73	262.66	267.59	272.77
	±SD	16.35	15.94	16.15	18.71	22.25	33.33	34.22	27.97	24.17	21.70
	n	5	5	5	5	5	5	5	5	5	5

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

Table 7.1. Summary of percent change in body weight (%) with respect to day 1. Males

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight (%) during Day
Group, Sex & Dose (mg/kg body weight/day)		1 to 8	1 to 15	1 to 22	1 to 28	1 to 35
G1, M & 0	Mean	3.05	8.56	11.25	14.38	16.34
	±SD	1.74	2.38	4.35	5.07	6.23
	n	12	12	12	12	12
G2, M & 2	Mean	3.62	9.33	11.53	15.46	18.22
	±SD	2.94	4.45	6.66	9.26	9.48
	n	12	12	12	12	12
G3, M & 4.3	Mean	2.58	7.74	9.92	14.67	16.81
	±SD	1.95	3.18	4.80	6.45	7.68
	n	12	12	12	12	12
G4, M & 9.5	Mean	3.76	8.25	9.64	9.03	9.51
	±SD	2.02	2.98	4.86	6.23	6.82
	n	12	12	12	12	12

Table 7.2. Summary of percent change in body weight (%) with respect to day 1. Females

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight (%) during Day
Group, Sex & Dose (mg/kg body weight/day)		1 to 8	1 to 15	1 to 22^#	1 to 28^#
G1, F & 0	Mean	2.06	6.09	8.43	14.57
	±SD	1.65	2.32	3.45	-
	n	12	12	7	1
G2, F & 2	Mean	3.04	6.92	10.13	13.02
	±SD	3.27	5.09	4.96	-
	n	12	12	4	1
G3, F & 4.3	Mean	2.08	4.99	7.49	12.14
	±SD	1.02	1.93	2.35	-
	n	12	12	6	1
G4, F & 9.5	Mean	1.95	6.05	7.49	10.12
	±SD	1.46	1.88	1.97	-
	n	12	12	4	1

#: Presented mean values are obtained from the females in cohabitation period. Data of females confirmed with mating considered for gestation body weight

Table 7.3. Summary of percent change in body weight (%) with respect to day 1. Recovery groups

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight (%) during Day
Group, Sex & Dose (mg/kg body weight/day)		1 to 8	1 to 15	1 to 22	1 to 28	1 to 35	1 to 42	1 to 49	1 to 56	1 to 63
G1R, M & 0	Mean	3.76	8.55	11.82	17.38	20.84	22.64	24.82	28.97	30.57
	±SD	1.04	2.02	2.35	6.75	5.06	3.90	4.35	5.83	5.25
	n	5	5	5	5	5	5	5	5	5
G4R, M & 9.5	Mean	1.45*	7.03	9.17	9.77	7.40*	6.06*	8.77*	13.86*	17.14*
	±SD	1.21	1.26	1.72	3.41	4.44	6.75	8.10	8.93	9.39
	n	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	2.17	6.12	8.63	10.61	12.26	12.89	14.27	17.72	19.57
	±SD	1.45	2.33	2.95	1.82	2.15	2.64	2.59	1.84	2.37
	n	5	5	5	5	5	5	5	5	5
G4R, F & 9.5	Mean	2.30	4.85	6.34	2.90*	0.75*	1.00*	3.87*	5.90*	8.01*
	±SD	2.11	2.47	3.04	3.19	7.96	8.55	5.45	3.79	2.69
	n	5	5	5	5	5	5	5	5	5

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

Table 8.1. Summary of average feed consumption (g/animal/day) record. Males

Group, Sex & Dose (mg/kg body weight/day)		Feed Consumption (g/animal/day) during Pre-mating Period
Group, Sex & Dose (mg/kg body weight/day)		Week 1	Week 2
G1, M & 0	Mean	23.99	25.23
	±SD	2.20	1.91
	n	12	12
G2, M & 2	Mean	23.14	24.85
	±SD	1.75	2.04
	n	12	12
G3, M & 4.3	Mean	22.64	24.69
	±SD	2.63	2.06
	n	12	12
G4, M & 9.5	Mean	22.08	24.28
	±SD	1.03	1.54
	n	12	12

Table 8.2. Summary of average feed consumption (g/animal/day) record. Females

Group, Sex & Dose (mg/kg body weight/day)		Feed Consumption (g/animal/day) during Pre-mating Period
Group, Sex & Dose (mg/kg body weight/day)		Week 1	Week 2
G1, F & 0	Mean	16.20	20.55
	±SD	1.64	2.35
	n	12	12
G2, F & 2	Mean	17.23	21.03
	±SD	1.43	1.88
	n	12	12
G3, F & 4.3	Mean	16.76	20.68
	±SD	1.06	1.29
	n	12	12
G4, F & 9.5	Mean	16.10	20.32
	±SD	1.31	1.91
	n	12	12

Table 8.3. Summary of average feed consumption (g/animal/day) record. Recovery groups

Group, Sex & Dose (mg/kg body weight/day)		Feed Consumption (g/animal/day)
Group, Sex & Dose (mg/kg body weight/day)		Week 1	Week 2	Week 3	Week 4	Week 5	Week 6	Week 7	Week 8	Week 9
G1R, M & 0	Mean	22.48	24.88	26.99	28.00	29.15	29.76	30.17	30.58	29.57
	±SD	1.31	3.64	3.32	4.22	3.85	3.92	4.25	2.57	1.61
	n	5	5	5	5	5	5	5	5	5
G4R, M & 9.5	Mean	22.21	24.46	24.95	25.44	20.32*	17.90*	18.99*	21.66*	24.12*
	±SD	1.12	1.06	3.06	3.83	3.10	1.83	0.51	0.87	1.76
	n	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	17.98	21.28	22.56	23.52	25.78	27.50	27.69	27.87	29.37
	±SD	2.32	1.53	2.90	3.00	4.55	4.72	3.16	6.88	3.86
	n	5	5	5	5	5	5	5	5	5
G4R, F & 9.5	Mean	17.99	20.95	20.90	19.32	17.90	16.07*	20.93	23.17	25.33
	±SD	2.18	0.91	1.75	3.17	3.77	3.21	4.54	2.65	2.58
	n	5	5	5	5	5	5	5	5	5

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

Table 9.1. Summary of neurological/functional observation battery record. Males

Day 37
Parameters↓	Group & Sex		G1 & M	G2 & M	G3 & M	G4 & M
	Dose (mg/kg body weight/day)		0	2	4.3	9.5
	Number of Animals (randomly selected)		5	5	5	5
Home Cage Observations
Home cage posture			1	1	1	1
Respiratory pattern			1	1	1	1
Clonic involuntary movements			1	1	1	1
Tonic involuntary movements			1	1	1	1
Vocalization			1	1	1	1
Palpebral closure			1	1	1	1
Handling Observations
a. Ease of removal from the cage			2	2	2	2
b. Ease of handling animal in hand			2	2	2	2
Red or crusty deposits		Eyes	1	1	1	1
		Nose	1	1	1	1
		Mouth	1	1	1	1
Lacrimation			1	1	1	1
Salivation			1	1	1	1
Fur appearance			1	1	1	1
Piloerection			1	1	1	1
Eye prominence			1	1	1	1
Muscle tone			1	1	1	1
Home Cage Observations:Home cage posture - 1=Normal; Respiratory Pattern - 1=Normal; Clonic involuntary movements - 1=Normal; Tonic involuntary movements - 1=Normal; Vocalization - 1=Present; Palpebral closure - 1=Normal; Handling Observations: Ease of removal from the cage - 2=Normal; Ease of handling animal in hand - 2=Normal; Red or crusty depsits - 1=Absent; Lacrimation - 1=None; Salivation- 1=Normal; Fur appearance - 1=Normal; Piloerection - 1=None; Eye Prominence - 1=Normal; Muscle tone - 1=Normal

Day 37
Parameters↓	Group & Sex		G1 & M	G2 & M	G3 & M	G4 & M
	Dose (mg/kg body weight/day)		0	2	4.3	9.5
	Number of Animals (randomly selected)		5	5	5	5
Open Field Observations
Mobility			1	1	1	1
Gait			1	1	1	1
Arousal			3	3	3	3
Number of rearing		Mean	4.6	4.6	4.6	3.8
Number of rearing		±SD	0.9	0.9	1.1	1.6
Number of urination		Mean	2.4	2.4	2.2	2.2
Number of urination		±SD	0.5	0.5	0.4	0.8
Number of defecation		Mean	1.4	2.6	1.8	2.2
Number of defecation		±SD	0.5	0.9	0.8	0.8
Clonic involuntary movement			1	1	1	1
Tonic involuntary movement			1	1	1	1
Stereotype behaviour			1	1	1	1
Excessive grooming		Mean	2.2	2.2	2.4	2.2
Excessive grooming		±SD	0.4	0.4	0.5	0.8
Sensory Observations
Approach response			2	2	2	2
Auditory response			2	2	2	2
Touch response			2	2	2	2
Pupil reflex			2	2	2	2
Tail pinch response			2	2	2	2
Righting reflex			1	1	1	1
Physiological Observation
Body temperature (°F)		Mean	98.3	98.2	98.4	98.6
Body temperature (°F)		±SD	0.5	0.7	0.6	0.6
Open Field Observations: Mobility- 1=Normal;Gait- 1=Normal;Arousal- 3=Normal;Clonic involuntary movement- 1=None;Tonic involuntary movement -1=None;Stereotype behavior -1=Absent;Sensory Observations: Approach response- 2=Normal; Auditory response- 2=Normal;Touch response -2=Normal; Pupil reflex- 2=Normal;Tail pinch response- 2=Normal;Righting reflex- 1=Present

Table 9.2. Summary of neurological/functional observation battery record. Females

Lactation Day 13
Parameters↓	Group & Sex		G1 & F	G2 & F	G3 & F	G4 & F
	Dose (mg/kg body weight/day)		0	2	4.3	9.5
	Number of Animals (randomly selected)		5	5	5	5
Home Cage Observations
Home cage posture			1	1	1	1
Respiratory pattern			1	1	1	1
Clonic involuntary movements			1	1	1	1
Tonic involuntary movements			1	1	1	1
Vocalization			1	1	1	1
Palpebral closure			1	1	1	1
Handling Observations
a. Ease of removal from the cage			2	2	2	2
b. Ease of handling animal in hand			2	2	2	2
Red or crusty deposits		Eyes	1	1	1	1
		Nose	1	1	1	1
		Mouth	1	1	1	1
Lacrimation			1	1	1	1
Salivation			1	1	1	1
Fur appearance			1	1	1	1
Piloerection			1	1	1	1
Eye prominence			1	1	1	1
Muscle tone			1	1	1	1
Home Cage Observations:Home cage posture - 1=Normal; Respiratory Pattern - 1=Normal; Clonic involuntary movements - 1=Normal; Tonic involuntary movements - 1=Normal; Vocalization - 1=Present; Palpebral closure - 1=Normal; Handling Observations: Ease of removal from the cage - 2=Normal; Ease of handling animal in hand - 2=Normal; Red or crusty depsits - 1=Absent; Lacrimation - 1=None; Salivation- 1=Normal; Fur appearance - 1=Normal; Piloerection - 1=None; Eye Prominence - 1=Normal; Muscle tone - 1=Normal

Lactation Day 13
Parameters↓	Group & Sex		G1 & F	G2 & F	G3 & F	G4 & F
	Dose (mg/kg body weight/day)		0	2	4.3	9.5
	Number of Animals (randomly selected)		5	5	5	5
Open Field Observations
Mobility			1	1	1	1
Gait			1	1	1	1
Arousal			3	3	3	3
Number of rearing		Mean	3.8	3.6	3.2	4.0
Number of rearing		±SD	0.8	0.9	0.4	1.2
Numbers of urination		Mean	3.0	2.8	3.2	2.4
Numbers of urination		±SD	0.7	0.4	0.4	0.9
Number of defecation		Mean	2.4	2.2	2.4	2.0
Number of defecation		±SD	0.5	0.4	0.5	0.7
Clonic involuntary movement			1	1	1	1
Tonic involuntary movement			1	1	1	1
Stereotype behaviour			1	1	1	1
Excessive grooming		Mean	3.0	2.6	2.8	2.4
Excessive grooming		±SD	0.7	0.5	0.8	0.5
Sensory Observations
Approach response			2	2	2	2
Auditory response			2	2	2	2
Touch response			2	2	2	2
Pupil reflex			2	2	2	2
Tail pinch response			2	2	2	2
Righting reflex			1	1	1	1
Physiological Observation
Body temperature (°F)		Mean	98.6	98.5	98.3	98.7
Body temperature (°F)		±SD	0.7	0.6	0.5	0.6
Open Field Observations: Mobility- 1=Normal;Gait- 1=Normal;Arousal- 3=Normal;Clonic involuntary movement- 1=None;Tonic involuntary movement -1=None;Stereotype behavior -1=Absent;Sensory Observations: Approach response- 2=Normal; Auditory response- 2=Normal;Touch response -2=Normal; Pupil reflex- 2=Normal;Tail pinch response- 2=Normal;Righting reflex- 1=Present

Table 9.3. Summary of neurological/functional observation battery record. Recovery groups

Day 64
Parameters↓	Group & Sex		G1R & M	G4R & M	G1R & F	G4R & F
	Dose (mg/kg body weight/day)		0	9.5	0	9.5
	Number of Animals		5	5	5	5
Home Cage Observations
Home cage posture			1	1	1	1
Respiratory pattern			1	1	1	1
Clonic involuntary movements			1	1	1	1
Tonic involuntary movements			1	1	1	1
Vocalization			1	1	1	1
Palpebral closure			1	1	1	1
Handling Observations
a. Ease of removal from the cage			2	2	2	2
b. Ease of handling animal in hand			2	2	2	2
Red or crusty deposits		Eyes	1	1	1	1
		Nose	1	1	1	1
		Mouth	1	1	1	1
Lacrimation			1	1	1	1
Salivation			1	1	1	1
Fur appearance			1	1	1	1
Piloerection			1	1	1	1
Eye prominence			1	1	1	1
Muscle tone			1	1	1	1
Home Cage Observations:Home cage posture - 1=Normal; Respiratory Pattern - 1=Normal; Clonic involuntary movements - 1=Normal; Tonic involuntary movements - 1=Normal; Vocalization - 1=Present; Palpebral closure - 1=Normal; Handling Observations: Ease of removal from the cage - 2=Normal; Ease of handling animal in hand - 2=Normal; Red or crusty depsits - 1=Absent; Lacrimation - 1=None; Salivation- 1=Normal; Fur appearance - 1=Normal; Piloerection - 1=None; Eye Prominence - 1=Normal; Muscle tone - 1=Normal

Day 64
Parameters↓	Group & Sex		G1R & M	G4R & M	G1R & F	G4R & F
	Dose (mg/kg body weight/day)		0	9.5	0	9.5
	Number of Animals		5	5	5	5
Open Field Observations
Mobility			1	1	1	1
Gait			1	1	1	1
Arousal			3	3	3	3
Number of rearing		Mean	3.6	3.6	3.6	3.6
Number of rearing		±SD	0.9	0.9	0.9	0.9
Numbers of urination		Mean	2.4	2.8	2.8	2.6
Numbers of urination		±SD	0.5	0.4	0.4	0.5
Number of defecation		Mean	2.2	2.2	2.4	2.4
Number of defecation		±SD	0.4	0.4	0.5	0.5
Clonic involuntary movement			1	1	1	1
Tonic involuntary movement			1	1	1	1
Stereotype behaviour			1	1	1	1
Excessive grooming		Mean	2.6	2.6	2.4	2.4
Excessive grooming		±SD	0.5	0.5	0.5	0.5
Sensory Observations
Approach response			2	2	2	2
Auditory response			2	2	2	2
Touch response			2	2	2	2
Pupil reflex			2	2	2	2
Tail pinch response			2	2	2	2
Righting reflex			1	1	1	1
Physiological Observation
Body temperature (°F)		Mean	98.2	98.3	98.2	98.5
Body temperature (°F)		±SD	0.5	0.7	0.4	0.6
Open Field Observations: Mobility- 1=Normal;Gait- 1=Normal;Arousal- 3=Normal;Clonic involuntary movement- 1=None;Tonic involuntary movement -1=None;Stereotype behavior -1=Absent;Sensory Observations: Approach response- 2=Normal; Auditory response- 2=Normal;Touch response -2=Normal; Pupil reflex- 2=Normal;Tail pinch response- 2=Normal;Righting reflex- 1=Present

Table 10.1. Summary of haematology record. Males

Group, Sex & Dose (mg/kg body weight/day)		Total Leucocyte Count	Total Erythrocyte Count	Haemoglobin	Haematocrit	Mean Corpuscular Volume	Mean Corpuscular Haemoglobin
		(WBC)	(RBC)	(HGB)	(HCT)	(MCV)	(MCH)
		(10³cells/µL)	(10⁶cells/µL)	(g/dL)	(%)	(fL)	(pg)
G1, M & 0	Mean	12.73	6.77	13.22	43.04	64.62	19.64
	±SD	2.46	1.11	1.95	2.92	9.06	1.37
	n	5	5	5	5	5	5
G2, M & 2	Mean	10.23	7.61	14.96	45.34	59.66	19.72
	±SD	2.21	0.24	0.83	2.47	4.43	1.47
	n	5	5	5	5	5	5
G3, M & 4.3	Mean	11.00	6.01	11.76	39.98	72.14	20.42
	±SD	2.18	2.34	2.97	4.89	18.42	3.01
	n	5	5	5	5	5	5
G4, M & 9.5	Mean	6.55*	6.85	13.50	41.42	61.24	19.94
	±SD	1.26	1.11	1.52	4.10	6.33	2.03
	n	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Mean Corpuscular Haemoglobin Concentration	Platelet Count	Mean Platelet Volume	Reticulocyte Count	Neutrophils	Lymphocytes
		(MCHC)	(PLT)	(MPV)	(Retic)	(Neut)	(Lymph)
		(g/dL)	(10³cells/µL)	(fL)	(%)	(%)	(%)
G1, M & 0	Mean	30.66	824.60	6.40	7.61	18.72	75.92
	±SD	3.05	116.04	1.54	8.75	4.41	4.70
	n	5	5	5	5	5	5
G2, M & 2	Mean	33.06	761.80	6.00	2.01	22.02	72.58
	±SD	0.38	97.20	0.54	0.84	3.04	2.52
	n	5	5	5	5	5	5
G3, M & 4.3	Mean	29.04	862.80	8.10	24.46	17.38	75.96
	±SD	3.76	176.98	1.92	23.34	6.86	8.05
	n	5	5	5	5	5	5
G4, M & 9.5	Mean	32.60	616.60	6.64	2.63	14.48	79.88
	±SD	0.92	256.07	0.45	2.28	2.65	2.12
	n	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Monocytes	Eosinophils	Basophils	Absolute Reticulocyte Count	Absolute Neutrophils	Absolute Lymphocytes
		(Mono)	(Eos)	(Baso)	(Retic)	(Neut)	(Lymph)
		(%)	(%)	(%)	(10⁹cells/L)	(10³cells/µL)	(10³cells/µL)
G1, M & 0	Mean	2.92	1.24	0.34	440.52	2.34	9.70
	±SD	0.43	0.57	0.05	465.83	0.57	2.17
	n	5	5	5	5	5	5
G2, M & 2	Mean	2.82	1.46	0.28	152.56	2.28	7.41
	±SD	0.96	0.34	0.04	62.48	0.70	1.52
	n	5	5	5	5	5	5
G3, M & 4.3	Mean	4.28	1.02	0.44	1064.60	1.97	8.29
	±SD	1.53	0.82	0.05	939.34	1.03	1.49
	n	5	5	5	5	5	5
G4, M & 9.5	Mean	1.90	0.10*	0.38	163.70	0.92*	5.25*
	±SD	0.73	0.00	0.08	126.00	0.09	1.12
	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)
		(10³cells/µL)	(10³cells/µL)	(10³cells/µL)	(Seconds)	(Seconds)
G1, M & 0	Mean	0.37	0.15	0.05	24.46	28.66
	±SD	0.11	0.08	0.02	1.53	4.14
	n	5	5	5	5	5
G2, M & 2	Mean	0.27	0.15	0.03	21.38	24.48
	±SD	0.05	0.06	0.01	2.84	1.76
	n	5	5	5	5	5
G3, M & 4.3	Mean	0.46	0.12	0.05	21.98	32.94
	±SD	0.18	0.10	0.01	1.84	8.33
	n	5	5	5	5	5
G4, M & 9.5	Mean	0.12*	0.00*	0.02*	23.48	26.02
	±SD	0.04	0.00	0.01	1.59	2.09
	n	5	5	5	5	5

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

Table 10.2. Summary of haematology record. Females

Group, Sex & Dose (mg/kg body weight/day)		Total Leucocyte Count	Total Erythrocyte Count	Haemoglobin	Haematocrit	Mean Corpuscular Volume	Mean Corpuscular Haemoglobin
		(WBC)	(RBC)	(HGB)	(HCT)	(MCV)	(MCH)
		(10³cells/µL)	(10⁶cells/µL)	(g/dL)	(%)	(fL)	(pg)
G1, F & 0	Mean	11.60	6.87	14.36	43.18	62.96	20.92
	±SD	2.26	0.31	0.49	1.83	3.42	1.17
	n	5	5	5	5	5	5
G2, F & 2	Mean	12.52	7.15	14.40	43.18	60.48	20.14
	±SD	3.54	0.22	0.85	1.83	3.13	1.25
	n	5	5	5	5	5	5
G3, F & 4.3	Mean	10.27	7.14	14.34	42.84	60.12	20.12
	±SD	2.88	0.55	0.81	2.51	2.83	1.02
	n	5	5	5	5	5	5
G4, F & 9.5	Mean	7.47	6.08*	13.06*	39.00*	64.46	21.54
	±SD	3.50	0.58	0.86	1.79	4.95	1.22
	n	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Mean Corpuscular Haemoglobin Concentration	Platelet Count	Mean Platelet Volume	Reticulocyte Count	Neutrophils	Lymphocytes
		(MCHC)	(PLT)	(MPV)	(Retic)	(Neut)	(Lymph)
		(g/dL)	(10³cells/µL)	(fL)	(%)	(%)	(%)
G1, F & 0	Mean	33.26	812.20	7.38	2.76	28.22	64.68
	±SD	0.64	162.21	0.38	0.43	10.10	10.49
	n	5	5	5	5	5	5
G2, F & 2	Mean	33.34	935.00	7.02	2.62	32.80	60.48
	±SD	0.71	212.99	0.36	1.01	11.16	9.68
	n	5	5	5	5	5	5
G3, F & 4.3	Mean	33.48	899.60	7.22	2.78	33.38	60.14
	±SD	0.70	126.40	0.59	1.16	8.27	7.36
	n	5	5	5	5	5	5
G4, F & 9.5	Mean	33.42	792.00	7.32	5.15	23.78	72.76
	±SD	0.75	148.99	0.51	5.10	14.07	14.59
	n	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Monocytes	Eosinophils	Basophils	Absolute Reticulocyte Count	Absolute Neutrophils	Absolute Lymphocytes
		(Mono)	(Eos)	(Baso)	(Retic)	(Neut)	(Lymph)
		(%)	(%)	(%)	(10⁹cells/L)	(10³cells/µL)	(10³cells/µL)
G1, F & 0	Mean	4.20	1.28	0.84	188.92	3.33	7.46
	±SD	1.74	0.94	0.65	27.49	1.44	1.69
	n	5	5	5	5	5	5
G2, F & 2	Mean	3.54	1.14	1.02	187.04	4.30	7.39
	±SD	0.98	0.33	0.95	71.65	2.38	1.37
	n	5	5	5	5	5	5
G3, F & 4.3	Mean	3.94	1.16	0.58	193.26	3.45	6.13
	±SD	1.66	0.63	0.24	62.25	1.17	1.77
	n	5	5	5	5	5	5
G4, F & 9.5	Mean	1.88*	0.46	0.28	291.02	1.79	5.43
	±SD	0.83	0.26	0.15	253.48	1.26	2.97
	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)
		(10³cells/µL)	(10³cells/µL)	(10³cells/µL)	(Seconds)	(Seconds)
G1, F & 0	Mean	0.48	0.14	0.10	30.36	36.14
	±SD	0.19	0.11	0.07	6.22	27.82
	n	5	5	5	5	5
G2, F & 2	Mean	0.44	0.15	0.12	28.44	32.34
	±SD	0.16	0.08	0.09	4.96	22.35
	n	5	5	5	5	5
G3, F & 4.3	Mean	0.43	0.12	0.06	23.96	24.44
	±SD	0.26	0.07	0.03	4.32	9.12
	n	5	5	5	5	5
G4, F & 9.5	Mean	0.13*	0.03	0.02	21.74*	23.10
	±SD	0.05	0.01	0.02	4.86	7.56
	n	5	5	5	5	5

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

Table 10.3. Summary of haematology record. Recovery groups

Group, Sex & Dose (mg/kg body weight/day)		Total Leucocyte Count	Total Erythrocyte Count	Haemoglobin	Haematocrit	Mean Corpuscular Volume	Mean Corpuscular Haemoglobin
		(WBC)	(RBC)	(HGB)	(HCT)	(MCV)	(MCH)
		(10³cells/µL)	(10⁶cells/µL)	(g/dL)	(%)	(fL)	(pg)
G1R, M & 0	Mean	11.36	7.73	14.72	44.62	58.24	19.22
	±SD	3.13	1.07	1.05	3.22	6.09	1.83
	n	5	5	5	5	5	5
G4R, M & 9.5	Mean	9.83	6.95	14.86	44.30	63.90	21.44
	±SD	0.83	0.36	0.48	1.88	3.17	1.40
	n	5	5	5	5	5	5
G1R, F & 0	Mean	8.85	7.17	13.64	40.04	55.86	19.04
	±SD	1.97	0.18	0.69	1.47	1.99	0.88
	n	5	5	5	5	5	5
G4R, F & 9.5	Mean	7.63	6.26*	13.14	38.70	61.96*	21.06*
	±SD	2.11	0.42	0.40	1.14	3.38	1.24
	n	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Mean Corpuscular Haemoglobin Concentration	Platelet Count	Mean Platelet Volume	Reticulocyte Count	Neutrophils	Lymphocytes
		(MCHC)	(PLT)	(MPV)	(Retic)	(Neut)	(Lymph)
		(g/dL)	(10³cells/µL)	(fL)	(%)	(%)	(%)
G1R, M & 0	Mean	33.02	891.20	6.92	1.96	22.78	71.40
	±SD	0.33	99.39	0.22	0.44	2.28	2.88
	n	5	5	5	5	5	5
G4R, M & 9.5	Mean	33.54	873.80	7.22	3.99*	23.88	65.46*
	±SD	0.76	313.03	1.07	1.20	3.45	3.76
	n	5	5	5	5	5	5
G1R, F & 0	Mean	34.06	1009.00	6.72	1.59	20.66	73.00
	±SD	0.50	181.21	0.25	0.48	1.31	1.95
	n	5	5	5	5	5	5
G4R, F & 9.5	Mean	33.96	941.20	6.66	3.34*	20.76	72.64
	±SD	0.49	112.29	0.11	0.77	8.11	8.76
	n	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Monocytes	Eosinophils	Basophils	Absolute Reticulocyte Count	Absolute Neutrophils	Absolute Lymphocytes
		(Mono)	(Eos)	(Baso)	(Retic)	(Neut)	(Lymph)
		(%)	(%)	(%)	(10⁹cells/L)	(10³cells/µL)	(10³cells/µL)
G1R, M & 0	Mean	3.30	1.52	0.44	150.92	2.58	8.12
	±SD	1.08	0.44	0.18	33.84	0.73	2.32
	n	5	5	5	5	5	5
G4R, M & 9.5	Mean	4.96*	4.46*	0.52	274.24*	2.37	6.42
	±SD	0.81	1.28	0.11	69.40	0.50	0.32
	n	5	5	5	5	5	5
G1R, F & 0	Mean	3.28	1.74	0.30	113.82	1.82	6.47
	±SD	0.54	0.38	0.10	34.81	0.40	1.48
	n	5	5	5	5	5	5
G4R, F & 9.5	Mean	3.20	2.32	0.34	208.76*	1.46	5.68
	±SD	0.76	0.97	0.09	46.00	0.19	1.97
	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)
		(10³cells/µL)	(10³cells/µL)	(10³cells/µL)	(Seconds)	(Seconds)
G1R, M & 0	Mean	0.38	0.17	0.05	27.68	17.52
	±SD	0.18	0.04	0.03	1.22	0.68
	n	5	5	5	5	5
G4R, M & 9.5	Mean	0.49	0.44*	0.05	24.36*	18.76
	±SD	0.08	0.14	0.01	1.93	2.16
	n	5	5	5	5	5
G1R, F & 0	Mean	0.29	0.15	0.03	22.62	22.30
	±SD	0.09	0.04	0.01	2.08	2.43
	n	5	5	5	5	5
G4R, F & 9.5	Mean	0.24	0.18	0.03	24.36	18.16*
	±SD	0.06	0.10	0.01	3.63	2.21
	n	5	5	5	5	5

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

Table 11.1. Summary of clinical chemistry record. Males

Group, Sex & Dose (mg/kg body weight/day)		Glucose	Urea	Creatinine	Total Cholesterol	Triglycerides
		(GLU)	-	(CRE)	(CHO)	(TRI)
		(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)
G1, M & 0	Mean	94.80	29.14	0.61	46.60	49.00
	±SD	3.27	1.24	0.07	6.69	6.28
	n	5	5	5	5	5
G2, M & 2	Mean	100.00	34.74*	0.60	47.20	58.80
	±SD	10.46	4.18	0.03	8.29	26.44
	n	5	5	5	5	5
G3, M & 4.3	Mean	92.20	33.88*	0.59	36.40	48.60
	±SD	13.86	1.59	0.02	6.35	14.08
	n	5	5	5	5	5
G4, M & 9.5	Mean	92.60	29.56	0.59	40.60	68.80
	±SD	9.45	3.12	0.04	5.22	15.99
	n	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Total Protein	Albumin	Alanine aminotransferase	Aspartate aminotransferase	Alkaline phosphatase
		(TPR)	(ALB)	(ALT)	(AST)	(ALP)
		(g/dL)	(g/dL)	(U/L)	(U/L)	(U/L)
G1, M & 0	Mean	6.66	3.12	72.60	122.40	136.20
	±SD	0.34	0.17	17.11	14.12	28.86
	n	5	5	5	5	5
G2, M & 2	Mean	6.64	3.10	50.00*	106.20	145.00
	±SD	0.29	0.12	11.66	16.48	52.78
	n	5	5	5	5	5
G3, M & 4.3	Mean	6.72	3.01	56.20	106.40	160.00
	±SD	0.23	0.17	9.47	14.17	68.60
	n	5	5	5	5	5
G4, M & 9.5	Mean	6.52	3.07	41.20*	84.60*	134.60
	±SD	0.29	0.16	7.26	4.93	70.21
	n	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Total Bilirubin	Calcium	Phosphorous	Globulin	Blood Urea Nitrogen
		(BIT)	(CAL)	(PHO)	(GLO)	(BUN)
		(mg/dL)	(mg/dL)	(mg/dL)	(g/dL)	(mg/dL)
G1, M & 0	Mean	0.07	7.68	6.06	3.54	13.60
	±SD	0.08	0.64	0.63	0.21	0.58
	n	5	5	5	5	5
G2, M & 2	Mean	0.02	7.92	6.20	3.54	16.21*
	±SD	0.04	0.44	0.72	0.29	1.95
	n	5	5	5	5	5
G3, M & 4.3	Mean	0.08	7.76	6.38	3.71	15.81*
	±SD	0.08	0.35	0.78	0.30	0.75
	n	5	5	5	5	5
G4, M & 9.5	Mean	0.04	8.02	6.14	3.45	13.79
	±SD	0.02	0.64	0.64	0.21	1.46
	n	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Sodium	Potassium	Chloride
		(Na)	(K)	(CLO)
		(mmol/L)	(mmol/L)	(mmol/L)
G1, M & 0	Mean	144.60	3.71	109.66
	±SD	1.13	0.20	2.99
	n	5	5	5
G2, M & 2	Mean	145.24	3.88	110.10
	±SD	1.07	0.11	1.34
	n	5	5	5
G3, M & 4.3	Mean	144.92	3.70	109.76
	±SD	1.32	0.31	2.00
	n	5	5	5
G4, M & 9.5	Mean	145.84	3.91	112.00
	±SD	0.86	0.27	1.01
	n	5	5	5

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

Table 11.2. Summary of clinical chemistry record. Females

Group, Sex & Dose (mg/kg body weight/day)		Glucose	Urea	Creatinine	Total Cholesterol	Triglycerides
		(GLU)		(CRE)	(CHO)	(TRI)
		(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)
G1, F & 0	Mean	85.40	49.18	0.52	65.80	48.60
	±SD	12.95	15.76	0.06	25.09	19.99
	n	5	5	5	5	5
G2, F & 2	Mean	92.00	47.12	0.60	73.00	114.20*
	±SD	23.79	10.64	0.07	14.68	45.25
	n	5	5	5	5	5
G3, F & 4.3	Mean	91.20	42.90	0.52	67.00	101.00
	±SD	14.62	9.17	0.07	7.65	34.52
	n	5	5	5	5	5
G4, F & 9.5	Mean	106.20	30.72*	0.54	71.00	70.80
	±SD	15.85	7.22	0.04	19.17	25.88
	n	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Total Protein	Albumin	Alanine aminotransferase	Aspartate aminotransferase	Alkaline phosphatase
		(TPR)	(ALB)	(ALT)	(AST)	(ALP)
		(g/dL)	(g/dL)	(U/L)	(U/L)	(U/L)
G1, F & 0	Mean	6.72	3.29	84.40	102.00	243.20
	±SD	0.65	0.38	29.25	18.75	138.36
	n	5	5	5	5	5
G2, F & 2	Mean	6.56	3.41	120.20	129.80	388.00
	±SD	0.29	0.35	36.46	42.74	302.67
	n	5	5	5	5	5
G3, F & 4.3	Mean	6.32	3.16	124.20	127.20	385.80
	±SD	0.55	0.35	29.95	37.41	67.22
	n	5	5	5	5	5
G4, F & 9.5	Mean	6.50	3.41	91.20	111.60	278.80
	±SD	0.45	0.40	66.95	57.69	67.20
	n	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Total Bilirubin	Calcium	Phosphorous	Globulin	Blood Urea Nitrogen
		(BIT)	(CAL)	(PHO)	(GLO)	(BUN)
		(mg/dL)	(mg/dL)	(mg/dL)	(g/dL)	(mg/dL)
G1, F & 0	Mean	0.13	8.84	6.10	3.43	22.95
	±SD	0.10	0.73	1.26	0.35	7.36
	n	5	5	5	5	5
G2, F & 2	Mean	0.10	9.40	6.66	3.15	21.99
	±SD	0.08	0.77	2.01	0.26	4.97
	n	5	5	5	5	5
G3, F & 4.3	Mean	0.09	9.10	6.50	3.16	20.02
	±SD	0.09	0.54	0.83	0.36	4.28
	n	5	5	5	5	5
G4, F & 9.5	Mean	0.16	9.48	5.28	3.09	14.33*
	±SD	0.14	0.69	1.65	0.22	3.37
	n	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Sodium	Potassium	Chloride
		(Na)	(K)	(CLO)
		(mmol/L)	(mmol/L)	(mmol/L)
G1, F & 0	Mean	141.36	3.27	103.92
	±SD	3.01	0.54	4.31
	n	5	5	5
G2, F & 2	Mean	140.12	3.34	103.34
	±SD	2.51	0.37	3.78
	n	5	5	5
G3, F & 4.3	Mean	139.92	3.21	102.52
	±SD	1.74	0.29	2.83
	n	5	5	5
G4, F & 9.5	Mean	141.28	3.44	103.80
	±SD	2.63	0.38	4.86
	n	5	5	5

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

Table 11.3. Summary of clinical chemistry record. Recovery groups

Group, Sex & Dose (mg/kg body weight/day)		Glucose	Urea	Creatinine	Total Cholesterol	Triglycerides
		(GLU)	-	(CRE)	(CHO)	(TRI)
		(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)
G1R, M & 0	Mean	95.80	31.34	0.49	43.40	30.60
	±SD	6.83	2.80	0.01	6.35	3.65
	n	5	5	5	5	5
G4R, M & 9.5	Mean	98.60	28.08	0.45*	40.40	35.20
	±SD	20.60	3.19	0.02	9.61	5.02
	n	5	5	5	5	5
G1R, F & 0	Mean	106.60	32.32	0.54	65.60	34.00
	±SD	9.02	4.63	0.06	5.86	6.63
	n	5	5	5	5	5
G4R, F & 9.5	Mean	107.40	27.32	0.75*	53.00*	47.20
	±SD	11.37	2.46	0.17	4.30	12.99
	n	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Total Protein	Albumin	Alanine aminotransferase	Aspartate aminotransferase	Alkaline phosphatase
		(TPR)	(ALB)	(ALT)	(AST)	(ALP)
		(g/dL)	(g/dL)	(U/L)	(U/L)	(U/L)
G1R, M & 0	Mean	6.56	3.05	69.40	113.80	120.40
	±SD	0.23	0.16	12.54	9.98	28.59
	n	5	5	5	5	5
G4R, M & 9.5	Mean	6.68	3.07	66.80	110.60	152.00
	±SD	0.36	0.13	12.07	12.88	44.86
	n	5	5	5	5	5
G1R, F & 0	Mean	7.04	3.61	47.60	105.80	57.80
	±SD	0.28	0.16	15.68	25.06	18.77
	n	5	5	5	5	5
G4R, F & 9.5	Mean	6.98	3.76	44.40	89.80	65.80
	±SD	0.61	0.30	11.01	16.90	29.42
	n	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Total Bilirubin	Calcium	Phosphorous	Globulin	Blood Urea Nitrogen
		(BIT)	(CAL)	(PHO)	(GLO)	(BUN)
		(mg/dL)	(mg/dL)	(mg/dL)	(g/dL)	(mg/dL)
G1R, M & 0	Mean	0.15	9.12	4.98	3.51	14.63
	±SD	0.10	0.19	0.23	0.35	1.31
	n	5	5	5	5	5
G4R, M & 9.5	Mean	0.17	9.78*	5.60*	3.61	13.10
	±SD	0.09	0.40	0.34	0.23	1.49
	n	5	5	5	5	5
G1R, F & 0	Mean	0.11	10.04	4.22	3.43	15.08
	±SD	0.08	0.42	0.41	0.34	2.16
	n	5	5	5	5	5
G4R, F & 9.5	Mean	0.22*	9.94	4.16	3.22	12.75
	±SD	0.05	0.54	0.25	0.37	1.15
	n	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Sodium	Potassium	Chloride
		(Na)	(K)	(CLO)
		(mmol/L)	(mmol/L)	(mmol/L)
G1R, M & 0	Mean	143.74	3.37	108.70
	±SD	1.15	0.16	1.86
	n	5	5	5
G4R, M & 9.5	Mean	143.40	3.40	106.86
	±SD	0.69	0.19	1.67
	n	5	5	5
G1R, F & 0	Mean	142.80	3.33	108.14
	±SD	0.79	0.42	0.31
	n	5	5	5
G4R, F & 9.5	Mean	144.44*	3.12	108.10
	±SD	0.80	0.23	1.02
	n	5	5	5

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

Table 12. Summary of urinalysis record

Examination	Group, Sex & Dose (mg/kg body weight/day)		G1, M & 0	G2, M & 2	G3, M & 4.3	G4, M & 9.5
Examination	Number of Animals (randomly selected)		5	5	5	5
Physical Examination	Color	Pale Yellow	3	4	3	4
	Color	Yellow	2	1	2	1
	Appearance	Clear	3	4	3	3
	Appearance	Turbidity	2	1	2	2
	Volume (mL)	Mean	8.9	9.2	7.8	5.9
	Volume (mL)	±SD	2.2	1.4	2.8	2.3
Chemical Examination	pH	Mean	8.1	8.4	8.4	8.5
	pH	±SD	0.5	0.2	0.2	0.0
	Specific Gravity	Mean	1.007	1.006	1.006	1.006
	Specific Gravity	±SD	0.003	0.002	0.002	0.002
	Urobilinogen (mg/dL)	Mean	0.2	0.2	0.6	0.4
	Urobilinogen (mg/dL)	±SD	0.0	0.0	0.8	0.4
	Bilirubin (mg/dL)	Neg	5	5	4	5
	Bilirubin (mg/dL)	1	-	-	1	-
	Ketones (mg/dL)	5	2	3	3	3
	Ketones (mg/dL)	Neg	3	2	2	2
	Blood (Ery/µL)	Ca25	-	2	1	-
		Neg	2	2	2	3
		>=Ca200	2	1	1	1
		Ca10	1	-	1	1
	Protein (mg/dL)	Trace	-	3	1	1
		100	2	1	-	3
		>=300	-	1	2	-
		30	1	-	2	1
		Neg	2	-	-	-
	Nitrite	Neg	2	1	3	1
	Nitrite	Pos	3	4	2	4
	Leucocytes	Neg	3	3	3	2
		Ca15	2	1	2	2
		Ca70	-	1	-	1
	Glucose (mg/dL)	Neg	5	5	5	5
	Microalbumin (mg/dL)	15	1	-	-	-
		>15	3	5	5	5
		Neg	1	-	-	-
Microscopic Examination	Epi Cells	0	4	2	4	4
		0-1	1	2	-	-
		1-2	-	1	1	1
	Casts	Absent	5	5	5	5
	Crystals	Present	5	5	5	5

Examination	Group, Sex & Dose (mg/kg body weight/day)		G1R, M & 0	G4R, M & 9.5	G1R, F & 0	G4R, F & 9.5
Examination	Number of Animals		5	5	5	5
Physical Examination	Color	Pale Yellow	5	4	5	5
	Color	Yellow	-	1	-	-
	Appearance	Clear	2	3	5	4
	Appearance	Turbidity	3	2	-	1
	Volume (mL)	Mean	7.6	8.1	7.5	7.8
	Volume (mL)	±SD	2.3	1.8	1.3	3.4
Chemical Examination	pH	Mean	8.4	8.3	7.0	8.1*
	pH	±SD	0.2	0.4	0.5	0.4
	Specific gravity	Mean	1.007	1.006	1.017	1.007*
	Specific gravity	±SD	0.004	0.002	0.003	0.003
	Urobilinogen (mg/dL)	Mean	0.2	0.2	0.2	0.2
	Urobilinogen (mg/dL)	±SD	0.0	0.0	0.0	0.0
	Bilirubin (mg/dL)	Neg	5	5	5	5
	Ketones (mg/dL)	5	4	2	1	1
	Ketones (mg/dL)	Neg	1	3	4	4
	Blood (Ery/µL)	Ca25	-	2	2	2
		Ca80	-	2	-	2
		Neg	-	-	-	1
		>=Ca200	3	1	1	-
		Ca10	2	-	2	-
	Protein (mg/dL)	Trace	-	3	2	-
		30	3	2	-	2
		100	1	-	-
		Neg	1	-	3	3
	Nitrite	Neg	2	1	4	2
	Nitrite	Pos	3	4	1	3
	Leucocytes	Neg	5	3	5	5
	Leucocytes	Ca15	-	2	-	-
	Glucose (mg/dL)	Neg	5	5	5	5
	Microalbumin (mg/dL)	15	-	-	3	2
		>15	4	5	2	2
		Neg	1	-	-	1
Microscopic Examination	Epi Cells	0	3	3	3	3
		0-1	1	1	2	1
		1-2	1	1	-	1
	Casts	Absent	5	5	5	5
	Crystals	Present	5	5	5	5

M: Male; F: Female; R: Recovery; SD: Standard Deviation; Pos: Positive; Neg: Negative

Ca: Calculated; >=: Greater than or equal to; >: Greater than

Table 13. Summary of vaginal smear examination 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)
G1, F & 0	12	n	12	0	Mean	4.96
		%	100.00	0.00	±SD	0.14
					n	12
G2, F & 2	12	n	12	0	Mean	4.92
		%	100.00	0.00	±SD	0.19
					n	12
G3, F & 4.3	12	n	12	0	Mean	4.96
		%	100.00	0.00	±SD	0.14
					n	12
G4, F & 9.5	12	n	12	0	Mean	5.00
		%	100.00	0.00	±SD	0.00
					n	12

Table 14. Summary record of gestation body weight (g)

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Gestation Day (GD)
Group, Sex & Dose (mg/kg body weight/day)		0	7	14	20
G1, F & 0	Mean	278.31	293.77	322.21	382.79
	±SD	23.53	26.12	26.55	33.83
	n	11	11	11	11
G2, F & 2	Mean	275.69	292.67	325.49	388.21
	±SD	16.80	17.63	23.01	23.29
	n	12	12	12	12
G3, F & 4.3	Mean	268.09	286.56	316.82	375.04
	±SD	16.98	18.05	21.33	24.66
	n	11	11	11	11
G4, F & 9.5	Mean	268.90	279.30	292.77*	315.74*
	±SD	20.52	21.48	24.39	28.38
	n	9	9	9	9

n: Number of Litters

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

Table 15. Summary record of percent change in body weight (%) during gestation period

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight (%) during Gestation Day (GD)
Group, Sex & Dose (mg/kg body weight/day)		0 to 7	7 to 14	14 to 20
G1, F & 0	Mean	5.52	9.76	18.81
	±SD	1.01	2.30	3.72
	n	11	11	11
G2, F & 2	Mean	6.18	11.17	19.41
	±SD	1.61	2.33	4.18
	n	12	12	12
G3, F & 4.3	Mean	6.90	10.55	18.48
	±SD	1.54	1.80	5.04
	n	11	11	11
G4, F & 9.5	Mean	3.87	4.81*	7.82*
	±SD	1.93	2.73	3.18
	n	9	9	9

n: Number of Litters

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

Table 16. Summary of lactation body weight (g) record

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Lactation Day (LD)
Group, Sex & Dose (mg/kg body weight/day)		1	4	7	13
G1, F & 0	Mean	307.86	310.70	318.55	331.25
	±SD	27.73	29.72	30.50	29.30
	n	11	11	11	11
G2, F & 2	Mean	311.03	317.21	323.91	335.95
	±SD	23.47	23.94	23.20	21.92
	n	12	12	12	12
G3, F & 4.3	Mean	302.13	309.03	317.06	331.58
	±SD	21.59	22.31	22.33	23.37
	n	11	11	11	11
G4, F & 9.5	Mean	282.79	280.88*	281.10*	282.55*
	±SD	28.29	29.58	30.91	30.44
	n	9	9	9	9

n: Number of Litters

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

Table 17. Summary of percent change in body weight (%) during lactation

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight (%) during Lactation Day (LD)
Group, Sex & Dose (mg/kg body weight/day)		1 to 4	4 to 7	7 to 13
G1, F & 0	Mean	0.89	2.53	4.05
	±SD	1.38	1.20	1.25
	n	11	11	11
G2, F & 2	Mean	2.00	2.15	3.76
	±SD	1.89	1.26	1.26
	n	12	12	12
G3, F & 4.3	Mean	2.29	2.61	4.58
	±SD	1.28	0.84	1.06
	n	11	11	11
G4, F & 9.5	Mean	-0.70	0.04*	0.55*
	±SD	2.21	1.41	1.02
	n	9	9	9

n: Number of dams

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

Table 18.1. Summary of absolute organ weights (g) record. Males

Group, Sex & Dose (mg/kg body weight/day)		Adrenals	Thymus	Spleen	Testes	Epididymes	Heart	Kidneys	Brain	Liver
G1, M & 0	Mean	0.0573	0.3336	1.2240	3.4297	1.4279	1.3142	2.4930	1.9968	9.5906
	±SD	0.0059	0.0745	0.1263	0.1785	0.1078	0.1285	0.2223	0.1735	0.9855
	n	5	5	5	12	12	5	5	5	5
G2, M & 2	Mean	0.0580	0.3343	1.3110	3.4437	1.4561	1.4206	2.6853	2.0335	10.7475
	±SD	0.0015	0.0258	0.1323	0.2288	0.1167	0.1636	0.2341	0.1507	1.1692
	n	5	5	5	12	12	5	5	5	5
G3, M & 4.3	Mean	0.0573	0.3630	1.2304	3.5510	1.4691	1.3602	2.5710	2.0455	10.0981
	±SD	0.0052	0.0915	0.3067	0.2816	0.1595	0.1507	0.2624	0.0927	0.8477
	n	5	5	5	12	12	5	5	5	5
G4, M & 9.5	Mean	0.0622	0.2103*	1.0243	3.2469	1.4406	1.3828	2.4998	2.0719	10.2933
	±SD	0.0058	0.0219	0.1794	0.3584	0.1728	0.1683	0.3065	0.1339	1.7572
	n	5	5	5	12	12	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Prostate	Seminal vesicles with coagulating glands	Lungs	Thyroid along with parathyroid^W
G1, M & 0	Mean	1.4901	1.6335	2.2906	0.0201
	±SD	0.2713	0.3206	0.2613	0.0023
	n	12	12	5	12
G2, M & 2	Mean	1.4601	1.7087	2.3882	0.0211
	±SD	0.2423	0.4194	0.2229	0.0042
	n	12	12	5	12
G3, M & 4.3	Mean	1.3257	1.7352	2.3277	0.0208
	±SD	0.3358	0.3346	0.2665	0.0032
	n	12	12	5	12
G4, M & 9.5	Mean	1.4384	1.5831	2.4727	0.0215
	±SD	0.3947	0.4769	0.3709	0.0031
	n	12	12	5	12

n: Number of Animals (Adrenals, Thymus, Spleen, Heart, Kidneys, Brain & Liver weighed for 5 randomly selected animals; Testes, Epididymis, Prostate, Seminal vesicles with coagulating glands & Thyroid along with parathyroid weighed for all animals); w: Weighed post fixation.

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

Table 18.2. Summary of absolute organ weights (g) record. Females

Group, Sex & Dose (mg/kg body weight/day)		Adrenals	Thymus	Spleen	Heart	Kidneys	Brain	Liver	Lungs	Thyroid along with parathyroid^W
G1, F & 0	Mean	0.0901	0.2217	1.1327	1.2036	2.3634	2.1352	12.9287	2.1487	0.0205
	±SD	0.0102	0.0339	0.0932	0.1093	0.2518	0.0693	1.7899	0.2039	0.0029
	n	5	5	5	5	5	5	5	5	11
G2, F & 2	Mean	0.0820	0.2382	1.1168	1.2173	2.4303	2.0116	13.3214	1.9219	0.0215
	±SD	0.0058	0.0402	0.1109	0.0309	0.2635	0.1630	1.8291	0.0902	0.0027
	n	5	5	5	5	5	5	5	5	12
G3, F & 4.3	Mean	0.0840	0.2104	1.1447	1.1633	2.4926	2.1440	13.5957	2.0602	0.0221
	±SD	0.0040	0.0316	0.0650	0.1158	0.2005	0.1109	0.4648	0.1476	0.0023
	n	5	5	5	5	5	5	5	5	11
G4, F & 9.5	Mean	0.0764	0.1436*	0.9023*	1.1161	2.1686	2.0183	10.0050*	2.1364	0.0189
	±SD	0.0139	0.0455	0.1127	0.2021	0.2380	0.1481	2.1592	0.2409	0.0020
	n	5	5	5	5	5	5	5	5	9

n: Number of Animals (Adrenals, Thymus, Spleen, Heart, Kidneys, Brain & Liver weighed for 5 randomly selected animals; Thyroid along with parathyroid weighed for all pregnant females); w: Weighed post fixation

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

Table 19.1. Summary of terminal body weight (g) and organ weight (%) relative to terminal body weight record. Males

Group, Sex & Dose (mg/kg body weight/day)		Fasting Body Weight (g)	Adrenals	Thymus	Spleen	Testes	Epididymes	Heart	Kidneys	Brain	Liver
G1, M & 0	Mean	379.18	0.0157	0.0911	0.3352	0.9098	0.3791	0.3603	0.6816	0.5463	2.6199
	±SD	34.78	0.0022	0.0183	0.0346	0.0763	0.0419	0.0397	0.0398	0.0368	0.1617
	n	12	5	5	5	12	12	5	5	5	5
G2, M & 2	Mean	383.55	0.0150	0.0870	0.3398	0.9040	0.3819	0.3663	0.6946	0.5267	2.7859
	±SD	35.09	0.0012	0.0119	0.0384	0.0940	0.0396	0.0209	0.0523	0.0418	0.3293
	n	12	5	5	5	12	12	5	5	5	5
G3, M & 4.3	Mean	380.70	0.0161	0.1003	0.3408	0.9494	0.3900	0.3788	0.7206	0.5730	2.8115
	±SD	46.81	0.0025	0.0192	0.0772	0.1640	0.0541	0.0333	0.1154	0.0672	0.1383
	n	12	5	5	5	12	12	5	5	5	5
G4, M & 9.5	Mean	358.52	0.0170	0.0571*	0.2763	0.9101	0.4031	0.3738	0.6767	0.5630	2.7772
	±SD	38.07	0.0022	0.0054	0.0266	0.0953	0.0406	0.0148	0.0472	0.0368	0.2655
	n	12	5	5	5	12	12	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Prostate	Seminal vesicles with coagulating glands	Lungs	Thyroid along with parathyroid
G1, M & 0	Mean	0.3949	0.4328	0.6252	0.0053
	±SD	0.0759	0.0845	0.0408	0.0006
	n	12	12	5	12
G2, M & 2	Mean	0.3843	0.4516	0.6204	0.0056
	±SD	0.0834	0.1252	0.0815	0.0012
	n	12	12	5	12
G3, M & 4.3	Mean	0.3497	0.4565	0.6463	0.0055
	±SD	0.0816	0.0722	0.0253	0.0010
	n	12	12	5	12
G4, M & 9.5	Mean	0.4027	0.4475	0.6694	0.0060
	±SD	0.1107	0.1531	0.0767	0.0007
	n	12	12	5	12

*: Statistically significant than the control group (p<0.05)

Table 19.2. Summary of terminal body weight (g) and organ weight (%) relative to terminal body weight record. Females

Group, Sex & Dose (mg/kg body weight/day)		Fasting Body Weight (g)	Adrenals	Thymus	Spleen	Heart	Kidneys	Brain	Liver	Lungs	Thyroid along with parathyroid
G1, F & 0	Mean	317.65	0.0288	0.0711	0.3637	0.3847	0.7545	0.6852	4.1349	0.6897	0.0065
	±SD	29.29	0.0020	0.0125	0.0444	0.0254	0.0484	0.0561	0.5037	0.0817	0.0008
	n	11	5	5	5	5	5	5	5	5	11
G2, F & 2	Mean	322.35	0.0255	0.0739	0.3468	0.3788	0.7587	0.6249	4.1373	0.5973	0.0067
	±SD	22.14	0.0019	0.0103	0.0247	0.0152	0.1145	0.0370	0.4862	0.0087	0.0006
	n	12	5	5	5	5	5	5	5	5	12
G3, F & 4.3	Mean	317.65	0.0263	0.0655	0.3581	0.3625	0.7794	0.6706	4.2480	0.6447	0.0070
	±SD	23.71	0.0033	0.0083	0.0381	0.0309	0.0934	0.0684	0.3391	0.0744	0.0007
	n	11	5	5	5	5	5	5	5	5	11
G4, F & 9.5	Mean	266.69*	0.0275	0.0508*	0.3207	0.3946	0.7701	0.7192	3.5311	0.7648	0.0071
	±SD	25.13	0.0066	0.0149	0.0294	0.0507	0.0517	0.0559	0.4982	0.1233	0.0009
	n	9	5	5	5	5	5	5	5	5	9

*: Statistically significant than the control group (p<0.05)

Table 20. Summary of serum thyroxine (T4) hormone levels (ng/mL) record - adults

Group, Sex & Dose (mg/kg body weight/day)		Serum T4 Levels (ng/mL)
Group, Sex & Dose (mg/kg body weight/day)		Serum T4 Levels (ng/mL)
G1, M & 0	Mean	51.383
	±SD	6.023
	n	12
G2, M & 2	Mean	51.333
	±SD	3.501
	n	12
G3, M & 4.3	Mean	52.324
	±SD	5.705
	n	12
G4, M & 9.5	Mean	55.676
	±SD	7.908
	n	12

Group, Sex & Dose (mg/kg body weight/day)		Serum T4 Levels (ng/mL)
Group, Sex & Dose (mg/kg body weight/day)		Serum T4 Levels (ng/mL)
G1, F & 0	Mean	45.222
	±SD	3.897
	n	11
G2, F & 2	Mean	43.690
	±SD	5.786
	n	12
G3, F & 4.3	Mean	45.895
	±SD	3.977
	n	11
G4, F & 9.5	Mean	50.490
	±SD	7.106
	n	9

Table 21. Summary of serum thyroxine (T4) hormone levels (ng/ml) record - PND 4 pups

Group, Sex & Dose (mg/kg body weight/day)		Serum Thyroxine Hormone (T4) Levels (ng/mL)
Group, Sex & Dose (mg/kg body weight/day)		Serum Thyroxine Hormone (T4) Levels (ng/mL)
G1, F & 0	Mean	34.666
	±SD	8.503
	n	4
G2, F & 2	Mean	29.118
	±SD	3.612
	n	5
G3, F & 4.3	Mean	31.232
	±SD	4.869
	n	7
G4, F & 9.5	Mean	-
	±SD	-
	n	-

n: Number of Litters.

No pups were sacrificed from group G4, as the litter size is equal to or less than 10.

Table 22. Summary of serum thyroxine (T4) hormone levels (ng/ml) record - PND 13 pups

Group, Sex & Dose (mg/kg body weight/day)		Serum T4 Levels (ng/mL)
Group, Sex & Dose (mg/kg body weight/day)		Serum T4 Levels (ng/mL)
G1, F & 0	Mean	42.055
	±SD	3.692
	n	11
G2, F & 2	Mean	44.330
	±SD	4.328
	n	12
G3, F & 4.3	Mean	43.180
	±SD	4.290
	n	11
G4, F & 9.5	Mean	46.427
	±SD	11.722
	n	4

n: Number of Litters.

Table 23. Summary of gross pathology findings: adults

Sex		Males						Females
Route of administration		Oral Gavage
Vehicle/Test item		Vehicle Control	Test item			Vehicle Control	Test item	Vehicle Control	Test item			Vehicle Control	Test item
Parameters	Group	G1	G2	G3	G4	G1R	G4R	G1	G2	G3	G4	G1R	G4R
	Dose (mg/kg body weight/day)	0	2	4.3	9.5	0	9.5	0	2	4.3	9.5	0	9.5
	Number of animals	12	12	12	12	5	5	12	12	12	12	5	5
No. of dead rats during treatment or recovery period		0	0	0	0	0	0	0	0	0	0	0	0
No. of moribund sacrificed rats		0	0	0	0	0	0	0	0	0	0	0	0
No. of terminally sacrificed rats		12	12	12	12	5	5	12	12	12	12	5	5
No. of rats showing external gross pathologyfindings		0	0	0	4	0	0	0	0	0	9	0	0
Wet perineum		-	-	-	4	-	-	-	-	-	7	-	-
Wet perineum with blood stains around vaginal region		-	-	-	-	-	-	-	-	-	2	-	-

No. of rats showing internal gross pathologyfindings		0	0	0	0	0	0	0	0	0	2	0	0
Uterus with resorptions/dead fetuses		-	-	-	-	-	-	-	-	-	2	-	-

Table 24. Summary of gross pathology findings :pups

Group		G1		G2		G3		G4
Dose (mg/kg body weight/day)		0		2		4.3		9.5
Sex		Male	Female	Male	Female	Male	Female	Male	Female
Total No. of Live Pups at Birth		61	56	55	70	56	63	36	31

a. Total No. of Dead Pups at Birth		1	-	1		1	-	3	3
External Gross Pathology Findings	NAD	1	-	-	-	1	-	1	-
	Cannibalized	-	-	1	-	-	-	-	-
	Pale Colored	-	-	-	-	-	-	2	3
Internal Gross Pathology Findings	NAD	1	-	NA	-	1	-	3	3

b. Total No. of Dead Pups during postnatal Period		-	-	-	-	-	-	19	23
External Gross Pathology Findings	NAD	-	-	-	-	-	-	3	5
	Cannibalized	-	-	-	-	-	-	-	1
	Pale Colored	-	-	-	-	-	-	16	16
	Nose Discoloration, red	-	-	-	-	-	-	-	1
Internal Gross Pathology Findings	NAD	-	-	-	-	-	-	19	23

c. Total No. of pups sacrificed on PND 4		-	6	-	9	-	11	-	-
External Gross Pathology Findings	NAD	-	6	-	9	-	11	-	-
Internal Gross Pathology Findings	NAD	-	6	-	9	-	11	-	-

d. Total No. of pups sacrificed on PND 13		61	50	55	61	56	52	17	8
External Gross Pathology Findings	NAD	61	50	55	61	56	52	17	8
Internal Gross Pathology Findings	NAD	61	50	55	61	56	52	17	8

NAD: No Abnormality Detected; PND: Post-natal Day; - : No incidence; NA: Not Applicable

Table 25. Summary of histopathology findings: Adult animals (randomly selected animals)

Route of Administration		Oral Gavage
Vehicle /Test item		Control		Test item
Dose (mg/kg body weight/day)		0		9.5
Group		G1		G4
Sex		M	F	M	F
Number of Animals		5	5	5	5
Adrenals	Number examined	5	5	5	5
Adrenals	Within normal limits	4	4	5	5
Accessory cortical tissue	Present	1	-	-	-
Angiectasis	Minimal	-	1	-	-
Brain	Number examined	5	5	5	5
Brain	Within normal limits	5	5	5	5
Caecum	Number examined	5	5	5	5
Caecum	Within normal limits	5	5	5	5
Cervix	Number examined	X	5	X	5
Cervix	Within normal limits	-	5	-	5
Coagulating glands	Number examined	5	X	5	X
Coagulating glands	Within normal limits	5	-	5	-
Cowper’s glands	Number examined	5	X	5	X
Cowper’s glands	Within normal limits	5	-	5	-
Colon	Number examined	5	5	5	5
Colon	Within normal limits	5	5	5	5
Duodenum	Number examined	5	5	5	5
Duodenum	Within normal limits	5	5	5	5
Epididymides	Number examined	5	X	5	X
Epididymides	Within normal limits	5	-	5	-
Eyes with Optic nerve	Number examined	5	5	5	5
Eyes with Optic nerve	Within normal limits	5	5	5	5
Femur bone with joint	Number examined	5	5	5	5
Femur bone with joint	Within normal limits	5	5	-	-
Cellularity, decreased, bone marrow	Mild	-	-	5	5
Glans penis	Number examined	5	X	5	X
Glans penis	Within normal limits	5	-	5	-
Heart	Number examined	5	5	5	5
Heart	Within normal limits	3	3	5	5
Infiltrations, mononuclear cells, Ventricle	Minimal	2	2	-	-
Ileum with Peyer’s patches	Number examined	5	5	5	5
Ileum with Peyer’s patches	Within normal limits	5	5	5	5
Jejunum	Number examined	5	5	5	5
Jejunum	Within normal limits	5	5	5	5
Kidneys	Number examined	5	5	5	5
Kidneys	Within normal limits	4	5	3	4
Mineralization, Cortex	Minimal	1	-	-	-
Infiltrations, mononuclear cells, interstitial	Minimal	-	-	1	-
Dilatation, tubules	Minimal	-	-	-	1
Cast(s), medulla	Minimal	-	-	1	-
Levator ani plus bulbocavernous muscle complex	Number examined	5	X	5	X
Levator ani plus bulbocavernous muscle complex	Within normal limits	5	-	5	-

Route of administration		Oral Gavage
Vehicle /Test item		Control		Test item
Dose (mg/kg body weight/day)		0		9.5
Group		G1		G4
Sex		M	F	M	F
Number of Animals		5	5	5	5
Liver	Number examined	5	5	5	5
Liver	Within normal limits	2	3	4	3
Infiltrations, mononuclear cells	Minimal	3	2	1	2
Lungs	Number examined	5	5	5	5
Lungs	Within normal limits	3	4	5	5
Macrophages, increased, alveoli	Minimal	1	-	-	-
Infiltrations, mononuclear cells	Minimal	1	1	-	-
Mammary glands	Number examined	5	5	5	5
Mammary glands	Within normal limits	5	5	5	5
Mandibular lymph nodes	Number examined	5	5	5	5
Mandibular lymph nodes	Within normal limits	5	5	5	5
Mesenteric lymph nodes	Number examined	5	5	5	5
Mesenteric lymph nodes	Within normal limits	5	5	5	5
Ovaries	Number examined	X	5	X	5
Ovaries	Within normal limits	-	5	-	5
Parathyroid	Number examined	5	5	5	5
Parathyroid	Within normal limits	5	5	5	5
Pituitary gland	Number examined	5	5	5	5
Pituitary gland	Within normal limits	5	5	5	5
Prostate gland	Number examined	5	X	5	X
Prostate gland	Within normal limits	5	-	5	-
Rectum	Number examined	5	5	5	5
Rectum	Within normal limits	5	5	5	5
Sciatic nerve	Number examined	5	5	5	5
Sciatic nerve	Within normal limits	5	5	5	5
Seminal vesicles	Number examined	5	X	5	X
Seminal vesicles	Within normal limits	5	-	5	-
Skeletal muscle	Number examined	5	5	5	5
Skeletal muscle	Within normal limits	5	5	5	5
Spinal cord	Number examined	5	5	5	5
Spinal cord	Within normal limits	5	5	5	5
Spleen	Number examined	5	5	5	5
Spleen	Within normal limits	5	5	3	3
Cellularity, decreased, white pulp	Mild	-	-	2	2
Stomach	Number examined	5	5	5	5
Stomach	Within normal limits	4	4	4	4
Dilatation, gastric glands, glandular	Minimal	1	1	1	1
Testes	Number examined	5	X	5	X
Testes	Within normal limits	5	-	5	-

Route of administration		Oral Gavage
Vehicle /Test item		Control		Test item
Dose (mg/kg body weight/day)		0		9.5
Group		G1		G4
Sex		M	F	M	F
Number of Animals		5	5	5	5
Thymus	Number examined	5	5	5	5
Thymus	Within normal limits	5	5	3	3
Cellularity, decreased, lymphocyte, cortex	Mild	-	-	2	2
Thyroid	Number examined	5	5	5	5
Thyroid	Within normal limits	4	4	3	5
Ultimobranchial cyst (s)	Present	1	1	2	-
Trachea	Number examined	5	5	5	5
Trachea	Within normal limits	5	5	5	5
Urinary bladder	Number examined	5	5	5	5
Urinary bladder	Within normal limits	5	5	5	5
Uterus	Number examined	X	5	X	5
Uterus	Within normal limits	-	5	-	5
Vagina	Number examined	X	5	X	5
Vagina	Within normal limits	-	5	-	5

-: no incidence; X: not applicable.

Table 26. Summary of histopathology findings: Males. Reproductive organs and thyroid with parathyroid glands

Route of administration		Oral Gavage
Vehicle /Test item		Control	Test item
Dose (mg/kg body weight/day)		0	9.5
Group		G1	G4
Number of Animals		7	7
Thyroid	Number examined	7	7
Thyroid	Within normal limits	4	7
Ultimobranchial cyst(s)	Present	3	-
Parathyroid	Number examined	7	7
Parathyroid	Within normal limits	7	7
Coagulating glands	Number examined	7	7
Coagulating glands	Within normal limits	7	7
Cowper’s glands	Number examined	7	7
Cowper’s glands	Within normal limits	7	7
Glans penis	Number examined	7	7
Glans penis	Within normal limits	7	7
Levator ani plus bulbocavernous muscle complex	Number examined	7	7
Levator ani plus bulbocavernous muscle complex	Within normal limits	7	7
Epididymides	Number examined	7	7
Epididymides	Within normal limits	7	7
Prostate gland	Number examined	7	7
Prostate gland	Within normal limits	7	7
Seminal vesicles	Number examined	7	7
Seminal vesicles	Within normal limits	7	7
Testes	Number examined	7	7
Testes	Within normal limits	7	7

-: no incidence

Table 27. Summary of histopathology findings: Females. Reproductive organs and thyroid with parathyroid glands

Route of administration		Oral Gavage
Vehicle /Test item		Control	Test item
Dose (mg/kg body weight/day)		0	9.5
Group		G1	G4
Number of Animals		7	7
Thyroid	Number examined	7	7
Thyroid	Within normal limits	5	3
Ultimobranchial cyst(s)	Present	2	4
Parathyroid	Number examined	7	7
Parathyroid	Within normal limits	7	7
Ovaries	Number examined	7	7
Ovaries	Within normal limits	7	7
Uterus with Cervix	Number examined	7	7
Uterus with Cervix	Within normal limits	7	7

Table 28. Summary of histopathology findings: Record of lower doses and recovery groups

Route of administration		Oral Gavage
Sex		Male				Female
Dose (mg/kg body weight/day)		2	4.3	0	9.5	2	4.3	0	9.5
Group		G2	G3	G1R	G4R	G2	G3	G1R	G4R
Number of Animals		5	5	5	5	5	5	5	5
Spleen	Number examined	5	5	5	5	5	5	5	5
Spleen	Within normal limits	5	5	5	5	5	5	5	5
Thymus	Number examined	5	5	5	5	5	5	5	5
Thymus	Within normal limits	5	5	5	5	5	5	5	5
Femur bone with joint	Number examined	5	5	5	5	5	5	5	5
Femur bone with joint	Within normal limits	5	5	5	5	5	5	5	5

Conclusions:

Based on the findings of this study, the NOAEL of the test substance after a repeated oral exposure of 37 days in males and 64 days in females was determined to be 4.3 mg/kg bw/day.

Executive summary:

A combined repeated dose toxicity study with the reproduction /developmental toxicity screening test of the test substance POLY-U by oral administration in rats was conducted according to OECD guideline 422, in accordance with GLP principles. A total of 116 (58 males + 58 females) Sprague Dawley rats were distributed to four main groups and two recovery groups to detect delayed occurrence and recovery from toxic effects. Each main group (G1, G2, G3 and G4) consisted of 12 males and 12 females and each recovery group (G1R and G4R) consisted of 5 males and 5 females. The animals in G1/G1R group were administered with vehicle (Corn oil), and the animals in G2, G3 and G4/G4R groups were administered with test item at the dose levels of 2, 4.3 and 9.5 mg/kg bw/d, based on the results of two consecutive dose range finding studies performed with the same species for a period of 14 days at doses of 50, 100 and 300 mg/kg bw /d and 2.7, 8.3 and 25 mg/kg bw /d respectively. The test item was administered to the main group males for a period of 37 days (two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period), to the main group females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13 and to the recovery group animals for a period of 50 days with a 14 days recovery. The vehicle and test item formulations were administered orally by gavage at the dose volume of 5 mL/kg body weight. The stability of test item formulations in corn oil was established before initiation of the treatment. Dose formulation analysis for homogeneity and concentration verification was performed during weeks 1 and 5 of the treatment period and the results were within acceptable limits.

All the main group and recovery group animals were observed for clinical signs, mortality and morbidity, detailed clinical examination, body weight, feed consumption and ophthalmological and neurological/functional examinations. The clinical pathological (haematology, clinical chemistry and urinalysis) examinations were conducted for 5 randomly selected animals from each group per sex for main group and from all the animals for recovery group at termination. The gross pathology and organ weighing were performed on the day of termination for all the main and recovery group animals. All the main group females were observed for maternal body weight and feed consumption during gestation and lactation. The main group females were evaluated for oestrus cyclicity. Each dam was observed for mating performance, fertility performance, gestation length, litter size, number and percentage of live/dead pups, live birth index, sex ratio and observation of litter throughout the lactation period. Histopathological examination was conducted on all the tissues collected from the main group vehicle control and high dose group animals sacrificed at termination with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. The pups were observed once daily for clinical signs and external examinations, weighed individually on postnatal day (PND) 1, 4, 7 and 13, 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. The analysis of thyroxine hormone (T4) levels in serum collected at termination was performed for main group males and for PND 4 (from available pups) and PND 13 pups.

In groups G2 and G3, there were no indication of test item-related effects in any of the parameters assessed for systemic or reproductive toxicity.

In group G4/G4R, the animals were noted with test item-related clinical signs of toxicity such as, lethargic, rough hair coat, wet perineum, chromodacryorrhea, hair thinning or blood stains around vagina, as well as test item-related reduction in body weight and feed consumption. Also, test item-related reduced motor activity assessments, effects on clinical pathology investigations and reduced thymus spleen and liver weights were found at the dose level of 9.5 mg/kg bw/d. During gross pathology, test item-related changes like wet pernineum (4/12 males and 7/12 females), wet pernineum along with blood stains around vagina (2/12 females) and uterus with resorptions/dead fetuses (2/12 females) were noted from group G4. Microscopically, test item-related changes were observed in spleen, thymus and bone marrow of femur in both sexes at this dose level. Thus, the investigations were extended to the lower dose and recovery groups for these organs. Effects were not observed in these groups. All these changes in thymus, spleen and femoral bone marrow were considered to be reversible, secondary effects of reduced food consumption and decreased body weight gain (Everds et.al., 2013). The high dose recovery group animals treated with 9.5 mg/kg bw/day were recovered from the systemic toxicity effects during recovery period.

In group G4, test item-related reduced gestation index, increased post-implantation loss, increased postnatal loss, reduced litter size, reduced live birth index per litter, increased number of dead pups at birth / during postnatal period and reduced pup survival index were noted. Also, test item-related external anomalies in pups, reduced mean pup weight and ano-genital distance ratio per litter in either sex were noted. However, these reproductive effects were considered as secondary non-specific effects occurred as a result of systemic toxicity due to absence of adverse effects or no effects noted on major reproductive end points at this dose.

Therefore, based on the above results discussed, the NOAEL for the test item POLY-U is considered to be 4.3 mg/kg bw/day.

Cross-reference 4

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

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 February 2020 – 22 July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

GLP compliance:

yes (incl. QA statement)

Limit test:

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

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:

yes

Details on analytical verification of doses or concentrations:

Duration of treatment / exposure:

Main group males: two weeks pre-mating, during mating and up to the day before sacrifice during the post-mating period (total of 37 days of treatment).
Main group females main group: two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13.
Recovery animals: same period as for the main group females until the first scheduled sacrifice of dams and kept without treatment for a further 14 days observation.

Frequency of treatment:

Once a day

Details on study schedule:

F1 animals were not mated.

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

G1 - Vehicle control + G1R - Vehicle Control Recovery Group

Dose / conc.:

2 mg/kg bw/day (actual dose received)

Remarks:

G2 - Low dose

Dose / conc.:

4.3 mg/kg bw/day (actual dose received)

Remarks:

G3 - Mid dose

Dose / conc.:

9.5 mg/kg bw/day (actual dose received)

Remarks:

G4 - High dose + G4R - High dose Recovery Group

No. of animals per sex per dose:

Main Group: 12
Recovery Group: 5

Control animals:

yes, concurrent vehicle

Details on study design:

Positive control:

None

Parental animals: Observations and examinations:

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
prior to the treatment on day 1 and weekly thereafter during treatment for all the animals. Signs noted were included, but not be limited to, 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:
The main group animals were weighed at receipt, on the first day of dosing, weekly thereafter and at termination. The main group females were weighed on gestation days 0, 7, 14 and 20 during pregnancy and on days 1, 4, 7 and 13 during lactation period. The recovery group animals (both males and females) were weighed at receipt, on the first day of dosing, weekly thereafter and at termination.

FOOD CONSUMPTION:
yes
-feed consumption was measured for main group animals (both males and females) once a week during premating and weekly once for main group males during the post mating period. Feed consumption was not measured during the mating period for both males and females. Thereafter, feed consumption for main group 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. Feed consumption was measured for recovery group animals (both males and females) once a week throughout the experimental period. Average feed intake per rat (g/rat/day) was calculated using the amount of feed given and left over in each cage and the number of rats surviving in each cage.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once before treatment for all animals, at the end of the dosing period for males (shortly prior to scheduled sacrifice) and during the lactation period for females (shortly prior to scheduled sacrifice) of vehicle control and high dose main group animals and during the last week for the recovery group animals.
- Dose groups that were examined: vehicle control and high dose main group.

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
on the day of scheduled terminal sacrifice (for main group males on day 38, for main group females on lactation day 14 and for recovery group animals on the day 66).
- 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 and all recovery group animals.
- Parameters checked: Haemoglobin concentration (HGB), Haematocrit (HCT), Erythrocyte count (RBC), Total leukocyte count (WBC), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet count (PLT), Mean platelet volume (MPV), Reticulocyte count (Retic), Absolute reticulocyte count, 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 (for main group males on day 38, for main group females on lactation day 14 and for recovery group animals on the day 66).
- Animals fasted: Yes (water provided ad libitum)
- How many animals:
5 males and 5 females randomly selected from each main group and all recovery group animals.
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Cholinesterase, Total Protein, Albumin, Total bilirubin, Glucose, Total Cholesterol, Creatinine, Urea, Blood urea nitrogen (BUN), Triglycerides, Phosphorous, Calcium, Globulin. Sodium (mmol/L), Potassium (mmol/L) and Chloride (mmol/L) were estimated using a Na/K/Cl analyzer.

URINALYSIS: Yes
- Time schedule for collection of urine:
on the day of scheduled terminal sacrifice.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 5 randomly selected males of each main group and all recovery animals.
- Parameters checked: Blood, Bilirubin, Urobilinogen, Ketones, Protein, Glucose, Microalbumin, Leucocytes. In addition pH, nitrite 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.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Towards the end of the dosing period for males (on day 37) and during lactation period for females (on lactation day 13). Towards the end of the recovery period (shortly prior to scheduled sacrifice on day 65) for the recovery group.
- Dose groups that were examined: all main and recovery groups.
- Battery of functions tested: Home Cage Observations (convulsions, tremors and palpebral closure), Handling Observations, Open Field Observations, Sensory Observations, Neuromuscular Observations, Physiological Observation (Rectal temperature), Grip strength assessment and Motor activity assessment.

OTHER:

Thyroxine Hormone (T4) Level estimation: Blood samples were collected for measurement of serum T4 levels on the following schedule:
a. Two pups per litter on lactation day 4 based on the following conditions:
- Two female pups in order to retain more male pups for nipple retention on PND 13.
- No pups were eliminated when litter size dropped below 10 pups/litter.
- Only one pup was eliminated and used for blood collection in case of only one pup was available above ten.
b. All main group females (dams) at termination (lactation day 14).
c. Two pups per litter (same sex) at termination (lactation day 13).
d. All adult main group males, at termination (after completion of 37 days of treatment).

Oestrous cyclicity (parental animals):

Estrous cycles were monitored during the acclimatization to evaluate its normal oestrous cyclicity (4 to 5 days). Only females with normal oestrous cyclicity were selected for the treatment. For the main group females, the vaginal smears were monitored daily from the beginning of the treatment period until evidence of mating. Oestrous cyclicity was also monitored on the day of sacrifice for main group females.

Sperm parameters (parental animals):

Parameters examined in all parental male in the control and high dose groups: testis and epididymis weight. Also, a detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen or any cell or stage specificity of testicular findings.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- maximum of 10 pups/litter as nearly as possible; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
The number of pups born (dead and live) in a litter, sex and external observations were recorded at birth. Individual body weight of live pups on lactation day 1 (within 24 hours of parturition), 4, 7 and 13 was recorded. The anogenital distance of each pup was measured on postnatal day 4 (lactation day 4). The number of nipples/areolae in male pups was counted on postnatal day 13 (lactation day 13). Fertility index for dams and sires, pup survival index and sex ratio at birth were calculated.
Also, blood samples were collected for measurement of serum T4 levels on the following schedule:
1. Two pups per litter on lactation day 4 based on the following conditions:
- Two female pups in order to retain more male pups for nipple retention on PND 13.
- No pups were eliminated when litter size dropped below 10 pups/litter.
- Only one pup was eliminated and used for blood collection in case of only one pup was available above ten.
2. Two pups per litter (same sex) at termination (lactation day 13).

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after completion of 36 days of treatment.
- Maternal animals: All surviving animals on lactation day 14
- Recovery animals: All surviving animals after completion of 14 days observation from the first scheduled sacrifice of dams.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Tables 1 and 2 were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring was sacrificed on lactation day 4 (those pups selected for blood collection) and on lactation day 13 (the rest of pups)
- These animals were subjected to postmortem examinations (macroscopic examination) as follows:

GROSS NECROPSY
- The dead pups and pups which were sacrificed on PND 4/13 were examined for gross abnormalities (external and internal examinations) with particular attention to the external reproductive genitals.

Statistics:

Reproductive indices:

See table 3 below.

Offspring viability indices:

See table 3 below.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

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, 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 at groups G2 and G3 in both sexes throughout the experimental period.

In group G4, there were no changes noted in mean body weight and percent change in mean body weight gain with respect to day 1 till week 3 in both sexes. However, reduction in mean body weight and percent change in mean body weight gain was noted from week 4 onwards and continued til l termination. Even though these reductions were not statistically significant, these changes are considered as test item-related due to noted test item-related clinical signs and test item-related effects in neurological observations during these periods.

In group G4R, there were no changes noted in mean body weight and percent change in mean body weight gain with respect to day 1 till week 3 in both sexes. However, reduction in mean body weight and percent change in mean body weight gain was noted from week 4 onwards and continued till end of dosing period. Furthermore, some of these reductions were found to be statistically significant compared to the control group. These changes are considered as test item-related due to noted test item-related clinical signs and test item-related decreased mean feed consumption during these periods. However, the body weight and body weight gains were started to recover during recovery period in both sexes.

In group G4, reduction in mean body weight and percent change in mean body weight gain was noted throughout the gestation period which is statitistically significant on gestation day (GD) 14 and 20 for mean body weight and statitistically significant during GD 7 to 14 and 14 to 20 for percent change in mean body weight gain when compared with vehicle control group. These reductions are considered as test item-related due to noted test item-related clinical signs and test item-related reduction in feed consumption during these periods.

In group G4, reduction in mean body weight and percent change in mean body weight gain was noted throughout the lactation period which is statitistically significant on lactation day (LD) 4, 7 and 13 for mean body weight and statitistically significant during LD 4 to 7 and 7 to 13 for percent change in mean body weight gain when compared with vehicle control group. These reductions are considered as test item-related due to noted test item-related clinical signs and test item-related reduction in feed consumption during these periods.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There were no changes noted in mean feed consumption at groups G2 and G3 in both sexes during pre-mating, gestation and lactation periods when compared with vehicle control group.

In group G4, there were no changes noted in mean feed consumption in both sexes during pre-mating period when compared with vehicle control group. However, reductions in mean feed consumption were noted during gestation and lactation periods which are statitistically significant during GD 14 to 20 and during LD 1 to 4, 4 to 7 and 7 to 13, when compared with vehicle control group. These reductions are considered as test item-related due to noted test item-related clinical signs and test item related reduction in body weights during these periods.

In group G4R, there were no changes noted in mean feed cosnumption till week 3 in both sexes. However, reduction in mean feed consumption was noted from week 4 onwards and continued till end of dosing period. These reductions are statistically significant during week 5 to 9 in G4R males; statistically significant during week 6 in G4R females when compared with respective vehicle control group. These changes are considered as test item-related due to noted test item-related clinical signs, decreased mean body weight and or percent body weight gain during these periods in both sexes. However, the mean feed consumption was started to recover during recovery period in both sexes.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Serum Thyroxine (T4) Levels: There were no changes noted in T4 levels of males and dams of main groups when compared with vehicle control group. The obtained T4 levels are within the normal range of this species and strain.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no irregularities observed in the oestrus cyclicity of main group females during pre-mating period at any of the tested dose groups. The mean length of oestrus cycle (days) per dam during pre-mating period was unaffected at all the tested dose groups when compared with vehicle control group.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No effects were detected during examination of spermatogenesis stages in the male gonads and histopathology of interstitial testicular cell structure.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Gestation Index (%): A total of 11 (out of 11), 12 (out of 12), 11 (out of 11), and 9 (out of 11) females were confirmed with live born pups with a gestation index of 100.0%, 100.0%, 100.0% and 81.8% from group G1, G2, G3 and G4, respectively. There were no statistically significant differences noted for this parameter in any of the tested dose groups when compared with the vehicle control group. However, in group G4 the noted reduction in gestation index is considered as test item-related, as 2 out of 11 fertile females were noted with total litter loss with evidence of implantation sites only and also this can be correlated with increased pup mortalities and reduced pup survival index from other littered females of same dose group.

Post-Implantation Loss (%): A mean number of 0.18, 0.25, 0.18 and 5.11 post-implantation losses with a percentage of 1.82%, 2.68%, 1.53% and 38.27% were noted from groups G1, G2, G3 and G4, respectively. In group G4, the post-implantation loss was statistically significant in both number and percentage when compared with vehicle control group. This noted change is considered as test item-related, due to noted increase in number of resorptions, test item-related effects in litter observations or test item-related maternal effects during gestation and lactation periods.

Post-Natal Loss (%): In group G4, the mean number of post-natal loss was noted as 4.67 per litter (0 in control group) with a percentage of 62.47% (0% in control group). These noted increase in post-natal losses were statistically significant in both number and percentage when compared with vehicle control group. These effects are considered as test item-related, due to noted test item-related increase in number of pup mortalities, increased resorptions, effects in litter observations during lactation period, reduced pup survival index, reduced pup weights and also due to noted test item-related maternal effects during gestation and lactation periods.

The reproductive effects observed at the highest dose level of 9.5 mg/kg bw/day were considered as secondary non-specific effects occurred as a result of systemic toxicity due to absence of adverse effects or no effects noted on major reproductive end points at this dose.

Key result

Dose descriptor:

LOAEL

Effect level:

9.5 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive performance

Key result

Dose descriptor:

NOAEL

Effect level:

4.3 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive performance

Remarks on result:

other: Effects observed at the highest tested dose of 9.5 mg/kg bw/day.

Key result

Critical effects observed:

Lowest effective dose / conc.:

9.5 mg/kg bw/day (actual dose received)

System:

immune system

Organ:

bone marrow

spleen

thymus

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no external anomalies or no behaviour changes noted in any of the pups during daily observation of pups from tested dose groups G2, G3 and vehicle control group litters during post-natal period. All the pups were noted with normal behaviour during daily observations.

In group G4, test item-related external anomalies and mortalities were noted during post-natal period. These changes are considered as test item-related due to noted effects on maternal body weights and feed consumption of litters at this dose level and also these findings can be correlate with reduced mean pup weights.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

In group G4, reduced litter size at birth, reduced live birth index per litter, reduced number of live pups during lactation period in either sex, increased number of dead pups at birth and during lactation period and reduced pup survival index during lactation period were noted when compared with vehicle control group. These noted changes are statistically significant for number of live born female pups, number of survived male pups per litter during LD 1 to 4, number of survived female pups per litter during LD 1 to 4, 5 to 7 and 8 to 13, number of dead pups per litter during LD 1 to 4 and 5 to 7 and pup survival index between LD 1 to 4, 5 to 7 and 8 to 13 when compared with vehicle control group. These noted changes at group G4 are considered as test item-related due to noted test item-related clinical signs, test item-related reduction in maternal body weights and feed consumption during gestation and lactation periods.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In group G4, statistically significant reduction in mean pup (both male and female) weight per litter on PND 1, 4, 7 and 13 were noted. These changes are considered as test item-related due to increased pup mortalities, occurrence of external changes in pups and decreased pup survival index per litter at this dose level. These effects can also correlate with test item-related maternal effects on litters during gestation and lactation periods.

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):

effects observed, treatment-related

Description (incidence and severity):

In group G4, reduction in mean pup (both male and female) anogenital distance measurement (mm) and ratio per litter were noted. Changes in AGD ratio were found statistically significant in both sexes. These changes are considered as test item-related due to increased pup mortalities, decreased growth/weight of pups, occurrence of external changes in pups and decreased pup survival index per litter at this dose level. These effects can also correlate with test item-related maternal effects on litters during gestation and lactation periods.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

There were no occurrences of nipple/arealaes in any of the male pups examined on their PND 13 from any of the tested dose group litters and vehicle control group litters.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At necropsy, the dead pups which were found dead by birth and during lactation period noted with pale discoloration and red discoloration at nasal region from group G4 litters. No gross pathological changes were noted in any of the survived pups from group G4 and all pups from other dose group litters.

Histopathological findings:

not examined

Description (incidence and severity):

The histopathological examination of the thyroid gland pups was not conducted as there were no changes noted in this organ during miscoscopic examination and no effects noted in absolute or relative weight in adults of both sexes.

Other effects:

no effects observed

Description (incidence and severity):

Serum Thyroxine (T4) Levels: There were no changes noted in T4 levels of PND 13 pups in any of the tested dose groups when compared with vehicle control group. The obtained T4 levels of PND 13 pups are within the normal range of this species and strain. There were no changes noted in T4 levels of PND 4 pups from the tested dose groups G2 and G3 when compared with vehicle control group. In group G4, serum T4 levels of PND 4 pups was not conducted as there were no surplus pups remained from this dose level on PND 4. However, the obtained T4 levels of PND 4 pups from groups G2 and G3 are within the normal range of this species and strain.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

The reproductive effects observed at the highest dose level of 9.5 mg/kg bw/day were considered as secondary non-specific effects occurred as a result of systemic toxicity in parental animals due to absence of adverse effects or no effects noted on major reproductive end points at this dose.

Key result

Dose descriptor:

LOAEL

Generation:

Effect level:

9.5 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

body weight and weight gain

gross pathology

Key result

Dose descriptor:

NOAEL

Generation:

Effect level:

4.3 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

body weight and weight gain

gross pathology

Remarks on result:

other: Effects observed at the highest tested dose of 9.5 mg/kg bw/day.

Key result

Critical effects observed:

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

9.5 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Table 5. Summary of vaginal smear examination 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)
G1, F & 0	12	n	12	0	Mean	4.96
		%	100.00	0.00	±SD	0.14
					n	12
G2, F & 2	12	n	12	0	Mean	4.92
		%	100.00	0.00	±SD	0.19
					n	12
G3, F & 4.3	12	n	12	0	Mean	4.96
		%	100.00	0.00	±SD	0.14
					n	12
G4, F & 9.5	12	n	12	0	Mean	5.00
		%	100.00	0.00	±SD	0.00
					n	12

Table 6. 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 Fertiltiy Index (%)
G1, M & 0	11 (12)	91.7	11 (12)	91.7
G2, M & 2	11 (12)	91.7	11 (12)	91.7
G3, M & 4.3	11 (12)	91.7	11 (12)	91.7
G4, M & 9.5	11 (12)	91.7	11 (12)	91.7

Group, Sex & Dose (mg/kg body weight/day)		Cohabitation Record and Pre-coital Interval (Mean Time to Mating)				Gestational Length (duration of pregnancy)
Group, Sex & Dose (mg/kg body weight/day)		Pre-coital Interval (Days)		Concieving Days (1 to 5)	Concieving Days (More than 5 days)	Gestation Length (Days)
G1, F & 0	Mean	9.08	n	5	7	22.09
	±SD	6.05	%	41.67	58.33	0.54
	n	12	%	41.67	58.33	11
G2, F & 2	Mean	6.75	n	7	5	22.17
	±SD	5.33	%	58.33	41.67	0.39
	n	12	%	58.33	41.67	12
G3, F & 4.3	Mean	7.58	n	6	6	22.18
	±SD	5.70	%	54.55	54.55	0.60
	n	12	%	54.55	54.55	11
G4, F & 9.5	Mean	6.33	n	7	5	22.11
	±SD	5.38	%	58.33	41.67	0.33
	n	12	%	58.33	41.67	9

n: Number of dams

Group, Sex & Dose (mg/kg body weight/day)	Female Mating Index (%)		Female Fertiltiy Index (%)
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 Fertiltiy Index (%)
G1, F & 0	12 (12)	100.0	11 (12)	91.7
G2, F & 2	12 (12)	100.0	12 (12)	100.0
G3, F & 4.3	12 (12)	100.0	11 (12)	91.7
G4, F & 9.5	12 (12)	100.0	11 (12)	91.7

Group, Sex & Dose (mg/kg body weight/day)	Female Fecundity or Pregnancy Index (%)			Gestation Index (%)
Group, Sex & Dose (mg/kg body weight/day)	No. of Pregnant Females	No. of Females Confirmed with Mating	Female Fecundity or Pregnancy Index (%)	Females with Live Born Pups at Parturation	No. of Females with Evidence of Pregnancy	Gestation Index (%)
G1, F & 0	11	12	91.7	11	11	100.0
G2, F & 2	12	12	100.0	12	12	100.0
G3, F & 4.3	11	12	91.7	11	11	100.0
G4, F & 9.5	11	12	91.7	9	11	81.8

Group, Sex & Dose (mg/kg body weight/day)		Post-implantation Loss (%)				Post-natal 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	Post-natal Loss (%)
G1, F & 0	Mean	10.82	10.64	0.18	1.82	0.00	0.00
	±SD	1.54	1.69	0.40	4.05	0.00	0.00
	n	11	11	11	11	11	11
G2, F & 2	Mean	10.67	10.42	0.25	2.68	0.00	0.00
	±SD	1.78	1.98	0.62	7.32	0.00	0.00
	n	12	12	12	12	12	12
G3, F & 4.3	Mean	11.00	10.82	0.18	1.53	0.00	0.00
	±SD	1.67	1.60	0.40	3.41	0.00	0.00
	n	11	11	11	11	11	11
G4, F & 9.5	Mean	12.56	7.44	5.11*	38.27*	4.67*	62.47*
	±SD	2.07	3.13	4.37	28.23	4.30	44.96
	n	9	9	9	9	9	9

n: Number of dams

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

Table 7. Summary of delivery and litter observation record during lactation period

Group, Sex & Dose (mg/kg body weight/day)		Litter Size (No.)	Live Pups (No.)			Dead Pups (No.)			Cannibalized Pups (No.)				Sex Ratio (m/f) at Birth	Live Birth Index (%)
Group, Sex & Dose (mg/kg body weight/day)		Litter Size (No.)	Male	Female	Total	Male	Female	Total	Undetermined	Male	Female	Total	Sex Ratio (m/f) at Birth	Live Birth Index (%)
G1, F & 0	Mean	10.73	5.55	5.09	10.64	0.09	0.00	0.09	0.00	0.00	0.00	0.00	1.34	99.09
	±SD	1.62	2.11	1.64	1.69	0.30	0.00	0.30	0.00	0.00	0.00	0.00	1.01	3.02
	n	11	11	11	11	11	11	11	11	11	11	11	11	11
G2, F & 2	Mean	10.50	4.58	5.83	10.42	0.00	0.00	0.00	0.00	0.08	0.00	0.08	1.15	99.40
	±SD	2.11	0.90	2.08	1.98	0.00	0.00	0.00	0.00	0.29	0.00	0.29	1.28	2.06
	n	12	12	12	12	12	12	12	12	12	12	12	12	12
G3, F & 4.3	Mean	10.91	5.09	5.73	10.82	0.09	0.00	0.09	0.00	0.00	0.00	0.00	0.99	99.30
	±SD	1.70	1.30	1.68	1.60	0.30	0.00	0.30	0.00	0.00	0.00	0.00	0.44	2.32
	n	11	11	11	11	11	11	11	11	11	11	11	11	11
G4, F & 9.5	Mean	8.11	4.00	3.44*	7.44	0.33	0.33	0.67	0.00	0.00	0.00	0.00	1.17	90.43
	±SD	2.67	1.73	2.24	3.13	0.71	0.71	1.32	0.00	0.00	0.00	0.00	0.86	21.83
	n	9	9	9	9	9	9	9	9	9	9	9	9	9

n: Number of Dams; m/f: Male/Female;

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

Note: A total of 9 dams were considered for mean calculations of all end points/parameters at birth from group G4 as 1 out of 12 dams confirmed as non-pregnant; 2 out of 12 dams were confirmed with total litter loss [postimplantation loss].

Group, Sex & Dose (mg/kg body weight/day)		During LD 1 to 4									Sex Ratio (m/f) on LD 4	Pup Survival Index (%) LD 1 to 4
		Live Pups (No.)			Dead Pups (No.)			Cannibalized (No.)
		Male	Female	Total	Male	Female	Total	Male	Female	Total
G1, F & 0	Mean	5.55	5.09	10.64	0.00	0.00	0.00	0.00	0.00	0.00	1.34	100.00
	±SD	2.11	1.64	1.69	0.00	0.00	0.00	0.00	0.00	0.00	1.01	0.00
	n	11	11	11	11	11	11	11	11	11	11	11
G2, F & 2	Mean	4.58	5.83	10.42	0.00	0.00	0.00	0.00	0.00	0.00	1.15	100.00
	±SD	0.90	2.08	1.98	0.00	0.00	0.00	0.00	0.00	0.00	1.28	0.00
	n	12	12	12	12	12	12	12	12	12	12	12
G3, F & 4.3	Mean	5.09	5.73	10.82	0.00	0.00	0.00	0.00	0.00	0.00	0.99	100.00
	±SD	1.30	1.68	1.60	0.00	0.00	0.00	0.00	0.00	0.00	0.44	0.00
	n	11	11	11	11	11	11	11	11	11	11	11
G4, F & 9.5	Mean	1.89*	1.22*	3.11*	2.11*	2.11*	4.22*	0.00	0.11	0.11	0.85	40.89*
	±SD	2.42	1.39	3.72	2.47	2.37	4.29	0.00	0.33	0.33	1.13	46.69
	n	9	9	9	9	9	9	9	9	9	9	9

n: Number of Dams; m/f: Male/Female;

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

Note: A total of 9 dams were considered for mean calculations of all end points/parameters during LD 1 to 4 from group G4 as 1 out of 12 dams confirmed as non-pregnant;

2 out of 12 dams were confirmed with total litter loss [postimplantation loss].

Group, Sex & Dose (mg/kg body weight/day)	Animal No.	Pups Sacrificed for Blood Collection on LD 4 (No.)			Live Pups (No.) on LD 4 after Sacrificed for Blood Collection			During Lactation Day 5 to 7									Sex Ratio (m/f) on LD 7	Pup Survival Index (%) during LD 5 to 7
		Pups Sacrificed for Blood Collection on LD 4 (No.)						Live Pups per litter (No.)			Dead Pups per litter (No.)			Cannibilized Pups per litter (No.)
		Male	Female	Total	Male	Female	Total	Male	Female	Total	Male	Female	Total	Male	Female	Female
G1, F & 0	Mean	0.00	0.55	0.55	5.55	4.55	10.09	5.55	4.55	10.09	0.00	0.00	0.00	0.00	0.00	0.00	1.52	100.00
	±SD	0.00	0.82	0.82	2.11	1.44	1.04	2.11	1.44	1.04	0.00	0.00	0.00	0.00	0.00	0.00	1.14	0.00
	n	11	11	11	11	11	11	11	11	11	11	11	11	11	11	11	11	11
G2, F & 2	Mean	0.00	0.75	0.75	4.58	5.08	9.67	4.58	5.08	9.67	0.00	0.00	0.00	0.00	0.00	0.00	1.25	100.00
	±SD	0.00	0.97	0.97	0.90	1.62	1.30	0.90	1.62	1.30	0.00	0.00	0.00	0.00	0.00	0.00	1.26	0.00
	n	12	12	12	12	12	12	12	12	12	12	12	12	12	12	12	12	12
G3, F & 4.3	Mean	0.00	1.00	1.00	5.09	4.73	9.82	5.09	4.73	9.82	0.00	0.00	0.00	0.00	0.00	0.00	1.30	100.00
	±SD	0.00	0.89	0.89	1.30	1.42	0.98	1.30	1.42	0.98	0.00	0.00	0.00	0.00	0.00	0.00	0.95	0.00
	n	11	11	11	11	11	11	11	11	11	11	11	11	11	11	11	11	11
G4, F & 9.5	Mean	0.00	0.00	0.00	3.40	2.20	5.60	3.40	1.80*	5.20	0.00	0.40*	0.40*	0.00	0.00	0.00	1.70	77.50*
	±SD	0.00	0.00	0.00	2.30	1.10	3.21	2.30	1.30	3.42	0.00	0.55	0.55	0.00	0.00	0.00	1.20	43.66
	n	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5

n: Number of Dams; LD: Lactation Day; m/f: Male/Female

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

Note: A total of 5 dams were considered for mean calculations of all end points/parameters during LD 5 to 7 from group G4 as 4 out of 9 dams were noted with total postnatal pup loss during LD 1 to 4.

Group, Sex & Dose (mg/kg body weight/day)	Animal No.	During Lactation Day 8 to 13									Sex Ratio (m/f) at 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
G1, F & 0	Mean	5.55	4.55	10.09	0.00	0.00	0.00	0.00	0.00	0.00	1.52	100.00
	±SD	2.11	1.44	1.04	0.00	0.00	0.00	0.00	0.00	0.00	1.14	0.00
	n	11	11	11	11	11	11	11	11	11	11	11
G2, F & 2	Mean	4.58	5.08	9.67	0.00	0.00	0.00	0.00	0.00	0.00	1.25	100.00
	±SD	0.90	1.62	1.30	0.00	0.00	0.00	0.00	0.00	0.00	1.26	0.00
	n	12	12	12	12	12	12	12	12	12	12	12
G3, F & 4.3	Mean	5.09	4.73	9.82	0.00	0.00	0.00	0.00	0.00	0.00	1.30	100.00
	±SD	1.30	1.42	0.98	0.00	0.00	0.00	0.00	0.00	0.00	0.95	0.00
	n	11	11	11	11	11	11	11	11	11	11	11
G4, F & 9.5	Mean	4.25	2.00*	6.25	0.00	0.25	0.25	0.00	0.00	0.00	2.38	97.22*
	±SD	1.50	0.82	1.71	0.00	0.50	0.50	0.00	0.00	0.00	0.95	5.56
	n	4	4	4	4	4	4	4	4	4	4	4

n: Number of Dams; LD: Lactation Day; m/f: Male/Female;

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

Note: A total of 4 dams were considered for mean calculations of all end points/parameters during LD 8 to 13 from group G4 as 1 out of 5 dams were noted with total postnatal pup loss during LD 5 to 7.

Table 8. Summary record of pup observations during post-natal period

Group, Sex & Dose (mg/kg body weight/day)		At Birth (PND 1)	PND 1 to 4	Pups Sacrificed for Blood Collection on PND 4 (No.)*	PND 5 to 7^$	PND 8 to 13^$
G1, F & 0	No. of Dams^#	11	11	4	11	11
	No. of Live Pups	117	117	6	111	111
	Pup Observation/ No. of Pups observed	N/117	N/117	-	N/111	N/111
G2, F & 2	No. of Dams^#	12	12	5	12	12
	No. of Live Pups	125	125	9	116	116
	Pup Observation/ No. of Pups observed	N/125	N/125	-	N/116	N/116
G3, F & 4.3	No. of Dams^#	11	11	7	11	11
	No. of Live Pups	119	119	11	108	108
	Pup Observation/ No. of Pups observed	N/119	N/119	-	N/108	N/108
G4, F & 9.5	No. of Dams^#	9	9	0	9	9
	No. of Live Pups	67	28	0	26	25
	Pup Observation/ No. of Pups observed	P/28; HS/1; N/38	L/2; N/26	-	L, P/1; N/25	N/25

N: Normal; PND: Post-natal Day; #: Dams confirmed with littering; P: Pale colored; HS: Haemorrhagic spot; L: Lethargic

*: Pups selected for blood collection from dams with litter size of more than 10; $: Pups sacrificed on PND 4 were excluded

Table 9. 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
G1, F & 0	Mean	6.75	6.44	11.42	10.64	16.25	15.26	29.26	27.87
	±SD	0.09	0.17	0.36	0.20	0.47	0.35	0.51	0.82
	n	11	11	11	11	11	11	11	11
G2, F & 2	Mean	6.82	6.57	11.58	10.81	16.51	15.60	29.58	28.56
	±SD	0.14	0.16	0.37	0.19	0.37	0.40	0.25	0.40
	n	12	12	12	12	12	12	12	12
G3, F & 4.3	Mean	6.78	6.48	11.50	10.72	16.54	15.44	29.47	28.13
	±SD	0.12	0.18	0.32	0.23	0.30	0.33	0.33	0.65
	n	11	11	11	11	11	11	11	11
G4, F & 9.5	Mean	3.94*	3.73*	6.95*	5.70*	10.88*	9.11*	19.99*	17.88*
	±SD	0.89	0.80	1.79	1.91	3.28	2.75	4.47	4.49
	n	9	8	4	5	4	4	4	4

n: Number of litters

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

Table 10. Summary record of mean pup ano-genital distance (AGD) measurement (mm) and ano-genital distance (AGD) ratio per litter on post-natal day 4

Group, Sex & Dose (mg/kg body weight/day)		Mean Male Pup AGD Measurement (mm)	Mean Female Pup AGD Measurement (mm)	Mean Male Pup AGD Ratio	Mean Female Pup AGD Ratio
G1, F & 0	Mean	5.43	2.69	2.41	1.22
	±SD	0.22	0.12	0.08	0.05
	n	11	11	11	11
G2, F & 2	Mean	5.42	2.77	2.39	1.25
	±SD	0.25	0.13	0.09	0.05
	n	12	12	12	12
G3, F & 4.3	Mean	5.34	2.70	2.37	1.23
	±SD	0.19	0.13	0.07	0.05
	n	11	11	11	11
G4, F & 9.5	Mean	4.07	1.78	2.14*	1.00*
	±SD	0.61	0.41	0.15	0.15
	n	4	5	4	5

n: Number of litters

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

Table 11. Summary of male pup nipple/areolae retention (no.) record

Group, Sex & Dose (mg/kg body weight/day)		Mean No. of Pups with Retention of Nipples/ Areolae on Post-natal Day 13
G1, F & 0	Mean	0.00
	±SD	0.00
	n	11
G2, F & 2	Mean	0.00
	±SD	0.00
	n	12
G3, F & 4.3	Mean	0.00
	±SD	0.00
	n	11
G4, F & 9.5	Mean	0.00
	±SD	0.00
	n	9

n: Number of litters

Conclusions:

The NOAEL of the test substance for parental and reproductive toxicity in rats by oral route was determined to be 4.3 mg/kg bw/day. The reproductive effects observed at the highest dose level of 9.5 mg/kg bw/day were considered as secondary non-specific effects occurred as a result of systemic toxicity in parental animals due to absence of adverse effects or no effects noted on major reproductive end points at this dose.

Executive summary:

A combined repeated dose toxicity study with the reproduction /developmental toxicity screening test of the test substance POLY-U by oral administration in rats was conducted according to OECD guideline 422, in accordance with GLP principles. A total of 116 (58 males + 58 females) Sprague Dawley rats were distributed to four main groups and two recovery groups to detect delayed occurrence and recovery from toxic effects. Each main group (G1, G2, G3 and G4) consisted of 12 males and 12 females and each recovery group (G1R and G4R) consisted of 5 males and 5 females. The animals in G1/G1R group were administered with vehicle (Corn oil), and the animals in G2, G3 and G4/G4R groups were administered with test item at the dose levels of 2, 4.3 and 9.5 mg/kg bw/d based on the results of two consecutive dose range finding studies performed with the same species for a period of 14 days. at doses of 50, 100 and 300 mg/kg bw /d and 2.7, 8.3 and 25 mg/kg bw /d respectively. The test item was administered to the main group males for a period of 37 days (two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period), to the main group females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13 and to the recovery group animals for a period of 50 days with a 14 days recovery. The vehicle and test item formulations were administered orally by gavage at the dose volume of 5 mL/kg body weight. The stability of test item formulations in corn oil was established before initiation of the treatment. Dose formulation analysis for homogeneity and concentration verification was performed during weeks 1 and 5 of the treatment period and the results were within acceptable limits.

In groups G2 and G3, there were no indication of test item-related effects in any of the parameters assessed for systemic or reproductive toxicity.

In group G4, test item-related reduced gestation index, increased post-implantation loss, increased postnatal loss, reduced litter size, reduced live birth index per litter, increased number of dead pups at birth / during postnatal period and reduced pup survival index were noted. Also, test item-related external anomalies in pups, test item-related reduced mean pup weight and ano-genital distance ratio per litter in either sex were noted. However, there were no occurrences of nipples in male pups examined on PND 13 from all the litters. The pups which were found dead by birth and during postnatal period were noted with gross pathological changes like pale coloured and nose discoloration. No gross pathological changes were noted in any of the survived pups. There were no changes noted in serum thyroxine (T4) hormone levels estimated for PND 13 pups.

However, these reproductive effects were considered as secondary non-specific effects occurred as a result of systemic toxicity due to absence of adverse effects or no effects noted on major reproductive end points at this dose.

Therefore, based on the above results discussed, the NOAEL of the test item POLY-U for parental and reproductive toxicity is considered to be 4.3 mg/kg bw/day.

Data source

Reference

Reference Type:: study report
Title:: Unnamed
Year:: 2020
Report date:: 2020

Materials and methods

Test guideline

Qualifier:: according to guideline
Guideline:: other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:: no

GLP compliance:: yes (incl. QA statement)
Limit test:: no

Test material

Test material information

Test material form:: solid: particulate/powder

Test animals

Species:: rat
Strain:: Sprague-Dawley
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: 343.19-343.99 g (range of average values of each group of males at first day of dosing); 254.10-255.22 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.
Acclimatization: maximum of 2 animals of same sex per cage.
Pre mating: 2 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.
Recovery animals: 2 animals of same sex per cage.
- Diet (e.g. ad libitum): Altromin maintenance diet for rats and mice (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. Females were screened for normal oestrous cycles (4 to 5 days) in a two weeks pre-treatment period before initiation of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6-23.2ºC
- Humidity (%): 46-65 %
- Air changes (per hr): 12-15
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:: oral: gavage
Vehicle:: corn oil
Details on exposure:: PREPARATION OF DOSING SOLUTIONS:
The required quantity of test item was weighed into a clean beaker and a little volume of vehicle was added into the beaker and mixed well with glass rod. The formulation was transferred into a measuring cylinder, the beaker was rinsed with some more amount of vehicle also transferred into the measuring cylinder. This procedure was repeated until the entire quantity of the test item formulation was transferred into the measuring cylinder. Finally, the volume was made up to the required quantity with vehicle to get the desired concentration for the different dose levels.
Test item formulations were prepared daily before administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item was found insoluble in distilled water but formed uniform suspension with corn oil at the concentration of 1.9 mg/mL (considering highest dose of 9.5 mg/kg bw with a dose volume of 5 mL/kg bw) based on the in-house suspendibility test results. Hence, corn oil was selected as vehicle for test item formulation preparations. Corn oil is universally accepted and routinely used vehicle in oral toxicity studies.
- Concentration in vehicle: 0.4, 0.86 and 1.9 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): L12017004, L32011001.
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 at the concentrations of 0.4 mg/mL and 1.9 mg/mL in corn oil. 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 HPLC method (study nº BIO-ANM 1513). Sampling and analysis of formulations were performed during week 1 (16-03-2020) and week 5 (14-04-2020) of the treatment. Samples were collected in duplicates from top, middle and bottom layers from low, mid and high dose concentrations and in duplicates from single 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%.
Details on mating procedure:: - Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: No
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy.
- Any other deviations from standard protocol: No
Duration of treatment / exposure:: Main group males: two weeks pre-mating, during mating and up to the day before sacrifice during the post-mating period (total of 37 days of treatment).
Main group females main group: two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13.
Recovery animals: same period as for the main group females until the first scheduled sacrifice of dams and kept without treatment for a further 14 days observation.
Frequency of treatment:: Once a day
Duration of test:: Main group Males: 17 March - 23 April 2020
Main group Females: 17 March - 23 May 2020
Recovery Animals: 17 March - 21 May 2020

Doses / concentrationsopen all close all

Doses / concentrations 1

Dose / conc.:: 0 mg/kg bw/day (actual dose received)
Remarks:: G1 - Vehicle control + G1R - Vehicle Control Recovery Group

Doses / concentrations 2

Dose / conc.:: 2 mg/kg bw/day (actual dose received)
Remarks:: G2 - Low dose

Doses / concentrations 3

Dose / conc.:: 4.3 mg/kg bw/day (actual dose received)
Remarks:: G3 - Mid dose

Doses / concentrations 4

Dose / conc.:: 9.5 mg/kg bw/day (actual dose received)
Remarks:: G4 - High dose + G4R - High dose Recovery Group

No. of animals per sex per dose:: Main Group: 12
Recovery Group: 5
Control animals:: yes, concurrent vehicle
Details on study design:: - Dose selection rationale:
Doses of 0, 50, 100 and 300 mg/kg bw /day were selected in a first DRF study (BIO-CTX 139) based on the available results for an acute oral toxicity study conducted as per OECD 423 guideline. Treatment related effects including mortalities were found at the lowest level tested. Thus, it was not possible to properly decide on doses for the main study and a second DRF study (BIO-CTX 171) was performed at doses of 0, 2.7, 8.3 and 25 mg/kg bw /day. The dose of 25 mg/kg bw /d was the lowest dose level at which treatment related adverse effects were found and based on these results the doses 2, 4.3 and 9.5 mg/kg bw /day were considered for present main study.
- Fasting period before blood sampling for clinical biochemistry: yes, overnight (water allowed).

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
prior to the treatment on day 1 and weekly thereafter during treatment for all the animals. Signs noted were included, but not be limited to, 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:
The main group animals were weighed at receipt, on the first day of dosing, weekly thereafter and at termination. The main group females were weighed on gestation days 0, 7, 14 and 20 during pregnancy and on days 1, 4, 7 and 13 during lactation period. The recovery group animals (both males and females) were weighed at receipt, on the first day of dosing, weekly thereafter and at termination.

FOOD CONSUMPTION:
yes
-feed consumption was measured for main group animals (both males and females) once a week during premating and weekly once for main group males during the post mating period. Feed consumption was not measured during the mating period for both males and females. Thereafter, feed consumption for main group 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. Feed consumption was measured for recovery group animals (both males and females) once a week throughout the experimental period. Average feed intake per rat (g/rat/day) was calculated using the amount of feed given and left over in each cage and the number of rats surviving in each cage.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once before treatment for all animals, at the end of the dosing period for males (shortly prior to scheduled sacrifice) and during the lactation period for females (shortly prior to scheduled sacrifice) of vehicle control and high dose main group animals and during the last week for the recovery group animals.
- Dose groups that were examined: vehicle control and high dose main group.

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
on the day of scheduled terminal sacrifice (for main group males on day 38, for main group females on lactation day 14 and for recovery group animals on the day 66).
- 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 and all recovery group animals.
- Parameters checked: Haemoglobin concentration (HGB), Haematocrit (HCT), Erythrocyte count (RBC), Total leukocyte count (WBC), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet count (PLT), Mean platelet volume (MPV), Reticulocyte count (Retic), Absolute reticulocyte count, 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 (for main group males on day 38, for main group females on lactation day 14 and for recovery group animals on the day 66).
- Animals fasted: Yes (water provided ad libitum)
- How many animals:
5 males and 5 females randomly selected from each main group and all recovery group animals.
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Cholinesterase, Total Protein, Albumin, Total bilirubin, Glucose, Total Cholesterol, Creatinine, Urea, Blood urea nitrogen (BUN), Triglycerides, Phosphorous, Calcium, Globulin. Sodium (mmol/L), Potassium (mmol/L) and Chloride (mmol/L) were estimated using a Na/K/Cl analyzer.

URINALYSIS: Yes
- Time schedule for collection of urine:
on the day of scheduled terminal sacrifice.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 5 randomly selected males of each main group and all recovery animals.
- Parameters checked: Blood, Bilirubin, Urobilinogen, Ketones, Protein, Glucose, Microalbumin, Leucocytes. In addition pH, nitrite 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.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Towards the end of the dosing period for males (on day 37) and during lactation period for females (on lactation day 13). Towards the end of the recovery period (shortly prior to scheduled sacrifice on day 65) for the recovery group.
- Dose groups that were examined: all main and recovery groups.
- Battery of functions tested: Home Cage Observations (convulsions, tremors and palpebral closure), Handling Observations, Open Field Observations, Sensory Observations, Neuromuscular Observations, Physiological Observation (Rectal temperature), Grip strength assessment and Motor activity assessment.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice:
Male animals: All surviving animals after completion of 36 days of treatment.
Maternal animals: All surviving animals on lactation day 14
Recovery animals: All surviving animals after completion of 14 days observation from the first scheduled sacrifice of dams.
- Organs examined: See tables 1 and 2 below.

OTHER:
Thyroxine Hormone (T4) Level estimation: Blood samples were collected for measurement of serum T4 levels on the following schedule:
a. Two pups per litter on lactation day 4 based on the following conditions:
- Two female pups in order to retain more male pups for nipple retention on PND 13.
- No pups were eliminated when litter size dropped below 10 pups/litter.
- Only one pup was eliminated and used for blood collection in case of only one pup was available above ten.
b. All main group females (dams) at termination (lactation day 14).
c. Two pups per litter (same sex) at termination (lactation day 13).
d. All adult main group males, at termination (after completion of 37 days of treatment).
Ovaries and uterine content:: The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes.
- Number of implantations: Yes.
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:
Ovaries and uterus were examined for histopathology.
Fetal examinations:: - External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: No data
- Head examinations: No data
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.
Indices:: See table 3 below.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:: effects observed, treatment-related
Description (incidence and severity):: There were no clinical signs of toxicity observed at groups G1/G1R, G2 and G3 animals of either sex throughout the experimental period.

Animals in groups G4/G4R were noted with test item-related clinical signs of toxicity such as, lethargic, rough hair coat, wet perineum, chromodacryorrhea, hair thinning or blood stains around vagina. However, all the animals from group G4R started to recover from the above mentioned clinical signs during recovery period and found normal at termination. In group G4 all the animals did not reveal any clinical signs till day 23 of treatment period. However, from day 24 onwards the animals started to reveal clinical signs. In group G4R all the animals did not reveal any clinical signs till day 25 of treatment period. However, from day 26 onwards the animals started to reveal clinical signs.
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, 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 at groups G2 and G3 in both sexes throughout the experimental period.

In group G4, there were no changes noted in mean body weight and percent change in mean body weight gain with respect to day 1 till week 3 in both sexes. However, reduction in mean body weight and percent change in mean body weight gain was noted from week 4 onwards and continued til l termination. Even though these reductions were not statistically significant, these changes are considered as test item-related due to noted test item-related clinical signs and test item-related effects in neurological observations during these periods.

In group G4R, there were no changes noted in mean body weight and percent change in mean body weight gain with respect to day 1 till week 3 in both sexes. However, reduction in mean body weight and percent change in mean body weight gain was noted from week 4 onwards and continued till end of dosing period. Furthermore, some of these reductions were found to be statistically significant compared to the control group. These changes are considered as test item-related due to noted test item-related clinical signs and test item-related decreased mean feed consumption during these periods. However, the body weight and body weight gains were started to recover during recovery period in both sexes.

In group G4, reduction in mean body weight and percent change in mean body weight gain was noted throughout the gestation period which is statitistically significant on gestation day (GD) 14 and 20 for mean body weight and statitistically significant during GD 7 to 14 and 14 to 20 for percent change in mean body weight gain when compared with vehicle control group. These reductions are considered as test item-related due to noted test item-related clinical signs and test item-related reduction in feed consumption during these periods.

In group G4, reduction in mean body weight and percent change in mean body weight gain was noted throughout the lactation period which is statitistically significant on lactation day (LD) 4, 7 and 13 for mean body weight and statitistically significant during LD 4 to 7 and 7 to 13 for percent change in mean body weight gain when compared with vehicle control group. These reductions are considered as test item-related due to noted test item-related clinical signs and test item-related reduction in feed consumption during these periods.
Food consumption and compound intake (if feeding study):: effects observed, treatment-related
Description (incidence and severity):: There were no changes noted in mean feed consumption at groups G2 and G3 in both sexes during pre-mating, gestation and lactation periods when compared with vehicle control group.

In group G4, there were no changes noted in mean feed consumption in both sexes during pre-mating period when compared with vehicle control group. However, reductions in mean feed consumption were noted during gestation and lactation periods which are statitistically significant during GD 14 to 20 and during LD 1 to 4, 4 to 7 and 7 to 13, when compared with vehicle control group. These reductions are considered as test item-related due to noted test item-related clinical signs and test item related reduction in body weights during these periods.

In group G4R, there were no changes noted in mean feed cosnumption till week 3 in both sexes. However, reduction in mean feed consumption was noted from week 4 onwards and continued till end of dosing period. These reductions are statistically significant during week 5 to 9 in G4R males; statistically significant during week 6 in G4R females when compared with respective vehicle control group. These changes are considered as test item-related due to noted test item-related clinical signs, decreased mean body weight and or percent body weight gain during these periods in both sexes. However, the mean feed consumption was started to recover during recovery period in both sexes.
Food efficiency:: not examined
Water consumption and compound intake (if drinking water study):: not examined
Ophthalmological findings:: no effects observed
Description (incidence and severity):: There were no ocular changes observed in any of the animals from all the tested dose and vehicle control main groups in both sexes during ophthalmological examination conducted towards end of the dosing period. Likewise, there were no ocular changes observed in any of the animals from both recovery groups in either sex during ophthalmological examination conducted towards end of the recovery period.
Haematological findings:: effects observed, treatment-related
Description (incidence and severity):: There were no changes noted in mean haematology parameters in groups G2 and G3 of both sexes when compared with vehicle control group. In group G4/G4R, the following statistically significant changes were noted: decrease in mean absolute differential leucocyte count (nuetrophills, lymphocytes, monocytes, eosinophils, basophils), eosinophils (%), WBC count of group G4 males; decrease in mean RBC count, HGB, HCT, monocytes (absolute and %), prothrombin time of group G4 females; increase in mean reticulocyte count (absolute and %), eosinophils (absolute and %) and monocytes (%), decrease in mean lymphocytes (%) and prothrombin time of group G4R males; and decrease in RBC count and Activated Prothrombin Time, increase in MCV, MCH, reticulocyte count (absolute and %) of group G4R females.
Clinical biochemistry findings:: effects observed, treatment-related
Description (incidence and severity):: There were no test item-related changes noted in mean clinical chemistry parameters of groups G2 and G3 in both sexes when compared with vehicle control group. In group G4/G4R, the following statistically significant changes were noted: decrease in mean alanine aminotransferase (ALT), aspartate aminotransferase (AST) levels of group G4 males; decrease in mean urea and blood urea nitrogen levels of group G4 females; decrease in mean creatinine levels and increase in mean calcium and phosphorous levels of group G4R males; increase in mean creatinine, sodium and total bilirubin levels and decrease in mean total cholesterol levels of group G4R females.
Urinalysis findings:: effects observed, non-treatment-related
Description (incidence and severity):: There were no test item-related changes observed in urinalysis parameters in any of the tested dose levels of both main and recovery groups when compared with vehicle control group. Some of the sporadically occurring statistically significant differences like, increase in mean urine pH levels and decrease in urine specific gravity levels in group G4R females were considered incidental and therefore of no toxicological relevance.
Behaviour (functional findings):: effects observed, treatment-related
Description (incidence and severity):: There were no changes noted in neurological/functional examinations like home cage/ handling/open-field/sensory/neuromuscular (hind limb foot splay)/physiological observations and grip strength or motor activity assessments in any of the animals from groups G2 and G3 when compared with vehicle control groups.

In group G4, there were no changes noted in home cage/handling/open-field/sensory/ neuromuscular (hind limb foot splay)/physiological observations when compared with vehicle control group in both sexes. However, a slight (non-statistically significant) reduction in mean movements count during motor activity assessment in G4 males, statistically significant reduction in mean fore/hind limb grip strengths in G4 males were noted when compared with vehicle control group. These changes can be considered as secondary effects to the test item exposure, noted due to test item-related clinical signs, decreased mean body weight, percent body weight gain and mean feed consumption during this period.

There were no changes noted in neurological/functional examinations like home cage/ handling/open-field/sensory/neuromuscular (hind limb foot splay)/physiological observations and grip strength or motor activity assessments in any of the animals from group G4R in both sexes when compared with vehicle control group.
Immunological findings:: not examined
Organ weight findings including organ / body weight ratios:: effects observed, treatment-related
Description (incidence and severity):: There were no changes noted in both mean absolute and relative organ weights of groups G2 and G3 in either sex when compared with vehicle control group.

In group G4, statistically significant decrease in mean thymus weight (absolute and relative) of males, statistically significant decrease in mean spleen weight (absolute), mean liver weight (absolute) and thymus weight (absolute and relative) of females was noted when compared with vehicle control group. These observed changes can be considered as test item-related due to noted mild decrease in lymphocytes in white pulp of spleen and cortical area of thymus during microscopic examination of these organs at this dose level. These changes can also correlate with test item-related and statistically significant reduction in mean terminal body weight at this dose level.
Gross pathological findings:: effects observed, treatment-related
Description (incidence and severity):: There were no gross pathological changes observed in any of the adult animals from groups G1/G1R, G2 and G3.

The animals of group G4 were noted with test item related external gross pathological changes like wet pernineum (4 males and 7 females), wet pernineum along with blood stains around vagina (2 females) and test item related internal gross pathological changes like uterus with resorptions/dead fetuses during necropsy. However, the group G4R animals of both sexes treated with high dose did not reveal any external or internal gross pathological changes during necropsy. These observations are because of recovery of animals from clinical signs during recovery period and hence all the animals were found normal at termination.
Neuropathological findings:: not examined
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Description (incidence and severity):: Test item-related changes were observed in spleen, thymus and bone marrow of femur in both sexes at group G4. The microscopic changes were characterized by mild decrease in lymphocytes in white pulp of spleen, cortical area of thymus and also mild decrease in marrow cellularity of femoral bone. Similar changes were not observed in lower and recovery group of animals. All these changes in thymus, spleen and femoral bone marrow were considered to be reversible, secondary effects of reduced food consumption and decreased body weight gain (Everds et.al., 2013). Few of the other microscopic findings observed in the study such as accessory cortical tissue in adrenal glands, ultimobranchial cysts(s) in thyroid gland 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.
Histopathological findings: neoplastic:: no effects observed
Other effects:: no effects observed
Description (incidence and severity):: Serum Thyroxine (T4) Levels: There were no changes noted in T4 levels of males and dams of main groups when compared with vehicle control group. The obtained T4 levels are within the normal range of this species and strain.

Maternal developmental toxicity

Number of abortions:: no effects observed
Description (incidence and severity):: No abortions ocurred in any dose group.
Pre- and post-implantation loss:: effects observed, treatment-related
Description (incidence and severity):: A mean number of 0.18, 0.25, 0.18 and 5.11 post-implantation losses with a percentage of 1.82%, 2.68%, 1.53% and 38.27% were noted from groups G1, G2, G3 and G4, respectively. In group G4, the post-implantation loss was statistically significant in both number and percentage when compared with vehicle control group. This noted change is considered as test item-related, due to noted increase in number of resorptions, test item-related effects in litter observations or test item-related maternal effects during gestation and lactation periods.
Total litter losses by resorption:: effects observed, treatment-related
Description (incidence and severity):: In Group 4, 1 out of 11 fertile females were confirmed with total litter loss with evidence of resorptions at necropsy.
Early or late resorptions:: effects observed, treatment-related
Description (incidence and severity):: In Group 4, 1 out of 11 fertile females were confirmed with early/late resorptions.
Dead fetuses:: effects observed, treatment-related
Description (incidence and severity):: In Group 4, 1 out of 11 fertile females were confirmed with total litter loss with evidence of dead fetuses at necropsy.
Changes in pregnancy duration:: no effects observed
Description (incidence and severity):: The mean gestation length (day of confirmed as mated to day of parturition) was 22.09, 22.17, 22.18 and 22.11 days for groups G1, G2, G3 and G4, respectively. There were no statistically significant differences noted for this parameter in any of the tested dose groups when compared with the control group.
Changes in number of pregnant:: no effects observed
Description (incidence and severity):: A total of 11 (out of 12), 12 (out of 12), 11 (out of 12), and 11 (out of 12), females were confirmed as pregnants / with evidence of implantation sites with a fecundity index of 91.7%, 100.0%, 91.7% and 91.7% from group G1, G2, G3 and G4, respectively. There were no statistically significant differences noted for this parameter in any of the tested dose groups when compared with the control group.
Other effects:: effects observed, treatment-related
Description (incidence and severity):: Oestrous cycle: There were no irregularities observed in the oestrus cyclicity of main group females during pre-mating period at any of the tested dose groups. The mean length of oestrus cycle (days) per dam during premating period was unaffected at all the tested dose groups when compared with control group.

Gestation Index (%): A total of 11 (out of 11), 12 (out of 12), 11 (out of 11), and 9 (out of 11) females were confirmed with live born pups with a gestation index of 100.0%, 100.0%, 100.0% and 81.8% from group G1, G2, G3 and G4, respectively. There were no statistically significant differences noted for this parameter in any of the tested dose groups when compared with the vehicle control group. However, in group G4 the noted reduction in gestation index is considered as test item-related, as 2 out of 11 fertile females were noted with total litter loss with evidence of implantation sites only and also this can be correlated with increased pup mortalities and reduced pup survival index from other littered females of same dose group.
Details on maternal toxic effects:: The reproductive effects observed at the highest dose level of 9.5 mg/kg bw/day were considered as secondary non-specific effects occurred as a result of systemic toxicity due to absence of adverse effects or no effects noted on major reproductive end points at this dose.

Effect levels (maternal animals)

open all close all

1

: Key result
Dose descriptor:: LOAEL
Effect level:: 9.5 mg/kg bw/day (actual dose received)
Based on:: test mat.
Basis for effect level:: body weight and weight gain; clinical biochemistry; clinical signs; food consumption and compound intake; gross pathology; haematology; histopathology: non-neoplastic; organ weights and organ / body weight ratios; pre and post implantation loss

2

: Key result
Dose descriptor:: NOAEL
Effect level:: 4.3 mg/kg bw/day (actual dose received)
Based on:: test mat.
Basis for effect level:: body weight and weight gain; clinical biochemistry; clinical signs; food consumption and compound intake; gross pathology; haematology; histopathology: non-neoplastic; organ weights and organ / body weight ratios; pre and post implantation loss
Remarks on result:: other: Effects observed at the highest tested dose of 9.5 mg/kg bw/day.

Maternal abnormalities

: Key result
Abnormalities:: no effects observed

Results (fetuses)

Fetal body weight changes:: effects observed, treatment-related
Description (incidence and severity):: In group G4, statistically significant reduction in mean pup (both male and female) weight per litter on PND 1, 4, 7 and 13 were noted. These changes are considered as test item-related due to increased pup mortalities, occurrence of external changes in pups and decreased pup survival index per litter at this dose level. These effects can also correlate with test item-related maternal effects on litters during gestation and lactation periods.
Reduction in number of live offspring:: effects observed, treatment-related
Description (incidence and severity):: In group G4, reduced live birth index per litter (90.43%) was noted when compared with vehicle control group (99.09%). This noted change was statistically significant for the mean number of live born female pups and is considered as test item-related due to noted test item-related clinical signs, test item-related reduction in maternal body weights and feed consumption during gestation and lactation periods.
Changes in sex ratio:: no effects observed
Description (incidence and severity):: There were no statistically significant differences observed in the sex ratio of pups between the treated groups and the vehicle control group.
Changes in litter size and weights:: effects observed, treatment-related
Description (incidence and severity):: In group G4, reduced mean litter size at birth (8.11) was noted when compared with vehicle control group (10.73). This noted change is considered as test item-related due to noted test item-related clinical signs, test item-related reduction in maternal body weights and feed consumption during gestation and lactation periods.
Changes in postnatal survival:: effects observed, treatment-related
Description (incidence and severity):: In group G4, reduced number of live pups in either sex and pup survival index during lactation period were noted when compared with vehicle control group. These noted changes are statistically significant for number of survived male pups per litter during LD 1 to 4, number of survived female pups per litter during LD 1 to 4, 5 to 7 and 8 to 13, number of dead pups per litter during LD 1 to 4 and 5 to 7 and pup survival index between LD 1 to 4, 5 to 7 and 8 to 13 when compared with vehicle control group. These effects are considered as test item-related due to noted test item-related clinical signs, test item-related reduction in maternal body weights and feed consumption during gestation and lactation periods.
External malformations:: effects observed, treatment-related
Description (incidence and severity):: At necropsy, the dead pups which were found dead by birth and during lactation period were noted with pale discoloration and red discoloration at nasal region from group G4 litters. No gross pathological changes were noted in any of the survived pups from group G4 and all pups from other dose group litters.
Skeletal malformations:: not specified
Visceral malformations:: no effects observed
Description (incidence and severity):: No visceral abnormalities were detected in any of the pups sacrificed.
Other effects:: effects observed, treatment-related
Description (incidence and severity):: Serum Thyroxine (T4) Levels: There were no changes noted in T4 levels of PND 13 pups in any of the tested dose groups when compared with vehicle control group. The obtained T4 levels of PND 13 pups are within the normal range of this species and strain. There were no changes noted in T4 levels of PND 4 pups from the tested dose groups G2 and G3 when compared with vehicle control group. In group G4, serum T4 levels of PND 4 pups was not conducted as there were no surplus pups remained from this dose level on PND 4. However, the obtained T4 levels of PND 4 pups from groups G2 and G3 are within the normal range of this species and strain.

Anogenital distance (AGD): In group G4, reduction in mean pup (both male and female) anogenital distance measurement (mm) and ratio per litter were noted. Changes in AGD ratio were found statistically significant in both sexes. These changes are considered as test item-related due to increased pup mortalities, decreased growth/weight of pups, occurrence of external changes in pups and decreased pup survival index per litter at this dose level. These effects can also correlate with test item-related maternal effects on litters during gestation and lactation periods.

Nipple retention in male pups: There were no occurrences of nipple/arealaes in any of the male pups examined on their PND 13 from any of the tested dose group litters and vehicle control group litters.
Details on embryotoxic / teratogenic effects:: The reproductive effects observed at the highest dose level of 9.5 mg/kg bw/day were considered as secondary non-specific effects occurred as a result of systemic toxicity in maternal animals due to absence of adverse effects or no effects noted on major reproductive end points at this dose.

Effect levels (fetuses)

open all close all

1

: Key result
Dose descriptor:: LOAEL
Effect level:: 9.5 mg/kg bw/day (actual dose received)
Based on:: test mat.
Sex:: male/female
Basis for effect level:: reduction in number of live offspring; fetal/pup body weight changes; changes in litter size and weights; changes in postnatal survival; external malformations

2

: Key result
Dose descriptor:: NOAEL
Effect level:: 4.3 mg/kg bw/day (actual dose received)
Based on:: test mat.
Sex:: male/female
Basis for effect level:: reduction in number of live offspring; fetal/pup body weight changes; changes in litter size and weights; changes in postnatal survival; external malformations
Remarks on result:: other: Effects observed at the highest tested dose of 9.5 mg/kg bw/day.

Fetal abnormalities

open all close all

1

: Key result
Abnormalities:: effects observed, treatment-related
Localisation:: external: face
Description (incidence and severity):: Dead pups which were found dead by birth and during lactation period in Group 4 were noted with pale discoloration and red discoloration at nasal region.

2

: Key result
Abnormalities:: effects observed, treatment-related
Localisation:: external: anogenital distance
Description (incidence and severity):: Reduction in mean pup (both male and female) anogenital distance measurement (mm) and ratio per litter were noted in group G4. Changes in AGD ratio were found statistically significant.

Overall developmental toxicity

: Key result
Developmental effects observed:: yes
Lowest effective dose / conc.:: 9.5 mg/kg bw/day (actual dose received)
Treatment related:: yes
Relation to maternal toxicity:: developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:: yes
Relevant for humans:: not specified

Any other information on results incl. tables

Table 5. Summary of vaginal smear examination 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)
G1, F & 0	12	n	12	0	Mean	4.96
		%	100.00	0.00	±SD	0.14
					n	12
G2, F & 2	12	n	12	0	Mean	4.92
		%	100.00	0.00	±SD	0.19
					n	12
G3, F & 4.3	12	n	12	0	Mean	4.96
		%	100.00	0.00	±SD	0.14
					n	12
G4, F & 9.5	12	n	12	0	Mean	5.00
		%	100.00	0.00	±SD	0.00
					n	12

Table 6. 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 Fertiltiy Index (%)
G1, M & 0	11 (12)	91.7	11 (12)	91.7
G2, M & 2	11 (12)	91.7	11 (12)	91.7
G3, M & 4.3	11 (12)	91.7	11 (12)	91.7
G4, M & 9.5	11 (12)	91.7	11 (12)	91.7

Group, Sex & Dose (mg/kg body weight/day)		Cohabitation Record and Pre-coital Interval (Mean Time to Mating)				Gestational Length (duration of pregnancy)
Group, Sex & Dose (mg/kg body weight/day)		Pre-coital Interval (Days)		Concieving Days (1 to 5)	Concieving Days (More than 5 days)	Gestation Length (Days)
G1, F & 0	Mean	9.08	n	5	7	22.09
	±SD	6.05	%	41.67	58.33	0.54
	n	12	%	41.67	58.33	11
G2, F & 2	Mean	6.75	n	7	5	22.17
	±SD	5.33	%	58.33	41.67	0.39
	n	12	%	58.33	41.67	12
G3, F & 4.3	Mean	7.58	n	6	6	22.18
	±SD	5.70	%	54.55	54.55	0.60
	n	12	%	54.55	54.55	11
G4, F & 9.5	Mean	6.33	n	7	5	22.11
	±SD	5.38	%	58.33	41.67	0.33
	n	12	%	58.33	41.67	9

n: Number of dams

Group, Sex & Dose (mg/kg body weight/day)	Female Mating Index (%)		Female Fertiltiy Index (%)
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 Fertiltiy Index (%)
G1, F & 0	12 (12)	100.0	11 (12)	91.7
G2, F & 2	12 (12)	100.0	12 (12)	100.0
G3, F & 4.3	12 (12)	100.0	11 (12)	91.7
G4, F & 9.5	12 (12)	100.0	11 (12)	91.7

Group, Sex & Dose (mg/kg body weight/day)	Female Fecundity or Pregnancy Index (%)			Gestation Index (%)
Group, Sex & Dose (mg/kg body weight/day)	No. of Pregnant Females	No. of Females Confirmed with Mating	Female Fecundity or Pregnancy Index (%)	Females with Live Born Pups at Parturation	No. of Females with Evidence of Pregnancy	Gestation Index (%)
G1, F & 0	11	12	91.7	11	11	100.0
G2, F & 2	12	12	100.0	12	12	100.0
G3, F & 4.3	11	12	91.7	11	11	100.0
G4, F & 9.5	11	12	91.7	9	11	81.8

Group, Sex & Dose (mg/kg body weight/day)		Post-implantation Loss (%)				Post-natal 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	Post-natal Loss (%)
G1, F & 0	Mean	10.82	10.64	0.18	1.82	0.00	0.00
	±SD	1.54	1.69	0.40	4.05	0.00	0.00
	n	11	11	11	11	11	11
G2, F & 2	Mean	10.67	10.42	0.25	2.68	0.00	0.00
	±SD	1.78	1.98	0.62	7.32	0.00	0.00
	n	12	12	12	12	12	12
G3, F & 4.3	Mean	11.00	10.82	0.18	1.53	0.00	0.00
	±SD	1.67	1.60	0.40	3.41	0.00	0.00
	n	11	11	11	11	11	11
G4, F & 9.5	Mean	12.56	7.44	5.11*	38.27*	4.67*	62.47*
	±SD	2.07	3.13	4.37	28.23	4.30	44.96
	n	9	9	9	9	9	9

n: Number of dams

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

Table 7. Summary of delivery and litter observation record during lactation period

Group, Sex & Dose (mg/kg body weight/day)		Litter Size (No.)	Live Pups (No.)			Dead Pups (No.)			Cannibalized Pups (No.)				Sex Ratio (m/f) at Birth	Live Birth Index (%)
Group, Sex & Dose (mg/kg body weight/day)		Litter Size (No.)	Male	Female	Total	Male	Female	Total	Undetermined	Male	Female	Total	Sex Ratio (m/f) at Birth	Live Birth Index (%)
G1, F & 0	Mean	10.73	5.55	5.09	10.64	0.09	0.00	0.09	0.00	0.00	0.00	0.00	1.34	99.09
	±SD	1.62	2.11	1.64	1.69	0.30	0.00	0.30	0.00	0.00	0.00	0.00	1.01	3.02
	n	11	11	11	11	11	11	11	11	11	11	11	11	11
G2, F & 2	Mean	10.50	4.58	5.83	10.42	0.00	0.00	0.00	0.00	0.08	0.00	0.08	1.15	99.40
	±SD	2.11	0.90	2.08	1.98	0.00	0.00	0.00	0.00	0.29	0.00	0.29	1.28	2.06
	n	12	12	12	12	12	12	12	12	12	12	12	12	12
G3, F & 4.3	Mean	10.91	5.09	5.73	10.82	0.09	0.00	0.09	0.00	0.00	0.00	0.00	0.99	99.30
	±SD	1.70	1.30	1.68	1.60	0.30	0.00	0.30	0.00	0.00	0.00	0.00	0.44	2.32
	n	11	11	11	11	11	11	11	11	11	11	11	11	11
G4, F & 9.5	Mean	8.11	4.00	3.44*	7.44	0.33	0.33	0.67	0.00	0.00	0.00	0.00	1.17	90.43
	±SD	2.67	1.73	2.24	3.13	0.71	0.71	1.32	0.00	0.00	0.00	0.00	0.86	21.83
	n	9	9	9	9	9	9	9	9	9	9	9	9	9

n: Number of Dams; m/f: Male/Female;

*: 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 (%) LD 1 to 4
		Live Pups (No.)			Dead Pups (No.)			Cannibalized (No.)
		Male	Female	Total	Male	Female	Total	Male	Female	Total
G1, F & 0	Mean	5.55	5.09	10.64	0.00	0.00	0.00	0.00	0.00	0.00	1.34	100.00
	±SD	2.11	1.64	1.69	0.00	0.00	0.00	0.00	0.00	0.00	1.01	0.00
	n	11	11	11	11	11	11	11	11	11	11	11
G2, F & 2	Mean	4.58	5.83	10.42	0.00	0.00	0.00	0.00	0.00	0.00	1.15	100.00
	±SD	0.90	2.08	1.98	0.00	0.00	0.00	0.00	0.00	0.00	1.28	0.00
	n	12	12	12	12	12	12	12	12	12	12	12
G3, F & 4.3	Mean	5.09	5.73	10.82	0.00	0.00	0.00	0.00	0.00	0.00	0.99	100.00
	±SD	1.30	1.68	1.60	0.00	0.00	0.00	0.00	0.00	0.00	0.44	0.00
	n	11	11	11	11	11	11	11	11	11	11	11
G4, F & 9.5	Mean	1.89*	1.22*	3.11*	2.11*	2.11*	4.22*	0.00	0.11	0.11	0.85	40.89*
	±SD	2.42	1.39	3.72	2.47	2.37	4.29	0.00	0.33	0.33	1.13	46.69
	n	9	9	9	9	9	9	9	9	9	9	9

n: Number of Dams; m/f: Male/Female;

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

Note: A total of 9 dams were considered for mean calculations of all end points/parameters during LD 1 to 4 from group G4 as 1 out of 12 dams confirmed as non-pregnant;

2 out of 12 dams were confirmed with total litter loss [postimplantation loss].

Group, Sex & Dose (mg/kg body weight/day)	Animal No.	Pups Sacrificed for Blood Collection on LD 4 (No.)			Live Pups (No.) on LD 4 after Sacrificed for Blood Collection			During Lactation Day 5 to 7									Sex Ratio (m/f) on LD 7	Pup Survival Index (%) during LD 5 to 7
		Pups Sacrificed for Blood Collection on LD 4 (No.)						Live Pups per litter (No.)			Dead Pups per litter (No.)			Cannibilized Pups per litter (No.)
		Male	Female	Total	Male	Female	Total	Male	Female	Total	Male	Female	Total	Male	Female	Female
G1, F & 0	Mean	0.00	0.55	0.55	5.55	4.55	10.09	5.55	4.55	10.09	0.00	0.00	0.00	0.00	0.00	0.00	1.52	100.00
	±SD	0.00	0.82	0.82	2.11	1.44	1.04	2.11	1.44	1.04	0.00	0.00	0.00	0.00	0.00	0.00	1.14	0.00
	n	11	11	11	11	11	11	11	11	11	11	11	11	11	11	11	11	11
G2, F & 2	Mean	0.00	0.75	0.75	4.58	5.08	9.67	4.58	5.08	9.67	0.00	0.00	0.00	0.00	0.00	0.00	1.25	100.00
	±SD	0.00	0.97	0.97	0.90	1.62	1.30	0.90	1.62	1.30	0.00	0.00	0.00	0.00	0.00	0.00	1.26	0.00
	n	12	12	12	12	12	12	12	12	12	12	12	12	12	12	12	12	12
G3, F & 4.3	Mean	0.00	1.00	1.00	5.09	4.73	9.82	5.09	4.73	9.82	0.00	0.00	0.00	0.00	0.00	0.00	1.30	100.00
	±SD	0.00	0.89	0.89	1.30	1.42	0.98	1.30	1.42	0.98	0.00	0.00	0.00	0.00	0.00	0.00	0.95	0.00
	n	11	11	11	11	11	11	11	11	11	11	11	11	11	11	11	11	11
G4, F & 9.5	Mean	0.00	0.00	0.00	3.40	2.20	5.60	3.40	1.80*	5.20	0.00	0.40*	0.40*	0.00	0.00	0.00	1.70	77.50*
	±SD	0.00	0.00	0.00	2.30	1.10	3.21	2.30	1.30	3.42	0.00	0.55	0.55	0.00	0.00	0.00	1.20	43.66
	n	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5

n: Number of Dams; LD: Lactation Day; m/f: Male/Female

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

Note: A total of 5 dams were considered for mean calculations of all end points/parameters during LD 5 to 7 from group G4 as 4 out of 9 dams were noted with total postnatal pup loss during LD 1 to 4.

Group, Sex & Dose (mg/kg body weight/day)	Animal No.	During Lactation Day 8 to 13									Sex Ratio (m/f) at 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
G1, F & 0	Mean	5.55	4.55	10.09	0.00	0.00	0.00	0.00	0.00	0.00	1.52	100.00
	±SD	2.11	1.44	1.04	0.00	0.00	0.00	0.00	0.00	0.00	1.14	0.00
	n	11	11	11	11	11	11	11	11	11	11	11
G2, F & 2	Mean	4.58	5.08	9.67	0.00	0.00	0.00	0.00	0.00	0.00	1.25	100.00
	±SD	0.90	1.62	1.30	0.00	0.00	0.00	0.00	0.00	0.00	1.26	0.00
	n	12	12	12	12	12	12	12	12	12	12	12
G3, F & 4.3	Mean	5.09	4.73	9.82	0.00	0.00	0.00	0.00	0.00	0.00	1.30	100.00
	±SD	1.30	1.42	0.98	0.00	0.00	0.00	0.00	0.00	0.00	0.95	0.00
	n	11	11	11	11	11	11	11	11	11	11	11
G4, F & 9.5	Mean	4.25	2.00*	6.25	0.00	0.25	0.25	0.00	0.00	0.00	2.38	97.22*
	±SD	1.50	0.82	1.71	0.00	0.50	0.50	0.00	0.00	0.00	0.95	5.56
	n	4	4	4	4	4	4	4	4	4	4	4

n: Number of Dams; LD: Lactation Day; m/f: Male/Female;

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

Table 8. Summary record of pup observations during post-natal period

Group, Sex & Dose (mg/kg body weight/day)		At Birth (PND 1)	PND 1 to 4	Pups Sacrificed for Blood Collection on PND 4 (No.)*	PND 5 to 7^$	PND 8 to 13^$
G1, F & 0	No. of Dams^#	11	11	4	11	11
	No. of Live Pups	117	117	6	111	111
	Pup Observation/ No. of Pups observed	N/117	N/117	-	N/111	N/111
G2, F & 2	No. of Dams^#	12	12	5	12	12
	No. of Live Pups	125	125	9	116	116
	Pup Observation/ No. of Pups observed	N/125	N/125	-	N/116	N/116
G3, F & 4.3	No. of Dams^#	11	11	7	11	11
	No. of Live Pups	119	119	11	108	108
	Pup Observation/ No. of Pups observed	N/119	N/119	-	N/108	N/108
G4, F & 9.5	No. of Dams^#	9	9	0	9	9
	No. of Live Pups	67	28	0	26	25
	Pup Observation/ No. of Pups observed	P/28; HS/1; N/38	L/2; N/26	-	L, P/1; N/25	N/25

N: Normal; PND: Post-natal Day; #: Dams confirmed with littering; P: Pale colored; HS: Haemorrhagic spot; L: Lethargic

*: Pups selected for blood collection from dams with litter size of more than 10; $: Pups sacrificed on PND 4 were excluded

Table 9. 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
G1, F & 0	Mean	6.75	6.44	11.42	10.64	16.25	15.26	29.26	27.87
	±SD	0.09	0.17	0.36	0.20	0.47	0.35	0.51	0.82
	n	11	11	11	11	11	11	11	11
G2, F & 2	Mean	6.82	6.57	11.58	10.81	16.51	15.60	29.58	28.56
	±SD	0.14	0.16	0.37	0.19	0.37	0.40	0.25	0.40
	n	12	12	12	12	12	12	12	12
G3, F & 4.3	Mean	6.78	6.48	11.50	10.72	16.54	15.44	29.47	28.13
	±SD	0.12	0.18	0.32	0.23	0.30	0.33	0.33	0.65
	n	11	11	11	11	11	11	11	11
G4, F & 9.5	Mean	3.94*	3.73*	6.95*	5.70*	10.88*	9.11*	19.99*	17.88*
	±SD	0.89	0.80	1.79	1.91	3.28	2.75	4.47	4.49
	n	9	8	4	5	4	4	4	4

n: Number of litters

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

Table 10. Summary record of mean pup ano-genital distance (AGD) measurement (mm) and ano-genital distance (AGD) ratio per litter on post-natal day 4

Group, Sex & Dose (mg/kg body weight/day)		Mean Male Pup AGD Measurement (mm)	Mean Female Pup AGD Measurement (mm)	Mean Male Pup AGD Ratio	Mean Female Pup AGD Ratio
G1, F & 0	Mean	5.43	2.69	2.41	1.22
	±SD	0.22	0.12	0.08	0.05
	n	11	11	11	11
G2, F & 2	Mean	5.42	2.77	2.39	1.25
	±SD	0.25	0.13	0.09	0.05
	n	12	12	12	12
G3, F & 4.3	Mean	5.34	2.70	2.37	1.23
	±SD	0.19	0.13	0.07	0.05
	n	11	11	11	11
G4, F & 9.5	Mean	4.07	1.78	2.14*	1.00*
	±SD	0.61	0.41	0.15	0.15
	n	4	5	4	5

n: Number of litters

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

Table 11. Summary of male pup nipple/areolae retention (no.) record

Group, Sex & Dose (mg/kg body weight/day)		Mean No. of Pups with Retention of Nipples/ Areolae on Post-natal Day 13
G1, F & 0	Mean	0.00
	±SD	0.00
	n	11
G2, F & 2	Mean	0.00
	±SD	0.00
	n	12
G3, F & 4.3	Mean	0.00
	±SD	0.00
	n	11
G4, F & 9.5	Mean	0.00
	±SD	0.00
	n	9

n: Number of litters

Applicant's summary and conclusion

Conclusions:

The NOAEL of the test substance for maternal and developmental toxicity in rats by oral route was determined to be 4.3 mg/kg bw/day. The reproductive effects observed at the highest dose level of 9.5 mg/kg bw/day were considered as secondary non-specific effects occurred as a result of systemic toxicity in maternal animals due to absence of adverse effects or no effects noted on major reproductive end points at this dose.

Executive summary:

A combined repeated dose toxicity study with the reproduction /developmental toxicity screening test of the test substance POLY-U by oral administration in rats was conducted according to OECD guideline 422, in accordance with GLP principles. A total of 116 (58 males + 58 females) Sprague Dawley rats were distributed to four main groups and two recovery groups to detect delayed occurrence and recovery from toxic effects. Each main group (G1, G2, G3 and G4) consisted of 12 males and 12 females and each recovery group (G1R and G4R) consisted of 5 males and 5 females. The animals in G1/G1R group were administered with vehicle (Corn oil), and the animals in G2, G3 and G4/G4R groups were administered with test item at the dose levels of 2, 4.3 and 9.5 mg/kg bw/d based on the results of two consecutive dose range finding studies performed with the same species for a period of 14 days. at doses of 50, 100 and 300 mg/kg bw /d and 2.7, 8.3 and 25 mg/kg bw /d respectively. The test item was administered to the main group males for a period of 37 days (two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period), to the main group females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13 and to the recovery group animals for a period of 50 days with a 14 days recovery. The vehicle and test item formulations were administered orally by gavage at the dose volume of 5 mL/kg body weight. The stability of test item formulations in corn oil was established before initiation of the treatment. Dose formulation analysis for homogeneity and concentration verification was performed during weeks 1 and 5 of the treatment period and the results were within acceptable limits.

In groups G2 and G3, there were no indication of test item-related effects in any of the parameters assessed for systemic or reproductive toxicity.

In group G4, test item-related reduced gestation index, increased post-implantation loss, increased postnatal loss, reduced litter size, reduced live birth index per litter, increased number of dead pups at birth / during postnatal period and reduced pup survival index were noted. Also, test item-related external anomalies in pups, test item-related reduced mean pup weight and ano-genital distance ratio per litter in either sex were noted. However, there were no occurrences of nipples in male pups examined on PND 13 from all the litters. The pups which were found dead by birth and during postnatal period were noted with gross pathological changes like pale coloured and nose discoloration. No gross pathological changes were noted in any of the survived pups. There were no changes noted in serum thyroxine (T4) hormone levels estimated for PND 13 pups.

Therefore, based on the above results discussed, the NOAEL of the test item POLY-U for maternal and developmental toxicity is considered to be 4.3 mg/kg bw/day.

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N,N'-(methylenedi-p-phenylene)bis(aziridine-1-carboxamide)

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