Fatty acids, C16-18 (even numbered), esters with 1,2,3-propanetriol and oligomers, ethoxylated

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-09-16 to 2018-04-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted on 29 July 2016

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN:
- Premating exposure duration for parental (P0) animals: Yes (14 days)
- Basis for dose level selection: The doses of 0, 100, 300 and 1000 mg/kg body weight for vehicle control, low dose, mid dose and high dose respectively were selected in consultation with the sponsor based on the results of Dose Range Finding Study for Reproduction/Developmental Toxicity Screening Test of Hostacerin DGSB by Oral Gavage in Sprague Dawley Rats (Bioneeds Study No.: BIO-TX 2516).
These doses were selected as the test item Hostacerin DGSB when administered orally to Sprague Dawley males for two weeks prior to mating, during mating and up to the day before sacrifice during post-mating period (total of at 30 days treatment) and females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 after which the pups were sacrificed on lactation day 13, females (dams) were sacrificed on lactation day 14.
- Route of administration: oral (gavage) route
- Other considerations:
choice of species and strain: Rat (Rattus norvegicus) and Sprague Dawley (standard laboratory rodent species and strain used for toxicity assessment and also recommended by various regulatory authorities)
vehicle: The test item was in-soluble in distilled water and forms clear homogenous suspension in 0.5 % w/v Carboxy Methyl Cellulose at the concentration of 100 mg/mL (highest dose concentration) as evidenced by the in-house solubility and suspendibility test results. Hence, 0.5 % w/v Carboxy Methyl Cellulose was selected for the test item formulation preparation.
number of animals: 96 (48 Males + 48 Females); 12 Males + 12 Females/group as per OECD 421 guideline specification

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C16-18 (even numbered), esters with 1,2,3-propanetriol and oligomers, ethoxylated

IUPAC Name:

Fatty acids, C16-18 (even numbered), esters with 1,2,3-propanetriol and oligomers, ethoxylated

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: Clariant Produkte (Deutschland) GmbH
84504 Burgkirchen, Germany and DEG4338635

- Expiration date of the batch: 04.10.2018
- Purity test date: 04.10.2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Stability under test conditions: The stability of the test item in dose formulations was established under Bioneeds Study No.: BIO-ANM 450. The test item formulations were stable for 6 hours at room temperature and for 24 hours at 2 to 8 oC at the concentrations of 10 mg/mL and 100 mg/mL.
- Solubility of the test substance in the solvent/vehicle: 0.5 % w/v Carboxy Methyl Cellulose was used as vehicle for the preparation of formulations as the test item formed clear homogenous suspension in 0.5 % w/v Carboxy Methyl Cellulose at the concentration of 100 mg/mL (highest dose concentration) as evidenced by the in-house solubility and suspendibility test results.

FORM AS APPLIED IN THE TEST: Liquid (Suspension)

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Rat is one of the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-house bred animals
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks
- Weight at study initiation: Males: 220.17 g to 255.97 g; Females: 201.51 g to 231.96 g
- Housing: Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted food and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
i. Pre mating
Two animals of same sex and group per cage were housed.
ii. Mating
During mating, two animals (one male and one female) of same group were housed.
iii. 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.
- Diet: Altromin Maintenance diet for rats and mice 1324 manufactured by Altromin Spezialfutter GmbH & Co.
- Water: Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period (Including Oestrus Cycle Evaluation): 18 September 2017 to 06 October 2017.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.8 to 23.4°C
- Humidity (%): 43 to 69%
- Air changes (per hr): 12 to 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle.

IN-LIFE DATES: From: To: 18 September 2017 to 10 December 2017 (till end of last female necropsy)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5 % w/v Carboxy Methyl Cellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle : The test item was in-soluble in distilled water and formed clear homogenous suspension in 0.5 % w/v Carboxy Methyl Cellulose at the concentration of 100 mg/mL (highest dose concentration) as evidenced by the in-house solubility and suspendibility test results. Hence, 0.5 % w/v Carboxy Methyl Cellulose was selected for the test item formulation preparation.
- Concentration in vehicle: G1 (vehicle control): 0 mg/mL; G2 (low dose): 10 mg/mL; G3 (mid dose): 30 mg/mL; G4 (high dose): 100 mg/mL
- Amount of vehicle: 10 mL/kg body weight
- Batch no.: BCBN1690V

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 14 days.
- 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: individually
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and dose formulation analysis for dose concentration verification was done by Analytical Chemistry department of Bioneeds India Private Limited.
The analysis was done as per methods detailed in the Study Plan No. BIO-ANM 450. Sampling and analysis of formulations was performed during first week of treatment and during week 4 of the treatment. The samples were collected in duplicates (20 mL each) from top, middle and bottom layer from low and mid dose concentrations, in duplicates (5 mL each) from top, middle and bottom layer from high dose concentrations and from middle layer in duplicate sets from vehicle control.
The collected samples were transferred to Analytical Chemistry department of Bioneeds India Private Limited for dose concentration analysis. One set of aliquot of each formulation was analyzed. The second aliquot was stored as a backup purpose at 2 to 8°C conditions. The second set of samples was discarded, as the analysis results of first set of samples were within the limits. 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) is ≤10%.

Duration of treatment / exposure:

Males: two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 34 days of treatment).
Females: two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13.

Frequency of treatment:

Once daily

Details on study schedule:

- Age at mating of the mated animals in the study: 14 to 15 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

vehicle control

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day

Remarks:

low dose

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day

Remarks:

mid dose

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

high dose

No. of animals per sex per dose:

12 animals per sex per dose (group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The doses of 0, 100, 300 and 1000 mg/kg body weight for vehicle control, low dose, mid dose and high dose respectively were selected in consultation with the sponsor based on the results of Dose Range Finding Study for Reproduction/Developmental Toxicity Screening Test of Hostacerin DGSB by Oral Gavage in Sprague Dawley Rats (Bioneeds Study No.: BIO-TX 2516).
These doses were selected as the test item Hostacerin DGSB when administered orally to Sprague Dawley males for two weeks prior to mating, during mating and up to the day before sacrifice during post-mating period (total of at 30 days treatment) and females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 after which the pups were sacrificed on lactation day 13, females (dams) were sacrificed on lactation day 14 did not reveal any treatment related effects at all the tested doses (100, 300 and 1000 mg/kg body weight).
- Rationale for animal assignment:
The animals were weighed and arranged in ascending order of their body weights. These body weight stratified animals were distributed to all the groups using Microsoft Excel Spreadsheet, such that body weight variation of animals selected for the study did not exceed ± 20% (+12.02% and -11.65% for males and +12.09% and -10.47% for females) of the mean body weight of each sex. The grouping was done one day prior to the initiation of treatment. Body weight of the animals was analyzed statistically for mean body weight to rule out the statistical significant difference between groups within each sex.

Positive control:

not applicable

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations checked in table 1 were included.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly once were included in (table 1)

BODY WEIGHT: Yes
- Time schedule for examinations: first day of dosing, weekly thereafter and at termination. The
females were weighed on gestation days 0, 7, 14 and 20 during pregnancy and on days 1, 4, 7 and 1
3 during lactation period. (table 2, 3, 9, 10, 12 & 13)

FOOD CONSUMPTION: Food consumption for each animal determined and mean daily diet consumption calculated as
g food/kg body weight/day: Yes; weekly once; gestation days 0 to 7, 7 to 14 and 14 to 20 during
pregnancy and day 1 to 4, 4 to 7, 7 to 13 during lactation period. (table 4, 11 & 14)

Oestrous cyclicity (parental animals):

Two weeks after five days of acclimatization to evaluate its normal oestrus cyclicity (4 to 5 days), daily from the beginning of the treatment period until evidence of mating and on the day of sacrifice for females.

Sperm parameters (parental animals):

Parameters examined: testis weight, epididymis weight
Special emphasis on stages of spermatogenesis and histology of interstitial testicular cell structure.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

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

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

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All animals [after completion of mating period (34 days of treatment)]
- Maternal animals: All animals [on lactation day 14]

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [26 and 27] (i.e. Epidymides, Prostate and Seminal vesicles and coagulating glands, Testes Uterus with cervix Thyroid along with parathyroid) were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The offspring were sacrificed on lactation day 13.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

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

HISTOPATHOLOGY / ORGAN WEIGTHS
Thyroid along with parathyroid was collected from one male and one female pup per litter on lactation day 13 and were prepared for microscopic examination

Statistics:

The data was subjected to various 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 below table: Body weight, Change in body weight, Feed consumption, Copulatory interval, Gestation length, Organ
weights, Anogenital distance, Mean pup weight, Pre implantation loss, Pre natal loss, Post natal loss, Post implantation loss:- Parametric - One-way ANOVA with Dunnett’s post test.
Corpora lutea/dam, Implantations/dam, No. of pups/dam, Sex ratio, Litter size, Pup weight:- Non Parametric - Kruskal-Wallis followed by the Mann-Whitney test. Pregnancy rate. Dams with live pups, Dams with dead pups, No. of litters with/without dead pups, No.of litters with/without resorptions:- Cross Tabs - Chi-square test/ Fischer's Exact Test.

Reproductive indices:

Male Fertility Index (%) = Number of Males Impregnating Females
----------------------------------------------------------- x 100
Number of Males used for Mating

Female Fertility Index (%) = Number of Females confirmed with Pregnancy
------------------------------------------------------------------ x 100
Number of Females used for Mating

Parturition (%) = Number of Parturitions
--------------------------------- x 100
Number of Pregnancies

Copulatory Interval (Days) = Date of Initiation of Cohabitation - Date of Confirmation of Mating
Gestation Length (days) = Date of Delivery - Date of Confirmation of Mating

Offspring viability indices:

Male/Female Sex Ratio = Number Male Pups Per Dam
------------------------------------------
Number Female Pups Per Dam

Live Birth Index (%) = Number of Viable Pups Born
----------------------------------------- x 100
Number of Pups Born
Lactation Day 4
Pup Survival Index (%) = Number of Viable Pups on Lactation Day 4
---------------------------------------------------------------x 100
Number of Pups Born
Lactation Day 7
Pup Survival Index (%) = Number Of Viable Pups on Lactation Day 7
--------------------------------------------------------------- x 100
Number Of Viable Pups on Lactation Day 4

Lactation Day 13
Pup Survival Index (%) = Number Of Viable Pups on Lactation Day 13
---------------------------------------------------------------- x 100
Number Of Viable Pups on Lactation Day 7

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs of toxicity observed at any of the tested dose group animals of either sex during the experimental period.

Dermal irritation (if dermal study):

not specified

Description (incidence and severity):

not applicable

Mortality:

no mortality observed

Description (incidence):

There were no mortality/morbidity observed at any of the tested dose group animals of either sex during the experimental period

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no changes observed in mean body weight and percent change in body weight with respect to day 1 at any of the tested dose group of either sex during the experimental period.
There were no treatment related changes observed in the gestation body weight and percent change in gestation body weight during gestation period at any of the tested dose group animals when compared with vehicle control group animals. However, statistical significant increase in mean body weight on gestation day 14 at G3 group animals was observed when compared with vehicle control group animals. This change is considered as incidental as there were no changes noted during the other gestation periods and there were no effects noted in feed consumption at this period.
There were no treatment related changes observed in the lactation body weight and percent change in lactation body weight at any of the tested dose group animals when compared with vehicle control group animals.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no changes observed in feed consumption at any of the tested dose group animals of either sex during the experimental period.
There were no changes observed in the feed consumption during gestation period at any of the tested dose group animals when compared with vehicle control group animals. However, statistical significant increase in mean feed consumption during gestation day 0 to 7 was noted at low and high dose group animals when compared with vehicle control group animals. This change is considered as incidental and not treatment related as there were no effects noted in mean body weight at this duration and the change is inconsistent.
There were no changes observed in the feed consumption during lactation period at any of the tested dose group animals when compared with vehicle control group animals.

Food efficiency:

not examined

Description (incidence and severity):

not applicable

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

not examined

Description (incidence and severity):

not applicable

Ophthalmological findings:

not examined

Description (incidence and severity):

not applicable

Haematological findings:

not examined

Description (incidence and severity):

not applicable

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes observed in serum T4 levels in males, females (pregnant), lactation day 4 and 13 pups at any of the tested dose groups when compared with vehicle control group.
However, statistically significant decrease in T4 levels at all the tested dose group males when compared with vehicle control group males and statistically significant decrease in T4 levels at mid and high dose group females when compared with vehicle control group females was noted.
This significant variation could be due to two males in G1 group (Animal No. Rc3917 and Rc3922) with a serum T4 level of 139.88 and 134.97 respectively which are significantly more than other animals of same group. In case of females the variation is not dose dependant. Hence, the variations observed in the T4 levels are considered to be incidental.

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

Behaviour (functional findings):

not examined

Description (incidence and severity):

not applicable

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment related histopathological findings noticed in ovaries, testes and epididymides (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure) of the animals from the high dose group during histopathological examination.
Minimal grade and focally distributed interstitial mononuclear cell [MNC] infiltration, unilateral was observed in epididymis of one male animal of G4 group. This lesion was considered as incidental finding because of lack of consistency and similar finding was observed in concurrent control animal.
Testes, which were screened with special emphasis on stages of spermatogenesis and interstitial testicular cell structure, revealed normal progression of the spermatogenic cycle and presence of all expected associations.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

not applicable

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no changes observed in the oestrus cyclicity at any of the tested dose group females during pre-mating treatment, mating treatment and on lactation day 14.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

no effects were noted.

Reproductive performance:

no effects observed

Description (incidence and severity):

No effects were noted. There were no treatment related changes observed in the copulatory interval and number of conceiving days at any of the tested dose group animals when compared with vehicle control group animals. There were no changes observed in the gestation length, implantation index, pre-implantation loss, post-implantation loss, number of pups delivered, sex ratio and live birth index of each litter at any of the tested dose group animals when compared with vehicle control group animals.

Details on results (P0)

no treatment elated effects were noted in reproductive and developmental end points at all the tested dose groups

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Mean live birth index is 99.3, 87.85, 98.52 and 100 % for dose groups G1, G2, G3, and G4, respectively. Total number of dead pups at birth were 1, 3, 2 and 0 for dose groups G1, G2, G3, and G4, respectively.
Mean pup survival index (LD 1-4) is 98.48, 100, 100, and 100 % for dose groups G1, G2, G3, and G4, respectively. Total number of dead pups were 2, 0, 0 and 0 for dose groups G1, G2, G3, and G4, respectively.

Body weight and weight changes:

no effects observed

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:

no effects observed

Description (incidence and severity):

There were no changes observed in anogenital distance ratio of both male and female pups, recorded on lactation day 4 at any dose group litters when compared with vehicle control group litters.
There were no occurrences of nipples in male pups of dams of the tested dose group litters when compared with vehicle control group litters.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

There were no treatment related changes observed in serum T4 levels in lactation day 4 and 13 pups at any of tested dose groups when compared with vehicle control group.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect s observed

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

TABLE 1.        SUMMARYOF CLINICAL SIGNSOF TOXICITY, DETAILED CLINICAL EXAMINATIONAND MORTALITY RECORD

                                                                                                          

Group, Sex & Dose (mg/kg body weight/day)	No. of Animals	Clinical Signs of Toxicity/ Detailed Clinical Examination	Mortality (No. of Mortality/ No. of Animals dosed)
G1, M & 0	12	N	0/12
G2, M & 100	12	N	0/12
G3, M & 300	12	N	0/12
G4, M & 1000	12	N	0/12

M: Male; N: Normal

TABLE 1 (Contd..,). SUMMARYOF CLINICAL SIGNSOF TOXICITY, DETAILED CLINICAL EXAMINATIONAND

MORTALITY RECORD

      

Group, Sex & Dose (mg/kg body weight/day)		No. of Animals		Clinical Signs of Toxicity/ Detailed Clinical Examination		Mortality (No. of Mortality/ No. of Animals dosed)
G1, F & 0		12		N		0/12
G2, F & 100		12		N		0/12
G3, F & 300		12		N		0/12
G4, F & 1000		12		N		0/12

F: Female; N: Normal

TABLE 2.        SUMMARY OF BODY WEIGHT (g)RECORD

    

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Days
		1	7	14	21	28	34
G1, M & 0	Mean	331.48	355.57	372.35	392.61	408.21	429.21
	±SD	21.09	25.46	26.61	30.62	31.33	35.64
	n	12	12	12	12	12	12
G2, M & 100	Mean	333.49	348.67	366.07	382.71	394.48	414.61
	±SD	20.91	24.45	30.62	31.50	33.73	39.05
	n	12	12	12	12	12	12
G3, M & 300	Mean	333.86	349.97	361.35	384.41	399.83	423.79
	±SD	20.50	18.44	22.25	23.24	26.15	27.88
	n	12	12	12	12	12	12
G4, M & 1000	Mean	331.99	357.47	378.38	396.73	411.93	426.47
	±SD	18.56	18.42	19.85	21.46	21.62	23.80
	n	12	12	12	12	12	12

  M: Male; SD: Standard Deviation; n: Number of Animals

 

TABLE 2 (Contd..,). SUMMARY OF BODY WEIGHT (g) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Days
		1	7	14	21^#
G1, F & 0	Mean	263.38	269.72	279.18	292.40
	±SD	16.71	16.23	19.53	22.58
	n	12	12	12	4
G2, F & 100	Mean	263.21	269.81	281.42	292.27
	±SD	16.04	13.15	13.93	21.16
	n	12	12	12	5
G3, F & 300	Mean	263.38	269.54	281.01	281.16
	±SD	15.85	13.80	16.37	21.01
	n	12	12	12	6
G4, F & 1000	Mean	262.96	269.34	286.13	284.89
	±SD	14.76	14.63	15.25	19.99
	n	12	12	12	5

F: Female; SD: Standard Deviation; n: Number of Animals

#: The statistical analysis is not performed for day 21 body weight as the presented mean and standard deviations were calculated only for the females in cohabitation.

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

                                      

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight (%) with respect to Day 1 during Days
		1 to 7	1 to 14	1 to 21	1 to 28	1 to 34
G1, M & 0	Mean	7.23	12.33	18.43	23.16	29.51
	±SD	2.62	4.24	5.26	5.83	7.60
	n	12	12	12	12	12
G2, M & 100	Mean	4.55	9.74	14.78	18.31	24.26
	±SD	2.94	5.40	6.29	6.98	7.56
	n	12	12	12	12	12
G3, M & 300	Mean	4.94	8.42	15.37	19.94	27.21
	±SD	3.94	6.65	7.45	7.38	9.22
	n	12	12	12	12	12
G4, M & 1000	Mean	7.79	14.23	19.91	24.49	28.83
	±SD	4.61	7.57	9.96	10.01	9.93
	n	12	12	12	12	12

  M: Male; SD: Standard Deviation; n: Number of Animals

 

TABLE 3 (Contd..,). SUMMARY OF PERCENTCHANGE IN BODY WEIGHT (%) WITH RESPECT TO DAY 1RECORD

 

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight (%) with respect to Day 1 during Days
		1 to 7	1 to 14	1 to 21^#
G1, F & 0	Mean	2.45	6.02	9.31
	±SD	1.97	3.71	5.11
	n	12	12	4
G2, F & 100	Mean	2.59	7.10	11.07
	±SD	2.35	5.26	4.99
	n	12	12	5
G3, F & 300	Mean	2.42	6.86	8.90
	±SD	3.00	6.13	6.84
	n	12	12	6
G4, F & 1000	Mean	2.45	8.87	11.94
	±SD	1.38	3.28	8.06
	n	12	12	5

   F: Female; SD: Standard Deviation; n: Number of Animals

  #: The statistical analysis is not performed for day 21 body weight as the presented mean and standard deviations were calculated only for the females in cohabitation.

TABLE 4.  SUMMARY OFFEED CONSUMPTION (g/rat/day)RECORD

Group, Sex & Dose (mg/kg body weight/day)		Feed Consumption (g)
		Week 1 (Day 1 to 7)	Week 2 (Day 7 to 14)	Post Mating (Day 28 to 34)
G1, M & 0	Mean	23.12	22.81	29.41
	±SD	1.34	2.28	0.67
	n	12	12	12
G2, M & 100	Mean	22.76	23.08	28.95
	±SD	1.71	1.95	0.76
	n	12	12	12
G3, M & 300	Mean	22.66	23.23	29.49
	±SD	1.48	2.35	0.66
	n	12	12	12
G4, M & 1000	Mean	24.16	24.26	29.68
	±SD	1.12	1.73	0.66
	n	12	12	12

M: Male;SD: Standard Deviation; n: Number ofanimals

 

TABLE 4 (Contd..,). SUMMARY OF FEED CONSUMPTION (g/rat/day)RECORD

Group, Sex & Dose (mg/kg body weight/day)		Feed Consumption (g)
		Week 1 (Day 1 to 7)	Week 2 (Day 7 to 14)
G1, F & 0	Mean	18.19	18.43
	±SD	1.13	1.55
	n	12	12
G2, F & 100	Mean	18.85	18.65
	±SD	1.93	1.58
	n	12	12
G3, F & 300	Mean	17.63	18.21
	±SD	1.98	0.86
	n	12	12
G4, F & 1000	Mean	18.33	18.60
	±SD	1.18	0.80
	n	12	12

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

TABLE 5.           SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex & Dose (mg/kg body weight/day)				Epididymes		Testes		Prostate+Seminal vesicles with coagulating glands (PSC)		Thyroid along with parathyroid^#
G1, M & 0		Mean		1.7248		3.3488		3.8053		0.0357
		±SD		0.1932		0.2725		0.5705		0.0033
		n		12		12		12		12
G2, M & 100		Mean		1.7994		3.3460		3.7837		0.0370
		±SD		0.1389		0.2603		0.3461		0.0022
		n		12		12		12		12
G3, M & 300		Mean		1.6893		3.2506		4.1135		0.0359
		±SD		0.1685		0.2056		0.2480		0.0027
		n		12		12		12		12
G4, M & 1000		Mean		1.7526		3.3840		4.1498		0.0363
		±SD		0.1925		0.2029		0.4138		0.0020
		n		12		12		12		12

M: Male;SD: Standard Deviation; n: Number of animals: #: Weighed post fixation

TABLE 5 (Contd..,). SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

                                                                                                       

Group, Sex & Dose (mg/kg body weight/day)				Uterus		Thyroid along with parathyroid^#
G1, F & 0		Mean		0.6154		0.0291
		±SD		0.0395		0.0031
		n		11		11
G2, F & 100		Mean		0.5932		0.0253
		±SD		0.0270		0.0033
		n		11		11
G3, F & 300		Mean		0.6264		0.0279
		±SD		0.0764		0.0056
		n		11		11
G4, F & 1000		Mean		0.5864		0.0306
		±SD		0.0420		0.0048
		n		12		12

 F: Female;SD: Standard Deviation; n: Number of dams;

#: Weighed post fixation (excluding non-pregnant animals)

TABLE 6.           SUMMARY OF FASTING BODY WEIGHT (g) AND
ORGAN WEIGHT (%) RELATIVE TO FASTING BODY WEIGHT RECORD

Group, Sex & Dose (mg/kg body weight/day)				Fasting Body Weight (g)		Epididymes		Testes		Prostate+Seminal vesicles with coagulating glands (PSC)		Thyroid along with parathyroid
G1, M & 0		Mean		403.62		0.4300		0.8341		0.9467		0.0089
		±SD		34.33		0.0578		0.0859		0.1466		0.0011
		n		12		12		12		12		12
G2, M & 100		Mean		391.26		0.4644		0.8645		0.9795		0.0095
		±SD		37.96		0.0638		0.1260		0.1606		0.0010
		n		12		12		12		12		12
G3, M & 300		Mean		397.31		0.4275		0.8208		1.0396		0.0091
		±SD		26.57		0.0570		0.0674		0.0932		0.0008
		n		12		12		12		12		12
G4, M & 1000		Mean		399.57		0.4393		0.8503		1.0407		0.0091
		±SD		24.64		0.0473		0.0805		0.1104		0.0008
		n		12		12		12		12		12

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

TABLE 6 (Contd..,). SUMMARY OF FASTING BODY WEIGHT (g) AND
ORGAN WEIGHT (%) RELATIVE TO FASTING BODY WEIGHT RECORD

Group, Sex & Dose (mg/kg body weight/day)				Fasting Body Weight (g)		Uterus		Thyroid along with parathyroid
G1, F & 0		Mean		304.24		0.2035		0.0096
		±SD		21.64		0.0224		0.0013
		n		11		11		11
G2, F & 100		Mean		306.66		0.1943		0.0083
		±SD		23.92		0.0148		0.0012
		n		11		11		11
G3, F & 300		Mean		324.21		0.1933		0.0087
		±SD		28.55		0.0177		0.0022
		n		11		11		11
G4, F & 1000		Mean		306.69		0.1921		0.0101
		±SD		24.23		0.0182		0.0020
		n		12		12		12

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

TABLE 7.           SUMMARY RECORD OF VAGINAL SMEAR EXAMINATION FOR DETERMINATION OF OESTRUS CYCLICITY

Group & Dose (mg/kg body weight/day)		No. of Females		No. of Females with Regular Oestrus Cyclicity during Pre-mating, mating and on Lactation day 14		No. of Females with Irregular Oestrus Cyclicity during Pre-mating, mating and on Lactation day 14
G1 & 0		12		12		0
G2 & 100		12		12		0
G3 & 300		12		12		0
G4 & 1000		12		12		0

TABLE 8.           SUMMARY OF COHABITATION RECORD

Group & Dose (mg/kg body weight/day)				Copulatory Interval (Days)				Conceiving Days (1 to 5)		Conceiving Days (6 to 20)
G1 & 0		Mean		5.83		n		8		4
		±SD		4.63
		n		12		%		66.7		33.3
G2 & 100		Mean		7.00		n		6		6
		±SD		4.24
		n		12		%		50.0		50.0
G3 & 300		Mean		7.42		n		5		7
		±SD		4.60
		n		12		%		41.7		58.3
G4 & 1000		Mean		7.58		n		6		6
		±SD		4.80
		n		12		%		50.0		50.0

SD: Standard Deviation; n: Number of animals; %: Percent

TABLE 9.           SUMMARY RECORD OF BODY WEIGHT (g) DURINGGESTATION PERIOD

Group & Dose (mg/kg body weight/day)		Body weight (g) on Gestation Days
		0	7	14	20
G1 & 0	Mean	283.86	297.35	342.70	421.08
	±SD	21.63	21.53	28.88	20.97
	n	11	11	11	11
G2 & 100	Mean	291.15	304.38	349.07	422.35
	±SD	15.19	13.65	19.69	17.65
	n	11	11	11	11
G3 & 300	Mean	291.93	307.30	364.18*	440.73
	±SD	11.96	11.79	12.56	22.28
	n	11	11	11	11
G4 & 1000	Mean	292.90	305.82	358.55	429.48
	±SD	14.68	15.66	17.10	24.61
	n	12	12	12	12

SD: Standard Deviation; n: Number of dams

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

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

Group & Dose (mg/kg body weight/day)		Percent Change in body weight (%) during Gestation
		0 to 7	7 to 14	14 to 20
G1 & 0	Mean	4.78	15.24	23.31
	±SD	0.82	4.95	6.98
	n	11	11	11
G2 & 100	Mean	4.59	14.70	21.17
	±SD	1.38	4.36	5.18
	n	11	11	11
G3 & 300	Mean	5.28	18.57	21.02
	±SD	1.78	3.46	4.51
	n	11	11	11
G4 & 1000	Mean	4.41	17.30	19.82
	±SD	0.92	2.71	4.74
	n	12	12	12

SD: Standard Deviation; n: Number of dams

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

Group, Sex & Dose (mg/kg body weight/day)		Feed Consumption (g) during Gestation
		GD 0 to 7	GD 7 to 14	GD 14 to 20
G1 & 0	Mean	22.64	26.85	30.28
	±SD	1.35	1.46	1.12
	n	11	11	11
G2 & 100	Mean	23.92*	27.62	30.43
	±SD	0.79	0.96	0.91
	n	11	11	11
G3 & 300	Mean	23.19	27.05	30.00
	±SD	0.89	1.49	1.06
	n	11	11	11
G4 & 1000	Mean	23.92*	26.79	30.47
	±SD	0.78	1.12	0.69
	n	12	12	12

SD: Standard Deviation; n: Number of dams
*: Statistically significant than the control group (p<0.05)

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

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Lactation Day
		1	4	7	13
G1 & 0	Mean	309.57	313.98	320.23	327.06
	±SD	21.76	22.19	24.05	21.94
	n	11	11	11	11
G2 & 100	Mean	307.28	313.82	318.42	329.52
	±SD	30.13	28.65	28.85	23.41
	n	11	11	11	11
G3 & 300	Mean	327.46	335.18	341.73	346.58
	±SD	35.21	31.80	31.26	28.52
	n	11	11	11	11
G4 & 1000	Mean	306.28	313.07	318.52	327.51
	±SD	33.02	28.48	27.29	24.06
	n	12	12	12	12

SD: Standard Deviation; n: Number of animals

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

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in body weight (%) during Lactation
		1 to 4	4 to 7	7 to 13
G1 & 0	Mean	1.42	1.97	2.20
	±SD	0.46	1.22	1.28
	n	11	11	11
G2 & 100	Mean	2.20	1.48	3.70
	±SD	1.85	0.49	3.39
	n	11	11	11
G3 & 300	Mean	2.50	1.98	1.50
	±SD	2.26	0.89	1.15
	n	11	11	11
G4 & 1000	Mean	2.42	1.79	2.93
	±SD	2.73	0.64	1.70
	n	12	12	12

SD: Standard Deviation; n: Number of dams    

                                                                      

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

Group & Dose (mg/kg body weight/day)		Feed Consumption (g) during Lactation
		LD 1 to 4	LD 4 to 7	LD 7 to 13
G1 & 0	Mean	23.34	27.53	32.34
	±SD	1.26	1.55	1.22
	n	11	11	11
G2 & 100	Mean	24.65	29.18	33.01
	±SD	2.32	2.43	1.04
	n	11	11	11
G3 & 300	Mean	22.80	28.77	33.44
	±SD	1.89	3.14	2.17
	n	11	11	11
G4 & 1000	Mean	24.67	28.77	33.64
	±SD	3.09	2.52	1.33
	n	12	12	12

SD: Standard Deviation; n: Number of dams; LD: Lactation Day

 TABLE 15.       SUMMARY RECORD OF GESTATION LENGTH (DAYS) AND DELIVERY DATA

Group & Dose (mg/kg body weight/day)		Gestation Length (Days)		Delivery Data
				Litter Size (No.)	Live Pups (No.)				Dead Pups (No.)				Sex Ratio (M/F) at Birth	Live Birth Index (%)
					Total (No.)	Male (No.)	Female (No.)		Total (No.)	Male (No.)	Female (No.)
G1 & 0	Mean	22.64		12.64	12.55	5.91	6.64		0.09	0.00	0.09		1.13	99.30
	±SD	1.03		2.06	2.07	1.76	2.84		0.30	0.00	0.30		0.71	2.32
	n	11		11	11	11	11		11	11	11		11	11
G2 & 100	Mean	22.55		13.73	13.45	7.00	6.45		0.27	0.09	0.18		1.14	97.85
	±SD	1.13		2.15	2.30	1.95	1.51		0.47	0.30	0.40		0.42	3.74
	n	11		11	11	11	11		11	11	11		11	11
G3 & 300	Mean	22.73		13.00	12.82	6.36	6.45		0.18	0.00	0.18		1.16	98.52
	±SD	1.42		1.95	2.04	1.86	2.21		0.40	0.00	0.40		0.69	3.31
	n	11		11	11	11	11		11	11	11		11	11
G4 & 1000	Mean	22.83		11.83	11.83	5.58	6.25		0.00	0.00	0.00		0.92	100.00
	±SD	1.27		2.17	2.17	2.15	0.87		0.00	0.00	0.00		0.38	0.00
	n	12		12	12	12	12		12	12	12		12	12

  M: Male; F: Female;SD: Standard Deviation; n: Number of dams; NAD: No abnormality detected; LD: Lactation Day

 

TABLE 16.       SUMMARY OF LITTER OBSERVATION RECORD DURING LACTATION PERIOD

Group & Dose (mg/kg body weight/day)		No. of Live Pups At Birth	During LD 1 to 4							Sex Ratio (M/F) at LD 4	No. of Survived Pups during LD 1 to 4	Pup Survival Index (%) LD 1 to 4
			Live Pups (No.)				Dead Pups (No.)
			Total (No.)	Male (No.)	Female (No.)		Total (No.)	Male (No.)	Female (No.)
G1 & 0	Mean	12.55	12.36	5.91	6.45		0.18	0.00	0.18	1.15	11.33	98.48
	±SD	2.07	2.20	1.76	2.81		0.60	0.00	0.60	0.70	4.14	5.03
	n	11	11	11	11		11	11	11	11	12	11
G2 & 100	Mean	13.45	13.45	7.00	6.45		0.00	0.00	0.00	1.14	12.33	100.00
	±SD	2.30	2.30	1.95	1.51		0.00	0.00	0.00	0.42	4.46	0.00
	n	11	11	11	11		11	11	11	11	12	11
G3 & 300	Mean	12.82	12.82	6.36	6.45		0.00	0.00	0.00	1.16	11.75	100.00
	±SD	2.04	2.04	1.86	2.21		0.00	0.00	0.00	0.69	4.18	0.00
	n	11	11	11	11		11	11	11	11	12	11
G4 & 1000	Mean	11.83	11.83	5.58	6.25		0.00	0.00	0.00	0.92	11.83	100.00
	±SD	2.17	2.17	2.15	0.87		0.00	0.00	0.00	0.38	2.17	0.00
	n	12	12	12	12		12	12	12	12	12	12

M: Male: F: Female;SD: Standard Deviation; n: Number of dams; LD: Lactation Day

TABLE 16 (Contd..,). SUMMARY OF LITTER OBSERVATION RECORD DURING LACTATION PERIOD

Group & Dose (mg/kg body weight/ day)		Live Pups (No.) on LD 4				Pups Sacrificed for Blood Collection on LD 4 (No.)				Live Pups (No.) on LD 4 after Sacrificed for Blood Collection			During LD 4 to 7								Sex Ratio (M/F) at LD 7	No. of Survived Pups during LD 4-7	Pup Survival Index (%) during LD 4-7
													Live Pups (No.)
		Male (No.)	Female (No.)	Total (No.)		Male (No.)	Female (No.)	Total (No.)		Male (No.)	Female (No.)	Total (No.)	Male (No.)	Female (No.)	Total (No.)		Male (No.)	Female (No.)	Total (No.)
G1 & 0	Mean	5.91	6.45	12.36		0.00	1.36	1.36		5.91	5.09	11.00	5.91	5.09	11.00		0.00	0.00	0.00		1.62	11.00	100.00
	±SD	1.76	2.81	2.20		0.00	0.81	0.81		1.76	2.47	1.55	1.76	2.47	1.55		0.00	0.00	0.00		1.32	1.55	0.00
	n	11	11	11		11	11	11		11	11	11	11	11	11		11	11	11		11	11	11
G2 &  100	Mean	7.00	6.45	13.45		0.00	1.64	1.64		7.00	4.82	11.82	7.00	4.82	11.82		0.00	0.00	0.00		1.65	11.82	100.00
	±SD	1.95	1.51	2.30		0.00	0.67	0.67		1.95	1.47	1.83	1.95	1.47	1.83		0.00	0.00	0.00		0.81	1.83	0.00
	n	11	11	11		11	11	11		11	11	11	11	11	11		11	11	11		11	11	11
G3 &  300	Mean	6.36	6.45	12.82		0.00	1.55	1.55		6.36	4.91	11.27	6.36	4.91	11.27		0.00	0.00	0.00		1.75	11.27	100.00
	±SD	1.86	2.21	2.04		0.00	0.82	0.82		1.86	2.12	1.49	1.86	2.12	1.49		0.00	0.00	0.00		1.29	1.49	0.00
	n	11	11	11		11	11	11		11	11	11	11	11	11		11	11	11		11	11	11
G4 & 1000	Mean	5.58	6.25	11.83		0.00	1.00	1.00		5.58	5.25	10.83	5.58	5.25	10.83		0.00	0.00	0.00		1.17	10.83	100.00
	±SD	2.15	0.87	2.17		0.00	0.95	0.95		2.15	1.06	1.40	2.15	1.06	1.40		0.00	0.00	0.00		0.64	1.40	0.00
	n	12	12	12		12	12	12		12	12	12	12	12	12		12	12	12		12	12	12

M: Male: F: Female;SD: Standard Deviation; n: Number of dams; LD: Lactation Day

TABLE 16 (Contd..,). SUMMARY OF LITTER OBSERVATION RECORD DURING LACTATION PERIOD

Group & Dose (mg/kg body weight/day)		Live Pups (No.) on LD 7				During LD 7 to 13								Sex Ratio (M/F) at LD 7	No. of Survived Pups during LD 7 to 13	Pup Survival Index (%) during LD 7 to 13
						Live Pups (No.)
		Male (No.)	Female (No.)	Total (No.)		Male (No.)	Female (No.)	Total (No.)		Male (No.)	Female (No.)	Total (No.)
G1 & 0	Mean	5.91	5.09	11.00		5.91	5.09	11.00		0.00	0.00	0.00		1.62	11.00	100.00
	±SD	1.76	2.47	1.55		1.76	2.47	1.55		0.00	0.00	0.00		1.32	1.55	0.00
	n	11	11	11		11	11	11		11	11	11		11	11	11
G2 & 100	Mean	7.00	4.82	11.82		7.00	4.82	11.82		0.00	0.00	0.00		1.65	11.82	100.00
	±SD	1.95	1.47	1.83		1.95	1.47	1.83		0.00	0.00	0.00		0.81	1.83	0.00
	n	11	11	11		11	11	11		11	11	11		11	11	11
G3 & 300	Mean	6.36	4.91	11.27		6.36	4.91	11.27		0.00	0.00	0.00		1.75	11.27	100.00
	±SD	1.86	2.12	1.49		1.86	2.12	1.49		0.00	0.00	0.00		1.29	1.49	0.00
	n	11	11	11		11	11	11		11	11	11		11	11	11
G4 & 1000	Mean	5.58	5.25	10.83		5.58	5.25	10.83		0.00	0.00	0.00		1.17	10.83	100.00
	±SD	2.15	1.06	1.40		2.15	1.06	1.40		0.00	0.00	0.00		0.64	1.40	0.00
	n	12	12	12		12	12	12		12	12	12		12	12	12

M: Male: F: Female;SD: Standard Deviation; n: Number of dams; LD: Lactation Day

 

TABLE 17.       SUMMARY OF PUP OBSERVATIONS RECORD DURING LACTATION PERIOD

Group & Dose (mg/kg body weight/day)	Pup Observation on LD 1	Pup Observation on LD 4	Pup Observation on LD 7	Pup Observation on LD 13
G1 & 0	N	N	N	N
G2 & 100	N	N	N	N
G3 & 300	N	N	N	N
G4 & 1000	N	N	N	N

LD: Lactation Day; N: Normal

 

 TABLE 18.          SUMMARY RECORD OF MEAN PUP WEIGHT (g) DURING LACTATION PERIOD

Group& Dose (mg/kg body weight/day)		Mean Pup weight (g) on LD 1			Mean Pup weight (g) on LD 4			Mean Pup Weight (g) on LD 7			Mean Pup weight (g) on LD 13
		Male	Female		Male	Female		Male	Female		Male	Female
G1 & 0	Mean	6.68	5.98		9.99	8.52		14.55	12.15		25.65	21.90
	±SD	0.49	0.66		0.76	0.74		1.12	1.26		1.63	2.36
	n	11	11		11	11		11	11		11	11
G2 & 100	Mean	6.24	5.30		10.26	8.89		14.99	13.13		27.22	22.26
	±SD	0.68	0.83		1.15	1.11		1.54	1.82		2.26	2.16
	n	11	11		11	11		11	11		11	11
G3 & 300	Mean	6.39	5.76		10.22	8.65		15.63	13.13		27.11	21.93
	±SD	0.59	0.66		1.41	0.83		1.14	1.62		1.48	3.26
	n	11	11		11	11		11	11		11	11
G4 & 1000	Mean	6.52	5.60		11.19*	9.06		16.03*	13.22		27.29	21.68
	±SD	0.64	0.44		1.22	1.36		1.19	1.61		1.90	3.61
	n	12	12		12	12		12	12		12	12

SD: Standard Deviation; n: Number of dams;LD: Lactation Day

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

 

 TABLE 19.          SUMMARY OF UTERI OBSERVATIONS RECORD

Group & Dose (mg/kg body weight/day)		No. of Corporaluetea	No. of Implantations	Pre-Implantation Loss (%)	Post-Implantation Loss (%)	Pre-natal Loss (No.)	Post-natal Loss (%)	Post-natal Loss (No.)	No. of Early Resorptions	No. of Late Resorptions
G1 & 0	Mean	12.82	12.64	1.22	0.70	0.09	1.52	0.18	0.00	0.00
	±SD	2.23	2.06	2.72	2.32	0.30	5.03	0.60	0.00	0.00
	n	11	11	11	11	11	11	11	11	11
G2 & 100	Mean	13.82	13.73	0.57	2.15	0.27	0.00	0.00	0.00	0.00
	±SD	2.23	2.15	1.88	3.74	0.47	0.00	0.00	0.00	0.00
	n	11	11	11	11	11	11	11	11	11
G3 & 300	Mean	13.18	13.00	1.36	1.48	0.18	0.00	0.00	0.00	0.00
	±SD	1.99	1.95	3.11	3.31	0.40	0.00	0.00	0.00	0.00
	n	11	11	11	11	11	11	11	11	11
G4 & 1000	Mean	12.00	11.83	1.28	0.00	0.00	0.00	0.00	0.00	0.00
	±SD	2.26	2.17	3.05	0.00	0.00	0.00	0.00	0.00	0.00
	n	12	12	12	12	12	12	12	12	12

SD: Standard Deviation; n: Number of dams

 

 TABLE 20.          SUMMARY RECORD OF ANO-GENITAL DISTANCE [AGD] RATIO ON LACTATION DAY 4

Group & Dose (mg/kg body weight/day)		Mean Male AGD Ratio	Mean Female AGD Ratio
G1 & 0	Mean	2.53	1.17
	±SD	0.18	0.07
	n	11	11
G2 & 100	Mean	2.66	1.17
	±SD	0.16	0.09
	n	11	11
G3 & 300	Mean	2.65	1.18
	±SD	0.11	0.07
	n	11	11
G4 & 1000	Mean	2.69	1.23
	±SD	0.21	0.17
	n	12	12

 SD: Standard Deviation; n: Number of dams

 

 TABLE 21.          SUMMARY RECORD OF MALE PUP NIPPLE/AREOLAE RETENTION (no.) ON LACTATION DAY 13

Group & Dose (mg/kg body weight/day)		No. of Nipples/Areolae
G1 & 0	Mean	0.00
	±SD	0.00
	n	12
G2 & 100	Mean	0.00
	±SD	0.00
	n	11
G3 & 300	Mean	0.00
	±SD	0.00
	n	11
G4 & 1000	Mean	0.00
	±SD	0.00
	n	11

SD: Standard Deviation; n: Number of dams

TABLE 22.          SUMMARY OF SERUM T4 LEVELS (ng/mL) RECORD - MALES

Group, Sex & Dose (mg/kg body weight/day)		Serum T4 Levels (ng/mL)
G1, M & 0	Mean	101.492
	±SD	18.172
	n	12
G2, M & 100	Mean	84.759*
	±SD	5.744
	n	12
G3, M & 300	Mean	82.176*
	±SD	8.380
	n	12
G4, M & 1000	Mean	73.659*
	±SD	5.815
	n	12

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

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

                   

TABLE 23.          SUMMARY OF SERUM T4 LEVELS (ng/mL) RECORD - FEMALES

Group, Sex & Dose (mg/kg body weight/day)		Serum T4 Levels (ng/mL)
G1, F & 0	Mean	78.821
	±SD	11.933
	n	11
G2, F & 100	Mean	73.067
	±SD	7.682
	n	11
G3, F & 300	Mean	64.148*
	±SD	5.037
	n	11
G4, F & 1000	Mean	67.385*
	±SD	8.707
	n	12

SD: Standard Deviation; n: Number of dams

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

 

TABLE 24.          SUMMARY OF SERUM T4 LEVELS (ng/mL) RECORD - LACTATION DAY 4 PUPS

Group & Dose (mg/kg body weight/day)		Serum T4 Levels (ng/mL)
G1 & 0	Mean	70.985
	±SD	5.708
	n	9
G2 & 100	Mean	69.599
	±SD	3.939
	n	10
G3 & 300	Mean	69.114
	±SD	3.762
	n	9
G4 & 1000	Mean	70.672
	±SD	5.016
	n	7

SD: Standard Deviation; n: Number of dams (Serum collected from pups with litter size of more than ten per dam pooled for analysis)

TABLE 25.          SUMMARY OF SERUM T4 LEVELS (ng/mL) RECORD - LACTATION DAY 13 PUPS

Group & Dose (mg/kg body weight/day)		Serum T4 Levels (ng/mL)
G1 & 0	Mean	73.460
	±SD	8.044
	n	11
G2 & 100	Mean	71.633
	±SD	7.751
	n	11
G3 & 300	Mean	71.633
	±SD	7.751
	n	11
G4 & 1000	Mean	75.088
	±SD	7.169
	n	12

SD: Standard Deviation; n: Number of dams (Serum collected from pups per dam pooled for analysis 

 

 TABLE 26.          SUMMARY OF GROSS PATHOLOGY RECORD

Organs/Lesions	Group	G1		G2		G3		G4
	Dose (mg/kg body weight/day)	0		100		300		1000
	Sex	M	F	M	F	M	F	M	F
	No. of Animals	12	12	12	12	12	12	12	12
No Abnormality Detected		12	12	12	12	12	12	12	12

Note: The numeral indicates number of animal/s without lesion. M: Male; F: Female  

TABLE 27.          SUMMARY OF HISTOPATHOLOGY RECORD

 

Group No.	G1		G4
Dose (mg/kg body weight/day)	0		1000
Sex	M	F	M	F
No. of Animals	12	12	12	12
ORGANS AND OBSERVATIONS
EPIDIDYMIDES
Interstitial MNC infiltration ; unilateral	1	X	1	X

Note: The numeral indicates number of animal/s with lesion; M: Male; F: Female; X: Organs not subjected for histopathological examination due to sex difference.

 

Applicant's summary and conclusion

Conclusions:

Based on the results of the OECD 421 screening study in rats discussed, the No Observed Adverse Effect Level (NOAEL) of the test item Hostacerin DGSB was found to be 1000 mg/kg body weight when administered to the males for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 34 days), to the females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13 observation under the experimental conditions employed in the study.

Executive summary:

The test item, Hostacerin DGSB was evaluated for possible adverse effects following repeated oral dosing to males for 34 days, to females for two weeks pre-mating period, during mating period, during pregnancy (gestation) and up to lactation day 13 to evaluate effects of test item on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus, parturition, and early neonatal development.

A total of 96 (48 males + 48 females) Sprague Dawley rats were distributed to four groups. Each group (G1, G2, G3and G4) consisted of 12 males and 12 females. The animals in G1 group were administered with vehicle[0.5% w/v Carboxy Methyl Cellulose],animals in G2, G3 and G4groups were administered withtest itemat dose levels of100, 300 and 1000 mg/kg body weight for low dose, mid dose and high dose groups respectively. The vehicle and test item formulations were administered orally by gavage at the dose volume of 10 mL/kg body weight. Males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 34 days of treatment). The females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 after which the pups were sacrificed on lactation Day 13 and females (dams) were sacrificed on lactation day 14 after overnight fasting (water allowed). Dose formulation analysis for homogeneity and concentration verification was performed during week 1 and 4 of the treatment period and the results are found to be within the acceptable range of 85 to 115 % the nominal concentration and the % RSD is less than 10.0%.All animals were observed for clinical signs once daily, mortality and morbidity twice daily, detailed clinical examination weekly once, body weight and feed consumption weekly once.The serum collected from adult males and females and from lactation day 4 and 13 pups representing each per litter was screened for T4 levels. Females were observed for oestrus cyclicity during pre-mating treatment and mating treatment period and the dams on lactation day 14 prior to sacrifice. The females were observed for copulatory interval and all the adult animals were observed for mating and fertility index. Each litter was examined after delivery (lactation day 1) and the number and sex of pups (litter size), stillbirths (dead pups born on day 1) and live births were recorded. The dams were observed for body weights and feed consumption during gestation and lactation periods, gestation length, live birth index, number of pups, sex ratio and pup survival indexthroughout the lactation period. The pups were observed forclinical signs and once daily from PND 1 to 13. The both male and female pupweights were recorded separately on lactation days 1, 4, 7 and 13.The anogenital distance of each pup was measured on lactation day 4. The numbers of nipples/areolae in male pups were counted on lactation day 13.Gross pathology and organ weighing were performed on day 35 for males. The gross pathology was performed on lactation day 13 for pups, lactation day 14 for dams. The number of corpora lutea and implantation sites for dams were recorded during necropsy.

All animals did not reveal any clinical signs of toxicity, no mortality or morbidity observed throughout the experimental period. No treatment related changes in body weight, percent change in body weight, feed consumption, organ weights (both absolute and relative) were observed in both the sex of all tested dose group animals. There were no treatment related changes observed in serum T4 levels of adult animals and in serum T4 levels of pups at any of the tested dose groups.

Females did not reveal any treatment related changes in oestrus cyclicity, copulatory interval, fertility index, body weights and feed consumption during gestation and lactation periods, gestation length, implantation index, pre-implantation loss, post-impalntation loss, number of pups delivered, live birth index of each litter, sex ratio and pup survival index at all the tested dose groups throughout the lactation periodwhen compared with vehicle control group animals.

All pups did not reveal any clinical signs or external anomalies throughout the lactation period. No treatment related changes in pup weights, anogenital distance ratio were noted. No occurrences of nipples in male pups at any of the tested dose groups and vehicle control group.

All animals of all dose groups did not reveal any treatment related gross pathological and histopathological findings compared to vehicle control group.