Reaction products of alcohols, C11-14-iso, C13 rich and phosphorus pentoxide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

- Dose range finding study for reproduction/developmental toxicity screening test: NOAEL: 1000 mg/kg bw.

- Reproduction/developmental toxicity screening test (OECD 421): NOAEL: 1000 mg/kg bw.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 January 2018 to 05 September 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

TESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out: Hostacor ITD [Phosphoric acid, isotridecyl ester]

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:

yes

Justification for study design:

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

- Premating exposure duration for parental (P0) animals: 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 based on the results of Dose Range Finding Study for Reproduction/ Developmental Toxicity Screening Test of Hostacor ITD by Oral Gavage in Sprague Dawley Rats (Bioneeds Study No.: BIO-TX 2704).
These doses were selected as the test item Hostacor ITD 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 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 were did not reveal any treatment related effects at all the tested doses (100, 300 and 1000 mg/kg body weight).

- Route of administration: Oral gavage
- Other considerations,
choice of species & strain: Rat (Rattus norvegicus) & Sprague Dawley
vehicle and number of animals: Corn oil and 12 animals per group per sex

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot No.of test material: Clariant Produkte (Deutschland) GmbH
- Expiration date of the lot: 22.02.2022
- Purity test date: 29.03.2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Stability under test conditions: 6 hours at Ambient (21 to 29°C) conditions
- Solubility and stability of the test substance in the solvent/vehicle: Corn oil was used as vehicle for the preparation of formulations as evidenced by the in-house miscibility test results and stable for 6 hours at Ambient (21 to 29°C) conditions


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Formulated in corn oil


FORM AS APPLIED IN THE TEST: Liquid

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Rat and Sprague-Dawley is one of the standard laboratory rodent species and strain 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
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9 to 10 weeks
- Weight at study initiation: Males: 239.19 g to 273.99 g; Females: 211.08 g to 244.79 g
- Housing: 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 (ad libitum): Altromin Maintenance diet for rats and mice 1324 manufactured by Altromin Spezialfutter GmbH & Co. KG
- Water (ad libitum): Deep bore-well water passed through reverse osmosis unit
- Acclimation period: 11 January 2018 to 05 February 2018 [including Including Estrous Cycle Evaluation]
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.4 to 23.9 degree C
- Humidity (%): 40 to 69%,
- Air changes (per hr): 12 to 15
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle
IN-LIFE DATES: From: 11 January 2018; To: 12 April 2018

Route of administration:

oral: gavage

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

The required quantity of test item was weighed into a clean beaker and there by adding little volume of corn oil (vehicle) in to the beaker, mixed well with glass rod and transferred into the measuring cylinder and the beaker was rinsed using a small volume of corn oil and the volume was transferred to measuring cylinder. The rinsing procedure was repeated until entire quantity of the test item formulation was transferred into a measuring cylinder. Finally, the volume was made up to the required quantity with vehicle to get a desired concentration of 10, 30 and 100 mg/mL of test item for low, mid and high dose groups respectively as mentioned in the table below

VEHICLE
- Justification for use and choice of vehicle: Corn oil was used as vehicle for the preparation of formulations as evidenced by the in-house miscibility test results
- Concentration in vehicle: 10, 30 and 100 mg/mL of test item for low, mid and high dose groups respectively
- Amount of vehicle: 10 mL/kg body weight
- batch no.: A1708001
- Purity: 100%

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: minimum: 1 day; maximum: 17 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 such incidence
- After successful mating each pregnant female was caged (how): individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity and dose formulation analysis was performed during week 1 and 5 of treatment period and the results were found to be within the acceptable range of
85 to 115% the nominal concentration.

The Concentrations: G1: 0 mg/mL; G2: 10 mg/mL; G3: 30 mg/mL; G4: 100 mg/mL

Duration of treatment / exposure:

Males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 37 days of treatment). The females were treated for 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: 13 to 14 weeks

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle Control

Dose / conc.:

100 mg/kg bw/day

Remarks:

Low Dose

Dose / conc.:

300 mg/kg bw/day

Remarks:

Mid Dose

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

High Dose

No. of animals per sex per dose:

12 animals per sex per dose

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 selectedbased on the results of Dose Range Finding Study for Reproduction/ Developmental Toxicity Screening Test of Hostacor ITD by Oral Gavage in Sprague Dawley Rats (Bioneeds Study No.: BIO-TX 2704).

These doses were selected as the test item Hostacor ITD 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 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 were did not reveal any treatment related effects at all the tested doses (100, 300 and 1000 mg/kg body weight).

- Rationale for animal assignment (if not random): 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% to +8.89% for males and -11.85% to +11.20% for females) of the mean body weight of each sex.

Positive control:

Not applicable

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once Daily
- Cage side observations checked in table [No.1] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly once

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly once and at termination. For females on gestation days 0, 7, 14 and 20 during pregnancy and on days 1, 4, 7 and 13 during lactation period.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

Oestrous cyclicity (parental animals):

Estrous cycles were monitored for two weeks after five days of acclimatization to evaluate its normal estrous cyclicity (4 to 5 days). Only females with normal estrous cyclicity were selected for the treatment. Vaginal smears were monitored daily from the beginning of the treatment period until evidence of mating. Estrous cyclicity was also monitored on the day of sacrifice.

Sperm parameters (parental animals):

Parameters examined in male parental generations:
testis weight, epididymis weight

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, 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 as soon as possible after the last litters in each generation were produced.]
- 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 [5, 6 and 27] were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The offspring were subjected to postmortem examinations (macroscopic examination) as follows:

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

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

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

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 to day 35 at all the tested group animals of either sex during the experimental period. However, a temporary statistical significant reduction in percent change in body weight was observed in G4 males during day 1 to 8 and day 1 to day 14 when compared with vehicle control group males.

This change is not considered treatment related but incidental and inconsistent as all animals have gained weight in absolute terms and no significant reduction in percent body weight change has been noted when considering the entire experimental period from day 1 to 35, compared to all other groups.

Additionally, no changes in feed consumption and no clinical signs of toxicity were noted during the experimental period.

Food consumption and compound intake (if feeding study):

no effects observed

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.

Description (incidence and severity):

Not applicable

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:

not examined

Description (incidence and severity):

Not applicable

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

Description (incidence and severity):

No treatment related histopathological findings noticed.

Lesions considered spontaneous and incidental were observed in treated and control rats. These lesions consisted of unilateral interstitial mononuclear cell (MNC) infiltration in epididymis.

Uterus showed yellow brown colour pigmentation. This pigmentation was observed in G4 and concurrent control group (G1) females. Pigmentation might be from decidualized myometrium because of pregnancy.

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.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

Not applicable

Other effects:

no effects observed

Description (incidence and severity):

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

Serum Estrogen and Progesterone Levels:

There were no treatment related changes observed in serum Estrogen and Progesterone Levels of dams sacrificed on lactation day 14 at any of the tested dose groups when compared with vehicle control group.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no changes observed in the estrous 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 effect of spermatogenesis during histopathology

Reproductive performance:

no effects observed

Description (incidence and severity):

No effect of Mating and Fertiltiy of the animals at all the tested dose groups

The test item Hostacor ITD did not reveal any effects on reproductive performance of male and female rats.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

other: Hormone lelvels [T4, Serum Estrogen and Progesterone Levels]

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs and external anomalies observed in any of the pups of tested dose group animals during lactation period

Description (incidence and severity):

Not applicable

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There were mortalities were noticed in the newborn pups from G2 and G3 groups. This mortalities not considered to the treatment related because gross pathology examination did not revealed any pathological changes

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no changes observed in mean pup (male and female) weight on lactation day 1, 4, 7 and 13 at any of the tested dose groups when compared with vehicle control group dams. However, statistical significant reduction in mean female pup weight on lactation day 13 in G3 group was noted when compared with vehicle control group. This change is considered as incidental but not treatment related as there were no abnormalities were noted in this group pups during lactation period and no gross pathological findings noted during necropsy

Food consumption and compound intake (if feeding 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:

not examined

Description (incidence and severity):

Not applicable

Urinalysis findings:

not examined

Description (incidence and severity):

Not applicable

Sexual maturation:

not examined

Description (incidence and severity):

Not applicable

Organ weight findings including organ / body weight ratios:

not examined

Description (incidence and severity):

Not applicable

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological lesions noticed at necropsy of males and females as well as pups (both live and dead)

Histopathological findings:

not examined

Description (incidence and severity):

Not applicable

No treatment related effects noted at F1 generation pups at all the tested dose groups

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

gross pathology

other: Srum T4 Levels of PND13 pups

Key result

Reproductive effects observed:

no

SUMMARY OF THE STUDY

Parameters ↓		Group & Dose (mg/kg body weight/day)
		G1 & 0	G2 & 100	G3 & 300	G4 & 1000
Reproductive Indices
Mating Indices
Pairs started (No.)	12		12	12	12
Males showing evidence of mating (No.) within 14 days	9		10	10	9
Male Mating Index (%) within 14 days	75.00		83.33	83.33	75.00
Females showing evidence of copulation (No.)	12		12	12	12
Female Mating Index (%)	100.00		100.00	100.00	100.00
Fertility Indices
Females achieving pregnancy (No.)	11		12	11	12
Female Fertility Index (%)	91.67		100.00	91.67	100.00
Copulatory Indices
Conceiving days 1 to 5 (No.)	4		5	7	4
Conceiving days >6 (No.)	8		7	5	8
Mean Precoital Interval (Days)	10.33		7.67	6.50	9.92
Gestation Indices
Pregnancy≤21 days (No.)	0		1	0	0
Pregnancy = 22 days (No.)	4		5	4	4
Pregnancy≥23 days (No.)	7		6	7	8
Mean Gestation length (Days)	22.82		22.50	22.64	22.83
Gestation Index (%)	100.00		100.00	100.00	100.00
Dams with live young born (No.)	11		12	11	12
Dams with live young at day 4 post-partum (No.)	11		12	11	12
Dams with live young at day 13 post-partum (No.)	11		12	11	12
Implantation Index and Pre and Post implantation Losses
Implants/dam (Mean)	11.00		11.83	11.82	10.17
Corpora luetea/dam (Mean)	11.09		11.83	11.82	10.17
Implantation Index (%)	98.99		100.00	100.00	100.00
Pre-Implantation Loss (%)	1.01		0.00	0.00	0.00
Post-Implantation Loss (%)	0.00		0.64	0.76	0.00
Offspring Viability Indices
Live Birth Indices and Sex Ratio at Birth
Live pups/dam at birth (mean)	11.00		11.75	11.73	10.08
Litter Size (Total No. of pups born/dam) at birth (mean)	11.00		11.75	11.73	10.08
Mean Live Birth Index/dam (%)	100.00		99.36	99.24	100.00
Male Live pups/dam at birth (mean)	5.36		6.00	6.36	5.75
Female Live pups/dam at birth (mean)	5.64		5.75	5.36	4.33
Sex Ratio (male/female)	1.00		1.17	1.27	1.54

SUMMARY OF THE STUDY (Contd..,).

Parameters ↓	Group & Dose (mg/kg body weight/day)
	G1 & 0	G2 & 100	G3 & 300	G4 & 1000
Pup Survival Indices and Sex Ratio during lactation
Mean No. of Pups survived per dam ( LD1 to 4)	11.00	11.75	11.73	10.08
Mean No. of Pups dead per dam ( LD1 to 4)	0.00	0.00	0.00	0.00
Mean Pup Survival Index (%) per dam (LD1 to 4)	100.00	100.00	100.00	100.00
Sex Ratio (male/female) per dam at LD 4	1.00	1.17	1.27	1.54
Mean No. of Pups Sacrificed for Blood Collection on LD4	1.00	1.25	1.27	0.33
Mean No. of Pups survived per dam (LD4 to 7)	10.00	10.50	10.45	9.75
Mean No. of Pups dead per dam (LD4 to 7)	0.00	0.00	0.00	0.00
Mean Pup Survival Index (%) per dam (LD4 to 7)	100.00	100.00	100.00	100.00
Sex Ratio (male/female) per dam at LD 7	1.25	1.51	1.83	1.80
Mean No. of Pups survived per dam (LD7 to 13)	10.00	10.50	10.45	9.75
Mean No. of Pups dead per dam (LD7 to 13)	0.00	0.00	0.00	0.00
Mean Pup Survival Index (%) per dam ( LD7 to 13)	100.00	100.00	100.00	100.00
Sex Ratio (male/female) per dam at LD 13	1.25	1.51	1.83	1.80
Pre and Postnatal loss
Mean Pre-natal loss (implantations minus live births) (No.)	0.00	0.08	0.09	0.00
Females with 0 (No.)	11	11	10	12
Females with ≥ 1 (No.)	0	1	1	0
Post-natal loss (live births minus alive at post natal day 13)
Females with 0 (No.)	11	12	11	12
Females with ≥ 1 (No.)	0	0	0	0
Litter Observations
Male Pup weight at birth (mean) in gram	7.18	7.21	7.22	7.19
Female Pup weight at birth (mean) in gram	6.86	6.70	6.84	6.61
Male Pup weight on LD4 (mean) in gram	12.02	11.95	12.05	12.27
Female Pup weight on LD4 (mean) in gram	11.27	11.26	11.36	11.49
Male Pup weight on LD7 (mean) in gram	18.00	17.62	17.56	18.29
Female Pup weight on LD7 (mean) in gram	17.35	16.93	16.44	16.94
Male Pup weight on LD13 (mean) in gram	27.83	27.89	27.68	28.16
Female Pup weight on LD13 (mean) in gram	27.03	26.32	25.93	26.46

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	8	14	21	28	35
G1, M & 0	Mean	361.58	377.04	392.59	396.99	404.48	411.57
	±SD	18.23	16.73	19.62	19.39	23.07	24.96
	n	12	12	12	12	12	12
G2, M & 100	Mean	357.09	372.40	384.39	387.97	398.77	407.93
	±SD	24.60	26.64	27.79	28.95	32.21	31.15
	n	12	12	12	12	12	12
G3, M & 300	Mean	360.53	369.07	383.84	390.53	400.83	408.85
	±SD	24.29	28.11	30.11	30.99	29.43	30.63
	n	12	12	12	12	12	12
G4, M & 1000	Mean	360.82	364.19	377.79	384.19	394.37	403.99
	±SD	19.98	22.62	28.98	31.06	32.03	32.38
	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	8	14	21#	28#
G1, F & 0	Mean	267.24	272.29	283.41	286.21	314.07
	±SD	15.90	15.44	16.07	20.13	12.43
	n	12	12	12	8	3
G2, F & 100	Mean	266.21	272.72	285.28	299.95	326.83
	±SD	15.12	15.70	16.86	13.74	17.88
	n	12	12	12	6	2
G3, F & 300	Mean	265.22	270.61	277.81	270.58	280.42
	±SD	15.91	16.93	20.19	15.94	1.50
	n	12	12	12	4	2
G4, F & 1000	Mean	264.11	267.45	279.09	276.09	279.41
	±SD	15.27	16.69	20.22	19.01	2.13
	n	12	12	12	8	3

F: Female; SD: Standard Deviation; n: Number of Animals; -: Not applicable

#: The statistical analysis is not performed for day 22 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 (%) during Days
		1 to 8	1 to 14	1 to 21	1 to 28	1 to 35
G1, M & 0	Mean	4.32	8.61	9.84	11.88	13.84
	±SD	1.71	2.68	3.15	3.81	4.24
	n	12	12	12	12	12
G2, M & 100	Mean	4.30	7.68	8.67	11.67	14.29
	±SD	2.63	3.73	3.63	4.45	4.97
	n	12	12	12	12	12
G3, M & 300	Mean	2.33	6.44	8.30	11.20	13.43
	±SD	2.03	3.53	3.91	4.13	4.47
	n	12	12	12	12	12
G4, M & 1000	Mean	0.91*	4.64*	6.38	9.21	11.88
	±SD	1.50	4.18	4.40	4.81	4.99
	n	12	12	12	12	12

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

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

 

TABLE 3 (Contd..,). 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 (%) during Days
		1 to 8	1 to 14	1 to 21#	1 to 28#
G1, F & 0	Mean	1.91	6.10	7.51	16.00
	±SD	1.01	2.88	3.84	4.32
	n	12	12	8	3
G2, F & 100	Mean	2.45	7.20	10.21	17.39
	±SD	1.45	3.47	5.02	3.26
	n	12	12	6	2
G3, F & 300	Mean	2.03	4.69	3.95	7.75
	±SD	2.00	2.21	1.76	1.86
	n	12	12	4	2
G4, F & 1000	Mean	1.24	5.67	6.72	11.85
	±SD	0.98	4.29	3.70	3.60
	n	12	12	8	3

   F: Female; SD: Standard Deviation; n: Number of Animals; -: Not applicable

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

 

Note: The data obtained during pre-mating period for all the females was considered for mean calculations.

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

Group, Sex & Dose (mg/kg body weight/day)		Feed Consumption (g)
		Week 1 (Day 1 to 8)	Week 2 (Day 8 to 14)	Post Mating (Day 31 to 35)	Post Mating (Day 35 to 37)
G1, M & 0	Mean	23.74	26.23	30.43	32.65
	±SD	0.47	0.50	1.21	1.68
	n	12	12	12	12
G2, M & 100	Mean	23.68	26.18	30.15	32.01
	±SD	0.52	0.54	1.32	0.90
	n	12	12	12	12
G3, M & 300	Mean	23.97	26.60	29.97	32.74
	±SD	0.60	0.46	1.60	2.08
	n	12	12	12	12
G4, M & 1000	Mean	23.91	26.21	30.26	32.89
	±SD	0.51	0.61	3.59	3.13
	n	12	12	12	12

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

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

Group, Sex & Dose (mg/kg body weight/day)		Feed Consumption (g)
		Week 1 (Day 1 to 8)	Week 2 (Day 8 to 14)
G1, F & 0	Mean	20.31	21.98
	±SD	0.45	0.48
	n	12	12
G2, F & 100	Mean	20.36	21.93
	±SD	0.65	0.48
	n	12	12
G3, F & 300	Mean	20.66	22.27
	±SD	0.54	0.41
	n	12	12
G4, F & 1000	Mean	20.82	22.29
	±SD	0.48	0.33
	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	1.5812	3.5987	3.6165	0.0300
	±SD	0.0816	0.3419	0.1841	0.0022
	n	12	12	12	12
G2, M & 100	Mean	1.6136	3.4643	3.4524	0.0292
	±SD	0.1238	0.2595	0.2526	0.0025
	n	12	12	12	12
G3, M & 300	Mean	1.5383	3.4801	3.4760	0.0295
	±SD	0.1027	0.2353	0.2963	0.0032
	n	12	12	12	12
G4, M & 1000	Mean	1.4543*	3.3486	3.4098	0.0294
	±SD	0.0746	0.1950	0.5073	0.0023
	n	12	12	12	12

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

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

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	0.4658	0.0251
	±SD	0.1017	0.0019
	n	11	11
G2, F & 100	Mean	0.4849	0.0258
	±SD	0.0867	0.0020
	n	12	12
G3, F & 300	Mean	0.4393	0.0259
	±SD	0.0933	0.0022
	n	11	11
G4, F & 1000	Mean	0.4400	0.0253
	±SD	0.0760	0.0018
	n	12	12

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

#: 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	403.76	0.3925	0.8911	0.8986	0.0074
	±SD	24.51	0.0256	0.0623	0.0676	0.0002
	n	12	12	12	12	12
G2, M & 100	Mean	395.35	0.4101	0.8808	0.8793	0.0074
	±SD	31.43	0.0418	0.0911	0.1034	0.0002
	n	12	12	12	12	12
G3, M & 300	Mean	399.40	0.3868	0.8751	0.8774	0.0074
	±SD	31.41	0.0329	0.0786	0.1166	0.0003
	n	12	12	12	12	12
G4, M & 1000	Mean	396.20	0.3699	0.8498	0.8644	0.0074
	±SD	34.11	0.0414	0.0743	0.1423	0.0003
	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	308.01	0.1505	0.0082
	±SD	17.08	0.0285	0.0008
	n	11	11	11
G2, F & 100	Mean	306.35	0.1587	0.0084
	±SD	26.63	0.0284	0.0004
	n	12	12	12
G3, F & 300	Mean	317.81	0.1387	0.0082
	±SD	23.75	0.0295	0.0006
	n	11	11	11
G4, F & 1000	Mean	309.35	0.1425	0.0082
	±SD	23.55	0.0235	0.0003
	n	12	12	12

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

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

Group & Dose (mg/kg body weight/day)	No. of Females	No. of Females with Regular Estrous Cyclicity during Pre-mating and Mating	No. of Females with Regular Estrous Cyclicity on Lactation day 14	No. of Females with Irregular Estrous Cyclicity during Pre-mating, Mating and on Lactation day 14
G1 & 0	12	12	11	0
G2 & 100	12	12	12	0
G3 & 300	12	12	11	0
G4 & 1000	12	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 14)
G1 & 0	Mean	10.33	N	4	8
	±SD	5.63
	n	12	%	33.3	66.7
G2 & 100	Mean	7.67	n	5	7
	±SD	5.58
	n	12	%	41.7	58.3
G3 & 300	Mean	6.50	n	7	5
	±SD	5.44
	n	12	%	58.3	41.7
G4 & 1000	Mean	9.92	n	4	8
	±SD	5.48
	n	12	%	33.3	66.7

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

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

Group & Dose (mg/kg body weight/day)		Body weight (g) on Gestation Days
		0	7	14	20
G1 & 0	Mean	290.15	304.33	338.92	394.70
	±SD	16.21	17.10	21.73	19.17
	n	11	11	11	11
G2 & 100	Mean	296.25	309.37	340.03	397.88
	±SD	23.35	21.84	23.69	30.89
	n	12	12	12	12
G3 & 300	Mean	283.53	300.41	337.60	408.43
	±SD	18.96	20.10	26.73	33.66
	n	11	11	11	11
G4 & 1000	Mean	288.32	305.31	339.42	402.08
	±SD	17.90	17.71	21.19	28.72
	n	12	12	12	12

SD: Standard Deviation; n: Number of dams

TABLE 10.           SUMMARY RECORD OF PERENT 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	4.89	11.35	16.74
	±SD	0.84	2.86	7.10
	n	11	11	11
G2 & 100	Mean	4.49	9.98	16.96
	±SD	1.26	3.62	2.23
	n	12	12	12
G3 & 300	Mean	5.98	12.32	21.03
	±SD	2.31	3.06	4.52
	n	11	11	11
G4 & 1000	Mean	5.93	11.19	18.48
	±SD	1.80	3.13	4.25
	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	22.16	22.91	26.69
	±SD	1.44	1.43	1.57
	n	11	11	11
G2 & 100	Mean	22.47	23.38	27.37
	±SD	1.17	1.30	1.81
	n	12	12	12
G3 & 300	Mean	21.32	22.47	28.14
	±SD	3.28	2.82	1.42
	n	11	11	11
G4 & 1000	Mean	22.13	23.07	26.28
	±SD	1.41	1.10	2.09
	n	12	12	12

SD: Standard Deviation; n: Number of animals

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, F & 0	Mean	296.88	303.85	310.79	322.15
	±SD	13.08	15.29	17.44	15.75
	n	11	11	11	11
G2, F & 100	Mean	294.96	302.70	308.61	319.75
	±SD	25.11	26.15	25.62	24.74
	n	12	12	12	12
G3, F & 300	Mean	307.76	316.52	321.99	333.35
	±SD	26.53	26.94	26.32	24.61
	n	11	11	11	11
G4, F & 1000	Mean	299.22	304.10	311.84	323.96
	±SD	22.07	22.71	22.07	22.66
	n	12	12	12	12

SD: Standard Deviation; n: Number of dams

TABLE 13.           SUMMARY RECORD OF 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, F & 0	Mean	2.33	2.26	3.70
	±SD	1.16	1.32	1.62
	n	11	11	11
G2, F & 100	Mean	2.62	1.99	3.66
	±SD	1.44	1.18	1.56
	n	12	12	12
G3, F & 300	Mean	2.87	1.76	3.59
	±SD	2.39	1.05	1.50
	n	11	11	11
G4, F & 1000	Mean	1.64	2.58	3.93
	±SD	1.52	1.30	3.26
	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	22.60	26.27	26.16
	±SD	3.05	2.28	4.39
	n	11	11	11
G2 & 100	Mean	24.93	27.72	27.06
	±SD	4.13	4.50	3.69
	n	12	12	12
G3 & 300	Mean	25.80	28.46	28.03
	±SD	2.21	5.32	1.40
	n	11	11	11
G4 & 1000	Mean	24.41	25.53	26.31
	±SD	3.81	2.45	1.24
	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	22.82		11.00	11.00	5.36	5.64		0.00	0.00	0.00		1.00	100.00
	±SD	0.75		1.79	1.79	1.29	1.43		0.00	0.00	0.00		0.31	0.00
	n	11		11	11	11	11		11	11	11		11	11
G2 & 100	Mean	22.50		11.83	11.75	6.00	5.75		0.08	0.00	0.08		1.17	99.36
	±SD	0.80		2.69	2.67	2.00	2.26		0.29	0.00	0.29		0.48	2.22
	n	12		12	12	12	12		12	12	12		12	12
G3 & 300	Mean	22.64		11.82	11.73	6.36	5.36		0.09	0.09	0.00		1.27	99.24
	±SD	0.50		2.14	2.15	1.69	1.43		0.30	0.30	0.00		0.48	2.51
	n	11		11	11	11	11		11	11	11		11	11
G4 & 1000	Mean	22.83		10.08	10.08	5.75	4.33		0.00	0.00	0.00		1.54	100.00
	±SD	0.72		1.78	1.78	1.91	1.44		0.00	0.00	0.00		0.82	0.00
	n	12		12	12	12	12		12	12	12		12	12

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

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	11.00	11.00	5.36	5.64		0.00	0.00	0.00	1.00	11.00	100.00
	±SD	1.79	1.79	1.29	1.43		0.00	0.00	0.00	0.31	1.79	0.00
	n	11	11	11	11		11	11	11	11	11	11
G2 & 100	Mean	11.75	11.75	6.00	5.75		0.00	0.00	0.00	1.17	11.75	100.00
	±SD	2.67	2.67	2.00	2.26		0.00	0.00	0.00	0.48	2.67	0.00
	n	12	12	12	12		12	12	12	12	12	12
G3 & 300	Mean	11.73	11.73	6.36	5.36		0.00	0.00	0.00	1.27	11.73	100.00
	±SD	2.15	2.15	1.69	1.43		0.00	0.00	0.00	0.48	2.15	0.00
	n	11	11	11	11		11	11	11	11	11	11
G4 & 1000	Mean	10.08	10.08	5.75	4.42		0.00	0.00	0.00	1.54	10.08	100.00
	±SD	1.78	1.78	1.91	1.44		0.00	0.00	0.00	0.82	1.78	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)	Animal No.	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 to 7	Pup Survival Index (%) during          LD 4 to 7
														Live Pups (No.)				Dead 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	5.36	5.64	11.00		0.00	1.00	1.00		5.36	4.64	10.00		5.36	4.64	10.00		0.00	0.00	0.00		1.25	10.00	100.00
	±SD	1.29	1.43	1.79		0.00	1.00	1.00		1.29	1.03	1.00		1.29	1.03	1.00		0.00	0.00	0.00		0.53	1.00	0.00
	n	11	11	11		11	11	11		11	11	11		11	11	11		11	11	11		11	11	11
G2 & 100	Mean	6.00	5.75	11.75		0.00	1.25	1.25		6.00	4.50	10.50		6.00	4.50	10.50		0.00	0.00	0.00		1.51	10.50	100.00
	±SD	2.00	2.26	2.67		0.00	0.97	0.97		2.00	1.73	1.88		2.00	1.73	1.88		0.00	0.00	0.00		0.67	1.88	0.00
	n	12	12	12		12	12	12		12	12	12		12	12	12		12	12	12		12	12	12
G3 & 300	Mean	6.36	5.36	11.73		0.00	1.27	1.27		6.36	4.09	10.45		6.36	4.09	10.45		0.00	0.00	0.00		1.83	10.45	100.00
	±SD	1.69	1.43	2.15		0.00	0.90	0.90		1.69	1.45	1.51		1.69	1.45	1.51		0.00	0.00	0.00		1.06	1.51	0.00
	n	11	11	11		11	11	11		11	11	11		11	11	11		10	10	11		11	11	11
G4 & 1000	Mean	5.75	4.33	10.08		0.00	0.33	0.33		5.75	4.00	9.75		5.75	4.00	9.75		0.00	0.00	0.00		1.80	9.75	100.00
	±SD	1.91	1.44	1.78		0.00	0.78	0.78		1.91	1.48	1.14		1.91	1.48	1.14		0.00	0.00	0.00		1.26	1.14	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)	Animal No.	Live Pups (No.) on LD 7				During LD 7 to 13									Sex Ratio (M/F) at LD 13	No. of Survived Pups during LD 7 to 13	Pup Survival Index (%) during LD 7 to 13
						Live Pups (No.)					Dead Pups (No.)
		Male (No.)	Female (No.)	Total (No.)		Male (No.)	Female (No.)	Total (No.)			Male (No.)	Female (No.)	Total (No.)
G1 & 0	Mean	5.36	4.64	10.00		5.36	4.64	10.00			0.00	0.00	0.00		1.25	10.00	100.00
	±SD	1.29	1.03	1.00		1.29	1.03	1.00			0.00	0.00	0.00		0.53	1.00	0.00
	n	11	11	11		11	11	11			11	11	11		11	11	11
G2 & 100	Mean	6.00	4.50	10.50		6.00	4.50	10.50			0.00	0.00	0.00		1.51	10.50	100.00
	±SD	2.00	1.73	1.88		2.00	1.73	1.88			0.00	0.00	0.00		0.67	1.88	0.00
	n	12	12	12		12	12	12			12	12	12		12	12	12
G3 & 300	Mean	6.36	4.09	10.45		6.36	4.09	10.45			0.00	0.00	0.00		1.83	10.45	100.00
	±SD	1.69	1.45	1.51		1.69	1.45	1.51			0.00	0.00	0.00		1.06	1.51	0.00
	n	11	11	11		11	11	11			11	11	11		11	11	11
G4 & 1000	Mean	5.75	4.00	9.75		5.75	4.00	9.75			0.00	0.00	0.00		1.80	9.75	100.00
	±SD	1.91	1.48	1.14		1.91	1.48	1.14			0.00	0.00	0.00		1.26	1.14	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

 

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	7.18	6.86		12.02	11.27		18.00	17.35		27.83	27.03
	±SD	0.25	0.12		0.32	0.49		0.87	0.79		0.40	0.92
	n	11	11		11	11		11	11		11	11
G2 & 100	Mean	7.21	6.70		11.95	11.26		17.62	16.93		27.89	26.32
	±SD	0.51	0.56		0.75	0.86		1.16	1.22		0.99	0.98
	n	12	12		12	12		12	12		12	12
G3 & 300	Mean	7.22	6.84		12.05	11.36		17.56	16.44		27.68	25.93*
	±SD	0.60	0.60		0.34	0.51		1.33	1.25		0.96	0.96
	n	11	11		11	11		11	11		11	11
G4 & 1000	Mean	7.19	6.61		12.27	11.49		18.37	16.94		28.16	26.46
	±SD	0.28	0.37		0.28	0.54		0.46	0.68		0.43	0.81
	n	12	12		12	12		12	12		12	12

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

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

TABLE 19.              SUMMARY OF UTERI OBSERVATIONS RECORD

Group & Dose (mg/kg body weight/day)		No. of Corpora lutea	No. of Implantations	Implantation Index	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	11.09	11.00	98.99	1.01	0.00	0.00	0.00	0.00	0.00	0.00
	±SD	1.64	1.79	3.35	3.35	0.00	0.00	0.00	0.00	0.00	0.00
	n	11	11	11	11	11	11	11	11	11	11
G2 & 100	Mean	11.83	11.83	100.00	0.00	0.64	0.08	0.00	0.00	0.00	0.00
	±SD	2.69	2.69	0.00	0.00	2.22	0.29	0.00	0.00	0.00	0.00
	n	12	12	12	12	12	12	12	12	12	12
G3 & 300	Mean	11.82	11.82	100.00	0.00	0.76	0.09	0.00	0.00	0.00	0.00
	±SD	2.14	2.14	0.00	0.00	2.51	0.30	0.00	0.00	0.00	0.00
	n	11	11	11	11	11	11	11	11	11	11
G4 & 1000	Mean	10.17	10.17	100.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
	±SD	1.80	1.80	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
	n	12	12	12	12	12	12	12	12	12	12

SD: Standard Deviation; n: Number of animals

 

TABLE 20.              SUMMARY RECORD OF ANO-GENITAL DISTANC RATIO ON LACTATION DAY 4

Group & Dose (mg/kg body weight/day)		Mean Male AGD Ratio	Mean Female AGD Ratio
G1 & 0	Mean	2.36	1.00
	±SD	0.10	0.05
	n	11	11
G2 & 100	Mean	2.32	1.02
	±SD	0.11	0.07
	n	12	12
G3 & 300	Mean	2.37	1.02
	±SD	0.13	0.04
	n	11	11
G4 & 1000	Mean	2.31	1.03
	±SD	0.09	0.10
	n	12	12

 SD: Standard Deviation; n: Number of animals

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	11
G2 & 100	Mean	0.00
	±SD	0.00
	n	12
G3 & 300	Mean	0.00
	±SD	0.00
	n	11
G4 & 1000	Mean	0.00
	±SD	0.00
	n	12

SD: Standard Deviation; n: Number of animals

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	87.144
	±SD	9.811
	n	12
G2, M & 100	Mean	94.818
	±SD	7.447
	n	12
G3, M & 300	Mean	91.817
	±SD	8.802
	n	12
G4, M & 1000	Mean	84.652
	±SD	5.674
	n	12

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

TABLE 23.              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	59.709
	±SD	13.496
	n	11
G2 & 100	Mean	62.633
	±SD	7.849
	n	12
G3 & 300	Mean	56.212
	±SD	5.039
	n	11
G4 & 1000	Mean	58.045
	±SD	9.753
	n	12

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

TABLE 24.              SUMMARY OF SERUM ESTROGEN (pg/mL) RECORD - DAMS

Group & Dose (mg/kg body weight/day)		Serum Estrogen Levels (pg/mL)
G1 & 0	Mean	237.529
	±SD	25.797
	n	11
G2 & 100	Mean	254.767
	±SD	20.425
	n	11^$
G3 & 300	Mean	251.143
	±SD	32.806
	n	11
G4 & 1000	Mean	209.784
	±SD	42.035
	n	12

SD: Standard Deviation; n: Number of Dams

 $: The data of animal No. Rc6339 is excluded due to its lower obtained value.

TABLE 25.              SUMMARY OF SERUM PROGESTERONE (ng/mL) RECORD - DAMS

Group & Dose (mg/kg body weight/day)		Serum Progesterone Levels (ng/mL)
G1 & 0	Mean	160.411
	±SD	109.496
	n	11
G2 & 100	Mean	143.256
	±SD	106.684
	n	12
G3 & 300	Mean	153.732
	±SD	113.631
	n	11
G4 & 1000	Mean	116.492
	±SD	119.809
	n	12

SD: Standard Deviation; n: Number of Dams

TABLE 26.     SUMMARY OF GROSS PATHOLOGY FINDINGS

Organs/ Lesions	Group	G1		G2		G3		G4
	Dose (mg/kg body weight)	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 26 (Contd..,). SUMMARY OF GROSS PATHOLOGY FINDINGS OF PUPS

Organs/ Lesions	Group	G1		G2		G3		G4
	Dose (mg/kg body weight)	0		100		300		1000
	Sex	M	F	M	F	M	F	M	F
	No. of Dead pups (At birth)	0	0	0	1	1	0	0	0
No Abnormality Detected		0	0	0	1	1	0	0	0
	No. of pups sacrificed on Lactation Day 4	0	11	0	15	0	14	0	4
No Abnormality Detected		0	11	0	15	0	14	0	4
	No. of pups sacrificed on Lactation Day 13	59	51	72	54	70	45	69	48
No Abnormality Detected		59	51	72	54	70	45	69	48

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

TABLE 27.     SUMMARY OF HISTOPATHOLOGY FINDINGS

Group		G1	G4	G1	G4
Dose (mg/kg body weight)		0	1000	0	1000
No. of Animals		12	12	12	12
Sex		Male		Female
ORGANS AND OBSERVATIONS/SEVERITY
Epididymides
Interstitial MNC infiltration; unilateral	Minimal	1	1	X	X
Uterus
Yellow-brown pigmentation	Minimal	X	X	6	7
	Mild	X	X	1	2

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

 

Conclusions:

The test item Hostacor ITD was evaluated for possible adverse effects on reproductive performance following repeated oral administration to male and female rats according OECD Test Guideline 421.

Based under the experimental conditions employed, no effect on any parameter investigated was observed. Thus, the No Observed Adverse Effect Level (NOAEL) of Hostacor ITD was found to be 1000 mg/kg body weight per day for male and female rats.

Executive summary:

Hostacor ITDwas evaluated for possible adverse effects on reproductive performance such as gonadal function, mating behavior, conception, development of conceptus, parturition and early neonatal development following repeated oral administration to male and female rats. The study was conducted according to OECD Test Guideline 421 and followed the principles of GLP.

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[corn oil],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 vehicleand test item formulationswere administered orally by gavageat 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 37 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 weeks 1 and 5 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 lactation day 13 pups representing each per litter was screened for T4 levels and serum collected from dams on lactation day 14 was screened for Estrogen and Progesterone levels.

Females were observed for estrous cyclicityduring pre-mating treatment and mating treatment period and the dams on lactation day 14 prior to sacrifice.The females were observed forcopulatory 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 external examinations once daily from lactation day 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 male pups were observed for retention of nipples/areolae on lactation day 13.

Gross pathology and organ weighing were performed on day 37 for males and on lactation day 14 for dams. Gross pathology was performed on lactation day 4/13 for pups. The number of corpora lutea and implantation sites for dams were recorded during necropsy.

All the tested dose group animals of either sex did not reveal any clinical signs of toxicity and no mortality/morbidity observed. There were no changes observed in mean body weight, percent change in body weight with respect to day 1 to day 35 and feed consumption at all the tested dose group animals of either sex during the experimental period. 

No treatment related changes were observed in organ weights (both absolute and relative) at all the tested dose group animals. There were no treatment related changes observed in serum T4 levels of adult males and lactation day 13 pups at any of the tested dose groups.

Dams did not reveal any treatment related changes in estrous cyclicity, copulatory interval, body weights and feed consumption during gestation and lactation periods, gestation length, live birth index, number of pups, sex ratio and pup survival index at all the tested dose groups throughout the lactation period. There were no treatment related changes observed in serum Estrogen and Progesterone levels of dams at any of the tested dose groups.

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

There were no gross pathological changes (both external and internal) observed at all the tested dose group adult animals and pups.

There were no treatment related histopathological findings noticed during the microscopic examination.Testes were screened with special emphasis on stages of spermatogenesis and interstitial testicular cell structure, which revealed normal progression of the spermatogenic cycle and presence of all germ layers (cells). In addition to this, ovaries did not show any pathological findings/lesions.                   

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

high qualidy, GLP study according to OECD TG Guideline

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The subsmission substance was evaluated for possible adverse effects on reproductive performance such as gonadal function, mating behavior, conception, development of conceptus, parturition and early neonatal development following repeated oral administration to male and female rats. The study was conducted according to OECD Test Guideline 421 and followed the principles of GLP. 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[corn oil],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 vehicleand test item formulationswere administered orally by gavageat 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 37 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).  

All the tested dose group animals of either sex did not reveal any clinical signs of toxicity and no mortality/morbidity observed. There were no changes observed in mean body weight, percent change in body weight with respect to day 1 to day 35 and feed consumption at all the tested dose group animals of either sex during the experimental period. 

No treatment related changes were observed in organ weights (both absolute and relative) at all the tested dose group animals. There were no treatment related changes observed in serum T4 levels of adult males and lactation day 13 pups at any of the tested dose groups.

Dams did not reveal any treatment related changes in estrous cyclicity, copulatory interval, body weights and feed consumption during gestation and lactation periods, gestation length, live birth index, number of pups, sex ratio and pup survival index at all the tested dose groups throughout the lactation period. There were no treatment related changes observed in serum Estrogen and Progesterone levels of dams at any of the tested dose groups.

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

There were no gross pathological changes (both external and internal) observed at all the tested dose group adult animals and pups.

There were no treatment related histopathological findings noticed during the microscopic examination.Testes were screened with special emphasis on stages of spermatogenesis and interstitial testicular cell structure, which revealed normal progression of the spermatogenic cycle and presence of all germ layers (cells). In addition to this, ovaries did not show any pathological findings/lesions.                   

Based under the experimental conditions employed, no effect on any parameter investigated was observed. Thus, the No Observed Adverse Effect Level (NOAEL) of Hostacor ITD was found to be 1000 mg/kg body weight per day for male and female rats.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Species:

rat

Justification for classification or non-classification

Based on the available data no classification for reproductive and developmental effects is warranted according to Regulation (EC) No 1272/2008 and Council Directive 67/548/EEC.