MoVNbTe mixed oxide

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 March 2021 to 01 October 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD Guideline for Testing of Chemicals, Number 421, "Reproduction/Developmental Toxicity Screening Test", adopted on 29 July 2016.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

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

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Justification for study design:

SPECIFICATION OF STUDY DESIGN:

- Premating exposure duration for parental (P0) animals: 2 weeks
- Basis for dose level selection: dose range finding study [BIO-DTX 046]
- Route of administration: Oral gavage
- Other considerations, e.g. on choice of species, strain, vehicle and number of animals [if applicable]:

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

The test item was insoluble in water at the concentrations of 80 and 100 mg/mL. The test item formed uniform and homogeneous suspension in 0.5% w/v Carboxymethyl cellulose at the concentration of 100 mg/mL as per in-house solubility/suspendibility test results.
Hence, 0.5% w/v Carboxymethyl cellulose was used as vehicle for test item formulations.

Total = 96 (48 Males + 48 Females); 12 males + 12 females per group

Test material

Test material information

Constituent 1

Reference substance name:

niobium trivanadium decamolybdenum tellurium dotetracontaoxide

EC Number:

888-364-4

Cas Number:

146569-48-4

Molecular formula:

Mo10V3Nb1Te1O42

IUPAC Name:

niobium trivanadium decamolybdenum tellurium dotetracontaoxide

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Clariant Produkte (Deutschland) GmbH
83052 Bruckmühl/Heufeld, Germany; Batch no.: EX.14402.600

- Expiration date of the lot/batch: No change of properties known over time as specified by sponsor

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Stability under test conditions: The test item was stable up to 6 hours at room temperature and up to 48 hours at 2 to 8°C in 0.5% w/v Carboxymethyl cellulose with the concentrations of 10 mg/mL and 100 mg/mL.

- Solubility and stability of the test substance in the solvent/vehicle: The test item was insoluble in water at the concentrations of 80 and 100 mg/mL. The test item formed uniform and homogeneous suspension in 0.5% w/v Carboxymethyl cellulose at the concentration of 100 mg/mL as per in-house solubility/suspendibility test results.

The test item was stable up to 6 hours at room temperature and up to 48 hours at 2 to 8°C in 0.5% w/v Carboxymethyl cellulose with the concentrations of 10 mg/mL and 100 mg/mL.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Formulations in 0.5% w/v Carboxymethyl cellulose.
- Final preparation of a solid: As a liquid formulation (suspension)

FORM AS APPLIED IN THE TEST: Formulations

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 is 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 (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 to 9 weeks
- Weight at study initiation: (P) Males: 242.20 to 261.71 g, Females: 204.38 to 232.61 g
- Housing: During acclimatization, two animals of same sex were housed, Pre-mating - Per cage, two animals of same sex and group, were housed, Cohabitation Period (mating) - Per cage, two animals (one male and one female) of same group were housed, Post-mating - After confirming the 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.
- Diet : Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was made available ad libitum to the animals throughout the acclimatization and experimental periods.
- Water : Water was made available ad libitum throughout the acclimatization and experimental periods. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: Healthy and young adult animals were acclimatized to experimental room conditions for five days. Females were screened for regular oestrus cycles (4 to 5 days) in a two- weeks pre-treatment period before initiation of treatment and were observed for clinical signs once daily.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.4 to 23.0oC
- Humidity (%): 43 to 66%
- Air changes (per hr): 12 to 15 air changes per hour
IN-LIFE DATES: From: To: 26 March 2021 to 02 August 2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% w/v

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The required quantity of test item was weighed in aluminium foil, transferred to mortar and triturated well using pestle. A small quantity of vehicle was added and ground well until a homogenous suspension was formed and thereafter the entire quantity of the formulation was transferred into measuring cylinder. A small quantity of vehicle was added to rinse the mortar and transferred into the measuring cylinder. The rinsing procedure of mortar and pestle was repeated to ensure the transfer of the contents to the measuring cylinder. Finally, the volume was made up to required quantity with vehicle to get desired concentration of 20, 40 and 80/50 mg/mL of test item for low, mid and high dose groups respectively.


VEHICLE
- Justification for use and choice of vehicle : The test item was insoluble in water at the concentrations of 80 and 100 mg/mL. The test item formed uniform and homogeneous suspension in 0.5% w/v Carboxymethyl cellulose at the concentration of 100 mg/mL as per in-house solubility/suspendibility test results.
Hence, 0.5% w/v Carboxymethyl cellulose was used as vehicle for test item formulations and the details were recorded in the raw data and presented in the study report.
Carboxymethyl cellulose is a routinely used vehicle of choice for the oral toxicity studies.

- Concentration in vehicle: G1- Vehicle control 0 mg/mL, G2- Low Dose 20 mg/mL, G3- Mid Dose 40 mg/mL, G4- High Dose 80/50 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg body weight
- Lot/batch no. (if required): BCBN1690V and SLCD3996

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: Sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility: No
- After successful mating each pregnant female was caged : Housed 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 the Analytical Chemistry department of Bioneeds India Private Limited. The analysis was done as per methods detailed in the Study Plan No. BIO-ANM 1660. Sampling and analysis of formulations were performed during week 1 and 4 of the treatment. The samples were collected in duplicates (5 mL each) from the top, middle and bottom layers for low, mid and high dose concentrations and in duplicate from a single layer for vehicle control.

The prepared test item formulations were stirred using magnetic stirrer during sampling.

The collected samples were transferred to the Analytical Chemistry Department of Bioneeds India Private Limited for dose formulation analysis. One set of aliquots of each formulation was analyzed. The second aliquot was stored for backup purpose at established stability conditions. The second set of samples were discarded as the analysis results of first set of samples were within the limits. Formulations were considered acceptable, since the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) was <10%.

Duration of treatment / exposure:

The test item or vehicle were administered to animals through oral (gavage) route once daily.
Males: The males were treated for a period of two weeks (14-days) during pre-mating, during cohabitation period and up to the day before scheduled sacrifice (total of completion of 35 days of treatment).

Females:
-The females were treated for a two-week pre-mating period (14-days), during mating, pregnancy (gestation) and up to lactation day 13, with a total period of 49 (for the first littered female) to 63 (for the last littered female) days.

-The non-pregnant females were treated during two-week pre-mating period, during mating until confirmed as mated and further 25 days from the day of confirmation of mating (group G1 - one female until experimental day 44; group G2 - one female until experimental day 47, one female until experimental day 45; G4 - one female until experimental day 52).

-In tested dose group G4 two females found dead on experimental day11 were treated till experimental day 10 and one female found dead on experimental day 10 was treated till experimental day 9

Frequency of treatment:

Once daily.

Details on study schedule:

- Age at mating of the mated animals in the study: 13 to 14 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle control

Doses / concentrations 2

Dose / conc.:

200 mg/kg bw/day

Remarks:

Low dose

Doses / concentrations 3

Dose / conc.:

400 mg/kg bw/day

Remarks:

Mid dose

Doses / concentrations 4

Dose / conc.:

500 mg/kg bw/day

Remarks:

High dose.
From day 11 -end of study, changed from 800 mg/kg bw/d beforehand due to noted adverse treatment related clinical signs of toxicity and mortalities.

Doses / concentrations 5

Dose / conc.:

800 mg/kg bw/day

Remarks:

From day 1 -11, changed to 500 mg/kg bw/d afterwards due to noted adverse treatment related clinical signs of toxicity and mortalities.

No. of animals per sex per dose:

12 Males + 12 Females per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The toxicity information on the test item was not available. Hence, the doses of 0, 35, 110, 330 and 1000 mg/kg body weight/day were selected control, low dose, low-mid dose, high-mid dose and high dose groups respectively for adose range finding study (BIO-DTX 046) in consultation with the sponsor. The doses of 35, 110 and 330 did not produce any indication of systemic, reproduction and developmental toxicity in either sex in the dose range finding study.

The dose of 1000 mg/kg body weight/day revealed test item-related clinical signs of toxicity in both sexes, mortalities in females (33%), reduced body weight and feed consumption in both sexes and irregularities in oestrus cycles of females. The animals from this dose level were found recovered from test-item related effects once the dose was reduced from 1000 to 650 mg/kg body weight/day. All the males from this dose level were treated with test item for a total period of 29 days (with 1000 mg/kg body weight/day for the first 15 days and with 650 mg/kg body weight/day for the next 14 days) with an average dose of approximately 830 mg/kg body weight/day. All the females from this dose level were treated with test item for a maximum period of 63 days (with 1000 mg/kg body weight/day for the first 15 days and with 650 mg/kg body weight/day until termination) with an average dose of approximately 730 mg/kg body weight/day.
Based on the results obtained from the dose range finding study (BIO-DTX 046), the doses of 200, 400 and 800 mg/kg body weight/day were selected as low, mid and high dose levels respectively, for the present Reproduction/Developmental Toxicity Screening Test in consultation with the sponsor.
However, the high dose was reduced from 800 mg/kg body weight/day to 500 mg/kg body weight/day from treatment day 11 onwards, due to noted adverse treatment related clinical signs of toxicity and mortalities.

- Rationale for animal assignment (if not random): Females were screened for regular oestrus cycles (4 to 5 days) in a two- weeks pre-treatment period before initiation of treatment and were observed for clinical signs once daily. Veterinary examination of all animals was performed on the day of receipt and the day of randomization and grouping. The animals were weighed and arranged in ascending order of their bodyweights. These animals, stratified for bodyweight, were distributed to all the groups using Microsoft Excel Spreadsheet, such that bodyweight variation of animals selected for this study does not exceed ±20% (Males: -5.19 to 5.08%; Females: -6.95 to 7.18%) of the mean body weight of each sex. The grouping was done one day before the initiation of treatment. The bodyweight of animals was analyzed statistically for the mean body weight to rule out any statistically significant difference between groups within each sex and there were no such incidences noted in both sexes.

Examinations

Parental animals: Observations and examinations:

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

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

FOOD CONSUMPTION :
Feed consumption (cage wise) was measured during following occasions:
Males: Cage wise feed consumption was measured for all males once in a week during premating period coinciding with body weight recording. The feed consumption was measured for males once in a week during post-mating.

Females:
- Cage wise feed consumption was measured for all females once in a week during premating period coinciding with body weight recording.
- Cage wise feed consumption was measured for all pregnant animals during
GD 0 to 7, 7 to 14 and 14 to 20, during LD 1 to 4, 4 to 7 and 7 to 13.
- Cage wise feed consumption was measured for all non-pregnant animals once in a week coinciding with body weight recording.

Oestrous cyclicity (parental animals):

Oestrus cyclicity was monitored for two weeks after five days of acclimatization to evaluate the normal oestrus cycle (4 to 5 days). Only females with normal oestrus cyclicity were selected for treatment. Vaginal smears were monitored daily from the beginning of the treatment period until the evidence of mating. When obtaining vaginal/cervical cells, care was taken to avoid disturbance of mucosa.

The status of oestrus cyclicity of all females was determined on the day of scheduled termination.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no
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.
Blood collection for total T4 (Thyroxine) Level estimation: Blood samples were collected from all the animals for measurement of serum total T4 levels on the following schedule:
a.Two pups per litter on PND 4 based on the following conditions:
- two female pups in order to retain more male pups for assessment of nipple retention on PND 13.
- no pups were eliminated when litter size dropped below or equal to
10 pups/litter.
- only one pup was eliminated and used for blood collection for possible serum T4 assessments in case of availability of only one pup above the normal litter size.


GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities, determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: Males were sacrificed after completion of 35 days of treatment. During necropsy, the males were randomized to avoid sampling bias.
- Maternal animals: All the pregnant females were sacrificed on lactation day 14. The females confirmed with mating but not littered were sacrificed after 25 days from the day of confirmed as m ated.

GROSS NECROPSY
All the surviving animals were fasted overnight; water was made available ad libitum during fasting. The next day, the bodyweight of all fasted animals was recorded prior to necropsy. The animals were humanely sacrificed using deep CO2 asphyxiation and subjected to detailed gross necropsy, which included careful examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents as well as organs and tissues of each animal with special emphasis on reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
Epididymides, Testes, Ovaries and Vagina were subjected for microscopic examination.A detailed histopathological examination was performed on the ovaries, testes and epididymides. Part icular emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure of the animals was given. This detailed histopathological examination was performed in all animals fr om both control and treatment dose groups sacrificed at termination.
Epididymides, Testes, Prostate, Seminal vesicles with coagulating glands, Ovaries, Uterus with Cervix and Thyroid along with parathyroid were weighed.

Postmortem examinations (offspring):

SACRIFICE
- Dead pups and pups which were sacrificed on PND 4/13, examined for gross abnormalities with particular attention to the external reproductive genitals and the findings were recorded.

GROSS NECROPSY
Dead pups and pups which were sacrificed on PND 4/13, examined for gross abnormalities with particular attention to the external reproductive genitals and the findings were recorded.

Statistics:

The raw data was subjected to computer statistical processing. The computer printout of the data (in the form of the appendix) was verified with the raw data. After verification, the data was subjected to various statistical analyses using SPSS software version 22.

All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05), indicated by the aforementioned tests were designated by the superscripts throughout the report, as stated below:
*: Statistically significant (P<0.05) change than the vehicle control group.

Note: Data of non-pregnant females is presented in the individual animal data but excluded for mean calculations and statistical analysis of reproductive endpoints.

Parametric - One-way ANOVA with Dunnett's post-test: Bodyweight (weekly/gestation/lactation body weights), Percent change in body weight (weekly/gestation/lactation), Feed consumption (weekly/gestation/lactation), Copulatory interval, Gestation length, Absolute/relative organ weights, Mean pup weight per litter, Mean pup anogenital distance ratio per litter, Serum T4 values

Non-parametric - Kruskal-Wallis:Implantations/litter, No. of pups/litter, Sex ratio/litter at birth and during the lactation period, Litter size at birth and during the lactation period, Post-implantation loss/litter, Postnatal loss/litter

Cross Tabs – Frequency comparison Chi-square test: mated/non-mated; fertile/infertile; pregnant/non-pregnant; with/without live pups; with/without dead pups

Reproductive indices:

The male / female mating and fertility indices were calculated as mentioned below:

Male Mating Index (%)= No. of males with confirmed mating/Total no. of males cohabited X 100
Female Mating Index (%) = No. of sperm-positive females/Total no. of females cohabited X 100
Male Fertility Index (%) = No. of males impregnating a female/Total no. of males confirmed as mated X 100
Female Fertility Index (%) = No. of pregnant females/No. of sperm-positive females X 100

Female pre-coital interval was calculated as mentioned below:

Pre-coital Interval (Days) = Date of confirmation of mating - Date of initiation of cohabitation
gestation length per litter was calculated as mentioned below:
Gestation Length (days) = Date of parturition – Date of evidence of mating (GD 0)
The gestation index (%) per group was calculated as mentioned below:
Gestation Index (%) = No. of females with live born/No. of females with evidence of pregnancy X 100
The parturition index (%) per group was calculated as mentioned below:
Parturition Index (%) = No. of females littered/No. of females with evidence of pregnancy X 100
The pregnancy index (%) per group was calculated as mentioned below:
Pregnancy Index (%) = No. of pregnant females/No. of females with confirmed mating X 100

Offspring viability indices:

The day of littering was considered as lactation day (LD) 1 for dams and postnatal day (PND) 1 for pups. The number of male/female pups born (live/dead) per litter were recorded and sex ratio (m/f) and live birth index were calculated per litter as mentioned below:

Sex ratio (m/f) = No. of male offspring/Number of female offspring
Live Birth Index (%) per litter = No. of pups born alive/Total No. of pups born X 100
The number of pups survived/dead per litter were recorded during lactation period and pup survival index (%) per litter between lactation day 1 to 4, 4 to 7 and 7 to 13 were calculated per litter as mentioned below:
Pup Survival index (%) on LD 4/7/13 = Total No. of live pups on LD 4/7/13/No. of pups born/4/7 X 100
The sex ratio (m/f) on LD 4, 7, and 13 were calculated per litter, as mentioned below:
Sex ratio (m/f) on LD 4/7/13 = No. of male offspring on LD 4/7/13/No. of female offspring on LD 4/7/13

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinical signs of toxicity and no mortality/morbidity noted in any of the animals of both sexes from the tested dose groups G2 (200 mg/kg body weight/day), G3 (400 mg/kg body weight/day) and vehicle control group [0 mg/kg body weight/day] throughout the experimental period. In group G4, there were no clinical signs of toxicity noted for the first four days of the treatment in both sexes when treated with 800 mg/kg body weight/day. However, the females from this dose level started to reveal test item-related clinical signs from day 5 and males from day 6. The detailed observed clinical signs of toxicity were as mentioned below.
Day 5: Lethargy (2 females); Lethargy and chromodacryorrhea (2 females).
Day 6 and 7: Lethargy, soft stools and wet perineum (1 female); lethargy, soft stools, chromodacryorrhea and wet perineum (3 females); soft stools and wet perineum
(1 male).
Day 8: Lethargy, soft stools and wet perineum (1 female); lethargy, soft stools, chromodacryorrhea and wet perineum (2 females); lethargy, dehydration, soft stools, ataxia, chromodacryorrhea and wet perineum (1 female); soft stools and wet perineum (1 male); wet perineum (1 male).
Day 9: Lethargy, dehydration, soft stools, ataxia, chromodacryorrhea and wet perineum (2 females); lethargy, soft stools, ataxia, chromodacryorrhea and wet perineum (2 females); lethargy (2 females); lethargy, chromodacryorrhea and wet perineum (1 female); lethargy and soft stools (1 female); lethargy, soft stools, chromodacryorrhea and wet perineum (2 males).
Day 10: Death (1 female); lethargy (1 female); chromodacryorrhea (1 female); lethargy and chromodacryorrhea (1 female); lethargy, soft stools and wet perineum (1 female); lethargy, soft stools, chromodacryorrhea and wet perineum (1 female); lethargy, soft stools, ataxia, chromodacryorrhea and wet perineum (1 female); lethargy, dehydration, soft stools, ataxia, chromodacryorrhea and wet perineum (2 females); lethargy, soft stools, chromodacryorrhea and wet perineum (2 males); lethargy, soft stools and wet perineum (2 males).
Note: The dose level was reduced to 500 mg/kg body weight/day from 800 mg/kg body weight/day for group G4 from day 11onwards due to severe clinical signs of toxicity and mortalities.
Day 11: Death (2 females); lethargy (1 female); chromodacryorrhea (1 female); lethargy and chromodacryorrhea (1 female); lethargy, soft stools and wet perineum (1 female); lethargy, soft stools, chromodacryorrhea and wet perineum (1 female); lethargy, soft stools, ataxia, chromodacryorrhea and wet perineum (1 female); lethargy, soft stools, chromodacryorrhea and wet perineum (2 males); soft stools and wet perineum (2 males).
Day 12: Chromodacryorrhea (2 females); soft stools and wet perineum (1 female); lethargy, soft stools, chromodacryorrhea and wet perineum (1 female); lethargy, ataxia, chromodacryorrhea and wet perineum (1 female); chromodacryorrhea and wet perineum (2 males); wet perineum (2 males).
Day 13: Chromodacryorrhea (1 female); wet perineum (1 female); chromodacryorrhea and wet perineum (2 females); chromodacryorrhea and wet perineum (2 males); wet perineum (2 males).
Day 14: Wet perineum (1 female); chromodacryorrhea and wet perineum (2 females); chromodacryorrhea (1 male); wet perineum (1 male); chromodacryorrhea and wet perineum (1 male).
Day 15: Wet perineum (2 females); chromodacryorrhea (1 female); chromodacryorrhea (2 males).
Day 16: Wet perineum (2 females); chromodacryorrhea (1 female); chromodacryorrhea (1 male).
Day 17: Wet perineum (1 female).
All the males from day 17 and all the survived females from day 18 were recovered completely from clinical signs and found normal until termination.

Mortality:

mortality observed, treatment-related

Description (incidence):

A total of 3 out of 12 females from high dose which were found dead while treated with 800 mg/kg body weight/day noted with wet perineum as an external gross finding and internally autolyzed during conduct the necropsy. These mortalities are due to noted adverse treatment related clinical signs of toxicity, reduced body weight and reduced feed consumption at this dose level.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no changes noted in mean body weight and percent change in mean body weight gain with respect to day 1 in the tested dose groups G2 and G3 of both sexes throughout the experimental period when compared with vehicle control group.

in group G3 males statistically significant reduction in percent change in mean body weight gain was noted on day 35 with respect to day 1.

In group G4 males, statistically significant reduction in mean body weight and percent change in mean body weight with respect to day 1 on all the days until termination was noted when compared with vehicle control group.

Gestation body weight: There were no changes noted in mean body weight and percent change in mean body weight gain during gestation period in the tested dose groups G2 and G3 when compared with vehicle control group. However, the noted statistically significant decrease in mean body weight on gestation day 14 in group G3 when compared with the vehicle control group is considered as incidental and also the noted mean value is within in-house historical control range of same species and strain.

In group G4, a statistically significant decrease in mean gestation body weight on all the days (gestation day 0, 7, 14 and 20) was noted when compared with the vehicle control group.

Lactation Body weight: There were no changes noted in mean body weight and percent change in mean body weight gain during lactation period in the tested dose groups G2 and G3 when compared with vehicle control group.

In group G4, a statistically significant decrease in mean lactation body weight on all the days (lactation day 1, 4, 7 and 13) was noted when compared with the vehicle control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There were no changes noted in mean feed consumption (grams/day/animal) in the tested dose groups G2 and G3 of both sexes when compared with the vehicle control group during premating period.

In group G4, statistically significant reduction in mean feed consumption was noted during pre-mating (week 1 and 2) in both sexes. This reduction is considered as test item-related due to noted treatment related clinical signs of toxicity, reduction in mean body weights and percent change in body weight gain and also due to noted mortalities in females at this dose level. Also, statistically significant reduction in mean feed consumption was noted in G4 males during post-mating (week 5). However, an increase in mean feed consumption was noted when compared with pre-mating period consumption of same group as the dose was reduced to 500 mg/kg body weight.

Gestation Feed consumption: There were no changes noted in mean feed consumption during gestation period in any of the tested dose groups when compared with the vehicle control group.

Lactation Feed consumption:There were no changes noted in mean feed consumption during lactation period in any of the tested dose groups when compared with vehicle control group.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no microscopic changes noted in the organs (epididymides and testes in males; ovaries and vagina in females) subjected to histopathological examinations from high dose group animals of both sexes

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

There were no changes noted in mean serum T4 levels in any of the tested dose group males when compared with the vehicle control group. The examination was not extended to adult females as there were no changes noted in serum Thyroxine (T4) hormone levels of adult males.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

There were no test item-related irregularities observed in oestrus cyclicity of females and also the mean length of oestrus cycle per female during pre-mating and mating treatment period was unaffected by the test item administration and the mean length was comparable with the vehicle control group in the tested dose groups G2 and G3. In group G2, an incidental occurring i.e. 1 out of 12 females noted with one irregular cycle (6-days cycle), but noted as mated and confirmed as pregnant.

In group G4, 3 out of 12 females which were found dead during pre-mating noted with prolonged diestrus when treated with 800 mg/kg body weight/day. 1 out of 12 females which was recovered and survived later during the treatment noted with prolonged diestrus. 1 out of 12 females which was recovered and survived later during the treatment noted with one irregular cycle (6-days cycle), but noted with regular cycles later in the treatment period.

These noted irregularities in group G4 are considered as test item-related due to noted treatment related clinical signs of toxicity, mortalities, reduced mean body weight, reduced percent change in mean body weight gain, reduced mean feed consumption when treated with 800 mg/kg body weight/day.

However, all the survived females (9 out of 12) were recovered and confirmed as mated later in the study when the dose level reduced to 500 mg/kg body weight/day. The noted increase in mean length of oestrus cycle (days) per dam i.e. 5.02 days at this dose level was due to initial toxicity effects of test item when treated with 800 mg/kg body weight/day.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Male Mating Index: All the males (12 out of 12) were confirmed with mating during cohabitation period with a mating index of 100.0% from all the tested dose groups and vehicle control group.

Male Fertility Index: A total of 11 (out of 12), 10 (out of 12), 12 (out of 12) and 10 (out of 12) mated males were confirmed as fertile by impregnating a female or siring a litter with a fertility index of 91.7%, 83.3%, 100.0% and 83.3% from groups G1, G2, G3 and G4, respectively. There were no statistically significant differences noted for male fertility index in any of the tested dose groups when compared with vehicle control group.

Female Mating Index: All the females left for cohabitation were confirmed as sperm positive during vaginal smear examination with a mating index of 100.0% from all the tested dose groups and vehicle control group.

Female Fertility Index: A total of 11 (out of 12), 10 (out of 12), 12 (out of 12) and 8 (out of 9) mated females were confirmed with presence of implantations / presence of live or dead pups / evidence of parturition with a fertility index of 91.7%, 83.3%, 100.0% and 88.9% from groups G1, G2, G3 and G4, respectively.
There were no statistically significant differences noted for female fertility index in any of the tested dose groups when compared with vehicle control group.
Pre-coital Interval/Copulatory Interval/Mean time to Mating: The mean pre-coital interval was 4.58, 5.08, 2.67 and 7.22 days for groups G1, G2, G3 and G4, respectively.

There were no statistically significant differences noted for mean pre-coital interval in any of the tested dose groups when compared with the vehicle control group. Gestation Length/Duration of Pregnancy: The mean gestation length [day of confirmed as successfully mated to day of parturition] was 22.73, 22.70, 23.00 and 23.00 days for groups G1, G2, G3 and G4, respectively.

There were no statistically significant differences noted for mean gestation length in any of the tested dose groups when compared with the vehicle control
group.

Fecundity or Pregnancy Index: A total of 11 (out of 12), 10 (out of 12), 12 (out of 12) and 8 (out of 9) mated females were confirmed as pregnant / with evidence of implantation sites with a fecundity index of 91.7%, 83.3%, 100.0% and 88.9% from groups G1, G2, G3 and G4, respectively. There were no statistically significant differences noted for fecundity index in any of the tested dose groups when compared with the vehicle control group.

Parturition Index: A total of 11 (out of 11), 10 (out of 10), 12 (out of 12), and 8 (out of 8) pregnant females were confirmed with parturition with a parturition indexof 100.0% for all the tested dose groups and vehicle control group.

Gestation Index: A total of 11 (out of 11), 10 (out of 10), 12 (out of 12) and 8 (out of 8) pregnant females were confirmed with live born pups with a gestation index of 100.0% for all the tested dose groups and vehicle control group.

Implantation sites and Viable Pups: A mean number of 13.73, 13.00, 12.67 and 12.38 implantation sites and a mean number of 13.36, 12.60, 12.17 and 11.75 viable pups were noted from groups G1, G2, G3 and G4, respectively. There were no statistically significant changes noted for both mean implantation sites and viable pups in any of the tested dose groups when compared with the vehicle control group.

Post-Implantation Loss: A mean number of 0.36, 0.40, 0.50 and 0.63 post-implantation losses with a percentage of 3.00, 2.71, 3.74 and 4.74 were noted from groups G1, G2, G3 and G4 respectively. There were no statistically significant changes noted for mean number and percentage of occurred post-implantation losses in any of the tested dose groups when compared with the vehicle control group.

Postnatal Loss: There were no incidences of postnatal loss noted in any of the tested dose groups and vehicle control group during lactation period.

Details on results (P0)

In conclusion, the oral administration of test item Mo10V3TeNbO42 over a relatively limited period of time (for males with a total of 35 days and for females ranging from 49 to 63 days) did not produce any indication of systemic toxicity at the dose levels of 200 and 400 mg/kg body weight/day under experimental conditions employed. The dose level of 800 mg/kg body weight/day indicated adverse systemic toxicity and did not produce any reproductive and developmental toxicity indications once reduced to 500 mg/kg body weight/day.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

ca. 500 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

water consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive performance

other: Serum T4 levels

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no external abnomalities or behavioural changes noted in any of the pups during daily observation from all the tested dose group and vehicle control group litters during postnatal period. All the pups were noted with normal behaviour during daily observations

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no treatment related pup mortalities noted in any of the tested dose groups.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

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

However, statistically significant increase in mean male pup weight on PND 4 in group G2; statistically significant increase in mean male and female pup weight on PND 4 and 7 in group G3; statistically significant increase in mean female pup weight on PND 13 in groups G3 and G4 were noted when compared with vehicle control group. These noted changes are incidental and toxicologically irrelevant as the obtained mean values are within the in-house historical control range and also no developmental changes noted in any of the tested dose group litters.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant decrease in mean absoluteuterus weight (G4 females), increase in mean relative thyroid weight (G4 males) and increase in mean relative ovaries weight (G4 females) were noted when compared with the vehiclecontrol group. These noted changes are considered as incidental and un-related to treatment due to lack of dose dependency and also the obtained mean values are within in-house historical control range of same species and strain.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological changes observed in any of the pups (in both ‘found dead at birth' and ‘sacrificed at termination on PND 4 / 13’) of both sexes from all the treated dose groups and vehicle control group during conduct the necropsy.

Histopathological findings:

no effects observed

Details on results (F1)

The oral administration of test item over a relatively limited period of time (for males with a total of 35 days and for females ranging from 49 to 63 days) did not produce any indication of reproductive and developmental toxicity at the dose levels of 200 and 400 mg/kg body weight/day under experimental conditions employed. The dose level of 800 mg/kg body weight/day indicated adverse systemic toxicity and did not produce any reproductive and developmental toxicity indications once reduced to 500 mg/kg body weight/day.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

500 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: Anogenital distance, observation of male pups for nipple retention and Serum T4 lelves

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

 

TABLE 1.      SUMMARYOF CLINICAL SIGNS OF TOXICITY AND MORTALITY RECORD

                                                                                                                                                                                                                   

Group, Sex & Dose (mg/kg body weight/day)	Total No. of Animals	Clinical Signs of Toxicity^a: Observation (No. of Animals revealed)	Mortality^b: No. of Mortalities (Total No. of Animals)
G1, M & 0	12	N (12)	0 (12)
G2, M & 200	12	N (12)	0 (12)
G3, M & 400	12	N (12)	0 (12)
G4, M & 800/500	12	N (8); 1 (4); 37 (4); 82 (2); 110 (4)	0 (12)

M: Male; N: Normal; 1: Lethargy; 37: Soft stool; 82: Chromodacryorrhea; 110: Wet perineum.
a: observed daily once; b: observed twice daily.

 

 TABLE 1 (Contd…). SUMMARY OF CLINICAL SIGNS OF TOXICITY AND MORTALITY RECORD

                        

Group, Sex & Dose (mg/kg body weight/day)		Total No. of Animals		Clinical Signs of Toxicity^a: Observation (No. of Animals revealed)		Mortality^b No. of Mortalities (Total No. of Animals)
G1, F & 0		12		N (12)		0 (12)
G2, F & 200		12		N (12)		0 (12)
G3, F & 400		12		N (12)		0 (12)
G4, F & 800/500		12		N(5); 1 (7); 2 (3); 37 (6); 47 (4); 82 (6); 110 (6)		3 (12)

F: Female; N: Normal; 1: Lethargy; 2: Dehydration; 37: Soft stool; 47: Ataxia; 82: Chromodacryorrhea; 110: Wet perineum
a: observed daily once; b: observed twice daily.

 

TABLE 2.      SUMMARY OF BODY WEIGHT (g) RECORD

  

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Day
		1	7	14	21	28	35
G1, M & 0	Mean	362.71	391.56	409.57	426.12	450.80	476.35
	±SD	10.53	10.72	14.73	19.56	21.51	24.74
	n	12	12	12	12	12	12
G2, M & 200	Mean	364.07	387.15	409.13	422.63	445.19	470.10
	±SD	9.03	14.30	14.43	18.36	21.82	22.51
	n	12	12	12	12	12	12
G3, M & 400	Mean	364.13	383.51	400.12	414.11	434.82	454.57
	±SD	10.06	16.78	21.26	23.92	27.20	32.32
	n	12	12	12	12	12	12
G4, M & 800/500	Mean	363.09	366.51*	367.15*	393.63*	422.34*	440.39*
	±SD	7.67	21.11	38.24	36.49	27.39	26.49
	n	12	12	12	12	12	12

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

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

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

 

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Day
		1	7	14	21^#
G1, F & 0	Mean	258.09	273.31	278.75	285.26
	±SD	10.60	11.68	12.20	19.86
	n	12	12	12	2
G2, F & 200	Mean	258.80	268.66	274.84	275.24
	±SD	10.81	12.72	10.14	8.38
	n	12	12	12	3
G3, F & 400	Mean	257.90	265.05	270.04	-
	±SD	9.46	6.61	8.17	-
	n	12	12	12	0
G4, F & 800/500	Mean	258.78	244.78*	242.89*	259.58
	±SD	6.45	16.20	33.66	21.60
	n	12	12	9	3

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

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

^#: The variation in “n” is due to consideration of data obtained from females in cohabitation only for mean calculations. The data of day 21 body weights were not subjected to statistical analysis due to uneven number of variables, but presented in individual animal data.

 

TABLE 3. SUMMARY OF GESTATION BODY WEIGHT (g) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Gestation Day (GD)
		0	7	14	20
G1, F & 0	Mean	283.54	299.67	334.69	393.31
	±SD	11.68	12.22	14.14	22.74
	n	11	11	11	11
G2, F & 200	Mean	279.93	295.52	328.64	392.78
	±SD	9.74	9.37	9.05	15.01
	n	10	10	10	10
G3, F & 400	Mean	272.31	285.85	315.26*	381.02
	±SD	7.78	6.45	10.88	16.35
	n	12	12	12	12
G4, F & 800/500	Mean	254.00*	268.89*	302.59*	364.90*
	±SD	25.36	25.48	27.85	31.56
	n	8	8	8	8

F: Female; SD: Standard Deviation; n: Number of Dams. *: Statistically significant (P<0.05) change than the vehicle control group.

Note: Excluded the data of non-pregnant females for mean calculations and statistical analysis, but presented in individual animal data.

 

   TABLE 4.      SUMMARY OF LACTATION BODY WEIGHT (g)RECORD

 

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Lactation Day (LD)
		1	4	7	13
G1, F & 0	Mean	305.93	308.90	324.53	342.62
	±SD	13.13	16.25	14.32	15.79
	n	11	11	11	11
G2, F & 200	Mean	304.08	308.10	322.88	341.36
	±SD	11.54	11.15	11.97	12.31
	n	10	10	10	10
G3, F & 400	Mean	296.54	301.54	319.25	338.53
	±SD	11.50	12.10	13.62	15.27
	n	12	12	12	12
G4, F & 800/500	Mean	275.52*	278.60*	294.79*	311.54*
	±SD	25.88	24.43	22.39	22.09
	n	8	8	8	8

 

F: Female; SD: Standard Deviation; n: Number of Dams.

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

 

TABLE 5.      SUMMARY OFPERCENT CHANGE IN BODY WEIGHT GAIN (%) WITH RESPECT TO DAY 1 RECORD

                                                                                   

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight (%) Gain during Day
		1 to 7	1 to 14	1 to 21	1 to 28	1 to 35
G1, M & 0	Mean	7.97	12.93	17.48	24.29	31.35
	±SD	1.66	2.81	4.30	4.90	6.04
	n	12	12	12	12	12
G2, M & 200	Mean	6.33	12.37	16.08	22.29	29.13
	±SD	2.26	2.62	3.98	5.27	5.40
	n	12	12	12	12	12
G3, M & 400	Mean	5.29	9.85	13.69	19.37	24.79*
	±SD	2.50	4.18	5.07	5.91	7.43
	n	12	12	12	12	12
G4, M & 800/500	Mean	0.92*	1.09*	8.37*	16.31*	21.28*
	±SD	4.99	10.05	9.27	7.12	6.74
	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 5 (Contd…). SUMMARYOF PERCENT CHANGE IN BODY WEIGHT GAIN (%) WITH RESPECT TO DAY 1 RECORD

 

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight (%) during Day
		1 to 7	1 to 14	1 to 21^#
G1, F & 0	Mean	5.90	8.01	11.26
	±SD	1.53	1.83	3.14
	n	12	12	2
G2, F & 200	Mean	3.80	6.23	8.83
	±SD	1.83	1.96	2.07
	n	12	12	3
G3, F & 400	Mean	2.82	4.75	-
	±SD	1.79	2.47	-
	n	12	12	-
G4, F & 800/500	Mean	-5.31*	-5.58*	-1.65
	±SD	7.34	13.92	10.04
	n	12	9	3

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

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

^#: The data obtained from females in cohabitation only considered for mean calculations. The data of day 21 body weight gain was not subjected to statistical analysis due to uneven number of variables, but presented in individual animal data.  

TABLE 6.      SUMMARY OFPERCENT CHANGE IN GESTATION BODY WEIGHT GAIN (%) RECORD

 

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight Gain (%) during Gestation Day (GD)
		0 to 7	7 to 14	14 to 20
G1, F & 0	Mean	5.69	11.68	17.53
	±SD	0.76	1.13	5.12
	n	11	11	11
G2, F & 200	Mean	5.58	11.24	19.52
	±SD	0.67	1.99	3.12
	n	10	10	10
G3, F & 400	Mean	5.00	10.28	20.86
	±SD	1.02	2.41	3.04
	n	12	12	12
G4, F & 800/500	Mean	5.91	12.58	20.68
	±SD	0.83	2.39	2.87
	n	8	8	8

 

F: Female; SD: Standard Deviation; n: Number of Dams.

Note: Excluded the data of non-pregnant females for mean calculations and statistical analysis, but presented in individual animal data. 

 

TABLE 7.      SUMMARY OFPERCENT CHANGE IN LACTATION BODY WEIGHT (g)

 

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight Gain (%) during Lactation Day (LD)
		1 to 4	4 to 7	7 to 13
G1, F & 0	Mean	0.93	5.11	5.57
	±SD	1.42	1.33	0.85
	n	11	11	11
G2, F & 200	Mean	1.34	4.80	5.73
	±SD	1.34	1.29	0.55
	n	10	10	10
G3, F & 400	Mean	1.68	5.87	6.03
	±SD	0.75	1.03	0.75
	n	12	12	12
G4, F & 800/500	Mean	1.19	5.92	5.72
	±SD	1.80	2.03	1.36
	n	8	8	8

F: Female; SD: Standard Deviation; n: Number of Dams.

                                                                                                              
           TABLE 8.      SUMMARY OF FEED CONSUMPTION (g/animal/day) RECORD

 

Group, Sex & Dose (mg/kg body weight/day)		Feed Consumption (g/animal/day) during
		Pre-mating period			Post-mating period
		Week 1	Week 2		Week 5
G1, M & 0	Mean	25.86	27.06		24.20
	±SD	0.94	1.33		0.99
	n	12 (6)	12 (6)		12 (6)
G2, M & 200	Mean	25.57	26.84		23.55
	±SD	1.70	1.20		1.01
	n	12 (6)	12 (6)		12 (6)
G3, M & 400	Mean	23.43	24.92		23.07
	±SD	1.65	2.34		1.10
	n	12 (6)	12 (6)		12 (6)
G4, M & 800/500	Mean	18.91*	17.11*		22.42*
	±SD	3.27	3.61		0.55
	n	12 (6)	12 (6)		12 (6)

M: Male; SD: Standard Deviation; n: Number of Animals(Number of Cages).

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

                           

 

   TABLE 8 (Contd...).      SUMMARY OF FEED CONSUMPTION (g/animal/day) RECORD

 

Group, Sex & Dose (mg/kg body weight/day)		Feed Consumption (g/animal/day) during Pre-mating period
		Week 1	Week 2
G1, F & 0	Mean	19.94	20.41
	±SD	1.20	1.15
	n	12 (6)	12 (6)
G2, F & 200	Mean	18.74	20.02
	±SD	1.49	1.16
	n	12 (6)	12 (6)
G3, F & 400	Mean	17.87	19.16
	±SD	1.23	1.46
	n	12 (6)	12 (6)
G4, F & 800/500	Mean	12.22*	12.14*
	±SD	2.51	2.02
	n	12 (6)	12 (6)

F: Female; SD: Standard Deviation; n: Number of Animals (Number of Cages).

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

 

TABLE 9.      SUMMARY RECORD OF FEED CONSUMPTION (g/animal/day) DURING GESTATION PERIOD

 

Group, Sex & Dose (mg/kg body weight/day)		Feed Consumption (g/animal/day) during Gestation Day (GD)
		0 to 7	7 to 14	14 to 20
G1, F & 0	Mean	22.49	23.40	26.95
	±SD	0.70	0.40	0.48
	n	11	11	11
G2, F & 200	Mean	22.24	23.14	27.10
	±SD	0.87	0.60	0.90
	n	10	10	10
G3, F & 400	Mean	22.09	23.00	26.71
	±SD	0.85	0.76	0.59
	n	12	12	12
G4, F & 800/500	Mean	21.75	22.88	26.90
	±SD	0.31	0.38	0.85
	n	8	8	8

F: Female; SD: Standard Deviation; n: Number of Dams.

Note: Excluded the data of non-pregnant females for mean calculations and statistical analysis, but presented in individual animal data.

 

TABLE 10.      SUMMARY RECORD OF FEED CONSUMTION (g/animal/day) DURING LACTATION PERIOD

 

Group, Sex & Dose (mg/kg body weight/day)		Feed Consumption (g/animal/day) during Lactation Day (LD)
		1 to 4	4 to 7	7 to 13
G1, F & 0	Mean	28.20	29.99	36.90
	±SD	1.38	1.06	0.89
	n	11	11	11
G2, F & 200	Mean	28.14	29.92	36.64
	±SD	1.16	1.49	0.76
	n	10	10	10
G3, F & 400	Mean	27.94	29.81	36.76
	±SD	1.18	1.05	0.80
	n	12	12	12
G4, F & 800/500	Mean	27.54	29.28	36.81
	±SD	1.65	1.13	0.99
	n	8	8	8

F: Female; SD: Standard Deviation; n: Number of Dams.

 

TABLE 11.      SUMMARY RECORD OF VAGINAL SMEAR EXAMINATION FOR DETERMINATION OF OESTRUS CYCLICITY

 

Determination of Oestrus Cyclicity during Pre-Mating Treatment Period
Group, Sex & Dose (mg/kg body weight/day)	Total No. of Females Evaluated		No. of Females with Complete Regular Oestrus Cycle	No. of Females with at least one Irregular Oestrus Cycle	Average Length of Oestrus Cycle (Days)
G1, F & 0	12	n	12	-	Mean	4.91
		%	100.00	-	±SD	0.16
					n	12
G2, F & 200	12	n	11	1	Mean	4.97
		%	91.67	8.33	±SD	0.18
					n	12
G3, F & 400	12	n	12	-	Mean	5.00
		%	100.00	-	±SD	0.00
					n	12
G4, F & 800/500	12	n@	7	2	Mean	5.02
		%	77.78	22.22	±SD	0.49
					n	9

F: Female; SD: Standard Deviation; n: Number of Animals. %: Percent.

@: In group G4, 3 out of 12 females which were found dead during pre-mating noted with prolonged diestrus stage when treated with 800 mg/kg body weight/day. The data of these females is excluded for interpretation, but presented in individual animal data.

 

TABLE 12.      SUMMARY OF DELIVERYDATA (AT BIRTH) RECORD PER LITTER

 

Group, Sex & Dose (mg/kg body weight/day)		Delivery Record At birth
		Litter Size (No.)		Live Pups (No.)				Dead Pups (No.)				Cannibalized Pups (No.)					Sex Ratio (m/f) at Birth		Live Birth Index (%)
				Male	Female	Total		Male	Female	Total		Undetermined	Male	Female	Total
G1, F & 0	Mean	13.45		7.36	6.00	13.36		0.09	0.00	0.09		0.00	0.00	0.00	0.00		1.37		99.47
	±SD	2.88		1.69	2.10	2.77		0.30	0.00	0.30		0.00	0.00	0.00	0.00		0.54		1.77
	n	11		11	11	11		11	11	11		11	11	11	11		11		11
G2, F & 200	Mean	12.60		6.20	6.40	12.60		0.00	0.00	0.00		0.00	0.00	0.00	0.00		1.13		100.00
	±SD	1.58		1.99	2.01	1.58		0.00	0.00	0.00		0.00	0.00	0.00	0.00		0.65		0.00
	n	10		10	10	10		10	10	10		10	10	10	10		10		10
G3, F & 400	Mean	12.25		5.67	6.50	12.17		0.08	0.00	0.08		0.00	0.00	0.00	0.00		0.98		99.24
	±SD	2.38		2.27	1.88	2.44		0.29	0.00	0.29		0.00	0.00	0.00	0.00		0.56		2.62
	n	12		12	12	12		12	12	12		12	12	12	12		12		12
G4, F & 800/500	Mean	12.00		6.13	5.63	11.75		0.00	0.25	0.25		0.00	0.00	0.00	0.00		1.37		97.90
	±SD	2.07		1.73	2.56	2.12		0.00	0.46	0.46		0.00	0.00	0.00	0.00		0.80		3.90
	n	8		8	8	8		8	8	8		8	8	8	8		8		8

F: Female; SD: Standard Deviation; n: Number of Dams; m/f: male/female

 

TABLE 13.      SUMMARY OF LITTER OBSERVATION RECORD DURING LACTATION PERIOD

.

Group, Sex & Dose (mg/kg body weight/day)		LD 1 to 4												Sex Ratio (m/f) on LD 4		Pup Survival Index (%) [LD 1 to 4]
		Live Pups (No.)				Dead Pups (No.)				Cannibalized Pups (No.)
		Male	Female	Total		Male	Female	Total		Male	Female	Total
G1, F & 0	Mean	7.36	6.00	13.36		0.00	0.00	0.00		0.00	0.00	0.00		1.37		100.00
	±SD	1.69	2.10	2.77		0.00	0.00	0.00		0.00	0.00	0.00		0.54		0.00
	n	11	11	11		11	11	11		11	11	11		11		11
G2, F & 200	Mean	6.20	6.40	12.60		0.00	0.00	0.00		0.00	0.00	0.00		1.13		100.00
	±SD	1.99	2.01	1.58		0.00	0.00	0.00		0.00	0.00	0.00		0.65		0.00
	n	10	10	10		10	10	10		10	10	10		10		10
G3, F & 400	Mean	5.67	6.50	12.17		0.00	0.00	0.00		0.00	0.00	0.00		0.98		100.00
	±SD	2.27	1.88	2.44		0.00	0.00	0.00		0.00	0.00	0.00		0.56		0.00
	n	12	12	12		12	12	12		12	12	12		12		12
G4, F & 800/500	Mean	6.13	5.63	11.75		0.00	0.00	0.00		0.00	0.00	0.00		1.37		100.00
	±SD	1.73	2.56	2.12		0.00	0.00	0.00		0.00	0.00	0.00		0.80		0.00
	n	8	8	8		8	8	8		8	8	8		8		8

F: Female; SD: Standard Deviation; n: Number of Dams; m/f: male/female; LD: Lactation Day.

 

TABLE 13 (Contd…). SUMMARY OF LITTER OBSERVATION RECORD DURING LACTATION PERIOD

Group, Sex & Dose (mg/kg body  weight/day)		Pups Sacrificed for Litter Standardization / Hormonal analysis (No.)				Live Pups (No.) on LD 4 after Litter Standardization / Hormonal analysis (No.)				LD 5 to 7												Sex Ratio (m/f) on LD 7		Pup Survival Index (%) [LD 5 to 7]
										Live Pups (No.)				Dead Pups (No.)				Cannibalized (No.)
		Male	Female	Total		Male	Female	Total		Male	Female	Total		Male	Female	Total		Male	Female	Total
G1, F & 0	Mean	0.00	1.55	1.55		7.36	4.45	11.82		7.36	4.45	11.82		0.00	0.00	0.00		0.00	0.00	0.00		1.84		100.00
	±SD	0.00	0.82	0.82		1.69	1.57	2.09		1.69	1.57	2.09		0.00	0.00	0.00		0.00	0.00	0.00		0.80		0.00
	n	11	11	11		11	11	11		11	11	11		11	11	11		11	11	11		11		11
G2, F & 200	Mean	0.00	1.60	1.60		6.20	4.80	11.00		6.20	4.80	11.00		0.00	0.00	0.00		0.00	0.00	0.00		1.69		100.00
	±SD	0.00	0.84	0.84		1.99	1.75	0.94		1.99	1.75	0.94		0.00	0.00	0.00		0.00	0.00	0.00		1.37		0.00
	n	10	10	10		10	10	10		10	10	10		10	10	10		10	10	10		10		10
G3, F & 400	Mean	0.00	1.25	1.25		5.67	5.25	10.92		5.67	5.25	10.92		0.00	0.00	0.00		0.00	0.00	0.00		1.43		100.00
	±SD	0.00	0.97	0.97		2.27	1.96	1.68		2.27	1.96	1.68		0.00	0.00	0.00		0.00	0.00	0.00		1.14		0.00
	n	12	12	12		12	12	12		12	12	12		12	12	12		12	12	12		12		12
G4, F & 800/500	Mean	0.00	1.13	1.13		6.13	4.50	10.63		6.13	4.50	10.63		0.00	0.00	0.00		0.00	0.00	0.00		1.70		100.00
	±SD	0.00	0.99	0.99		1.73	1.85	1.30		1.73	1.85	1.30		0.00	0.00	0.00		0.00	0.00	0.00		1.11		0.00
	n	8	8	8		8	8	8		8	8	8		8	8	8		8	8	8		8		8

F: Female; SD: Standard Deviation; n: Number of Dams; LD: Lactation Day; m/f: male/female.

 

TABLE 13 (Contd…). SUMMARY OF LITTER OBSERVATION RECORD DURING LACTATION PERIOD

 

Group, Sex & Dose (mg/kg body weight/day)		LD 8 to 13												Sex Ratio (m/f) on LD 13		Pup Survival Index (%) [LD 8 to 13]
		Live Pups (No.)				Dead Pups (No.)				Cannibalized (No.)
		Male	Female	Total		Male	Female	Total		Male	Female	Total
G1, F & 0	Mean	7.36	4.45	11.82		0.00	0.00	0.00		0.00	0.00	0.00		1.84		100.00
	±SD	1.69	1.57	2.09		0.00	0.00	0.00		0.00	0.00	0.00		0.80		0.00
	n	11	11	11		11	11	11		11	11	11		11		11
G2, F & 200	Mean	6.20	4.80	11.00		0.00	0.00	0.00		0.00	0.00	0.00		1.69		100.00
	±SD	1.99	1.75	0.94		0.00	0.00	0.00		0.00	0.00	0.00		1.37		0.00
	n	10	10	10		10	10	10		10	10	10		10		10
G3, F & 400	Mean	5.67	5.25	10.92		0.00	0.00	0.00		0.00	0.00	0.00		1.43		100.00
	±SD	2.27	1.96	1.68		0.00	0.00	0.00		0.00	0.00	0.00		1.14		0.00
	n	12	12	12		12	12	12		12	12	12		12		12
G4, F & 800/500	Mean	6.13	4.50	10.63		0.00	0.00	0.00		0.00	0.00	0.00		1.70		100.00
	±SD	1.73	1.85	1.30		0.00	0.00	0.00		0.00	0.00	0.00		1.11		0.00
	n	8	8	8		8	8	8		8	8	8		8		8

 F: Female; SD: Standard Deviation; n: Number of Dams; LD: Lactation Day; m/f: male/female.

 

TABLE 14.      SUMMARY OF REPRODUCTIVE PERFORMANCERECORD

 

Group, Sex & Dose (mg/kg body weight/day)	No. of Males with Evidence of Mating (Total No. of Males used for Mating)	Male Mating Index (%)		No. of Males Capable of Impregnating a Female (No. of Males confirmed as mated)	Male Fertility Index (%)
G1, M & 0	12 (12)	100.0		11 (12)	91.7
G2, M & 200	12 (12)	100.0		10 (12)	83.3
G3, M & 400	12 (12)	100.0		12 (12)	100.0
G4, M & 800/500	12 (12)	100.0		10 (12)	83.3

M: Male.

 

TABLE 14 (Contd…). SUMMARY OF REPRODUCTIVE PERFORMANCERECORD

 

Group, Sex & Dose (mg/kg body weight/day)		Pre-coital Interval / Mean Time to Mating (Days)				Gestation Length / Duration of Pregnancy (Days)
		Pre-Coital Interval / Copulatory Interval		Conceiving Days (1 to 5)	Conceiving Days (5 to More)	Gestation Length (Days)
G1, F & 0	Mean	4.58	n	9	3	22.73
	±SD	3.40	%	75.00	25.00	0.47
	n	12				11
G2, F & 200	Mean	5.08	n	7	5	22.70
	±SD	3.58	%	58.33	41.67	0.48
	n	12				10
G3, F & 400	Mean	2.67	n	12	0	23.00
	±SD	1.23	%	100.00	0.00	0.60
	n	12				12
G4, F & 800/500	Mean	7.22	n	5	4	23.00
	±SD	4.71	%	55.56	44.44	0.00
	n	9				8

F: Female; SD: Standard Deviation.

n: Number of Females confirmed with mating (in case of Pre-Coital Interval) / Number of Females confirmed with pregnancy (in case of gestation length).

 

TABLE 14 (Contd…). SUMMARY OF REPRODUCTIVE PERFORMANCE RECORD

 

Group, Sex & Dose (mg/kg body weight/day)	Female Mating Index (%)				Female Fertility Index (%)				Female Fecundity or Pregnancy Index (%)
	No. of Females with Evidence of Mating	No. of Females used for Mating	Female Mating Index (%)		No. of Females confirmed as Fertile	No. of Females used for Mating	Female Fertility Index (%)		No. of Pregnant Females	No. of Females with confirmed Mating	Female Fecundity or Pregnancy Index (%)
G1, F & 0	12	12	100.0		11	12	91.7		11	12	91.7
G2, F & 200	12	12	100.0		10	12	83.3		10	12	83.3
G3, F & 400	12	12	100.0		12	12	100.0		12	12	100.0
G4, F & 800/500	9	9	100.0		8	9	88.9		8	9	88.9

F: Female.

 

TABLE 14 (Contd…). SUMMARY OF REPRODUCTIVE PERFORMANCERECORD

 

Group, Sex & Dose (mg/kg body weight/day)	Gestation index (%)				Parturition index (%)
	With Live born Pups at Parturition	No. of Females with evidence of pregnancy	Gestation index (%)		No. of Females Littered	No. of Females with evidence of pregnancy	Parturition index (%)
G1, F & 0	11	11	100.0		11	11	100.0
G2, F & 200	10	10	100.0		10	10	100.0
G3, F & 400	12	12	100.0		12	12	100.0
G4, F & 800/500	8	8	100.0		8	8	100.0

F: Female.

 

TABLE 14 (Contd…). SUMMARY OF REPRODUCTIVE PERFORMANCERECORD

 

Group, Sex & Dose (mg/kg body weight/day)		Post-implantation Loss (%)					Post-natal Loss (%)
		No. of Implantations	No. of Viable (Live) Pups	Post-implantation Loss (No.)	Post-implantation Loss (%)		Total No. of Pups Found Dead/ Cannibalized during lactation period	Post-natal Loss (%)
G1, F & 0	Mean	13.73	13.36	0.36	3.00		0.00	0.00
	±SD	2.53	2.77	0.67	6.22		0.00	0.00
	n	11	11	11	11		11	11
G2, F & 200	Mean	13.00	12.60	0.40	2.71		0.00	0.00
	±SD	1.89	1.58	0.70	4.71		0.00	0.00
	n	10	10	10	10		10	10
G3, F & 400	Mean	12.67	12.17	0.50	3.74		0.00	0.00
	±SD	2.39	2.44	1.17	8.46		0.00	0.00
	n	12	12	12	12		12	12
G4, F & 800/500	Mean	12.38	11.75	0.63	4.74		0.00	0.00
	±SD	2.13	2.12	1.06	8.13		0.00	0.00
	n	8	8	8	8		8	8

F: Female; SD: Standard Deviation; n: Number of Dams.

 

 

TABLE 15.      SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

 

Group, Sex & Dose (mg/kg body weight/day)		Testes	Epididymides	Prostate	Seminal vesicles with coagulating glands	Thyroid along with parathyroid^WP
G1, M & 0	Mean	3.4622	1.4351	1.2356	1.9661	0.0253
	±SD	0.2812	0.1430	0.1985	0.3329	0.0029
	n	12	12	12	12	12
G2, M & 200	Mean	3.4078	1.4409	1.2133	2.0594	0.0263
	±SD	0.1806	0.0980	0.3439	0.2185	0.0021
	n	12	12	12	12	12
G3, M & 400	Mean	3.3748	1.4142	1.3173	1.8457	0.0253
	±SD	0.3487	0.1444	0.1789	0.3414	0.0038
	n	12	12	12	12	12
G4, M & 800/500	Mean	3.2204	1.3094	1.3775	1.8211	0.0261
	±SD	0.1708	0.1418	0.5036	0.3148	0.0019
	n	12	12	12	12	12

M: Male; SD: Standard Deviation; n: Number of Animals; WP: Weighed post-fixation.

 

 

TABLE 15 (Contd…). SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Ovaries	Uterus with cervix	Thyroid along with parathyroid^WP
G1, F & 0	Mean	0.1524	0.5707	0.0202
	±SD	0.0095	0.0564	0.0023
	n	12	12	12
G2, F & 200	Mean	0.1541	0.5434	0.0213
	±SD	0.0053	0.0574	0.0021
	n	12	12	12
G3, F & 400	Mean	0.1532	0.5271	0.0204
	±SD	0.0064	0.0512	0.0015
	n	12	12	12
G4, F & 800/500	Mean	0.1519	0.5065*	0.0191
	±SD	0.0049	0.0115	0.0027
	n	9	9	9

F: Female; SD: Standard Deviation; n: Number of Animals; WP: Weighed post-fixation.

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

 

 

TABLE 16.      SUMMARY OF TERMINAL BODY WEIGHT (g) AND ORGAN WEIGHT RELATIVE TO
TERMINAL BODY WEIGHT (%) RECORD

 

Group, Sex & Dose (mg/kg body weight/day)		Terminal Body Weight (g)	Testes	Epididymides	Prostate	Seminal vesicles with coagulating glands	Thyroid along with parathyroid
G1, M & 0	Mean	452.44	0.7671	0.3189	0.2728	0.4363	0.0056
	±SD	25.46	0.0722	0.0423	0.0393	0.0780	0.0006
	n	12	12	12	12	12	12
G2, M & 200	Mean	448.34	0.7611	0.3218	0.2710	0.4602	0.0059
	±SD	20.87	0.0444	0.0232	0.0789	0.0519	0.0004
	n	12	12	12	12	12	12
G3, M & 400	Mean	430.99	0.7859	0.3285	0.3066	0.4298	0.0059
	±SD	32.55	0.0905	0.0279	0.0430	0.0805	0.0007
	n	12	12	12	12	12	12
G4, M & 800/500	Mean	414.66*	0.7797	0.3154	0.3302	0.4397	0.0063*
	±SD	27.60	0.0664	0.0237	0.1125	0.0723	0.0005
	n	12	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 16 (Contd…). SUMMARY OF TERMINAL BODY WEIGHT (g) AND ORGAN WEIGHT RELATIVE TO TERMINAL BODY WEIGHT (%) RECORD

 

Group, Sex & Dose (mg/kg body weight/day)		Terminal Body Weight (g)	Ovaries	Uterus	Thyroid along with parathyroid
G1, F & 0	Mean	328.93	0.0464	0.1741	0.0061
	±SD	16.53	0.0034	0.0209	0.0006
	n	12	12	12	12
G2, F & 200	Mean	325.10	0.0475	0.1676	0.0066
	±SD	16.93	0.0026	0.0202	0.0008
	n	12	12	12	12
G3, F & 400	Mean	326.48	0.0470	0.1620	0.0063
	±SD	15.81	0.0027	0.0195	0.0003
	n	12	12	12	12
G4, F & 800/500	Mean	298.54*	0.0511*	0.1705	0.0064
	±SD	22.49	0.0036	0.0125	0.0005
	n	9	9	9	9

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

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

 

TABLE 17.      SUMMARY RECORD OF SERUM THYROXINE (T4) HORMONE LEVELS (ng/mL) RECORD -
MALES

Group, Sex & Dose (mg/kg body weight/day)		Serum T4 Levels (ng/mL)
G1, M & 0	Mean	74.777
	±SD	15.488
	n	12
G2, M & 200	Mean	70.779
	±SD	5.168
	n	12
G3, M & 400	Mean	69.416
	±SD	2.464
	n	12
G4, M & 800/500	Mean	67.976
	±SD	7.147
	n	12

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

 

TABLE 18.      SUMMARY OF PUP OBSERVATIONS RECORD DURING POSTNATAL PERIOD

Group, Sex & Dose (mg/kg body weight/day)				At Birth (PND 1)		PND 1 to 4		Pups Sacrificed for Blood Collection on PND 4 (No.)*		PND 5 to 7^$		PND 8 to 13^$
G1, F & 0		No. of Dams / Litters^#		11		11		9		11		11
		No. of Live Pups		147		147		17		130		130
		Pup Observation/ No. of Pups observed		N/147		N/147		-		N/130		N/130
G2, F & 200		No. of Dams / Litters^#		10		10		8		10		10
		No. of Live Pups		126		126		16		110		110
		Pup Observation/ No. of Pups observed		N/126		N/126		-		N/110		N/110
G3, F & 400		No. of Dams / Litters^#		12		12		8		11		11
		No. of Live Pups		146		146		15		131		131
		Pup Observation/ No. of Pups observed		N/146		N/146		-		N/131		N/131
G4, F & 800/500		No. of Dams / Litters^#		8		8		5		11		11
		No. of Live Pups		94		94		9		85		85
		Pup Observation/ No. of Pups observed		N/94		N/94		-		N/85		N/85

F: Female; N: Normal; PND: Postnatal Day; #: confirmed with live pups.

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

 

TABLE 19.      SUMMARY OF MEAN PUP WEIGHT (g) PER LITTER RECORD

Group & Dose (mg/kg body weight/day)		PND 1			PND 4			PND 7			PND 13
		Mean Pup Weight (g)			Mean Pup Weight (g)			Mean Pup Weight (g)			Mean Pup Weight (g)
		Male	Female		Male	Female		Male	Female		Male	Female
G1 & 0	Mean	6.80	6.38		11.52	10.27		14.88	13.26		27.41	25.55
	±SD	0.21	0.17		0.47	0.39		0.43	0.37		0.58	0.58
	n	11	11		11	11		11	11		11	11
G2 & 200	Mean	6.87	6.41		11.98*	10.46		15.35	13.35		27.77	25.77
	±SD	0.16	0.19		0.39	0.46		0.36	0.40		0.35	0.50
	n	10	10		10	10		10	10		10	10
G3 & 400	Mean	6.92	6.61		11.91*	11.12*		15.58*	13.97*		27.84	26.34*
	±SD	0.19	0.30		0.28	0.62		0.71	0.82		0.70	0.73
	n	12	12		12	12		12	12		12	12
G4 & 800/500	Mean	6.89	6.45		11.85	10.56		15.00	13.40		27.89	26.37*
	±SD	0.15	0.25		0.34	0.44		0.38	0.67		0.39	0.69
	n	8	8		8	8		8	8		8	8

F: Female; SD: Standard Deviation; n: Number of Litters; PND: Postnatal Day.

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

 

TABLE 20.      SUMMARY OF MEAN PUP ANOGENITAL DISTANCE (AGD) MEASUREMENT (mm) AND ANOGENITAL DISTANCE (AGD) RATIO PER LITTER RECORD ON POSTNATAL DAY 4

Group & Dose (mg/kg body weight/day)		Mean Anogenital Distance Measurement (mm)			Mean Anogenital Distance Ratio
		Male	Female		Male	Female
G1 & 0	Mean	4.56	2.52		2.02	1.16
	±SD	0.09	0.08		0.02	0.03
	n	11	11		11	11
G2 & 200	Mean	4.53	2.49		1.98*	1.14
	±SD	0.09	0.06		0.05	0.02
	n	10	10		10	10
G3 & 400	Mean	4.60	2.58		2.01	1.16
	±SD	0.06	0.07		0.02	0.01
	n	12	12		12	12
G4 & 800/500	Mean	4.58	2.49		2.01	1.13*
	±SD	0.08	0.07		0.03	0.02
	n	8	8		8	8

Female; SD: Standard Deviation; n: Number of Litters; AGD: Anogenital Distance.

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

 

 

TABLE 21.      SUMMARY OF MALE PUP NIPPLE/AREOLAE RETENTION (no.) RECORDPER LITTER

 

Group & Dose (mg/kg body weight/day)		Mean No. of Pups with Retention of Nipples/ Areolae on Postnatal Day 13
G1 & 0	Mean	0.00
	±SD	0.00
	n	11
G2 & 200	Mean	0.00
	±SD	0.00
	n	10
G3 & 400	Mean	0.00
	±SD	0.00
	n	12
G4 & 800/500	Mean	0.00
	±SD	0.00
	n	8

SD: Standard Deviation; n: Number of Litters.

 

 

TABLE 22.      SUMMARY OF SERUM THYROXINE (T4) HORMONE LEVELS (ng/mL) RECORD - POSTNATAL DAY 13

 

Group, Sex & Dose (mg/kg body weight/day)		Serum T4 Levels (ng/mL)
G1 & 0	Mean	63.138
	±SD	5.360
	n	11
G2 & 200	Mean	60.905
	±SD	4.086
	n	10
G3 & 400	Mean	60.588
	±SD	4.603
	n	12
G4 & 800/500	Mean	63.614
	±SD	2.673
	n	8

SD: Standard Deviation; n: Number of Litters; PND: Postnatal Day.

TABLE 23.      SUMMARY OF GROSS PATHOLOGICAL FINDINGS RECORD - ADULTS   Sex Male Female Route of administration Oral Gavage Treatment Vehicle Control Mo10V3TeNbO42 Vehicle Control Mo10V3TeNbO42 Group G1 G2 G3 G4 G1 G2 G3 G4 Dose (mg/kg body weight/day) 0  200  400  800/500 0  200  400  800/500 Number of animals 12 12 12 12 12 12 12 12 No. of dead rats during treatment 0 0 0 0 0 0 0 3 No. of moribund sacrificed rats 0 0 0 0 0 0 0 0 No. of terminally sacrificed rats 12 12 12 12 12 12 12 9 No of rats showing gross pathology - - - - - - - 3 External -Wet perineum - - - - - - - 3 Internal- Autolysis precluded evaluation - - - - - - - 3

 

TABLE 24.      SUMMARY OF GROSS PATHOLOGICAL FINDINGS RECORD - PUPS

 

Group		G1		G2		G3		G4
Dose (mg/kg body weight/day)		0		200		400		800/500
Sex		Male	Female	Male	Female	Male	Female	Male	Female
No. of Dead Pups at Birth		1	-	-	-	-	1	-	2
External Gross Pathology Findings	NAD	1	-	-	-	-	1	-	2
Internal Gross Pathology Findings	NAD	1	-	-	-	-	1	-	2
						-
No. of pups sacrificed on PND 4		-	17	-	16	-	15	-	9
External Gross Pathology Findings	NAD	-	17	-	16	-	15	-	9
Internal Gross Pathology Findings	NAD	-	17	-	16	-	15	-	9
No. of pups sacrificed on PND 13		81	49	62	48	68	63	49	36
External Gross Pathology Findings	NAD	81	49	62	48	68	63	49	36
Internal Gross Pathology Findings	NAD	81	49	62	48	68	63	49	36

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

 

 

TABLE 25.      SUMMARY OF HISTOPATHOLOGICAL FINDINGS RECORD -MALES

Route of administration		Oral Gavage
Vehicle /Test item		Control	Test item
Dose (mg/kg body weight/day)		0	800/500
Group		G1	G4
Number of Animals		12	12
Epididymides	Number examined	12	12
	Within normal limits	12	12
Testes	Number examined	12	12
	Within normal limits	12	12

 

TABLE 26.      SUMMARY OF HISTOPATHOLOGICAL FINDINGS RECORD -MALES

 

Route of administration		Oral Gavage
Vehicle /Test item		Control	Test item
Dose (mg/kg body weight/day)		0	800/500
Group		G1	G4
Number of Animals		12	12
Ovaries	Number examined	12	09
	Within normal limits	12	09
Stages of estrus cycle	Proestrus	05	01
	Estrus	01	-
	Metestrus	01	-
	Diestrus	05	08
Vagina	Number examined	12	09
	Within normal limits	12	09

 

Applicant's summary and conclusion

Conclusions:

In conclusion, the oral administration of test item over a relatively limited period of time (for males with a total of 35 days and for females ranging from 49 to 63 days) did not produce any indication of systemic, reproduction and developmental toxicity at the dose levels of 200 and 400 mg/kg body weight/day under experimental conditions employed. The dose level of 800 mg/kg body weight/day indicated adverse systemic toxicity and did not produce any reproductive and developmental toxicity indications once reduced to 500 mg/kg body weight/day. The systemic toxicity observed at 800 mg/kg bw/d recovered after reduction to 500 mg/kg bw/d.

Therefore, the no-observed-adverse-effect-level (NOAEL) of test item is considered as 500 mg/kg body weight for systemic, reproduction and developmental toxicity end points.

Executive summary:

The objective of this Reproduction/Developmental Toxicity Screening Test of the test item by oral gavage in Sprague Dawley Rats was to determine the possible health hazards likely to arise from repeated exposure to the test item over a relatively limited period of time. This study was also conducted to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition and an estimate of the No Observed Adverse Effect Level (NOAEL).

 

A total of 96 (48 males + 48 females) Sprague Dawley rats were selected for the study and distributed to four (G1, G2, G3 and G4) groups. Each group consisted of 12 males and 12 females. The animals in group G1 were administered with vehicle [0.5% w/v Carboxymethyl Cellulose], animals in groups G2 and G3 were administered with test item at the dose levels of 200 and 400 mg/kg body weight/day for low and mid dose groups, respectively. The animals in group G4 were administered with test item at a dose level of 800 mg/kg body weight/day as a high dose initially and was reduced to 500 mg/kg body weight/day from day 11 onwards due to occurrence of test item-related clinical signs of toxicity and mortalities. Vehicle and test item formulations were administered orally by gavage with the dose volume of 10 mL/kg body weight/day once daily at similar times on each day for 7 days a week.

 

All the males were administered with test item during pre-mating (14-days), mating and post-mating period (total of 35 days). The pregnant females were administered with test item during pre-mating (14-days), mating, pregnancy (gestation) and up to lactation day 13 ranging from 49 to 63 days.The non-pregnant femaleswere administered with test itemduringpre-mating (14-days),mating until confirmed as mated and further 24 days from the day of confirmation of mating.

  

In groups G2 and G3, there were no clinical signs of toxicity and no mortality/morbidity noted. No changes were noted in mean body weight, percent change in mean body weight gain and mean feed consumption of both sexes. The estimated serum T4 levels did not reveal any changes. The absolute and relative organ weights of both sexes did not reveal any changes. No gross pathological changes noted in any of the animals during conduct of necropsy.

 

In group G4, the animals were noted with test item-related clinical signs of toxicity and mortalities (3 out 12 females). The mean body weight, percent change in mean body weight gain and mean feed consumption were also reduced. All the males and survived females were recovered and found normal after reducing the dose from 800 to 500 mg/kg body weight/day from day 11 onwards.

 

In group G4, the estimated serum T4 levels, absolute and relative organ weights did not reveal any changes. No gross pathological changes noted in any of the animals. No microscopic findings noted in the tissues/organs subjected for histopathological evaluation from high dose group animals of both sexes.

 

There were no effects noted on reproductive performance of both sexes in all the tested dose groups (G2, G3 and G4). No changes in at birth (delivery) and or litter observations noted during postnatal period in all the tested dose groups. No effects on live birth index and pup survival index were noted in all the tested dose groups. No test item-related post-implantation losses were noted in all the tested dose groups.

 

There were no developmental/external behavioural changes noted and no test item-related mortalities noted during postnatal period in any of pups in all the tested dose groups. The mean pup weight, mean anogenital distance and its ratio in either sex of pups per litter were un-affected by the test item in all the tested dose groups. There were no incidences of retention of nipples in male pups examined on PND 13 in all the tested dose groups. The estimated serum T4 levels of PND 13 (from all litters) pups did not reveal any changes in all the tested dose groups. There were no gross pathological changes noted in any of the pups during scheduled sacrifice in all the tested dose groups.