2-[(4-methyl-2-nitrophenyl)azo]-3-oxo-N-phenylbutyramide

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Remarks:

Prenatal Developmental Toxicity Study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 July 2021 to 04 April 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD Test Guideline No. 414, “Prenatal Developmental Toxicity Study”, adopted on 25 June 2018

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted on 25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

1

Reference substance name:

3-hydroxy-N-(o-tolyl)-4-[(2,4,5-trichlorophenyl)azo]naphthalene-2-carboxamide

EC Number:

229-440-3

EC Name:

3-hydroxy-N-(o-tolyl)-4-[(2,4,5-trichlorophenyl)azo]naphthalene-2-carboxamide

Cas Number:

6535-46-2

Molecular formula:

C24H16Cl3N3O2

IUPAC Name:

3-hydroxy-N-(2-methylphenyl)-4-[(2,4,5-trichlorophenyl)diazenyl]-2-naphthamide

Test material form:

solid: nanoform, no surface treatment

Details on test material:

- Density: 1.48 g/cm3 (20 degree celcius)

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hylasco Biotechnology India Pvt. Ltd, Charles River Technology Licensee,
CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) Registration No.: 1808/PO/RcBt/S/15/CPCSEA

- Age at study initiation: 11 weeks
- Weight at study initiation: Males: 240.25 to 298.41 g, Females: 200.65 to 249.44 g
- Housing: Sterilized standard polypropylene cage of Size: L 430 × B 285 × H 150 mm
- Diet (e.g. ad libitum): Altromin maintenance diet for rats and mice manufactured by Altromin Spezialfutter GmbH & Co. KG
- Water (e.g. ad libitum): Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: Minimum of five days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1 to 23.2
- Humidity (%): 49 to 64%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark cycle and 12 hours fluorescent light

IN-LIFE DATES: From:30 July 2021 To:23 September 2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Justification for use and choice of vehicle : The test item was uniformly suspended in 1% w/v Carboxymethyl cellulose (CMC) at the concentration of 100 mg/mL the highest dose concentration selected for the study considering the dose volume of 10 mL/kg body weight as per in-house solubility/suspendibility test results. Hence, 1% w/v Carboxymethyl Cellulose was used as vehicle for test item formulations. 1% w/v Carboxymethyl Cellulose is a routinely used and universally accepted vehicle of choice for the oral toxicity studies.

- Concentration in vehicle: G2 (11.1 mg/mL), G3 (33.3 mg/mL) and G4 (100.0 mg/mL)
- Amount of vehicle: 10 mL/kg
- Lot no.: SLCH1520
- Purity: Analytical grade.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sampling and analysis of formulations were performed during first week and last week of the treatment. Approximately, 5 mL of samples were collected in duplicates from top, middle and bottom layers from low, mid and high dose concentrations and in duplicates from single layer from vehicle control. The prepared test item formulations were stirred using magnetic stirrer during sampling.

The collected samples were transferred to 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 as a backup purpose at established stability conditions. The second set of samples was discarded, as the analysis results of first set of samples were within the limits.

Formulations were considered acceptable, as the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is ≤10% (treatment day 1: mean recovery (%) is 99.15%, 99.17% and 99.06% with % RSD of 0.06, 0.06 and 0.03 for groups G2, G3 and G4, respectively; treatment day 38: mean recovery (%) is 100.10%, 100.16% and 100.09% with % RSD of 0.06, 0.05 and 0.09 for groups G2, G3 and G4, respectively).

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:1:2 ratio
- M/F ratio per cage: 1:2 ratio (one male and two females)
- Length of cohabitation: 14 days, Females not mated within 14 days of pairing with the first male were placed with a second proven male for one week
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as gestation day 0 of pregnancy
- Any other deviations from standard protocol: No

Duration of treatment / exposure:

The test item/vehicle was administered to respective group of female rats once daily from gestation day 5 to 19.

Frequency of treatment:

Once Daily

Duration of test:

15 days per animal starting from implantation to one day prior to scheduled delivery (GD 5 to GD 19)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

111 mg/kg bw/day

Remarks:

Low dose

Doses / concentrations 2

Dose / conc.:

333 mg/kg bw/day

Remarks:

Mid dose

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

High dose

No. of animals per sex per dose:

25 presumed pregnant females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: There was no information or data available from repeated dose/reproducti on toxicity and teratology studies for agrocer red 112, hence The dose levels of 0, 111, 333 and 1000 mg/kg body weight/day were selected as vehicle control, low, mid and high dose groups, respectively as consultation with sponsor.

- Rationale for animal assignment: The body weight of mated females on each day of gestation day ‘0’ (GD 0) was recorded and arranged in the ascending order of their body weight. These mated females were evenly distributed to all the groups based on their body weights so as to maintain comparable mean body weights for all groups, permanent identification numbers were assigned.

Examinations

Maternal examinations:

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

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on gestation days (GD) 0, 3, 5, 8, 11, 14, 17, 19 and on 20 (day of caesarean section).

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined as g food/kg body weight/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: All visceral organs, along with uterus, ovaries and thyroid along with parathyroid.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [ half per litter]
- Skeletal examinations: Yes: [ half per litter]
- Head examinations: Yes: [ half per litter]

Statistics:

Parametric: One-way ANOVA with Dunnett’s post test:
Maternal body weight,Percent change in maternal body weight, Corrected body weight for maternal increase, Gravid uterus weight, Maternal Feed consumption,Absolute / relative thyroid weights, Mean fetal weight per dam, Mean fetal crown rump length per dam.

Non-Parametric: Kruskal-Wallis test:
No. of corpora lutea per dam, No. of implantations per dam, Litter size per dam, No. of live fetuses per dam, Percent of live fetuses per dam, No. of early/late resorptions per dam, Percent of early/late resorptions per dam, Sex ratio (m/f) per dam, Pre/Post implantation losses (%) per dam, Fetal external / visceral / skeletal anomalies per dam.

Frequencies Comparison: Cross Tabs - Chi-square test: No. of Pregnant / Non-pregnant females (Pregnancy status), No. of dams with / without live fetuses, No. of dams with / without dead fetuses, No. of dams with / without resorptions.

Indices:

Corrected Body Weight (g) = (Gestation day 20 body weight - Gestation day 5 body weight) - Gravid uterus weight
Pre-implantation Loss (%) = Total Number of Corpora lutea - Number of Implantation Sites / Total Number of Corpora lutea x 100
Post-implantation Loss (%) = Number of Implantation sites - Number of Viable fetuses / Number of Implantation sites x 100

Historical control data:

In-house historical data available for the same species and strain and referred to end points where required in the study report.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs of toxicity noted in any of the animals from vehicle control and all the tested dose groups throughout the treatment period

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no changes noted in mean gestation (maternal) body weight and percent change in mean gestation (maternal) body weight gain in all the tested dose groups when compared with the vehicle control group. There were no changes noted in mean body weight change corrected for gravid uterus weight in all the tested dose groups when compared with the vehicle control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no changes noted in mean gestation (maternal) feed consumption in all the tested dose groups when compared with the vehicle control group.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes noted for mean serum T3 levels in any of the tested dose groups when compared to vehicle control group. The noted statistically significant increase in mean serum T3 levels (ng/mL) in group G3 is considered as incidental and un-related to treatment with test item as the obtained mean values are within normal range of same species and strain.

There were no changes noted for mean serum T4 levels in any of the tested dose groups when compared to vehicle control group.

There were no changes noted for mean serum TSH levels in any of the tested dose groups when compared to vehicle control group.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The thyroid along with parathyroid was collected, preserved and weighed post fixation from all the dams of each dose group. There were no changes noted for mean absolute and relative weight for this organ in all the tested dose groups when compared to the vehicle control group.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological changes noted in any of the animals from all the tested dose groups and vehicle control group during conduct of the necropsy.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item related histopathological findings noted in thyroid and parathyroid glands of all treated animals.
However, ultimobranchial cysts and ectopic thymus in thyroid gland were noted in this study. These changes were considered as incidental findings as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats.

Histopathological findings: neoplastic:

no effects observed

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

No abortions were noted

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The mean percentage of pre-implantation loss per litter was 5.77, 7.29, 10.86 and 12.44 for groups G1, G2, G3 and G4 respectively.
There were no changes noted and no statistically significant differences noted for percentage of pre-implantation loss per litter in all the teste dose groups.

The mean percentage of post-implantation loss per litter was 9.61, 9.96, 6.94 and 11.62 for groups G1, G2, G3 and G4 respectively.
There were no changes or no statistically significant differences noted for mean percentage of post-implantation loss per litter in all the tested dose groups when compared to the vehicle control group.

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

Two incidental findings with total implantation loss (with total empty implantation sites/early resorptions) during conduct of necropsy was noted in 1 out of 25 females from group G1 and 1 out of 25 females from group G3.

Early or late resorptions:

no effects observed

Description (incidence and severity):

The mean number of early resorptions per litter was 0.71, 0.71, 0.62 and 0.95 with a percentage of early resorptions was 6.53, 9.20, 6.62 and 8.43 for groups G1, G2, G3 and G4 respectively. There were no changes or no statistically significant differences noted for mean number and percentage of early resorptions per dam across dose groups when compared to the vehicle control group.

The mean number of late resorptions per litter was 0.14, 0.00, 0.00 and 0.00 and the percentage of late resorptions was 1.30, 0.00, 0.00 and 0.00 for groups G1, G2, G3 and G4 respectively. There were no changes or no statistically significant differences noted for mean number and percentage of late resorptions per dam across dose groups when compared to the vehicle control group.

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

The mean number of dead fetuses per litter was 0.05, 0.10, 0.05 and 0.00 with a percentage of 0.79, 0.84, 0.34 and 0.00. There were no statistically significant differences noted for mean number of dead fetuses in all the tested dose groups when compared to the vehicle control group.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

No early deliveries noted before caesarean sectioning.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): A total of 21 (out of 25), 21 (out of 25), 21 (out of 25) and 22 (out of 25) females from group G1 (0 mg/kg body weight/day), G2 (111 mg/kg body weight/day),
G3 (333 mg/kg body weight/day) and G4 (1000 mg/kg body weight/day) were found with implantations with live fetuses yielding to pregnancy with rates of 84%, 84%, 84% and 88% respectively.

No female was littered during gestation period in all the dose groups and control group before caesarean sectioning.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

A total of 21 (out of 25), 21 (out of 25), 21 (out of 25) and 22 (out of 25) females from group G1 (0 mg/kg body weight/day), G2 (111 mg/kg body weight/day),
G3 (333 mg/kg body weight/day) and G4 (1000 mg/kg body weight/day) were found with implantations with live fetuses yielding to pregnancy with rates of 84%, 84%, 84% and 88% respectively. One incidental finding with total implantation loss (with empty implantation sites) during conduct of necropsy was noted in 1 out of 25 females from group G1 and 1 out of 25 females from group G3.

Details on maternal toxic effects:

The oral administration of test item Agrocer Red 112 by gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 did not produce any indication of maternal toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

changes in number of pregnant

changes in pregnancy duration

clinical signs

dead fetuses

early or late resorptions

effects on pregnancy duration

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

maternal abnormalities

mortality

necropsy findings

number of abortions

organ weights and organ / body weight ratios

pre and post implantation loss

total litter losses by resorption

Remarks on result:

not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

amniotic fluid

cervix

mammary gland

ovary

oviduct

placenta

umbilical cord

uterus

vagina

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no changes noted in mean fetal weight of either sex across the dose groups when compared with the vehicle control group.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): There were no changes noted in mean fetal weight of either sex across the dose groups when compared with the vehicle control group.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex ratio per litter calculated as the mean of male/female fetuses per litter was 1.49, 1.26, 1.21 and 0.92 for groups G1, G2, G3 and G4 respectively. There were changes or no statistically significant differences noted for mean male/female sex ratio across the dose groups when compared to the control group.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The mean litter sizes, assessed as the total number of fetuses in uteri [live plus dead] per dam, was 10.90, 10.05, 10.33 and 9.41 for groups G1, G2, G3 and G4, respectively.
There were no changes or no statistically significant differences noted in all the tested dose groups when compared with vehicle control group for mean litter size.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related fetal structural and developmental external malformations or variations noted for any of the fetuses examined from all the tested dose group litters and also no statistically significant differences occurred.

However, some of the sporadic fetal external alterations were noted across the dose groups and vehicle control group such as Subcutaneous hemorrhagic spot beneath the skin on different regions of the body, Skin discoloured / pale coloured, Bent tail, Kinked tail, Hyperextension (unilateral) of forelimb, Hyperextension (unilateral) of hind limb and Protrusion of tongue.These developmental and structural alterations are considered incidental as these observations are comparable with the vehicle control group and or also these developmental alterations are common for this species and strain.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test item-related fetal structural malformations or variations noted for any of the fetuses examined from all the tested dose group litters during skeletal examination. The noted statistically significant decrease in number of incidences of unossified sternebra no. 6 in group G3 when compared with vehicle control group is considered as incidental and toxicologically in-signficant. These structural and skeletal alterations are considered incidental as these observations are comparable with the vehicle control group and also these alterations are common for this species and strain. Also, the noted alterations in ossifications, i.e. delayed ossification, incomplete ossification, dumbbell shaped ossification from various regions from all the tested dose groups and control group are considered as transient findings and are reported to resolve shortly with the development of fetus (refer table no. 16 in study report).

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related fetal structural and developmental visceral/soft tissue malformations or variations noted for any of the fetuses examined from all the tested dose group litters and also no statistically significant differences occurred. However, some of the sporadic fetal visceral/soft tissue alterations were noted across the dose groups and vehicle control group such as Pale colored liver, Renal pelvis dilation (bilateral) of kidneys, Dilatation of Ureters (bilateral), Dilated lateral ventricles of brain and Nasal conchae enlarged.These developmental soft tissue alterations are considered incidental as these observations are comparable with the vehicle control group and also these developmental alterations are common for this species and strain.

Other effects:

no effects observed

Description (incidence and severity):

There were no changes noted in mean fetal anogenital distance/mean fetal anogenital distance ratio and mean fetal crown rump length per litter of either across the dose groups when compared with the vehicle control group.

Details on embryotoxic / teratogenic effects:

The oral administration of test item Agrocer Red 112 by gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 did not produce any indication of fetal/developmental toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

external malformations

skeletal malformations

visceral malformations

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

external: ear

external: eye

external: face

external: limb

external: paw

external: tail

external: trunk

external: anogenital distance

external: anus

external: genital tubercle

external: large intestine

external: thorax

external: umbilicus

external: pelvic region

skeletal: skull

skeletal: skull, fontanelles

skeletal: skull sutures

skeletal: clavicle

skeletal: scapule

skeletal: forelimb

skeletal: sternum

skeletal: rib

skeletal: supernumerary rib

skeletal: vertebra

skeletal: pelvic girdle

skeletal: hindlimb

visceral/soft tissue: integumentary

visceral/soft tissue: gastrointestinal tract

visceral/soft tissue: hepatobiliary

visceral/soft tissue: urinary

visceral/soft tissue: cardiovascular

visceral/soft tissue: heamatopoietic

visceral/soft tissue: immune system

visceral/soft tissue: musculoskeletal system

visceral/soft tissue: nervous system

visceral/soft tissue: central nervous system

visceral/soft tissue: peripheral nervous system

visceral/soft tissue: somatic nervous system

visceral/soft tissue: autonomic nervous system

visceral/soft tissue: endocrine system

visceral/soft tissue: respiratory system

visceral/soft tissue: male reproductive system

visceral/soft tissue: female reproductive system

visceral/soft tissue: eye

visceral/soft tissue: ear

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

In conclusion, the oral administration of test item Agrocer Red 112 by gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 did not produce any indication of maternal and developmental toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.
Based on the obtained results from this pre-natal developmental toxicity study conducted in Sprague Dawley Rats, the NOAEL (No observed adverse effect level) of Agrocer Red 112 for both maternal and fetal toxicity is estimated as 1000 mg/kg body weight/day under experimental conditions employed.

Executive summary:

The objective of this prenatal developmental toxicity study inSprague Dawley rats conducted as per OECD test guidelines 414,wasto provide general information concerning the effects of prenatal exposure of test itemAgrocer Red 112on the pregnant females and in the developing organisms. This study was also conducted to assess the maternal toxicity in pregnant females and also structural abnormalities or altered growth in the fetuses. The aim ofthis study was to estimate no-observed-adverse-effect-level (NOAEL) of the test itemfor both maternal as well as fetal end points.

A total of 100 mated female Sprague Dawley rats were distributed to four groups. Each group (G1, G2, G3 and G4) consisted of 25 presumed pregnant females. The animals in group G1 were administered with vehicle [1% w/v Carboxymethyl cellulose], the animals in groups G2, G3 and G4 were administered with test item at the dose levels of111, 333 and 1000mg/kg body weight/dayfor low, mid and high dose groups respectively. The vehicle and test item formulations were administered orally by gavage at the dose volume of 10 mL/kg body weight to mated and presumed-pregnant females from Gestation Day [GD] 5 to 19. The end points of assessment for dams were maternal death, maternal body weight and clinical signs of maternal toxicity and the end points of assessment for fetuses were fetal weights, growth and development, structural variations and malformations or altered growth.

Homogeneity and dose formulation analysis for dose concentration verification was performed during the first and last week of the treatment. The analysis results of test samples were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is ≤10%.

A total of 21 (out of 25), 21 (out of 25), 21 (out of 25) and 22 (out of 25) females from group G1, G2, G3 and G4 were found with implantations and live fetuses yielding to pregnancy with rates of 84%, 84%, 84% and 88% respectively. In groups G1 and G3, 1 out of 25 females each was noted with total implantation loss with no evidence of live fetuses.

All the dams from each group were observed for clinical signs of toxicity once daily, for mortalities twice daily during the experimental period. The body weight was recorded on Gestation Day (GD) 0, 3, 5, 8, 11, 14, 17, 19 and on 20 (day of caesarean section). The feed consumption was measured from GD 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17, 17 to 19 and 19 to 20. All the dams were euthanized on GD 20 by exposing to CO₂asphyxiation and subjected to detailed gross pathological examination. All the females were observed for status of pregnancy and the gravid uterus weight for all the dams was recorded on the day of caesarean section. Each dam was observed for number of live fetuses, litter size, sex ratio, number of implantation sites and number of resorptions. The ovaries of all the dams were observed for number of corpora lutea. The pre-and post-implantation losses per dam were calculated based on number of corpora lutea and number of implantation sites. The weight of thyroid along with the parathyroid was recorded post-fixation for all the group animals. The assessment of thyroid hormones such as, thyroxine (T4), triiodothyronine (T3) and thyroid stimulating hormone (TSH) was conducted for all the group dams. All the dams from each group were evaluated for histopathology of thyroid along with the parathyroid.

All the fetuses collected from each litter were weighed and measured for its crown rump length and anogenital distance. The mean fetal weight, mean crown rump length, and mean anogenital distance measurement/ratio per litter was calculated. All the fetuses were subjected to external examination on the day of caesarean section. All the even numbered fetuses from each litter were subjected to fresh visceral (soft tissue) examination and fixed head sections examination. All the odd numbered fetuses from each litter were stained with Alizarin Red S stain and subjected to skeletal examination.

For maternal toxicity assessment, there were no clinical signs of toxicity and no mortality/morbidity noted in any of the dams in all the tested dose groups. There were no differences noted between the groups in mean maternal body weight, percent change in maternal body weight gain, body weight corrected for maternal increase and maternal feed consumption. There were no effects noted in mean number of live fetuses per litter, litter size, sex ratio, number of corpora lutea, number of implantation sites, number of incidences of resorptions, pre-and post-implantation losses per dam from all the tested dose groups. There were no differences noted between the dose groups in mean gravid uterus weight. The mean absolute and relative thyroid along with the parathyroid weight did not reveal any changes when weighed post-fixation and no test item-related changes were noted in mean thyroid hormonal levels (T3, T4 and TSH). There were no gross pathological changes noted in any of the dams during caesarean section and no test item-related microscopic changes were noted in thyroid along with the parathyroid of all dams during histopathological examination.

For fetal (pre-natal developmental) toxicity assessment, there were no test item-related changes noted in mean fetal weight, mean fetal crown rump length and mean fetal anogenital distance measurement / ratio per litter in either sex in all the tested dose groups. There was no test item-related fetal developmental and or structural alterations noted from all the tested dose group litters when subjected to fetal external, visceral and skeletal examinations. The observed fetal external/visceral/skeletal developmental variations and or malformations are considered as incidental and unrelated to treatment as these findings occurred infrequently or at a frequency similar to the vehicle control group and did not occur in a dose-dependent manner. Also, the observed mean litter/fetal proportions were within the in-house historical control range of this species and strain.