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

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Remarks:

Prenatal Developmental Toxicity Study, Adopted on 25 June 2018

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 July 2021 to 25 March 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Constituent 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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Clariant Plastics & Coatings Deutschland
- Expiration date of the batch: 18-November-2027
- Purity test date: 95.5% (w/w)


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Stability under test conditions: for 6 hours and 48 hours at room temperature
- Solubility and stability of the test substance in the solvent/vehicle: uniformly suspended in 1% w/v Carboxymethyl cellulose (CMC) at the concentration of 100 mg/mL, the highest dose concentration.


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: formulation using 1% w/v Carboxymethyl cellulose (CMC) at the concentration 11.1, 33.3 and 100.0 of G2, G3 and G4 respectively
- Final preparation of a solid: formulation in vehicle

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Adita Biosys Private Limited, Plot No. SPL 26, 2nd Stage, KSSIDC, Industrial Estate, Antharasanahalli, Tumakuru, Karnataka 572106. CPCSEA Registration No. 1868/PO/RcBt/S/16/CPCSEA
- Age at study initiation: 4 to 5 Months
- Weight at study initiation: Males: 2.15468 kg to 2.64927 kg;
Females: 2.00849 kg to 2.28642 kg
- Housing: stainless steel wire mesh cage (Size: L 24 x B 18 x H 18 inches)
- Diet (e.g. ad libitum): Altromin maintenance diet for rabbits (manufactured by Altromin Spezialfutter GmbH & Co. KG)
- Water (e.g. ad libitum): Deep bore-well water passed through reverse osmosis unit
- Acclimation period: (Minimum of five days) Start: 05 August 2021; End: 10 August 2021

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.8 to 20.9oC
- Humidity (%): 44 to 61%
- Air changes (per hr): 12 hours fluorescent light and 12 hours dark cycle


IN-LIFE DATES: From: 05 August 2021 To: 05 October 2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1% w/v in water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Justification for use and choice of vehicle: 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 suspendibility test results.
Hence, 1% w/v Carboxymethyl cellulose was used as vehicle for test item formulations. Carboxymethyl cellulose is a routinely used 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 (if gavage): 10 mL/kg
body weight
- Lot no. (if required): SLCH1520
- Purity: Analytical grade

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sampling and analysis of formulations was performed during first week and last week of the treatment. Approximately, 5 mL of samples was 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 were 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%.

Details on mating procedure:

- M/F ratio per cage: 1:1 (M:F)
- Length of cohabitation: Until visual confirmation of mating
- Verification of same strain and source of both sexes: Yes
- Proof of pregnancy: [visual observation] referred to as [day 0] of pregnancy
- Any other deviations from standard protocol: no

Duration of treatment / exposure:

The test item/vehicle was administered by oral (gavage) route to respective group of animals once daily from gestation day (GD) 6 to 28.

Frequency of treatment:

once daily

Duration of test:

22 days per animal starting from implantation to one day prior to scheduled delivery (GD 6 to GD 28)

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/reproduction 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 (if not random):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 GD 0, 3, 6, 9, 12, 15, 18, 21, 24, 26, 28 and on 29 (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 29
- Organs examined: all visceral organs along with uterus and ovaries

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: [all per litter]
- Skeletal examinations: Yes: [all 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 doe, Mean fetal crown rump length per doe

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

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

Indices:

Corrected Body Weight (g) = (Gestation day 29 body weight - Gestation day 6 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:

All the obtained Data within the historical control range

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test item related clinical signs of toxicity noted at all the tested dose groups and vehicle control group animals during the experimental period.

Mortality:

no mortality observed

Description (incidence):

There were no morbidity/mortality noted at all the tested dose groups and vehicle control group animals during the experimental period.

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.

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 does 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 test item-related histopathological findings in thyroid and parathyroid glands of all treated animals.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related histopathological findings in thyroid and parathyroid glands of all treated animals.
However, ultimobranchial cysts in thyroid gland was 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 rabbits (Elizabeth McInnes, 2012).

Details on results:

The oral administration of test item Agrocer Red 112 by gavage to presumed pregnant female New Zealand White Rabbits from Gestation Day 6 to 28 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.

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

No abortions were noted

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

The mean percentage of pre-implantation loss per litter was 13.78, 13.10, 13.58 and 15.76 for groups G1, G2, G3 and G4 respectively.
There were no statistically significant differences noted for percentage of pre-implantation loss per litter at all the tested dose groups when compared to the vehicle control group.
The mean percentage of post-implantation loss per litter was 0.00, 0.00, 0.00 and 4.05 for groups G1, G2, G3 and G4 respectively.
There were no statistically significant differences noted for 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):

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 G2.

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

The mean number of early resorptions per litter was 0.00, 0.00, 0.00 and 0.10 and the percentage of early resorptions was 0.00, 0.00, 0.00 and 3.57 for groups G1, G2, G3 and G4 respectively. There were no statistically significant differences in the number and percentage of early resorptions per doe across dose groups when compared to the vehicle control group.
The mean number of late resorptions per litter was 0.00, 0.00, 0.00 and 0.05 and the percentage of late resorptions was 0.00, 0.00, 0.00 and 0.48 for groups G1, G2, G3 and G4 respectively. There were no statistically significant differences in the number and percentage of late resorptions per doe across dose groups when compared to the vehicle control group.

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no dead fetuses noted in any of the litters from all the tested dose and vehicle control groups during caesarean section.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

No early deliveries noted.
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, 20, 21 and 21 females from groups 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 yielding to pregnancy with rates of 84%, 80%, 84% and 84% respectively.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

A total of 21(out of 25), 20(out of 25), 21(out of 25) and 21(out of 25) females from groups 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 yielding to pregnancy with rates of 84%, 80%, 84% and 84% 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 G2.

Details on maternal toxic effects:

The oral administration of Agrocer Red 112 by gavage to presumed pregnant female New Zealand White Rabbits from Gestation Day 6 to 28 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

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): No such incidences noted in any of the tested dose groups.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No such incidences noted in any of the tested dose groups.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No such incidences noted in any of the tested dose groups.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

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 external malformations or developmental variations for any of the fetuses examined from all the tested dose group litters.
General / developmental variations such as subcutaneous hemorrhagic spot/s on different regions of the body and noted external malformations such as hyperextension of forelimb and hyperextension of hindlimb across the tested dose group litters are considered incidental as these incidences are comparable with the vehicle control group and also these developmental alterations are common for this species and strain. Also, no remarkable differences or statistically significant changes noted for these alterations in any of the tested dose groups when compared with vehicle control group (refer table no. 10 in study report).

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test item-related fetal visceral/soft tissue malformations or developmental variations for any of the fetuses examined from all the tested dose group litters.
These noted skeletal developmental variations and skeletal malformations are considered as incidental and un-related 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 occurred mean litter/fetal proportions were within the in-house historical control range of this species and strain (refer table no. 12 in study report).

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test item-related fetal visceral/soft tissue malformations or developmental variations for any of the fetuses examined from all the tested dose group litters.
Developmental variations such as discoloured liver/thymus/lungs/spleen, dilatation of renal pelvis and dilatation of ureters across the tested dose group litters are considered incidental as these incidences are comparable with the vehicle control group and also these developmental variations are common for this species and strain.
developmental malformations lateral/third ventricular dilatation of brain, retinal fold, misshapen liver lobes and retrocaval ureters across the tested dose group litters are considered incidental as these incidences are comparable with the vehicle control group and also these developmental alterations are common for this species and strain(refer table no. 11 in study report).

Other effects:

no effects observed

Description (incidence and severity):

There were no changes noted in 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:

oral administration of test item Agrocer Red 112 by gavage to presumed pregnant female New Zealand White Rabbits from Gestation Day 6 to 28 did not produce any indication of 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

changes in postnatal survival

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: cranium

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 New Zealand White Rabbits from Gestation Day 6 to 28 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 New Zealand White Rabbits, 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 study was to provide general information concerning the effects of prenatal exposure of test itemAgrocer Red 112on the pregnant rabbits and in the developing organisms; this included the assessment of maternal toxicity as well as death, structural abnormalities, or altered growth in the fetuses.

A total of 100 mated female New Zealand White rabbits distributed to four groups. Each group (G1, G2, G3 and G4) consisted of 25 presumed pregnant rabbits. 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 of 111, 333 and 1000 mg/kg body weight for 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] 6 to 28. The end points of assessment for does 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 were performed during the first and last week of treatment. The prepared formulations were considered acceptable and the mean results were within the range of 85 to 115% of nominal concentration and the relative standard deviation (% RSD) is less than 10%(mean recovery was between 98.62% and 99.97% at all measurements).

 A total of 21, 20, 21 and 21 females from group G1, G2, G3 and G4 were found with implantations yielding pregnancy rates of 84%, 80%, 84% and 84% respectively.

All the animals 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, 6, 9, 12, 15, 18, 21, 24, 26, 28 and on 29 (day of caesarean section). The feed consumption was measured from GD 0 to 3, 3 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18, 18 to 21, 21 to 24, 24 to 26, 26 to 28 and 28 to 29. All the animals were euthanized on gestation Day 29 by intravenous injection of sodium thiopentone and subjected to detailed gross pathological examination. All the females were observed for status of pregnancy and the gravid uterus weight for all the does was recorded on the day of caesarean section. Each doe was observed for number of live or dead fetuses, litter size, sex ratio, number of implantation sites and number of resorptions. The ovaries of all the does were observed for number of corpora lutea. The pre-and post-implantation losses per doe 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 animals. All the does were evaluated for histopathology of thyroid along with the parathyroid.

All the fetuses collected from each litter were weighed and measured for their crown rump length. The mean fetal weight and mean crown rump length per litter was calculated. All the fetuses were subjected to external examination on the day of caesarean section. All the fetuses from each litter were subjected for fresh soft tissue (visceral) examination and fixed head sections examination, except the heads of fetuses which were allotted for skeletal evaluation. The bodies of fetuses without heads and all other intact fetuses from each litter were double stained with alcian blue for cartilage and Alizarin Red S for bones and subjected to skeletal examination.

For maternal toxicity assessment, there were no clinical signs of toxicity and no mortality/morbidity noted during the experimental period at all the tested dose and vehicle control group animals. No changes were noted in mean maternal body weight, percent change in mean maternal body weight gain and mean maternal feed consumption at all the tested dose groups. There were no gross pathological changes noted during caesarean section of all the tested dose and vehicle control group animals. There were no effects noted in pregnancy rate and mean gravid uterus weight in all the tested dose groups. There were no effects noted in mean number of live fetuses, mean litter size, mean sex ratio, mean number of implantation sites, mean number of resorptions, mean number of corpora lutea at all the tested dose groups. The mean pre- and post-implantation losses per doe were unaffected at all the tested dose groups. The mean absolute and relative thyroid along with the parathyroid weight did not reveal any changes when weighed post-fixation at all the tested dose groups. No test item-related microscopic changes were noted in thyroid along with the parathyroid of all the does during histopathological examination from all the tested dose groups.

For fetal (pre-natal developmental) toxicity assessment, there were no test item-related changes noted in mean fetal weight and mean fetal crown rump length per litter in either sex at 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-dependant manner.