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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

No test item related effects were detected in studies according to OECD TG 414 in rats and rabbits after oral exposure to doses of 111 to 1000 mg/kg bw /day.

Link to relevant study records

Referenceopen allclose all

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
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
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


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
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)
Dose / conc.:
111 mg/kg bw/day
Remarks:
Low dose
Dose / conc.:
333 mg/kg bw/day
Remarks:
Mid dose
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
High dose
No. of animals per sex per dose:
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.
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
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.

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.
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
Key result
Abnormalities:
effects observed, non-treatment-related
Localisation:
amniotic fluid
cervix
mammary gland
ovary
oviduct
placenta
umbilical cord
uterus
vagina
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.
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
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
Key result
Developmental effects observed:
no
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.

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
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
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
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)
Dose / conc.:
111 mg/kg bw/day
Remarks:
Low dose
Dose / conc.:
333 mg/kg bw/day
Remarks:
Mid dose
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
High dose
No. of animals per sex per dose:
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.
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.
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
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.
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
Key result
Abnormalities:
effects observed, non-treatment-related
Localisation:
amniotic fluid
cervix
mammary gland
ovary
oviduct
placenta
umbilical cord
uterus
vagina
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.
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
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
Key result
Developmental effects observed:
no
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.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Additional information