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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 July 2020 to 04 November 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to other study
Remarks:
DRF study
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
13 March 2020 to 08 May 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Reason / purpose for cross-reference:
reference to other study
Remarks:
main study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
DRF study, only 10 pregnant females.
GLP compliance:
no
Remarks:
Following the OECD Principles of Good Laboratory Practices [C (97) 186/Final], however GLP status will not be claimed for the study.
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: In-house bred animals
- Age at study initiation: 9 to 10 weeks
- Weight at study initiation: Males: 259.90 to 289.32 g, Females: 202.35 to 263.47 g
- Fasting period before study: no
- Housing: Acclimatization - Maximum of three animals of same sex per cage were housed in sterilized standard polypropylene cage (Size: L 430 × B 285 × H 150 mm).
- Housing: Cohabitation Period - During cohabitation, three animals (one male and two females) were housed in standard polypropylene cage (Size: L 430 × B 285 × H 150 mm).
- Housing: Post-mating - After confirming presence of sperm in the vaginal smear and / or vaginal plugs (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually in polypropylene cage (Size: L 430 × B 285 × H 150 mm).
- Diet (e.g. ad libitum): not specified
- Water (e.g. ad libitum): not specified
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.0 to 23.2°C
- Humidity (%): 46 to 65%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle

IN-LIFE DATES: From: 13 March 2020 To: 08 May 2020
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item formulations were freshly prepared before dose administration on each treatment day. The required quantity of test item was weighed and triturated well in a mortar with a small quantity of vehicle until a homogenous suspension was formed and thereafter the entire quantity of the formulation was transferred into measuring cylinder. A small quantity of vehicle was added to rinse the mortar and this was transferred into the measuring cylinder. The rinsing procedure of mortar and pestle was repeated (many times) to ensure the transfer of the contents to the measuring cylinder. Finally, the volume was made up to required quantity with vehicle to get desired concentration of 10, 30 and 100 mg/mL of test item for low, mid and high dose groups respectively. The test formulations were maintained under stirring conditions using magnetic stirrer to maintain homogeneity of the test item formulations.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item was in-soluble in distilled water and uniformly suspended in 0.5% w/v Carboxy Methyl Cellulose 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, 0.5% w/v Carboxy Methyl Cellulose was used as vehicle for test item formulations and the details were recorded in the raw data and presented in the study report.
- Concentration in vehicle: 10 - 100mg/mL
- Amount of vehicle (if gavage): 10mL/kg bw
- Lot/batch no. (if required): BCBN1690V
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
The stability and homogeneity of the test item in dose formulations was not established under this dose range finding study. However, freshly prepared test item formulations were administered to the animals.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:2
- Length of cohabitation: 2 weeks or until confirmation of mating
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: not detailed
- Verification of same strain and source of both sexes: yes, in-house bred line
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
Gestation Day 5 to Day 19
Frequency of treatment:
once daily
Duration of test:
20 days
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
10 females in each group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: LD50 of similar test item Silicic acid, aluminum sodium salt, sulfurized, EC number 309-928-3 is >2000 mg/kg obtained from acute oral toxicity study in rats conducted as per OECD423 TG, The NOAEL of test item is 50 mg/kg when tested at 50, 500 and 5000 mg/kg obtained from a 90-day repeated oral toxicity study in rats conducted similar to ECD408 TG, The NOAEL for maternal toxicity is >=300 mg/kg, NOAEL for reproductive toxicity>=1000 mg/kg and NOAEL for offspring development >=1000 mg/kg when tested at 100, 300 and 1000 mg/kg body weight obtained from a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening study in rats conducted as per OECD422 TG, The NOAEL for maternal toxicity is >100 mg/kg and the NOAEL for fetal toxicity (teratogenicity) is >10000 mg/kg when tested at 10, 100, 1000, 10000 mg/kg obtained from a Prenatal Developmental Toxicity Study in rats conducted in lines with OECD414 TG. Based on the above available information, the doses of 100, 300 and 1000 mg/kg body weight for vehicle control, low dose, mid dose and high dose groups, respectively were selected.
- Rationale for animal assignment (if not random): The body weight of mated females on its GD 0 was recorded and arranged in the ascending order of their body weight until required number of mated females acquired for each group. These mated females were distributed to all the groups based on their body weights so as to maintain comparable mean body weight for all groups
- Fasting period before blood sampling for (rat) dam thyroid hormones: n/a
- Time of day for (rat) dam blood sampling: n/a
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily for clinical signs of toxicity and twice daily
for mortality and morbidity


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily for clinical signs of toxicity and twice daily
for mortality and morbidity

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 3, 5, 8, 11, 14, 17, 19 and on 20

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): no feeding study


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): no drinking study


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: Ovaries, Uterus with cervix, Thyroid with Parathyroid glands

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
Blood sampling:
- Plasma: No
- Serum: No
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: No
- Head examinations: No
- Anogenital distance of all live rodent pups: Yes
Statistics:
Parametric: One-way ANOVA with Dunnett’s post test: Gestation body weight (g), Percent change in gestation body weight (%), Corrected body weight (g), Percent change in corrected body weight (%), Gravid uterus weight (g), Feed consumption (g), Mean Fetal weight (g) per dam, Mean Fetal crown rump length (mm) per dam, Mean Fetal ano-genital distance (mm) and ratio per dam, Organ weight. Non-Parametric: Kruskal-Wallis: No. of corpora lutea per dam, No. of implantations per dam, Litter size per dam, No. of live/dead fetuses per dam, No. of early/late resorptions per dam, Percent of live/dead fetuses per dam, Percent of early/late resorptions per dam, Sex ratio (m/f) per dam, Pre/Post-implantation losses (%) per dam, Fetal external anomalies per dam. Frequencies Comparison: Cross Tabs - Chi-square test: Pregnancy rate, No. of dams with/without live fetuses, No. of dams with/without dead fetuses, No. of litters with/without resorptions
Indices:
- Corrected Body weight (g) = (Gestation day 20 body weight - Gestation day 5 body weight) -
Gravid uterus weigh
- Percent of Live Fetuses per dam = (Number of Live Fetuses / Litter Size) x 100
- Percent of Dead Fetuses per dam = (Number of Dead Fetuses/ Litter Size) x 100
- Percent of Early Resorptions (%) per dam = (Number of Early Resorptions/Number of Implantation sites) x 100
- Percent of Late Resorptions (%) per dam = (Number of Late Resorptions / Number of Implantation sites)x 100
- Pre-implantation Loss (%) per dam = [(Number of Corpora lutea - Number of Implantation sites)/ Number of Corpora lutea]x 100
- Post-implantation Loss (%) per Dam = [(Number of Implantation sites - Number of Viable fetuses )/ Number of Implantation sites]x 100
- Sex Ratio (m/f) = Number of live male fetuses / Number of live female fetuses
- Male/Female Fetuses (%) = (Number of live male/female fetuses/ Total number of live fetuses)x 100
- Ano-genital Distance Ratio = Cube root of Fetal weight (g) /Ano-genital distance measurement (mm)
- Fetal incidence (%) = (Number of Fetuses with particular observation per group/Total number of Fetuses examined per group)x 100
- Litter incidence (%) = (Number of Litters/dams with particular observation per group/Total number of Litters per group) x 100










Historical control data:
not available
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
A statistically significant reduction in the percent change in mean maternal body weight gain was noted during Gestation Day (GD) 8 to 11 at all the tested dose groups when compared with the vehicle control group. However, this noted change is considered as incidental and unrelated to treatment as there were no changes noted in feed consumption during this period and also no clinical signs were noted at any time point of treatment.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
A statistically significant reduction in mean maternal feed consumption was noted during GD 5 to 8 at all the tested dose groups when compared with the vehicle control group. However, this noted change is considered as incidental and unrelated to treatment as there were no effects noted in mean body weight and percent change in mean body weight gain during this period and also the change was inconsistent during treatment period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The absolute and relative thyroid along with the parathyroid weight did not reveal any changes when weighed post-fixation at all the tested dose groups.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no effects noted in mean number of implantation sites at all the tested dose groups. The pre-and post-implantation losses per dam were unaffected at all the tested dose groups.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no effects noted in mean number of resorptions.
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no effects noted in mean live or dead fetuses at all the tested dose groups.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
There were no effects noted in pregnancy rate at all the tested dose groups.
Other effects:
no effects observed
Description (incidence and severity):
There were no effects noted in mean gravid uterus weight at all the tested dose groups. There were no effects noted in mean number of corpora lutea at all the tested dose groups.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No adverse effects observed up to the highest dose tested
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no changes noted in mean fetal weight at all the tested dose groups.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no effects noted in mean sex ratio at all the tested dose groups.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no effects noted in mean litter size at all the tested dose groups.
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
There were no changes noted in mean ano-genital distance measurement / ratio per dam at all the tested dose groups.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
A total of 103 (9), 104 (10), 90 (8) and 100 (9) fetuses (litters) were available for gross external examination from G1, G2, G3 and G4 respectively. There were no test itemrelated external bnormalities noted in fetuses of all the tested dose groups and the vehicle control group. However, subcutaneous haemorrhagic spots on skin were noted as 1 (1), 1(1) and 3(3) [fetal incidences (litter incidences)] from vehicle control group G1 and tested dose groups G2 and G3 respectively. This noted occurrence is considered as incidental and unrelated to treatment.
Skeletal malformations:
not examined
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no test item-related changes noted in any of the group fetuses subjected to visceral (soft tissue) examination on the day of caesarean section.
Other effects:
no effects observed
Description (incidence and severity):
There were no changes noted in mean crown rump length at all the tested dose groups.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed up to the highest dose tested
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

TABLE 1 SUMMARY OF PREGNANCY STATUS, CLINICAL SIGNS OF TOXICITY AND MORTALITY RECORD                                                             



















































Group, Sex


& Dose


(mg/kg body weight/day)



Pregnancy Status



 



Clinical Signs and Mortality Record



No. of Presumed


Pregnant Animals / Group



No. of Females


confirmed with


Pregnancy



Rate of Pregnancy (%)



Clinical Signs of


Toxicity revealed


(No. of Animals)



No. of Mortalities /


Total No. of animals



G1, F & 0



10



9



90.0



N (10)



0/10



G2, F & 100



10



10



100.0



N (10)



0/10



G3, F & 300



10



8



80.0



N (10)



0/10



G4, F & 1000



10



9



90.0



N (10)



0/10



N: Normal


TABLE 2 SUMMARY OF GESTATION BODY WEIGHT (g) RECORD










































































































































































Group, Sex & Dose


(mg/kg body weight/day)


 

Body Weight (g) on Gestation Day (GD)



0



3



5



8



11



14



17



19



20



G1, F & 0



Mean



247.08



255.53



263.45



274.18



293.15



311.72



335.72



358.76



371.63



±SD



24.92



25.58



25.13



26.33



32.82



33.92



32.85



39.43



40.92



n



9



9



9



9



9



9



9



9



9



G2, F & 100



Mean



243.54



250.26



259.01



266.90



279.13



293.08



315.48



339.71



349.73



±SD



16.73



16.60



18.19



19.08



21.37



20.17



23.26



29.33



31.50



n



10



10



10



10



10



10



10



10



10



G3, F & 300



Mean



256.13



262.40



270.75



278.41



288.26



299.95



323.46



347.08



359.33



±SD



17.14



17.20



18.86



18.56



20.59



20.02



22.82



22.87



25.41



n



8



8



8



8



8



8



8



8



8



G4, F & 1000



Mean



248.64



256.81



264.03



271.91



282.68



299.23



324.77



346.72



360.42



±SD



19.65



18.66



18.02



18.86



20.44



27.61



30.08



35.63



34.04



n



9



9



9



9



9



9



9



9



9



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


TABLE 3 SUMMARY OF PERCENT CHANGE IN GESTATION BODY WEIGHT (%) GAIN RECORD





























































































































































Group, Sex & Dose


(mg/kg body weight/day)


 

Percent Change in Body Weight (%) during Gestation Day (GD)



0 to 3



3 to 5



5 to 8



8 to 11



11 to 14



14 to 17



17 to 19



19 to 20



G1, F & 0



Mean



3.43



3.15



4.08



6.80



6.38



7.86



6.76



3.59



±SD



0.83



1.28



1.86



2.73



2.48



3.96



1.88



1.58



n



9



9



9



9



9



9



9



9



G2, F & 100



Mean



2.78



3.48



3.04



4.56*



5.06



7.64



7.61



2.94



±SD



1.09



1.16



1.40



1.78



1.47



2.92



2.37



1.94



n



10



10



10



10



10



10



10



10



G3, F & 300



Mean



2.46



3.16



2.85



3.52*



4.09



7.83



7.34



3.51



±SD



0.80



0.86



1.06



1.21



1.27



1.71



1.49



0.81



n



8



8



8



8



8



8



8



8



G4, F & 1000



Mean



3.33



2.84



2.98



3.95*



5.75



8.55



6.69



4.03



±SD



1.06



0.83



0.90



1.58



2.94



1.71



2.07



1.56



n



9



9



9



9



9



9



9



9



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


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


 


TABLE 4 SUMMARY OF GRAVID UTERUS WEIGHT (g) AND MATERNAL BODY WEIGHT CHANGE CORRECTED FOR GRAVID UTERINE WEIGHT (g)






































































































Group, Sex & Dose


(mg/kg body weight/day)


 

Body Weight Change (g)


GD 5 to 20



Gravid Uterus Weight (g)



Corrected Body weight (Gram)



Corrected Body weight (Percentage)



G1, F & 0



Mean



108.18



74.97



33.21



12.45



±SD



23.74



15.41



20.50



7.76



n



9



9



9



9



G2, F & 100



Mean



90.71



61.14



29.57



11.49



±SD



21.65



15.04



10.60



4.36



n



10



10



10



10



G3, F & 300



Mean



88.59



63.58



25.00



9.19



±SD



12.54



6.60



10.74



3.56



n



8



8



8



8



G4, F & 1000



Mean



96.39



65.18



31.21



11.65



±SD



25.48



16.10



15.35



5.31



n



9



9



9



9



F: Female; SD: Standard Deviation; n: No. of Animals


 


 


TABLE 5 SUMMARY OF AVERAGE GESTATION FEED CONSUMPTION (g/animal/day) RECORD





























































































































































Group, Sex & Dose


(mg/kg body weight/day)



 



Average Feed Consumption (g/animal/day) during Gestation Day



0 to 3



3 to 5



5 to 8



8 to 11



11 to 14



14 to 17



17 to 19



19 to 20



G1, F & 0



Mean



16.58



21.27



19.78



21.17



22.46



25.39



22.06



24.38



±SD



1.82



1.72



1.46



2.86



1.52



1.90



3.33



4.92



n



9



9



9



9



9



9



9



9



G2, F & 100



Mean



17.24



19.20



17.99*



19.72



22.50



24.32



22.56



23.91



±SD



1.83



2.69



1.15



1.32



1.80



3.39



3.62



5.17



n



10



10



10



10



10



10



10



10



G3, F & 300



Mean



17.45



19.58



17.80*



18.99



22.29



23.59



21.46



25.89



±SD



1.08



1.58



2.18



2.82



2.16



1.83



2.34



6.53



n



8



8



8



8



8



8



8



8



G4, F & 1000



Mean



19.28



19.34



17.32*



19.14



22.25



24.61



20.84



26.48



±SD



4.09



0.74



1.00



1.68



1.30



1.78



2.10



5.34



n



9



9



9



9



9



9



9



9



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


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


TABLE 6 SUMMARY OF UTERI OBSERVATIONS PER LITTER DURING CAESAREAN SECTION RECORD



















































































































Group, Sex & Dose


(mg/kg body weight/day)


 

No. of Corpora lutea



No. of Implantations



Litter size



No. of Live Fetuses



No. of Male Live Fetuses



G1, F & 0



Mean



12.89



12.44



11.56



11.44



6.33



±SD



1.36



1.59



2.19



2.35



3.00



n



9



9



9



9



9



G2, F & 100



Mean



11.70



11.40



10.40



10.40



5.20



±SD



2.50



2.46



3.13



3.13



2.62



n



10



10



10



10



10



G3, F & 300



Mean



12.00



11.63



11.25



11.25



5.13



±SD



1.77



1.51



1.67



1.67



0.64



n



8



8



8



8



8



G4, F & 1000



Mean



12.22



11.89



11.11



11.11



4.56



±SD



1.99



2.26



2.76



2.76



1.51



n



9



9



9



9



9



F: Female; SD: Standard Deviation; n: No. of Animals


 


TABLE 6 (Contd…). SUMMARY OF UTERI OBSERVATIONS PER LITTER DURING CAESAREAN SECTION RECORD






































































































Group, Sex & Dose


(mg/kg body weight/day)


 

No. of Female Live Fetuses



No. of Dead Fetuses



No. of Early Resorptions



No. of Late Resorptions



G1, F & 0



Mean



5.11



0.11



0.56



0.33



±SD



1.17



0.33



1.01



0.71



n



9



9



9



9



G2, F & 100



Mean



5.20



0.00



0.90



0.10



±SD



1.87



0.00



1.29



0.32



n



10



10



10



10



G3, F & 300



Mean



6.13



0.00



0.38



0.00



±SD



1.73



0.00



0.74



0.00



n



8



8



8



8



G4, F & 1000



Mean



6.56



0.00



0.78



0.00



±SD



2.35



0.00



1.39



0.00



n



9



9



9



9



F: Female; SD: Standard Deviation; n: No. of Animals


 


 


TABLE 7 SUMMARY OF MATERNAL DATA PER LITTER RECORD



















































































































Group, Sex & Dose


(mg/kg body weight/day)


 

Pre-Implantation Loss (%)



Post-Implantation Loss (%)



Percent of Dead Fetus



Percent of Early Resorptions



Percent of Late Resorptions



G1, F & 0



Mean



3.52



8.32



1.23



4.75



2.65



±SD



5.53



12.91



3.70



9.09



5.76



n



9



9



9



9



9



G2, F & 100



Mean



2.37



10.01



0.00



9.24



0.77



±SD



5.27



12.25



0.00



12.64



2.43



n



10



10



10



10



10



G3, F & 300



Mean



2.76



3.23



0.00



3.23



0.00



±SD



5.61



6.61



0.00



6.61



0.00



n



8



8



8



8



8



G4, F & 1000



Mean



3.17



6.80



0.00



6.80



0.00



±SD



4.93



12.65



0.00



12.65



0.00



n



9



9



9



9



9



F: Female; SD: Standard Deviation; n: No. of Animals


 


 


TABLE 7 (Contd…). SUMMARY OF MATERNAL DATA PER LITTER RECORD






































































































Group, Sex & Dose


(mg/kg body weight/day)



 



Male/Female


Sex ratio



Male Fetuses (%)



Female Fetuses (%)



Percent of


Live Fetuses



G1, F & 0



Mean



1.41



52.45



47.55



98.77



±SD



0.91



18.79



18.79



3.70



n



9



9



9



9



G2, F & 100



Mean



1.16



48.80



51.20



100.00



±SD



0.78



15.06



15.06



0.00



n



10



10



10



10



G3, F & 300



Mean



0.94



46.43



53.57



100.00



±SD



0.46



9.53



9.53



0.00



n



8



8



8



8



G4, F & 1000



Mean



0.79



41.93



58.07



100.00



±SD



0.41



11.52



11.52



0.00



n



9



9



9



9



F: Female; SD: Standard Deviation; n: No. of Animals


 


TABLE 8 SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD































































Group, Sex & Dose


(mg/kg body weight/day)


 

Thyroid along with parathyroid #



G1, F & 0



Mean



0.0192



±SD



0.0034



n



9



G2, F & 100



Mean



0.0206



±SD



0.0014



n



10



G3, F & 300



Mean



0.0198



±SD



0.0019



n



8



G4, F & 1000



Mean



0.0211



±SD



0.0020



n



9



F: Female; SD: Standard Deviation; n: No. of Animals; #: Weighed Post fixation


TABLE 9 SUMMARY OF TERMINAL BODY WEIGHT (g) AND ORGAN WEIGHT (%) RELATIVE TO TERMINAL BODY WEIGHT RECORD












































































Group, Sex & Dose


(mg/kg body weight/day)


 

Terminal Body Weight (g)



Thyroid along


with parathyroid



G1, F & 0



Mean



371.63



0.0052



±SD



40.92



0.0012



n



9



9



G2, F & 100



Mean



349.73



0.0060



±SD



31.50



0.0009



n



10



10



G3, F & 300



Mean



359.33



0.0055



±SD



25.41



0.0005



n



8



8



G4, F & 1000



Mean



360.42



0.0059



±SD



34.04



0.0008



n



9



9



F: Female; SD: Standard Deviation; n: No. of Animals


 


TABLE 10 SUMMARY OF MEAN FETAL WEIGHT (g) PER LITTER, MEAN FETAL CROWN RUMP LENGTH (mm) PER LITTER AND MEAN ANOGENITAL DISTANCE (AGD) RATIO PER LITTER RECORD



































































































































































Group, Sex


& Dose


(mg/kg body weight/day)


 

Mean Fetal Weight (g)


 

Mean Crown Rump Length (mm)


 

Mean Ano-genital Distance Measurement (mm)


 

Mean Ano-genital Distance Ratio



Male



Female



Male



Female



Male



Female



Male



Female



G1, F & 0



Mean



4.12



3.86



37.62



35.83



3.56



2.47



2.22



1.58



±SD



0.18



0.17



1.18



1.06



0.32



0.14



0.21



0.08



n



9



9



9



9



9



9



9



9



G2, F & 100



Mean



4.16



3.87



37.81



36.19



3.49



2.50



2.17



1.60



±SD



0.13



0.07



0.86



0.32



0.16



0.14



0.11



0.09



n



10



10



10



10



10



10



10



10



G3, F & 300



Mean



4.12



3.85



37.72



36.33



3.54



2.44



2.21



1.55



±SD



0.16



0.14



0.78



0.48



0.12



0.14



0.07



0.09



n



8



8



8



8



8



8



8



8



G3, F & 1000



Mean



4.17



3.88



38.13



36.34



3.56



2.47



2.21



1.58



±SD



0.21



0.19



0.34



0.54



0.07



0.14



0.07



0.08



n



9



9



9



9



9



9



9



9



F: Female; SD: Standard Deviation; n: No. of Animals


 


TABLE 11 SUMMARY RECORD OF FETAL EXTERNAL EXAMINATION PER LITTER













































































































































Parameters



Group



G1



G2



G3



G4



Dose
(mg/kg body weight/day)



0



100



300



1000



Number of Dams



9



10



8



9



Total Number of fetuses evaluated for
External Examination



103



104



90



100



 



 



 



 



 



No. of Fetuses (No. of Litters) noted with
No Abnormality Detected



102 (8)



103 (9)



87 (5)



100 (9)



Percentage of Fetuses (Percentage of Dams) with No Abnormality Detected



99.03 (88.89)



99.04 (90.00)



96.67 (62.50)



100.00 (100.00)



 



 



 



 



 



No. of Fetuses (No. of Dams) noted with Malformations



0 (0)



0 (0)



0 (0)



0 (0)



Percentage of Fetuses (Percentage of Dams) with Malformations



0.0 (0.0)



0.0 (0.0)



0.0 (0.0)



0.0 (0.0)



 



 



 



 



 



No. of Fetuses (No. of Dams) with Variations



1 (1)



1 (1)



3 (3)



0 (0)



Percentage of Fetuses (Percentage of Dams) with Variations



0.97 (11.11)



0.96 (10.00)



3.33 (37.50)



0.00 (0.00)



VARIATIONS



HAEMORRHAGIC SPOTS [EXTERNAL]



Haemorrhagic spot
(occurrence on different parts of the fetus)



Fetal Incidences



1



0.97



1



0.96



3



3.33



0



0.00



Litter Incidences



1



11.11



1



10.00



3



37.50



0



0.00


            

 





















 



 



No. of fetuses with abnormality



% of Abnormality



:



------------------------------------------ X 100



 



 



  Total No. of fetuses examined



 


TABLE 12 SUMMARY OF FETAL VISCERAL EXAMINATION PER LITTER RECORD


































































































Parameters



Group



G1



G2



G3



G4



Dose


(mg/kg body weight/day)



0



100



500



1000



Number of Dams



9



10



8



9



Total Number of Fetuses evaluated for
Soft Tissue (Visceral) Examination



103



104



90



100



 



 



 



 



 



No. of Fetuses (No. of Litters) noted with
No Abnormality Detected



103 (9)



104 (10)



90 (8)



100 (9)



Percentage of Fetuses (Percentage of Dams) with
No Abnormality Detected



100.00 (100.00)



100.00 (100.00)



100.00 (100.00)



100.00 (100.00)



 



 



 



 



 



No. of Fetuses (No. of Dams) with Malformations



0 (0)



0 (0)



0 (0)



0 (0)



Percent of Fetuses (Percent of Dams) with Malformations



0.00 (0.00)



0.00 (0.00)



0.00 (0.00)



0.00 (0.00)



 



 



 



 



 



No. of Fetuses (No. of Dams) with Variations



0 (0)



0 (0)



0 (0)



0 (0)



Percentage of Fetuses (Percentage of Dams) with Variations



0.00 (0.00)



0.00 (0.00)



0.00 (0.00)



0.00 (0.00)



 





















 



 



No. of fetuses with abnormality



% of Abnormality



:



------------------------------------------ X 100



 



 



  Total No. of fetuses examined



 


TABLE 13 SUMMARY OF GROSS PATHOLOGY FINDINGS RECORD





































































 


Parameters ↓



Sex



Female



Group & Dose (mg/kg body weight/day)



G1 & 0



G2 & 100



G3 & 300



G4 & 1000



No. of Animals
(both Pregnant and Non-pregnant)



10



10



10



10



No. of Animals found dead during treatment period



0



0



0



0



No. of Animals moribund sacrificed during treatment period



0



0



0



0



No. of Animals sacrificed terminally



10



10



10



10



Gross Pathology Findings



External



No. of animals with No Abnormality Detected



10



10



10



10



Internal



No. of animals with No Abnormality Detected



10



10



10



10


Conclusions:
The oral administration of test item 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 100, 300 and 1000 mg/kg body weight/day under experimental conditions employed.
Executive summary:

To establish doses for a definitive pre-natal developmental toxicity study a dose range finding study was performed similar to OECD 414, non GLP using Sprague Dawley Rats. Doses of 100mg/kg, 300mg/kg and 1000mg/kg were chosen in this study based on available toxicological data in similar substance. 10 pregnant rats were allocated to each dose group and a concurrent vehicle control group of 10 animals was added. Carboxy methyl cellulose was chosen as vehicle and the test item was applied via oral (gavage) from gestation day 5 to 19 once daily. During the experiment, any clinical signs, mortality, body weight development, feed consumption was recorded. On GD20 the animals were euthanized and were subject to gross necropsy and relevant organs like ovaries, uterus with cervix and thyroid and parathyroid glands extracted. The sex, weight, crown-rump lengths, Ano-genital distance of the fetuses were determined and their soft tissue examined. Particular attention was paid to their reproductive tract and if external and internal sex matched. There were no signs of toxiticy in maternal animals for any endpoint. Similar, for pre-natal developmental toxicity no signs of toxicity could be noted besides some non test-item related alterations recorded during the external examination. The oral administration of test item 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 100, 300 and 1000 mg/kg body weight/day under experimental conditions employed and the same are then considered appropriate for the definitive test.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium polysulfide aluminosilicate with a SOD-type framework structure
EC Number:
701-340-9
Molecular formula:
|Na+6-x+y+z (S2•-)y(S3•-)z|[Al6-x Si6+x O24] - SOD Where: 6 ≤ 6-x+y+z ≤ 8 0 ≤ x ≤ 1.2 1 ≤ y+z ≤ 2
IUPAC Name:
Sodium polysulfide aluminosilicate with a SOD-type framework structure
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: In-house bred animals
- Age at study initiation: Minimum of 10 weeks
- Weight at study initiation: Males: 251.54 g to 289.11 g & Females: 204.23 g to 250.27 g
- Fasting period before study: No
- Housing: Pre-mating/Acclimatization - Maximum of three animals of same sex per cage were housed in sterilized standard polypropylene cage (Size: L 430 × B 285 × H 150 mm). Steam sterilized clean paddy husk was used as bedding material and was changed along with the cage at least twice a week
- Housing: Mating - During mating, three rats (one male and two females) were housed in standard polypropylene cages.
- Housing: Post mating - After confirming presence of sperm in the vaginal smear (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually in polypropylene cages.
- Diet (e.g. ad libitum): ad libitum - Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG)
- Water (e.g. ad libitum): ad libitum - Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: for a minimum period of five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1ºC to 23.5 ºC
- Humidity (%): relative humidity 50 to 62%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark

IN-LIFE DATES: From: To: 28 July 2020 to 04 November 2020

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required quantity of test item was weighed and triturated well in a mortar with a small quantity of vehicle until a homogenous suspension was formed and thereafter the entire quantity of the formulation was transferred into measuring cylinder. A small quantity of vehicle was added to rinse the mortar and this was transferred into the measuring cylinder. The rinsing procedure of mortar and pestle was repeated many times to ensure the transfer of the contents to the measuring cylinder. Finally, the volume was made up to required quantity with vehicle to get desired concentration of 10, 30 and 100 mg/mL of test item for low, mid and high dose groups respectively.

VEHICLE
- Justification for use and choice of vehicle (if other than water): 0.5% w/v Carboxy Methyl Cellulose, test item was insoluble in distilled water
- Concentration in vehicle: 100 mg/mL (maximum)
- Amount of vehicle (if gavage): 10mL/kg
- Lot/batch no. (if required): BCBN1690V
- Purity: n/a
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Verification of concentration and homogonetiy of the test item was conduced by BIONEEDS INDIA PRIVATE LIMITED DEVARAHOSAHALLY, SOMPURA HOBLI, NELAMANGALA TALUK, BANGALORE RURAL DISTRICT, PIN - 562 111, KARNATAKA, INDIA which hold the Good Laboratory Practice Certificate (GLP). 0.1 mL of sample was pipetted out into a digestion tube, a volume of 6 mL of concentrated nitric acid was added and placed in microwave sample preparation system and digested by the selected method. After digestion, the digestion tubes were cooled to room temperature and the contents were transferred to suitable volumetric flasks and diluted with Milli-Q water.


TECHNIQUE AND TEST METHOD: ICP-MS


TEST PARAMETERS: Timings Sweeps : 30 Seconds, Reading : 1, Replicates : 3, Analyte : Aluminium, Mass : 26.9815, Sample Flush : 35 seconds, Read Delay : 15 Seconds, Wash : 45 seconds, Mode : KED, Flow (Helium) : 4.0 mL, Vehicle: 0.5% Carboxymethyl Cellulose


ACCEPTANCE CRITERIA: Homogeneity and dose formulation analysis of prepared test formulations for the dose concentration verification were performed during first and last week of the treatment and the obtained 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:
- Impregnation procedure: Cohoused
- If cohoused:
- M/F ratio per cage: 1:2 ratio (one male and two females)
- Length of cohabitation: until evidence of copulation was observed or for two weeks.
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility. yes
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: In-house bred animals
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: no
Duration of treatment / exposure:
Gestation days 5 to 19
Frequency of treatment:
once daily
Duration of test:
Acclimatization: 28 July 2020 to 20 August 2020
Cohabitation: 04 August 2020 to 03 September 2020
Treatment: 10 August 2020 to 23 September 2020
Necropsy: 25 August 2020 to 24 September 2020
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose range finding study, conducted with the doses of 100, 300 and 1000 mg/kg body weight based on the available literature. Oral administration of test item to pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 did not produce any indication of maternal and prenatal-developmental toxicity at all these tested dose levels in the dose range finding study.
- Rationale for animal assignment (if not random): evenly distributed to all the groups based on their body weights so as to maintain comparable mean body weight for all groups
- Fasting period before blood sampling for (rat) dam thyroid hormones: 0
- Time of day for (rat) dam blood sampling: within 2 hours before necropsy.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily for clinical signs of toxicity and twice daily for mortality and morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

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 AND COMPOUND INTAKE (if feeding study): not feeding study
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Recorded as g/animal/day during gestation days 0-3, 3-5, 5-8, 8-11, 11-14, 14-17, 17-19 and 19-20
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No applicable

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not drinking water study


POST-MORTEM EXAMINATIONS: Yes / No / No data
- Sacrifice on gestation day 20
- Organs examined: Ovaries, Uterus with cervix, Thyroid and Parathyroid glands
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
- Other: observation of uterine content.
Blood sampling:
- Plasma: No
- Serum: Yes
- Volume collected - n/a
- Other: blood samples were collected as follows:
- From all dams at termination for mandatory assessment of thyroid hormones T4, T3 and thyroid stimulating hormone (TSH) within 2 hours before necropsy.
- Blood samples from non-pregnant females were also collected but were not pooled with the pregnant dams.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No
- Anogenital distance of all live rodent pups: Individual fetus was measured for its anogenital distance on the day of caesarean section
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, Mean fetal weight per dam, Mean fetal crown rump length per dam, Mean fetal anogenital distance per dam, Serum T3, T4 and TSH levels, Organ weight

Non-Parametric: Kruskal-Wallis: No. of corpora lutea per dam, No. of implantations per dam, Litter size per dam, No. of live/dead fetuses per dam, Percent of live/dead 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 weigh
- Percent of Live Fetuses per dam = (Number of Live Fetuses / Litter Size) x 100
- Percent of Dead Fetuses per dam = (Number of Dead Fetuses/ Litter Size) x 100
- Percent of Early Resorptions (%) per dam = (Number of Early Resorptions/Number of Implantation sites) x 100
- Percent of Late Resorptions (%) per dam = (Number of Late Resorptions / Number of Implantation sites)x 100
- Pre-implantation Loss (%) per dam = [(Number of Corpora lutea - Number of Implantation sites)/ Number of Corpora lutea]x 100
- Post-implantation Loss (%) per Dam = [(Number of Implantation sites - Number of Viable fetuses )/ Number of Implantation sites]x 100
- Sex Ratio (m/f) = Number of live male fetuses / Number of live female fetuses
- Male/Female Fetuses (%) = (Number of live male/female fetuses/ Total number of live fetuses)x 100
- Ano-genital Distance Ratio = Ano-genital distance (mm) / Cube root of Fetal weight (g)
- Fetal incidence (%) = (Number of Fetuses with particular observation per group/Total number of Fetuses examined per group)x 100
- Litter incidence (%) = (Number of Litters/dams with particular observation per group/Total number of Litters per group) x 100










Historical control data:
Historical control data not included in report. Historical control data from in-house of the same species and strain.

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 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 at 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 at all the tested dose groups when compared with the vehicle control group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid hormone T3: There were no changes noted for this parameter across the dose groups when compared to the vehicle control group. The statistically significant increase in serum T3 levels (ng/mL) at groups G3 and G4 is considered as incidental and un-related to treatment with test item due to lack of a dose-response relationship and also the obtained values were within the historical control range of same species and strain.

Thyroid hormone T4: There were no changes noted for this parameter across the dose groups when compared to the vehicle control group.

Thyroid stimulating hormone THS: There were no statistically significant differences noted for this parameter across the dose groups when compared to the vehicle control group due to noted larger standard deviations from groups G3 and G4. However, the noted differences in mean value from groups G3 and G4 when compared with vehicle control group are considered as incidental and also the obtained mean values from all the tested dose groups are within in-house historical control range.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
no effects observed
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 and no significant differences 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.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related changes noted in thyroid along with the parathyroid subjected to histopathological examination at all the tested dose group animals. The noted ultimobranchial cyst/s in thyroid gland is considered as incidental finding as they occurred randomly across the dose groups including concurrent controls and/or were expected from laboratory rats of this age.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no statistically significant differences noted for percentage of pre- or post-implantation loss per litter at all the tested dose groups when compared to the vehicle control group.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in the number and percentage of early or late resorptions per dam across dose groups when compared to the vehicle control group.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no statistically significant differences noted for number of dead fetuses at all the tested dose groups when compared to the vehicle control group.
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
pregnancy with rates of 88%, 92%, 88% and 92% in groups G1, G2, G3 and G4 respectively
Other effects:
no effects observed
Description (incidence and severity):
The mean number of corpora lutea per litter was 13.00, 13.22, 13.50 and 13.48 for groups G1, G2, G3 and G4 respectively. There were no changes or no statistically significant differences noted at all the tested dose groups when compared with vehicle control group for number of corpora lutea.

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:
other: Highest concentration tested, no adverse effects observed

Maternal abnormalities

Key result
Abnormalities:
no effects observed

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.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There were no statistically significant differences noted for number of live fetuses at all the tested dose groups when compared to the vehicle control group.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no statistically significant differences noted for 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 utero (live plus dead) per dam, was 11.64, 11.83, 11.86 and 12.13 for groups G1, G2, G3 and G4, respectively. There were no changes or no statistically significant differences noted at all the tested dose groups when compared with vehicle control group for litter size.
Anogenital distance of all rodent fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
The mean male fetal anogenital distance per litter was 4.25 mm, 4.30 mm, 4.20 mm and 4.28 mm and the mean female anogenital distance per litter was 2.47 mm, 2.51 mm, 2.57 mm and 2.59 mm for groups G1, G2, G3 and G4 respectively. The mean male fetal anogenital distance ratio per litter was 2.61, 2.67, 2.60 and 2.65 and the mean female anogenital distance ratio per litter was 1.55, 1.58, 1.62 and 1.64 for groups G1, G2, G3 and G4 respectively.

Statistically significant increase in mean female fetal anogenital distance and mean fetal anogenital distance ratio per litter was noted at groups G3 and G4 when compared with the vehicle control group. These changes are considered as incidental and un-related to treatment as the obtained range is within in-house historical control range.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The
noted general / developmental variations such as subcutaneous hemorrhagic spot/s beneath the skin on different regions of the body, pale skin colored fetuses, kinked tail and noted external malformations such as unilateral supernumerary digit of hindlimb, unilateral fused digits of hindlimb paw unilateral, hyperextension of forelimb unilateral, misshappened/flat head, short forelimb unilateral 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 were noted for these alterations in any of the tested dose groups when compared with vehicle control group.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The below mentioned occasional incidences of skeletal developmental variations were
noted across the tested dose group litters.
- incomplete ossification of skull bones (parietal/interparietal/supraoccipital/ex-occipital/ mandibular/zygoma);
- incomplete or bipartite or unossification of sternabral bones (sternabra 2, 3, 4, 5 and 6);
- wavy ribs (both unilateral and bilateral);
- extra ossification site/s near to first lumbar vertebra i.e. immediately after 13th rib (both unilateral and bilateral);
- dumbbell or semi-bipartite ossification of thoracic verterbral centrum (centrum no. 8, 9, 10, 11, 12 and 13) and lumbar vertebral centrum (centrum no. 2);
- hemicentric ossification of thoracic verterbral centrum (centrum no. 11 and 12);
- incomplete or unossification of metacarpal no. 5 or proximal phalanges of forelimb;
- incomplete ossification of pubis bone unilateral.

The below mentioned occasional incidences of skeletal malformation were noted across
the tested dose group litters.
- fused skull bones (parietal) - single incidence for a fetus from vehicle control group;
- missing rib unilateral (rib no. 12) - single incidence for a fetus from vehicle control group;
- fused rib no. 11 and 12 - single incidence for a fetus from group G3;
- fused rib no. 12 and 13 - single incidence for a fetus from group G1;
- rudimentary rib (14th rib) - single incidences for a fetus from group G2, G3 and G4 each;
- split centrum no. 10 of thoracic vertebra - 1, 2, 0 and 1 fetal and litter incidences from group G1, G2, G3 and G4 respectively.
- split centrum no. 11 of thoracic vertebra - 0, 2, 2 and 3 fetal and litter incidences from group G1, G2, G3 and G4 respectively.
- split centrum no. 12 of thoracic vertebra - 1, 1, 1 and 2 fetal and litter incidences from group G1, G2, G3 and G4 respectively.
- split centrum no. 13 of thoracic vertebra - 0, 0, 1 and 0 fetal and litter incidences from group G1, G2, G3 and G4 respectively.
These 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-dependant manner. Also, the occurred mean litter/fetal proportions were within the in-house historical control range of this species and strain.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were 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. The noted developmental variations such as pale or discolored adrenals/liver lobes/lung lobes, fused liver lobes, 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. There were no remarkable differences or statistically significant changes noted for these variations in any of the tested dose groups when compared with vehicle control group.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
The mean male fetal crown rump length per litter was 37.28 mm, 37.40 mm, 37.56 mm and 37.67 mm and the female mean crown rump length per litter was 36.61 mm, 36.96 mm, 36.79 mm and 36.92 mm for groups G1, G2, G3 and G4 respectively. Statistically significant increase in mean male fetal crown rump length per litter was noted at group G4 when compared with the vehicle control group. This change is considered as incidental and un-related to treatment as the obtained range is within in-house historical control range.

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:
other: Highest dose tested, no adverse effects observed

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

TABLE 1. SUMMARY OF PREGNANCY STATUS, CLINICAL SIGNS OF TOXICITY AND MORTALITY RECORD                                                                                                                     
















































Group
& Dose
(mg/kg body
weight/day)		Pregnancy StatusClinical Signs and Mortality Record
No. of Presumed
Pregnant Animals / Group		No. of Presumed
Pregnant Animals / Group		Rate of Pregnancy (%)Clinical Signs of
Toxicity revealed
(No. of Animals)		No. of Mortalities /
Total No. of animals
G1 & 0252288.0N (25)0/25
G2 & 100252392.0N (25)0/25
G3 & 300252288.0N (25)0/25
G4 & 1000252392.0N (25)0/25

TABLE 2. SUMMARY OF GESTATION BODY WEIGHT (g) RECORD










































































































































































Group & Dose (mg/kg body weight/day)



 



Body Weight (g) on Gestation Day (GD)



0



3



5



8



11



14



17



19



20



G1  & 0



Mean ±SD



251.79



258.46



265.3



274.34



285.93



300.86



323.93



343.93



358.5



13.58



12.74



12.08



12.31



15.43



15.91



19.21



24.16



28.5



 



n



22



22



22



22



22



22



22



22



22



G2 & 100



Mean ±SD



257.16



264.34



271.41



281.48



292.19



305.77



328.69



350.58



366.59



12.26



10.56



10.52



12.39



13.49



14.67



17.94



21.18



27.52



 



n



23



23



23



23



23



23



23



23



23



G3 & 300



Mean ±SD



252.48



259.66



266.37



275.52



287.86



301.9



326.49



349.47



365.92



16.2



16.12



16.59



16.32



16.93



19.56



21.46



24.03



27.12



 



n



22



22



22



22



22



22



22



22



22



G4 & 1000



Mean ±SD



257.53



265.91



273.18



283.86



295.36



309.37



336.11



359.99



372.98



17.79



16.62



15.73



15.79



15.18



16.49



19.04



24.34



25.84



 



n



23



23



23



23



23



23



23



23



23



Table 3 SUMMARY OF PERCENT CHANGE IN GESTATION BODY WEIGHT (%) RECORD
































































































































































Group & Dose (mg/kg body weight/day)



 



Percent Change in Body Weight (%) during Gestation Day (GD)



0 to 3



3 to 5



5 to 8



8 to 11



11 to 14



14 to 17



17 to 19



19 to 20



G1 & 0



Mean ±SD



2.68



2.67



3.42



4.2



5.24



7.7



6.14



4.19



1.05



1.03



1.34



1.77



1.37



4.19



2.74



2.26



 



n



22



22



22



22



22



22



22



22



G2 & 100



Mean ±SD



2.83



2.68



3.7



3.8



4.65



7.5



6.65



4.49



1.38



1.01



1.19



0.77



1.46



3.16



2.19



1.96



 



n



23



23



23



23



23



23



23



23



G3 & 300



Mean ±SD



2.86



2.59



3.46



4.49



4.85



8.18



7.04



4.68



1.1



0.78



1.03



1.45



1.32



2.85



2.33



1.62



 



n



22



22



22



22



22



22



22



22



G4 & 1000



Mean ±SD



3.31



2.77



3.93



4.08



4.75



8.66



7.08



3.61



1.58



1.25



1.39



1.19



1.45



2.92



2.95



1.85



 



n



23



23



23



23



23



23



23



23



SD: Standard Deviation; n: Number of Dams.



TABLE 4. SUMMARY OF GRAVID UTERUS WEIGHT (g) AND MATERNAL BODY WEIGHT CHANGE CORRECTED FOR GRAVID UTERINE WEIGHT (g)





















































































































Group & Dose (mg/kg body weight/day)



 



Body Weight Change (g) from GD 5 to 20



Gravid Uterus Weight (g)



 Corrected Body Weight


 

g



%



G1 & 0



Mean ±SD



93.21



70.87



22.34



8.43



21.52



19.66



10.17



3.81



 



n



22



22



22



22



G2 & 100



Mean ±SD



95.21



71.6



23.61



8.67



21.61



20.26



9.42



3.37



 



n



23



23



23



23



G3 & 300



Mean ±SD



99.55



74



25.55



9.62



18.69



15.96



11.52



4.35



 



n



22



22



22



22



G4 & 1000



Mean ±SD



99.8



73.06



26.74



9.89



23.15



21.85



10.02



3.9



 



n



23



23



23



23


      

SD: Standard Deviation; n: Number of Dams; GD: Gestation Day. *: Statistically significant (P<0.05) change than the control group.



TABLE 5. SUMMARY OF AVERAGE GESTATION FEED CONSUMPTION (g/animal/day) RECORD





























































































































































Group & Dose (mg/kg body weight/day)



 



Feed Consumption (g/animal/day) during Gestation Day (GD)



0 to 3



3 to 5



5 to 8



8 to 11



11 to 14



14 to 17



17 to 19



19 to 20



G1 & 0



Mean ±SD



14.98



18.97



15.96



19.76



22.45



24.77



26.64



28.78



1.31



1.13



1.05



1.57



1.52



1.69



2.22



2.01



 



n



22



22



22



22



22



22



22



22



G2 & 100



Mean ±SD



14.91



19.19



16.26



19.53



22.39



24.6



26.8



29.19



1.23



1.17



0.97



1.89



1.41



2.27



2.45



2.38



 



n



23



23



23



23



23



23



23



23



G3 & 300



Mean ±SD



14.71



19.52



16.83



19.62



22.28



24.85



25.79



28.62



1.59



1.76



1.27



1.55



1.23



1.33



2.65



2.37



 



n



22



22



22



22



22



22



22



22



G4 & 1000



Mean ±SD



15.51



19.75



16.94



19.9



22.88



25.5



26.02



28.06



1.69



1.62



2.17



1.54



0.89



0.89



2.17



2.63



 



n



23



23



23



23



23



23



23



23



 


TABLE 6. SUMMARY OF UTERI OBSERVATIONS PER LITTER RECORD











































































































































































Group & Dose (mg/kg body weight/day)


 

No. of Corpora lutea



No. of Implantations



Litter size



No. of Live Fetuses



No. of Dead Fetuses



No. of Early Resorptions



No. of Late Resorptions


 

Total



Male



Female



G1 & 0



Mean ±SD



13



12.27



11.64



11.64



5.5



6.14



0



0.64



0



2.94



2.88



3.39



3.39



2.3



2.59



0



1.05



0



 



n



22



22



22



22



22



22



22



22



22



G2 & 100



Mean ±SD



13.22



12.57



11.83



11.83



6.57



5.26



0



0.74



0



3.48



3.76



4.05



4.05



2.89



2.68



0



1.05



0



 



n



23



23



23



23



23



23



23



23



23



G3 & 300



Mean ±SD



13.5



12.73



11.86



11.77



5.59



6.18



0.09



0.86



0



2.86



2.6



2.95



2.91



1.76



2.17



0.43



1.25



0



 



n



22



22



22



22



22



22



22



22



22



G4 & 1000



Mean ±SD



13.48



12.96



12.13



12.09



6.3



5.78



0.04



0.78



0.04



3.57



3.36



3.81



3.8



2.55



2.5



0.21



0.9



0.21



 



n



23



23



23



23



23



23



23



23



23



TABLE 7. SUMMARY OF MATERNAL DATA PER LITTER RECORD


















































































































































































































Group  & Dose  (mg/kg body weight/day)



 



Pre-Implantation Loss (%)



Post-Implantation Loss (%)



Percent of Dead Fetus (%)



Percent of Early Resorptions (%)



Percent of Late Resorptions (%)



Male/Female Sex Ratio



Male  Fetuses (%)



Female Fetuses (%)



Percent of Live Fetuses



 


 
  
  
  

G1 & 0



Mean ±SD



5.57



6.23



0



6.23



0



1.06



47.55



52.45



100


  

6.11



10.88



0



10.88



0



0.61



14.03



14.03



0


  

 



n



22



22



22



22



22



22



22



22



22


  

G2 & 100



Mean ±SD



6.13



7.32



0



7.32



0



1.69



56.19



43.81



100


  

8.5



11.88



0



11.88



0



1.28



15.94



15.94



0


  

 



n



23



23



23



23



23



23



23



23



23


  

G3 & 300



Mean ±SD



5.51



8.04



0.65



7.4



0



1.02



48



52



99.35


  

4.82



11.95



3.05



11.98



0



0.46



11.41



11.41



3.05


  

 



n



22



22



22



22



22



22



22



22



22


  

G4 & 1000



Mean ±SD



3.71



8.28



0.33



7.61



0.33



1.14



53.02



46.98



99.67


  

5.74



12.48



1.6



12.5



1.6



0.65



16.95



16.95



1.6


  

 



n



23



23



23



23



23



23



23



23



23


  

SD: Standard Deviation; n: Number of Dams.


  

TABLE 8. SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

































































Group & Dose (mg/kg body weight/day)  Absolute Thyroid along with Parathyroid Weight (g)# 
G1 & 0 Mean ±SD 0.0183
0.0027
 n 22
G2 & 100 Mean ±SD 0.0177
0.0025
 n 23
G3 & 300 Mean ±SD 0.0185
0.0025
 n 22
G4 & 1000 Mean ±SD 0.019
0.0019
 n 23
SD: Standard Deviation; n: Number of Dams; #: Weighed post fixation. 

TABLE 9. SUMMARY OF TERMINAL BODY WEIGHT (g) AND ORGAN
WEIGHT (%) RELATIVE TO TERMINAL BODY WEIGHT RECORD














































































Group & Dose (mg/kg body weight/day)  Terminal Body Weight (g) Relative Thyroid along with Parathyroid weight (%) 
G1 & 0 Mean ±SD 358.50.0051
28.50.0008
 n 2222
G2 & 100 Mean ±SD 366.590.0049
27.520.0007
 n 2323
G3 & 300 Mean ±SD 365.920.0051
27.120.0006
 n 2222
G4 & 1000 Mean ±SD 372.980.0051
25.840.0006
 n 2323
SD: Standard Deviation; n: Number of Dams. 

TABLE 10. SUMMARY OF SERUM TRIIODOTHYRONINE (T3) LEVELS
(ng/mL) RECORD


































































Group & Dose (mg/kg body weight/day)



 



Serum T3 Levels (ng/mL)



G1 & 0



Mean ±SD



2.587



0.249



 



n



22



G2 & 100



Mean ±SD



2.659



0.273



 



n



23



G3 & 300



Mean ±SD



3.082*



0.225



 



n



22



G4 & 1000



Mean ±SD



2.849*



0.535



 



n



23



SD: Standard Deviation;   n: Number of Dams considered for mean calculations.



TABLE 11. SUMMARY OF SERUM THYROXINE (T4) LEVELS (ng/mL)
RECORD































































Group & Dose  (mg/kg body weight/day)



 



Serum T4 Levels (ng/mL)



G1 & 0



Mean ±SD



72.608



16.419



 



n



22



G2 & 100



Mean ±SD



71.998



13.244



 



n



23



G3 & 300



Mean ±SD



67.222



15.074



 



n



22



G4 & 1000



Mean ±SD



62.908



11.753



 



n



23



TABLE 12. SUMMARY OF SERUM THYROID STIMULATING HORMONE
(TSH) LEVELS (µIU/mL) RECORD































































Group & Dose (mg/kg body weight/day)



 



Serum TSH Levels (µIU/mL)



G1 & 0



Mean ±SD



3.853



4.033



 



n



20



G2 & 100



Mean ±SD



3.441



3.896



 



n



20



G3 & 300



Mean ±SD



7.357



6.235



 



n



21



G4 & 1000



Mean ±SD



6.445



5.991



 



n



21



TABLE 13. SUMMARY OF MEAN FETAL WEIGHT, MEAN FETAL CROWN RUMP LENGTH, MEAN FETAL ANOGENITAL DISTANCE AND RATIO RECORD


























































































































































































































Group & Dose (mg/kg body weight/day)


 

Fetal Weight (g)



Crown Rump Length (mm)



Anogenital Distance
Measurement (mm)



Anogenital Distance Ratio



 


 
 

Male



Female



Male



Female



Male



Female



Male



Female



 


 
  
  

G1 & 0



Mean ±SD



4.24



4.06



37.28



36.61



4.25



2.47



2.61



1.55


  

0.33



0.31



0.57



0.67



0.18



0.12



0.18



0.07


  
 

n



22



22



22



22



22



22



22



22


  
  
  

G2 & 100



Mean ±SD



4.2



4.01



37.4



36.96



4.3



2.51



2.67



1.58


  

0.38



0.4



0.56



0.59



0.23



0.1



0.14



0.08


  
 

n



23



23



23



23



23



23



23



23


  
  
  

G3 & 300



Mean ±SD



4.25



4.02



37.56



36.79



4.2



2.57*



2.6



1.62*


  

0.33



0.21



0.5



0.65



0.27



0.12



0.18



0.07


  
 

n



22



22



22



22



22



22



22



22


  
  
  

G4 & 1000



Mean ±SD



4.22 0.30



3.93 0.28



37.67*



36.92



4.28



2.59*



2.65 0.17



1.64*


  

0.49



0.6



0.24



0.1



0.07


  

 



n



23



22



23



22



23



22



23



22


  

Table 14 SUMMARY RECORD OF FETAL EXTERNAL EXAMINATION PER LITTER











































































































































































































































































































































Group



G1



G2



G3



G4



Dose (mg/kg body weight/dry)



0



100



300



1000


            

Litters Evaluated for External Examination



No



22



23



22



23



Fetuses Evaluated for External Examination



No



256



272



259



278



Variations



Region/Tissue



Alteration


         

General



Subcutaneous hemorrhagic spot/s beneach the skin on different regions of the body


 

Litter Incidences



No (%)



7



(31.8)



10



(43.5)



6



(27.3)



8



(34.8)


 

Fetal Incidences



No (%)



9



(3.5)



14



(5.1)



8



(3.1)



10



(3.6)



General



Pale colored skin


 

Litter Incidences



No (%)



2



(9.1)



1



(4.3)



1



(4.5)



3



(13.0)


 

Fetal Incidences



No (%)



2



(0.8)



1



(0.4)



1



(0.4)



3



(1.1)



Tail



Kinked


 

Litter Incidences



No (%)



0



(0.0)



0



(0.0)



0



(0.0)



1



(4.3)


 

Fetal Incidences



No (%)



0



(0.0)



0



(0.0)



0



(0.0)



1



(0.4)



Malformations



Region/Tissue



Alteration


         

Hindlimb



Supernumerary digit (unilateral)


 

Litter Incidences



No (%)



0



(0.0)



1



(4.3)



0



(0.0)



0



(0.0)


 

Fetal Incidences



No (%)



0



(0.0)



1



(0.4)



0



(0.0)



0



(0.0)



Hindlimb



Fused digits (unilateral)


 

Litter Incidences



No (%)



0



(0.0)



1



(4.3)



0



(0.0)



1



(4.3)


 

Fetal Incidences



No (%)



0



(0.0)



1



(0.4)



0



(0.0)



1



(0.4)



Forelimb



Hyperextension (unilateral)


 

Litter Incidences



No (%)



0



(0.0)



0



(0.0)



1



(4.5)



0



(0.0)


 

Fetal Incidences



No (%)



0



(0.0)



0



(0.0)



2



(0.8)



0



(0.0)



Head



Misshappen (flat shaped)


 

Litter Incidences



No (%)



0



(0.0)



0



(0.0)



0



(0.0)



1



(4.3)


 

Fetal Incidences



No (%)



0



(0.0)



0



(0.0)



0



(0.0)



1



(0.4)



Forelimb



Short (unilateral)


         
 

Litter Incidences



No (%)



0



(0.0)



0



(0.0)



0



(0.0)



1



(4.3)


 

Fetal Incidences



No (%)



0



(0.0)



0



(0.0)



0



(0.0)



1



(0.4)



TABLE 15. SUMMARY OF FETAL VISCERAL EXAMINATION PER LITTER
RECORD
































































































































































































































































Group



G1



G2



G3



G4



Dose (mg/kg body weight/dry)



0



100



300



1000


            

Litters Evaluated for External Examination



No



22



23



22



23



Fetuses Evaluated for External Examination



No



123



129



124



133



Variations



Region/Tissue



Alteration


         

Adrenals



Pale/Discoloration


         
 

Litter Incidences



No (%)



1



4.5



0



0


 

0



0



0


 

Fetal Incidences



No (%)



1



0.8



0



0


 

0



0



0



Kidneys



Dilatation of renal pelvis
(bilateral)


 

Litter Incidences



No (%)



4



18.2



2



8.7



0



13.6



3



13


 

Fetal Incidences



No (%)



6



4.8



4



3.1



0



4



5



3.8



Liver



Pale/Discoloration


 

Litter Incidences



No (%)



4



18.2



3



13



3



4.5



2



8.7


 

Fetal Incidences



No (%)



5



4



3



2.3



5



0.8



3



2.3



Liver



Fused Lobes


 
 

Litter Incidences



No (%)



1



4.5



0



0



1



0



0



0


 

Fetal Incidences



No (%)



1



0.8



0



0



1



0



0



0



Lungs



Pale/Discoloration


 

Litter Incidences



No (%)



0



0



1



4.3



0



0



1



4.3


 

Fetal Incidences



No (%)



0



0



1



0.8



0



0



2



1.5



Ureters



Dilatation


 

Litter Incidences



No (%)



2



9.1



4



17.4



4



18.2



3



13


 

Fetal Incidences



No (%)



3



2.4



5



3.9



4



3.2



5



3.8



TABLE 16. SUMMARY OF SKELETAL EXAMINATION OF FETUSES PER LITTER RECORD









































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































Group



G1



G2



G3



G4



Dose (mg/kg body weight/dry)



0



100



300



1000


            

Litters Evaluated for External Examination



No



22



23



22



23



Fetuses Evaluated for External Examination



No



132



143



136



146



Variations



Region/Tissue



Alteration


         

Skull



Parietal bones  Incomplete ossification


       

Litter Incidences



No (%)



2



(9.1)



1



(4.3)



1



(4.5)



0



(0.0)



Fetal Incidences



No (%)



2



(1.5)



1



(0.7)



1



(0.7)



0



(0.0)



Interparietal bones  Incomplete ossification
(bilateral)


  

 



 



 


 

 



 



 



 



Litter Incidences



No (%)



1



(4.5)



4



(17.4)



2



(9.1)



1



(4.3)



Fetal Incidences



No (%)



1



(0.8)



5



(3.5)



2



(1.5)



1



(0.7)



Supraoccipital bones  Incomplete ossification


          

Litter Incidences



No (%)



0



(0.0)



1



(4.3)



2



(9.1)



2



(8.7)



Fetal Incidences



No (%)



0



(0.0)



1



(0.7)



2



(1.5)



2



(1.4)



Exoccipital bones  Incomplete ossification


      

Litter Incidences



No (%)



0



(0.0)



2



(8.7)



1



(4.5)



1



(4.3)



Fetal Incidences



No (%)



0



(0.0)



2



(1.4)



1



(0.7)



1



(0.7)



Mandibular bones  Incomplete ossification


      

Litter Incidences



No (%)



0



(0.0)



0



(0.0)



0



(0.0)



1



(4.3)



Fetal Incidences



No (%)



0



(0.0)



0



(0.0)



0



(0.0)



1



(0.7)



Zygomatic Arch  Incomplete ossification


       

Litter Incidences



No (%)



0



(0.0)



1



(4.3)



0



(0.0)



2



(8.7)



Fetal Incidences



No (%)



0



(0.0)



1



(0.7)



0



(0.0)



2



(1.4)



Sternum



Sternebra No. 3  Bipartite ossification


       

Litter Incidences



No (%)



0



(0.0)



1



(4.3)



0



(0.0)



0



(0.0)



Fetal Incidences



No (%)



0



(0.0)



1



(0.7)



0



(0.0)



0



(0.0)



Sternebra No. 4  Bipartite ossification


       

Litter Incidences



No (%)



0



(0.0)



1



(4.3)



0



(0.0)



1



(4.3)



Fetal Incidences



No (%)



0



(0.0)



1



(0.7)



0



(0.0)



1



(0.7)



Sternebra No. 5  Bipartite ossification


       

Litter Incidences



No (%)



1



(4.5)



1



(4.3)



2



(9.1)



1



(4.3)



Fetal Incidences



No (%)



1



(0.8)



1



(0.7)



2



(1.5)



1



(0.7)



Sternebra No. 6  Bipartite ossification



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



1



(4.3)



1



(4.5)



0



(0.0)



Fetal Incidences



No (%)



0



(0.0)



1



(0.7)



1



(0.7)



0



(0.0)



Sternebra No. 2  Bipartite ossification



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



0



(0.0)



0



(0.0)



1



(4.3)



Fetal Incidences



No (%)



0



(0.0)



0



(0.0)



0



(0.0)



1



(0.7)



Sternebra No. 2  Incomplete ossification


       

Litter Incidences



No (%)



0



(0.0)



1



(4.3)



0



(0.0)



0



(0.0)



Fetal Incidences



No (%)



0



(0.0)



1



(0.7)



0



(0.0)



0



(0.0)



Sternebra No. 5  Incomplete ossification



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



10



(45.5)



12



(52.2)



8



(36.4)



4



(17.4)



Fetal Incidences



No (%)



14



(10.6)



15



(10.5)



11



(8.1)



5



(3.4)



Sternebra No. 6  Incomplete ossification



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



8



(36.4)



15



(65.2)



12



(54.5)



14



(60.9)



Fetal Incidences



No (%)



14



(10.6)



25



(17.5)



18



(13.2)



22



(15.1)



Sternebra No. 5  Unossified



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



2



(9.1)



6



(26.1)



6



(27.3)



6



(26.1)



Fetal Incidences



No (%)



2



(1.5)



8



(5.6)



7



(5.1)



6



(4.1)



Sternebra No. 6  Unossified



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



2



(9.1)



5



(21.7)



4



(18.2)



5



(21.7)



Fetal Incidences



No (%)



2



(1.5)



10



(7.0)



4



(2.9)



5



(3.4)



Ribs



Rib No. 9 Wavy (Unilateral)



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



0



(0.0)



1



(4.5)



0



(0.0)



Fetal Incidences



No (%)



0



(0.0)



0



(0.0)



1



(0.7)



0



(0.0)



Rib No. 10 Wavy (Unilateral)



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



0



(0.0)



1



(4.5)



0



(0.0)



Fetal Incidences



No (%)



0



(0.0)



0



(0.0)



1



(0.7)



0



(0.0)



Rib No. 5 Wavy (bilateral)



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



0



(0.0)



1



(4.5)



0



(0.0)



Fetal Incidences



No (%)



0



(0.0)



0



(0.0)



1



(0.7)



0



(0.0)



Rib No. 6 Wavy (bilateral)



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



0



(0.0)



1



(4.5)



0



(0.0)



Fetal Incidences



No (%)



0



(0.0)



0



(0.0)



1



(0.7)



0



(0.0)



Rib No. 7 Wavy (bilateral)



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



0



(0.0)



1



(4.5)



0



(0.0)



Fetal Incidences



No (%)



0



(0.0)



0



(0.0)



1



(0.7)



0



(0.0)



Rib No. 8 Wavy (bilateral)



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



0



(0.0)



1



(4.5)



0



(0.0)



Fetal Incidences



No (%)



0



(0.0)



0



(0.0)



1



(0.7)



0



(0.0)



Rib No. 9 Wavy (bilateral)


        

Litter Incidences



No (%)



0



(0.0)



1



(4.3)



1



(4.5)



0



(0.0)



Fetal Incidences



No (%)



0



(0.0)



1



(0.7)



1



(0.7)



0



(0.0)



Rib No. 10 Wavy (bilateral)



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



1



(4.3)



1



(4.5)



0



(0.0)



Fetal Incidences



No (%)



0



(0.0)



1



(0.7)



1



(0.7)



0



(0.0)



Rib No. 11 Wavy (bilateral)



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



1



(4.3)



1



(4.5)



0



(0.0)



Fetal Incidences



No (%)



0



(0.0)



1



(0.7)



1



(0.7)



0



(0.0)



Rib No. 12 Wavy (bilateral)



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



1



(4.3)



1



(4.5)



0



(0.0)



Fetal Incidences



No (%)



0



(0.0)



1



(0.7)



1



(0.7)



0



(0.0)



Rib No. 13 Wavy (bilateral)



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



1



(4.5)



2



(8.7)



3



(13.6)



0



(0.0)



Fetal Incidences



No (%)



1



(0.8)



3



(2.1)



3



(2.2)



0



(0.0)



Ossification Site at First Lumbar Vertebra (Unilateral)



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



2



(8.7)



3



(13.6)



4



(17.4)



4



(18.2)



Fetal Incidences



No (%)



2



(1.5)



4



(2.8)



5



(3.7)



5



(3.4)



Ossification Site at First Lumbar Vertebra (Bilateral)



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



2



(9.1)



2



(8.7)



2



(9.1)



Fetal Incidences



No (%)



0



(0.0)



2



(1.4)



3



(2.2)



3



(2.1)



Thoracic Vertebra



Centrum No. 8  Dumbbellshaped



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



1



(4.3)



0



(0.0)



0



(0.0)



0



(0.0)



Fetal Incidences



No (%)



1



(0.8)



0



(0.0)



0



(0.0)



0



(0.0)



Centrum No. 9  Dumbbellshaped



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



2



(9.1)



3



(13.0)



3



(13.6)



Fetal Incidences



No (%)



0



(0.0)



2



(1.4)



3



(2.2)



3



(2.1)



Centrum No. 10  Dumbbellshaped



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



6



(26.1)



7



(31.8)



9



(39.1)



9



(40.9)



Fetal Incidences



No (%)



6



(4.5)



11



(7.7)



9



(6.6)



9



(6.2)



Centrum No. 11  Dumbbellshaped



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



7



(30.4)



2



(9.1)



8



(34.8)



8



(36.4)



Fetal Incidences



No (%)



10



(7.6)



3



(2.1)



10



(7.4)



12



(8.2)



Centrum No. 12  Dumbbellshaped



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



7



(30.4)



8



(36.4)



9



(39.1)



10



(45.5)



Fetal Incidences



No (%)



10



(7.6)



10



(7.0)



9



(6.6)



14



(9.6)



Centrum No. 13  Dumbbellshaped



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



4



(17.4)



4



(18.2)



2



(8.7)



6



(27.3)



Fetal Incidences



No (%)



4



(3.0)



4



(2.8)



2



(1.5)



8



(5.5)



Centrum No. 11  Hemicentric



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.00



0



(0.0)



1



(4.3)



0



(0.0)



Fetal Incidences



No (%)



0



(0.0)



0



(0.0)



1



(0.7)



0



(0.0)



Centrum No. 12  Hemicentric



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



0



(0.0)



1



(4.3)



0



(0.0)



Fetal Incidences



No (%)



0



(0.0)



0



(0.0)



1



(0.7)



0



(0.0)



Sacral Vertebra



Arch No. 1  Ossification Site



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



0



(0.0)



0



(0.0)



1



(4.5)



Fetal Incidences



No (%)



0



(0.0)



0



(0.0)



0



(0.0)



1



(0.7)



Lumbar Vertebra



Centrum No. 2  Dumbbellshaped



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.00



1



(4.5)



0



(0.0)



0



(0.0)



Fetal Incidences



No (%)



0



(0.0)



1



(0.7)



0



(0.0)



0



(0.0)



Forelimb



Metacarpal No. 5  Unossified (Bilateral)



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



3



(13.6)



2



(8.70



3



(13.6)



Fetal Incidences



No (%)



0



(0.0)



4



(2.8)



2



(1.5)



3



(2.1)



Metacarpal No. 5  Incomplete
ossification (Bilateral) Litter



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



1



(4.3)



2



(9.1)



0



(0.0)



4



(18.2)



Fetal Incidences



No (%)



1



(0.8)



2



(1.4)



0



(0.0)



4



(2.7)



Proximal Phalanges  Unossified
(Bilateral)



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



18



(78.3)



12



(54.5)



10



(43.5)



15



(68.2)



Fetal Incidences



No (%)



20



(15.2)



18



(12.6)



14



(10.3)



18



(12.3)



Proximal Phalanges  Incomplete
ossification (Bilateral) Litter



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



1



(4.3)



3



(13.6)



3



(13.0)



1



(4.5)



Fetal Incidences



No (%)



1



(0.8)



3



92.1)



4



(2.9)



1



(0.7)



Pelvic Girdle



Pubis  Incomplete ossification
(Unilateral)



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



0



(0.0)



0



(0.0)



1



(4.5)



Fetal Incidences



No (%)



0



(0.0)



0



(0.0)



0



(0.0)



1



(0.7)



Malformations



Skull



Parietal bones - fused



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



1



(4.3)



0



(0.0)



0



(0.0)



0



(0.0)



Fetal Incidences



No (%)



1



(0.8)



0



(0.0)



0



(0.0)



0



(0.0)



Ribs



Rib No. 12 - Absent (unilateral)



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



1



(4.3)



0



(0.0)



0



(0.0)



0



(0.0)



Fetal Incidences



No (%)



1



(0.8)



0



(0.0)



0



(0.0)



0



(0.0)



Rib No. 11 and 12 - Fused



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



0



(0.0)



1



(4.3)



0



(0.0)



Fetal Incidences



No (%)



0



(0.0)



0



(0.0)



1



(0.7)



0



(0.0)



Rib No. 12 and 13 - Fused



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



1



(4.3)



0



(0.0)



0



(0.0)



0



(0.0)



Fetal Incidences



No (%)



1



(0.8)



0



(0.0)



0



(0.0)



0



(0.0)



Rudimentary (bilateral)



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



1



(4.5)



1



(4.3)



1



(4.5)



Fetal Incidences



No (%)



0



(0.0)



1



(0.7)



1



(0.7)



1



(0.7)



Thoracic Vertebra



Centrum No. 10 - Split



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



1



(4.3)



2



(9.1)



0



(0.0)



1



(4.5)



Fetal Incidences



No (%)



1



(0.8)



2



(1.4)



0



(0.0)



1



(0.7)



Centrum No. 11 - Split



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



2



(9.1)



2



(8.7)



3



(13.6)



Fetal Incidences



No (%)



0



(0.0)



2



(1.4)



2



(1.5)



3



(2.1)



Centrum No. 12 - Split



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



1



(4.3)



1



(4.5)



1



(4.3)



2



(9.1)



Fetal Incidences



No (%)



1



(0.8)



1



(0.7)



1



(0.7)



2



(1.4)



Centrum No. 13 - Split



 



 



 



 



 



 



 



 



Litter Incidences



No (%)



0



(0.0)



0



(0.0)



1



(4.3)



0



(0.0)



Fetal Incidences



No (%)



0



(0.0)



0



(0.0)



1



(0.7)



0



(0.0)



 

Applicant's summary and conclusion

Conclusions:
In a pre-natal developmental toxicity study conducted in Sprague Dawley Rats, the NOAEL of the test substance for both maternal and fetal toxicity was determined to be 1000 mg/kg body weight/day under the experimental conditions employed.
Executive summary:

The developmental toxicity of the test item was determined using OECD Guideline 414 under GLP conditions. The study used spraque dawley rats from an in-house line to test the daily oral administration via gavage of the test item using Carboxymethyl cellulose as vehicle. Based on a preliminary dose range study doses of 100, 300 and 1000 mg/kg bw/day were set with a concurrent vehicle control. On day 5 until day 19 of gestation, the test item was administered orally once daily to 25 pregnant females per group. During this time weight, food consumption, clinical signs of toxicity, mortality were observed. On day 20 all animals were euthanized, and tissue, organs and fetuses extracted and examined. There were no clinical signs of toxicity, mortality, morbidity, changes in body weight, food consumption changes, gross pathological or visceral changes in any group of maternal animals. Pregnancy rate, mean gravid uterus weight, live fetuses, mean litter size, mean sex ratio, mean number of implantation sites, mean number of resorptions, mean number of corpora lutae were similarly not affected by the treatment. Also unaffected were pre-and post- implantation losses, mean absolute and relative thyroid along with parathyropid weight, histopathology of the thryoid along with the parathyroid. The fetal toxicity assessment concluded no effect on mean fetal weight or crown-rump length in either sex. No test item related effects on developmental or structural alterations could be observed. Some visceral and skeletal alterations are considered incidental and not related to the test item as they occur in all groups, including the control and are not dose dependent. During the study no test item related effects in maternal or offspring animals could be observed. Therefore the "no observed adverse effect level (NOAEL) for reproduction and developmental toxicity is the highest dose used in this study of 1000mg/kg bw /day for maternal animals and offspring in rats.