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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:
4 Oct 2019 - 10 Nov 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
25th June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
Cinnamaldehyde
EC Number:
203-213-9
EC Name:
Cinnamaldehyde
Cas Number:
104-55-2
Molecular formula:
C9H8O
IUPAC Name:
3-phenylacrylaldehyde
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Animals were procured from CPCSEA approved vendor (Cadila Pharmaceuticals Limited, Ahmedabad; CPCSEA registration no. 161/PO/RcBiBt/S/99/CPCSEA)
- Age at study initiation: 10 - 12 weeks
- Housing: 1 - 3 rats in polycarbonate cage, during mating 2 females and 1 males per cage, pregnant femaleswere housed individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days prior to estrous cycle evaluation

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.2 - 23.9
- Humidity (%): 46.2 - 66.5
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was weighed and dissolved in corn oil to achieve the desired concentration of the test item at each dose level. Formulations were prepared on the same day or one day prior to dose administration and stored at room temperature until usage as the test item stability was proven up to 24 h in a previous validation study. At the time of dosing, dose formulations were kept on a magnetic stirrer for maintaining homogeneity of test formulation.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil was selected as vehicle based on a previously performed study. Corn oil is widely used as vehicle in oral toxicity studies and it is well tolerated at the selected dose volume
- Concentration in vehicle: 0, 31.25, 62.5, 125 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dose formulation samples from all dose groups were analysed. Two replicates of approximately 2 mL samples from each upper, middle and lower level were analysed during the first and the last week of treatment using a validated analytical method.

The obtained concentrations of the analysed formulations were 31.333, 62.081 and 124.428 mg/mL for the first week of treatment and 31.493, 61.284 and 122.898 mg/mL for the last week of treatment for low, mid and high dose levels, respectively. All these values fall in the acceptance limits of ±15% of the nominal concentrations for all the formulations.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:2
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
Pregnant females were exposed to test item from GD5 to GD19
Frequency of treatment:
once daily
Duration of test:
Until GD20
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
125 mg/kg bw/day (nominal)
Remarks:
The substance was given by oral gavage to 25 pregnant Wistar rats at 0 (vehicle), 250, 500 and 1000 mg/kg bw/day from GD 5 to 19. Due to unexpected mortality at 1000 mg/kg, an additional group of 25 pregnant Wistar rats treated at 125 mg/kg bw/day from GD 5 to 19 was included.
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
The substance was given by oral gavage to 25 pregnant Wistar rats at 0 (vehicle), 250, 500 and 1000 mg/kg bw/day from GD 5 to 19. Due to unexpected mortality at 1000 mg/kg, an additional group of 25 pregnant Wistar rats treated at 125 mg/kg bw/day from GD 5 to 19 was included.
No. of animals per sex per dose:
25 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: in consultation with the sponsor based on available information on the test substance
- Rationale for animal assignment (if not random): Randomization was done based on body weight of the pregnant females on GD0. The animals were assigned in an unbiased manner to control and treatment groups manually as per internal SOP. Females inseminated from the same male were evenly distributed across the groups. Individual body weights were within ±20% of the respective group mean after randomization. The mean group body weights on the day of randomization were analyzed by One-way ANOVA and it was found that the group means were statistically comparable to each group.
- Time of day for (rat) dam blood sampling: morning

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily, approx. 1 h after dose administration

BODY WEIGHT: Yes
- Time schedule for examinations: at time of receipt, GD0, GD5, GD8, GD11, GD14, GD17, GD20

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
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: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
- Anogenital distance of all live rodent pups: yes
Statistics:
Raw data were processed using statistical software "Sigma Plot 14.0" (Supplied by Cranes Software International Ltd. Bangalore). The mean and standard deviation were calculated using the software and all data were summarized in tabular form. All continuous data (body weight, food consumption, hormone estimation, absolute and relative organ weights, maternal and pup parameters) were checked for normality using Shapiro Wilk test. All homogenous data was analysed using ANOVA and data showing significance in their variances was subjected to Dunnett's t-test, Dunn's Test, Kruskal-Wallis, ANOVA on ranks, Gross, skeletal and visceral abnormalities were represented as both, the total no. of fetuses and litters affected and the percentage of fetuses and litters affected. Further, the data of % incidences of abnormalities were analyzed for dose dependency by Pearson's correlation. P values of < 0.05 were deemed to be statistically significant.
Indices:
Pre-implantations loss = [(no. of corpora lutea − no. of implantations)/ no. of corpora lutea] x 100

Post-implantation loss = [(no. of resorptions)/ no. of total implantations] x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No treatment-related clinical signs or symptoms were observed in any animal up to 250 mg/kg bw/d. Symptoms of hypothermia, lethargy, prostration and excessive salivation were observed in all animals of the 500 mg/kg bw/d group starting from the first day of dosing until the termination. These clinical signs were evident from 1 h after dosing until the evening observation. However, no clinical signs were observed at the morning observation. This indicates that these signs were reversible in nature. No treatment-related clinical signs were observed in nesting/pregnancy behaviour in any of the animals.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One animal from the high dose group was found dead during morning observation on GD9 Although gross lesions on lung and stomach were observed in this animal, the exact cause of death could not be determined.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weight of pregnant and non-pregnant females remained camparable on GD 0, 5, 8, 11, 14, 17 and 20 for the control, 125 and 250 mg/kg bw/d group. The mean body weights of pregnant females on GD17 was significantly reduced in the 500 mg/kg bw/d group as compared to the control (p<0.05), whereas it remained comparable to the control on GD 0, 5, 8, 11 and 14. The % change in body weights with respect to GD0 was also statistically comparable among all the dose groups of pregnant and non-pregnant females, except for the decreased body weight gain in the 500 mg/kg bw/d group as compared to the control on GD17 (p<0.05). A trend of decrease in body weight and body weight gain was observed in pregnant females of the 500 mg/kg bw/d group compared to the control on GD20. Although it remained statistically insignificant due to high variation in the groups, the change in body weight and body weight gain at 500 mg/kg bw/d was considered as an effect of the test item.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The mean food consumed per day was significantly reduced in the 125 and 500 mg/kg bw/d groups on GD8 and GD11 when compared to the control group, while it remained statistically comparable among the groups at all other time points. Although the observed decrease in the food consumption of the 500 mg/kg bw/d group was considered to be an effect of the test item administration, it lacks toxicological significance due to its transient occurrence (significant reduction only on GD8 and GD11 and not on GD14, 17 and 20). On the contrary, the significant change observed in the 125 mg/kg bw/d group lacked dose-dependency and was therefore considered to be an inconsequential effect.
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:
no effects observed
Description (incidence and severity):
Thyroid hormones (T3 and T4) and thyroid stimulating hormone (TSH) levels in the sera were found to be statistically comparable among the control and treatment groups.
Endocrine findings:
no effects observed
Description (incidence and severity):
Thyroid hormones (T3 and T4) and thyroid stimulating hormone (TSH) levels in the sera were found to be statistically comparable among the control and treatment groups. No significant difference in the absolute and relative weight of thyroid and parathyroid between the treatment groups and the control group were found. Three females in the 500 mg/kg bw/d group exhibited unilateral and bilateral congenital cysts in the thyroid gland. These were considered to be spontaneous in nature and not attributable to the test item
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No treatment-related clinical signs were observed in nesting/pregnancy behaviour in any of the animals.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No significant difference in the absolute and relative weight of uterus, ovary, thyroid and parathyroid between the treatment groups and the control group were found.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
No gross findings were observed at 0 or 125 mg/kg. At 250 mg/kg, two animals showed pathological alterations in stomach which included white spots of muscular portion, increased size/swelling of muscular portions, and swelling of glandular portion. At 500 mg/kg, a total of 15 female animals showed pathological alterations in stomach which included white spots of muscular tissue, increased size of muscular portion, and increased size/swelling of glandular portion and red discolouration of the lung.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):

Microscopic findings at 250 mg/kg included focal hyperkeratosis in stomach in one animal (minimal in severity). At 500 mg/kg, microscopic findings included focal/multifocal hyperkeratosis in stomach; focal, erosion and oedema of muscular tissue in stomach, lymphocyte infiltration in muscular tissue in stomach, diffuse degeneration of glandular tissue in stomach and focal alveolar haemorrhage in lung in 7 animals. Minimal to moderate hyperkeratosis of stomach observed in most of the animals from the 500 mg/kg bw/d group were considered to be test item-related. This can be due to local irritation of the gastric mucosa.
Three females in the 500 mg/kg bw/d group exhibited unilateral and bilateral congenital cysts in the thyroid gland. These were considered to be spontaneous in nature and not attributable to the test item.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Maternal developmental toxicity

Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The number of implantation sites and the pre-and post-implantation loss (%) remained unchanged in treatment groups when compared to control.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No significant changes in the number of resorptions were observed in any dose groups.
Early or late resorptions:
no effects observed
Description (incidence and severity):
The early and late resorptions were found to be comparable among all the treatment groups and control group.
Dead fetuses:
no effects observed
Description (incidence and severity):
No dead foetuses were observed in any dose groups.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
effects observed, treatment-related
Description (incidence and severity):
At termination 18/25, 24/25, 17/25 and 13/25 females were found to be pregnant from the control 125 mg/kg bw, 250 mg/kg bw, and 500 mg/kg bw dose groups, respectively. Pregnancy rates were calculated as 72%, 96%, 68% and 52% for the control, 125 mg/kg bw, 250 mg/kg bw/day, and 500 mg/kg dose groups, respectively. This observation indicates a statistically significant and dose-dependent effect of the test item on the pregnancy rate.
Other effects:
no effects observed

Effect levels (maternal animals)

open allclose all
Dose descriptor:
LOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
changes in number of pregnant
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
changes in number of pregnant
Dose descriptor:
LOAEL
Remarks:
local
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
gross pathology
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
125 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
gross pathology
histopathology: non-neoplastic

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 500 mg/kg, a significant decrease in female foetal weight (by 8.2%) and a significant decrease in sex-combined foetal weight (by 6.4%) were observed as compared to the control group. No other significant changes in foetal weight were observed.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No reduction in number of live offspring was observed in any dose group when compared to control.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex ratios were comparable in all groups.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No significant differences in litter size were observed in any groups.
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
No significant differencein AGD were observed in any groups.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Gross external observation of foetuses revealed that 2.09, 3.79, 5.00 and 6.60 percentage of foetuses from control (G1), 125 (G5), 250 (G2) and 500 (G3) mg/kg bw/d, respectively, showed malformations/variations. The variation included haemorrhage:1.57 (G1), 2.07 (G5), 3.89 (G2), 4.72 (G3) % of foetuses and the malformations include: retarded growth (Runt): 0.52 (G1), 1.38 (G5), 0.56 (G2) and 1.89 (G3) % of foetuses; dome-shaped head: 1.03 (G5) and 0.94 (G3) % of foetuses; Anury (absence of tail): 0.56 (G2) % of foetuses. A significant correlation was found for the doses of test item and the total % of fetuses with malformations/variations (P<0.05). Further, when individual malformations/variations were compared, a significant increase in incidences of haemorrhage was observed in fetuses of the 500 mg/kg bw/d group as compared to the control group (P<0.05). However, no significant correlation was observed when litter was used as the unit for comparing the malformations/variations or when data of individual malformations/variations (other than haemorrhage) were compared. These observations were found to be independent of the treatment and rather seem to be spontaneous in nature.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
A number of skeletal variations/malformations were observed, however none of them were attributed to the test chemical since they lacked dose dependency and (or) were considered common to developing foetuses.
The following skeletal malformations/variations were observed in the skull: incomplete ossification of Frontal and Parietal observed in 1 /57 foetus at 500 mg/kg, dome-shaped Frontal and Parietal found in 1/150 foetus at 125 mg/kg, 1/57 at 500 mg/kg, flattened Parietal observed in 1/150 at 125 mg/kg and 1/57 at 500 mg/kg, fused Nasal, Premaxilla and Maxilla found in 1/150 at 250 mg/kg, elongated and bipartite Interparietal in 1/94 foetus at 250 mg/kg, small triangular-shaped Interparietal observed in 1/150 at 125 mg/kg, absence of Zygomatic in 2/94 at 250 mg/kg, absence of Hyoid in 2/100 foetuses at 0 mg/kg. Fused ribs were observed in 1/94 foetuses at 250 mg/kg, branched ribs in 1/57 foetuses at 500 mg/kg, misaligned ribs at 1/94 at 250 mg/kg and 2/57 foetuses at 500 mg/kg. Misaligned ribs were seen in 2/57 foetuses at 500 mg/kg, absent Thoracic, Lumbar, Sacral and caudal Vertebrae in 1/94 foetuses at 250 mg/kg.
Visceral malformations:
no effects observed
Description (incidence and severity):
No variations/malformations were observed during visceral and head razor examinations in any of the groups.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
A significant difference in crown to rump length was observed in the 125 mg/kg bw/d group when compared to the control (P<0.001). Such change was not observed in any other of the dose group and therefore it was considered to be an incidental finding.

Effect levels (fetuses)

open allclose all
Dose descriptor:
LOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Table 1. Table for Maternal Evaluation


 








































































































Groups



Control



Low Dose



Mid Dose



High Dose



Dose (mg/kg body weight)



0



125



250



500



Initial animals per group



25



25



25



25



Confirmed Pregnancy at necropsy



18



24



17



13



Pregnancy rate (%)



72



96



68



52



No. of Corpora lutea



14.50 ± 2.62



13.33 ±1.97



13.94 ± 2.73



13.15 ± 2.34



No. of implantation sites



12.44 ± 3.17



12.92 ± 1.84



12.24 ± 3.17



11.08 ± 3.80



No. of resorptions



1.83 ± 2.81



0.83 ± 1.01



1.65 ± 2.26



1.92 ± 3.48



Pre-implantation loss (%)



13.15 ± 20.98



2.91 ± 5.41



12.82 ±15.23



16.66 ± 25.53



Post-implantation loss (%)



14.11 ± 22.04



6.19 ± 6.77



15.23 ± 22.21



18.66 ± 34.90



Litter size



10.61 ± 3.81



12.08 ± 1.69



10.59 ± 4.18



8.83 ± 4.95



No of live foetuses



10.61 ± 3.81



12.08 ± 1.69



10.59 ± 4.18



8.83 ± 4.95



No of dead foetuses



0



0



0



0



Live Foetuses (%)



100



100



100



100



Values are represented as Mean± SD.


Pre-implantations loss = [(no. of corpora lutea − no. of implantations)/ no. of corpora lutea] x 100


Post-implantation loss = [(no. of resorptions)/ no. of total implantations] x 100


 


Table 2. Table for Body Weight


 
























































































































































Pregnant Females


 



Group



Dose (mg/kg)



 


 



Day of gestation and body weight in grams



0



5



8



11



14



17



20



Control



0



Mean



215.56



236.39



245.11



257.50



272.56



298.06



332.00



SD



13.26



13.14



13.53



14.36



14.97



17.06



25.65



N



18



18



18



18



18



18



18



 Low Dose



125



Mean



214.25



234.92



243.00



256.83



273.13



298.08



334.58



SD



12.81



9.66



10.44



10.87



12.16



14.10



15.37



N



24



24



24



24



24



24



24



  Mid Dose



250



Mean



213.94



237.00



244.94



258.06



272.41



294.35



330.18



SD



12.23



14.83



16.37



17.83



21.72



25.19



32.90



N



17



17



17



17



17



17



17



 High Dose



500



Mean



215.54



237.54



241.69



247.67



263.83



279.08↓



309.00



SD



11.64



15.23



13.93



10.80



13.03



21.94



30.36



N



13



13



13



12



12



12



12



↓: Decreased as compared to control (P<0.05)


 
























































































































































Non-Pregnant Female


 



Group



Dose (mg/kg)



 



Day of gestation and body weight in grams



0



5



8



11



14



17



20



 Control



0



Mean



215.14



233.57



240.29



244.29



244.14



246.14



250.14



SD



11.96



12.22



10.27



17.04



16.93



17.14



20.45



N



7



7



7



7



7



7



7



 Low Dose



125



Mean



211.00



219.00



219.00



223.00



225.00



230.00



238.00



SD



./.



./.



./.



./.



./.



./.



./.



N



1



1



1



1



1



1



1



 Mid Dose



250



Mean



222.00



240.38



242.25



249.88



247.75



250.25



253.00



SD



6.72



16.10



10.65



11.21



9.38



9.91



11.30



N



8



8



8



8



8



8



8



 High Dose



500



Mean



223.33



242.58



242.50



245.33



242.83



242.25



248.33



SD



15.20



22.44



19.64



20.83



15.80



15.78



16.74



N



12



12



12



12



12



12



12



 


Table 3. Table for Body Weight Change


 











































































































































Pregnant Females


 



Group



Dose (mg/kg)



 



Gestation Day and body weight change (%)



5



8



11



14



17



20



 Control



0



Mean



9.74



13.80



19.61



26.62



38.48



54.15



SD



2.76



3.32



5.27



5.92



6.98



9.50



N



18



18



18



18



18



18



 Low Dose



125



Mean



9.86



13.64



20.12



27.74



39.43



56.56



SD



5.12



5.43



6.03



6.64



7.85



9.72



N



24



24



24



24



24



24



 Mid Dose



250



Mean



10.80



14.51



20.64



27.32



37.55



54.29



SD



3.85



4.48



5.23



7.07



8.19



12.13



N



17



17



17



17



17



17



 High Dose



500



Mean



10.19



12.14



15.92



23.48



30.60↓



44.72



SD



3.27



2.70



2.86



4.04



8.72



14.17



N



13



13



12



12



12



12



↓: Decreased as compared to control (P<0.05)


 











































































































































Non-Pregnant Females


 



Group



Dose  (mg/kg)



 



Gestation Day and body weight change (%)



5



8



11



14



17



20



 Control



0



Mean



8.66



11.85



13.66



13.51



14.43



16.27



SD



4.35



5.23



7.35



5.65



5.52



7.20



N



7



7



7



7



7



7



 Low Dose



125



Mean



3.79



3.79



5.69



6.64



9.00



12.80



SD



./.



./.



./.



./.



./.



./.



N



1



1



1



1



1



1



 Mid Dose



250



Mean



8.19



9.12



12.55



11.60



12.73



13.95



SD



4.42



3.38



3.52



2.32



3.06



3.15



N



8



8



8



8



8



8



 High Dose



500



Mean



8.88



8.92



10.15



9.00



8.65



11.34



SD



9.71



9.58



9.78



7.38



5.89



5.80



N



12



12



12



12



12



12



 


Table 4. Table for External Gross Evaluation of Foetuses


 

















































































































































































































Group



 Control



 Low Dose



 Mid Dose



 High Dose



Dose level (mg/kg body weight)



0



125



250



500



Total No. of Litters with live foetuses



18



24



17



11



Total No. of live Foetuses



191



290



180



106



Total No. of Male Foetuses



98



133



89



61



Total No. of Female Foetuses



93



157



91



45



Weight of foetuses (g)



3.74 ± 0.59



3.69 ± 0.35



3.57 ± 0.38



3.50 ± 0.37↓



Weight of male foetuses (g)



3.82 ± 0.63



3.82 ± 0.30



3.64 ± 0.35



3.62 ± 0.32



Weight of female foetuses (g)



3.65± 0.54



3.57 ± 0.35



3.51 ± 0.39



3.35 ± 0.40↓



Weight of Placenta (g)



0.47 ± 0.09



0.49 ±0.07



0.50 ± 0.09



0.45 ± 0.09



Crown to rump length (cm)



3.68 ± 0.23



3.41 ± 0.31↓↓↓



3.68 ± 0.28



3.67 ± 0.28



AGD of Male foetuses (mm)



3.20 ± 0.33



3.10 ± 0.25



3.17 ± 0.26



3.10 ± 0.28



AGD of Female foetuses (mm)



1.30 ± 0.21



1.28 ± 0.16



1.30 ± 0.35



1.27 ± 0.14



Normalised AGD of Male foetuses



2.06 ± 0.23



1.98 ± 0.18



2.06 ± 0.16



2.02 ± 0.16



Normalised AGD of Female foetuses



0.85 ± 0.14



0.84 ± 0.10



0.86 ± 0.27



0.85 ± 0.10



Male/Female sex ratio (per dam)



1.31 ± 1.28



0.94 ± 0.45



1.25 ± 1.03



1.53 ± 1.28



External observations



Total no. of Foetuses with No Abnormality Detected



187



279



171



99



Total no. of Foetuses with abnormalities



4



11



9



7



Foetuses with abnormalities (%)



2.09



3.79



5.00



6.60 ↑



Litters with abnormalities (%)



5.56



33.33



35.29



27.27


 

Abnormalities



Haemorrhage (V)



3(1)



6(5)



7(5)



5(3)



Haemorrhage (%)



1.57(5.56)



2.07(20.83)



3.89(29.41)



4.72(27,27) ↑



Runt (M)



1(1)



4(4)



1(1)



2(2)



Runt (%)



0.52(5.56)



1.38(16.67)



0.56(5.88)



1.89(18.18)



Anury (M)



0(0)



0(0)



1(1)



0(0)



Anury (%)



0(0)



0(0)



0.56(5.88)



0(0)



Dome-shaped Head (M)



0(0)



3(3)



0(0)



1(1)



Dome-shaped Head (%)



0(0)



1.03(12.50)



0(0)



0.94(9.09)



Values are represented as Mean± SD; AGD: Anogenital distance; Normalised AGD: AGD/ Body weight(1/3); the incidence of the individual defect is presented as a number/ Percent of foetuses (number/ percent of litters); Abnormalities: includes malformations and variations; M: Malformations; V: Variations ↓:Decreased as compared to control (P<0.05); ↓↓↓:Decreased as compared to control (P<0.001); ↑:Increased as compared to control (P<0.05)


 


Table 5. Table for Skeletal Anomalies of the Foetuses


 
























































































































































































































































Groups



 Control



 Low Dose



 Mid Dose



 High Dose



Dose (mg/kg body weight)



0



125



250



500



 



No.



%



No.



%



No.



%



No.



%



Foetuses examined



100



150



94



57



No abnormality detected



83



134



86



41



Total and % Foetuses with abnormalities



17



17.00



16



10.67



8



8.51



16



28.07



 



Skull



Incomplete ossification of Frontal & Parietal (V)



0



0



0



0



0



0



1



1.75



Dome-shaped frontal and Parietal (M)



0



0



1



0.67



0



0



1



1.75



Flattened Parietal (M)



0



0



1



0.67



0



0



0



0



Fused Nasal, Premaxilla & Maxilla (M)



0



0



1



0.67



0



0



0



0



Elongated, Bipartite Interparietal (M)



0



0



0



0



1



1.06



0



0



Small Triangular-shaped Interparietal (M)



0



0



1



0.67



0



0



0



0



Absence of Zygomatic (M)



0



0



0



0



2



2.13



0



0



Absence of Hyoid (M)



2



2



0



0



0



0



0



0



 



Ribs



Fused (M)



0



0



0



0



1



1.06



0



0



Branched (M)



0



0



0



0



0



0



1



1.75



Misaligned (M)



0



0



0



0



1



1.06



2



3.51



Absent (M)



0



0



0



0



0



0



0



0



 



Sternebrae



Unossified/Incompletely ossified Sternebrae (1 or more) (V)



13



13.0



12



8.0



5



5.32



13



22.81



Misaligned (M)



1



1



0



0



0



0



2



3.51



 



Vertebrae



Unossified Caudal vertebrae (V)



1



1



0



0



0



0



0



0



Thoracic, Lumbar, Sacral and caudal Vertebrae absent (M)



0



0



0



0



1



1.06



0



0



The incidence of the individual defect is presented as a number and percentages of litters


Abnormalities: includes malformations and variations; M: Malformations; V: Variations


 


Table 6. Table for Skeletal Anomalies of the Foetuses(with respect to Litters)


 













































































































































































































































Groups



 Control



 Low Dose



 Mid Dose



 High Dose



Dose (mg/kg body weight)



0



125



250



500



 



No.



%



No.



%



No.



%



No.



%



Litters examined



18



24



17



11



No abnormality detected



7



11



13



2



Total and % Litters with abnormalities



11



61.11



13



54.17



4



23.53



9



81.82



 



Skull



Incomplete ossification of Frontal & Parietal (V)



0



0



0



0



0



0



1



9.09



Dome-shaped frontal and Parietal (M)



0



0



1



4.17



0



0



1



9.09



Flattened Parietal (M)



0



0



1



4.17



0



0



0



0



Fused Nasal, Premaxilla & Maxilla (M)



0



0



1



4.17



0



0



0



0



Elongated, Bipartite Interparietal (M)



0



0



0



0



1



5.88



0



0



Small Triangular-shaped Interparietal (M)



0



0



1



4.17



0



0



0



0



Absence of Zygomatic (M)



0



0



0



0



2



11.76



0



0



Absence of Hyoid (M)



2



11.11



0



0



0



0



0



0



 



Ribs



Fused (M)



0



0



0



0



1



5.88



0



0



Branched (M)



0



0



0



0



0



0



1



9.09



Misaligned (M)



0



0



0



0



1



5.88



2



18.18



 



Sternebrae



Unossified/Incompletely ossified Sternebrae (1 or more) (V)



9



50.0



11



45.83



3



17.65



6



54.55



Misaligned (M)



1



5.56



0



0



0



0



2



18.18



 



Vertebrae



Unossified Caudal vertebrae (V)



1



5.56



0



0



0



0



0



0



Thoracic, Lumbar, Sacral and caudal Vertebrae absent (M)



0



0



0



0



1



5.88



0



0



The incidence of the individual defect is presented as a number and percentages of litters


Abnormalities: includes malformations and variations; M: Malformations; V: Variations


 


Table 7. Table for Absolute Organ Weight


 

















































































































Pregnant



Group



Dose (mg/ Kg)



 



Body weight



Uterus



Ovaries



Right



Left



Control



0



Mean



332.00



61.73



0.0878



0.0818



SD



25.65



20.64



0.0152



0.0208



N



18



18



18



18



Low dose



125



Mean



334.58



66.61



0.0791



0.0828



SD



15.37



8.57



0.0197



0.0145



N



24



24



24



24



Mid dose



250



Mean



330.18



62.60



0.0860



0.0854



SD



32.90



25.27



0.0138



0.0183



N



17



17



17



17



High dose



500



Mean



309.00



49.70



0.0861



0.0753



SD



30.36



24.55



0.0202



0.0112



N



12



12



12



12



 

















































































































Non-Pregnant




Group



Dose (mg/ Kg)



 



Body weight



Uterus



Ovaries



Right



Left



Control



0



Mean



250.14



0.87



0.0634



0.0706



SD



20.45



0.14



0.0053



0.0083



N



7



7



7



7



Low dose



125



Mean



238.00



0.82



0.0655



0.0539



SD



./.



./.



./.



./.



N



1



1



1



1



Mid dose



250



Mean



253.00



0.88



0.1388



0.0712



SD



11.30



0.14



0.2047



0.0074



N



8



8



8



8



High dose



500



Mean



248.33



1.10



0.07



0.08



SD



16.74



0.41



0.0102



0.0100



N



12



12



12



12



 


Table 8. Table for Relative Organ weight to Body weight


 

















































































































Pregnant



Group



Dose (mg/ Kg)



 



Body weight



Uterus



Ovaries



Right



Left



Control



0



Mean



332.00



18.41



0.0264



0.0249



SD



25.65



5.63



0.0042



0.0073



N



18



18



18



18



Low dose



125



Mean



334.58



19.88



0.0237



0.0248



SD



15.37



2.13



0.0059



0.0043



N



24



24



24



24



Mid dose



250



Mean



330.18



18.68



0.0261



0.0259



SD



32.90



7.16



0.0040



0.0050



N



17



17



17



17



High dose



500



Mean



309.00



15.57



0.0278



0.0245



SD



30.36



7.21



0.0053



0.0035



N



12



12



12



12



 

















































































































Non Pregnant



Group



Dose (mg/ Kg)



 



Body weight



Uterus



Ovaries



Right



Left



Control



0



Mean



250.14



0.35



0.0255



0.0283



SD



20.45



0.07



0.0027



0.0033



N



7



7



7



7



Low dose



125



Mean



238.00



0.35



0.0275



0.0226



SD



./.



./.



./.



./.



N



1



1



1



1



Mid dose



250



Mean



253.00



0.35



0.0534



0.0282



SD



11.30



0.06



0.0761



0.0030



N



8



8



8



8



High dose



500



Mean



248.33



0.44



0.0277



0.0317



SD



16.74



0.16



0.0045



0.0048



N



12



12



12



12



 


Table 9. Table for Gross Pathology Observation


 




















































































































































































Pregnant Females



Group



Control



Low dose



Mid dose



High dose



Dose (mg/kg)



0



125



250



500



Total No. of TS Animals Observed



18



24



17



13



Organ & Lesion



No. of animals with abnormalities



0



0



0



7



No. of animals with no abnormalities



18



24



17



6



Stomach: Muscular portion



X



X



X


 

White Spot



X



X



X


 

Minimal



X



X



X



1



 Mild



X



X



X



1



Moderate



X



X



X



1



Severe



X



X



X


 

Increased size / swollen



X



X



X


 

Minimal



X



X



X



1



Mild



X



X



X



1



Moderate



X



X



X



1



Glandular portion



X



X



X


 

Increased size / Swollen



X



X



X


 

Moderate



X



X



X



1



Red Spot



X



X



X


 

Minimal



X



X



X



1



Lung: Red discoloration



X



X



X


 

Minimal



X



X



X



1



Mild



X



X



X


 

Moderate



X



X



X


 

Note:All organs/tissues as plan were observed for study


 




















































































































































































Non-Pregnant Females



Group



Control



Low dose



Mid dose



High dose



Dose (mg/kg)



0



125



250



500



Total No. of TS Animals Observed



7



1



8



12



Organ & Lesion



Abnormality Detected



0



0



2



8



No Abnormality Detected



7



1



6



4



Stomach: Muscular portion



X



X


  

White Spot



X



X


  

Minimal



X



X


  

 Mild



X



X



1



1



Moderate



X



X


 

4



Severe



X



X


 

1



Increased size / swollen



X



X


  

Minimal



X



X


  

Mild



X



X


 

1



Moderate



X



X



1


 

Glandular portion



X



X


  

Increased size / Swollen



X



X


  

Moderate



X



X



1



1



Red Spot



X



X


  

Minimal



X



X


 

1



Lung: Red discoloration



X



X


  

Minimal



X



X


 

1



Mild



X



X


  

Moderate



X



X


  

Note:All organs/tissues as plan were observed for study


 


Table 10. Table for Microscopic Observation


 



























































































































































































Pregnant Females



Group



Control



Low dose



Mid dose



High dose



Dose (mg/kg)



0



125



250



500



Total No. of TS Animals Observed



18



24



17



13



Organ & Lesion



Abnormality Detected



0



0



0



7



No Abnormality Detected



18



24



17



6



Stomach:Muscular Stomach


 

 


  

Hyperkeratosis


 

 


  

Focal Minimal



X



X



X



1



Focal Mild



X



X



X



2



Multifocal Mild



X



X



X



2



Diffuse Minimal



X



X



X



1



Erosion


 

 


  

Focal Mild



X



X



X



1



Focal Moderate



X



X



X



1



Edema


 

 


  

Diffuse minimal



X



X



X



1



Lymphocyte Infiltration


 

 


  

Focal Minimal



X



X



X



1



Glandular Stomach


 

 


  

Degeneration


 

 


  

Diffuse minimal



X



X



X



1



Lung: Haemorrhage Alveolar


 

 


  

Focal Minimal



X



X



X



1



Thyroid:Congenital Cyst 


 

 


  

Unilateral



X



X



X



1



 

















































































































































Non-Pregnant Females



Group



Control



Low dose



Mid dose



High dose



Dose (mg/kg)



0



125



250



500



Total No. of TS Animals Observed



7



1



8



12



Organ & Lesion



Abnormality Detected



0



0



1



8



No Abnormality Detected



7



1



1



4



Stomach:Muscular Stomach


 

 


  

Hyperkeratosis


 

 


  

Focal Minimal



X



X



1



1



Focal Mild



X



X


 

1



Focal moderate



X



X


 

1



Multifocal Minimal



X



X


 

1



Multifocal Mild



X



X


 

3



Diffuse Minimal



X



X


 

1



Glandular Stomach


 

 


  

Infiltration lymphocyte Mucosal


 

 


  

Focal Mild



X



X


 

1



Thyroid:Congenital Cyst 


 

 


  

   Bilateral



X



X


 

1



Unilateral



X



X


 

1



 


Table 11. Table for Head Razor and Visceral Observation Record of Foetuses


 









































Group



Control



Low dose



Mid dose



High dose



Dose (mg/kg)



0



125



250



500



Total No. of Foetuses Observed



63



132



86



50



Abnormality Detected



00



00



00



00



No Abnormality Detected



63



132



86



50


Applicant's summary and conclusion

Conclusions:
Based on the results obtained under the experimental conditions used in this study, it is concluded that the No Observed Adverse Effect Level (NOAEL) and Lowest Observed Adverse Effect Level (LOAEL) of Cinnamaldehyde (CAS No. 104-55-2) were 125 mg/kg bw/day and 250 mg/kg bw/day, respectively for general maternal toxicity. At 500 mg/kg bw/day of cinnamaldehyde (CAS No. 104-55-2) significant reductions were observed in pregnancy rate and mean foetal body weights. Therefore, the NOAEL and LOAEL of cinnamaldehyde (CAS No. 104-55-2) were 250 mg/kg bw/day and 500 mg/kg bw/day, respectively for developmental toxicity for both maternal and F1 generations.
Executive summary:

The present study aimed to provide information on general and developmental toxicological endpoints associated with the repeated oral administration of graduated doses of Cinnamaldehyde (CAS No.104-55-2) to Wistar rats. For this purpose, the OECD test guideline 414 was followed. The study was performed in compliance with GLP.
A total of 125 pregnant female rats were randomised into 5 experimental groups each containing 25 animals. The groups, viz., G1, G5, G2, G3 and G4 received 0, 125, 250, 500 and 1000 mg/kg bw/day, respectively. The initial doses of 0, 250, 500 and 1000 mg/kg bw (G1, G2, G3 and G4) were selected based on the available data in the literature and a limited dose range finding study. The additional dose level of 125 mg/kg bw (G5) was included due to unexpected mortality at 1000 mg/kg bw (G4). Dose administration was through an oral gavage and the vehicle used in this study was corn oil. All groups were administered with dose formulation once daily from gestation day (GD) 5 to 19 (one day prior to scheduled sacrifice).
To ensure the accurate administration of the test item, the concentrations and homogeneity of the dose formulation were verified through formulation analyses in the first and last weeks of treatment. The results of these were found within the acceptable limits in both occasions.
Observations on the animals encompassed mortality/morbidity, onset of any clinical signs/symptoms, body weight, body weight changes, feed consumption, pregnancy observations, foetal developmental parameters, serum hormone levels, gross pathology and histopathology.
The results showed that Cinnamaldehyde (CAS No. 104-55-2) does not cause mortality up to doses of 250 mg/kg bw/d when administered orally to pregnant rats. However, mortality was observed for all animals treated at 1000 mg/kg bw/d and for one animal treated at 500 mg/kg bw/d.
No clinical signs or symptoms were observed in animals up to doses of 250 mg/kg bw/d (G2) throughout the duration of the experiment. At least one of the following symptoms was observed in all animals treated at 500 mg/kg bw (G3) from GD 5, 6 or 7 and onwards: hypothermia, lethargy, prostration and excessive salivation. At 1000 mg/kg bw (G4) at least one of the following symptoms were observed in most of the animals from GD 5 and onwards: abdominal breathing, abnormal gait, bradycardia, hypothermia, lethargy, prostration and excessive salivation. The mean body weight and percent change in body weight with respect to GD 0 remained comparable for G1, G5, G2 and G3 for all the time-points recorded with the exception of GD 17 wherein significant reductions in both the parameters were observed for G3 (500 mg/kg) with respect to the control group. Also, a trend of decrease in body weight and body weight gain was observed in pregnant females of G3 (500 mg/kg bw) as compared to G1 (Control) group on GD 20. Significant decrease in body weight and body weight gain on GD 8 was observed for G4 (1000 mg/kg bw) when compared with G1 (control). Further, feed consumption was reduced significantly on GD 8 for G5 (125 mg/kg bw), G3 (500 mg/kg bw) and G4 (1000 mg/kg bw) and on GD11, it was reduced significantly in G5 (125 mg/kg bw) and G3 (500 mg/kg bw) groups as compared to G1 (control) group. It remained statistically comparable among the experimental groups at every other point of observation. The changes in body weight, body weight gain and feed consumption were neither consistent nor did they seem dose-dependent for the dose levels up to 250 mg/kg bw and hence presume to be toxicologically insignificant. However, the significant difference observed in body weight and body weight gain in G3 (500 mg/kg body weight) and G4 (1000 mg/kg body weight) group was considered to be an effect of test item administration.
Thyroid hormones (T3 and T4) and thyroid stimulating hormone (TSH) levels remained comparable across the groups for pregnant and non-pregnant females.
At termination 18/25, 24/25, 17/25, 13/25 and 9/25 females were found to be pregnant from G1 (control), G5 (125 mg/kg bw), G2 (250 mg/kg bw), G3 (500 mg/kg bw) and G4 (1000 mg/kg bw) group respectively. This observation indicates statistically significant and dose-dependent adverse effect of Cinnamaldehyde (CAS No. 104-55-2) on the pregnancy rate at 500 mg/kg bw and 1000 mg/kg bw. However, other maternal evaluation parameters including numbers of corpora lutea, implantation sites, resorptions and percentage of pre- and post-implantation loss remained comparable between all the treatment groups with control group. Further, no significant difference was observed for mean litter size or sex ratio between control and treatment groups.
No treatment-related changes were observed in absolute or relative weights of uterus, ovary, thyroid and parathyroid of pregnant and non-pregnant females.
The weights of placenta also remained comparable amongst all the groups. A statistically significant change, which was observed in crown-rump lengths of G5 (125 mg/kg bw) foetuses, was not found to be dose-dependent or treatment-related. However, significant decrease observed in the weights of female foetuses and combined weights of foetuses (male and female) in G3 (500 mg/kg bw) group as compared to control group were deemed to be treatment-related. Anogenital distance (AGD) and normalised AGD remained comparable with control for both male and female foetuses up to 500 mg/kg bw. No treatment-related gross, skeletal, or visceral malformations or variations were observed in the study.
Gross pathological and microscopic examination of females revealed lesions of the stomach in G2 (250 mg/kg bw), G3 (500 mg/kg bw) and G4 (1000 mg/kg bw) group and of the lung in G3 (500 mg/kg bw) and G4 (1000 mg/kg bw) group. These lesions were found to be dose-dependent and hence, related to Cinnamaldehyde (CAS No. 104-55-2) exposure.

Based on the results obtained under the experimental conditions used in this study, it is concluded that the No Observed Adverse Effect Level (NOAEL) and Lowest Observed Adverse Effect Level (LOAEL) of Cinnamaldehyde (CAS No. 104-55-2) were 125 mg/kg bw/day and 250 mg/kg bw/day, respectively for general maternal toxicity. At 500 mg/kg bw/day of cinnamaldehyde (CAS No. 104-55-2) significant reductions were observed in pregnancy rate and mean foetal body weights. Therefore, the NOAEL and LOAEL of cinnamaldehyde (CAS No. 104-55-2) were 250 mg/kg bw/day and 500 mg/kg bw/day, respectively for developmental toxicity for both maternal and F1 generations.