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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from a study report.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
[As per OECD Guideline No. 421, “Reproduction/Developmental Toxicity Screening Test”, adopted on 29 July 2016]
Deviations:
no
Principles of method if other than guideline:
According to OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Remarks:
White to yellow
Details on test material:
Name: Cinnamyl alcohol
IUPAC Name: 3-Phenyl-2-propen-1-ol
CAS No. 104-54-1
Molecular Formula: C9-H10-O
Molecular Weight: 134.177 g/mol
SMILES: OC\C=C\c1ccccc1
Specific details on test material used for the study:
Name of Test Item: Cinnamyl Alcohol
Chemical Name (IUPAC): (2E)-3-phenylprop-2-en-1-ol
CAS No.: 104-54-1
Physical Appearance (with colour): White to yellow solid
Lot No.: L241381803
Date of Manufacture : March 2018
Date of Expiry: February 2023
Purity (As per Certificate of Analysis): GC - 98.53%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: In-house bred animals
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 9 wks
- Weight at study initiation: (P) Males: 250.25 to 297.51 g; Females: 200.85 to 249.90 g
- Fasting period before study: No Data Available
- Housing: Housing was done as follows:
- Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted food and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
- During acclimatization, two animals of same sex were housed.
- Pre mating:
- Per cage two animals of the same sex and group were housed.
- Cohabitation Period (mating) - Per cage two animals (one male and one female) of the same group were housed.
- 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. Sterilized paper shreds were provided as a nesting material for mated females from gestation day 20 onwards.
- Use of restrainers for preventing ingestion (if dermal): no
- Diet (e.g. ad libitum):
Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to the animals throughout the experimental period.
- Water (e.g. ad libitum):
Water was provided ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period:
Healthy and young adult animals were acclimatized for five days to experimental room conditions and females were screened for normal oestrous cycles (4 to 5 days) in a two weeks pre-treatment period before initiation of treatment and were observed for clinical signs once daily.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 to 23.2 deg 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: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test chemical formulations were prepared before dose administration and administered within stability period. The required quantity of test chemical 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 17.5, 35 and 70 mg/mL of test chemical 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 chemical formulations.

DIET PREPARATION
- Rate of preparation of diet (frequency): No Data Available
- Mixing appropriate amounts with (Type of food): No Data Available
- Storage temperature of food: No Data Available

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 17.5, 35 and 70 mg/mL
- Amount of vehicle (if gavage): 5 ml/kg bw
- Lot/batch no. (if required): L12017004 and L32011001
- Purity: No data available
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Column: Zorbax Eclipse plus C18, 4.6×250 mm ,5µm
Flow rate: 1.0 mL/min
Injection volume: 10 µL
Wavelength: 242 nm
Run time: 10 minutes
Column Oven Temperature: 25°C
Mobile Phase: Acetonitrile: Milli-Q Water (50:50 %, v/v)
Diluent: Acetonitrile
Method validation parameters evaluated for the test chemical met the acceptance criteria. The results obtained are within the specified limits. Thus, this method is suitable for the analysis of the test chemical for dose formulations in the proposed Toxicology Studies.
Details on mating procedure:
- M/F ratio per cage: The males and females were placed in 1:1 ratio.
- Length of cohabitation: The female was placed with the same male until pregnancy occured by evidence of sperm in the vaginal smear until two weeks.
- Proof of pregnancy: [sperm in vaginal smear] referred to as [day 0] of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [no]
- After successful mating each pregnant female was caged (how):Individually
- Any other deviations from standard protocol: No Data Available
Duration of treatment / exposure:
Males: 34 days
Females: Approximately 70 days
Frequency of treatment:
Once Daily
Duration of test:
133 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control Group
Dose / conc.:
87.5 mg/kg bw/day (actual dose received)
Remarks:
Low Dose Group
Dose / conc.:
175 mg/kg bw/day (actual dose received)
Remarks:
Mid Dose Group
Dose / conc.:
350 mg/kg bw/day (actual dose received)
Remarks:
High Dose Group
No. of animals per sex per dose:
12 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses were selected based upon the available literature and previously performed studies of the test chemicals and similar source chemicals.
- Rationale for animal assignment (if not random): The animals were randomly assigned to the groups on the basis of their body weights.
- Fasting period before blood sampling for clinical biochemistry: No Data Available
- Other: No Data Available

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: The animals were weighed at receipt, on the first day of dosing, weekly thereafter and at termination. The females were weighed on gestation days 0, 7, 14 and 20 during pregnancy and on days 1, 4, 7 and 13 during lactation period and on day 14 (fasting body weight).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations: No Data available

POST-MORTEM EXAMINATIONS: Yes
- Organs examined: All reproductive organs as well as visceral organs were grossly examined.

OTHER: Oestrus cyclicity was monitored for two weeks after the five days of acclimatization to evaluate the normal oestrus cycle (4 to 5 days). Only females with normal oestrus cyclicity were selected for the treatment. Vaginal smears were monitored daily from the beginning of the treatment period until evidence of mating. When obtaining vaginal/cervical cells, care was taken to avoid disturbance of mucosa, which may induce pseudopregnancy. The status of oestrus cyclicity of females was determined on termination day (lactation day 14).
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: No Data Available
Fetal examinations:
The day of littering was considered as lactation day 1 for the dam and postnatal day 1 for pups. The number of male/female pups born (live/dead/cannibalized) per litter were recorded and the mean litter size is reported. The sex ratio (m/f) at birth were calculated per litter. The live birth index (%) per litter was calculated. The number of pups survived/dead per litter were recorded during lactation period and presented as pup survival index (%) per litter between lactation day 1 to 4, 4 to 7 and 7 to 13. The sex ratio on lactation day 4, 7 and 13 was calculated per litter.
Statistics:
The statistical analysis includes One-way ANOVA with Dunnett’s post test, Kruskal-Wallis and Chi-square test. The computer printout of the data (in the form of appendix) was verified with the raw data. After verification, the data was subjected to various statistical analyses using SPSS software version 22. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05).
Indices:
Male Mating Index (%), Male Fertility Index (%), Female Mating Index (%), Female Fertility Index (%), Pre-coital Interval (Days), Pre-coital Interval (Days), Gestation Index (%), Parturition Index (%) and Mean number of Implantations (%), Post-Implantation Loss, Post-implantation Loss/litter (No.), Post-natal Loss on lactation day 13 (No.), Post natal loss on lactation day 13 (%), Live Birth Index (%) per dam, Pup Survival index (%) on lactation day 4/7/13, Sex ratio (M/F), Percentage of male/female offspring (%) and Ano-genital Distance to cube root of the body weight Ratio (AGD ratio).
Historical control data:
No Data Available

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no test chemical related clinical signs were observed in maternal animals at all the dose groups, tested till 350 mg/kg bw (high dose groups).
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
No case of mortality/morbidity was observed in maternal animals at all the dose groups, tested till 350 mg/kg bw (high dose groups).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no changes noted in mean body weight and percent change in mean body weight with respect to day 1 in maternal animals during the experimental period in dosed groups when compared with vehicle control group. Also, there were no changes noted in mean body weight and percent change in mean body weight gain during gestation period in any of the tested dose groups when compared with vehicle control group. There were no test item-related changes noted in mean body weight and percent change in mean body weight gain during lactation period in any of the dosed groups when compared with vehicle control group.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant decreases in the mean feed consumption were noted during gestation day 7 to 14 and 14 to 20 in group G4 when compared with vehicle control group. There were no changes noted in mean feed consumption during lactation period in any of the the tested dosed groups when compared with vehicle control group.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no changes observed in serum Thyroxine (T4) hormone levels at all the dose groups in either sexes when compared with vehicle control group.
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test chemical-related changes observed in both absolute and relative organ weights at any of the tested dose group maternal animals when compared with concurrent vehicle control group.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross pathological changes observed during necropsy in any of maternal animals in the tested dose groups and vehicle control group.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related histopathological findings noted in high dose group maternal animals.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
There were no test chemical related clinical signs were observed in maternal animals at all the dose groups, tested till 350 mg/kg bw (high dose groups). No case of mortality/morbidity was observed in maternal animals at all the dose groups, tested till 350 mg/kg bw (high dose groups). There were no changes noted in mean body weight and percent change in mean body weight with respect to day 1 in maternal animals during the experimental period in dosed groups when compared with vehicle control group. Also, there were no changes noted in mean body weight and percent change in mean body weight gain during gestation period in any of the tested dose groups when compared with vehicle control group. There were no test item-related changes noted in mean body weight and percent change in mean body weight gain during lactation period in any of the dosed groups when compared with vehicle control group. Statistically significant decreases in the mean feed consumption were noted during gestation day 7 to 14 and 14 to 20 in group G4 when compared with vehicle control group. There were no changes noted in mean feed consumption during lactation period in any of the the tested dosed groups when compared with vehicle control group. There were no changes observed in serum Thyroxine (T4) hormone levels at all the dose groups in either sexes when compared with vehicle control group. There were no test chemical-related changes observed in both absolute and relative organ weights at any of the tested dose group maternal animals when compared with concurrent vehicle control group. There were no gross pathological changes observed during necropsy in any of maternal animals in the tested dose groups and vehicle control group. There were no test item-related histopathological findings noted in high dose group maternal animals.

Maternal developmental toxicity

Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
One female aborted each from high dose group, mid dose group and low dose group, respectively. However, all the other females in these three groups acheived pregnancy, thus this occurence was considered to be non-treatment related.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There was no statistically significant difference in numper of implantation and post implantation losses in any of the dosed groups when compared with the vehicle control group.
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
no effects observed
Description (incidence and severity):
No mortalities were observed after the administration of the test chemical.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
No changes in the pregnancy duration was observed due to the aadministration of the test chemical.
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
One female each from low dose, mid dose and high dose groups, respectively did not completed pregnancy despite being confirmed mated. However, all the other females acheived pregnancy and thus this occurrence was not considered as test-chemical related.
Other effects:
not specified
Details on maternal toxic effects:
One female aborted each from high dose group, mid dose group and low dose group, respectively. However, all the other females in these three groups acheived pregnancy, thus this occurence was considered to be non-treatment related. There was no statistically significant difference in numper of implantation and post implantation losses in any of the dosed groups when compared with the vehicle control group. No mortalities were observed after the administration of the test chemical. No changes in the pregnancy duration was observed due to the aadministration of the test chemical. One female each from low dose, mid dose and high dose groups, respectively did not completed pregnancy despite being confirmed mated. However, all the other females acheived pregnancy and thus this occurrence was not considered as test-chemical related.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
350 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
maternal abnormalities
number of abortions
pre and post implantation loss
dead fetuses
changes in pregnancy duration
changes in number of pregnant
necropsy findings
Remarks on result:
not determinable due to absence of adverse toxic effects

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no changes observed in mean pup [both male and female] weight per litter recorded on post-natal day (PND) 1, 4, 7 and 13 in any of the tested dose group litters when compared with vehicle control group.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No test chemical-related mortalities were noted during daily observation of pups from treated and vehicle control group litters during post-natal period.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No test chemical-related changes in sex ratios were noted during daily observation of pups from treated and vehicle control group litters during post-natal period.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No test chemical-related changes were observed in litter sizes and weights during observation of pups from treated and vehicle control group litters during post-natal period.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
No changes in the post-natal survival were observed during observation of pups from treated and vehicle control group litters during post-natal period.
External malformations:
no effects observed
Description (incidence and severity):
No external anomalies were noted due to the administration of the test chemical during treated and vehicle control group litters during post-natal period.
Skeletal malformations:
not specified
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no gross pathological changes observed during visceral examination in necropsy in any of the adult animals and pups of both sexes in any of the tested dose groups and vehicle control group.
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
There were no changes observed in mean pup [both male and female] weight per litter recorded on post-natal day (PND) 1, 4, 7 and 13 in any of the tested dose group litters when compared with vehicle control group. No test chemical-related mortalities were noted during daily observation of pups from treated and vehicle control group litters during post-natal period. No test chemical-related changes in sex ratios were noted during daily observation of pups from treated and vehicle control group litters during post-natal period. No test chemical-related changes were observed in litter sizes and weights during observation of pups from treated and vehicle control group litters during post-natal period. No changes in the post-natal survival were observed during observation of pups from treated and vehicle control group litters during post-natal period. No external anomalies were noted due to the administration of the test chemical during treated and vehicle control group litters during post-natal period. There were no gross pathological changes observed during visceral examination in necropsy in any of the adult animals and pups of both sexes in any of the tested dose groups and vehicle control group.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
350 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
external malformations
visceral malformations
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no
Treatment related:
no

Applicant's summary and conclusion

Conclusions:
Based on all the available data, it was observed that the test chemical did not cause any maternal toxicity and also toxicity to fetus in the developmental stage and also after birth. Therefore, based on all the observations and obtained results, it was concluded that the NOAEL for the test chemical was 350 mg/kg bw.
Executive summary:

A reproduction/developmental toxicity screening test as per the GLP compliance and in accordance with OECD TG 421 was performed using male and female Sprague Dawley rats for assessing the potential of the test chemical to cause adverse effect on the reproductive potential and effects on the developing fetuses. In this study 12 animals per sex per group were included which were grouped into 4 groups i.e. 0 mg/kg bw (Vehicle Control Group), 87.5 mg/kg bw (Low Dose Group), 175 mg/kg bw (Mid Dose Group) and 350 mg/kg bw (High Dose Group), respectively. Males were dosed for a period of 35 days (two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period, while females were dosed for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13 (day 49 to day 67 ). The vehicle and test chemical formulations were administered orally by gavage at the dose volume of 5 mL/kg body weight. All the animals were observed once daily for clinical signs, twice daily for mortality and morbidity. The weekly body weight and weekly feed consumption was recorded once weekly (except during cohabitation) for all the animals. The serum thyroxine hormone (T4) levels were estimated for all males by ELISA method. The gross pathology and organ weighing were performed on the day of termination for all animals. Detailed histopathological examination was conducted on ovaries, testes and epididymis from the groups G1 and G4 animals with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure. All the males were evaluated for reproductive performance such as, mating and fertility index. All the dams were evaluated for reproductive performance such as, mating index, fertility index, gestation index, parturition index, pre-coital interval and gestation length. All the females were evaluated for oestrus cyclicity during premating, mating treatment periods and at termination. The body weight and feed consumption was recorded for all the females during gestation day 0, 7, 14 and 20 and on lactation days 1, 4, 7 and 13. The terminal body weight for all the animals was measured on the day of the termination (LD 14). At birth/litter observation parameters such as, number of live/dead pups born, litter size, sex ratio, live birth index per litter and pup survival index were observed / calculated for all the litters. The total number of implantations per litter was recorded during necropsy and the post-implantation and post-natal losses were calculated. The pups were observed once daily for external examinations and twice daily for mortalities till termination [post-natal day (PND) 13], weighed individually on PND 1, 4, 7 and 13, measured for ano-genital distance (AGD) to calculate AGD ratio on PND 4, observed for retention of any nipples/areolae in male pups on PND 13. All the pups were observed for gross pathological observations at termination and analyzed the serum collected from PND 13 pups for thyroxine hormone (T4) levels by using ELISA method. There were no clinical signs of toxicity and no mortality/morbidity noted at all in any of the tested dose groups of either sex animals. There were no changes noted in mean body weight, percent change in mean body weight gain and mean feed consumption at all thein any of the tested dose groups of both the either sex. There were no changes noted in mean serum thyroxine hormone (T4) levels at all in any of the tested dose group of males. The absolute and relative organ weights did not reveal any changes at all thein any of the tested dose groups. No macroscopic changes noted during conduct of necropsy in at all the vehicle control and tested dosed groups of both either the sex. Histopathological examination conducted for group G4 animals of both sexes did not reveal any test chemical related microscopic changes. For reproduction toxicity end points, there were no effects noted in male mating and fertility indexes of all tested dose groups. No effects were noted in female mating index, fertility index, gestation index, parturition index, pre-coital interval and gestation length in at all tested all dosed groups. For maternal toxicity end points, there were no irregularities noted in oestrus cyclicity and no changes in mean cycle length, no changes in mean body weight, percent change in mean body weight gain and mean feed consumption were observed all dosed groups during gestation and lactation periods at all the tested dose groups. There were no changes observed in birth parameters and litter observations during lactation period. The number of implantations, post-implantation and post-natal losses were unaffected by the test chemical at all the tested in all treated dose groups. For developmental toxicity end points, there were no external anomalies noted and no test chemical-related mortalities among pups noted were observed in pups during post-natal period at doses tested. at all the tested dose group litters. There were no test chemical-related changes noted in mean pup weight and mean pup ano-genital distance ratio per litter in either sex at all the tested dose groups.at doses tested There were no occurrences of nipples in male pups of vehicle control and treated groups on PND 13 examined on PND 13 from all the tested dose and vehicle control group litters. There were no gross pathological changes noted in any of the pups during scheduled sacrifice from all the tested and control group litters at any doses tested. There were no changes noted in serum thyroxine (T4) hormone levels estimated for PND 13 (from all litters) pups at all the tested dose groups up litters. Based on all the available data, it was observed that the test chemical did not cause any maternal toxicity and also toxicity to fetus in the developmental stage and also after birth.  Therefore, based on all the observations and obtained results, it was concluded that the NOAEL for the test chemical was 350 mg/kg bw.