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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
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 an experimental 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:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Principles of method if other than guideline:
According to OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Name: Cinnamyl Alcohol
CAS No. 104-54-1
IUPAC Name: 3-Phenyl-2-propen-1-ol
Molecular Formula: C9-H10-O
Molecular Weight: 134.177 g/mol
SMILES: OC\C=C\c1ccccc1

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Rats are the standard laboratory rodent species used as a reliable test model to assess the Toxicity of the test item and recommended by the guidelines The strain Wistar was chosen on account of available historical data.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Sipra Labs Limited, Plot No.13, IDA, Balanagar, Hyderabad, India.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6-7 Weeks
- Weight at study initiation:
Male: 128.1g -136.9 g
Female: 125.7 g -134.4 g
- Fasting period before study: No Data Available
- Housing: Animals were housed in an autoclaved polycarbonate cages with stainless steel top grill having facilities for holding pellet feed and drinking water in polycarbonate bottle. Autoclaved corn cob was used as bedding material and it was changed at least once in a week.
- Diet (e.g. ad libitum): Rodent feed from a certified vendor was provided as ad libitum.
- Water (e.g. ad libitum): Aquaguard purified water was provided ad libitum by a water dispensing bottle.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1°C – 23.4°C.
- Humidity (%): 51 – 61 %.
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle per day

IN-LIFE DATES: From: To: No Data Available

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Distilled water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test chemical was weighed in pre-weighed glass beakers on an appropriate weighing balance. The test chemical was transferred to mortar and triturated with pestle. 10 mL of the vehicle was added to mortar and again triturated. This was transferred to a measuring cylinder. Sufficient quantity of vehicle was added to make up the required volume of formulation. This was transferred back to beaker. Homogeneity of the test chemical in the vehicle was maintained during administration using a magnetic stirrer.

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): Distilled water was used as a vehicle.
- Concentration in vehicle: 0 mg/ml, 25 mg/ml, 50 mg/ml and 100 mg/ml for control, low dose, mid dose and high dose groups, respectively.
- Amount of vehicle (if gavage): 10 ml/kg
- Lot/batch no. (if required): No Data Available
- Purity: No Data Available
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of dose formulation was checked before treatment start day at 0 and 6 hours. Test item was checked for its stability in the dose formulation with samples collected immediately (0 hours) and 6 hours post dose formulation preparation. Samples of the dose formulations were taken immediately after dilution of the concentrates with the diluents (vehicle) on treatment start date and treatment end date for homogeneity (mean of homogeneity was given as dose concentration) analyses. On week 3, samples of all dose formulations were taken for dose concentration analysis.

Linearity: Linearity was carried out by injecting a minimum of 5 different test concentrations (three replications from each concentration).The correlation coefficient and regression was calculated (r=0.9982).

Accuracy: Accuracy was performed at three levels ie., 50, 100, 150%. The mean recovery values of samples for 50% level is 102.35% , for 100% level is 100.18% and for 150% level is 97.14%.

Precision: The relative standard deviation of analysis of five independent fortifications samples were not exceed ±20% of the mean value.

Specificity: No interference was observed above 30 % of total peak area for target analyte.

The dose concentration verification was carried out for test item fortified in Distilled water pertaining to low dose (25mg/mL), mid dose (50mg/mL) and high dose (100mg/mL). The recovery of test item in the prepared dose formulation was within the guideline specification.
The test item was found to be homogeneously dispersed in Distilled water, which was evinced from the recovery of test item from different layers (Top, middle and bottom layers of Distilled water).
Duration of treatment / exposure:
28 days consecutively.
Frequency of treatment:
Once Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control Group (G1)
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control Group (Recovery) (G1R)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
Low Dose Group (G2)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
Intermediate Dose Group (G3)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
High Dose Group (G4)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
High Dose Group (Recovery) (G4R)
No. of animals per sex per dose:
5 animals per sex per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses for the study were selected based on the dose range finding study.
- Rationale for animal assignment (if not random): Animals were selected and grouped based on stratified randomization by using body weights taken before start of treatment. Computerized statistical analysis was used for randomization.
- Fasting period before blood sampling for clinical biochemistry: Yes, the animals were fasted before the blood sampling.
- Rationale for selecting satellite groups: Recovery groups for vehicle control and high dose were included to know the reversibility of treatment related effects.
- Post-exposure recovery period in satellite groups: 2 weeks
- Section schedule rationale (if not random): No Data Available
- Other: No Data Available
Positive control:
No Data Available

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily on first 3 days of treatment; once daily thereafter

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to the first exposure and weekly thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: Once Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, Once weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not specified
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

FOOD EFFICIENCY: No Data Available
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified
- Time schedule for examinations: No Data Available

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During week 4
- Dose groups that were examined: All dose groups were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 4 for main groups and Week 6 for recovery group
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes
- How many animals: All animals were fasted.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 4 for main groups and Week 6 for recovery group
- Animals fasted: Yes

URINALYSIS: Yes
- Time schedule for collection of urine: Week 4 for main groups and Week 6 for recovery group
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Before sacrifice
- Dose groups that were examined: All the dose groups
- Battery of functions tested: sensory activity / grip strength / motor activity /

IMMUNOLOGY: Not specified
- Time schedule for examinations: No Data Available
- How many animals: No Data Available
- Dose groups that were examined: No Data Available

OTHER: No Data Available
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, All animals in main study (Group 1 to Group 4) was sacrificed at the end of week 4 and animals in recobvery group (Group 1R and Group 4R) were sacrificed at the end of week 6 of experimental period by Carbon dioxide asphyxiation. All animals were weighed and sent to necropsy.

HISTOPATHOLOGY: Yes, full histopathology was performed on the preserved organs and tissues of all animals in the control and high dose groups. Histopathology examination was extended to the organs such as liver, testes and epididymides in low dose and intermediate dose group.
Other examinations:
No Data Available
Statistics:
The following statistical methods were used to analyze the body weight, feed consumption, organ weights as well as clinical pathology data.
• Data was summarized in tabular form. Statistical analysis was performed using SAS/STAT® Software (Version 9.3).
• All the data were analysed for homogeneity using Levene’s test
• The difference between the means was analyzed using Dunnet’s t- test
• Significant differences between control and treated groups analyzed using one-way ANOVA statistics
• Values were summarized as mean ± standard deviation (SD)

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs were observed in any of the vehicle control(G1), low(G2), intermediate (G3), high dose(G4), vehicle control recovery (G1R) and recovery high dose (G4R) group animals.
Mortality:
no mortality observed
Description (incidence):
No mortality was observed in vehicle control (G1), low (G2), intermediate (G3), high dose (G4), recovery control (G1R) and recovery high dose (G4R) group animals.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No adverse effect on body weight was observed for treated groups when compared with control group. No significant and toxicological relevant changes in the body weight between treated and control group were observed.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No significance in the feed consumption was observed in any of the treated and recovery groups when compared with vehicle control (G1) group.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No adverse effects on opthalmological findings were observed during the study.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males in high dose group (G4) were showed decrease in WBC count when compared with control group. However, no changes in the females were observed. Treatment with the test chemical had no effects on both male and female animals with respect to hematological parameters viz RBC, Hb, MCV, MCHC, PCV, PLT, PT and APTT.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In males, when compared with control group, bilirubin levels increased in low dose group (G2), ALT levels decreased in high dose, total protein levels decreased in low dose and intermediate dose compared to control, albumin levels increased in low, intermediate and high dose, Globulin levels decreased in low dose, intermediate and high dose, cholesterol levels increased in low dose and high dose. In females, when compared with control group, creatinine levels decreased in intermediate dose group (G3), bilirubin levels decreased in high dose (G4), triglyceride levels increased in intermediate dose (G3). All the recovery group male and female animals showed no effect on biochemical parameters. This change in the biochemical parameters is considered to have arisen by chance or caused by slightly higher/lower control values and not to represent a change of any biological significance.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No significant changes in any of the treated groups and recovery groups were observed in urinalysis findings.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No significant and test chemical related findings were observed during functional observation battery.
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 in the in absolute and relative organ weights including that of reproductive organs in both sexes in main group and recovery group animals.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test chemical related effects were observed during the necropsy of the animals in any organs including that of reproductive organs in main groups or also in the recovery group.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No test chemical related changes in the histopathology was observed in animals of all dose groups in main groups or also in the recovery group.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
No clinical signs were observed in any of the vehicle control(G1), low(G2), intermediate (G3), high dose(G4), vehicle control recovery (G1R) and recovery high dose (G4R) group animals. No mortality was observed in vehicle control (G1), low (G2), intermediate (G3), high dose (G4), recovery control (G1R) and recovery high dose (G4R) group animals. No adverse effect on body weight was observed for treated groups when compared with control group. No significant and toxicologically relevant changes in the body weight between treated and control group were observed. No significant changes related to the test chemical in the feed consumption was observed in any of the treated and recovery groups when compared with vehicle control (G1) group. No adverse effects on opthalmological findings were observed during the study. Males in high dose group (G4) were showed decrease in WBC count when compared with control group. However, no changes in the females were observed. Treatment with the test chemical had no effects on both male and female animals with respect to hematological parameters viz RBC, Hb, MCV, MCHC, PCV, PLT, PT and APTT. In males, when compared with control group, bilirubin levels increased in low dose group (G2), ALT levels decreased in high dose, total protein levels decreased in low dose and intermediate dose compared to control, albumin levels increased in low, intermediate and high dose, Globulin levels decreased in low dose, intermediate and high dose, cholesterol levels increased in low dose and high dose. In females, when compared with control group, creatinine levels decreased in intermediate dose group (G3), bilirubin levels decreased in high dose (G4), triglyceride levels increased in intermediate dose (G3). All the recovery group male and female animals showed no effect on biochemical parameters. This change in the biochemical parameters is considered to have arisen by chance or caused by slightly higher/lower control values and not to represent a change of any biological significance. No significant changes in any of the treated groups and recovery groups were observed in urinalysis findings. No significant and test chemical related findings were observed during functional observation battery. There were no test chemical related changes in the in absolute and relative organ weights in both sexes in main group and recovery group animals. No test chemical related effects were observed during the necropsy of the animals in any organs in main groups or also in the recovery group. No test chemical related changes in the histopathology was observed in the animals of all dose groups in main groups or also in the recovery group.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
ophthalmological examination
haematology
clinical biochemistry
urinalysis
behaviour (functional findings)
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on all the available data, it was observed that the test chemical did not induce any systemic and local toxicity in the tested animals in main groups and recovery groups, respectively. Therefore, on the basis of all the observations and results, it was concluded that the NOAEL for the test chemical was 1000 mg/kg bw/day.
Executive summary:

A 28 days repeated dose toxicity study was performed in accordance with the OECD TG 407 under GLP conditions using male and female Wistar rats. In this study, the animals of age 6 -7 weeks were used. 5 animals per sex per dose were used in the study. A total of 6 dose groups were selected for the study viz. 0 mg/kg bw/day (control group), 250 mg/kg bw (Low Dose Group), 500 mg/kg bw (Intermediate Dose Group) and 1000 mg/kg bw (High Dose Group). Further, two recovery groups were also included in the study in order to check the reversibility of the effects that are observed due to the administration of the test chemical, namely, Control Group Recovery (0 mg/kg bw/day) and High Dose Recovery (1000 mg/kg bw/day). 5 animals per sex per dose were used in the study. The animals were treated daily once via oral gavage route. The vehicle used for the study was distilled water. The parameters that were checked in the study were clincal signs, clinical observations (behavioral parameters), body weight and feed consumption, hematological and clinical biochemistry parameters, urinalysis, absolute and relative organ weights, gross pathology and histopathology. The animals of main groups were dosed consecutively for 28 days and on day 29, they were sacrificed. The animals were kept in metabolic cages a day prior to sacrifice in order to collect urine for urinalysis. Before sacrifice, blood was collected from all the animals from all the dose groups, using isofluorane anesthesia for hematological and biochemistry parameters. The same procedure was followed for recovery group animals before sacrifice on day 43. The in-life observations showed that No clinical signs were observed in any of the vehicle control(G1), low(G2), intermediate (G3), high dose(G4), vehicle control recovery (G1R) and recovery high dose (G4R) group animals. No mortality was observed in vehicle control (G1), low (G2), intermediate (G3), high dose (G4), recovery control (G1R) and recovery high dose (G4R) group animals. No adverse effect on body weight was observed for treated groups when compared with control group. No significant and toxicologically relevant changes in the body weight between treated and control group were observed. No significant changes related to the test chemical in the feed consumption was observed in any of the treated and recovery groups when compared with vehicle control (G1) group. No adverse effects on opthalmological findings were observed during the study. In hematological parameters, males in high dose group (G4) were showed decrease in WBC count when compared with control group. However, no changes in the females were observed. Treatment with the test chemical had no effects on both male and female animals with respect to hematological parameters viz RBC, Hb, MCV, MCHC, PCV, PLT, PT and APTT. In males, when compared with control group, bilirubin levels increased in low dose group (G2), ALT levels decreased in high dose, total protein levels decreased in low dose and intermediate dose compared to control, albumin levels increased in low, intermediate and high dose, Globulin levels decreased in low dose, intermediate and high dose, cholesterol levels increased in low dose and high dose. In females, when compared with control group, creatinine levels decreased in intermediate dose group (G3), bilirubin levels decreased in high dose (G4), triglyceride levels increased in intermediate dose (G3). All the recovery group male and female animals showed no effect on biochemical parameters. This change in the biochemical parameters is considered to have arisen by chance or caused by slightly higher/lower control values and not to represent a change of any biological significance. No significant changes in any of the treated groups and recovery groups were observed in urinalysis findings. No significant and test chemical related findings were observed during functional observation battery. There were no test chemical related changes in the in absolute and relative organ weights in both sexes in main group and recovery group animals. No test chemical related effects were observed during the necropsy of the animals in any organs in main groups or also in the recovery group. No test chemical related changes in the histopathology was observed in the animals of all dose groups in main groups or also in the recovery group. Therefore, based on all the available data, it was observed that the test chemical did not induce any systemic and local toxicity in the tested animals in main groups and recovery groups, respectively. Therefore, on the basis of all the observations and results, it was concluded that the NOAEL for the test chemical was 1000 mg/kg bw/day.