Benzyl isobutyrate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Principles of method if other than guideline:

Repeated Dose 28-day Oral Toxicity Study ofwas performed for the test chemical in the Rats.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Benzyl propionate

EC Number:

204-559-3

EC Name:

Benzyl propionate

Cas Number:

122-63-4

Molecular formula:

C10H12O2

IUPAC Name:

Benzyl propionate

Test material form:

liquid

Details on test material:

- Name of test material: Benzyl Propionate
- Molecular formula: C10H12O2
- Molecular weight: 164.2 g/mol
- Substance type: Organic
- Physical state: Colourless Liquid
-Smiles:c1(COC(CC)=O)ccccc1
-InChI:1S/C10H12O2/c1-2-10(11)12-8-9-6-4-3-5-7-9/h3-7H,2,8H2,1H3

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: National Institute of Biosciences, Pune.
- Age at study initiation: 6 to 8 weeks old
- Weight at study initiation:
Male 169.50 gg - 175.6 g
Female 152.35 g- 162.7 g
- Fasting period before study: No data available
- Housing: Animals were housed in polycarbonate cages as 3/sex/cage. Three rats of same sex were housed together in each cage of size 39 cm X 28 cm X 14 cm. Paddy husk was used as bedding material.
- Diet (e.g. ad libitum): Rodent feed supplied by the Nutrivet Life Sciences, Pune, was provided ad libitum from individual feeders on cage top.
- Water (e.g. ad libitum): Water was provided ad libitum from individual bottles attached to the cages. Water was from a local source and passed through the reverse osmosis membrane before use.
- Acclimation period: 5 days prior to dosing.
- Quarantine period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C (actual range: 19.5 °C to 22.8 °C)
- Humidity (%):30% to 70% (actual range: 53.3% to 58.1%).
- Air changes (per hr): Ten air changes per hour of 100% fresh air that has been passed through the HEPA filters.
- Photoperiod (hrs dark / hrs light): An artificial light and dark cycle of 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test chemical was diluted with Corn oil for preparation of dosing solution(s). The solution(s) of test item was made at volumes suitable for daily use for 28 days. The solution(s) were prepared at concentrations of 0, 25, 50 and 100 mg/ml such that dosage of 0 (vehicle), 250, 500 and 1000 mg/kg body weight respectively were administered. The concentration of the test item was varied so as to maintain the dose volume constant at or upto 10 ml/kg body weight.


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): Corn oil
- Concentration in vehicle:
The solution(s) were prepared at concentrations of 0, 25, 50 and 100 mg/ml such that dosage of 0 (vehicle), 250, 500 and 1000 mg/kg bw/day.
- 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:

Analysis for concentration and stability of the test chemical were conducted at Subcontracted Laboratory. Test item formulation samples prepared day 1 (pre-dosing) were sent to Subcontracted Laboratory.

Duration of treatment / exposure:

28 days consecutively

Frequency of treatment:

Once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0 (vehicle), 250, 500 and 1000 mg/kg bw/day.
Basis:
actual ingested

No. of animals per sex per dose:

Control: 6 male, 6 female
250 mg/kg bw/day: 6 male, 6 female
500 mg/kg bw/day: 6 male, 6 female
100 0mg/kg bw/day: 6 male, 6 female

Control animals:

yes, concurrent vehicle

Details on study design:

Details on study design
- Dose selection rationale: The doses were selected based on the results of the Dose Range Finder study. The study was conducted at the dose levels of 0 mg/kg, 50 mg/kg, 100 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg body weight. Based on results from a preliminary 14-day study there was no change in the survival, body weight, Daily clinical observations and Gross pathological examination of 1000 mg/kg/bw/day group. Based on these results, the 28 day study dose levels were finalized as 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg body weight.
- Rationale for animal assignment (if not random): Animals were randomized by sex and body weight
- Rationale for selecting satellite groups: No data available
- Post-exposure recovery period in satellite groups: No data available
- Section schedule rationale (if not random): No data available

Positive control:

No data

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations checked in table [No.?] were included. : Rats were observed for Viability, Behavior, Alterations, Vocalizations, Respiration and Palpebral closure. The rats were observed for reaction to removal, reaction to handling, urination, defecation, prominence of eye, lacrimation, salivation, piloerection, examination of mucous membrane, examination of skin / fur, examination of natural orifices and animal appearance. The animals were also placed in the open field and its appearance and behavior were observed

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations were made for individual animal once before the start of dose administration and at least once a week thereafter until scheduled sacrifice.

BODY WEIGHT: Yes
- Time schedule for examinations: On the day of randomization, first day of dosing, weekly thereafter and a fasting body weight at scheduled sacrifice on day 29.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, The quantity of feed consumed by control and different treatment groups was recorded weekly until scheduled sacrifice
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data available

FOOD EFFICIENCY:
- 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: No data

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once a day
- Dose groups that were examined: 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg bw/day.

HAEMATOLOGY: Yes, By using Beckman Coulter haematology analyzer.
- Time schedule for collection of blood: At termination on day 29
- Anaesthetic used for blood collection: No data available
- Animals fasted: Yes, overnight fasted prior to sampling.
- How many animals: Blood collected from all rats of 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg bw/day group at termination.
- Parameters checked in table [No.?] were examined. : Hemoglobin, Red Blood Corpuscles, Hematocrit, Mean Corpuscular Volume, Mean Corpuscular Hemoglobin, Mean Corpuscular Hemoglobin Concentration, Platelets, White Blood Corpuscles, Neutrophils, Lymphocytes, Eosinophils, Monocytes, Basophil and Prothrombin time were checked

CLINICAL CHEMISTRY: Yes, By using Dimension XpandPlus and Acculyte 5P.
- Time schedule for collection of blood: At termination on day 29.
- Animals fasted: Yes, overnight fasted prior to sampling.
- How many animals: Blood collected from all rats of 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg bw/day group at termination.
- Parameters checked in table [No.?] were examined. : Total Protein, Blood Urea Nitrogen, Urea Nitrogen, Alanine Aminotransferase, Aspartate Aminotransferase, Alkaline Phosphatase, Gamma Glutamyl Transferase, Glucose, Calcium, Phosphorous, Albumin, Total Bilirubin, Creatinine , Total Cholesterol, Triglycerides, Globulin Calculated Sodium, Potassium, Chloride were checked, Table No.I; Appendix No.VIII.

URINALYSIS: No data available
- Time schedule for collection of urine: No data available
- Metabolism cages used for collection of urine: No data available
- Animals fasted: No data available
- Parameters checked in table [No.?] were examined. : No data available

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: No data available
- Dose groups that were examined: No data available
- Battery of functions tested: sensory activity / grip strength / motor activity / other: Yes

OTHER: Viability, Behavior in Home cage, Vocalizations, Respiration, Palpebral closure, Handling Observations, Urination, Defecation, Prominence of Eye, Lacrimation, Salivation, Piloerection, Examination of Skin / Fur, Stereotype Behaviour, Rearing (Rears), Clonic and Tonic Movements and Severity of Gait were observed.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, The rats were sacrificed by CO2 asphyxiatio and Gross necropsy was conducted. All the animals of 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg/bw/day group were sacrificed and gross lesions were noted. Liver, Kidneys, Adrenals, Epididymides, Prostate + Seminal Vesicle with Coagulation gland as whole, Thymus, Spleen, Brain, Heart, Lungs, Uterus, Testes/Ovaries were dissected free of fat and weighed. The paired organs were weighed together.

HISTOPATHOLOGY: Yes, From each rat, samples or the whole of the tissue were preserved. All tissues were fixed in 10% neutral buffered formalin except, eyes and testes of all animals were preserved in Davidson’s solution for 24 hours and transferred to 10% neutral buffered formalin. Control and treated at the highest dose level of 1000 mg/kg were subjected to sacrifice.

Organs examined: Adrenals, Aorta, Brain (cerebrum, cerebellum and pons), Caecum, Cervix, Colon, Duodenum, Epididymides, Eyes, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs, Mesenteric Lymphnodes, Oesophagus, Ovaries, Pancreas, Pituitary gland, Pharyngeal Lymphnodes, Prostate, Rectum, Skeletal Muscles, Skin with Mammary Gland, Spleen, Sternum with bone marrow, Sciatic Nerve, Spinal Cord (Cervical, mid thoracic and lumbar), Stomach, Seminal Vesicles with Coagulation Gland, Testes, Thymus, Thyroid, Trachea, Vagina, Urinary Bladder and Uterus of 1000 mg/kg/bw/day group.

Statistics:

Data were analyzed for reporting group means and standard deviations with significance between the controls and treated groups, using in-house developed and validated MS-Excel 2003 based statistical software. All the parameters characterized by continuous data such as body weight, per cent body weight change, feed consumption, organ weight, relative organ weight, haematological and clinical chemistry data were subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analyses of Variance (ANOVA) and Dunnett’s t-test. Where the data did not meet the homogeneity of variance, Student’s t-test was performed to calculate significance.

Significance was calculated at 1% as well as 5% level and indicated in the summary tables as follows:

* = Significant than control at 95% level of confidence (p≤ 0.05).
** = Significant than control at 99% level of confidence (p≤ 0.01).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

Clinical signs and mortality
Clinical signs :
Daily clinical observations did not reveal any signs of toxicity in male and female animals from of 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg bw/day dose groups . Home cage observations in animals from all treated groups and control group revealed normal behavior, alterations, vocalization, respiration and palpebral closure. During handling observation, handling of animals did not reveal any abnormality from all treated groups and control group. Open field observation of animals did not reveal any abnormality from all treated groups and control group. There was no change in animals before commencement of of treatment and during study period.

Mortality:
No mortality were observed in any of the traeted groups of 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg bw/day.

Body weight and weight gain All the rat of 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg/bw/day dose groups exhibited normal body weight gain at the end of the study period of 28 days.

Food consumption and compound intake Food intake of animals for control and 250 mg/kg, 500 mg/kg and 1000 mg/kg /bw/day dose groups were found to be normal throughout the study period of 28 days.

Food efficiency: No data available

Water consumption and compound intake No data available

Opthalmoscopic examination No data available

Haematology
Male rats: Increase in the values of Hb of male rats dosed at 500 mg/kg and 1000 mg/kg, MCHC of male rats dosed at 500 mg/kg, Total WBC of male rats dosed at 1000 mg/kg were observed.

Female rats: Plateles of female rats dosed at 1000 mg/kg were also increase. The increase in the values of different parameters is within the normal laboratory limits.

Clinical chemistry
Male and female rats: Increased level of Sodium in male rats dosed at 250 mg/kg and 1000 mg/kg of Benzyl Propionate. Chloride levels increased in male rats dosed at 250 mg/kg of Benzyl Propionate

Female rats: Elevated levels of calcium were observed in animals from 250 mg/kg dose group (p≤0.01). Increased bilirubin levels were found in male rats dosed 500 mg/Kg. Elevated levels od sodium were observed in animals from 250 mg/kg (p≤0.01), 500 mg/kg (p≤0.05) and 1000 mg/kg (p≤0.01) dose groups, Alkaline phosphatase leves were decreased in animals from 250 mg/kg dose group (p≤0.05), a decrease in potassium levels were observed in animals from 250 mg/kg and 1000 mg/kg dose groups (p≤0.05) and Decreased levels od chloride were observed in animals from 1000 mg/kg dose group (p≤0.05).

Urinanalysis: No data available

Neurobehaviour: All animals from control and different dose groups showed normal arousal level, visual response, touch response, auditory response, tail pinch response and visual placing response. Normal air righting reflex was observed in all animals from control and different dose groups in week 4. Grip strength values observed in male and female animals for control and different dose groups were comparable. Higher values were observed in male animals from 500 mg/kg dose group for first interval (p≤0.05). Lower values were observed in female animals from 500 mg/kg dose group for first interval (p≤0.01) and for second interval (p≤0.05). These changes were within laboratory range and were considered to be of no toxicological importance.

Organ weights: Organ weight of 100 mg/kg/bw/day dose group male animals sacrificed on day 29, was found to be comparable with that of controls.

Organ weight of female animals sacrificed on day 29, revealed increased relative weights of liver, ovaries and lungs of 1000 mg/kg dose group.The significant changes in organ weights were observed in female animals from high dose group, no related gross pathological or histological changes were seen and hence considered to be of no toxicological importance.

Gross pathology All the treatment groups of control and 250 mg/kg/bw/day, 500 mg/kg/bw/day and 1000 mg/kg/bw/day of Benzyl Propionate did not reveal any abnormality.

Histopathology: Minimal focal to multifocal periportal mononuclear cell infiltration in the liver; minimal interstitial haemorrhages in the kidneys; minimal alveolar haemorrhages and/or alveolar histiocytosis in the lungs; minimal multifocal haemosiderosis and/or diffused congestion in spleen; minimal eosinophilic infiltration and/or luminal dilatation in uterus; minimal luminal seminal coagulum in the urinary bladder; minimal dilatation of zona reticularis and/or presence of accessory adrenocortical tissue in adrenals; minimal multifocal haemorrhages in thymus; presence of ultimobranchial cysts in thyroid; in male or female animals of control and 1000 mg/kg/bw/day dose group. All the changes observed in the control and 1000 mg/kg/bw/day dose treatment group animals were similar and the effect observed was not considered to be treatment- related.

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on Clinical biochemistry, Organ weight, Gross pathological, Histopathology and survival.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg bw/day when Sprague-Dawley rats were exposed to the test chemical orally for 28 days.

Executive summary:

A subacute repeated dose oral toxicity study was conducted to evaluate the toxic effects of repeated administration of the test chemical in male and female Sprague-Dawley rats by gavage. The test chemical was administered to 6 animals/sex/species with vehicle as corn oil at doses of 0, 250, 500 and 1000 mg/kg/bw/day for 28 days. All rats of 250, 500 and 1000 mg/kg/bw/day dose group survive though-out the study, the test chemical have no effect on mortality. Blood samples for Clinical Biochemistry and Haematology were collected. No abnormalities occurred that could be directly attributed to test chemical treatment. Although significant change in relative weights of liver, ovaries and lungs of female were observed in 1000 mg/kg/bw/day dose groups. No treatment related gross pathological or histological changes were seen and findings were not considered to be test chemical dependent and hence considered to be of no toxicological importance. Therefore the No Observed Adverse Effect Level (NOAEL) for repeated dose toxicity study was considered to be 1000 mg/kg/bw/day in male and female Sprague-Dawley rats when exposed to the test chemical by oral route for 28 days.