Benzyl propionate

Endpoint summary

Description of key information

Repeated dose toxicity ,Oral : The no observed adverse effect level (NOAEL) for repeated dose toxicity study for benzyl propionate was estimated to be 520.25 mg/kg/day when administered orally in feed to rat in a subchronic study of 90 days.

Repeated dose toxicity, Inhalation: The presence of benzyl propionate in cigarette at 13 ppm did not significantly change the type or extent of toxicologic effects observed in rodents inhaling cigarette smoke.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

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 of Benzyl Propionate in the Rat.

GLP compliance:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Details on test animal
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
Female 152.35 g

- Fasting period before study: No data available

- Housing: Animals were housed in polycarbonate cages. 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.

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

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

Details on oral exposure
PREPARATION OF DOSING SOLUTIONS: Benzyl Propionate was diluted with Corn oil for
preparation of dosing solution(s).

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): No data available

- 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):
0.00 mg/ml/day, 26.60 mg/ml/day, 52.38 mg/ml/ day and 105.27 mg/ml/day.

- 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 Benzyl Propionate 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

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:
Based on results from a preliminary 14-day study there was no chenge in the survivel, 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

Observations and examinations performed and frequency:

Observations and examinations performed & frequency
CAGE SIDE OBSERVATIONS: Yes

- Time schedule: No data available

- Cage side observations checked in table [No.?] were included. : Rats were observed for Behavior, Alterations, Vocalizations, Respiration and Palpebral closure.

DETAILED CLINICAL OBSERVATIONS: Yes

- Time schedule: Once daily, At least once a week there after until

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): No data available

- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data available

- 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: Yes / No / 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.

- 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, Table No.H; Appendix No.VII.

CLINICAL CHEMISTRY: Yes, By using Dimension XpandPlus and Acculyte 5P.

- Time schedule for collection of blood: At termination.

- 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: No data available

- 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:

Sacrifice and pathology
GROSS PATHOLOGY: Yes
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.

HISTOPATHOLOGY: Yes
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).

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

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 normale 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 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.
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 Increase level of Sodium in male rats dosed at 250 mg/kg and 1000 mg/kg of Benzyl Propionate.

Chloride levels significantly increase in male rats dosed at 250 mg/kg of Benzyl Propionate.

Statistically significant increase of Calcium levels in female rats dosed at 250 mg/kg and increase Bilirubin levels in female rats dosed at 500 mg/kg of Benzyl Propionate were found well increase level of Sodium in female rats dosed at 250 mg/kg, 500 mg/kg and 1000 mg/kg were observed.

Statistically significant decrease of Alkaline Phosphatase levels in female rats dosed at 250 mg/kg, decrease level of Potassium in female rats dosed at 250 mg/kg and 1000 mg/kg and decrease Chloride levels in female rats dosed at 1000 mg/kg Benzyl Propionate were observed.

Although there was an increase/decrease in the values the deviations were within the range of normal laboratory limits.

Urinanalysis: No data available
Neurobehaviour: No data available

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, the effects are not related to Benzyl Propionate.
Gross pathology All the treatment groups control and 250 mg/kg/bw/day, 500 mg/kg/bw/day and 1000 mg/kg/bw/day of Benzyl Propionate dus not showe any chengs.

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 are not due to Benzyl Propionate.

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.

Critical effects observed:

not specified

Conclusions:

NOAEL was considered to be 1000 mg/kg bw/day when Sprague-Dawley rats exposed to Benzyl Propionate orally foe 28 days.

Executive summary:

A subacute study was conducted to evaluate the toxic effects of repeated administration of Benzyl Propionate in male and female Sprague-Dawley rats by gavage. Benzyl Propionate was administered to 6 animals/sex/species in 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, Benzyl Propionate have no effect on mortality. Blood samples for Clinical Biochemistry and Haematology were collected. No abnormalities occurred that could be directly attributed to Benzyl Propionate 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 Benzyl Propionate related gross pathological or histological changes were seen and findings were not Benzyl Propionate dependent and hence considered to be of no toxicological importance. Therefore NOEAL for repeated dose toxicity study was considered to be 1000 mg/kg/bw/day in male and female Sprague-Dawley rats when exposed to Benzyl Propionate by oral route for 28 days.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Qualifier:

according to guideline

Guideline:

other:

Principles of method if other than guideline:

Sprague-Dawley rats were exposed by nose-only inhalation for 1 h/day, 5 days/wk for 13 wk to smoke from the test or reference cigarettes already described, or to air only, and necropsied after 13 wk of exposure or following 13 wk of recovery from smoke exposure

GLP compliance:

not specified

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 6-7 wks
- Housing: During the 13-wk exposure period, the animals were housed in individual stainless-steel cages on open racks. During the recovery period, the animals were housed in individual polycarbonate cages bedded with ALPHA-dri alpha cellulose bedding.
- Acclimation period: 24 days

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

Route of administration:

inhalation

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Animal exposures were conducted in AMESA exposure units
- System of generating particulates/aerosols: The smoke exposure machines were designed to contain 30 cigarettes on a smoking head that rotated 1 revolution per minute. A vacuum port aligned with, and drew a puff from, one test or reference cigarette at a time as the head rotated.
- Temperature, humidity, pressure in air chamber:
- Air flow rate: Air was drawn through the vacuum port by a peristaltic pump operating at a flow rate of ∼1.05 L/min,creating a 2-s, 35-ml puff through each cigarette once each minute. The smoke vacuum flow rate was regulated by a concentration control unit consisting of a real-time aerosol monitor [(RAM)-1], a computer, and an electronic flow controller.


TEST ATMOSPHERE
- Brief description of analytical method used: The exposure units contained 3 tiers, each with 24 animal exposure ports. The exposure ports were connected to a delivery manifold, which transferred smoke to the animal breathing zone, and to an outer concentric manifold that drew the exhaled and excess smoke to an exhaust duct. Each cigarette was retained for seven puffs.
- Samples taken from breathing zone: yes/no

VEHICLE (if applicable)
- Justification for use and choice of vehicle:
- Composition of vehicle:
- Type and concentration of dispersant aid (if powder):
- Concentration of test material in vehicle:
- Lot/batch no. of vehicle (if required):
- Purity of vehicle:

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

1 h/day, 5 days/wk

Remarks:

Doses / Concentrations:
0 or 0.013 mg/L (13 ppm) as a part of 0, 0.06, 0.2 or 0.8 mg/L WTPM of smoke
Basis:
no data

No. of animals per sex per dose:

30

Control animals:

yes, sham-exposed

Details on study design:

Each group of 30 rats/sex was subdivided into 2 groups: 20 rats/sex scheduled for necropsy immediately after 13 wk of exposure (interim sacrifice) and up to 10 rats/sex scheduled for necropsy following 13 wk of recovery from smoke exposure (final sacrifice)

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All rats were observed twice daily for mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each rat was examined every 4 wk for clinical signs

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were measured during the randomization procedure, on exposure day 1, biweekly thereafter, and at necropsy

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

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

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During wk 2, 10 and on the day of the 13-wk interim sacrifice
- Anaesthetic used for blood collection: Yes (∼70% CO2)
- Animals fasted: No data
- How many animals: No data
- Parameters examined: white blood cell (WBC) count, red blood cell (RBC) count, hemoglobin (Hb) concentration, volume of packed red cells (VPRC), the red cell indices (mean corpuscular volume [MCV], mean corpuscular hemoglobin [MCH], and mean corpuscular hemoglobin concentration [MCHC]), platelet count, and WBC differential counts.
During wk 2 and 10, samples were analyzed for blood carboxyhemoglobin (COHb)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: During wk 2, 10 and on the day of the 13-wk interim sacrifice
- Animals fasted: No data
- How many animals: No data
- Parameters examined: urea nitrogen (BUN), creatinine, glucose, total protein, albumin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transpeptidase(GGT), sodium, potassium, chloride, calcium, phosphorus, total bilirubin, cholesterol, and triglycerides.
During wk 2 and 10, Plasma nicotine was quantitatively determined using gas chromatography/mass spectrometry (GC/MS) with selected ion monitoring

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: No

Sacrifice and pathology:

A complete necropsy was done on all 13-wk exposure groups and 13-wk recovery group animals

GROSS PATHOLOGY: Yes
All abnormalities were recorded on the individual animal necropsy forms. Lungs, liver, kidneys, testes, adrenals, spleen, brain, and heart from all scheduled sacrifice animals were weighed. These organ weights and the body weights at necropsy were used to calculate organ:body weight ratios. In addition, organ:brain weight ratios were calculated

HISTOPATHOLOGY: Yes
Duplicate slides of nasal tissues, larynx, lung, and trachea were stained with periodic acid-Schiff/Alcian blue (PAS/AB) stains for evaluation of goblet cell populations. The lungs, nasal cavity (four sections), nasopharynx, larynx (three cross sections), trachea (three transverse sections), tracheobronchial lymph nodes, mediastinal (thymic) lymph nodes, heart, and all gross lesions were examined microscopically.
In addition, sections of brain, adrenals, spleen, liver, kidneys, and gonads from animals
in the sham control and the groups exposed to 0.8 mg/L of smoke from the test or reference cigarettes were examined microscopically.

Other examinations:

Respiratory Function Measurements
Tidal volume (TV), respiratory rate (RR), and minute volume (MV), derived from flow signals from spontaneously breathing animals, were measured in 4 rats/sex/group during wk 2, 8, and 13 using whole-body phethysmography.
Each animal was monitored once during a single exposure period.

Evaluation of Cell Proliferation Rates of Respiratory-Tract Tissues
Cell proliferation rates were measured on respiratory tract tissues collected from 10 rats of each sex from each exposure group and the sham controls necropsied immediately after 13 wk of exposure, using a monoclonal antibody to 5-bromo-2_-
deoxyuridine (BrdU). Tissues evaluated using the BrdU assay included the respiratory epithelium lining the median nasal septum and distal portions of maxillary and nasal turbinates, the transitional epithelium at the base of the epiglottis, the luminal epithelium dorsolateral to the ventral pouch, the luminal epithelium lining the cranial trachea, the luminal epithelium of the mainstem bronchi and adjacent bronchioles, and selected areas of alveolar epithelium

Statistics:

Body weight, body weight gain, organ:body weight, and organ:brain weight ratios were statistically analyzed using the Xybion PATH/TOX system.
Data homogeneity was determined by Bartlett’s test.
Dunnett’s t-test was performed on homogeneous data to identify differences between each concentration group and the sham control group, and between corresponding concentrations of test and reference cigarette smoke-exposed groups. Nonhomogeneous data were analyzed using a modified t-test.
Respiratory physiology, clinical pathology, COHb, and plasma nicotine data parameters were statistically evaluated using SAS software (Statistical Analysis System, SAS, Inc., Cary, NC).
One-way analysis of variance (ANOVA) between exposure groups was first conducted, followed by Bartlett’s test for homogeneity of variance.
A two-sided Dunnett’s multiple comparison test was employed to determine which exposure groups were different from the controls. An unpaired two-sided t-test was used to compare equivalent exposure groups between cigarette types

Clinical signs:

no effects observed

Description (incidence and severity):

No significant mortality occurred and exposure related adverse clinical signs were absent

Mortality:

no mortality observed

Description (incidence):

No significant mortality occurred and exposure related adverse clinical signs were absent

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights were consistently decreased in male rats and all female smoke-exposed groups were comparable to sham control females throughout the study. Mean body weights of smoke-exposed groups were similar to sham control during the recovery period.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were statistically significant differences in several hematology parameters between test and reference cigarette smoke exposed groups. These differences are not considered to be of toxicologic significance, nor were they exposure related.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were statistically significant differences in several clinical chemistry parameters between test and reference cigarette smoke groups. These differences are not considered to be of toxicologic significance, nor were they exposure related.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant differences in organ weights in groups of smoke-exposed rats were primarily low mean organ weights compared to their respective sham controls.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Few gross lesions were observed, with no evidence of changes attributable to exposure to smoke from the test or the reference cigarettes

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Exposure to smoke from reference or test cigarettes induced concentration-related proliferative, metaplastic, and inflammatory microscopic lesions in the respiratory tract after 13 wk of exposure.

Histopathological findings: neoplastic:

not specified

Details on results:

BODY WEIGHT AND WEIGHT GAIN: Mean body weights were consistently decreased compared to sham controls during the exposure period in male rats exposed to 0.8 mg/L of reference cigarette smoke and in males exposed to all 3 concentrations of test cigarette smoke

HAEMATOLOGY: Whole-blood COHb levels were increased in a graded dose response fashion as a function of exposure concentration for all test and reference cigarette smoke-exposed groups

CLINICAL CHEMISTRY: Plasma nicotine levels increased in a graded dose-response fashion for test and reference males and female groups

HISTOPATHOLOGY: NON-NEOPLASTIC
Hyperplasia of respiratory epithelium lining the anterior nasal cavity was present but was not of statistical significance.
Minimal goblet-cell hyperplasia was observed in the mucosal epithelium lining the median nasal septum in some smoke-exposed and sham control rats. Although not statistically significant, the incidence of nasal goblet cell hyperplasia in male rats exposed to the 0.8-mg/L concentration of smoke from the reference or test cigarette were considered to be toxicologically significant.
Exposure to smoke from the reference or test cigarette induced squamous metaplasia, hyperplasia, and hyperkeratosis of the transitional epithelium lining the base of the epiglottis and the epithelium lining the dorsal border of the ventral pouch and the adjacent laryngeal lumen.

Dose descriptor:

NOAEL

Effect level:

13 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No alteration in the toxicity of cigarette

Critical effects observed:

not specified

Conclusions:

The presence of benzyl propionate in cigarette at 13 ppm did not significantly change the type or extent of toxicologic effects observed in rodents inhaling cigarette smoke.

Executive summary:

Flavoring ingredients such as benzyl propionate are added to tobacco during the manufacture of many types of commercial cigarettes. This study has been carried out to determine the effect of these added ingredients on the toxicity of the resultant smoke.

 

Benzyl propionate was added at an level of 13 ppm in the cigarette. Sprague-Dawley rats were exposed by nose-only inhalation for 1 h/day, 5 days/wk for 13 wk to smoke from the test or reference cigarettes already described, or to air only, and necropsied after 13 wk of exposure or following 13 wk of recovery from smoke exposure.

 

Exposure to smoke from reference or test cigarettes induced increases in blood carboxyhemoglobin (COHb) and plasma nicotine, decreases in minute volume, differences in body or organ weights compared to air controls, and a concentration-related hyperplasia, squamous metaplasia, and inflammation in the respiratory tract. All these effects were greatly decreased or absent following the recovery period.

Comparison of rats exposed to similar concentrations of test and reference cigarette smoke indicated no difference at any concentration. In summary, the results did not indicate any consistent differences in toxicologic effects between smoke from cigarettes containing the flavoring ingredients and reference cigarettes. Thus, addition of benzyl propionate did not result in an alteration in the toxicity of the cigarette

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

13

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity: oral route

Based on the various studies available with Klimish rating 2 and 4 for the read across substances for CAS: 122-63-4 based on the category approach of organic functional group along with similar mechanistic approach and having structural similarities defined by QSAR toolbox. This data is combined with the prediction done using the QSAR toolbox for the target chemical based on similar category approach, the results is summarized as follows:

Sr. No	End point	Value	Species	Route	Duration	Effects	Remarks
1	NOAEL	520.25 mg/ kg bw/ d	Male/female Rat	Oral: feed	90 days	No effect on body weight and feed consumption	Predicted data for target chemical
2	NOAEL           LOAEL	25000 ppm (1750 mg/kg bw/d for males and 1870 mg/kg bw/d for females)   50000 ppm (3900 mg/kg bw/d for males and 4500 mg/kg bw/d for females)	Male/female Rat	Oral: feed	13 weeks (91 days)	No effect feed consumption         Mortality, decreased feed consumption, tremors and ataxia.	Data from NTP report for CAS: 140-11-4
3	NOAEL           LOAEL	6000 ppm (460 mg/Kg bw /d for males and 480 mg/Kg bw /d for females)   12000 ppm (900 mg/Kg bw /d for males and 930 mg/Kg bw /d for females)	Male/female Rat	Oral: feed	2 years	No effect on body weight and feed consumption           Decreased body weight and feed consumption	Data from NTP report for CAS: 140-11-4

 

Based on the studies summarized in the above table with oral routes it can be observed that NOAEL values estimated was 520.25 mg/Kg bw/ d. Also the NOAEL value varies from 460 - 1870 mg/kg bw/d and the LOAEL varies from 900 – 4500 mg/kg bw/day. The effects observed at these doses are listed as follows:

· Effects on mortality, decreased feed consumption, tremors and ataxia.

. Decreased body weight and feed consumption

Thus based on above discussion it can be concluded that substance CAS: 122-63-4 is expected to show the similar toxicological effect based on the effects observed on the other category members. Since the no effective dose value (NOAEL) is 520 mg/Kg bw/d thus based on this value it can be concluded that substance CAS: 122-63-4 is considered to be not toxic.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

NOAEL was considered to be 1000 mg/kg bw/day when Sprague-Dawley rats exposed to   Benzyl Propionate orally foe 28 days.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:

The presence of benzyl propionate in cigarette at 13 ppm did not significantly change the type or extent of toxicologic effects observed in rodents inhaling cigarette smoke.

Justification for classification or non-classification

Based on the available data, the substance benzyl propionate is not classified as toxic by the repeated exposure via oral and inhalation route.