Benzyl propionate

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Data source

Reference

Reference Type:

publication

Title:

Effects of Flavoring and Casing Ingredients on the Toxicity of Mainstream Cigarette Smoke in Rats

Author:

Roger A. Renne, et.al

Year:

2006

Bibliographic source:

Inhalation Toxicology, 18: 685–706, 2006

Materials and methods

Test guideline

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

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 propanoate

Details on test material:

- Name of test material: benzyl propionate
- Molecular formula: C10H12O2
- Molecular weight: 164.20 g/mole
- Substance type: Organic
- Physical state: Liquid

Test animals

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):

Administration / exposure

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

Doses / concentrations

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)

Examinations

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

Results and discussion

Results of examinations

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.

Effect levels

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

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

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