Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer reviewed publication

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Effects of Flavoring and Casing Ingredients on the Toxicity of Mainstream Cigarette Smoke in Rats
Author:
Roger A. Renne, Hiroyuki Yoshimura, Kei Yoshino, George Lulham, Susumu Minamisawa, Albrecht Tribukait Dennis D. Dietz, Kyeonghee Monica Lee, and R. Bruce Westerberg
Year:
2006
Bibliographic source:
Inhalation Toxicology, 18:685–706, 2006
Reference Type:
publication
Title:
Repeated dose inhalation toxicity study of the test chemical
Author:
Renne et al
Year:
2006
Bibliographic source:
Inhalation Toxicology

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Refer below principle
Principles of method if other than guideline:
Effects of the test chemical on the toxicity of mainstream cigarette smoke in rats.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Raleigh, NC)
- Age at study initiation: 6-7 weeks
- Weight at study initiation: No data available
- Fasting period before study: No data available
- Housing: During the 13-week exposure period, the animals were individual in stainless-steel cages on open racks, while the animals were housed indvidual in polycarbonate cages bedded with ALPHA-dri alpha cellulose bedding during the recovery period.
- Diet (e.g. ad libitum): No data available
- Water (e.g. ad libitum): No data available
- Acclimatization period: Approx. 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data available
- Humidity (%): No data available
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): No data available

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: AMESA exposure units
- Method of holding animals in test chamber: No data available
- Source and rate of air: Air was drawn through the vacuum port by a peristaltic pump operating at a rate of approx. 1.05 l/min, creating a 2-s, 35 ml puff through each cigarette once each minute.
- Method of conditioning air: No data available
- System of generating particulates/aerosols: (RAM)-1 (MIE Inc., Bedford, MA)
- Temperature, humidity, pressure in air chamber: No data available
- Air flow rate: At a rate of approx. 1.05 l/min
- Air change rate: Air flows of at least No data available
- Method of particle size determination: No data available
- Treatment of exhaust air: No data available

TEST ATMOSPHERE
- Brief description of analytical method used: The WTPM:nicotine and CO:nicotine ratios were calculated for the exposure atmospheres. The concentration
of CO in the test and reference atmospheres was determined using Horiba PIR-2000 CO analyzers (Horiba Instruments, Inc., Irvine, CA), monitored by DOS-based computers. Particle size distribution of the smoke was measured using Mercer-style cascade impactors designed specifically for the size range of particles found in cigarette smoke.
- Samples taken from breathing zone: No data available

VEHICLE (if applicable)
- Justification for use and choice of vehicle: Air
- Composition of vehicle: No data available
- Type and concentration of dispersant aid (if powder): No data available
- Concentration of test material in vehicle: 0, 0.06, 0.2 or 0.8 mg/l
- Lot/batch no. of vehicle (if required): No data available
- Purity of vehicle: No data available
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
26 weeks (including a recovery period of 13 weeks)
Frequency of treatment:
1 hour per day, 5 days per week
Doses / concentrations
Remarks:
Doses /Concentrations: 0, 0.06, 0.2 or 0.8 mg/l
No. of animals per sex per dose:
Total: 240 rats
Control: 30 males, 30 females
0.06 mg/l: 30 males, 30 females
0.2 mg/l: 30 males, 30 females
0.8 mg/l: 30 males, 30 females
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). Target smoke concentrations were 0.06, 0.2, or 0.8 mg WTPM/L for the test and reference cigarettes. An additional group of 30 rats/sex served as sham controls.
Positive control:
No data available

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations included: Mortality and morbundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: IEvery 4 weeks

BODY WEIGHT: Yes
- Time schedule for examinations: During the randomization procedure, on exposure day 1, biweekly thereafter and at necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data available

FOOD EFFICIENCY: No data available

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

OPHTHALMOSCOPIC EXAMINATION: No data available

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day of the 13-wk interim sacrifice
- Anaesthetic used for blood collection: Yes, approx. 70% carbon dioxide
- Animals fasted: No data available
- How many animals: On all rats included to be sacrificed on week 13
- 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. In addition, reticulocytes were stained.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On the day of the 13-wk interim sacrific
- Animals fasted: No data available
- How many animals: On all rats included to be sacrificed on week 13
- 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.

URINALYSIS: No data available

NEUROBEHAVIOURAL EXAMINATION: No data available

OTHER: Respiratory function measurements incl. tidal volume (TV), respiratory rate (RR) and minute volume (MV). In addition, the level of Carboxyhemoglobin and plasma nicotine in blood was measured during week 2 and 10.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
A complete necropsy was done on all 13-wk exposure groups and 13-wk recovery group animals. 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
All tissues were fixed, sectioned and stained. 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. The lungs were sectioned to present a maximal section of the main-stem bronchi. Three transverse laryngeal sections were prepared from the base of the epiglottis, the ventral pouch, and through the caudal larynx at the level of the vocal folds. 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. Exposure-related microscopic lesions were observed in the tissues from the rats exposed to 0.8 mg/l; target organs were examined microscopically in the lower concentration groups to ascertain a no-effect concentration.
Other examinations:
Evaluations of cell proliferations rates of respiratory-tract tissues was also performed, where 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 for each sex by exposure concentration group 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. The statistical evaluation of incidence and severity of lesions was made using the Kolmogorov–Smirnov two-sample test. All treatment group means were compared to the sham control mean, and means of groups exposed to the test cigarette smoke were compared to the corresponding reference cigarette smoke-exposed group means. Cell proliferation data were compared statistically using Tukey’s studentized range test with SAS software.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
Exposure-related adverse clinical signs were absent. Clinical observations noted were minor in consequence and low in incidence.
Mortality:
no mortality observed
Description (incidence):
No significant mortality occurred.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The only consistent statistical difference in body weight changes between the test and reference cigarette smoke-exposed groups in either study was the decreased mean body weight in males exposed to 0.8 mg/L of reference cigarette smoke during the exposure period of the first experiment. Mean body weights of smoke-exposed groups were similar to sham control weights during the recovery period.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
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 in both studies. In the second experiment, rats bled during exposure wk 2, there was a statistically significant decrease in COHb levels in both sexes exposed to 0.8 mg/L of test cigarette smoke and in females exposed to 0.2 mg/L of test cigarette smoke, compared to groups exposed to reference cigarette smoke. There were no other clear differences in whole-blood COHb levels between the test and reference cigarette groups at equivalent exposure levels in either study.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Plasma nicotine levels increased in a graded dose-response fashion for test and reference males and female groups in both studies. In the second experiment, test female groups exposed to 0.8 mg/l had significantly lower plasma nicotine levels than the 0.8 mg/l reference females at both 2- and 10-wk sampling. Comparing males to females at all exposure levels for test and reference cigarettes, the females consistently had higher plasma nicotine levels in both studies.
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Statistically significant differences in organ weights in groups of smoke-exposed rats were primarily low mean organ weights compared to their respective sham controls. There was no clear pattern of differences in any absolute or relative organ weight in smoke-exposed groups compared to sham controls, or in groups exposed to test versus reference cigarette smoke at either the interim sacrifice or the recovery sacrifices.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Few gross lesions were observed in either study, with no evidence of changes attributable to exposure to smoke from the test or the reference cigarettes. Exposure to smoke from reference or test cigarettes in both studies induced concentration-related proliferative, metaplastic, and inflammatory microscopic lesions in the respiratory tract after 13 wk of exposure.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Hyperplasia of respiratory epithelium lining the anterior nasal cavity was present in all rats exposed to 0.8 mg/l in both studies. Areas most severely and most frequently affected were the distal portions of the nasal and maxillary turbinates in sections of nose just caudal to the incisor teeth. In affected rats, the epithelium in the distal turbinates was up to six cells thick. There was also a clear dose response in the severity of nasal respiratory epithelial hyperplasia, with severity ranging from minimal to moderate. Although not statistically significant compared to concurrent sham controls, the incidence of nasal goblet cell hyperplasia in male rats exposed to the 0.8-mg/L concentration of smoke from the reference cigarette or test cigarette in the first experiment were considered to be toxicologically significant. There was no clear difference in the incidence of goblet cell hyperplasia between groups exposed to similar concentrations of reference and test cigarette smoke in either study.
Exposure to smoke from the reference or test cigarette in both studies 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. In affected smoke-exposed rats, the base of the epiglottis was covered by a stratified squamous epithelium up to eight cells thick with a variably keratinized surface layer and a distinct basal cell layer. There was a concentration-related increase in severity of squamous metaplasia and hyperplasia of epiglottis epithelium in rats exposed to test or reference cigarette smoke.
Comparison of incidence/severity of hyperkeratosis in the epiglottis between test and reference cigarette smoke-exposed groups indicated a statistically significant difference only in the 0.06-mg/L groups from study 1, in which females exposed to test cigarette smoke had a higher incidence/severity than females exposed to reference cigarette smoke.
An incidence (7/20) of goblet-cell hyperplasia in males exposed to the 0.8-mg/L concentration of smoke from the test cigarette in both studies, although not statistically significant, was considered to be toxicologically significant. Examination of tissue sections from rats necropsied at the end of the recovery period demonstrated nearly complete regression of nasal and tracheal lesions and a substantial decrease in the incidence and severity of smoke-induced lesions in the larynx and lungs in rats exposed to smoke from test or reference cigarettes in both studies. There was no statistically significant difference in the incidence or severity of respiratory-tract lesions between recovery group rats previously exposed to similar concentrations of test and reference cigarette smoke in either study.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Reductions in RR and/or TV resulted in consistently lower MV in rats exposed to test or reference cigarette smoke compared to sham controls in both studies.

Effect levels

Dose descriptor:
NOAEL
Effect level:
0.06 mg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The No observed adverse effect level (NOAEL) for the test chemical was considered to be 0.06 mg/L when male and female Sprague-Dawley Crl:CD rats were exposed by inhalation of cigarette smoke for 13 weeks.
Executive summary:

In a repeated-dose inhalation toxicity study, groups of 30 Sprague-Dawley rats of each sex were exposed to concentrations of 0.06, 0.2 or 0.8 mg/l WTPM of smoke from test cigarettes containing Ethyl butyrate(as flavoring agent) together by over 100 other additives. The animals were exposed by nose-only inhalation for 1 h/day, 5 day/week for 13 consecutive weeks or for 13 weeks of recovery from smoke exposure. In addition, groups of 30 rats/sex were exposed to same concentration of smoke from reference cigarettes, similar to the test cigarettes but without flavoring agent or to filtered air only (shame control). Test animals were observed for mortality and moribundity as well as for clinical signs. Individual body weights were recorded biweekly. Respiratory function measurements including Tidal volume (TV), respiratory rate (RR) and minute volume (MV) were measured in 4 rats/sex/group. Samples for blood test and blood clinical chemistry was also collected. At the end of the study, test animals were subject to gross pathological and histopathological examination. In addition, cell proliferation rates were measured on respiratory tract tissues collected from 10 rats of each sex from each exposure group. As seen by the results, no significant mortality occurred, and the exposure-related adverse clinical signs were absent. Minor clinical observations were noted in low incidence. The mean body weights were consistently decreased 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 compared to sham controls. The mean body-weights of all female smoke-exposed was comparable to shame control females through the study, and at the end of 13 week of recovery period, the mean body weight of all the test- and reference smoke-exposed animals were similar to sham control weights. Comparison of organ weights in rats indicated no consistent differences between smoke-exposed group and shame control or between the test and reference smoke-exposed groups at either the interim sacrifice or the recovery sacrifices. Reductions in Respiratory rate (RR) and/or Tidal volume (TV) resulted in consistently lower Minute volume (MV) in rats exposed to test or reference cigarette smoke compared to sham controls, as excepted. Also, there was no consistent difference in MV between groups of rats exposed to test and reference cigarette smoke.Exposure to smoke from both reference or test cigarettes induced increases in blood carboxyhemoglobin (COHb)) and plasma nicotine level, 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. Thus, the results did not indicate any consistent differences in toxicological effects between smoke from cigarettes containing the flavoring or casing ingredients and reference cigarettes. Therefore, the NOAEL for the test chemical was considered to be 0.06 mg/L when male and female Sprague-Dawley rats were exposed by inhalation of cigarette smoke for 13 weeks.