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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic 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

Reference
Reference Type:
publication
Title:
Effects of Flavoring and Casing Ingredients on the Toxicity of Mainstream Cigarette Smoke in Rats
Author:
Renne RA, Yoshimura H, Yoshino K, Lulham G, Minamisawa S, Tribukait A, Dietz DD, Lee, KM, Westerberg RB
Year:
2006
Bibliographic source:
Inhalation Toxicology, 18:685–706, 2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Refer below principle
Principles of method if other than guideline:
Repeated dose oral toxicity study was performed to determine the toxic nature of the test chemical using rats for 90 days.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material: Reaction mass of 4-(2,6,6-trimethylcyclohex-2-ene-1-yl)-but-3-ene-2-one and 4-(2,6,6-trimethylcyclohex-1-ene-1-yl)-but-3-ene-2-one
- IUPAC name: Reaction mass of 4-(2,6,6-trimethylcyclohex-2-ene-1-yl)-but-3-ene-2-one and 4-(2,6,6-trimethylcyclohex-1-ene-1-yl)-but-3-ene-2-one
- Molecular formula: C13H20O
- Molecular weight: 192.3 g/mole
- Smiles : C1([C@@H](C(=CCC1)C)\C=C\C(C)=O)(C)C
- Inchl: 1S/C13H20O/c1-10-6-5-9-13(3,4)12(10)8-7-11(2)14/h6-8,12H,5,9H2,1-4H3/b8-7+
- Substance type: Organic
- Physical state: Solid (yellow)
Specific details on test material used for the study:
- Name of test material : Reaction mass of 4-(2,6,6-trimethylcyclohex-2-ene-1-yl)-but-3-ene-2-one and -(2,6,6-trimethylcyclohex-1-ene-1-yl)-but-3-ene-2-one
- Molecular formula : C13H20O
- Molecular weight : 192.3 g/mol
- Substance type: Organic
- Physical state: No data available
- Impurities (identity and concentrations): No data available

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
No data
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 6-7 weeks
- Weight at study initiation: No data available
- Fasting period before study: No data available
- Housing: 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.
- Diet (e.g. ad libitum): No data available
- Water (e.g. ad libitum): No data available
- Acclimatization period: 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

IN-LIFE DATES: From: To: No data available

Administration / exposure

Route of administration:
other: inhalation: smoke
Type of inhalation exposure:
nose only
Vehicle:
not specified
Remarks on MMAD:
MMAD / GSD: No data available
Details on inhalation exposure:
PREPARATION OF DOSING SOLUTIONS: The blends were cased with a mixture of glycerin and water (at a ratio of 2:1) to provide the necessary moisture for standard processing. In preparation of test cigarettes, the ingredients were applied at a rate of 10 kg/1000 kg leaf blend, that is, at 1% on the test cigarettes, and the casing was applied at a rate of 30 kg/1000 kg leaf blend.

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
- Concentration in vehicle: 0 or 0.13 mg/L wet total particulate matter (WTPM)
- Amount of vehicle (if gavage): No data available
- Lot/batch no. (if required): No data available
- Purity: No data available
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
1 h/day, 5 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
0 or 0.13 mg/L as a part of 0, 0.06, 0.2 or 0.8 mg/L WTPM of smoke
Basis:

No. of animals per sex per dose:
Control(filtered air only): 30 males, 30 females

Reference cigarette
0.06 mg/L of reference smoke: 30 males, 30 females
0.2 mg/L of reference smoke: 30 males, 30 females
0.8 mg/L of reference smoke: 30 males, 30 females

Cigarette with flavoring ingredients
0.06 mg/L WTPM of smoke: 30 males, 30 females
0.2 mg/L WTPM of smoke: 30 males, 30 females
0.8 mg/L WTPM of smoke: 30 males, 30 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: No data available
- Rationale for animal assignment (if not random): No data available
- 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. Mortality or morbundity was noted

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each rat was examined every 4 weeks 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): No data
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

FOOD EFFICIENCY: No data
- 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
- Time schedule for examinations: No data

OPHTHALMOSCOPIC EXAMINATION: No data
- Time schedule for examinations:
- Dose groups that were examined: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During week 2 and 10, and on the day of the 13-week interim sacrifice.
- Anaesthetic used for blood collection: Yes (using CO2 as anesthesia)
- Animals fasted: No data available
- How many animals: All control and treated animals in the study.
- Parameters examined: White blood cell (WBC) count, red blood cell (RBC) count, carboxyhemoglobin (COHb), hemoglobin (Hb) concentration, plasma nicotine, 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.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On the day of the 13-week interim sacrifice.
- Animals fasted: No data available
- How many animals: All control and treated animals in the study.
- Parameters examined: Urea nitrogen (BUN), creatinine, glucose, total protein, albumin, aspartate aminotransferase (AST),alanine aminotransferase (ALT), gamma-glutamyltranspeptidase (GGT), sodium, potassium, chloride, calcium, phosphorus,total bilirubin, cholesterol, and triglycerides.

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

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

OTHER:

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 week 2, 8, and 13 using whole-body phethysmography. Each animal was monitored once during a single exposure period.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
A complete necropsy was done on all 13-week exposure groups and 13-week recovery group animals. Rats designated for scheduled sacrifices or sacrificed due to moribund condition were weighed and anesthetized with 70% CO2 in air, followed by exsanguination before cessation of heartbeat. 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
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. 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.
Other examinations:
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 weeks 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. Data from both sides of bilaterally symmetrical tissues (nose, ventral pouch, mainstem bronchi) were combined for tabulation of results.

Statistics:
Body weight, body weight gain, organ:body weight, and organ:brain weight ratios were statistically analyzed for each sexby exposure concentration group using the Xybion PATH/TOXsystem.

Data homogeneity was determined by Bartlett’s test and Dunnett’st-test was performed to identifydifferences between each concentration group and the shamcontrol group, and between corresponding concentrations of testand reference cigarette smoke-exposed groups.

Nonhomogeneousdata were analyzed using a modified t-test. Respiratoryphysiology, clinical pathology, COHb, and plasma nicotine dataparameters were statistically evaluated using SAS software (StatisticalAnalysis System, SAS, Inc., Cary, NC).

One-way analysisof variance (ANOVA) between exposure groups was firstconducted, 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 compareequivalent exposure groups between cigarette types.

The statistical evaluationof incidence and severity of lesions was made using theKolmogorov–Smirnov two-sample test (Siegel, 1956). All treatmentgroup means were compared to the sham control mean, andmeans of groups exposed to the test cigarette smoke were comparedto the corresponding reference cigarette smoke-exposedgroup means.

Cell proliferation data were compared statisticallyusing Tukey’sstudentized range test with SAS software.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Exposure related adverse clinical signs were absent. Clinical observations noted were minor in consequence and low in incidence.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No significant mortality occurred
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
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. With the exception of day 71 (0.8 mg/L test), 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 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, non-treatment-related
Description (incidence and severity):
Occasional statistically significant differences in hematological parameters from control values were not considered to be of toxicologic significance, nor were they exposure related.

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. However, there were no other clear differences in whole blood COHb levels between the test and reference cigarette groups at equivalent exposure levels.

Plasma nicotine levels increased in a graded dose-response fashion for test and reference males and female groups. Comparing males to females in all exposure levels for test and reference cigarettes, the females consistently had higher plasma nicotine levels.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Occasional statistically significant differences in clinical chemistry parameters from control values were not considered to be of toxicologic significance, nor were they exposure related.
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
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, non-treatment-related
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.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
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 weeks of exposure.

Hyperplasia of respiratory epithelium lining the anterior nasal cavity was present in all rats exposed to 0.8 mg/L, a few rats exposed to 0.2 mg/L, and in 3/40 rats exposed to 0.06 mg/L.

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 were considered to be toxicologically significant.

Exposure to smoke from the reference or test cigarette in both 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.

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.

Chronic inflammation was present in the sub-mucosa of the epiglottis in some rats exposed to reference or test cigarette smoke, most frequently in rats exposed to the 0.8 mg/L smoke concentration. Squamous metaplasia, hyperplasia, and hyperkeratosis were also present in the epithelium lining of the opening of the ventral pouch and the adjacent laryngeal lumen in most rats exposed to smoke from the test or reference cigarette.

Exposure to smoke from reference or test cigarettes induced a dose-related increase in minimal hyperplasia of the mucosal epithelium lining the tracheal lumen in both sexes of rats.

There were increased numbers of macrophages diffusely scattered through the pulmonary alveoli of rats exposed to smoke from reference or test cigarettes, compared to concurrent controls.

There was a very low incidence of a variety of microscopic lesions in other tissues examined, with no evidence of an effect of exposure to smoke from the reference or test cigarette on these tissues.

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.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Effect levels

Dose descriptor:
NOAEC
Effect level:
0.13 mg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The results did not indicate any consistent differences in toxicologic effects between smoke from cigarettes containing the flavoring or casing ingredients and reference cigarettes
Remarks on result:
other: Not toxic

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The No Observed Adverse Effect Concentration (NOAEC) for the test chemical is considered to be 0.13 mg/L when Sprague-Dawley rats were exposed to test cigarette smoke by inhalation.
Executive summary:

A subchronic toxicity study was conducted to evaluate the toxic nature of repeated administration of the test chemical to Sprague-Dawley rats by an inhalation route of exposure.

 

Sprague dawley rats were exposed to 0 or 0.13 mg/L as a part of 0, 0.06, 0.2 or 0.8 mg/L WTPM of smoke 1 h/day, 5 days/week for 13 weeks.

 

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 or casing ingredients and reference cigarettes.

 

The No Observed Adverse Effect Concentration (NOAEC) for the test compound of the test chemical is found to be 0.13 mg/L when Sprague-Dawley rats were exposed to test cigarette smoke by inhalation.