Registration Dossier

Administrative data

Description of key information

Repeated dose toxicity: Oral

The chronic repeated dose toxicity study of the test chemical and the test chemical in rat was conducted to evaluate adverse effects by oral route. Groups of 10 male and 10 female rats were maintained for 17 weeks on diets containing the test chemical at levels of 0, 1000, 2500, and 10,000 ppm (approximately equivalent to 50, 125, and 500 mg/kg body weight). No adverse effects were observed on growth, appearance, food intake, haematology, final body weight, organ weights or macroscopic appearance of organs of rats on all levels of the test chemical in the diet. However, microscopic examination revealed swelling of the hepatic parenchymal cells at all dietary levels. This "swelling of parenchymal cells" was dose-dependent, being "slight to moderate" at the highest dietary level (500 mg/kg bw/day), "slight" at the intermediate level (125 mg/kg bw/day), and "very slight" at the lowest level (50 mg/kg bw/day). Thus, it was concluded that the no-observed adverse effect level (NOAEL) of the test chemical in this chronic study was considered to be 125.0 mg/kg bw.

Repeated dose toxicity: Inhalation

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 Reaction mass of the test chemical is found to be 0.13 mg/L when Sprague-Dawley rats were exposed to test cigarette smoke by inhalation.

Repeated dose toxicity: Dermal

The acute dermal toxicity value forReaction 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 Standard (60% alpha-ionone and 40% ß-ionone)(as provided in section 7.2.3) is >2000 mg/kg body weight. The substance was also found to be not irritating and sensitizing to the skin. Considering this, the end point for repeated dermal toxicity is considered as waiver.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Data is from peer-reviewed journal.
Qualifier:
no guideline available
Principles of method if other than guideline:
The chronic repeated dose toxicity study of the test chemical in rat was conducted to evaluate adverse effects by oral route.
GLP compliance:
not specified
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Substance tested: 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 Standard (60% alpha-ionone and 40% ß-ionone)
Species:
rat
Strain:
Osborne-Mendel
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: No data available
- Females (if applicable) nulliparous and non-pregnant: No data available
- Age at study initiation: Weanling rats used in the study.
- Weight at study initiation: No data available
- Fasting period before study: No data available
- Housing: The animals were housed individually in wire cages.
- Diet (e.g. ad libitum): Food, ad libitum
- Water (e.g. ad libitum): Water, ad libitum
- Acclimation period: No data available

DETAILS OF FOOD AND WATER QUALITY: No data available

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
Route of administration:
oral: feed
Vehicle:
other: Diet
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: No data available

DIET PREPARATION
- Rate of preparation of diet (frequency): Fresh diets were made and distributed weekly.
- 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): Diet
- Concentration in vehicle: 0, 50, 125, 500 mg/kg bw
- 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
Duration of treatment / exposure:
17 weeks
Frequency of treatment:
Daily
Dose / conc.:
0 ppm
Remarks:
(approximately equivalent to 0 mg/kg body weight)
Dose / conc.:
1 000 ppm
Remarks:
(approximately equivalent to 50 mg/kg body weight)
Dose / conc.:
2 500 ppm
Remarks:
(approximately equivalent to 125 mg/kg body weight)
Dose / conc.:
10 000 ppm
Remarks:
(approximately equivalent to 500 mg/kg body weight)
No. of animals per sex per dose:
0 ppm (approximately equivalent to 0 mg/kg body weight) : 10 male and 10 female
1000 ppm (approximately equivalent to 50 mg/kg body weight) : 10 male and 10 female
2500 ppm (approximately equivalent to 125 mg/kg body weight) : 10 male and 10 female
10,000 ppm (approximately equivalent to 500 mg/kg body weight) : 10 male and 10 female
Control animals:
yes
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Not specified
- Time schedule: Not specified
- Cage side observations checked in table [No.?] were included. Not specified

DETAILED CLINICAL OBSERVATIONS: Not specified
- Time schedule: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded every week.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not specified
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified
- The rat's food intake were recorded every week.

FOOD EFFICIENCY:Not specified
- 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: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified
- Time schedule for examinations: Not specified

OPHTHALMOSCOPIC EXAMINATION: Not specified
- Time schedule for examinations: Not specified
- Dose groups that were examined: Not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Not specified
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: Not specified
- Parameters checked in table [No.?] were examined: These examinations included white cell counts, red cell counts, haemoglobins and haematocrits.

CLINICAL CHEMISTRY: Not specified
- Time schedule for collection of blood: Not specified
- Animals fasted: Not specified
- How many animals:Not specified
- Parameters checked in table [No.?] were examined. Not specified

URINALYSIS: Not specified
- Time schedule for collection of urine:Not specified
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters checked in table [No.?] were examined. Not specified

NEUROBEHAVIOURAL EXAMINATION: Not specified
- Time schedule for examinations: Not specified
- Dose groups that were examined: Not specified
- Battery of functions tested: sensory activity / grip strength / motor activity / other: Not specified

IMMUNOLOGY: Not specified
- Time schedule for examinations: Not specified
- How many animals: Not specified
- Dose groups that were examined: Not specified
- Parameters checked in table [No.?] were examined: Not specified

OTHER: General condition of rats were recorded every week.
Sacrifice and pathology:
At the termination of the experiment the rats were sacrificed and exsanguinated.

GROSS PATHOLOGY: Yes
The tissues of all the rats were examined macroscopically at the time of sacrifice. The viscera were removed and the liver, kidneys, spleen, heart, and testes were weighed.

HISTOPATHOLOGY: Yes
These organs, the remaining abdominal and thoracic viscera, and one hind leg, for bone, bone marrow, and muscle, were preserved in 10 % buffered formalin-saline solution for histopathological examination.
Other examinations:
No data available
Statistics:
No data available
Clinical signs:
not specified
Mortality:
not specified
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No adverse effects were observed on final body weight of rats on all levels of the test chemical in the diet.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No adverse effects were observed on food intake of rats on all levels ofthe test chemical in the diet.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Description (incidence and severity):
No adverse effects were observed on haematology of rats on all levels of the test chemical in the diet.
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No adverse effects were observed on growth and appearance of rats on all levels of the test chemical in the diet.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No adverse effects were observed on organ weights of rats on all levels of the test chemical in the diet.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No adverse effects were observed on macroscopic appearance of organs of rats on all levels of the test chemical in the diet.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
A dose-dependent slight to moderate swelling of liver parenchyma was noted which occurred to a very slight degree at the lowest level.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Dose descriptor:
NOAEL
Effect level:
125 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
haematology
behaviour (functional findings)
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
other: No adverse effects were seen.
Critical effects observed:
no
Conclusions:
It was concluded that the no-observed adverse effect level (NOAEL) of the test chemical in this chronic study was considered to be 125.0 mg/kg bw.

Executive summary:

The chronic repeated dose toxicity study of the test chemical in rat was conducted to evaluate adverse effects by oral route. Groups of 10 male and 10 female rats were maintained for 17 weeks on diets containing the test chemical at levels of 0, 1000, 2500, and 10.000 ppm (approximately equivalent to 50, 125, and 500 mg/kg body weight). No adverse effects were observed on growth, appearance, food intake, haematology, final body weight, organ weights or macroscopic appearance of organs of rats on all levels of the test chemical in the diet. However, microscopic examination revealed swelling of the hepatic parenchymal cells at all dietary levels. This "swelling of parenchymal cells" was dose-dependent, being "slight to moderate" at the highest dietary level (500 mg/kg bw/day), "slight" at the intermediate level (125 mg/kg bw/day), and "very slight" at the lowest level (50 mg/kg bw/day). Thus, it was concluded that the no-observed adverse effect level (NOAEL) of the test chemical in this chronic study was considered to be 125.0 mg/kg bw.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
125 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Data is from K2 peer reviewed publication

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:
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
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
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
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
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
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
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.
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
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
Critical effects observed:
not specified
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
130 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
Data is from K2 peer reviewed publication

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: dermal, other
Data waiving:
other justification
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
Waiver

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Data available for the test chemical and its read across was reviewed to determine its toxic nature upon repeated exposure by oral and dermal route of exposure. The studies are as mentioned below:

 

Repeated dose toxicity: Oral

The chronic repeated dose toxicity study of the test chemical in rat was conducted to evaluate adverse effects by oral route. Groups of 10 male and 10 female rats were maintained for 17 weeks on diets containing the test chemical at levels of 0, 1000, 2500, and 10.000 ppm (approximately equivalent to 50, 125, and 500 mg/kg body weight). No adverse effects were observed on growth, appearance, food intake, haematology, final body weight, organ weights or macroscopic appearance of organs of rats on all levels of the test chemical in the diet. However, microscopic examination revealed swelling of the hepatic parenchymal cells at all dietary levels. This "swelling of parenchymal cells" was dose-dependent, being "slight to moder ate" at the highest dietary level (500 mg/kg bw/day), "slight" at the intermediate level (125 mg/kg bw/day), and "very slight" at the lowest level (50 mg/kg bw/day). Thus, it was concluded that the no-observed adverse effect level (NOAEL) of the test chemical in this chronic study was considered to be 125.0 mg/kg bw.

 

In another study, Repeated dose oral toxicity study was performed to determine the toxic nature of the test chemical using rats for 90 days. The test chemical was fed to Sprague-Dawley strain rats in the diet at levels to provide intakes of approximately 10 or 100 mg/kg body weight per day for 90 days. The study consisted of two test groups each of fifteen rats of each sex and one control group of thirty rats of each sex. Renal function and haematological studies were performed mid-way through the treatment period and at the end of the study. A reduction in weight gain, and food consumption and serum glucose concentrations, increased water intakes and mild renal functional changes were noted at 100 mg/Kg bw/day. No histological changes were evident in the kidneys or livers. On the basis of observations made, the No Observed adverse effect level (NOAEL) for the test chemical is considered to be 10 mg/Kg bw/day.

The purpose of the another study conducted was to evaluate the toxic effect of the test chemical on Osborne-Mendel rats. In a chronic 17 weeks study,rats were fed orally with the test chemical at dose levels of 0, 50, 125 or 500 mg/ kg bw/ day. No adverse effects were observed on growth, appearance, food intake, hematology, final body weight, organ weights or macroscopic appearance of organs of rats at all dose levels. However, microscopic examination revealed swelling of the hepatic parenchymal cells. This swelling was dose-dependent, being slight to moderate in the highest level, slight in the second dose level and very slight at the lowest level. Hence the lowest observed adverse effect level (LOAEL) of the test chemical was considered to be 50 mg/kg day.

In another toxicological study, male and female FDRL rats were fed diet containing 0 or 11.4 mg/kg/day of the test chemical,a concentration that was reported by conducted measurement to provide an average intake of 11.6 mg/kg/day for males and 13.1 mg/kg/day for females for 90 days. No exposure-related effects were observed based on evaluations of body weight, food intake, hematology, blood chemistry (endpoints not specified), liver and kidney weights and histopathology. Therefore, the No Observed Adverse Effect Level (NOAEL) for male FDRL rats was considered to be 11.6 mg/kg/day while NOAEL for female FDRL rats was considered to be 13.1 mg/kg/day when exposed to the test chemical.

In a toxicological study, male and female FDRL rats were fed diet containing 0 or 10.6 mg/kg/day with the test chemical, a concentration that was reported by conducted measurement to provide an average intake of 11.8 mg/kg/day for males and 11.1 mg/kg/day for females for 90 days. No exposure-related effects were observed based on evaluations of body weight, food intake, hematology, blood chemistry (endpoints not specified), liver and kidney weights and histopathology. Therefore, the no observed adverse effect level (NOAEL) for male FDRL rats was considered to be 11.8 mg/kg/day while NOAEL for female FDRL rats was considered to be 11.1 mg/kg/day when exposed to the test chemical.

Repeated dose toxicity: Inhalation

 

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 chemical is found to be 0.13 mg/L when Sprague-Dawley rats were exposed to test cigarette smoke by inhalation.

 

In another subchronic toxicity study was conducted to evaluate the selective degeneration caused by repeated administration of the test chemical to rats by inhalation route of exposure. Wistar rats were exposed to odours of Reaction mass of the test chemical for 1 week and 5 weeks respectively at dose levels of 0 or 0.0016 mg/L. Following treatment,the animals were perfused in buffered saline and fixed with a formaldehyde-glutaraldehyde mixture and the olfactory bulbs have been examined and compared with control litter-mates, which have been exposed to filtered room air only. Study emphasis was placed upon the distribution of changes in the mitral cells of the olfactory bulbs following exposure. A darkening and shrinkage of the cell bodies (both cytoplasm and nucleus) as seen by light microscopy of aldehyde-fixed material stained by the method of Richardson et al was noted. However, changes were, in general, better defined and more marked after 2 months than at earlier periods. The animals showed zones of change that had the same relative position in coronal section from anterior to posterior planes through the bulb; in the majority, degeneration was more marked anteriorly than posteriorly. However, the pattern and extent of this 'selective degeneration' did not appear to be correlated with the concentration of the odorant. On the basis of the observations made, the No observed adverse effect concentration (NOAEC) for the test chemical is considered to be 0.0016 mg/L

 

In a study, subchronic toxicity study was conducted to evaluate the toxic nature of repeated administration of fragrance mixture containing the test chemical to female CD rats by an inhalation route of exposure. Young female CD rats after 2 weeks of acclimation were exposed to the fragrance mix at dose level of 5 mg/L containing o or 5.7 X 10 -6 mg/L of the test chemical . The animals were exposed to the aerosol generated by whole body exposure in 500L inhalation chamber for 4hrs/day, 5 days/week for 28 exposures during the 6 weeks study period. The animals were observed for general appearance, behavioral observations, skin reactions if any, body weight changes, hematology, clinical chemistry, and the animals were subjected to gross and histopathology. No changes in the physical appearance as the result of their exposure to the fragrance mixtures. No skin reactions resulting from exposure of the animals were observed. No mortality was noted at the mentioned dose level of 5.7 X 10 -6 mg/L. Body weights and weight gains of treated animals did not differ significantly from controls. Values for all animals exposed for 6 weeks did not differ significantly from those of untreated control animals for hematological and clinical chemistry parameters. Animals showed no differences in organ weights or organ to body weight ratios that were judged significant toxicologically. No abnormal gross pathology findings were noted. No significant histopathological findings were observed in animals exposed to fragrances containing the test chemical. None of the animals exhibited any abnormal behavior. Based on the observations made, No Observed Adverse Effect concentration (NOAEC) for the test chemical is considered to be 5.7 X 10 -6 mg/L.

The objective of another study was to evaluate the safety of finished fragrance products via the inhalation route. 22 CD female rats were exposed by inhalation (whole body) to the mixture at 0, 5, 9 or 50 mg/m3for 4 h per day, 5 days per week for 6 weeks.No adverse effect were observed on body weight, appeareance, hematology, clinical chemistry, organ weight, gross and histopathology. Hence theNo Observed Adverse Effect Level (NOAEL) for the test chemical in 6 weeks study was considered to be at a dose concentration of 5 mg/m3.

The purpose of the study was to assess the influence of tobacco ingredients on cigarette smoke chemistry. 20 Sprague–Dawley rats were exposed to cigarette smoke containing the test chemical for a treatment of 13 weeks, 5 days per week at targeted chamber TPM concentrations of 1 mg/l of air. An increase in body weight and COHb concentration, some smoke treatment related effect were observed. No statistically significant differences between any of the mean histopathological parameters in nasal cavities, trachea, bronchi, larynx and lungs was observed. The No Observed Adverse Effect Level (NOAEL) of the test chemical on Sprague–Dawley rats in 13 weeks study was observed at dose concentration of 10 mg/l of air.

Another study was performed to assess the influence of tobacco ingredients on cigarette smoke chemistry. 20 Sprague–Dawley rats were exposed to cigarette smoke containing the test chemical 13 weeks, 5 days per week at targeted chamber TPMconcentrations of 1 mg/l of air.There was increase in body weight and COHb concentration, some smoke treatment related effect were also observed. No statistically significant differences between any of the mean histopathological parameters were noted innasal cavities, trachea, bronchi, larynx and lungs. On the basis of observations made, the No Observed Adverse Effect Level (NOAEL) was considered to be 7.5mg/l of air.

Repeated dose toxicity: Dermal

The acute dermal toxicity value forReaction 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 Standard (60% alpha-ionone and 40% ß-ionone)(as provided in section 7.2.3) is >2000 mg/kg body weight. The substance was also found to be not irritating and sensitizing to the skin. Considering this, the end point for repeated dermal toxicity is considered as waiver.

 

Based on the data available for the test chemical and its various read across chemicals, the test chemical is not likely to be toxic upon repeated exposure by oral, dermal and inhalation route of exposure as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the data available for the test chemical and its various read across chemicals, the test chemical is not likely to be toxic upon repeated exposure by oral, dermal and inhalation route of exposure as per the criteria mentioned in CLP regulation.