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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

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Diss Factsheets

Administrative data

Description of key information

Repeated dose toxicity study 1 (Oral):

In a study performed according to OECD 408 (1998), the study-derived NOAEL for the read across analogue was 30 mg/kg bw/day. This effect level was based on histopathological changes in the kidneys of male rats treated at 500 mg/kg bw/day. The observed effects in the kidneys in male rats treated at 500 mg/kg bw/day were not considered to be indicative of a hazard to human health.

Repeated dose toxicity study 2 (Oral):

In a 17-week repeated dose toxicity study, the study-derived LOAEL for the read-across analogue was 1000 ppm (approx. 50 mg/kg bw/day). This effect level was based on swelling of parenchyma hepatocytes at 1000 ppm (described as very slight in severity.

Repeated dose toxicity study (Dermal):

The acute dermal toxicity value for 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) (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.

Repeated dose toxicity study (Inhalation):

Study 1:

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.

Study 2:

The No observed adverse effect concentration (NOAEC) for the test chemical is considered to be 0.0016 mg/L.

Study 3:

The No Observed Adverse Effect concentration (NOAEC) for the test chemical is considered to be 5.7 X 10-6 mg/L.

Study 4:

The No Observed Adverse Effect Level (NOAEL) for the test chemical on CD rats in 6 weeks study was considered to be at a dose concentration of 5 mg/m3.

Study 5:

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.

Study 6:

The No Observed Adverse Effect Level (NOAEL) for the test chemical using Sprague–Dawley rats in 13 weeks study was considered to be 7.5 mg/l of air.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
1998
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD IGS BR strain
Details on species / strain selection:
Male and female Sprague-Dawley Crl:CD IGS BR strain rats were obtained from Charles River (UK) Ltd. (Margate, Kent)
Sex:
male/female
Details on test animals or test system and environmental conditions:
After 6 days of acclimatization, the animals were randomly allocated to treatment groups using a total randomization method and the group mean bodyweights were thereafter determined to ensure similarity between the test groups. Animals were housed according to sex in two groups of three and one group of four. At the initiation of treatment males weighed 131 to 172 g, females weighed 122 to 155 g, and were 6 to 8 weeks old. The study room was supplied with at least 15 air changes per hour. Temperature ranged from 19 to 23C and humidity from 40 to 70%. A 12:12-hour light-dark cycle period was used. Food was available ad lib, as was water. Wooden chew blocks and cardboard fun tunnels were provided for environmental enrichment.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
The dose volume was 4 mL/kg/d.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulations were stored in darkness at 4C and were tested for stability, homogeneity, and concentration. The formulations were observed to be stable for at least two weeks and to be within acceptable concentration limits (mean nominal concentrations of +/- 3%).
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily
Remarks:
0 (vehicle), 5, 30 and 500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Dose levels were selected based on the results of a 14-day dose range finding (DRF) study. In the DRF, 3 rats per sex per dose were treated at 0 and 1000 mg/kg bw/day. No deaths were observed, however increased salivation, some slightly reduced body weights, and some pale kidneys were observed at 1000 mg/kg. Therefore, 500 mg/kg bw/day was selected as top dose in the definitive study.
Positive control:
Not included.
Observations and examinations performed and frequency:
The animals were observed twice daily for mortality, morbidity, clinical signs of toxicity, and behavioral changes. Body weights were recorded on day 1 and weekly thereafter. Food intake was recorded weekly, and water intake daily (cage-wise) by visual inspection. During week 12, functional performance tests were performed on all rats. The examined parameters included motor activity, grip strength and sensory reactivity to auditory, visual and proprioceptive stimuli. Prior to the start of treatment and during week 12, the eyes of all rats treated at 0 and 500 mg/kg were examined for anterior structures of the eye, papillary, corneal blink reflex, and following dilatation, the internal structure of the eye. Blood samples were collected from all rats on day 90. Hematological parameters included hemoglobin, erythrocyte count, hematocrit, erythrocyte indices (mean corpuscular hemoglobin, mean corpuscular volume, and mean corpuscular hemoglobin concentration), total leukocyte count, differential leukocyte count, platelet count, reticulocyte count, prothrombin time, and activated partial thromboplastin time. Blood chemistry parameters included urea, glucose, total protein, albumin, albumin/globulin ratio, sodium, potassium, chloride, calcium, inorganic phosphorous, aspartate aminotransferase (AST), alanine aminotransferase, alkaline phosphatase, creatinine, total cholesterol, and total bilirubin.
Sacrifice and pathology:
All rats were sacrificed on day 91. The following organs were weighed at all dose levels: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, and uterus. The following tissues were examined microscopically at 0 and 500 mg/kg: adrenals, thoracic aorta, bone and bone marrow (femur including stifle joint and sternum), brain (cerebrum, cerebellum, and pons), cecum, colon, duodenum, epididymides, eyes, gross lesions, heart, ileum (including Peyer patches), jejunum, kidneys, liver, lungs (with bronchi), lymph nodes (cervical and mesenteric), mammary glands, muscle (skeletal), esophagus, ovaries, pancreas, pituitary, prostate, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles, skin (hind limb), spinal cord (cervical, midthoracic, and lumbar), spleen, stomach, testes, thymus, thyroid/parathyroid, tongue, trachea, urinary bladder, and uterus.
Statistics:
Homogeneity of means: ANOVA or ANCOVA and Bartlett test.
Transformed data: Williams test (parametric) or the Shirley test (nonparametric).
Data showing no dose-dependency, but homogeneity of the mean: Dunnett (parametric) or Steel (non-parametric).
Pairwise tests (student t test parametric or Mann-Whitney U test nonparametric) were used on a case-by-case basis.
Histopathology data: Chi-square analysis and Kruskal-Wallis one-way nonparametric analysis.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs included increased salivation ≥5 mg/kg, noisy breathing at ≥5 mg/kg, and hunched posture and tiptoe gait at 500 mg/kg. Such observations were often observed at the test facility following gavage treatment with unpalatable or slightly irritant test article formulations. On this basis, the clinical signs at ≥5 mg/kg were not considered to be indicative of systemic toxicity.One female each at 30 and 500 mg/kg showed isolated episodes of tail elevation, and the same female rat treated at 30 mg/kg showed ataxia. Due to the spontaneous nature of the findings and the lack of correlating behavioural effects indicative of neurotoxicity, they were not considered to be of toxicological significance. Isolated events of generalised fur loss, scab formation, and generalized red/brown stained fur were observed at all dose levels and were not attributed to the test chemical.
Mortality:
no mortality observed
Description (incidence):
All animals survived to planned death.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Female rats treated at 500 mg/kg showed sporadic decreases/increases in body weight gain compared to the controls. This effect was not considered to be of toxicological significance because mean terminal body weight of female rats treated at 500 mg/kg only differed by 1% compared to the control group. For the male rats, no significant effects on body weight gain were observed.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Female rats treated at ≥5 mg/kg showed reduced in plasma bilirubin levels compared to the controls. This effect was not dose-related and most of the individual values were within the historical control range. Hence, it was not considered to be of toxicological significance. Other effects on blood chemistry parameters at 500 mg/kg included increases in plasma creatinine, total protein and cholesterol (both sexes); increased plasma albumin (males only), decreased AST levels (both sexes); decreased alkaline phosphatase levels (females only); and decreases plasma chloride concentrations (males only). None of the observed effects on blood biochemistry were considered to be of toxicological significance in the study.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No treatment-related effects on behaviour, functional performance, or sensory reactivity were observed.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Notable changes in organ weight included significant increases in liver and kidney weights, both absolute and relative to terminal body weight, at 500 mg/kg (both genders).
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross pathological effects were observed at any dose level.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The microscopic examination revealed treatment-related changes in the liver, kidneys, thyroid and bone marrow. Alterations in the liver were limited to hepatocyte enlargement in 4 of 10 males and 9 of 10 females at 500 mg/kg. In the absence of associated inflammatory or degenerative changes, the finding was considered to be a normal adaptive response to chemical exposure. In the kidneys of the male rats, globular accumulation of eosinophilic material was observed with increased incidence and severity at ≥30 mg/kg. This effect was minimal in 6, 4, 5 and 3 rats at 0, 5, 30 and 500 mg/kg, respectively; slight in 0, 0, 4, 5 rats at 0, 5, 30 and 500 mg/kg, respectively; and moderate in 0, 0, 0, and 1 rats at 0, 5, 30 and 500 mg/kg, respectively. The authors attributed this effect to hydrocarbon nephropathy, which other mammals such as female rats, mice, dogs and monkeys are refractory to (Alden CL. Toxicol Pathol 1986; 14: 109-11). In the thyroid, follicular cell hypertrophy (minimal in nature) was observed at an increased incidence in male rats treated at 500 mg/kg compared to the controls. In the bone marrow, an increased incidence of adipose infiltration (minimal in nature) was reported in male rats treated at 500 mg/kg compared to the controls. In contrast, the incidence of slight adipose infiltration was significantly increased in male rats treated at 0 mg/kg compared to the 500 mg/kg dose group.
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
NOAEL
Effect level:
>= 30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
food efficiency
gross pathology
haematology
histopathology: neoplastic
histopathology: non-neoplastic
mortality
ophthalmological examination
organ weights and organ / body weight ratios
water consumption and compound intake
Dose descriptor:
LOAEL
Effect level:
<= 500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Effect level:
>= 500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
food efficiency
gross pathology
haematology
histopathology: neoplastic
histopathology: non-neoplastic
mortality
ophthalmological examination
organ weights and organ / body weight ratios
water consumption and compound intake
Critical effects observed:
no
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Conclusions:
Based on the histopathological changes in the kidneys in males, the study-derived NOAEL was 30 mg/kg bw/day. The observed kidney effects were not considered to be indicative of a hazard to human health in the study.
Executive summary:

The chemical was given by oral gavage to 10 rats per sex per dose level at 0 (vehicle), 5, 30 and 500 mg/kg/day for a total of 90 days. Dose levels were selected based on the results of a dose range-finding (DRF) study. In the DRF, three rats per sex per dose level were treated at 0 and 1000 mg/kg/day for 14 days. No unscheduled deaths were observed in the DRF, and clinical signs were limited to increased salivation at 1000 mg/kg. Male rats treated at 1000 mg/kg weighed slightly less than the controls on the 4th day of study. At necropsy, 2 males and 1 female treated at 1000 mg/kg showed pale kidneys. Based on these data, doses of 0, 5, 30 and 500 mg/kg/day were selected in the definitive 90-day study. The animals were observed twice daily for mortality, morbidity, clinical signs of toxicity, and behavioral changes. Body weights were recorded on day 1 and weekly thereafter. Food intake was recorded weekly, and water intake daily (cage-wise) by visual inspection. During week 12, functional performance tests were performed on all rats. The examined parameters included motor activity, grip strength and sensory reactivity to auditory, visual and proprioceptive stimuli. Prior to the start of treatment and during week 12, the eyes of all rats treated at 0 and 500 mg/kg were examined for anterior structures of the eye, papillary, corneal blink reflex, and following dilatation, the internal structure of the eye. Blood samples were collected from all rats on day 90. Hematological parameters includedhemoglobin, erythrocytecount, hematocrit, erythrocyte indices (meancorpuscular hemoglobin, mean corpuscular volume, and meancorpuscular hemoglobinconcentration), total leukocyte count,differential leukocyte count, platelet count, reticulocytecount, prothrombin time, and activated partial thromboplastintime. Blood chemistry parameters included urea,glucose, total protein, albumin, albumin/globulin ratio, sodium,potassium, chloride, calcium, inorganic phosphorous, aspartateaminotransferase (AST), alanine aminotransferase, alkalinephosphatase, creatinine, total cholesterol, and total bilirubin.All rats were sacrificed on day 91.Thefollowing organs were weighedat all dose levels:adrenals, brain, epididymides, heart, kidneys, liver, ovaries,spleen, testes, thymus, and uterus. The following tissues were examined microscopically at 0 and 500 mg/kg:adrenals, thoracic aorta, bone and bonemarrow (femur including stifle joint and sternum), brain (cerebrum,cerebellum, and pons), cecum, colon, duodenum,epididymides, eyes, gross lesions, heart, ileum (including Peyerpatches), jejunum, kidneys, liver, lungs (with bronchi), lymphnodes (cervical and mesenteric), mammary glands, muscle (skeletal),esophagus, ovaries, pancreas, pituitary, prostate, rectum,salivary glands (submaxillary), sciatic nerve, seminal vesicles,skin (hind limb), spinal cord (cervical, midthoracic, and lumbar),spleen, stomach, testes, thymus, thyroid/parathyroid, tongue,trachea, urinary bladder, and uterus.All animals survived to planned death. Clinical signs included increased salivation ≥5 mg/kg, noisy breathing at ≥5 mg/kg, and hunched posture and tiptoe gait at 500 mg/kg. Such observations were often observed at the test facility following gavage treatment with unpalatable or slightly irritant test article formulations. On this basis, the clinical signs at ≥5 mg/kg were not considered to be indicative of systemic toxicity. One female each at 30 and 500 mg/kg showed isolated episodes of tail elevation, and the same female rat treated at 30 mg/kg showed ataxia. Due to the spontaneous nature of the findings and the lack of correlating behavioural effects indicative of neurotoxicity, they were not considered to be of toxicological significance. Isolated events of generalised fur loss, scab formation, and generalized red/brown stained fur were observed at all dose levels and were not attributed to the test chemical. No treatment-related effects on behaviour, functional performance, or sensory reactivity were observed. Female rats treated at 500 mg/kg showed sporadic decreases/increases in body weight gain compared to the controls. This effect was not considered to be of toxicological significance because mean terminal body weight of female rats treated at 500 mg/kg only differed by 1% compared to the control group. For the male rats, no significant effects on body weight gain were observed. No adverse effects on food intake, food efficiency, or water intake were observed during the study period. The ophthalmoscopic examination revealed no treatment-related effects at 500 mg/kg. No treatment-related effects on haematology were observed. Female rats treated at ≥5 mg/kg showed reduced in plasma bilirubin levels compared to the controls. This effect was not dose-related and most of the individual values were within the historical control range. Hence, it was not considered to be of toxicological significance. Other effects on blood chemistry parameters at 500 mg/kg included increases in plasma creatinine, total protein and cholesterol (both sexes); increased plasma albumin (males only), decreased AST levels (both sexes); decreased alkaline phosphatase levels (females only); and decreases plasma chloride concentrations (males only). None of the observed effects on blood biochemistry were considered to be of toxicological significance in the study. No gross pathological effects were observed at any dose level. Notable changes in organ weight included significant increases in liver and kidney weights, both absolute and relative to terminal body weight, at 500 mg/kg (both genders). The microscopic examination revealed treatment-related changes in the liver, kidneys, thyroid and bone marrow. Alterations in the liver were limited to hepatocyte enlargement in 4 of 10 males and 9 of 10 females at 500 mg/kg. In the absence of associated inflammatory or degenerative changes, the finding was considered to be a normal adaptive response to chemical exposure. In the kidneys of the male rats, globular accumulation of eosinophilic material was observed with increased incidence and severity at ≥30 mg/kg. This effect was minimal in 6, 4, 5 and 3 rats at 0, 5, 30 and 500 mg/kg, respectively; slight in 0, 0, 4, 5 rats at 0, 5, 30 and 500 mg/kg, respectively; and moderate in 0, 0, 0, and 1 rats at 0, 5, 30 and 500 mg/kg, respectively. The authors attributed this effect to hydrocarbon nephropathy, which other mammals such as female rats, mice, dogs and monkeys are refractory to (Alden CL. Toxicol Pathol 1986; 14: 109-11). In the thyroid, follicular cell hypertrophy (minimal in nature) was observed at an increased incidence in male rats treated at 500 mg/kg compared to the controls. In the bone marrow, an increased incidence of adipose infiltration (minimal in nature) was reported in male rats treated at 500 mg/kg compared to the controls. In contrast, the incidence of slight adipose infiltration was significantly increased in male rats treated at 0 mg/kg compared to the 500 mg/kg dose group. Based on the histopathological changes in the kidneys in males, the study-derived NOAEL was 30 mg/kg bw/day. The observed kidney effects were not considered to be indicative of a hazard to human health in the study.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
30 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Klimisch 1 to 2 sources
System:
urinary
Organ:
kidney

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
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 or test system 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 a Klimisch 2 database.

Additional information

Repeated Dose Toxicity (Oral):

Study 1

The chemical was given by oral gavage to 10 rats per sex per dose level at 0 (vehicle), 5, 30 and 500 mg/kg/day for a total of 90 days. Dose levels were selected based on the results of a dose range-finding (DRF) study. In the DRF, three rats per sex per dose level were treated at 0 and 1000 mg/kg/day for 14 days. No unscheduled deaths were observed in the DRF, and clinical signs were limited to increased salivation at 1000 mg/kg. Male rats treated at 1000 mg/kg weighed slightly less than the controls on the 4th day of study. At necropsy, 2 males and 1 female treated at 1000 mg/kg showed pale kidneys. Based on these data, doses of 0, 5, 30 and 500 mg/kg/day were selected in the definitive 90-day study. The animals were observed twice daily for mortality, morbidity, clinical signs of toxicity, and behavioral changes. Body weights were recorded on day 1 and weekly thereafter. Food intake was recorded weekly, and water intake daily (cage-wise) by visual inspection. During week 12, functional performance tests were performed on all rats. The examined parameters included motor activity, grip strength and sensory reactivity to auditory, visual and proprioceptive stimuli. Prior to the start of treatment and during week 12, the eyes of all rats treated at 0 and 500 mg/kg were examined for anterior structures of the eye, papillary, corneal blink reflex, and following dilatation, the internal structure of the eye. Blood samples were collected from all rats on day 90. Hematological parameters includedhemoglobin, erythrocytecount, hematocrit, erythrocyte indices (meancorpuscular hemoglobin, mean corpuscular volume, and meancorpuscular hemoglobinconcentration), total leukocyte count,differential leukocyte count, platelet count, reticulocytecount, prothrombin time, and activated partial thromboplastintime. Blood chemistry parameters included urea,glucose, total protein, albumin, albumin/globulin ratio, sodium,potassium, chloride, calcium, inorganic phosphorous, aspartateaminotransferase (AST), alanine aminotransferase, alkalinephosphatase, creatinine, total cholesterol, and total bilirubin.All rats were sacrificed on day 91.Thefollowing organs were weighedat all dose levels:adrenals, brain, epididymides, heart, kidneys, liver, ovaries,spleen, testes, thymus, and uterus. The following tissues were examined microscopically at 0 and 500 mg/kg:adrenals, thoracic aorta, bone and bonemarrow (femur including stifle joint and sternum), brain (cerebrum,cerebellum, and pons), cecum, colon, duodenum,epididymides, eyes, gross lesions, heart, ileum (including Peyerpatches), jejunum, kidneys, liver, lungs (with bronchi), lymphnodes (cervical and mesenteric), mammary glands, muscle (skeletal),esophagus, ovaries, pancreas, pituitary, prostate, rectum,salivary glands (submaxillary), sciatic nerve, seminal vesicles,skin (hind limb), spinal cord (cervical, midthoracic, and lumbar),spleen, stomach, testes, thymus, thyroid/parathyroid, tongue,trachea, urinary bladder, and uterus.All animals survived to planned death. Clinical signs included increased salivation ≥5 mg/kg, noisy breathing at ≥5 mg/kg, and hunched posture and tiptoe gait at 500 mg/kg. Such observations were often observed at the test facility following gavage treatment with unpalatable or slightly irritant test article formulations. On this basis, the clinical signs at ≥5 mg/kg were not considered to be indicative of systemic toxicity. One female each at 30 and 500 mg/kg showed isolated episodes of tail elevation, and the same female rat treated at 30 mg/kg showed ataxia. Due to the spontaneous nature of the findings and the lack of correlating behavioural effects indicative of neurotoxicity, they were not considered to be of toxicological significance. Isolated events of generalised fur loss, scab formation, and generalized red/brown stained fur were observed at all dose levels and were not attributed to the test chemical. No treatment-related effects on behaviour, functional performance, or sensory reactivity were observed. Female rats treated at 500 mg/kg showed sporadic decreases/increases in body weight gain compared to the controls. This effect was not considered to be of toxicological significance because mean terminal body weight of female rats treated at 500 mg/kg only differed by 1% compared to the control group. For the male rats, no significant effects on body weight gain were observed. No adverse effects on food intake, food efficiency, or water intake were observed during the study period. The ophthalmoscopic examination revealed no treatment-related effects at 500 mg/kg. No treatment-related effects on haematology were observed. Female rats treated at ≥5 mg/kg showed reduced in plasma bilirubin levels compared to the controls. This effect was not dose-related and most of the individual values were within the historical control range. Hence, it was not considered to be of toxicological significance. Other effects on blood chemistry parameters at 500 mg/kg included increases in plasma creatinine, total protein and cholesterol (both sexes); increased plasma albumin (males only), decreased AST levels (both sexes); decreased alkaline phosphatase levels (females only); and decreases plasma chloride concentrations (males only). None of the observed effects on blood biochemistry were considered to be of toxicological significance in the study. No gross pathological effects were observed at any dose level. Notable changes in organ weight included significant increases in liver and kidney weights, both absolute and relative to terminal body weight, at 500 mg/kg (both genders). The microscopic examination revealed treatment-related changes in the liver, kidneys, thyroid and bone marrow. Alterations in the liver were limited to hepatocyte enlargement in 4 of 10 males and 9 of 10 females at 500 mg/kg. In the absence of associated inflammatory or degenerative changes, the finding was considered to be a normal adaptive response to chemical exposure. In the kidneys of the male rats, globular accumulation of eosinophilic material was observed with increased incidence and severity at ≥30 mg/kg. This effect was minimal in 6, 4, 5 and 3 rats at 0, 5, 30 and 500 mg/kg, respectively; slight in 0, 0, 4, 5 rats at 0, 5, 30 and 500 mg/kg, respectively; and moderate in 0, 0, 0, and 1 rats at 0, 5, 30 and 500 mg/kg, respectively. The authors attributed this effect to hydrocarbon nephropathy, which other mammals such as female rats, mice, dogs and monkeys are refractory to (Alden CL. Toxicol Pathol 1986; 14: 109-11). In the thyroid, follicular cell hypertrophy (minimal in nature) was observed at an increased incidence in male rats treated at 500 mg/kg compared to the controls. In the bone marrow, an increased incidence of adipose infiltration (minimal in nature) was reported in male rats treated at 500 mg/kg compared to the controls. In contrast, the incidence of slight adipose infiltration was significantly increased in male rats treated at 0 mg/kg compared to the 500 mg/kg dose group. Based on the histopathological changes in the kidneys in males, the study-derived NOAEL was 30 mg/kg bw/day. The observed kidney effects were not considered to be indicative of a hazard to human health in the study.

Study 2

The mixture was given by diet to 10 rats per sex per dose level at 0 (control group), 1000, 2500 and 10000 ppm for a total of 17 weeks. Clinical signs, body weights and food intake were recorded on a weekly basis. Blood samples were collected at the end of the study period. Haematological parameters included white blood cell counts, red blood cell counts, haemoglobins, and haematocrits. The tissues of all rats were examined grossly at termination. The liver, kidneys, spleen, heart, and testes were weighed. These organs, the remaining abdominal and thoracic viscera, one hind leg, bone marrow, and muscle were preserved for histopathological examination at 0 and 10000 ppm, and at lower doses in cases treatment-related effects were observed at 10000 ppm. No treatment-related effects on clinical signs, body weight, food intake, haematology, or organ weight were observed. Histopathological findings were limited to swelling of parenchyma hepatocytes at ≥1000 ppm. This effect was described as very slight at 1000 ppm, slight at 2500 ppm, and slight to moderate at 10000 ppm. LOAEL for the test chemical was considered to be 1000 ppm (approx. 50 mg/kg bw/day) in the study.

Repeated Dose Toxicity (Dermal):

The acute dermal toxicity value for 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) (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.

Repeated Dose Toxicity (Inhalation):

Study 1:

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.

Study 2:

A 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 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.

Study 3:

A 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 10-6mg/L 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 10-6mg/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 10-6mg/L.

Study 4:

The objective of this 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.

Study 5:

The purpose of this 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.

Study 6:

The purpose of this 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 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.

Justification for classification or non-classification

17 weeks of treatment with the 1st read across analogue in rats, at dietary concentrations up to 10000 ppm, did not produce any toxicological effects that would justify a classification for repeated dose toxicity by the oral route. While the study was limited in several aspects, the results of the study suggest a favourable toxicity profile of the registered substance in rats upon 17 weeks at doses up 10000 ppm (equivalent to 500 mg/kg bw/day, using a standard conversion factor of 20). In a study performed according to OECD 408 (1998), treatment with the 2nd read across analgue in rats, by oral gavage up to 500 mg/kg bw/day, did not produce any toxicological effects that would justify a classification for repeated dose toxicity by the oral route. By weight of evidence, with the exception of possible induction of hydrocarbon nephropathy in male rats (which is not a concern for human health), the registered substance can be assumed to be well-tolerated in rats up to 500 mg/kg bw/day upon 90 days of exposure by the oral route. The registered substance is therefore regarded to be classified as Not Classified for STOT RE by the oral route.