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Description of key information

Repeated dose toxicity: Oral

90 days repeated oral toxicity study was performed to determine the toxic nature of the test chemical. The study was performed using male and female Sprague Dawley rats at dose level of 10 or 100 mg/Kg bw/day. There were no mortalities, and no abnormal clinical signs in rats treated with the test chemical. No significant effects on mean body weights occurred in treated males. Sporadic, statistically significant, lower body weights were reported at various times in the last 6 weeks of the study in females treated with the test chemical, 100 mg/kg body weight/day. It is possible that the lower mean body weights at the high dose may have been related to an unpalatable diet. Food intake of both sexes treated with the test chemical, 100 mg/kg body weight/day, was significantly reduced, with the effect greater in females. It is possible that the reduced food consumption at the high dose may have been related to an unpalatable diet. Decreased erythrocyte counts were reported in 10 mg/kg body weight/day treated males. Males given 100 mg/kg body weight/day showed a slight, but significant, increase in neutrophil counts and a decrease in lymphocytes. No effects of the test chemical, at either 10 or 100 mg/kg body weight/day, were reported on hematological parameters in females. No hematological changes were observed in either sex after 13 weeks of treatment. The earlier abnormalities were considered to be of no toxicological significance. The serum chemistry analyses were normal except for significantly lower alkaline phosphatase values in males and lower glucose concentrations in females dosed with 100 mg/Kg bw of the test chemical. Given the small magnitude of these changes, they were considered to be of no biological significance. In high-dose animals treated with the test chemical, mild changes in urinary parameters were considered not to be of biological significance. Relative kidney weights were significantly increased in males but not females treated with the high dose of the test chemical. Females showed increased relative liver and brain weights at the high dose of the test chemical. Relative liver weights were significantly increased also in females given 10 mg/kg body weight/day. As a result, the only biologically significant finding at the low dose of either ionone was an increase in relative liver weight in females treated with the test chemical, a finding likely associated with enzyme induction. Necropsy revealed no effect of treatment on gross or microscopic pathology except for statistically significant ‘‘desquamation’’ of the thyroid epithelium in 3 females treated with 100 mg/kg body weight/day. There were no gross or histopathological correlates to increased absolute and/or relative kidney and/or liver weights. Based on the results of the study, the No Observed Adverse Effect Level (NOAEL) for the test chemical is considered to be 10 mg/Kg bw/day when male and female rats were exposed to the test chemical for 90 days.

 

Repeated dose toxicity: Inhalation

4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one (CAS no 127-41-3)has very low vapor pressure of3.61 Pa (0.0271 mm Hg), so the potential for the generation of inhalable vapours is very low, also the normal conditions of use of this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalatory route will be highly unlikely and therefore this end point for repeated dose toxicity by inhalation route of exposure was considered for waiver.

Repeated dose Toxicity: Dermal

The acute toxicity value for 4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one ( 127-41-3 ) (as provided in section 7.2.3) is >2000 mg/kg body weight. Also, used predominantly as a fragrance ingredient in cosmetic products especially in perfumery as well as in non-cosmetic products such as household cleaners and detergents. Its use worldwide is in the region of 100–1000 metric tonnes per annum. The maximum skin level that results from the use of alpha ionone in formulae that go into fine fragrances has been reported to be 1% (Belsito et al, 2007), assuming the dermal systemic exposure in cosmetic products to be 0.0512 mg/Kg/day. Since the exposure concentration related to the chemical is very less, hence the end point is considered for waiver. Hence this end point was considered for waiver.

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:
supporting 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 review article
Qualifier:
according to
Guideline:
other: Refer below principle
Principles of method if other than guideline:
90 days repeated oral toxicity study was performed to determine the toxic nature of the test chemical
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
No data
Route of administration:
oral: feed
Vehicle:
other: Feed
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test chemical was mixed with feed to give dose levels of 10 or 100 mg/Kg bw

DIET PREPARATION
- Rate of preparation of diet (frequency): No data
- Mixing appropriate amounts with (Type of food): No data
- Storage temperature of food: No data

VEHICLE
- Justification for use and choice of vehicle (if other than water): 10 or 100 mg/Kg bw
- Concentration in vehicle: No data
- Amount of vehicle (if gavage): No data
- Lot/batch no. (if required): No data
- Purity: No data
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily
Remarks:
10 or 100 mg/Kg bw
No. of animals per sex per dose:
Total: 30 males and 30 females
10 mg/Kg bw: 15 males and 15 females
100 mg/Kg bw: 15 males and 15 females
Control animals:
not specified
Details on study design:
No data
Positive control:
No data
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: No data
- Cage side observations checked in table [No.?] were included. Mortality and clinical signs

DETAILED CLINICAL OBSERVATIONS: No data
- Time schedule: No data

BODY WEIGHT: Yes
- Time schedule for examinations: No data

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No 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: No data
- Dose groups that were examined: No data

HAEMATOLOGY: yes
- Time schedule for collection of blood: After 13 weeks of treatment
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined. No data

CLINICAL CHEMISTRY: yes
- Time schedule for collection of blood: No data
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined. No data

URINALYSIS: Yes
- 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: No data
- Dose groups that were examined: No data
- Battery of functions tested: sensory activity / grip strength / motor activity / other: No data

OTHER: No data
Sacrifice and pathology:
GROSS PATHOLOGY: yes, organ weights were measured
HISTOPATHOLOGY: Yes, microscopic examination was performed
Other examinations:
No data
Statistics:
No data
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No significant effects on mean body weights occurred in treated males. Sporadic, statistically significant, lower body weights were reported at various times in the last 6 weeks of the study in females treated with the test chemical, 100 mg/kg body weight/day. It is possible that the lower mean body weights at the high dose may have been related to an unpalatable diet
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food intake of both sexes treated with the test chemical, 100 mg/kg body weight/day, was significantly reduced, with the effect greater in females. It is possible that the reduced food consumption at the high dose may have been related to an unpalatable diet
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):
Decreased erythrocyte counts were reported in 10 mg/kg body weight/day treated males. Males given 100 mg a-ionone/kg body weight/day showed a slight, but significant, increase in neutrophil counts and a decrease in lymphocytes. No effects of the test chemical, at either 10 or 100 mg/kg body weight/day, were reported on hematological parameters in females. No hematological changes were observed in either sex after 13 weeks of treatment. The earlier abnormalities were considered to be of no toxicological significance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The serum chemistry analyses were normal except for significantly lower alkaline phosphatase values in males and lower glucose concentrations in females dosed with 100 mg/Kg bw of the test chemical. Given the small magnitude of these changes, they were considered to be of no biological significance.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
In high-dose animals treated with the test chemical, mild changes in urinary parameters were considered not to be of biological significance
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Relative kidney weights were
significantly increased in males but not females treated with
the high dose of the test chemical. Females showed increased relative liver and brain weights at the high dose of the test chemical. Relative liver weights were significantly increased also in females given 10 mg/kg body weight/day.

There were no gross pathological correlates to increased absolute and/or relative kidney and/or liver weights.

As a result, the only biologically significant finding at the low dose of either ionone was an increase in relative liver weight in females treated with the test chemical, a finding likely associated with enzyme induction.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Necropsy revealed no effect of treatment on gross or microscopic pathology except for statistically significant ‘‘desquamation’’ of the thyroid epithelium in 3 females treated with 100 mg/kg body weight/day.

There were no histopathological correlates to increased absolute and/or relative kidney and/or liver weights.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Critical effects observed:
not specified
Conclusions:
The No Observed Adverse Effect Level (NOAEL) for the test chemical is considered to be 10 mg/Kg bw/day when male and female rats were exposed to the test chemical for 90 days.
Executive summary:

90 days repeated oral toxicity study was performed to determine the toxic nature of the test chemical. The study was performed using male and female Sprague Dawley rats at dose level of 10 or 100 mg/Kg bw/day. There were no mortalities, and no abnormal clinical signs in rats treated with the test chemical. No significant effects on mean body weights occurred in treated males. Sporadic, statistically significant, lower body weights were reported at various times in the last 6 weeks of the study in females treated with the test chemical, 100 mg/kg body weight/day. It is possible that the lower mean body weights at the high dose may have been related to an unpalatable diet. Food intake of both sexes treated with the test chemical, 100 mg/kg body weight/day, was significantly reduced, with the effect greater in females. It is possible that the reduced food consumption at the high dose may have been related to an unpalatable diet. Decreased erythrocyte counts were reported in 10 mg/kg body weight/day treated males. Males given 100 mg a-ionone/kg body weight/day showed a slight, but significant, increase in neutrophil counts and a decrease in lymphocytes. No effects of the test chemical, at either 10 or 100 mg/kg body weight/day, were reported on hematological parameters in females. No hematological changes were observed in either sex after 13 weeks of treatment. The earlier abnormalities were considered to be of no toxicological significance. The serum chemistry analyses were normal except for significantly lower alkaline phosphatase values in males and lower glucose concentrations in females dosed with 100 mg/Kg bw of the test chemical. Given the small magnitude of these changes, they were considered to be of no biological significance. In high-dose animals treated with the test chemical, mild changes in urinary parameters were considered not to be of biological significance. Relative kidney weights were significantly increased in males but not females treated with the high dose of the test chemical. Females showed increased relative liver and brain weights at the high dose of the test chemical. Relative liver weights were significantly increased also in females given 10 mg/kg body weight/day. As a result, the only biologically significant finding at the low dose of either ionone was an increase in relative liver weight in females treated with the test chemical, a finding likely associated with enzyme induction. Necropsy revealed no effect of treatment on gross or microscopic pathology except for statistically significant ‘‘desquamation’’ of the thyroid epithelium in 3 females treated with 100 mg/kg body weight/day. There were no gross or histopathological correlates to increased absolute and/or relative kidney and/or liver weights. Based on the results of the study, the No Observed Adverse Effect Level (NOAEL) for the test chemical is considered to be 10 mg/Kg bw/day when male and female rats were exposed to the test chemical for 90 days.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
10 mg/kg bw/day
Study duration:
chronic
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:
repeated dose toxicity: inhalation, other
Data waiving:
other justification
Justification for data waiving:
a short-term toxicity study does not need to be conducted because exposure of humans via inhalation in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment
Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
Waiver

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:
short-term repeated dose toxicity: dermal
Data waiving:
other justification
Justification for data waiving:
a short-term toxicity study does not need to be conducted because exposure of humans via the dermal route in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment
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

The data available for the test chemical was reviewed to determine the toxic nature of 4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one ( CAS no. 127-41-3 )repeated exposure by oral route. The studies are as mentioned below:

Repeated dose toxicity: Oral

90 days repeated oral toxicity study was performed to determine the toxic nature of the test chemical. The study was performed using male and female Sprague Dawley rats at dose level of 10 or 100 mg/Kg bw/day. There were no mortalities, and no abnormal clinical signs in rats treated with the test chemical. No significant effects on mean body weights occurred in treated males. Sporadic, statistically significant, lower body weights were reported at various times in the last 6 weeks of the study in females treated with the test chemical, 100 mg/kg body weight/day. It is possible that the lower mean body weights at the high dose may have been related to an unpalatable diet. Food intake of both sexes treated with the test chemical, 100 mg/kg body weight/day, was significantly reduced, with the effect greater in females. It is possible that the reduced food consumption at the high dose may have been related to an unpalatable diet. Decreased erythrocyte counts were reported in 10 mg/kg body weight/day treated males. Males given 100 mg a-ionone/kg body weight/day showed a slight, but significant, increase in neutrophil counts and a decrease in lymphocytes. No effects of the test chemical, at either 10 or 100 mg/kg body weight/day, were reported on hematological parameters in females. No hematological changes were observed in either sex after 13 weeks of treatment. The earlier abnormalities were considered to be of no toxicological significance. The serum chemistry analyses were normal except for significantly lower alkaline phosphatase values in males and lower glucose concentrations in females dosed with 100 mg/Kg bw of the test chemical. Given the small magnitude of these changes, they were considered to be of no biological significance. In high-dose animals treated with the test chemical, mild changes in urinary parameters were considered not to be of biological significance. Relative kidney weights were significantly increased in males but not females treated with the high dose of the test chemical. Females showed increased relative liver and brain weights at the high dose of the test chemical. Relative liver weights were significantly increased also in females given 10 mg/kg body weight/day. As a result, the only biologically significant finding at the low dose of either ionone was an increase in relative liver weight in females treated with the test chemical, a finding likely associated with enzyme induction. Necropsy revealed no effect of treatment on gross or microscopic pathology except for statistically significant ‘‘desquamation’’ of the thyroid epithelium in 3 females treated with 100 mg/kg body weight/day. There were no gross or histopathological correlates to increased absolute and/or relative kidney and/or liver weights. Based on the results of the study, the No Observed Adverse Effect Level (NOAEL) for the test chemical is considered to be 10 mg/Kg bw/day when male and female rats were exposed to the test chemical for 90 days.

Another repeated dose oral 90 days study was performed to determine the toxic nature of the test chemical. Male and female FDRL rats were fed diet containing 0 or 10.6 mg/kg/day 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. In addition to the usual observations (i.e. body weight and food consumption), haematologieal and blood chemical determinations were made on 8 rats of each sex at a 6-wk period, and in all rats at 12 wk. The tests were terminated at 0 days and at autopsy, liver and kidney weights were recorded and the following organs from half the animals in each group were taken for histological examination: liver, kidneys, stomach, small and large intestines, spleen, pancreas, heart, lungs, bone marrow, muscle, brain, spinal cord, bladder, adrenals, thyroid, pituitary, gonads, salivary glands, and lymph nodes. 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 test chemical

.

Repeated dose toxicity: Inhalation

4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one (CAS no 127-41-3)has very low vapor pressure of3.61 Pa (0.0271 mm Hg), so the potential for the generation of inhalable vapours is very low, also the normal conditions of use of this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalatory route will be highly unlikely and therefore this end point for repeated dose toxicity by inhalation route of exposure was considered for waiver.

Repeated dose Toxicity: Dermal

The acute toxicity value for 4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one ( 127-41-3 ) (as provided in section 7.2.3) is >2000 mg/kg body weight. Also, used predominantly as a fragrance ingredient in cosmetic products especially in perfumery as well as in non-cosmetic products such as household cleaners and detergents. Its use worldwide is in the region of 100–1000 metric tonnes per annum. The maximum skin level that results from the use of alpha ionone in formulae that go into fine fragrances has been reported to be 1% (Belsito et al, 2007), assuming the dermal systemic exposure in cosmetic products to be 0.0512 mg/Kg/day. Since the exposure concentration related to the chemical is very less, hence the end point is considered for waiver. Hence this end point was considered for waiver.

Based on the data available for the test chemical 4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one (CAS no. 127-41-3 ) is not likely to exhibit toxic nature upon repeated exposure by oral, inhalation and dermal route of exposure and hence is not likely to classify as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the data available for the test chemical 4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one (CAS no. 127-41-3 ) is not likely to exhibit toxic nature upon repeated exposure by oral, inhalation and dermal route of exposure and hence is not likely to classify as per the criteria mentioned in CLP regulation.