Registration Dossier

Administrative data

Description of key information

Repeated dose toxicity: Oral

Chronic toxicity study was performed for d- carvone using B6C3F1 mice. The test chemical was mixed with corn oil and dosed by the gavage route of exposure to male and female at dose levels of 0, 375 or 750 mg/kg. During the study period, the animals were observed for clinical signs, body weight changes, organ weight changes and histopathalogy.The survival of both 375 mg/Kg (after week 101) and 750 mg/Kg (after week 92) groups of female mice was significantly greater than that of the vehicle controIs. No significant differences were observed between any groups of male mice. No compound-related clinical signs were observed. Mean body weights of dosed and vehicle control male mice were similar throughout most of the studies; mean body weights of dosed female mice were within 7% of those of vehicle controls throughout most of the studies. No increase in neoplastic lesions was observed in dosed mice. No increases in tumor incidences were observed in mice administered d-carvone. In the current study, only nine primary neoplasms were seen in female vehicle control mice, each in a different animal. This low number may be related to the early deaths of female vehicle control mice. However, the incidences of male mice with primary neoplasms (vehicle control, 27/50; low dose, 15/50; high dose, 16/50) and the total numbers of primary neoplasms (vehicle control, 38; low dose, 18; high dose, 20) were significantly lower in dosed groups than in vehicle controls. It is not known if the low tumor yields are related to d-carvone administration. The only lesions considered possibly related to d-carvone in the 2-year studies in mice were atrophy of the olfactory epithelium and hyperplasia of the underlying Bowman's glands. These mayhave been related to reflux of d-carvone into the nose after the gavage needle was withdrawn, because inflammatory exudate and "foreign material" were often found in the nasal passage of dosed animals. Based on these considerations, the No observed Adverse Effect level (NOAEL) for d-carvone using male and female mice in 103 weeks study is considered to be 750 mg/kg/day.

Repeated dose toxicity: Inhalation

Based on the prediction done using the OECD QSAR toolbox version 3.3 with log kow as the primary descriptor and considering the five closest read across substances, repeated dose toxicity was predicted for d-carvone. The study assumed the use of male and female Wistar rats in a 90 days toxicity study. Since no significant treatment related effects were observed at the mentioned dose level, hence the No Observed Adverse Effect Concentration (NOAEC) for d-carvone is predicted to be 2364.870605469 mg/L.

 

Based on this value it can be concluded that the substance is considered to not toxic as per the criteria mentioned in CLP regulation.

Repeated dose toxicity: Dermal

The acute dermal toxicity value for d-carvone (as provided in section 7.2.3) is >2000 mg/kg body weight. The substance was also found to be not irritating to the skin. Based on these considerations, 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:
data from handbook or collection of data
Justification for type of information:
Data is from NTP report
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: Refer below principle
Principles of method if other than guideline:
Chronic toxicity study was performed for d- carvone using mice
GLP compliance:
not specified
Limit test:
no
Test material information:
Composition 1
Specific details on test material used for the study:
- Name of test material: D-Carvone- IUPAC name: (5S)-2-methyl-5-(prop-1-en-2-yl)cyclohex-2-en-1-one- Molecular formula: C10H14O- Molecular weight: 150.22 g/mol- Substance type: Organic- Physical state: No data- Purity: 96%- Impurities (identity and concentrations): 4%
Species:
mouse
Strain:
B6C3F1
Details on species / strain selection:
No data
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Frederick Cancer Research Facility (Frederick, MD)- Age at study initiation: 7 weeks- Weight at study initiation: No data available- Fasting period before study: No data available- Housing: Animals were housed five per cage in polycarbonate cages with beta chips and had Reemay spun-bonded polyester filters- Diet (e.g. ad libitum): NIH 07 Mouse Ration (ZeiglerBros., Inc., Gardners, PA); ad libitum- Water (e.g. ad libitum): automated watering system provided water ad libitum- Acclimation period: 13 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 15.5 ± 28.3 ˚C approx - Humidity (%): 23-90 %- Air changes (per hr): 6-12 room air changes/h- Photoperiod (hrs dark / hrs light): fluorescent light 12 h/d;IN-LIFE DATES: From: To: No data available
Route of administration:
oral: gavage
Details on route of administration:
The gavage route of administration was selected because the volatility of the chemical precluded its administration in feed. Because the feed blends of d-carvone were found to be unstable under the feed blending and simulated animal exposure conditions and because d-carvone is insoluble in water, corn oil gavage was selected as the route of administration for this study. The 21-day stability of d-carvone in corn oil at 0.5% (5 mg/g) stored at room temperature or at 5° C was determined. The corn oil solutions were extracted with methanol andanalyzed by high-performance liquid chromatography with a Brownlee RP-18 column and ultraviolet detection at 229 nm. The d-carvone/corn oil solutions were found to be stable for at least 21 days when stored in the dark at room temperature or at 5° C. The corn oil solutions were also stable under simulated dosing conditions for at least 3 hours.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test chemical was mixed with corn oil at dose level of 0, 375, or 750 mg/kgDIET PREPARATION- Rate of preparation of diet (frequency): No data- Mixing appropriate amounts with (Type of food): No data- Storage temperature of food: No dataVEHICLE- Justification for use and choice of vehicle (if other than water): Corn oil- Concentration in vehicle: 0, 375, or 750 mg/kg- Amount of vehicle (if gavage): 10 mL/Kg- Lot/batch no. (if required): No data available- Purity: No data available
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The chemical in corn oil was found by gas chromatography to be stable for at least 1 week in the dark at room temperature
Duration of treatment / exposure:
103 weeks
Frequency of treatment:
5 d/wk for 103 wk (some mice received 1 or 2 doses during wk 104)
Remarks:
0, 375, or 750 mg/kg
No. of animals per sex per dose:
Total: 150 males and 150 females0 mg/Kg bw: 50 males and 50 females375 mg/Kg bw: 50 males and 50 females750 mg/Kg bw: 50 males and 50 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: No data available- Rationale for animal assignment (if not random): Animals assigned to groups according to a table of random numbers- 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 and morbidityDETAILED CLINICAL OBSERVATIONS: No data- Time schedule: No dataBODY WEIGHT: Yes- Time schedule for examinations: Body weights were recorded once per week for the first 13 weeks of the study and at least once per month thereafterFOOD 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: - Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No dataFOOD 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 dataWATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data- Time schedule for examinations:OPHTHALMOSCOPIC EXAMINATION: No data- Time schedule for examinations: No data- Dose groups that were examined: No dataHAEMATOLOGY: No data- Time schedule for collection of blood: No data- Anaesthetic used for blood collection: No data- Animals fasted: No data- How many animals: No data- Parameters checked in table [No.?] were examined. CLINICAL CHEMISTRY: No data- 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 dataURINALYSIS: 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 dataNEUROBEHAVIOURAL EXAMINATION: No data No data- Time schedule for examinations:- Dose groups that were examined:- Battery of functions tested: sensory activity / grip strength / motor activity / other:OTHER: No data
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, all organs and tissues were examined for grossly visible lesionsHISTOPATHOLOGY: Yes, Tissues were preserved in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Adrenal glands, aorta, brain, cecum, colon, duodenum, epididymis/prostate/seminal vesicles/ testes or ovaries/ uterus, esophagus, gallbladder, gross lesions, heart, ileum, jejnum, kidneys, larynx, liver, lungs and bronchi, mammary gland, mandibular and mesenteric lymph nodes, nasal cavity and turbinates, pancreas, pancreatic islets, parathyroid glands, pituitary gland, preputial gland, rectum, salivary glands, spleen, sternebrae including marrow, stomach, thymus, thyroid gland, trachea, and urinary bladder were examined from the vehicle control, treated group animals
Other examinations:
No data
Statistics:
Refer below
Clinical signs:
no effects observed
Description (incidence and severity):
No compound-related clinical signs were observed.
Mortality:
mortality observed, treatment-related
Description (incidence):
The survival of both 375 mg/Kg (after week 101) and 750 mg/Kg (after week 92) groups of female mice was significantly greater than that of the vehicle controIs. No significant differences were observed between any groups of male mice.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights of dosed and vehicle control male mice were similar throughout most of the studies; mean body weights of dosed female mice were within 7% of those of vehicle controls throughout most of the studies
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:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Nasal Cavity: Foreign material, presumably the corn oil vehicle, was observed in the nasal cavity of male and female mice in dosed and vehicle control groups. It consisted of accumulations of pale yellow, translucent, foamy, or vacuolated material that was sometimes surrounded by an inflammatory exudate of mucus and neutrophils. Several lesions occurred in male and female mice with dose-related increased incidences and/or severity. Atrophy of the olfactory epithelium and hyperplasia of the underlying Bowman's glands occurred together. These lesions usually involved the mucosa along the dorsal meatus in the posterior region of the nose but extended to the septum and turbinates in the more severely affected animals. The olfactory epithelium was reduced in thickness because of the loss of the olfactory sensory epithelium and replacement by ciliated columnar cellis. The Bowman's glands were dilated and consisted of tall, columnar cells similar to those replacing the sensory epithelium on the surface. Acute, multifocal inflammation was characterized by accumulations of neutrophils and cellular debris, primarily in the lumina of the Bowman's glands of the turbinates. Since evidence of the corn oil vehicle was seen in over 50% of theanimals in all dosed and vehicle control groups, the Pathology Working Group felt that the lesions observed in the nasal mucosa were likely due to reflux of the gavage material into the nose after the gavage needle was withdrawn.Subcutaneous Tissue: Fibromas, sarcomas, fibrosarcomas, or neurofibrosarcomas (combined) were observed with a negative trend in male mice, and the reduced incidence was significant in low dose male mice (vehicle control, 9/50; low dose, 1/50; high dose, 3/50).Circulatory System: Three hemangiomas or hemangiosarcomas were observed in vehicle control male mice, but none was seen in dosed males; the difference was not significant.Urogenital System: Abscesses of the ovary and the uterus occurred at a high incidence in vehicle control female mice and at much lower incidences in dosed female mice (ovary: vehicle control, 26/50; low dose, 9/48; high dose, 1/48; uterus: 10/50; 3/50; 0/50). The lesions were similar to those observed in other studies associated with Krebsiella sp. infections and are believed to be the cause of reduced survival in vehicle control female mice relative to that of dosed female mice.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No increase in neoplastic lesions was observed in dosed mice.
Other effects:
not specified
Details on results:
No data
Dose descriptor:
NOAEL
Effect level:
750 other: mg/Kg
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxic treatment related clinical signs were noted
Critical effects observed:
not specified

Table 1. Survival of mice in the two year gavage studies of d-carvone

 

Vehicle control

375 mg/Kg

750 mg/Kg

Male

 

 

 

Animals initially in study

50

50

50

Natural deaths

6

6

7

Moribund kills

7

2

3

Killed accidentally

0

0

4

Animals surviving until study termination

37

42

36

Mean survival (days)

679

694

631

Survival P values

0.674

0.329

0.784

Female

 

 

 

Animals initially in study

50

50

50

Natural deaths

13

10

7

Moribund kills

23

10

4

Killed accidentally

0

2

1

Animals surviving until study termination

14

29

38

Mean survival (days)

639

652

676

Survival P values

<0.001

0.006

<0.001

 

Table: Number of mice with lesions of the nasal cavity in the two year gavage studies of d-carvone

Site/lesion

Male

Female

Vehicle control

375 mg/Kg

750 mg/Kg

Vehicle control

375 mg/Kg

750 mg/Kg

Number examined

50

50

49

49

49

50

Glands

 

 

 

 

 

 

Hyperplasia

3

**42

**44

19

**45

**49

Olfactory epithelium

 

 

 

 

 

 

Atrophy

11

**42

**44

25

**46

**49

Turbinate

 

 

 

 

 

 

Multifocal acute inflammation

0

3

**27

5

**22

**39

**P<0.01 vs. vehicle controls

Conclusions:
The No observed Adverse Effect level (NOAEL) for d-carvone using male and female mice in 103 weeks study is considered to be 750 mg/kg/day.
Executive summary:

Chronic toxicity study was performed for d- carvone using B6C3F1 mice. The test chemical was mixed with corn oil and dosed by the gavage route of exposure to male and female at dose levels of 0, 375 or 750 mg/kg. During the study period, the animals were observed for clinical signs, body weight changes, organ weight changes and histopathalogy.The survival of both 375 mg/Kg (after week 101) and 750 mg/Kg (after week 92) groups of female mice was significantly greater than that of the vehicle controIs. No significant differences were observed between any groups of male mice. No compound-related clinical signs were observed. Mean body weights of dosed and vehicle control male mice were similar throughout most of the studies; mean body weights of dosed female mice were within 7% of those of vehicle controls throughout most of the studies. No increase in neoplastic lesions was observed in dosed mice. No increases in tumor incidences were observed in mice administered d-carvone. In the current study, only nine primary neoplasms were seen in female vehicle control mice, each in a different animal. This low number may be related to the early deaths of female vehicle control mice. However, the incidences of male mice with primary neoplasms (vehicle control, 27/50; low dose, 15/50; high dose, 16/50) and the total numbers of primary neoplasms (vehicle control, 38; low dose, 18; high dose, 20) were significantly lower in dosed groups than in vehicle controls. It is not known if the low tumor yields are related to d-carvone administration. The only lesions considered possibly related to d-carvone in the 2-year studies in mice were atrophy of the olfactory epithelium and hyperplasia of the underlying Bowman's glands. These mayhave been related to reflux of d-carvone into the nose after the gavage needle was withdrawn, because inflammatory exudate and "foreign material" were often found in the nasal passage of dosed animals. Based on these considerations, the No observed Adverse Effect level (NOAEL) for d-carvone using male and female mice in 103 weeks study is considered to be 750 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
750 mg/kg bw/day
Study duration:
chronic
Species:
mouse
Quality of whole database:
Data is from NTP report

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: inhalation
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with limited documentation / justification
Justification for type of information:
Data is from OECD QSAR Toolbox version 3.3 and the supporting QMRF report has been attached
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: Refer below principle
Principles of method if other than guideline:
Prediction is done using OECD QSAR Toolbox version 3.3, 2018
GLP compliance:
not specified
Limit test:
no
Test material information:
Composition 1
Specific details on test material used for the study:
- Name of test material: D-Carvone- IUPAC name: (5S)-2-methyl-5-(prop-1-en-2-yl)cyclohex-2-en-1-one- Molecular formula: C10H14O- Molecular weight: 150.22 g/mol- Substance type: Organic
Species:
rat
Strain:
Wistar
Details on species / strain selection:
No data
Sex:
male/female
Details on test animals and environmental conditions:
No data
Route of administration:
inhalation
Type of inhalation exposure:
not specified
Vehicle:
not specified
Remarks on MMAD:
No data
Details on inhalation exposure:
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:
6 hours per workday
Remarks:
No data
No. of animals per sex per dose:
No data
Control animals:
not specified
Details on study design:
No data
Positive control:
No data
Observations and examinations performed and frequency:
No data
Sacrifice and pathology:
No data
Other examinations:
No data
Statistics:
No data
Clinical signs:
not specified
Mortality:
not specified
Body weight and weight changes:
not specified
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:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Dose descriptor:
NOAEC
Effect level:
2 364.871 mg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No significant alterations were noted at the mentioned dose level
Critical effects observed:
not specified

The prediction was based on dataset comprised from the following descriptors: "study NOEL"
Estimation method: Takes average value from the 8 nearest neighbours
Domain  logical expression:Result: In Domain

((((((("a" or "b" or "c" )  and ("d" and ( not "e") )  )  and ("f" and ( not "g") )  )  and "h" )  and "i" )  and "j" )  and ("k" and "l" )  )

Domain logical expression index: "a"

Referential boundary: The target chemical should be classified as Aliphatic Carbon [CH] OR Aliphatic Carbon [-CH2-] OR Aliphatic Carbon [-CH3] OR Carbonyl, aliphatic attach [-C(=O)-] OR Carbonyl, olefinic attach [-C(=O)-] OR Miscellaneous sulfide (=S) or oxide (=O) OR Olefinic carbon [=CH- or =C<] OR Olefinic carbon [=CH2] OR Tertiary Carbon by Organic functional groups (US EPA) ONLY

Domain logical expression index: "b"

Referential boundary: The target chemical should be classified as Allyl OR Cycloalkene OR Cycloketone OR Overlapping groups by Organic Functional groups (nested) ONLY

Domain logical expression index: "c"

Referential boundary: The target chemical should be classified as Alkene OR Allyl OR Cycloalkene OR Cycloketone by Organic Functional groups ONLY

Domain logical expression index: "d"

Referential boundary: The target chemical should be classified as No alert found by DNA binding by OASIS v.1.3

Domain logical expression index: "e"

Referential boundary: The target chemical should be classified as AN2 OR AN2 >> Nucleophilic addition to alpha, beta-unsaturated carbonyl compounds OR AN2 >> Nucleophilic addition to alpha, beta-unsaturated carbonyl compounds >> alpha, beta-Unsaturated Aldehydes OR AN2 >> Schiff base formation OR AN2 >> Schiff base formation >> alpha, beta-Unsaturated Aldehydes OR SN2 OR SN2 >> Alkylation, direct acting epoxides and related OR SN2 >> Alkylation, direct acting epoxides and related >> Epoxides and Aziridines by DNA binding by OASIS v.1.3

Domain logical expression index: "f"

Referential boundary: The target chemical should be classified as Michael Addition AND Michael Addition >> Michael addition on conjugated systems with electron withdrawing group AND Michael Addition >> Michael addition on conjugated systems with electron withdrawing group >> alpha,beta-Carbonyl compounds with polarized double bonds  by Protein binding by OASIS v1.3

Domain logical expression index: "g"

Referential boundary: The target chemical should be classified as Schiff base formation >> Schiff base formation with carbonyl compounds by Protein binding by OASIS v1.3

Domain logical expression index: "h"

Referential boundary: The target chemical should be classified as Alkene AND Allyl AND Cycloalkene AND Cycloketone by Organic Functional groups ONLY

Domain logical expression index: "i"

Referential boundary: The target chemical should be classified as Alkene AND Allyl AND Cycloalkene AND Cycloketone by Organic Functional groups ONLY

Domain logical expression index: "j"

Referential boundary: The target chemical should be classified as Alkene AND Allyl AND Cycloalkene AND Cycloketone by Organic Functional groups ONLY

Domain logical expression index: "k"

Parametric boundary:The target chemical should have a value of log Kow which is >= 1.27

Domain logical expression index: "l"

Parametric boundary:The target chemical should have a value of log Kow which is <= 3.78

Conclusions:
The No Observed Adverse Effect Concentration (NOAEC) for d-carvone is predicted to be 2364.870605469 mg/L.
Executive summary:

Based on the prediction done using the OECD QSAR toolbox version 3.3 with log kow as the primary descriptor and considering the five closest read across substances, repeated dose toxicity was predicted for d-carvone. The study assumed the use of male and female Wistar rats in a 90 days toxicity study. Since no significant treatment related effects were observed at the mentioned dose level, hence the No Observed Adverse Effect Concentration (NOAEC) for d-carvone is predicted to be 2364.870605469 mg/L.

 

Based on this value it can be concluded that the substance is considered to not toxic as per the criteria mentioned in CLP regulation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
2 364 870 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
Waiver

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

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

Mode of Action Analysis / Human Relevance Framework

Additional information

Repeated dose toxicity: Oral

Data available for the target chemical was reviewed to determine the toxic nature of D-carvone. The studies are as mentioned below:

Chronic toxicity study (NTP, 1999) was performed for d- carvone using B6C3F1 mice. The test chemical was mixed with corn oil and dosed by the gavage route of exposure to male and female at dose levels of 0, 375 or 750 mg/kg. During the study period, the animals were observed for clinical signs, body weight changes, organ weight changes and histopathalogy.The survival of both 375 mg/Kg (after week 101) and 750 mg/Kg (after week 92) groups of female mice was significantly greater than that of the vehicle controIs. No significant differences were observed between any groups of male mice. No compound-related clinical signs were observed. Mean body weights of dosed and vehicle control male mice were similar throughout most of the studies; mean body weights of dosed female mice were within 7% of those of vehicle controls throughout most of the studies. No increase in neoplastic lesions was observed in dosed mice. No increases in tumor incidences were observed in mice administered d-carvone. In the current study, only nine primary neoplasms were seen in female vehicle control mice, each in a different animal. This low number may be related to the early deaths of female vehicle control mice. However, the incidences of male mice with primary neoplasms (vehicle control, 27/50; low dose, 15/50; high dose, 16/50) and the total numbers of primary neoplasms (vehicle control, 38; low dose, 18; high dose, 20) were significantly lower in dosed groups than in vehicle controls. It is not known if the low tumor yields are related to d-carvone administration. The only lesions considered possibly related to d-carvone in the 2-year studies in mice were atrophy of the olfactory epithelium and hyperplasia of the underlying Bowman's glands. These mayhave been related to reflux of d-carvone into the nose after the gavage needle was withdrawn, because inflammatory exudate and "foreign material" were often found in the nasal passage of dosed animals. Based on these considerations, the No observed Adverse Effect level (NOAEL) for d-carvone using male and female mice in 103 weeks study is considered to be 750 mg/kg/day.

In another toxcity study mentoined in NTP (1990), chronic toxicity study was performed for d- carvone using B6C3F1 mice. The test chemical was mixed with corn oil and dosed by the gavage route of exposure to male and female at dose levels of0, 93, 187, 375, 750, or 1500 mg/kg. During the study period, the animals were observed for clinical signs, body weight changes, organ weight changes and histopathalogy.All 30 male and 9/10 female mice that received1,500 mg/kg and 1/10 males that received 93 mg/kg died before the end of the study. Hypoactivity, ataxia, and hypersensitivity to touch were seen at 1,500 mg/kg. The female that received 1,500 mg/kg and survived to the end of the study had body tremors, hypersensitivity to touch, and impaired hind limb function during the study. Final mean body weights of dosed and vehicle (control male mice were similar. The final mean body weight of the female survivor act 1,500 mg/kg was 12% lower than that of vehicle controls.The relative liver weights for mice that received 750 mg/kg were significantly greater than those for vehicle controls.No compound-related lesions were observed. Based on the observations made, the No observed Adverse Effect level (NOAEL) for d-carvone using male and female mice in 13 weeks study is considered to be 375 mg/kg/day.

Subacute toxicity study (NTP, 1990) was performed for d- carvone using B6C3F1 mice. The test chemical was mixed with corn oil and dosed by the gavage route of exposure to 5 male and 5 female at dose levels of 0, 150, 328, 723, 1590 or 3,500 mg/kg. During the study period, the animals were observed for clinical signs, body weight changes, ophthalmology, organ weight changes and histopathaology. All mice that received 1,590 or 3,500 mg/kg died on day 2 or 3. Ataxia, impaired grasping reflex, body tremors, prostration, gasping, clonic or tonic convulsions, or impaired righting reflex were observed at 1,590 and 3,500 mg/kg; lacrimation, piloerection, hypoactivity, bradypnea, or ptosis were observed at 723, 1590, and 3500 mg/kg. Mean necropsy body weights of dosed and vehicle control mice were similar. Ocular discharge and corneal opacity was noted at 1,590 and 3,500 mg/kg.Relative liver weights were increased for dosed male mice; relative thymus weights were decreased for dosed female mice.No compound related histologic changes were seen. Based on the observations made, the No observed Adverse Effect level (NOAEL) for d-carvone using male and female mice in 16 days study is considered to be 328 mg/kg/day.

Subacute toxicity study was also performed for d- carvone using Wistar Crl: (WI) BR rats (EFSA Journal, 2014). The test chemical was mixed with corn oil and dosed by the gavage route of exposure to 5 male and 5 female at dose levels of 0, 50, 200 or 1000 mg/kg. During the study period, the animals were observed for clinical signs, body weight changes and food intake changes, organ weight changes and histopathaology.Reduction in body weight and food consumption was noted at 1000 mg/kg bw/day. No haematology data were obtained for the highest (1000 mg/kg bw/day) dose group. Prothrombin time was significantly reduced in males at the 200 mg/kg bw/day dose, otherwise there was no indication of any treatment-related effects. No clinical chemistry data were obtained for the highest (1000 mg/kg bw/day) dose group. Mean relative kidney weight and heart weight of males was significantly increased compared to controls but there was no sign of a similar difference in females. Macroscopic examination at necropsy showed evidence of gastric irritation (thickening of the limiting ridge and alterations in forestomach epithelium). Based on the observations made, No observed Adverse Effect level (NOAEL) for d-carvone using male and female rats in 14 days study is considered to be 200 mg/kg/day.

In another study mentoined in EFSA Journal (2014) Subchronic toxicity study was performed for d- carvone using Wistar Crl: (WI) BR rats. The test chemical was mixed with corn oil and dosed by the gavage route of exposure to 10 male and 10 female at dose levels of 0, 5, 30 or 180 mg/kg. During the study period, the animals were observed for clinical signs, body weight changes and food intake changes, organ weight changes, opthalmological changes and histopathaology.No clinical observations that were considered to be related to treatment were noted. Slightly reduced body weight gain of males at the highest dose (180 mg/kg bw/day) was matched by a reduced food intake. No abnormalities were observed in the ophthalmoscopic examination. There were no treatment-related effects on the haematological results apart from reduction of prothrombin time (PT) of 180 mg/kg bw/day males (since a similar difference was present in the results of the 14-day study this may not be a chance finding) and a significant increase in partial thromboplastin time (PTT) for 180 mg/kg bw/day females. Organ weights and weights expressed relative to body weight were similar for treated and control groups apart from those for liver and kidney. The relative liver weights of both sexes were significantly higher than those of controls at the 180 mg/Kg bw/day and in females also at the 50 mg/Kg bw/day. Mean relative kidney weights were significantly increased compared with controls for both sexes at the 50 or 180 mg/kg bw/day. Although some statistically significant differences were seen between controls and treated animals in thymus and spleen weights these were confined to females and were not dose-related and are thus considered unlikely to be due to treatment.Microscopic examination revealed no histopathological changes related to treatment apart from those in the kidney which consisted of tubular necrosis, exclusively in male rats, and basophilic tubules in both sexes. However, the renal histopathological lesions observed in male animals of the 50 and 180 mg/Kg bw/day were most probably related to the renal accumulation ofα2u -globulin. This conclusion was confirmed by highly positive staining of the treated (high dose group rats with antibody againstα2u –globulin). Based on these observations it is concluded that the renal histopathological changes observed in male rats treated with 30 or 180 mg/kg bw per day carvone min. 95 % are caused by accumulation of α2u -globulin in the proximal tubular cells and hence are diagnosed as α2u -globulin in nephropathy”. Based on the observations made, the No observed Adverse Effect level (NOAEL) for d-carvone using male and female rats in 90 days study is considered to be 30 mg/kg/day.

In a study mentioned by Hagan et al (Food and Cosmetic Toxicology, 1967), chronic toxicity oral study for the test compound carvone was studied in male and female Osborne-Mendel rats. The test compound was fed through the diet at a concentration of 0 or 2500 ppm (0 or 125 mg/Kg bw) for 1 year. The animals were observed weekly for weight, food intake and general condition. Haematological examinations were made at termination. These examinations included white cell counts, red cell counts, haemoglobins and haematocrits. No toxic effects were noted at the mentioned dose level. Based on the observations made, the no observed Adverse Effect Level (NOAEL) for the test compound carvone in Osborne-Mendel rats is found to be 125 mg/Kg bw.

In the same study by Hagan et al (Food anc Cosmetic Toxicology, 1967), another chronic toxicity oral study for the test compound carvone was studied in male and female Osborne-Mendel rats. The test compound was fed through the diet at a concentration of 0 or 1000 ppm (0 or 50 mg/Kg/day) for 27-28 weeks. The animals were observed weekly for weight, food intake and general condition. Hematological examinations were made at termination. These examinations included white cell counts, red cell counts, haemoglobins and haematocrits. No toxic effects were noted at the mentioned dose level. Based on the observations made, the no observed Adverse Effect Level (NOAEL) for the test compound carvone in Osborne-Mendel rats is found to be 50 mg/Kg bw.

Yet another study was mentioned in the publication by hagan et al (Food and Cosmetic Toxicology, 1967), chronic toxicity oral study for the test compound carvone was studied in male and female Osborne-Mendel rats. The test compound was fed through the diet at a concentration of 0 or 10000 ppm (0 or 500 mg/Kg bw) for 16 weeks. The animals were observed weekly for weight, food intake and general condition. Haematological examinations were made at termination. These examinations included white cell counts, red cell counts, haemoglobins and haematocrits. Growth retardation and testicular atrophy was noted at 500 mg/Kg bw. Based on the observations made, the low observed Adverse Effect Level (LOAEL) for the test compound carvone in Osborne-Mendel rats is found to be 500 mg/Kg bw.

Repeated dose toxicity: Inhalation

Based on the prediction done using the OECD QSAR toolbox version 3.3 with log kow as the primary descriptor and considering the five closest read across substances, repeated dose toxicity was predicted for d-carvone. The study assumed the use of male and female Wistar rats in a 90 days toxicity study. Since no significant treatment related effects were observed at the mentioned dose level, hence the No Observed Adverse Effect Concentration (NOAEC) for d-carvone is predicted to be 2364.870605469 mg/L.

 

Based on this value it can be concluded that the substance is considered to not toxic as per the criteria mentioned in CLP regulation.

Further, the acute inhalation toxicity value for d-carvone (as provided in section 7.2.2) is >5 mg/L and the substance is considered to be not toxic as per the acute inhalation toxicity study. Also the predicted NOAEC value for d-carvone is considered to be 2364.87 mg/L. The lack of toxicity data for d-carvone does not allow for assessing the safety of this enantiomer for repeated inhalation toxicity end point. Given the use of the chemical, d-carvone is used as a flavouring agent, in personal care products and in pesticide. The total aggregate exposure estimates show that the highest exposure level to d-carvone from current uses (feed additive, pesticides, food flavourings, natural food occurrence and personal care products) is 0.59 mg/kg bw/day for high-level children consumers, which is marginally higher than that for high-level adult consumers (0.52 mg/kg bw/day). The highest exposure estimate is therefore at the level of the ADI established for d-carvone (0.6 mg/kg bw/day). When considering the anticipated exposure from intended pesticide uses, the highest exposure estimate would become 0.60 mg/kg bw/day, which is still at the level of the ADI for d-carvone. Based on these consideratons, the end point for repeated inhalation toxicity is considered for waiver.

Repeated dose toxicity: Dermal

The acute dermal toxicity value for d-carvone (as provided in section 7.2.3) is >2000 mg/kg body weight. The substance was also found to be not irritating to the skin. Based on these considerations, the end point for repeated dermal toxicity is considered as waiver.

Based on the data available for the target chemical, d-carvone is considered to be non toxic as per the criteria mentioned in CLP regulation. Majority of studies mentioned above represent non toxic nature of d-carvone. However the study that represents the toxic nature of carvone however does not specify the details to clearly judge its toxic nature. Hence considering this, the test chemical d-carvone is considered to be non-toxic.

Justification for classification or non-classification

Based on the data available for the target chemical, d-carvone (CAS no 2244 -16 -8) is considered to be non toxic as per the criteria mentioned in CLP regulation. Majority of studies mentioned above represent non toxic nature of d-carvone. However the study that represents the toxic nature of carvone however does not specify the details to clearly judge its toxic nature. Hence considering this, the test chemical d-carvone is considered to be non-toxic.