Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

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

Data source

Reference
Reference Type:
review article or handbook
Title:
Toxicology and Carcinogenesis Studies of d-Carvone (CAS No. 2244-16-8) in B6C3F1 Mice (Gavage Studies)
Author:
U.S. Department Of Health and Human Services
Year:
1990
Bibliographic source:
National Toxicology Program, NTP TR 381, NIH Publication No. 90-2836, 1990

Materials and methods

Test guideline
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

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- 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
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%

Test animals

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

Administration / exposure

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)
Doses / concentrations
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

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes- Time schedule: Twice daily- Cage side observations checked in table [No.?] were included. Mortality 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

Results and discussion

Results of examinations

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

Effect levels

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

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.