L-p-mentha-1(6),8-dien-2-one

Carcinogenicity

Administrative data

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1990

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

GLP compliance:

yes

Test material

Test material information

1

Reference substance name:

(S)-2-methyl-5-(1-methylvinyl)cyclohex-2-en-1-one

EC Number:

218-827-2

EC Name:

(S)-2-methyl-5-(1-methylvinyl)cyclohex-2-en-1-one

Cas Number:

2244-16-8

Molecular formula:

C10H14O

IUPAC Name:

5-isopropenyl-2-methylcyclohex-2-en-1-one

Test material form:

liquid

Specific details on test material used for the study:

- Source: Fritzsche, Dodge and Olcott Inc. (USA)
- Appearance: clear, pale yellow liquid
- Purity: 96.5% (as measured by titrimetric assay)
- Batch/Lot No.: K-332

Test animals

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Frederick Cancer Research Facility (USA)
- Age at study initiation: 7 wk
- Housing: 5 animals per polycarbonate cage (Hazleton Systems, Inc., Aberdeen, MD), bedded with beta chips
- Diet (e.g. ad libitum): ad libitum, NIH 07 Rat and Mouse Ration (Zeigler Bros, USA)
- Water (e.g. ad libitum): ad libitum, automatic watering system (Edstrom Industries, USA)
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature: 60-83 °F
- Humidity (%): 23-90
- Air changes (per hr): 6-12
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

VEHICLE
- Justification for use and choice of vehicle (if other than water): d-carvone is insoluble in water, corn oil was selected as vehicle
- Amount of vehicle (if gavage): 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

During the 2- year studies, the dose mixtures were analyzed at approximately 8-week intervals by extraction with methanol, followed by analysis with ultra-violet spectroscopy at 236 nm. All dose mixtures were found to be within 10% of the target concentrations by the study laboratory.

Duration of treatment / exposure:

103 weeks (some mice received 1 or 2 doses during week 104)

Frequency of treatment:

5 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

375 mg/kg bw/day (nominal)

Remarks:

Low conc.

Doses / concentrations 3

Dose / conc.:

750 mg/kg bw/day (nominal)

Remarks:

High conc.

No. of animals per sex per dose:

50

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on results from a previously conducted 13-week repeated dose toxicity study.
- Post-exposure period: no

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: two times per day

Sacrifice and pathology:

GROSS PATHOLOGY AND HISTOPATHOLOGY:
Necropsy and histologic exams performed on all animals; the following tissues were examined: adrenal glands, aorta, brain, cecum, colon, duodenm, epididymis/prostate/seminal vesicle/testes or ovaries/uterus, esophagus, femur, gall- bladder, gross lesions, Harderian gland, heart, ileum, jejunum, kidneys, larynx, liver, lungs and bronchi, mammary gland, mesenteric lymph nodes, nasal cavity and turbinates, pancreas, pancreatic islets, parathyroid and pituitary glands, preputial gland, rectum, salivary glands, skin, skeletal muscle, spinal cord, spleen, sternebrae including marrow, stomach, thymus, thyroid gland, trachea, and urinary bladder.

Other examinations:

No further data

Statistics:

According to Kaplan and Meier, method of Cox (1972) and Tarone's (1975) life table test, indicential tumor analysis, Fisher's exact/Cochrane-Armitage trend analysis.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No compound related clinical signs were observed.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Please refer to box "details on results".

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Please refer to box "details on results".

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Please refer to box "details on results".

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No neoplastic lesions attributed to d-carvone dosing were observed in mice.

Details on results:

BODY WEIGHTS AND CLINICAL SIGNS:
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 compound-related clinical signs were observed.

SURVIVAL:
The survival of both the low dose (after week 101) and high dose (after week 92) groups of female mice was significantly greater than that of the vehicle controls. No significant differences were observed between any groups of male mice.

NON-NEOPLASTIC LESIONS:
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 be-cause of the loss of the olfactory sensory epithelium and replacement by ciliated columnar cells. 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 de-bris, 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 the animals in all dosed and vehicle control groups, the Pathology Working Group considered 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, fi-brosarcomas, 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, 9/50;high dose, 1/48; uterus: 10150; 3/50; 0150). 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.

NEOPLASTIC LESIONS:
No increase in neoplastic lesions was observed in dosed mice.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment related effects observed.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Mean body weights of mice in the two-year gavage studies of d-carvone

Week on Study
	Vehicle control		375 mg/kg			750 mg/kg
	Av. Wt (gram).	Number Weighed	Av. Wt (gram).	Wt. (percent of vehicle controls)	Number Weighed	Av. Wt (gram).	Wt. (percent of vehicle controls)	Number Weighed
MALE
1	20.2	50	20.4	101.0	50	20.1	99.5	50
2	22.8	50	22.9	100.4	50	22.7	99.6	47
3	22.9	50	23.0	100.4	50	23.0	100.4	47
4	24.8	50	24.7	99.6	50	24.6	99.2	47
5	25.9	50	25.3	97.7	50	25.2	97.3	47
6	26.0	50	25.4	97.7	50	25.7	98.8	47
7	27.3	50	26.3	96.3	50	26.0	95.2	47
8	27.8	50	27.0	97.1	50	26.5	95.3	47
9	26.4	50	25.7	97.3	50	25.4	96.2	47
10	28.2	50	27.7	98.2	50	26.6	94.3	47
11	28.6	50	27.9	97.6	50	27.5	96.2	47
12	29.1	50	28.8	99.0	50	27.9	95.9	47
13	30.1	50	29.2	97.0	50	27.9	92.7	47
17	32.0	50	30.5	95.3	50	29.6	92.5	47
21	32.8	50	31.7	96.6	50	31.0	94.5	46
25	33.7	SO	32 7	97.0	50	32.3	95.8	45
29	33.1	49	33 1	100.0	50	32.2	97.3	45
33	35.0	49	34.0	97.1	49	33.9	96.9	45
37	34.7	49	34 8	100.3	49	34.1	98.3	45
41	35.3	49	35 7	101.1	49	34.3	97.2	45
45	35.3	48	35.7	101.1	49	34.5	97.7	45
49	34.8	47	35.0	100.6	48	35.0	100.6	44
54	35.2	47	35.3	100.3	48	33.7	95.7	44
57	34.4	47	35.0	101.7	48	34.1	99.1	44
61	35.5	47	35.8	100.8	47	35.0	98.6	43
65	35.2	47	36.1	102.6	47	34.8	98.9	43
69	35.2	46	35.8	101.1	47	35.2	100.0	42
73	35.2	45	35.1	99.7	47	35.0	99.4	41
77	34.8	45	36.0	103.4	46	35.4	101.7	41
81	35.5	45	36.4	102.5	45	35.6	100.3	4 1
85	35.0	43	36.9	105.4	45	35.6	101.7	40
89	34.6	42	37.2	107.5	45	35.5	102.6	39
9 1	33.2	4 1	36.2	109.0	45	34.6	104.2	39
93	35.1	4 1	36.9	105.1	45	35.5	101.1	39
95	35.0	4 1	37.3	106.6	44	35.6	101.7	39
97	34.1	40	37.0	108.5	43	35.7	104.7	39
99	34.8	38	37.4	107.5	43	36.4	104.6	39
101	34.5	37	36.1	104.6	43	35.1	101.7	38
103	34.2	37	36.1	105.6	42	34.4	100.6	36
Mean for weeks
1-13	26.2		25.7	98		25.3	97
17-49	34.1		33.7	99		33.0	97
54-103	34.8		36.3	104		35.1	101
FEMALE
1	16.3	50	16.3	100.0	50	16.3	100.0	50
2	17.6	50	18.2	103.4	49	18.3	104.0	49
3	17.8	50	18.4	103.4	49	19.0	106.7	49
4	20.0	50	19.9	99.5	49	19.9	99.5	49
5	20.0	50	20.4	102.0	49	20.6	103.0	49
6	20.0	50	19.7	98.5	49	20.9	104.5	49
7	20.8	50	20.9	100.5	49	20.7	99.5	49
8	21.3	50	21.4	100.5	49	21.3	100.0	49
9	19.3	50	19.8	102.6	49	20.6	106.7	49
10	21.5	50	21.7	100.9	49	21.5	100.0	49
11	21.7	50	21.9	100.9	49	21.7	100.0	49
12	21.9	50	22.3	101.8	49	22.1	100.9	49
13	22.6	50	22.5	99.6	49	22.1	97.8	49
17	23.0	50	23.3	101.3	49	23.2	100.9	49
21	24.1	50	24.3	100.8	49	24.1	100.0	49
25	25.0	50	25.1	100.4	49	24.8	99.2	49
29	24.7	50	25.6	103.6	49	25.1	101.6	49
33	26.7	50	26.3	98.5	49	26.7	100.0	49
37	27.7	50	27.1	97.8	49	27.3	98.6	49
4 1	28.6	50	28.1	98.3	49	27.4	95.8	49
45	28.6	50	27.4	95.8	48	28.1	98.3	49
49	28.5	50	28.3	99.3	48	27.9	97.9	49
54	28.9	50	27.9	96.5	48	27.3	94.5	49
57	29.4	50	28.1	95.6	47	27.9	94.9	47
6 1	29.7	4 1	28.7	96.6	47	28.1	94.6	47
65	29.8	46	28.5	95.6	46	28.6	96.0	47
69	29.8	45	28.4	95.3	46	28.5	95.6	47
73	29.6	43	27.7	93.6	44	28.3	95.6	47
77	29.0	40	28.0	96.6	44	29.1	100.3	46
81	29.1	39	28.6	98.3	42	28.9	99.3	43
85	29.3	36	28.9	98.6	40	28.7	98.0	42
89	28.9	33	29.2	101.0	37	29.0	100.3	40
9 1	29.3	32	28.9	98.6	35	29.1	99.3	40
93	29.6	29	29.0	98.0	33	29.2	98.6	40
95	29.7	27	28.7	96.6	32	29.3	98.7	40
97	29.6	25	28.7	97.0	31	29.8	100.7	40
99	30.4	22	29.1	95.7	3 1	30.1	99.0	38
101	30.5	18	28.9	94.8	29	29.1	95.4	38
103	30.7	14	28.5	92.8	29	20.1	94.8	38
Mean for weeks
1.13	20.1		20.3	101		20.4	101
17-49	26.3		26.2	100		26.1	100
54-103	29.6		28.6	97		28.8	97

 

Table 2: Survival of mice in the two-year gavage studies of d-carvone

	Vehicle Control	375 mg/kg	750 mg/kg
MALE (a)
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 (b)	0.675	0.329	0.784
FEMALE (a)
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	(c)29	38
Mean survival (days)	639	652	676
Survival P values (b)	<0.001	0.006	<0.001

(a) First day of termination period: 729

(b) The result of the life table trend test is in the vehicle control column, and the results of the life table pairwise comparisons with the vehicle controls are in the dosed columns.

(c) One animal died or was killed accidentally or in a moribund condition during the termination period and was combined for statistical purposes with those killed a t the end of the study.

 

Table 3: Numbers of mice with lesions of the nasal cavity in the two-year gavage studies of d-Carvone

	Male			Female
Site/Lesion	Vehicle Control	375 mg/kg	750 mg/kg	Vehicle Control	375 mg/kg	750 m g k g
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:

In a carcinogenicity study conducted equivalent to OECD TG 451 (supervised by NTP) male and female mice received 5 days per week for 103 weeks by gavage once daily 0, 375 and 750 mg/kg bw d-carvone in corn oil. No effects on mortality, body weight gain and and no compound-related clinical signs were observed throughout the study. No neoplastic and non-neoplastic lesions were observed, except atrophy of the olfactory epithelium and hyperplasia of the underlying Bowman´s glands. 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. Based on the results, the NOAEL is considered to be 750 mg/kg bw/day.

Executive summary:

In a carcinogenicity study conducted equivalent to OECD TG 451 (supervised by NTP) male and female mice received 5 days per week for 103 weeks by gavage once daily 0, 375 and 750 mg/kg bw d-carvone in corn oil. No effects on mortality, body weight gain and and no compound-related clinical signs were observed throughout the study. No neoplastic and non-neoplastic lesions were observed, except atrophy of the olfactory epithelium and hyperplasia of the underlying Bowman´s glands in all treatment groups. These lesions, as the authors of the NTP report suggested, may have 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 the results, the NOAEL is considered to be 750 mg/kg bw/day.

 

This carcinogenicity study in mice is acceptable and satisfies the fundamental requirements for a carcinogenicity study according to OECD 451 in mice.