2-methylcyclohexyl acetate

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral

Remarks:

other: Carcinogenicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1979

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD 451 Carcinogenicity study

Deviations:

yes

Remarks:

(2 dose levels tested)

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Menthol

EC Number:

201-939-0

EC Name:

Menthol

Cas Number:

89-78-1

IUPAC Name:

2-isopropyl-5-methylcyclohexanol

Details on test material:

- Name of test material (as cited in study report): dl-menthol
- Molecular formula (if other than submission substance): C10H20O
- Molecular weight (if other than submission substance):156.26
- Lot/batch No.: 4-HTP-6 (Glidden Organics Internation, Jaksonville, Florida), only 3 weeks in rats; and N11-26-74-2054 (Norda, Inc., New York).
- Impurities: 4-HTP-6: Two impurities estimated at 0.3% each and other impurity of about 1.3%. N11-26-74-2054: One minor impurity (0.2%).
- Water: 4-HTP-6: 0.22 ± 0.02%. N11-26-74-2054: <0.1%.
- Storage condition of test material: at 1 ºC in their original containers

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Frederick Cancer Research Center, Frederick, Maryland (USA)
- Age at study initiation: 9 weeks
- Fasting period before study: 4 weeks
- Housing: Polycarbonate cages covered with stainless steel cage lids and non-woven fiber filter bonnets. Cages were furnished with heat-treated hardwood chip bedding.
- Diet: Ad libitum
- Water: Ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 ºC
- Humidity (%): 45-55 %
- Air changes (per hr): 12 changes of room air per hour (filtered through 2-inch-thick disposable fiberglass filters).

Administration / exposure

Route of administration:

oral: feed

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The appropriate weight of the dl-menthol required for each dietary concentration was dissolved in corn oil by stirring over a low heat.

DIET PREPARATION
- Rate of preparation of diet (frequency): prepared each week (and used within 1 week of preparation).
- Mixing appropriate amounts with (Type of food): The test item solution was added to the Wayne Lab Blox animal meal and thoroughly mixed.
- Storage temperature of food: room temperature

VEHICLE
- Concentration: 2% by weight in the basal diets.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duplicate 10-g dosed feed samples were extracted with 20 ml of carbon disulphide, and aliquots of the extract analysed by gas chromatography (thermal conductivity detector). The average recoveries from these spiked samples were greater than 90%.

Duration of treatment / exposure:

103 weeks

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

3 750 ppm

Remarks:

~187 mg/kg bw/day, nominal in diet

Doses / concentrations 2

Dose / conc.:

7 500 ppm

Remarks:

~375 mg/kg bw/day
nominal in diet

No. of animals per sex per dose:

50 animals per sex and per dose

Control animals:

yes

Details on study design:

- Dose selection rationale: A subchronic feeding study was conducted to estimate the maximum tolerated dosed of dl-menthol (see study endpoint 07.05.01_03).

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day.
- Observations: sings of toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Every week
- Observations: clinical signs and the presence of palpable masses

BODY WEIGHT: Yes
- Time schedule for examinations: every 2 weeks for the first 12 weeks and every month thereafter.

FOOD CONSUMPTION: YES
- Time schedule: every 2 weeks for the first 12 weeks and every month thereafter.

Sacrifice and pathology:

Animals that were moribund and those that survived to the termination of the study were killed by exsanguination under sodium pentobarbital anaesthesia. The pathologic evaluation consisted of gross and microscopic examination of major tissues, major organs, and all gross lesions from killed animals and from animals found dead. The tissues were preserved in 10% buffered formalin, embedded in paraffin, sectioned, and stained with haematoxylin and eosin. The following tissues were examined microscopically: brain (frontal cortex and basal ganglia, parietal cortex and thalamus, and cerebellum and pons), pituitary, spinal cord (if neurologic signs were present), eyes (if grossly abnormal), oesophagus, trachea, salivary gland, mandibular lymph node, thyroid, parathyroid, heart, thymus, lungs and mainstem bronchi, liver, gallbladder (mice), pancreas, spleen, kidney, adrenal, stomach, small intestine, colon, urinary bladder, prostate or uterus, testes or ovaries, sternebrae femur, or vertebrae including marrow, mammary gland, tissue masses, and any macroscopic lesions.

Statistics:

Probabilities of survival were estimated by the product-limit procedure of Kaplan and Meier (1958). Statistical analyses for a possible dose-related effect on survival used the method of Cox (1972) for testing two groups for equality and Tarone's (1975) extensions of Cox's methods for testing for a dose-related trend. The one-tailed Fisher exact test (Cox, 1970) was used to compare the tumour incidence of a control group with that of a group of dosed animals at each dose level. The Cochran-Armitage test for linear trend in proportions, with continuity correction (Armitage, 1971), was also used to determine if the slope of the dose-response curve is different from zero at the one tailed 0.05 level of significance.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

(males and females)

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

MORTALITY:
The dose-related trend in mortality and the comparison between the survival of the control group with each dosed group are not significant in either sex. In male rats, 34/50 (68%) of the high-dose group, 33/50 (66%) of the low-dose group, and 31/50 (62%) of the controls were alive at week 105. In females, 38/50 (76%) of the high-dose group, 35/50 (70%) of the low-dose group, and 36/50 (72%) of the controls were alive at week 105. Sufficient numbers of rats of each sex were at risk for the development of late-appearing tumors.

CLINICAL SIGNS:
No clinical signs related to administration of test item were noted. Clinical signs commonly observed among rats of this strain were noted at comparable rates in the control and dosed groups, particularly during the second year of the bioassay, and increased in incidence as the animals aged. These signs included eye changes (redness, paleness, cloudiness, lacrimation, a red discharge or bloody crust, and an enlarged or protruding eye), a hunched and/or thin appearance, urine stains on the abdominal fur, and occasionally, nasal discharge, sores on the body or the extremities, soft feces, and enlarged testes. The incidence of palpable nodules and tissue masses in the dosed males was generally comparable to that in the control males, but was lower in the dosed females than in the control females.

BODY WEIGHT AND WEIGHT GAIN
Mean body weights of the dosed male and female rats were slightly lower than those of the corresponding controls throughout the bioassay.

HISTOPATHOLOGY: NON-NEOPLASTIC
Chronic inflammation of the kidney was observed with greater frequency in the dosed males than in the control males (29/49 controls, 41/50 low-dose, 41/50 high-dose); however, this finding is of questionable importance, since such lesions are often found in aged male Fischer 344 rats.

HISTOPATHOLOGY: NEOPLASTIC
Each of the tumour types observed has been encountered previously as a spontaneous lesion, and occurred with no appreciable differences in frequency between control and dosed rats with a few exceptions. In female rats, chromophobe adenomas of the pituitary gland and fibroadenomas of the mammary gland were observed with greater frequency in female control rats. Chromophobe adenomas occurred in 28/48 controls, 25/47 low-dose and 19/43 high-dose female rats. Mammary gland fibroadenomas were diagnosed in 20/50 female controls, 10/49 low-dose, and 7/49 high-dose rats. Mammary adenocarcinomas were seen in 1/50 controls, 3/49 low-dose, and 0/49 high-dose rats.

All other inflammatory, degenerative, and hyperplastic lesions that occurred were similar in incidence and kind to those naturally occurring lesions found in aged Fischer 344 rats.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

7 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Remarks on result:

other: (~375 mg/kg bw/day)

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Based on the histopathologic examination, 2 -isopropyl-5 -methylcyclohexanol was neither toxic nor carcinogenic to Fischer 344 rats under the conditions of this bioassay.

Applicant's summary and conclusion

Conclusions:

The NOAEL for 2-isopropyl-5-methylcyclohexanol when administered to rats for 103 consecutive weeks by oral route was determined to be 7500 ppm (~375 mg/kg bw/day).

Executive summary:

A 103 week oral (diet) carcinogenicity study was conducted with test item 2 -isopropyl-5 -methylcyclohexanol. Groups of 50 Fischer 344 rats of each sex were administered 0, 3750 or 7500 ppm (~ 0, 187, 375 mg/kg bw/day) dl-2-isopropy-5-methylcyclohexanol in their feed daily for 103 weeks. Animals were housed five per cage until week 48 when the male rats were divided into groups of two to three per cage. The animals were observed twice daily for signs of toxicity. Body weight and food consumption were recorded every two weeks for the first twelve weeks, and once a month thereafter. Necropsies and histological examinations were performed on all animals at the termination of the study and on those found dead during the study. The mean body weights of the male and female rats administered at 3750 or 7500 ppm (~187 or 375 mg/kg bw/day) were slightly lower when compared to the controls. Survival of the high- and low-dose groups of male (controls, 31/50; low-dose, 33/50; high-dose, 34/50) and female (controls, 36/50; low-dose, 35/50; high-dose, 38/50) rats was similar to the control animals. Chronic inflammation of the kidney observed in the dosed older males was not considered by the authors to be related to the administration of dl-2-isopropy-5-methylcyclohexanol since the effect is commonly observed in aged male Fischer 344 rats. There was no increase in the incidence of neoplasms of dosed females compared to that of control animals. In the low-dose (10/49) and high-dose (7/49) female groups, fibroadenomas of the mammary glands occurred at a lower incidence than in the control group (20/50). Alveolar/bronchiolar adenomas or carcinomas were reported only for the female control rats. Under the conditions of this study, it was concluded that 2-isopropy-5-methylcyclohexanol was neither carcinogenic nor toxic for either sex of Fischer 344 rats at dose levels of 3750 or 7500 ppm (~187 or 375 mg/kg bw/day).