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Toxicological information

Repeated dose toxicity: oral

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Endpoint:
chronic toxicity: oral
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Test procedure in accordance with generally accepted scientific standards and described in sufficient detail. Because study from 1979 it was not performed according to GLP.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reference
Endpoint:
chronic toxicity: oral
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: carcinogenicity study
Reason / purpose:
reference to same study
Related information:
Composition 1
Reason / purpose:
reference to same study
Related information:
Composition 1
Reason / purpose:
reference to same study
Related information:
Composition 1
Reason / purpose:
reference to same study
Related information:
Composition 1
Reason / purpose:
reference to same study
Related information:
Composition 1
Reference:
Composition 0
Principles of method if other than guideline:
A bioassay of menthol for possible carcinogenicity was conducted by administrating the test chemical in feed to B6C3F1 mice.
Groups of 50 mice of each sex were administrated dl-menthol at one of the following doses, either 3750 or 7500 ppm for 103 weeks, then observed for 1 or 2 additional weeks. Matched controls consisted of 50 untreated mice of each sex. All surviving mice were killed at 104 weeks.
GLP compliance:
not specified
Limit test:
no
Test material information:
Composition 1
Specific details on test material used for the study:
In the report the test substance is named as DL-menthol but identified with CAS 89-78-1. The EC name of CAS 89-78-1 is menthol.
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 6 weeks
- Weight at study initiation: 24 g
- Diet (e.g. ad libitum): Wayne Lab Chow Meal and water available ad libitum
- Housing: Polycarbonate cages covered with stainless steel cage lids and non-woven fiber filter bonnets (Filtek, Appleton, Wis.).
The mice were housed 5 per cage throughout the study.
- Diet : ad libitum
- Water : ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 45-55%
- Air changes (per hr): 12 changes per hour
- Photoperiod : 12 hours dark/ 12 hours light
Route of administration:
oral: feed
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): prepared each week and used within 1 week of preparation.
- Storage temperature of food: The containers were stored at room temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Duplicate 10g dosed feed sample were extracted with 20ml of carbon disulfide, and aliquots of the extract analyzed by gas chromatography (thermal conductivity detector).
(see attached illustration for values)
Duration of treatment / exposure:
The compound was administered in the diet for 103 weeks
Frequency of treatment:
daily
Dose / conc.:
2 000 ppm
Remarks:
Doses / Concentrations: 0.20 %
Basis: nominal in diet
Dose / conc.:
4 000 ppm
Remarks:
Doses / Concentrations: 0.40%
Basis: nominal in diet
No. of animals per sex per dose:
50 animals per dose per sex
Control animals:
yes, concurrent vehicle
Details on study design:
- On the basis of the results of a 14-day range-finding study, doses of 930, 1,870, 3,750, 7,500, and 15,000 ppm were selected to be administered in the diet in the subchronic studies.
- Dose selection rationale: Based on the results of 13-week study (see cross reference studies).
- Rationale for animal assignment: Animals were segregated by body weight so that a homogeneous distribution of mean weight ranges was obtained between groups.
Observations and examinations performed and frequency:
All animals were observed twice a day for signs of toxicity.
Clinical signs and the presence of palpable masses were recorded every week.
Mean body weights and food consumption were recorded 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 anesthesia (Diabutal, Diamond Laboratories, Inc., Des Moines, Iowa).

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 hematoxylin 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), esophagus, 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.
A few tissues from some animals were not examined, particularly from those animals that died early. Also, some animals may have been missing, cannibalized, or judged to be in such an advanced state of autolysis as to preclude histopathologic evaluation. Thus, the number of animals from which particular organs or tissues were examined microscopically varies, and does not necessarily represent the number of animals that were placed on study in each group.
Statistics:
Pertinent data on this experiment have been recorded in an automatic data processing system, the Carcinogenesis Bioassay Data System (Linhart et al., 1974).
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 2 groups for equality and Tarone's (1975) extensions of Cox's methods for testing dose related trend.
The one-tailed Fisher exact test (Cox, 1970) wa used to compare the tumor 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.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
slightly lower than those of the corresponding controls
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:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
A. Body Weights and Clinical Signs
Mean body weights of the dosed male and female mice were slightly lower than those of the corresponding controls throughout the bioassay.
The appearance and behavior of the dosed and control groups of animals were generally similar, and clinical signs usually associated with aging were noted at comparable rates in the control and dosed groups. These signs included alopecia (generalized or localized), sores on the back and other parts of the body, particularly in the males, anal and/or penile irritation, a hunched and/or thin appearance, and occasional abdominal distention.
The incidences of palpable nodules and tissue masses in the dosed male or female groups were generally comparable to those of corresponding control groups.

B. Survival
The Kaplan and Meier curves estimates the probabilities of survival for male and female mice administered menthol in the diet at the doses of this bioassay, together with those of the matched controls. In male mice, the result of the Tarone test for dose—related trend in mortality, and the results of the Cox test comparing the survival of the control group with each dosed group, are not significant. In females, the result of the Tarone test is significant (P =0.008). The result of the Cox test comparing the survival of the control group with the high—dose group is significant (P = 0.020), but the comparison between the control and low—dose groups is not significant.
In male mice, there were 35/50 (70%) of the high—dose group, 32/50 (64%) of the low—dose group, and 32/50 (64%) of the controls still alive at week 104. In female mice, there were 36/50 (72%) of the high—dose group, 40/50 (80%) of the low—dose group, and 45/50 (90%) of the controls still alive at week 104. Sufficient numbers of mice of each sex were at risk for the development of late—appearing tumors.

C. Pathology
A low incidence of neoplasia was observed in both control and dosed groups of mice. These neoplasms were of the usual number and type observed in mice of this age and strain. A slightly increased incidence of hepatocellular carcinomas was observed in the high—dose males (8/47 controls, 8/49 low—dose, 14/48 high— dose); however, the incidence was not increased over that observed occasionally in historical—control groups of mice of this age and strain. Alveolar/bronchiolar adenomas or carcinomas of the lung occurred primarily in the dosed females (1/49 controls, 3/47 low—dose, 5/48 high—dose). The incidence of lung neoplasms was not considered indicative of a carcinogenic effect, as this neoplasm has been commonly seen at a similar low incidence in historical—control groups.
Other degenerative, proliferative, and inflammatory lesions observed were also of the usual incidence and kind observed in aged B6C3F1 mice, and incidences in dosed groups were comparable with those in control groups.
Dose descriptor:
NOAEL
Effect level:
> 4 000 ppm
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: overall effects
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOAEL
Effect level:
> 667 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No effects were observed; 4000 ppm in diet applied to mice were converted to 667 mg/kg/day calculated for Mice with a mean body weight of 30 g and 5 g/day of food consumption
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
not specified
Conclusions:
Based on the histopathologic examination, menthol was neither toxic nor carcinogenic to B6C3F1 mice under the conditions of this bioassay.
Executive summary:

Mean body weights of the dosed rats and mice were slightly lower than those of corresponding controls. No other clinical signs related to administration of menthol were noted in any dosed groups of rats and mice. A dose-related trend in mortality was observed only in the female mice. Survival at the end of the bioassay was at least 62% in all dosed and control groups of animals, and sufficient numbers of animals were at risk for the development of late-appearing tumors.
In mice of either sex, no tumors occurred in dosed groups at incidences that were significantly different from those for corresponding control groups.


It is concluded that under the conditions of this bioassay, menthol was not carcinogenic for B6C3F1 mice.

Reason / purpose:
reference to same study
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
In the NCI carcinogenicity study an overview is given of the subchronic study performed to estimate the maximum tolerated doses of DL Menthol, on the basis of which two concentrations were determined for use in the chronic studies. Not clear if described study conditions of the carcinogenicity study are the same as the one used in the subchronic study.
Reason / purpose:
reference to same study
Related information:
Composition 1
Reason / purpose:
reference to same study
Related information:
Composition 1
Reason / purpose:
reference to same study
Related information:
Composition 1
Reason / purpose:
reference to same study
Related information:
Composition 1
Reason / purpose:
reference to same study
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
not specified
Limit test:
no
Test material information:
Composition 1
Specific details on test material used for the study:
In the report the test substance is named as DL-menthol but identified with CAS 89-78-1. The EC name of CAS 89-78-1 is menthol.
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Not specified
- Weight at study initiation: Not specified
- Diet (e.g. ad libitum): Wayne Lab Chow Meal and water available ad libitum
- Housing: Polycarbonate cages covered with stainless steel cage lids and non-woven fiber filter bonnets (Filtek, Appleton, Wis.).
Rats were housed 5 per cage until week 48, and then the males were divided into groups of 2 or 3 per cage.
- Diet : ad libitum
- Water : ad libitum
- Acclimation period: 4 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 45-55%
- Air changes (per hr): 12 changes per hour
- Photoperiod : 12 hours dark/ 12 hours light
Route of administration:
oral: feed
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): prepared each week and used within 1 week of preparation.
- Storage temperature of food: The containers were stored at room temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Duplicate 10g dosed feed sample were extracted with 20ml of carbon disulfide, and aliquots of the extract analyzed by gas chromatography (thermal conductivity detector).
Duration of treatment / exposure:
The compound was administered in the diet for 13 weeks.
Frequency of treatment:
daily
Dose / conc.:
930 ppm
Remarks:
Doses / Concentrations: 0.093%
Basis: nominal in diet
Dose / conc.:
1 870 ppm
Remarks:
Doses / Concentrations: 0.187%
Basis: nominal in diet
Dose / conc.:
3 750 ppm
Remarks:
Doses / Concentrations: 0.375 %
Basis: nominal in diet
Dose / conc.:
7 500 ppm
Remarks:
Doses / Concentrations: 0.75%
Basis: nominal in diet
Dose / conc.:
15 000 ppm
Remarks:
Doses / Concentrations: 1.5%
Basis: nominal in diet
No. of animals per sex per dose:
10 males and 10 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of a 14-day range-finding study.
- Rationale for animal assignment: Animals were segregated by body weight so that a homogeneous distribution of mean weight ranges was obtained between groups.
Observations and examinations performed and frequency:
Mean body weight and mortality was followed throughout the study.
Sacrifice and pathology:
At termination date, all animals were necropsied and histological examination was made.
Statistics:
Not specified
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
mean body weight gains in dosed groups were comparable to those in the control groups
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
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
slightly increased incidence of interstitial nephritis in the male rats in the highest dose groups which had been considered of questionable significance by the authors of the study.
Histopathological findings: neoplastic:
not specified
Details on results:
Menthol administered with the diet (up to 15,000 ppm) for 13 weeks to rats did not induce any effects on organ weights. Microscopic examination of a comprehensive range of tissues revealed a slight increase in the severity of spontaneous interstitial nephritis in the male rats at the highest dose level but this had not been considered of significance by the authors of the study.
Dose descriptor:
NOAEL
Effect level:
> 15 000 ppm
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: overall effects
Dose descriptor:
NOAEL
Effect level:
> 937 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No effects were observed; 15000 ppm in diet applied to rats corresponding to 937 mg/kg bw/day for male and 998 mg/kg bw/ day for female rats.
Critical effects observed:
not specified
Conclusions:
No death or clinical observation could be related to compound administration.
According to this study, the low and high doses for the chronic studies using rats can be set at 3 750 and 7 500 ppm.
Within this study the NOAEL was detrmined to be at 15000 ppm (equals 937 mg/kg bw/day)
Executive summary:

In rats the only effect recorded after 13 weeks of menthol exposure at 15000 ppm (corresponding to 937 mg/kg bw/d) was a minimal increase in the severity of spontaneous interstitial nephritis in males, which was considered by the authors of the study as of questionable significance. Thus the NOAEL for this study is 937 mg/kg bw/d for male/female rats.

Reason / purpose:
reference to same study
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
In the NCI carcinogenicity study an overview is given of the subchronic study performed to estimate the maximum tolerated doses of DL-Menthol, on the basis of which two concentrations were determined for use in the chronic studies. Not clear if described study conditions of the carcinogenicity study are the same as the one used in the subchronic study.
Reason / purpose:
reference to same study
Related information:
Composition 1
Reason / purpose:
reference to same study
Related information:
Composition 1
Reason / purpose:
reference to same study
Related information:
Composition 1
Reason / purpose:
reference to same study
Related information:
Composition 1
Reason / purpose:
reference to same study
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
not specified
Limit test:
no
Test material information:
Composition 1
Specific details on test material used for the study:
In the report the test substance is named as DL-menthol but identified with CAS 89-78-1. The EC name of CAS 89-78-1 is menthol.
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Not specified
- Weight at study initiation: Not specified
- Diet (e.g. ad libitum): Wayne Lab Chow Meal and water available ad libitum
- Housing: Polycarbonate cages covered with stainless steel cage lids and non-woven fiber filter bonnets (Filtek, Appleton, Wis.).
The mice were housed 5 per cage throughout the study.
- Diet : ad libitum
- Water : ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 45-55%
- Air changes (per hr): 12 changes per hour
- Photoperiod : 12 hours dark/ 12 hours light
Route of administration:
oral: feed
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): prepared each week and used within 1 week of preparation.
- Storage temperature of food: The containers were stored at room temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Duplicate 10g dosed feed sample were extracted with 20ml of carbon disulfide, and aliquots of the extract analyzed by gas chromatography (thermal conductivity detector).
Duration of treatment / exposure:
The compound was administered in the diet for 13 weeks.
Frequency of treatment:
daily
Dose / conc.:
930 ppm
Remarks:
Doses / Concentrations: 0.093%
Basis: nominal in diet
Dose / conc.:
1 870 ppm
Remarks:
Doses / Concentrations: 0.187%
Basis: nominal in diet
Dose / conc.:
3 750 ppm
Remarks:
Doses / Concentrations: 0.375 %
Basis: nominal in diet
Dose / conc.:
7 500 ppm
Remarks:
Doses / Concentrations: 0.75%
Basis: nominal in diet
Dose / conc.:
15 000 ppm
Remarks:
Doses / Concentrations: 1.5%
Basis: nominal in diet
No. of animals per sex per dose:
10 males and 10 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of a 14-day range-finding study.
- Rationale for animal assignment (if not random): Animals were segregated by body weight so that a homogeneous distribution of mean weight ranges was obtained between groups.
Observations and examinations performed and frequency:
Mean body weight and mortality was followed throughout the study.
Sacrifice and pathology:
At termination date, all animals were necropsied and histological examination was made.
Statistics:
Not specified
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
6 deaths, but could not be related to compound administration
Mortality:
mortality observed, treatment-related
Description (incidence):
6 deaths, but could not be related to compound administration
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
only females receiving 15000ppm gained 2g less than the controls.
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:
effects observed, treatment-related
Description (incidence and severity):
slightly increased incidence of perivascular lymphoid hyperplasia.
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
slightly increased incidence of interstitial nephritis among female mice in the 2 highest dose groups.
Histopathological findings: neoplastic:
not specified
Dose descriptor:
NOAEL
Effect level:
7 500 ppm
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: based on slightly reduced body weight gain
Dose descriptor:
NOAEL
Effect level:
1 250 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: based on slightly reduced body weight gain; 7500 ppm in diet applied to mice corresponding to 1250 mg/kg bw/day calculated for mice with a mean body weight of 30 g and 5 g/day of food consumption.
Critical effects observed:
not specified
Conclusions:
No death or clinical observation could be related to compound administration.
According to this study, the low and high doses for the chronic studies using mice was set to 4 000 and 2 000 ppm.
Executive summary:

In male and female mice a slight decrease in body weight gain was observed at 15000 ppm but not at 7500 ppm (corresponding to 1956 mg/kg bw/d in male and 2386 mg/kg bw/day in female). Histologic examination of tissues at the 7500 and 15000 ppm levels revealed no compound-related tissue alterations in any of the mice. Also minimal focal interstitial nephritis was noted as a spontaneous lesion which was observed in control and treated mice, and not related to the treatment with menthol. The NOAEL therefore is 1956 mg/kg bw/d for mice based on slightly reduced body weight gain in male mice.

Reason / purpose:
reference to same study
Reference
Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose:
reference to same study
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 451 (Carcinogenicity Studies)
Deviations:
yes
Remarks:
only two doses tested
GLP compliance:
no
Remarks:
test was performed prior to GLP
Test material information:
Composition 1
Specific details on test material used for the study:
In the report the test substance is named as DL-menthol but identified with CAS 89-78-1. The EC name of CAS 89-78-1 is menthol.
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 9 weeks
- Mean body weight at study initiation: male 175g, female 130g
- Housing: Polycarbonate cages covered with stainless steel cage lids and non-woven fiber filter bonnets (Filtek, Appleton, Wis.).
Rats were housed 5 per cage until week 48, and then the males were divided into groups of 2 or 3 per cage.
- Diet: Wayne Lab Chow Meal, ad libitum
- Water : ad libitum
- Acclimation period: 4 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 45-55%
- Air changes (per hr): 12 changes per hour
- Photoperiod : 12 hours dark/ 12 hours light
Route of administration:
oral: feed
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): prepared each week and used within 1 week of preparation.
- Storage temperature of food: The containers were stored at room temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Duplicate 10g dosed feed sample were extracted with 20ml of carbon disulfide, and aliquots of the extract analyzed by gas chromatography (thermal conductivity detector).
(see attached illustration for values)
Duration of treatment / exposure:
The compound was administered in the diet seven days a week for 103 weeks at the indicated level.
During the 2 post-treatment weeks (recovery), the animals were supplied with standard diet.
Frequency of treatment:
daily for 103 weeks
Post exposure period:
2 weeks
Dose / conc.:
3 750 ppm
Remarks:
Doses / Concentrations: 0.375 %
Basis: nominal in diet
Dose / conc.:
7 500 ppm
Remarks:
Doses / Concentrations: 0.75%
Basis: nominal in diet
No. of animals per sex per dose:
50 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of 13-week study (see cross reference studies).
- Rationale for animal assignment (if not random): Animals were segregated by body weight so that a homogeneous distribution of mean weight ranges was obtained between groups.
Positive control:
No data
Observations and examinations performed and frequency:
All animals were observed twice a day for signs of toxicity.
Clinical signs and the presence of palpable masses were recorded every week.
Mean body weights and food consumption were recorded 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 anesthesia (Diabutal, Diamond Laboratories, Inc., Des Moines, Iowa).

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 hematoxylin 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), esophagus, 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.
A few tissues from some animals were not examined, particularly from those animals that died early. Also, some animals may have been missing, cannibalized, or judged to be in such an advanced state of autolysis as to preclude histopathologic evaluation. Thus, the number of animals from which particular organs or tissues were examined microscopically varies, and does not necessarily represent the number of animals that were placed on study in each group.
Statistics:
Pertinent data on this experiment have been recorded in an automatic data processing system, the Carcinogenesis Bioassay Data System (Linhart et al., 1974).
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 2 groups for equality and Tarone's (1975) extensions of Cox's methods for testing dose related trend.
The one-tailed Fisher exact test (Cox, 1970) was used to compare the tumor 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.
Clinical signs:
no effects observed
Description (incidence and severity):
not significant in either sex
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
not significant in either sex
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
mean body weight gains in dosed groups were slightly lower than in the control groups
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:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
nephritis of questionable origin (see below "Details on results")
Histopathological findings: neoplastic:
no effects observed
Details on results:
A. Body Weights and Clinical Signs (Rats)
Mean body weights of the dosed male and female rats were slightly lower than those of the corresponding controls throughout the bioassay. No other clinical signs related to administration of the menthol 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, lacrimination, 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.

B. Survival (Rats)
The Kaplan and Meier curves estimates the probabilities of survival for male and female rats administered menthol in the diet at the doses of this bioassay, together with those of the matched controls. The results of the Tarone test for dose-related trend in mortality and the results of the Cox test comparing 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.

C. Pathology (Rats)
Each of the tumor types observed has been encountered previously as 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.
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.
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.
Relevance of carcinogenic effects / potential:
Not found
Dose descriptor:
NOAEL
Effect level:
> 7 500 ppm
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOAEL
Effect level:
> 375 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic
Remarks on result:
other:
Remarks:
7500 ppm in diet applied to mice were converted to 375 mg/kg/day calculated for rat with a mean body weight of 400 g and 20 g/day of food consumption
Conclusions:
Based on the histopathologic examination, menthol was neither toxic nor carcinogenic to Fischer 344 rats under the conditions of this bioassay.
Executive summary:

Mean body weights of the dosed rats and mice were slightly lower than those of corresponding controls. No other clinical signs related to administration of menthol were noted in any dosed groups of rats and mice. Survival at the end of the bioassay was at least 62% in all dosed and control groups of animals of each species, and sufficient numbers of animals were at risk for the development of late-appearing tumours.

In male rats, no tumours occurred at incidences which were considered to be associated with the administration of menthol.

In female rats, no tumours occurred at higher incidences in dosed groups than in control groups. Fibro adenomas of the mammary gland occurred at lower incidences in the low-dose (10/49) and high-dose (7/49) groups than in the control group (20/50), and alveolar/bronchiolar adenomas or carcinomas of the lung occurred only in the controls (3/50).

The acute oral LD50 of Menthol in Osborne-Mendel rats has been reported as 3,180 mg/kg body weight (Jenner et al., 1964) and as 2,900 mg/kg body weight (Herken, 1961).When administered in the diet to male and female rats for 5.5 weeks, D- or menthol at 100 or 200 mg/kg body weight caused no adverse effects on gain in weight (Herken, 1961). No long-term studies have been reported previous to the present bioassay.

It is concluded that under the conditions of this bioassay, menthol was not carcinogenic for Fischer 344 rats

Reason / purpose:
reference to same study
Reference
Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose:
reference to same study
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 451 (Carcinogenicity Studies)
Deviations:
yes
Remarks:
only two doses were used
GLP compliance:
no
Remarks:
study was performed before GLP
Test material information:
Composition 1
Specific details on test material used for the study:
In the report the test substance is named as DL-menthol but identified with CAS 89-78-1. The EC name of CAS 89-78-1 is menthol.
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 6 weeks
- Mean body weight at study initiation: male 24g, female 20g
- Housing: Polycarbonate cages covered with stainless steel cage lids and non-woven fiber filter bonnets (Filtek, Appleton, Wis.).
The mice were housed 5 per cage throughout the study.
- Diet: Wayne Lab Chow Meal, ad libitum
- Water : ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 45-55%
- Air changes (per hr): 12 changes per hour
- Photoperiod : 12 hours dark/ 12 hours light
Route of administration:
oral: feed
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of diet preparation (frequency): prepared each week and used within 1 week after preparation.
- Storage temperature of food: The containers were stored at room temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Duplicate 10g dosed feed sample were extracted with 20ml of carbon disulfide, and aliquots of the extract analyzed by gas chromatography (thermal conductivity detector).
(see attached illustration for values)
Duration of treatment / exposure:
The compound was administered in the diet seven days a week for 103 weeks at the indicated level.
During the 2 post-treatment weeks (recovery), the animals were supplied with standard diet.
Frequency of treatment:
daily for 103 weeks
Post exposure period:
2 weeks
Dose / conc.:
2 000 ppm
Remarks:
Doses / Concentrations: 0.20%
Basis: nominal in diet
Dose / conc.:
4 000 ppm
Remarks:
Doses / Concentrations: 0.40 %
Basis: nominal in diet
No. of animals per sex per dose:
50 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of a 13-week study (see cross reference studies).
- Rationale for animal assignment (if not random): Animals were segregated by body weight so that a homogeneous distribution of mean weight ranges was obtained between groups.
Positive control:
No data
Observations and examinations performed and frequency:
All animals were observed twice a day for signs of toxicity.
Clinical signs and the presence of palpable masses were recorded every week.
Mean body weights and food consumption were recorded 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 anesthesia (Diabutal, Diamond Laboratories, Inc., Des Moines, Iowa).

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 hematoxylin 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), esophagus, 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.
A few tissues from some animals were not examined, particularly from those animals that died early. Also, some animals may have been missing, cannibalized, or judged to be in such an advanced state of autolysis as to preclude histopathologic evaluation. Thus, the number of animals from which particular organs or tissues were examined microscopically varies, and does not necessarily represent the number of animals that were placed on study in each group.
Statistics:
Pertinent data on this experiment have been recorded in an automatic data processing system, the Carcinogenesis Bioassay Data System (Linhart et al., 1974).
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 2 groups for equality and Tarone's (1975) extensions of Cox's methods for testing dose related trend.
The one-tailed Fisher exact test (Cox, 1970) wa used to compare the tumor 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.
Clinical signs:
no effects observed
Description (incidence and severity):
not significant in either sex
Mortality:
no mortality observed
Description (incidence):
not significant in either sex
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
mean body weight gains in dosed groups were slightly lower than in the control groups
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:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
nephritis of questionable origin (see below "Details on results")
Histopathological findings: neoplastic:
no effects observed
Details on results:
A. Body Weights and Clinical Signs
Mean body weights of the dosed male and female mice were slightly lower than those of the corresponding controls throughout the bioassay.
The appearance and behavior of the dosed and control groups of animals were generally similar, and clinical signs usually associated with aging were noted at comparable rates in the control and dosed groups. These signs included alopecia (generalized or localized), sores on the back and other parts of the body, particularly in the males, anal and/or penile irritation, a hunched and/or thin appearance, and occasional abdominal distention.
The incidences of palpable nodules and tissue masses in the dosed male or female groups were generally comparable to those of corresponding control groups.

B. Survival
The Kaplan and Meier curves estimates the probabilities of survival for male and female mice administered menthol in the diet at the doses of this bioassay, together with those of the matched controls. In male mice, the result of the Tarone test for dose—related trend in mortality, and the results of the Cox test comparing the survival of the control group with each dosed group, are not significant. In females, the result of the Tarone test is significant (P =0.008). The result of the Cox test comparing the survival of the control group with the high—dose group is significant (P = 0.020), but the comparison between the control and low—dose groups is not significant.
In male mice, there were 35/50 (70%) of the high—dose group, 32/50 (64%) of the low—dose group, and 32/50 (64%) of the controls still alive at week 104. In female mice, there were 36/50 (72%) of the high—dose group, 40/50 (80%) of the low—dose group, and 45/50 (90%) of the controls still alive at week 104. Sufficient numbers of mice of each sex were at risk for the development of late—appearing tumors.

C. Pathology
A low incidence of neoplasia was observed in both control and dosed groups of mice. These neoplasms were of the usual number and type observed in mice of this age and strain. A slightly increased incidence of hepatocellular carcinomas was observed in the high—dose males (8/47 controls, 8/49 low—dose, 14/48 high— dose); however, the incidence was not increased over that observed occasionally in historical—control groups of mice of this age and strain. Alveolar/bronchiolar adenomas or carcinomas of the lung occurred primarily in the dosed females (1/49 controls, 3/47 low—dose, 5/48 high—dose). The incidence of lung neoplasms was not considered indicative of a carcinogenic effect, as this neoplasm has been commonly seen at a similar low incidence in historical—control groups.
Other degenerative, proliferative, and inflammatory lesions observed were also of the usual incidence and kind observed in aged B6C3F1 mice, and incidences in dosed groups were comparable with those in control groups.
Relevance of carcinogenic effects / potential:
Not found
Dose descriptor:
NOAEL
Effect level:
> 4 000 ppm
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOAEL
Effect level:
> 667 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Remarks on result:
other:
Remarks:
4000 ppm in diet applied to mice were converted to 667 mg/kg/day calculated for Mice with a mean body weight of 30 g and 5 g/day of food consumption
Conclusions:
Based on the histopathologic examination, menthol was neither toxic nor carcinogenic to mice under the conditions of this bioassay.
Executive summary:

Mean body weights of the dosed rats and mice were slightly lower than those of corresponding controls. No other clinical signs related to administration of menthol were noted in any dosed groups of rats and mice. Survival at the end of the bioassay was at least 62% in all dosed and control groups, and sufficient numbers of animals were at risk for the development of late-appearing tumours.

In mice of either sex , no tumours occurred in dosed groups at incidences that were significantly different from those for corresponding control group.


It is concluded that under the conditions of this bioassay, DL Menthol was not carcinogenic for B6C3F1 mice.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1979

Materials and methods

Principles of method if other than guideline:
A bioassay of menthol for possible carcinogenicity was conducted by administrating the test chemical in feed to Fisher 344 rats.
Groups of 50 rats of each sex were administrated dl-menthol at one of the following doses, either 3750 or 7500 ppm for 103 weeks, then observed for 1 or 2 additional weeks. Matched controls consisted of 50 untreated rats of each sex. All surviving rats were killed at 105 weeks.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
In the report the test substance is named as DL-menthol but identified with CAS 89-78-1. The EC name of CAS 89-78-1 is menthol.

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 9 weeks
- Weight at study initiation: 125 g
- Housing: Polycarbonate cages covered with stainless steel cage lids and non-woven fiber filter bonnets (Filtek, Appleton, Wis.).
Rats were housed 5 per cage until week 48, and then the males were divided into groups of 2 or 3 per cage.
- Diet : ad libitum: Wayne Lab Chow Meal
- Water : ad libitum
- Acclimation period: 4 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 45-55%
- Air changes (per hr): 12 changes per hour
- Photoperiod : 12 hours dark/ 12 hours light

Administration / exposure

Route of administration:
oral: feed
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): prepared each week and used within 1 week of preparation.
- Storage temperature of food: The containers were stored at room temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Duplicate 10g dosed feed sample were extracted with 20ml of carbon disulfide, and aliquots of the extract analyzed by gas chromatography (thermal conductivity detector).
(see attached illustration for values)
Duration of treatment / exposure:
The compound was administered in the diet for 103 weeks
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
3 750 ppm
Remarks:
Doses / Concentrations: 0.375%
Basis: nominal in diet
Dose / conc.:
7 500 ppm
Remarks:
Doses / Concentrations: 0.75%
Basis: nominal in diet
No. of animals per sex per dose:
50 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- On the basis of the results of a 14-day range-finding study, doses of 930, 1,870, 3,750, 7,500, and 15,000 ppm were selected to be administered in the diet in the subchronic studies.
- Dose selection rationale: Based on the results of 13-week study (see cross reference studies).
- Rationale for animal assignment: Animals were segregated by body weight so that a homogeneous distribution of mean weight ranges was obtained between groups.

Examinations

Observations and examinations performed and frequency:
All animals were observed twice a day for signs of toxicity.
Clinical signs and the presence of palpable masses were recorded every week.
Mean body weights and food consumption were recorded 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 anesthesia (Diabutal, Diamond Laboratories, Inc., Des Moines, Iowa).

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 hematoxylin 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), esophagus, 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.
A few tissues from some animals were not examined, particularly from those animals that died early. Also, some animals may have been missing, cannibalized, or judged to be in such an advanced state of autolysis as to preclude histopathologic evaluation. Thus, the number of animals from which particular organs or tissues were examined microscopically varies, and does not necessarily represent the number of animals that were placed on study in each group.
Statistics:
Pertinent data on this experiment have been recorded in an automatic data processing system, the Carcinogenesis Bioassay Data System (Linhart et al., 1974).
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 2 groups for equality and Tarone's (1975) extensions of Cox's methods for testing dose related trend.
The one-tailed Fisher exact test (Cox, 1970) was used to compare the tumor 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.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
not significant in either sex
Mortality:
no mortality observed
Description (incidence):
not significant in either sex
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
mean body weight gains in dosed groups were slightly lower than in the control groups
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:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
nephritis of questionable origin (see below "Details on results")
Histopathological findings: neoplastic:
no effects observed
Details on results:
A. Body Weights and Clinical Signs (Rats)
Mean body weights of the dosed male and female rats were slightly lower than those of the corresponding controls throughout the bioassay. No other clinical signs related to administration of menthol 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.

B. Survival (Rats)
The Kaplan and Meier curves estimates the probabilities of survival for male and female rats administered menthol in the diet at the doses of this bioassay, together with those of the matched controls. The results of the Tarone test for dose—related trend in mortality and the results of the Cox test comparing 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.

C. Pathology (Rats)
Each of the tumor 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.
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.
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

open allclose all
Dose descriptor:
NOAEL
Effect level:
> 7 500 ppm
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: overall effects
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOAEL
Effect level:
> 375 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No effects were observed; 7500 ppm in diet applied to rats were converted to 375 mg/kg/day calculated for rats with a mean body weight of 400 g and 20 g/day of food consumption
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the histopathologic examination, menthol was neither toxic nor carcinogenic to Fischer 344 rats under the conditions of this bioassay.
Executive summary:

Mean body weights of the dosed rats were slightly lower than those of corresponding controls. No other clinical signs related to administration of menthol were noted in any dosed groups of rats. Survival at the end of the bioassay was at least 62% in all dosed and control groups of animals, and sufficient numbers of animals were at risk for the development of late-appearing tumours.
In male rats, no tumours occurred at incidences which were considered to be associated with the administration of menthol.
In female rats, no tumours occurred at higher incidences in dosed groups than in control groups. Fibro adenomas of the mammary gland occurred at lower incidences in the low-dose (10/49) and high-dose (7/49) groups than in the control group (20/50), and alveolar/bronchiolar adenomas or carcinomas of the lung occurred only in the controls (3/50).