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Administrative data

Description of key information

Read-across compound DL-menthol is not toxic in rats when applied daily by oral route (feed) at a dose of 375 mg/kg bw/day for 103 weeks (corresponding to a NOEL for menthyl acetate of 476 mg/kg bw/day).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
chronic toxicity: oral
Remarks:
combined repeated dose and carcinogenicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Not GLP compliant study, no guideline specifically mentionned. However, the study is documented enough to be scientifically assessed and found reliable.
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
GLP compliance:
not specified
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Frederick Cancer Research Center, Frederick, Maryland
- Age at study initiation: 9 weeks
- 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).
(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 weeks of observation, the animal were supplied with standard diet.
Frequency of treatment:
Feed hoppers were sanitized once a week at 81°C.
Remarks:
Doses / Concentrations:
0.375 %
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0.75%
Basis:
nominal in diet
No. of animals per sex per dose:
50 animals per dose (sex ratio not mentioned)
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.
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
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 dl—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 dl—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.
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
Remarks:
no NOAEL identified
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
Remarks:
no NOAEL identified
Dose descriptor:
NOAEL
Effect level:
> 476 mg/kg bw/day (nominal)
Based on:
other: Menthyl acetate
Sex:
male/female
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Critical effects observed:
not specified
Conclusions:
Based on the histopathologic examination, DL-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 DL-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 DL-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 DL-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, DL-menthol was not carcinogenic for either Fischer 344 rats.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
476 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
Four reliable studies: chronic in rat and mouse and subchronic in rat and mouse are available for DL-menthol as read-across compound.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

- Repeated dose toxicity, oral:

There is no data on menthyl acetate.

According to reliable chronic (103-week) repeated dose toxicity studies in rat and mouse, the NOEL for the read-across compound DL-menthol was at the highest dose causing no adverse effects, i.e at 375 mg/kg bw/day (476 mg/kg bw/day for Menthyl acetate) in rat and at 667 mg/kg bw/day (846 mg/kg bw/day for Menthyl acetate) in mouse. In two reliable 90-day studies, basing on few examination parameters (clinical signs and mortality, body weight and weight gain, haematology and histopathology), the NOAEL of the read-across compound DL-menthol was identified: 750 mg/kg bw/day in rat and 1250 mg/kg bw/day in mouse. The available results show that toxicity of the compound does not lead to a RE-STOT classification according to CLP.

- Repeated dose toxicity, dermal: no studies available

- Repeated dose toxicity, inhalation: no studies available


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Read-across from DL-menthol of reliable 103-week study. This study in rats provides better profile on results of examinations comparing to 90-day DL-menthol study in rat.

Justification for classification or non-classification

- Repeated dose toxicity, oral:

Based on the above stated assessment of the specific target organ toxicity potential after repeated oral exposure of read-across compound DL-menthol, menthyl acetate does not need to be classified according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) or according to CLP (Regulation (EC) No 1272/2008 of the European Parliament and of the Council) as implementation of UN-GHS in the EU.

- Repeated dose toxicity, dermal:

As no data on of the specific target organ toxicity potential after repeated dermal exposure of menthyl acetate is available a classification is not possible according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) and according to CLP (Regulation (EC) No 1272/2008 of the European Parliament and of the Council) as implementation of UN-GHS in the EU.

- Repeated dose toxicity, inhalation:

As no reliable data on of the specific target organ toxicity potential after repeated inhalation exposure of menthyl acetate is available a classification is not possible according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) and according to CLP (Regulation (EC) No 1272/2008 of the European Parliament and of the Council) as implementation of UN-GHS in the EU.