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Description of key information

No effects were observed in life-time (103 weeks) studies in rats and mice  by using DL Menthol up to the highest tested dose equalling 375 mg/kg bw/day for the rat and 667 mg/kg bw/day for the mouse. In a 90-day feeding study with DL Menthol the NOAEL was 937 mg/kg bw/d for rats (highest dose tested). In a 90-day feeding study the NOAEL was 1250 mg/kg bw/d for mice based on slightly reduced body weight gain.
As no sytemic effects were seen in both 103 weeks repeated dose toxicity studies applied by oral route, repeated dose studies via dermal or inhalative route do not appear to be scientifically justified.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Dose descriptor:
NOAEL
375 mg/kg bw/day
Study duration:
chronic
Species:
rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: According to ECVAM ToxRTool reliability 3. Missing information: Testsubstance purity, source of the substance, the strain of test animal, the final concentrations of test material inhaled by animal
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Principles of method if other than guideline:
Ingredient was tested through addition at different concentrations to the tobacco of experimental cigarettes. Ingredient was tested in 90-day nose-only rat inhalation study using mainstream cigarette smoke.
GLP compliance:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
other: cigarette smoke
Remarks on MMAD:
MMAD / GSD: MMAD ranged from 0.27 to 0.64; The GSD ranged from 1.58 to 2.25
Details on inhalation exposure:
Nose-only smoke exposure was used to ensure maximal uptake of the test substance by inhalation route and to minimize non-inhalation routes of exposure. Male and female rats were randomly allocated to experimental groups (including air-sham) by body weight stratification and were exposed for 6h/day for 90 consecutive days. Exposure at the selected smoke concentration has previously been shown to be within the dynamic range of the respiratory tract histopathology and allows for evaluation of both increase and decrease in effect due to peppermint oil (Menthol). To assess the reversibility of changes in histopathology, sub-groups of rats were kept for a 42-day post inhalation period (recovery)
The continuous stream of mainstream cigarette smoke from the smoking machine was diluted (between 1:160 and 1:200) with filtered air to produce the target TPM concentration of 150 mg/m3. Nose-only exposures at PMRL initially used stainless steel, brass and glass IC88 exposure chambers which were subsequently changed to stainless steel and glass ECFPC nose-only exposure chambers (European Patent 2095791). Separate chambers were used for each experimental cigarette type.
Particle size distribution was measured after precipitation in an aerosol centrifuge by fluorometry. Temperature was measured with a thermistor probe, and RH was measured using a sling psychrometer or capacitive humidity sensor. Flow inside the exposure chamber was measured with either a float flow meter or by measuring pressure difference across a (calibrated) Venturi tube.
Minute ventilation was assessed by head-out plethysmography with a pneumotachograph. Measurements were typically made on five rats per sex per group, during weeks 4–6 of the inhalation part of the study. Blood carboxyhemoglobin (COHb) concentrations were measured during the last hour of exposure for one exposure day several times during the exposure period using a published metho. Nicotine and cotinine were measured in urine collected during the exposure.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Analytical verification of smoke exposure was performed. The final dose or concentration of peppermint oil (Menthol) in administered smoke was not determined.
The following smoke constituents were regularly measured throughout the study at one (or more) of the animal exposure ports: TPM (by weighing glass fiber Cambridge filter pads placed in the path of the smoke), CO (by nondispersive infra-red photometry), nicotine (by capillary GC, after trapping on sulfuric-acid impregnated silica gel), formaldehyde, acetaldehyde, acrolein, and propionaldehyde (by HPLC of DNPH derivatives after trapping in DNPH solution.
Blood carboxyhemoglobin (COHb) concentrations were measured during the last hour of exposure for one exposure day several times during the exposure period using a published metho. Nicotine and cotinine were measured in urine collected during the exposure.
Duration of treatment / exposure:
6h/day for 90 consecutive days
Frequency of treatment:
daily for 90 days
Remarks:
Doses / Concentrations:
509 (µg Menthol/g tobacco)
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
6960 (µg Menthol/g tobacco)
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
67200 (µg Menthol/g tobacco)
Basis:
analytical conc.
No. of animals per sex per dose:
10 animals per sex per dose and additional 10 animals per sex in the highest dose for recovery group
Control animals:
yes, concurrent vehicle
yes, sham-exposed
Details on study design:
Experimental cigarettes containing added peppermint oils at three inclusion levels, low, middle, and high, were compared with control cigarettes containing no added test ingredient (zero inclusion). Cigarettes (control and experimental) within a study were made concurrently with the same tobaccos and cigarette materials to avoid possible influence of disparate materials or processing.
Exposure at the selected smoke concentration (TPM 150 mg/m3) has previously been shown to be within the dynamic range of the respiratory tract histopathology and allows for evaluation of both increase and decrease in effect due to peppermint oil (Menthol). To assess the reversibility of changes in histopathology, sub-groups of rats were kept for a 42-day post inhalation period (recovery)
Observations and examinations performed and frequency:
Body weight and food consumption were measured once per week. Ophthalmologic evaluations were performed on all rats before the start of the study and on five rats per sex per group at the end of the inhalation part of the study. Clinical observations were made daily on each animal, before and after the exposures. Hematology and blood chemistry analyses were made on 10 rats per sex per group, at the end of the inhalation part of the study and at the recovery period. Standard analytical techniques were used.
Sacrifice and pathology:
Rats were not fasted before necropsy. On the day following the last exposure, selected rats were anesthetized by an intraperitoneal injection of sodium pentobarbital (30 mg/kg) and euthanized by exsanguination after severing the abdominal aorta. The carcasses were weighed and subjected to a complete gross examination under the supervision of a veterinary pathologist, with special attention paid to the respiratory tract. The following organ weights were determined at necropsy: lungs (with larynx and trachea attached), liver, kidneys, gonads, heart, adrenal glands, thymus, brain, and spleen. All tissues were preserved in 10% neutral buffered formalin (NBF), except the eyes (Davidson’s fixative) and testes (Bouin’s fixative). Lungs and urinary bladder were infused with NBF to ensure fixation. For histopathology, the skin, lower jaw, and brain were removed from the head and the nasal passages were gently flushed with NBF. The head was subsequently fixed in 10% NBF. Prior to trimming, the head was decalcified by immersion in a solution of sodium formate in formic acid (Kristensen solution) for up to 5 days. After decalcification, the nose was trimmed and transverse sections were cut to obtain four tissue slices: immediately posterior to the upper incisor teeth (“level 1”), posterior to the incisive papilla (“level 2”), at the second palatal ridge (“level 3”), and between the first and second molar teeth (“level 4”; Young, 1981). Using this technique, the respiratory epithelium of the nasoturbinates, maxilloturbinates, and walls of the nasal cavity were examined at four levels. The third section included the respiratory epithelium of the distal part of the nasoturbinate and maxilloturbinate, and the olfactory epithelium on the dorsal wall. The fourth section was primarily ethmoid turbinate covered by olfactory epithelium. The excised lungs, with trachea and larynx attached, were fixed by intratracheal instillation of NBF at an approximate pressure of 25 cm water column. For the larynx, three transverse sections were taken: at the arytenoid projections, at the base of the epiglottis, and at the vocal folds (Sagartz et al., 1992; Renne and Gideon, 2006). For the trachea, a single longitudinal section was taken to include the tracheal bifurcation and carina. The left lung was trimmed as described previously (Lamb and Reid, 1969). A longitudinal section was taken through the main bronchus of the left lung and a cross-section was taken through each of the cranial, middle, caudal, and accessory lobes of the right lung, as described previously (Dungworth et al., 1976). Individual sections were 5 to 6 μm thick; they were stained routinely with hematoxylin and eosin. In addition, sections of nose, lungs, and trachea were stained with periodic-acid-Schiff-Alcian blue to facilitate the recognition of mucus-producing goblet cells.
Statistics:
For continuous data in the inhalation study, ANOVA was applied for overall comparison followed by the Dunnett test for pairwise comparisons with the control group. Tests were conducted at the nominal level of significance of α = 0.05 (2-tailed). Results were considered statistically significant at P ≤ 0.05. No adjustment for multiple testing was made.
When assessing the degree of tissue change and significance of results a 5-point scale was used (i.e. 0 = none detected, 1 = slight, 2 = slight/moderate, 3 = moderate, 4 = moderate/ marked, 5 = marked). The generalized Cochran- Mantel-Haenszel (CMH) test was used for statistical comparisons of the severity scores between control and treated groups (Mantel and Haenszel, 1959). Pathologists noted that the differences between groups may or may not be a toxicologically significant difference depending upon expert interpretation which included analysis of factors such as dose response and coherence among findings.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
four males in the study. Non of the deaths were considered to be related to the peppermint oil inclusion
Mortality:
mortality observed, treatment-related
Description (incidence):
four males in the study. Non of the deaths were considered to be related to the peppermint oil inclusion
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
body weight gain in the smoke exposed groups of males were up to 28% smaller than the sham exposed groups but weight loss was similar in groups exposed to control smoke or smoke from cigarettes containing peppermint oil
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
sporadic statistically significant differences between groups, with no obvious trend
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:
no effects observed
Description (incidence and severity):
There was no significant group difference determined in the respiratory tract for either sex
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
other: Peppermint oil (Menthol determined as marker) in cigarette smoke tested in three levels didn't change the histopathological findings in the respiratory tract of rats (local effects) compared to smoke from similar control cigarettes without peppermint oil.
Effect level:
> 67 200 other: µg Menthol/g tobacco
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: rats were exposed to 150 mg/m3 total particular matter (TPM smoke) which contained an estimated Menthol content of 60mg/m3
Dose descriptor:
conc. level:
Effect level:
> 60 mg/m³ air
Based on:
other: calculation of transfered Menthol from tobacco to smoke
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified
Conclusions:
Peppermint oil (Menthol determined as marker) in cigarette smoke tested in three levels didn,t increase the histopathological findings in the respiratory tract of rats (local effects) compared to cigarette smoke without menthol.
Executive summary:

Peppermint oil was added to experimental cigarettes in three target concentrations (Low 1000 ppm, Middle 10000 ppm, High 100000 ppm). The Menthol level in tobacco after cigarette production was 509 µg/g (Low), 6960 µg/g (Middle) and 67200 µg/g (High). Smoke from each of the experimental cigarettes was evaluated in a 90 -day smoke inhalation study with rats and compared to smoke from experimental cigarettes produced from the same materials and to the same time but without peppermint oil. Selected smoke constituents in test smoke were determined and compared between test cigarette and control cigarette smoke ( no peppermint oil).

Cigarette smoke from the highest peppermint inclusion level showed consisitent reduction in smoke constituents, to approximately 60% of the control yields. Four male rats died during the study but none of the deaths were considered to be relevant to the ingredient inclusion. Body weight gain loss from animals exposed to control cigarettes was similar to the animals exposed to smoke from cigarettes containing peppermint oil. In necropsy , serum chemistry, hematology and organ weights there were occationally statistically significant differences in group mean values from means in the control groups, but these differences were sporadic and there were no overall trends. Exposure at the selected smoke concentration has previously been shown to be within the dynamic range of the respiratory tract histopathology and allows for evaluation of both increase and decrease in effect due to peppermint oil (Menthol). Histopathological changes produced as a result of smoke exposure were very similar to those reported in the literature, with no novel findings. In nose, larynx, trachea, and lung there were no significant group differences found for smoke from cigarettes with peppermint oil in differnt level or without , for either sex.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
subchronic
Species:
rat
Quality of whole database:
The study was performed to test the effect of peppermint oil in smoke to smoke exposed rats. The total particular matter (TPM) level was measured and used to adjust the exposure level. The data set would fullfill a reliability score of 2. The 3 level of peppermint oil in the experimental cigarettes were determined. But it is not clear what the final peppermint oil (Menthol) level in the exposed smoke was (reliability score of 3).

Additional information

There is no reliable repeated dose study on L Menthol available.

Justification for Read-across:

Based on the identical profiles of the different Menthol isomers we can use them for read across studies as laid out in the Read-Across Justification for Menthols (see file MentholsReadAcrossFinal.pdf in section 13).

Used isomers are L Menthol (CAS 2216-51-5), D Menthol (CAS 15356 -60 -2) and DL Menthol (CAS 89-78-1).

The comparative physico-chemical profile of these isomers confirms the similarity.

Details on the repeated dose studies :

- Two tests were performed with DL Menthol in rats for 13-weeks (males up to 998 mg/kg bw/day and females up to 937 mg/kg bw/day) and in mice (males up to 1956 mg/kg bw/d and females up to 2386 mg/kg bw/day) respectively. No toxicity was noted at the maximum dose tested in rat, while in mice a slight body weight effect was observed at the highest dose without any gross and microscopic pathological findings related to the treatment. The NOAELs derived from these studies were 937 mg/kg bw/d for rat and 1250 mg/kg bw/d for mouse.

- After 103 weeks oral administration of DL Menthol the 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.

The NOAEL resulted from this study was 7500 ppm (i.e. 375 mg/kg bw/day).

- After 103 weeks oral administration of DL Menthol the 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 behaviour 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.

The NOAEL resulted from this study was 4000 ppm (i.e. 667 mg/kg bw/day).



Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
No valid repeated dose Menthol inhalation studies are available. In one available inhalation studies the effect of peppermint oil was measured as cigarette ingredient in smoke (level meassured as Menthol on tobacco).

Justification for classification or non-classification

A 2 years feeding study on rats and mice revealed no signs of systemic toxicity or negative effects in a dose relevant for classification. Also these studie didn’t reveal any indication of tumor incidences in treated animals. Hence it can be concluded that the substance does not meet the criteria for classification and labeling for carcinogenicity or repeated dose toxicity (STOT), as set out in Regulation (EC) NO. 1272/2008 or in Directive 67/548/EEC respectively as indicated by study results on DL Menthol used for read across.