L-menthol

Repeated dose toxicity: inhalation

Administrative data

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

Data source

Reference

Reference 1

Reference Type:

publication

Title:

An evaluation of the toxicity of 95 ingredients added individually to experimental cigarettes: approach and methods

Author:

CL Gaworski, MJ Old, KA Wagner, CRE Coggins, and GJ Patskan

Year:

2011

Bibliographic source:

Inhalation Toxicology, 2011; 23(S1):1-12

Reference 2

Reference Type:

publication

Title:

A comprehensive evaluation of the toxicity of cigarette ingredients: essential oils and resins

Author:

CRE Coggins, JS Edminston, AM Jerome, TB Langston, EJ Sena, DC Smith, and MJ Oldham

Bibliographic source:

Inhalation Toxicology, 2011; 23(S1):41-69

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

peppermint oil

IUPAC Name:

peppermint oil

Constituent 2

Reference substance name:

8006-90-4

EC Number:

616-900-7

IUPAC Name:

8006-90-4

Constituent 3

Reference substance name:

2848

IUPAC Name:

2848

Constituent 4

Reference substance name:

Menthol

EC Number:

201-939-0

EC Name:

Menthol

Cas Number:

89-78-1

IUPAC Name:

2-isopropyl-5-methylcyclohexanol

Test material form:

aerosol dispenser: not specified

Remarks:

migrated information: aerosol

Details on test material:

Experimental cigarettes were prepared with components and construction processes consistent with commercial US cigarette manufacturing. Final tobacco weights of the finished cigarettes were intended to be 0.7g. Experimental and control cigarettes were produced at the same time and the same constant weight, irrespective of the inclusion level of the ingredient. This meant that as the inclusion level of the ingredient increased, the amount of tobacco in the experimental cigarettes decreased. Experimental cigarettes containing added peppermint oil at three inclusion levels, low, middle and high, where control cigarettes containing no added test ingredient. Cigarettes were made concurrently with the same tobaccos and cigarette material to avoid possible influence of disparate materials or processing. Cigarettes were conditioned and smoked to the appropriate ISO methods. The Menthol content as marker for peppermint oil in tobacco of test cigarettes was characterized by extraction in an appropriate solvent and analysed on a gas-chromatograph mass spectrometer. Menthol level on cigarette (µg/g tobacco): control: 0, low: 509, middle: 6960, high 67200.
Generated mainstream cigarette smoke from the smoking machine was diluted with filtered air to produce te target Total Paticular Matter (TPM) concentration of 150 mg/m3

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
509 (µg Menthol/g tobacco)
Basis:
analytical conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
6960 (µg Menthol/g tobacco)
Basis:
analytical conc.

Doses / concentrations 3

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)

Examinations

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.

Results and discussion

Results of examinations

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

Effect levels

1

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

2

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'

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

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.