Peppermint, ext.

Endpoint summary

Description of key information

28-d oral repeated dose toxicity study with peppermint oil: NOAEL 400 mg/kg bw/day based on absence of effects at the highest dose tested (Serota, 1990)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 November 1988 - 8 November 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study was conducted under GLP conditions and according to a reliable method, but not an official guideline. The study report is well-documented.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

no functional observations

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, North Carolina, USA
- Age at study initiation: 6 weeks
- Weight at study initiation:
Males: 198.4 - 226.1
Females: 139.3 - 170.5
- Housing: Individually
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.9 - 25.6
- Humidity (%): 50 +/- 20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Test material was weighed into a pre-calibrated beaker on an appropriate balance. Corn oil was added to achieve appropriate volume and stirred for 2-3 minutes. Test mixtures were prepared fresh weekly.

VEHICLE
- Justification for use and choice of vehicle (if other than water): No data
- Amount of vehicle (if gavage): 10 ml/kg
- Lot/batch no. (if required): BG 1314

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily

Remarks:

Doses / Concentrations:
100, 200, 400 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: No data
- Rationale for animal assignment: At random
- Section schedule rationale: Two-day period, fifty-fifty sacrificed and necropsied over the two days.

Positive control:

Not relevant

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Time schedule: Twice daily
- Cage side observations: mortality and moribundity

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Weekly

BODY WEIGHT:
- Time schedule for examinations: Weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined as g food/week.

HAEMATOLOGY:
- Time schedule for collection of blood: Prior to intitation of study and at termination
- Anaesthetic used for blood collection: Yes (ketamine)
- Animals fasted: Yes, overnight
- How many animals:
Prior to initiation: 10 healthy animals not selected for study
At termination: all surviving animals
- Parameters checked: corrected leukocyte count, leukocyte count, erythrocyte count, hemoglobin, hematocrit, platelet count, leukocyte differential count cell morphology, myeloid/erythroid ratio.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: Prior to intitation of study and at termination
- Anaesthetic used for blood collection: Yes (ketamine)
- Animals fasted: Yes, overnight
- How many animals:
Prior to initiation: 10 healthy animals not selected for study
At termination: all surviving animals
- Parameters checked: sodium, potassium, chloride, total protein, albumin, calcium, total carbon dioxide, total bilirubin, blood urea nitrogen, creatinine, glucose, alanine aminotransferase, aspartate aminotransferase, gamma glutamyltransferase, alkaline phosphatase.

Sacrifice and pathology:

GROSS PATHOLOGY: Necropsy was performed on all surviving animals and the following was examined: external surfaces, all orifices, cranial cavity, external surface of the brain (external surface of the spinal cord and the cut surfaces of the brain and spinal cord were examined at the time of tissue trimming), nasal cavity and paranasal sinuses, thoracic, abdominal and pelvic cavities and their viscera, cervical tissues and organs.

ORGAN WEIGTHS: Organ weights of the following organs were measured for each sacrificed animal: brain (including brainstem), spleen, liver, heart, kidneys, testes with epididymides, thyroid with parathyroids, adrenals, ovaries, pituitary.

HISTOPATHOLOGY: The following tissues were examined from all control and high-dose animals: femoral bone marrow, lung (with mainstem bronchi), ovaries, gross lesions, kidneys, adrenals, testes with epididymides, duodenum and jejunum and ileum, brain with brainstem (medulla/pons, cerebellar cortex, cerebral cortex), pancreas, urinary bladder, pituitary, uterus, thyroid (parathyroids), heart, liver, spleen, colon and cecum and rectum, stomach, mesenteric lymph node. From the low and middose group only the heart, liver, kidneys and gross lesions were examined microscopically.

Other examinations:

None performed.

Statistics:

ANOVA was used to determine significant differences in parameters between the test groups.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality was observed. An increase in urine stains was observed for the high-dose males as compared to the other test groups.

BODY WEIGHT AND WEIGHT GAIN
No significant differences in body weight (gain) were observed.

FOOD CONSUMPTION
No significant differences in food consumption were observed.

HAEMATOLOGY
A significant decrease in myeloid/erythroid ratio was observed for the high-dose males as compared to the control group.

CLINICAL CHEMISTRY
A significant decrease in glucose levels was observed for the high and mid-dose males as compared to the control group. A significant increase in alkaline phosphatase levels was observed for the high-dose group.

ORGAN WEIGHTS
A significant increase in absolute and relative liver weight was observed for females of the high-dose group as compared to the controls. Relative kidney weight was significantly higher for the high-dose males.

GROSS PATHOLOGY
An increased, non-significant, incidence in stomach dark area's was seen for the male dose groups as compared to control. This effect was also seen in the female groups, including the control group.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathological changes (chronic inflammation, regeneration of tubules, droplets in tubule cells) in the kidney were observed for the male dose groups and not in the control group. This is supported by the increase in relative kidney weight. The observed effects consisted of renal tubular protein droplets, thought to be related to lysosomal handling of alpha-2-µ globulin. This effects is a known male-specific effect in rats caused by exposure to hydrocarbons.
For females, the same changes were found as compared to controls, but to a lesser extent and no droplets were observed in the tubule cells. The differences in relative and absolute liver weight in the female high-dose group were not supported by histopathology. No other remarkable histopathological differences between control and test groups were noted.

Dose descriptor:

LOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Histopathological changes (kidney) in all dose group: renal tubular protein resorption droplets (male-rat specific)

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No observed adverse effects.

Critical effects observed:

not specified

No gross necrospy, organ weight and/or histopathological effects were observed for the brain stem (incl. medulla/pons, cerebellar cortex and cerebral cortex). No indication for neurotoxicity was found.

The individual result tables do not provide additional information on the effects that were observed (hematology, clinical chemistry, organ weights and histopathology).

Conclusions:

Under the conditions of this study, no treatment related considered effects were noted in females. In all male dose groups treatment related kidney effects were observed, resembling the male rat-specific effect hyalin droplet nephropathy. Based on these findings, the NOAEL for females was established to be 400 mg/kg bw/day, while for males a LOAEL of 100 mg/kg bw/day was found based on male rat-specific effects.

Executive summary:

Rats were exposed to the substance B100 for 28 days by gavage. Animals were observed for mortality, moribundity, body weight and food consumption. Additionally, several hematology and clinical chemistry parameters were studied and gross necropsy, organ weight measurements and histopathology were performed.

No mortality was observed in the study. An increase in urine stains was observed for the high-dose males as compared to the other test groups. No significant differences in body weight (gain) and food consumption were observed. A significant decrease in myeloid/erythroid ratio and increase in alkaline phosphatase was observed for the high-dose males and a significant decrease in glucose levels was observed for the high and mid-dose males as compared to the control group.

A significant increase in absolute and relative liver weight was observed for females of the high-dose group as compared to the controls. Relative kidney weight was significantly higher for the high-dose males. An increased, non-significant, incidence in stomach dark area's was seen for the male dose groups as compared to control. This effect was also seen in the female groups, including the control group.

Histopathological changes (chronic inflammation, regeneration of tubules, droplets in tubule cells) in the kidney were observed for the male dose groups and not in the control group. This is supported by the increase in relative kidney weight. The observed effects consisted of renal tubular protein droplets, thought to be related to lysosomal handling of alpha-2-µ globulin. This effects is a known male-specific effect in rats caused by exposure to hydrocarbons. The differences in relative and absolute liver weight in the female high-dose group were not supported by histopathology. No other remarkable histopathological differences between control and test groups were noted.

Under the conditions of this study, no treatment related considered effects were noted in females. However, in all male dose groups treatment related kidney effects were observed. Based on these findings, the NOAEL for females was established to be 400 mg/kg bw/day, while for males a LOAEL of 100 mg/kg bw/day was found.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

400 mg/kg bw/day

Study duration:

subacute

Species:

rat

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

Several repeated dose toxicity studies are available for peppermint oil, whereas no repeated dose toxicity studies are available with cornmint oil. However, cornmint oil can be considered as part of the mint-category together with the UVCB-substance peppermint oil, for the purpose of REACH-registration. The category is based on the common source of the oils, common methods of production, same dominant constituents, and same and/or similar constituents. A read across document has been prepared in which the category is further elaborated. The available key and supporting repeated dose toxicity studies with peppermint oil are summarized below.

 

The key study is an oral 28-day study with peppermint oil in rats exposed at dose levels of 0, 100, 200, and 400 mg/kg bw/day. No toxicologically relevant effects were observed in females. In male rats, alpha-2-microglobulin-associated hydrocarbon nephropathy was observed, which is not relevant to humans as it is a male rat-specific effect. No gross necropsy, organ weight and/or histopathological effects were observed for the brain stem (incl. medulla/pons, cerebellar cortex and cerebral cortex) and no indication for neurotoxicity was found. A NOAEL of 400 mg/kg bw/day was established for peppermint oil.(Serota, 1990)

 

Two supporting repeated dose toxicity studies with peppermint oil are available. In a 5-wk study in which rats were exposed to peppermint oil by gavage at concentrations of 20, 150, or 500 mg/kg bw/day, no toxicologically relevant effects were observed. Additionally, dogs were exposed to concentrations of 25 and 125 mg/kg bw/day by gavage. No toxicologically relevant effects were observed in dogs at these dose levels. The corresponding NOAELs are 500 mg/kg bw/day for rats and 125 mg/kg bw/day for dogs. (Mengs and Stotzem, 1989)

 

Available disregarded studies

Two studies with peppermint oil in which histopathological changes in the brain were observed were considered to be invalid as the effects could not be reproduced and were regarded to be artefacts of preparation and fixation of the brain tissue by the FEMA expert panel after review of the original brain slides.

 

In both, a 28-d study and a 90-d study, rats were exposed to peppermint oil by gavage, at concentrations of 0, 10, 40, and 100 mg/kg bw/day. In both studies histopathological changes in the white matter of the cerebellum (little brain) were observed, which consisted of cyst-like spaces scattered in the white matter. No cellular reactions in the surrounding tissue were observed, and demyelination could not be demonstrated after staining. Furthermore, no clinical symptoms were observed which could be attributed to encephalopathy. Based on the observed histopathological changes, a NOAEL of 10 mg/kg bw/day was derived in the 28-d study (Thorup et al., 1983), and 40 mg/kg bw/day in the 90-d study (Spindler and Madsen, 1992). Although the dosing period was considerably longer in the 90-d study, no increase in the extension of the cyst-like spaces was observed.

 

Similar histopathological effects in the brain were also observed in studies with pulegone, which is a minor constituent (2%) of both peppermint and cornmint oil. The available studies with pulegone were summarized by the Scientifc Committee on Food, which prepared an opinion on pulegone and major metabolite menthofuran (present in peppermint oil at 4%) in 2002. A NOAEL of 20 mg/kg bw/day was reported for pulegone based on the histopathological changes in the brain. (SCF, 2002)

 

With the aim of reproducing and supporting the histopathological findings in the brain after exposure to pulegone and peppermint oil, a study was performed by Mølck et al. (1998) to investigate whether the observed effects may be due to the used tissue fixation technique. Rats were exposed to pulegone at a dose of 0 or 160 mg/kg bw/day. Brain tissues were fixed using both, the earlier used technique (immersion tissue fixation) and another technique (perfusion tissue fixation). Results showed no occurrence of cyst-like spaces in the white matter of cerebellum using both techniques. The authors suggest that the observed histopathological changes in the earlier studies may have been caused by impurities in the test substance, drift in the genetic constitution of the animals, and/or small differences in processing brain tissue for histology. (Mølck et al., 1998).

Another publication is available in which the observed effects of both pulegone and peppermint oil on the brain are presented, however, the publication does not discuss the results any further. (Olsen and Thorup, 1984)

 

In the Opinion of the Scientific Committee on Food on pulegone and menthofuran, the different studies are mentioned, but no conclusion is drawn on the validity of the observed histological effects in the brain. The SCF concludes that based on the limited database for pulegone and menthofuran, no Acceptable Daily Intake (ADI) could be derived, and further studies were requested. (SCF, 2002)

 

The studies were also reviewed by the Flavor and Extract Manufacturers Association (FEMA) with respect to the GRAS substance menthone, a major constituent of peppermint oil. In this respect, the original rat brain histology slides from the three studies were reviewed by pathologists of the FEMA Expert Panel. They report that no evidence was found of cellular reaction in tissue adjacent to the cyst-like spaces in the white matter of the cerebellum in all studies. Furthermore, the appearance and extent of these cyst-like spaces was not significantly increased in the 90-d study with respect to the 28-d study. The FEMA concludes that, taking into account that the findings could not be reproduced upon attempt, and that a study in which a brain perfusion method was used could also not reproduce the finding, the observed cyst-like spaces in rat brain were artefacts resulting from inadequate preparation and fixation of the cerebellar tissue. Consequently, the GRAS-status of menthone was reaffirmed. (FEMA, 1996)

 

As mentioned above, encephalopathy was not observed in the available key and supporting repeated dose toxicity studies with peppermint oil. In the 5-wk repeated dose study with rats by Mengs and Stotzem (1989), special attention was given to the histopathology of the cerebellum. However, no cyst-like spaces in the white matter of the cerebellum were observed with light microscopy. They also observed no effects in dogs after 5 weeks of exposure. Also in the available 28-d study with rats by Serota (1990), no effects were observed. No histopathological effects were observed for the brain stem (incl. medulla/pons, cerebellar cortex and cerebral cortex), and no indication for neurotoxicity was found.

As the histopathological findings in the brain after exposure to peppermint oil (and pulegone) could not be reproduced in other studies, did not increase after extended dosing, were in the same range for pulegone and peppermint oil (while peppermint oil contains only 2% pulegone), and were considered to be artefacts of preparation and fixation of the brain tissue by the FEMA expert panel after review of the original brain slides, the studies by Thorup et al. (1983) and Spindler and Madsen (1992) are considered invalid and were not used for derivation of the DNEL.

 

Available information from constituents

Overview of available constituent data on repeated dose toxicity

In the table below the available supporting information on constituents is summarized. Information on sub-chronic/chronic studies is available for 71.1% of cornmint oil constituents and 70.6% of peppermint oil constituents. Observed effects in rats in the studies with constituents are decreased BW, clinical signs, increased organ weight (liver, kidney), mortality at high doses. Furthermore, in mice exposure to minor constituents alpha/beta pinene resulted in histopathological changes in the urinary bladder.

 

Constituent	Repeated dose tox	NOAEL
Major constituents (≥10%)
L-menthol	Chronic (2-yr, oral, rat)	≥375 mg/kg bw/d
Menthone	RA to L-menthol Chronic (2-yr, oral, rat)	≥375 mg/kg bw/d
Minor constituents (<10%)
Limonene	Subchronic (90-d, oral, rat)	600 mg/kg bw/d
Alpha-pinene	Subchronic (90-d, inhalation, rat) Subchronic (90-d, inhalation, mouse)	200 ppm or 170 mg/kg bw/d 50 ppm
Beta-pinene	RA to Alpha-pinene Subchronic (90-d, inhalation, rat) Subchronic (90-d, inhalation, mouse)	200 ppm or 170 mg/kg bw/d 50 ppm
Myrcene	Subchronic (90-d, oral, mouse) Subchronic (90-d, oral, rat)	250 mg/kg bw/d 500 mg/kg bw/d

 

References

 

ECETOC (2010). Guidance on Assessment Factors to Derive a DNEL, Technical Report No. 110, October 2010

 

FEMA (1996). Menthone (FEMA No. 2667). Food Technology, October 1996.

 

Mengs, U. and Stotzem, C.D. (1989). Toxicological evaluation of peppermint oil in rodents and dogs, Medical Science Research, Vol. 17, Nr. 1111, 499-500

 

Mølck, A-M, Poulsen, M., Tindgard Lauridsen, S., Olsen, P. (1998). Lack of histological cerebellar changes in Wistar rats given pulegone for 28 days. Comparison of immersion and perfusion tissue fixation. Toxicology Letters, 95, 117-122

 

Olsen, P. and Thorup, I. (1984). Neurotoxicity in rats dosed with peppermint oil and pulegone. Arch. Toxicol., Suppl. 7, 408-409

 

Scientific Committee on Food (2002). Opinion of the Scientific Committee on Food on pulegone and menthofuran. SCF/CS/FLAV/FLAVOUR/3 ADD2 Final, 25 July 2002

 

Serota, D.G. (1990) 28-Day oral toxicity study in rats. Peppermint oil (Report to FEMA, RIFM location 53796, sub-reference 11/08)

 

Spindler, P., and Madsen, C. (1992). Subchronic toxicity study of peppermint oil in rats. Toxicol. Lett. 62:215-220.

 

Thorup, I., Wurtzen, G., Carstensen, J., Olsen, P. (1983).Short term toxicity study in rats dosed with peppermint oil. Toxicol. Lett. 19:211-215

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The selected study is the key study for this endpoint.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
None of the available studies is considered a key study for this endpoint.

Justification for classification or non-classification

Based on the criteria outlined in 67/548/EEC and 1272/2008/EC cornmint oil does not have to be classified with regard to repeated toxicity.