Registration Dossier
Registration Dossier
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EC number: 290-058-5 | CAS number: 90063-97-1 Extractives and their physically modified derivatives such as tinctures, concretes, absolutes, essential oils, oleoresins, terpenes, terpene-free fractions, distillates, residues, etc., obtained from Mentha arvensis, Labiatae.
Endpoint summary
Administrative data
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
- 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: Read across from peppermint oil. 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
- 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:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- No data
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Read across from peppermint oil. Publication is concise, but the design of the experiment seems to be generally accepted. The test result is therefore of value for this endpoint.
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- Three male and three female dogs received 25 or 125 mg/kg daily as enteric-coated gelatin capsules for five weeks. Dogs were inspected daily for clinical signs throughout the study. Body weight and food consumption were recorded weekly. All hematological, blood biochemical and urinary parameters were determined and organ weight measurements and histopathology were performed.
- GLP compliance:
- not specified
- Limit test:
- no
- Species:
- dog
- Strain:
- Beagle
- Sex:
- male/female
- Route of administration:
- oral: capsule
- Vehicle:
- other: enteric-coated gelatin capsule
- Analytical verification of doses or concentrations:
- no
- Duration of treatment / exposure:
- 5 weeks
- Frequency of treatment:
- Daily
- Remarks:
- Doses / Concentrations:
25, 125 mg/kg/day
Basis:
actual ingested - No. of animals per sex per dose:
- 3 animals/sex/dose
- Control animals:
- yes
- Dose descriptor:
- NOAEL
- Effect level:
- 125 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: No toxicologically relevant effects
- Critical effects observed:
- not specified
- Conclusions:
- Under the conditions of this study, slight effects on alkaline phosphatase and urea levels were noted in dogs of high dose males after exposure to Peppermint oil. However, they were within normal ranges and considered not to be toxicologically relevant. Therefore, the NOAEL was established to be 125 mg/kg/day.
- Executive summary:
Three male and three female dogs received 25 or 125 mg/kg daily as enteric-coated gelatin capsules for five weeks. Dogs were inspected daily for clinical signs throughout the study. Body weight and food consumption were recorded weekly. All hematological, blood biochemical and urinary parameters were determined and organ weight measurements and histopathology were performed.
No effects on general condition, behaviour or body weight development were observed. An slight increase in alkaline phosphatase and urea levels was noted in high dose males. However, they were within normal ranges and considered not to be toxicologically relevant. Therefore, the NOAEL was established to be 125 mg/kg/day.
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- No data
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Read across from peppermint oil. Publication is concise, but the design of the experiment seems to be generally accepted. The test result is therefore of value for this endpoint.
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- 12 male rats received 20, 150 or 500 mg/kg daily by gavage for five weeks. The control group received olive oil. Rats were inspected daily for clinical signs throughout the study. Body weight and food consumption were recorded weekly. All hematological, blood biochemical and urinary parameters were determined and organ weight measurements and histopathology were performed.
- GLP compliance:
- not specified
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male
- Route of administration:
- oral: gavage
- Vehicle:
- not specified
- Analytical verification of doses or concentrations:
- no
- Duration of treatment / exposure:
- 5 weeks
- Frequency of treatment:
- Daily
- Remarks:
- Doses / Concentrations:
20, 150, 500 mg/kg/day
Basis:
actual ingested - No. of animals per sex per dose:
- 4
- Control animals:
- yes, sham-exposed
- Dose descriptor:
- NOAEL
- Effect level:
- 500 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: No toxicologically relevant effects
- Critical effects observed:
- not specified
- Conclusions:
- Under the conditions of this study, some effects were noted in rats of predominantly the high dose group after exposure to Peppermint oil. The authors considered these effects not to be of toxicologically relevance. Therefore, the NOAEL was established to be 500 mg/kg/day.
- Executive summary:
12 male rats received 20, 150 or 500 mg/kg daily by gavage for five weeks. The control group received olive oil. Rats were inspected daily for clinical signs throughout the study. Body weight and food consumption were recorded weekly. All hematological, blood biochemical and urinary parameters were determined and organ weight measurements and histopathology were performed.
No effects on general condition, behaviour or body weight development were observed. A higher water consumption was seen in the high dose group, while plasma glyceride levels were decreased. An increase in alkaline phosphatase levels was noted in all treatment groups. The mean relative weight of liver and kidneys were higher in the high dose group as compared to the control group, however, no histopathological abnormalities were recorded in any group.
Under the conditions of this study, some effects were noted in rats of predominantly the high dose group after exposure to Peppermint oil. The authors considered these effects not to be of toxicologically relevance. Therefore, the NOAEL was established to be 500 mg/kg/day.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- Jan 2001- May 2001
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: GLP study conducted equivalent or similar to OECD Guideline 408 with deviations: adrenals, spleen and brain weights not recorded, food consumption not followed, dosing 5 days/week instead of 7 days/week.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
- Deviations:
- yes
- Remarks:
- adrenals, spleen and brain weights not recorded, food consumption not followed, dosing 5 days/week instead of 7 days/week
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- mouse
- Strain:
- B6C3F1
- Sex:
- male/female
- Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Analytical verification of doses or concentrations:
- yes
- Duration of treatment / exposure:
- 14 weeks
- Frequency of treatment:
- 5 days/week
- Remarks:
- Doses / Concentrations:
0, 250, 500, 1000, 2000 and 4000 mg/kg
Basis:
actual ingested - No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Dose descriptor:
- NOAEL
- Effect level:
- 250 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- other: Statistically significant increase in relative and absolute liver weights and relative kidney weights associated with a statistically significant decrease in bodyweight at 500 mg/kg bw/day
- Dose descriptor:
- NOAEL
- Effect level:
- 250 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: Mortality and statistically significant decreased bodyweight at 1000 mg/kg bw/day. Slight increases in relative liver weights at 250 and 500 mg/kg bw/day not related with any other toxic effect (bodyweight, hematology, histopathology)
- Critical effects observed:
- not specified
- Conclusions:
- A NOAEL was set at 250 mg/kg/day in females based on statistically significant increase in relative and absolute liver weights and relative kidney weights associated with a statistically significant decrease in bodyweight at 500 mg/kg bw/day. A NOAEL was set at 500 mg/kg bw/day in males based on mortality and statistically significant decreased bodyweight at 1000 mg/kg bw/day. Slight increases in relative liver weights were observed at 250 and 500 mg/kg bw/day but they were not associated with any other toxic effect (bodyweight, hematology, histopathology).
- Executive summary:
A study was conducted to evaluate the cumulative toxic effects of repeated exposure toβ‑myrcene according to the method equivalent or similar to OECD Guideline 408 in compliance with Good Laboratory Practices.
Groups of 20 B6C3F1 mice (10/sex/dose) were administered 0, 250, 500, 1000, 2000 or 4000 mg/kg bw/day of β-myrcene in corn oil by gavage, 5 days per week for 14 weeks. Parameters evaluated included survival, clinical observations, body weight, hematological and biochemical estimations in blood, reproductive parameters, necropsy and histopathological examination in all animals.
All 4000 mg/kg bw/day (male and female mice) died during week 1; 9/10 males and 8/10 females in 2000 mg/kg bw/day group died by week 4. The mean body weights of 1000 mg/kg bw/day males and 500 and 2000 mg/kg bw/day females were significantly lower than those of the vehicle controls. Clinical findings in animals that did not survive to the end of the study included thinness, lethargy, and abnormal breathing.Absolute and relative right kidney weights of 1000 mg/kg bw/day females, and the relative kidney weights of 250 and 500 mg/kg bw/day females were significantly greater than those of the vehicle controls. Relative liver weights of 500 and 1000 mg/kg bw/day males and the absolute and relative liver weights of 250 mg/kg bw/day males and 500 and 1000 mg/kg bw/day females were significantly greater than those of the vehicle controls.No histopathology changes were observed in mice administered 1000 mg/kg bw/day or less. Mice in 2000 and 4000 mg/kg bw/day groups were not evaluated due to early deaths.
A NOAEL was set at 250 mg/kg/day in females based on statistically significant increase in relative and absolute liver weights and relative kidney weights associated with a statistically significant decrease in bodyweight at 500 mg/kg bw/day. A NOAEL was set at 500 mg/kg bw/day in males based on mortality and statistically significant decreased bodyweight at 1000 mg/kg bw/day. Slight increases in relative liver weights were observed at 250 and 500 mg/kg bw/day but they were not associated with any other toxic effect (bodyweight, hematology, histopathology).
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- Jan 2001- May 2001
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: GLP study conducted equivalent or similar to OECD Guideline 408 with deviations: adrenals, spleen and brain weights not recorded, food consumption not followed, dosing 5 days/week instead of 7 days/week.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
- Deviations:
- yes
- Remarks:
- adrenals, spleen and brain weights not recorded, food consumption not followed, dosing 5 days/week instead of 7 days/week
- Principles of method if other than guideline:
- Not applicable
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- other: F344/N
- Sex:
- male/female
- Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Analytical verification of doses or concentrations:
- yes
- Duration of treatment / exposure:
- - Core study: 14 weeks
- Special study: 22 days - Frequency of treatment:
- 5 days/week
- Remarks:
- Doses / Concentrations:
0, 250, 500, 1000, 2000 and 4000 mg/kg
Basis:
actual ingested - No. of animals per sex per dose:
- 20: 10 for core study + 10 for blood collection at Day 2320: 10 for core study + 10 for blood collection at Day 23
- Control animals:
- yes, concurrent vehicle
- Dose descriptor:
- LOAEL
- Effect level:
- 250 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Increase in liver and kidney weights associated with minimal renal tubule necrosis in males and females
- Critical effects observed:
- not specified
- Conclusions:
- A significant increase in liver and kidney weights in males and females, associated with minimal renal tubule necrosis and only in males, hyaline droplet accumulation, were observed at 250 mg/kg bw/day. Therefore, no NOAEL was identified and a LOAEL was set t 250 mg/kg bw/day.
- Executive summary:
A study was conducted to evaluate the cumulative toxic effects of repeated exposure to β‑myrcene according to the method equivalent or similar to OECD Guideline 408 in compliance with Good Laboratory Practices.
Groups of 20 F334/N rats (10/sex/dose) were administered 0, 250, 500, 1000, 2000 or 4000 mg/kg bw/day β-myrcene in corn oil by gavage, 5 days per week for 14 weeks. Additional groups of 10 male and 10 female special study rats were administered the same doses for 22 days. Parameters evaluated included survival, clinical observations, body weight, hematological and biochemical estimations in blood, reproductive parameters, necropsy and histopathological examination in all animals.
All rats exposed to 4000 mg/kg bw/day died and 2/10 males and 4/10 females died in the 2000 mg/kg bw/day group. Dose-related clinical findings in animals that died early included thinness, lethargy, abnormal breathing and ruffled fur. Mean body weights were significantly decreased in the500, 1000 and 2000mg/kg bw/day groups. A significant increase in liver and kidney weights was observed in all dosed animals as well as renal tubule necrosis. A significant decrease in creatinine was also detected in all dosed females and in males at 500 mg/kg bw/day and higher doses. The incidences and severities of olfactory epithelium degeneration were increased in males and females of the 2000 mg/kg bw/day group.The incidences of chronic inflammation in 1000 and 2000 mg/kg bw/day males and females were significantly increased. All 2000 mg/kg bw/day males and females had splenic atrophy. In the mesenteric lymph node, significantly increased incidences of atrophy occurred in 2000 mg/kg bw/day males and 1000 and 2000 mg/kg bw/day females. Acute inflammation of the forestomach occurred in four 2000 mg/kg bw/day females. The incidences of porphyrin pigmentation in the Harderian gland of males administered 500 mg/kg bw/day or greater were significantly increased.
As a significant increase in liver and kidney weights associated with minimal renal tubule necrosis in males and females, as well as hyaline droplet accumulation only in males, were observed at 250 mg/kg bw/day, no NOAEL could be identified in this study and a LOAEL was set at 250 mg/kg bw/day.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- From January 28 to April 30, 1980
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: GLP study performed similarly to OECD Guideline 408 but with deviations: dosing 5 days/week instead of 7 days/week; food consumption, haematological and clinical biochemical test not followed
- Reason / purpose for cross-reference:
- reference to other study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
- Deviations:
- yes
- Remarks:
- dosing 5 days/week instead of 7 days/week; food consumption, haematological and clinical biochemical test not followed
- Principles of method if other than guideline:
- Not applicable
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- mouse
- Strain:
- B6C3F1
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Breeding Laboratories (Portage, USA)
- Age at study initiation: 7-9 weeks
- Weight at study initiation: Males: 23.8-29.5 g; females: 20.2-21.5 g
- Housing: Housed in groups of five in polycarbonate cages
- Diet (e.g. ad libitum): Purina Lab Blox (Chesapeake Feed Co., Beltsville, USA) or NIH 07 Rat and Mouse Ration (Zeigler Bros., Inc., Gardners, PA, USA), ad libitum
- Water (e.g. ad libitum): Automatic watering system (Edstrom Industries, Waterford, USA), ad libitum
- Acclimation period: 18 days
ENVIRONMENTAL CONDITIONS
- Temperature (°F): 60-82 °F
- Humidity (%): 35-80%
- Air changes (per hour): 12-15/hour
- Photoperiod (hours dark / hours light): 12 hours dark / 12 hours light - Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS: Appropriate amount of test substance was weighed and mixed with corn oil by shaking in a volumetric flask.
VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg bw - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- - Apparatus: Periodic analysis for d-limonene in dose preparations was determined by extraction with methanol followed by gas chromatographic analysis of the extract with a 6-foot 3% OV-17 glass column, a nitrogen carrier at a flow rate of 30 mL/min, and a flame ionization detector.
- Sampling frequency: Once
- Results: 101-109% of the target concentrations - Duration of treatment / exposure:
- 13 weeks
- Frequency of treatment:
- Once per day; 5 days/week
- Remarks:
- Doses / Concentrations:
0, 125, 250, 500, 1000 or 2000 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: Doses were selected based on the mortalities observed at 3300 and 6600 mg/kg bw/day during a 16 day subacute toxicity study.
- Rationale for animal assignment (if not random): Random - Positive control:
- No
- Observations and examinations performed and frequency:
- CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily
BODY WEIGHT: Yes
- Time schedule for examinations: Initially and once per week thereafter - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes; necropsy performed on all animals
HISTOPATHOLOGY: Yes; performed on all vehicle control and high dose animals. Tissues examined include: adrenal glands, brain, colon, esophagus, eyes (if grossly abnormal), femur or sternebrae or vertebrae including marrow, gallbladder, gross lesions and tissue masses with regional lymph nodes, heart, kidneys, liver, lungs and mainstem bronchi, mammary gland, mandibular or mesenteric lymph nodes, pancreas, parathyroids, pituitary gland, prostate/testes or ovaries/uterus, salivary glands, small intestine, spinal cord (if neurologic signs present), spleen, stomach, thymus, thyroid gland, trachea and urinary bladder. - Other examinations:
- No
- Statistics:
- No data
- Clinical signs:
- effects observed, treatment-related
- Mortality:
- mortality observed, treatment-related
- Body weight and weight changes:
- effects observed, treatment-related
- Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not specified
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Histopathological findings: neoplastic:
- not examined
- Details on results:
- MORTALITY:
- 1/10 male and 2/10 females died at 2000 mg/kg bw/day
- 1/10 female died at 500 mg/kg bw/day
- Several animals in other groups died as a result of gavage error.
CLINICAL SIGNS:
- Rough hair coats and decreased activity were observed at 1000 and 2000 mg/kg bw/day.
BODY WEIGHT AND WEIGHT GAIN
- Final mean bodyweights of mice that received 1000 or 2000 mg/kg bw/day were 10% lower than that of the vehicle controls for males and 2% lower for females.
HISTOPATHOLOGY
- An alveolar cell adenoma was observed in the lung of 1/10 females that received 2000 mg/kg bw/day. - Dose descriptor:
- NOAEL
- Effect level:
- 500 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: mortality at 2000 mg/kg bw/day; decreased bodyweight gain and occurence of clinical signs of toxicity (rough hair coats and decreased activity) at 1000 and 2000 mg/kg bw/day
- Dose descriptor:
- LOAEL
- Effect level:
- 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: observation of clinical signs in both sexes and decreased bodyweights in males
- Critical effects observed:
- not specified
- Conclusions:
- Under the test conditions, the NOAEL was considered to be 500 mg/kg bw/day. The LOAEL was considered to be 1000 mg/kg bw/day for both female and male mice, based on observation of clinical signs in both sexes and decreased bodyweights in males.
- Executive summary:
In a 13-week subchronic toxicity study performed similarly to OECD Guideline 408 and in compliance with GLP, d-limonene was administered through gavage to groups of 10 B6C3F1 mice/sex/dose mixed in corn oil at dose levels of 0, 125, 250, 500, 1000 or 2000 mg/kg bw/day for 13 weeks (5 days/week). Animals were observed twice daily for clinical signs of toxicity and bodyweights were recorded weekly. Necropsy performed on all animals and microscopic examination of specified tissues was performed for all control and high dose animals scheduled to be killed at the end of the treatment period.
One of 10 males and 2/10 females that received 2000 mg/kg bw/day and 1/10 females that received 500 mg/kg bw/day died before the end of the studies. Several animals in other groups died as a result of gavage error. Clinical signs of rough hair coats and decreased activity were observed at the two highest doses. Final mean bodyweights of mice that received 1000 or 2000 mg/kg bw/day were 10% lower than that of the vehicle controls for males and 2% lower for females. An alveolar cell adenoma was observed in the lung of 1/10 females that received 2000 mg/kg bw/day.
Under the test conditions, the NOAEL was considered to be 500 mg/kg bw/day. The LOAEL was considered to be 1000 mg/kg bw/day for both female and male mice, based on observation of clinical signs in both sexes and decreased bodyweights in males.
- Endpoint:
- chronic toxicity: oral
- Remarks:
- combined repeated dose and carcinogenicity
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- 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.
- Qualifier:
- equivalent or similar to guideline
- 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 or test system 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 mentionned)
- 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) 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 (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
- 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 or B6C3F1 mice.- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- From January 28 to April 30, 1980
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: GLP study performed similarly to OECD Guideline 408 but with deviations: dosing 5 days/week instead of 7 days/week; food consumption, haematological and clinical biochemical test not followed
- Reason / purpose for cross-reference:
- reference to other study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
- Deviations:
- yes
- Remarks:
- dosing 5 days/week instead of 7 days/week; food consumption, haematological and clinical biochemical test not followed
- Principles of method if other than guideline:
- Not applicable
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- other: F344/N
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Breeding Laboratories (Portage, USA)
- Age at study initiation: 7-8 weeks
- Weight at study initiation: Males: 136-153 g; females: 101-120 g
- Housing: Housed in groups of five in polycarbonate cages
- Diet (e.g. ad libitum): Purina Lab Blox (Chesapeake Feed Co., Beltsville, USA) or NIH 07 Rat and Mouse Ration (Zeigler Bros., Inc., Gardners, PA, USA), ad libitum
- Water (e.g. ad libitum): Automatic watering system (Edstrom Industries, Waterford, USA), ad libitum
- Acclimation period: 18 or 19 days
ENVIRONMENTAL CONDITIONS
- Temperature (°F): 60-82 °F
- Humidity (%): 35-80%
- Air changes (per hour): 12-15/hour
- Photoperiod (hours dark / hours light): 12 hours dark / 12 hours light - Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS: Appropriate amount of test substance was weighed and mixed with corn oil by shaking in a volumetric flask.
VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg bw - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- - Apparatus: Periodic analysis for d-limonene in dose preparations was determined by extraction with methanol followed by gas chromatographic analysis of the extract with a 6-foot 3% OV-17 glass column, a nitrogen carrier at a flow rate of 30 mL/min, and a flame ionization detector.
- Sampling frequency: Once
- Results: 101-109% of the target concentrations - Duration of treatment / exposure:
- 13 weeks
- Frequency of treatment:
- Once per day; 5 days/week
- Remarks:
- Doses / Concentrations:
0, 150, 300, 600, 1200 and 2400 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: Doses were selected based on the mortalities observed at 3300 and 6600 mg/kg bw/day during a 16 day subacute toxicity study.
- Rationale for animal assignment (if not random): Random - Positive control:
- No
- Observations and examinations performed and frequency:
- CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily
BODY WEIGHT: Yes
- Time schedule for examinations: Initially and once per week thereafter - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes; necropsy performed on all animals
HISTOPATHOLOGY: Yes; performed on all vehicle control and high dose animals and all female rats in the 1200 mg/kg bw/day group. Tissues examined include: adrenal glands, brain, colon, esophagus, eyes (if grossly abnormal), femur or sternebrae or vertebrae including marrow, gross lesions and tissue masses with regional lymph nodes, heart, kidneys, liver, lungs and mainstem bronchi, mammary gland, mandibular or mesenteric lymph nodes, pancreas, parathyroids, pituitary gland, prostate/testes or ovaries/uterus, salivary glands, small intestine, spinal cord (if neurologic signs present), spleen, stomach, thymus, thyroid gland, trachea, and urinary bladder. Kidneys examined for all male rats. - Other examinations:
- No
- Statistics:
- No data
- Clinical signs:
- effects observed, treatment-related
- Mortality:
- mortality observed, treatment-related
- Body weight and weight changes:
- effects observed, treatment-related
- Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not specified
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Histopathological findings: neoplastic:
- not examined
- Details on results:
- MORTALITY:
- 5/10 males and 9/10 females at 2400 mg/kg bw/day died during week 1.
CLINICAL SIGNS:
- Rough hair coats, lethargy and excessive lacrimation were observed for rats that received 1200 or 2400 mg/kg bw/day.
BODY WEIGHT AND WEIGHT GAIN
- Final mean body weights of male rats in 600, 1200 or 2400 mg/kg bw/day groups were 6, 12 or 23% lower than that of the vehicle controls.
- Final body weight of the female rat that received 2400 mg/kg bw/day and lived to end of the study was 11% lower than the mean of the vehicle controls.
HISTOPATHOLOGY
- Nephropathy was identified in all groups of male rats, and there was a dose-related increased severity of the lesion in dosed groups.
- Nephropathy was characterized by degeneration of epithelium in the convoluted tubules, granular casts within tubular lumens, primarily in the outer stripe of the outer medulla, and regeneration of the tubular epithelium.
- Hyaline droplets (protein reabsorption droplets) were observed in the epithelium of proximal convoluted tubules in all groups of male rats, including vehicle controls. - Dose descriptor:
- NOAEL
- Sex:
- male
- Basis for effect level:
- other: male-rat specific nephrotoxicity at all dose levels (considered as not relevant for humans)
- Remarks on result:
- not determinable
- Remarks:
- no NOAEL identified
- Dose descriptor:
- NOAEL
- Effect level:
- 600 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: decrease of bodyweight gains at 1200 and 2400 mg/kg bw/day;
- Dose descriptor:
- LOAEL
- Effect level:
- 1 200 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: based on observation of clinical signs and nephropathy
- Dose descriptor:
- NOAEL
- Effect level:
- 600 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- other: mortality at 2400 mg/kg bw/day; occurrence of clinical signs of toxicity (rough hair coats, lethargy and excessive lacrimation) at 1200 and 2400 mg/kg bw/day
- Dose descriptor:
- LOAEL
- Effect level:
- 1 200 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- other: based on observation of clinical signs
- Critical effects observed:
- not specified
- Conclusions:
- Under the test conditions, the NOAEL for male and female rats was considered to be 600 mg/kg bw/day. The LOAEL for female and male rats were considered to be 1200 and 150 mg/kg bw/day, based on observation of clinical signs and nephropathy, respectively. As nephrotoxicity and accumulation of hyaline droplets were observed in male rats at all dose levels, no NOAEL for male rats could be identified in this study.
- Executive summary:
In a 13-week subchronic toxicity study performed similarly to OECD Guideline 408 and in compliance with GLP, d-limonene was administered through gavage to groups of 10 F344/N rats/sex/dose mixed in corn oil at dose levels of 0, 150, 300, 600, 1200 and 2400 mg/kg bw/day for 13 weeks (5 days/week). Animals were observed twice daily for clinical signs of toxicity and bodyweights were recorded weekly. Necropsy performed on all animals and microscopic examination of specified tissues was performed for all control and high dose animals scheduled to be killed at the end of the treatment period.
Five of 10 males and 9/10 female rats that received 2400 mg/kg bw/day died during week 1. Final mean body weights of male rats in 600, 1200 or 2400 mg/kg bw/day groups were 6, 12 or 23% lower than that of the vehicle controls. Final body weight of the female rat that received 2400 mg/kg bw/day and lived to end of the study was 11% lower than the mean of the vehicle controls. Rough hair coats, lethargy and excessive lacrimation were observed at 1200 or 2400 mg/kg bw/day. No treatment-related histopathologic lesions were observed in female rats. Nephropathy was identified in all groups of male rats, and there was a dose-related increased severity of the lesion in dosed groups. Nephropathy was characterized by degeneration of epithelium in the convoluted tubules, granular casts within tubular lumens, primarily in the outer stripe of the outer medulla, and regeneration of the tubular epithelium. Hyaline droplets (protein reabsorption droplets) were observed in the epithelium of proximal convoluted tubules in all groups of male rats, including vehicle controls. This mechanism of nephrocarcinogenicity has been proven as being male-rat specific and not relevant for humans.
Under the test conditions, the NOAEL for female rats was considered to be 600 mg/kg bw/day. When considering the non relevance of the nephrotoxic effects for humans, the NOAEL for male rats would be 600 mg/kg bw/day, based on decrease of bodyweight gains at 1200 and 2400 mg/kg bw/day. The LOAEL for female and male rats were considered to be 1200 and 150 mg/kg bw/day, based on observation of clinical signs and nephropathy, respectively. As nephrotoxicity and accumulation of hyaline droplets were observed in male rats at all dose levels, no NOAEL for male rats had primarily been identified in this study.
- Endpoint:
- sub-chronic toxicity: oral
- Adequacy of study:
- disregarded due to major methodological deficiencies
- Study period:
- No data
- Reliability:
- 3 (not reliable)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- Read across from peppermint oil. It was not indicated if the published test was performed according to an OECD guideline or under GLP conditions. As the results of the study could not be reproduced, they were carefully evaluated (extensive evaluation available in endpoint summary). The histopathological findings in the brain were considered to be artefacts of preparation and fixation of the brain tissue. Consequently, the study was considered invalid.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
- Deviations:
- yes
- Remarks:
- Not all required examinations performed and not all required haematology and clinical chemistry parameters checked
- GLP compliance:
- not specified
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Moellegaards Breeding Center Ltd., Lille Skensved, Denmark
- Age at study initiation: Four weeks
- Mean weight at study initiation:
Male: 143 g
Female: 123 g
- Housing: 2/cage, in stainless steelwire cages
- Diet (e.g. ad libitum): Ad libitum, pelleted diet
- Water (e.g. ad libitum): Ad libitum, acidified water
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 1
- Humidity (%): 60 +/- 5
- Air changes (per hr): 6-8
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: gavage
- Vehicle:
- other: soybean oil
- Details on oral exposure:
- VEHICLE
- Amount of vehicle (if gavage): 5 ml/kg - Analytical verification of doses or concentrations:
- not specified
- Details on analytical verification of doses or concentrations:
- No data available
- Duration of treatment / exposure:
- 90 days
- Frequency of treatment:
- Daily
- Remarks:
- Doses / Concentrations:
0, 10, 40 or 100
Basis:
nominal in diet - No. of animals per sex per dose:
- 14
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: No data
- Positive control:
- Not relevant
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Cage side observations: Twice daily (2 weeks predosing and during dosing period)
BODY WEIGHT: Yes
- Time schedule for examinations: Weekly
FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, weekly
WATER CONSUMPTION: Yes
- Time schedule for examinations: Weekly
HAEMATOLOGY: Yes
- Time schedule for collection of blood: On day 30 and 86
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 10 males and 10 females in each group
- Parameters checked: Red blood cell count, packed cell volume, white blood cell count, haemoglobin, reticulocyte count, white blood cell differential count.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On day 30 and 86
- Animals fasted: No data
- How many animals: 10 males and 10 females in each group
- Parameters checked: fasting glucose, creatinine, urea, bilirubin and ASAT, ASAT and ALP. - Sacrifice and pathology:
- GROSS PATHOLOGY:
Macroscopic examination on day 90 (after sacrifice).
HISTOPATHOLOGY:
Performed on the following tissues and all organs with macroscopic changes: brain, pituitary gland, thyroid, thymus, trachea and lungs, heart, aorta, liver, spleen, adrenals, kidneys, pancreas, ovaries, testes, uterus, oesophagus, gastrointestinal tract (seven levels), urinary bladder, axillaris lymh node, peripheral nerve, and sternum.
OTHER:
Additonally, extended liver histopathology was performed (Perl Prussian Blue and PAS) and brain sections from 12 animals from the control and high dose group were immunohistochemically stained for glia fibrillay acidic protein (GFAP). - Other examinations:
- Not relevant
- Statistics:
- - Two-way analysis of variance (ANOVA) followed by Dunnett's test
- Logistic regression (CATMOD) followed by Fisher's exact test (two-tail) - Clinical signs:
- no effects observed
- Description (incidence and severity):
- no test substance related effects
- Mortality:
- no mortality observed
- Description (incidence):
- no test substance related effects
- 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 specified
- Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- no effects observed
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- 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
- Histopathological findings: neoplastic:
- no effects observed
- Details on results:
- CLINICAL SIGNS AND MORTALITY
One animal of the control group and four animals of the highest dose group died due to accidental intratracheal dosing and were excluded from the study
HISTOPATHOLOGY: NON-NEOPLASTIC
Cyst-like spaces scattered in the white matter of the cerebellum were noted. No cellular reaction was observed in the surroundings. There was no statistical significant difference between sexes, but a significantly higher score (mild-moderate extension) of cyst-like space was observed in the highest dose group.
Staining for GFAP demonstrated no involvement of the astrocytes in the cyst-like spaces. No difference in intensity of GFAP staining noted.
The appearance of small intracytoplasmic hyaline droplets in the proximal tubular epithelia cells of the kidney of male rats of the highest dose group was statistically significant higher when compared to the other groups. However, there was no evidence of epithelial degeneration and as this effect is often seen in male rats it is considered negligible. - Dose descriptor:
- NOAEL
- Effect level:
- 40 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Higher incidence of cyst-like spaces in the highest dose group (100 mg/kg bw/day)
- Critical effects observed:
- not specified
- Conclusions:
- Under the conditions of this study, the number of cyst-like spaces in the brain of the high dose rats were found to be significantly higher as compared to other groups. No other treatment related effects were noted. Therefore, a NOAEL of 40 mg/kg bw/day was established.
- Executive summary:
This study was performed to determine the toxic potential of peppermint oil. Rats (14 males, 14 females per group) were exposed for 90 days to 0, 10, 40 or 100 mg/kg bw/day of peppermint oil in soybean oil. Clinical signs, body weight and food and water consumption were recorded, hematology, clinical chemistry, gross necropsy and histopathology were performed and organ weights were determined.
One animal of the control group and four animals of the highest dose group died due to accidental intratracheal dosing and were excluded from the study. Alterations in the brain, namely, cyst-like spaces scattered in the white matter of the cerebellum. No cellular reaction in the surroundings. There was no statistical significant difference between sexes, but a significantly higher score (mild-moderate extension) of cyst-like space in the highest dose group. Staining for GFAP demonstrated no involvement of the astrocytes in the cyst-like spaces. No difference in intesnity of GFAP staining noted.
Under the conditions of this study, the number of cyst-like spaces in the brain of the high dose rats were found to be significantly higher as compared to other groups. No other treatment related effects were noted. Therefore, a NOAEL of 40 mg/kg bw/day was established.
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- The read across justification is presented in the document attached to this record.
- Reason / purpose for cross-reference:
- read-across source
- 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
- Conclusions:
- The repeated dose toxicity of cornmint oil was assessed using read across from the source substance peppermint oil. 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:
The repeated dose toxicity of cornmint oil was assessed using read across from the source substance peppermint oil. 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:
- sub-chronic toxicity: oral
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- The read across justification is presented in the document attached to this record.
- Reason / purpose for cross-reference:
- read-across source
- Dose descriptor:
- NOAEL
- Effect level:
- 250 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- other: Statistically significant increase in relative and absolute liver weights and relative kidney weights associated with a statistically significant decrease in bodyweight at 500 mg/kg bw/day
- Dose descriptor:
- NOAEL
- Effect level:
- 250 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: Mortality and statistically significant decreased bodyweight at 1000 mg/kg bw/day. Slight increases in relative liver weights at 250 and 500 mg/kg bw/day not related with any other toxic effect (bodyweight, hematology, histopathology)
- Critical effects observed:
- not specified
- Conclusions:
- The repeated dose toxicity of cornmint oil was assessed using read across from the source substance β‑myrcene.
A NOAEL was set at 250 mg/kg/day in females based on statistically significant increase in relative and absolute liver weights and relative kidney weights associated with a statistically significant decrease in bodyweight at 500 mg/kg bw/day. A NOAEL was set at 500 mg/kg bw/day in males based on mortality and statistically significant decreased bodyweight at 1000 mg/kg bw/day. Slight increases in relative liver weights were observed at 250 and 500 mg/kg bw/day but they were not associated with any other toxic effect (bodyweight, hematology, histopathology). - Executive summary:
The repeated dose toxicity of cornmint oil was assessed using read across from the source substance β‑myrcene.
A study was conducted to evaluate the cumulative toxic effects of repeated exposure to β‑myrcene according to the method equivalent or similar to OECD Guideline 408 in compliance with Good Laboratory Practices.
Groups of 20 B6C3F1 mice (10/sex/dose) were administered 0, 250, 500, 1000, 2000 or 4000 mg/kg bw/day of β-myrcene in corn oil by gavage, 5 days per week for 14 weeks. Parameters evaluated included survival, clinical observations, body weight, hematological and biochemical estimations in blood, reproductive parameters, necropsy and histopathological examination in all animals.
All 4000 mg/kg bw/day (male and female mice) died during week 1; 9/10 males and 8/10 females in 2000 mg/kg bw/day group died by week 4. The mean body weights of 1000 mg/kg bw/day males and 500 and 2000 mg/kg bw/day females were significantly lower than those of the vehicle controls. Clinical findings in animals that did not survive to the end of the study included thinness, lethargy, and abnormal breathing.Absolute and relative right kidney weights of 1000 mg/kg bw/day females, and the relative kidney weights of 250 and 500 mg/kg bw/day females were significantly greater than those of the vehicle controls. Relative liver weights of 500 and 1000 mg/kg bw/day males and the absolute and relative liver weights of 250 mg/kg bw/day males and 500 and 1000 mg/kg bw/day females were significantly greater than those of the vehicle controls.No histopathology changes were observed in mice administered 1000 mg/kg bw/day or less. Mice in 2000 and 4000 mg/kg bw/day groups were not evaluated due to early deaths.
A NOAEL was set at 250 mg/kg/day in females based on statistically significant increase in relative and absolute liver weights and relative kidney weights associated with a statistically significant decrease in bodyweight at 500 mg/kg bw/day. A NOAEL was set at 500 mg/kg bw/day in males based on mortality and statistically significant decreased bodyweight at 1000 mg/kg bw/day. Slight increases in relative liver weights were observed at 250 and 500 mg/kg bw/day but they were not associated with any other toxic effect (bodyweight, hematology, histopathology).
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- The read across justification is presented in the document attached to this record.
- Reason / purpose for cross-reference:
- read-across source
- Sex:
- male/female
- Dose descriptor:
- LOAEL
- Effect level:
- 250 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Increase in liver and kidney weights associated with minimal renal tubule necrosis in males and females
- Critical effects observed:
- not specified
- Conclusions:
- The repeated dose toxicity of cornmint oil was assessed using read across from the source substance β‑myrcene.
A significant increase in liver and kidney weights in males and females, associated with minimal renal tubule necrosis and only in males, hyaline droplet accumulation, were observed at 250 mg/kg bw/day. Therefore, no NOAEL was identified and a LOAEL was set t 250 mg/kg bw/day. - Executive summary:
The repeated dose toxicity of cornmint oil was assessed using read across from the source substance β‑myrcene.
A study was conducted to evaluate the cumulative toxic effects of repeated exposure to β‑myrcene according to the method equivalent or similar to OECD Guideline 408 in compliance with Good Laboratory Practices.
Groups of 20 F334/N rats (10/sex/dose) were administered 0, 250, 500, 1000, 2000 or 4000 mg/kg bw/day β-myrcene in corn oil by gavage, 5 days per week for 14 weeks. Additional groups of 10 male and 10 female special study rats were administered the same doses for 22 days. Parameters evaluated included survival, clinical observations, body weight, hematological and biochemical estimations in blood, reproductive parameters, necropsy and histopathological examination in all animals.
All rats exposed to 4000 mg/kg bw/day died and 2/10 males and 4/10 females died in the 2000 mg/kg bw/day group. Dose-related clinical findings in animals that died early included thinness, lethargy, abnormal breathing and ruffled fur. Mean body weights were significantly decreased in the500, 1000 and 2000mg/kg bw/day groups. A significant increase in liver and kidney weights was observed in all dosed animals as well as renal tubule necrosis. A significant decrease in creatinine was also detected in all dosed females and in males at 500 mg/kg bw/day and higher doses. The incidences and severities of olfactory epithelium degeneration were increased in males and females of the 2000 mg/kg bw/day group.The incidences of chronic inflammation in 1000 and 2000 mg/kg bw/day males and females were significantly increased. All 2000 mg/kg bw/day males and females had splenic atrophy. In the mesenteric lymph node, significantly increased incidences of atrophy occurred in 2000 mg/kg bw/day males and 1000 and 2000 mg/kg bw/day females. Acute inflammation of the forestomach occurred in four 2000 mg/kg bw/day females. The incidences of porphyrin pigmentation in the Harderian gland of males administered 500 mg/kg bw/day or greater were significantly increased.
As a significant increase in liver and kidney weights associated with minimal renal tubule necrosis in males and females, as well as hyaline droplet accumulation only in males, were observed at 250 mg/kg bw/day, no NOAEL could be identified in this study and a LOAEL was set at 250 mg/kg bw/day.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- The read across justification is presented in the document attached to this record.
- Reason / purpose for cross-reference:
- read-across source
- Sex:
- male/female
- Vehicle:
- corn oil
- Clinical signs:
- effects observed, treatment-related
- Mortality:
- mortality observed, treatment-related
- Body weight and weight changes:
- effects observed, treatment-related
- Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not specified
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Histopathological findings: neoplastic:
- not examined
- Details on results:
- MORTALITY:
- 1/10 male and 2/10 females died at 2000 mg/kg bw/day
- 1/10 female died at 500 mg/kg bw/day
- Several animals in other groups died as a result of gavage error.
CLINICAL SIGNS:
- Rough hair coats and decreased activity were observed at 1000 and 2000 mg/kg bw/day.
BODY WEIGHT AND WEIGHT GAIN
- Final mean bodyweights of mice that received 1000 or 2000 mg/kg bw/day were 10% lower than that of the vehicle controls for males and 2% lower for females.
HISTOPATHOLOGY
- An alveolar cell adenoma was observed in the lung of 1/10 females that received 2000 mg/kg bw/day. - Dose descriptor:
- NOAEL
- Effect level:
- 500 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: mortality at 2000 mg/kg bw/day; decreased bodyweight gain and occurence of clinical signs of toxicity (rough hair coats and decreased activity) at 1000 and 2000 mg/kg bw/day
- Dose descriptor:
- LOAEL
- Effect level:
- 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: observation of clinical signs in both sexes and decreased bodyweights in males
- Critical effects observed:
- not specified
- Conclusions:
- The repeated dose toxicity of cornmint oil was assessed using read across from the source substance d-limonene. Under the test conditions, the NOAEL was considered to be 500 mg/kg bw/day. The LOAEL was considered to be 1000 mg/kg bw/day for both female and male mice, based on observation of clinical signs in both sexes and decreased bodyweights in males.
- Executive summary:
The repeated dose toxicity of cornmint oil was assessed using read across from the source substance d-limonene.
In a 13-week subchronic toxicity study performed similarly to OECD Guideline 408 and in compliance with GLP, d-limonene was administered through gavage to groups of 10 B6C3F1 mice/sex/dose mixed in corn oil at dose levels of 0, 125, 250, 500, 1000 or 2000 mg/kg bw/day for 13 weeks (5 days/week). Animals were observed twice daily for clinical signs of toxicity and bodyweights were recorded weekly. Necropsy performed on all animals and microscopic examination of specified tissues was performed for all control and high dose animals scheduled to be killed at the end of the treatment period.
One of 10 males and 2/10 females that received 2000 mg/kg bw/day and 1/10 females that received 500 mg/kg bw/day died before the end of the studies. Several animals in other groups died as a result of gavage error. Clinical signs of rough hair coats and decreased activity were observed at the two highest doses. Final mean bodyweights of mice that received 1000 or 2000 mg/kg bw/day were 10% lower than that of the vehicle controls for males and 2% lower for females. An alveolar cell adenoma was observed in the lung of 1/10 females that received 2000 mg/kg bw/day.
Under the test conditions, the NOAEL was considered to be 500 mg/kg bw/day. The LOAEL was considered to be 1000 mg/kg bw/day for both female and male mice, based on observation of clinical signs in both sexes and decreased bodyweights in males.
- Endpoint:
- chronic toxicity: oral
- Remarks:
- combined repeated dose and carcinogenicity
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- The read across justification is presented in the document attached to this record.
- Reason / purpose for cross-reference:
- read-across source
- 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
- Critical effects observed:
- not specified
- Conclusions:
- The repeated dose toxicity of cornmint oil was assessed using read across from the source substance DL-menthol. Based on the histopathologic examination, DL-menthol was neither toxic nor carcinogenic to Fischer 344 rats under the conditions of this bioassay.
- Executive summary:
The repeated dose toxicity of cornmint oil was assessed using read across from the source substance DL-menthol. 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 or B6C3F1 mice.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- The read across justification is presented in the document attached to this record.
- Reason / purpose for cross-reference:
- read-across source
- Clinical signs:
- effects observed, treatment-related
- Mortality:
- mortality observed, treatment-related
- Body weight and weight changes:
- effects observed, treatment-related
- Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not specified
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Histopathological findings: neoplastic:
- not examined
- Details on results:
- MORTALITY:
- 5/10 males and 9/10 females at 2400 mg/kg bw/day died during week 1.
CLINICAL SIGNS:
- Rough hair coats, lethargy and excessive lacrimation were observed for rats that received 1200 or 2400 mg/kg bw/day.
BODY WEIGHT AND WEIGHT GAIN
- Final mean body weights of male rats in 600, 1200 or 2400 mg/kg bw/day groups were 6, 12 or 23% lower than that of the vehicle controls.
- Final body weight of the female rat that received 2400 mg/kg bw/day and lived to end of the study was 11% lower than the mean of the vehicle controls.
HISTOPATHOLOGY
- Nephropathy was identified in all groups of male rats, and there was a dose-related increased severity of the lesion in dosed groups.
- Nephropathy was characterized by degeneration of epithelium in the convoluted tubules, granular casts within tubular lumens, primarily in the outer stripe of the outer medulla, and regeneration of the tubular epithelium.
- Hyaline droplets (protein reabsorption droplets) were observed in the epithelium of proximal convoluted tubules in all groups of male rats, including vehicle controls. - Dose descriptor:
- NOAEL
- Sex:
- male
- Basis for effect level:
- other: male-rat specific nephrotoxicity at all dose levels (considered as not relevant for humans)
- Remarks on result:
- not determinable
- Remarks:
- no NOAEL identified
- Dose descriptor:
- NOAEL
- Effect level:
- 600 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: decrease of bodyweight gains at 1200 and 2400 mg/kg bw/day;
- Dose descriptor:
- LOAEL
- Effect level:
- 1 200 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: based on observation of clinical signs and nephropathy
- Dose descriptor:
- NOAEL
- Effect level:
- 600 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- other: mortality at 2400 mg/kg bw/day; occurrence of clinical signs of toxicity (rough hair coats, lethargy and excessive lacrimation) at 1200 and 2400 mg/kg bw/day
- Dose descriptor:
- LOAEL
- Effect level:
- 1 200 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- other: based on observation of clinical signs
- Critical effects observed:
- not specified
- Conclusions:
- The repeated dose toxicity of cornmint oil was assessed using read across from the source substance d-limonene. Under the test conditions, the NOAEL for male and female rats was considered to be 600 mg/kg bw/day. The LOAEL for female and male rats were considered to be 1200 and 150 mg/kg bw/day, based on observation of clinical signs and nephropathy, respectively. As nephrotoxicity and accumulation of hyaline droplets were observed in male rats at all dose levels, no NOAEL for male rats could be identified in this study.
- Executive summary:
The repeated dose toxicity of cornmint oil was assessed using read across from the source substance d-limonene. In a 13-week subchronic toxicity study performed similarly to OECD Guideline 408 and in compliance with GLP, d-limonene was administered through gavage to groups of 10 F344/N rats/sex/dose mixed in corn oil at dose levels of 0, 150, 300, 600, 1200 and 2400 mg/kg bw/day for 13 weeks (5 days/week). Animals were observed twice daily for clinical signs of toxicity and bodyweights were recorded weekly. Necropsy performed on all animals and microscopic examination of specified tissues was performed for all control and high dose animals scheduled to be killed at the end of the treatment period.
Five of 10 males and 9/10 female rats that received 2400 mg/kg bw/day died during week 1. Final mean body weights of male rats in 600, 1200 or 2400 mg/kg bw/day groups were 6, 12 or 23% lower than that of the vehicle controls. Final body weight of the female rat that received 2400 mg/kg bw/day and lived to end of the study was 11% lower than the mean of the vehicle controls. Rough hair coats, lethargy and excessive lacrimation were observed at 1200 or 2400 mg/kg bw/day. No treatment-related histopathologic lesions were observed in female rats. Nephropathy was identified in all groups of male rats, and there was a dose-related increased severity of the lesion in dosed groups. Nephropathy was characterized by degeneration of epithelium in the convoluted tubules, granular casts within tubular lumens, primarily in the outer stripe of the outer medulla, and regeneration of the tubular epithelium. Hyaline droplets (protein reabsorption droplets) were observed in the epithelium of proximal convoluted tubules in all groups of male rats, including vehicle controls. This mechanism of nephrocarcinogenicity has been proven as being male-rat specific and not relevant for humans.
Under the test conditions, the NOAEL for female rats was considered to be 600 mg/kg bw/day. When considering the non relevance of the nephrotoxic effects for humans, the NOAEL for male rats would be 600 mg/kg bw/day, based on decrease of bodyweight gains at 1200 and 2400 mg/kg bw/day. The LOAEL for female and male rats were considered to be 1200 and 150 mg/kg bw/day, based on observation of clinical signs and nephropathy, respectively. As nephrotoxicity and accumulation of hyaline droplets were observed in male rats at all dose levels, no NOAEL for male rats had primarily been identified in this study.
- Endpoint:
- short-term repeated dose toxicity: oral
- Adequacy of study:
- disregarded due to major methodological deficiencies
- Study period:
- No data
- Reliability:
- 3 (not reliable)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- Read across from peppermint oil. It was not indicated if the published test was performed according to an OECD guideline or under GLP conditions. As the results of the study could not be reproduced, they were carefully evaluated (extensive evaluation available in endpoint summary). The histopathological findings in the brain were considered to be artefacts of preparation and fixation of the brain tissue. Consequently, the study was considered invalid.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
- Deviations:
- yes
- Remarks:
- Not all required examinations performed and not all required haematology and clinical chemistry parameters checked
- GLP compliance:
- not specified
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Mollegaards Breeding Centre Ltd., Ejby, DK-4623 L. Skensved
- Age at study initiation: 4 weeks
- Weight at study initiation: No data
- Housing: 2/cage, in stainless steelwire cages
- Diet (e.g. ad libitum): Ad libitum, pelleted diet
- Water (e.g. ad libitum): Ad libitum, acidified water
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +/- 1
- Humidity (%): 60 +/- 5
- Air changes (per hr): 6-8
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: gavage
- Vehicle:
- other: Soybean oil
- Details on oral exposure:
- VEHICLE
- Amount of vehicle (if gavage): 5 ml/kg - Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- 28 days
- Frequency of treatment:
- Once daily
- Remarks:
- Doses / Concentrations:
10, 40 and 100 mg/kg bw/day
Basis:
actual ingested - No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- No data available
- Positive control:
- Not relevant
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily (2 weeks predosing and during dosing period)
BODY WEIGHT: Yes
- Time schedule for examinations: Weekly
FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule: Weekly
WATER CONSUMPTION: Yes
- Time schedule: Weekly
HAEMATOLOGY: Yes
- Time schedule for collection of blood: On day 21 or 22
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 8 males and 8 females of each group
- Parameters checked: Hemoglobin, PCV, total erythrocyte count (RBC), total WBC, white blood cell differential count
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On day 21 or 22
- Animals fasted: No data
- How many animals: 8 males and 8 females of each group
- Parameters checked: Glucose (in blood) and creatinine, urea and activities of ASAT (in plasma) - Sacrifice and pathology:
- GROSS PATHOLOGY:
A thorough autopsy was performed after exsanguination after CO2 anesthesia.
HISTOPATHOLOGY:
Following organs were excised and weighed: kidneys, adrenals, heart, brain, liver and stomach with content.
Samples from these organs and from lungs, aorta, spleen, mesenterial lymph node, thymus, jejunum, colon, thyroid, parathyroid, pancreas, testis, ovary, uterus, spinal cord, ischiadic nerve, sternum, skeletal muscle and skin were studied by light microscopy (stainings: HE (all tissues), Oil Red O (liver), Perl (liver and spleen) and PAS (liver)). For special examination of the brain, frozen sections were stained with Luxol fast blue. - Other examinations:
- Not relevant
- Statistics:
- Student's t-test
- Clinical signs:
- no effects observed
- Mortality:
- no mortality observed
- 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):
- no effects observed
- Description (incidence and severity):
- not significant
- Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- no effects observed
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- 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
- Histopathological findings: neoplastic:
- no effects observed
- Details on results:
- CLINICAL SIGNS AND MORTALITY
No treatment related mortality or clinical signs were reported in the study.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
A non-significant increase in water consumption (about 10%) in all treatment groups as compared to the control group was observed.
HISTOPATHOLOGY: NON-NEOPLASTIC
Dose related alterations in the brain were observed. In the mid and high dose group cyst-like spaces appread scattered in the withe matter (especially cerebellum). No sign of any cellular reaction was seen in the adjacent tissue. The myelin sheets showed deviation around the cyst-like spaces (Luxol fast blue staining).
Some male and female rats in the mid and high dose group showed slight dissociation and vacuolisation of hepatocytes, but did not seen dose related as the changes were less marked in the high dose group. The vacuoles contained no fat. No difference in fat, glycogen or hemosiderin content in the liver was observed between treatment groups and the control group. - Dose descriptor:
- NOAEL
- Effect level:
- 10 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Cyst-like spaces in the white matter of the cerebellum in the mid and high dose group (dose related)
- Critical effects observed:
- not specified
- Conclusions:
- Under the conditions of this study, histopathology revealed cyst-like spaces in the cerebellum of the mid and high dose rats. No other significant treatment related effects were noted. Therefore, a NOAEL of 10 mg/kg bw/day was established.
- Executive summary:
This study was performed to determine the toxicity potential of peppermint oil. Rats (10 males, 10 females per group) were exposed for 28 days to 0, 10, 40 or 100 mg/kg bw/day of peppermint oil in soybean oil. Clinical signs, body weight and food and water consumption were recorded, hematology, clinical chemistry, gross necropsy and histopathology were performed and organ weights were determined.
No treatment related mortality or clinical signs were reported in the study. A non-significant increase in water consumption (about 10%) in all treatment groups as compared to the control group was observed. Dose related alterations in the brain were observed. In the mid and high dose group cyst-like spaces appread scattered in the withe matter (especially cerebellum). No sign of any cellular reaction was seen in the adjacent tissue. The myelin sheets showed deviation around the cyst-like spaces (Luxol fast blue staining). Some male and female rats in the mid and high dose group showed slight dissociation and vacuolisation of hepatocytes. The vacuoles contained no fat. No difference in fat, glycogen or hemosiderin content in the liver was observed between treatment groups and the control group.
Under the conditions of this study, histopathology revealed cyst-like spaces in the cerebellum of the mid and high dose rats. No other significant treatment related effects were noted. Therefore, a NOAEL of 10 mg/kg bw/day was established.
Referenceopen allclose all
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).
- No discernible effects on general condition, behaviour or body weight development
- Slightly raised alkaline phosphatase and urea levels in high dose males in week 5
- No histopathological abnormalities were recorded
The authors considered the slight increase in alkaline phosphatase and urea levels in high dose males not to be toxicologically relevant, bacause all values were within the normal range.
- No effects on general condition, behaviour or body weight development
- Higher water consumption for the high dose group
- Decreased plasma glyceride levels in high dose group
- Increased alkaline phosphatase levels in all treatment groups
- Relative mean weight of liver and kidneys were higher in the high dose group as compared to the control group
- No histopathological abnormalities were recorded
The authors considered all noted effects not to be toxicologically relevant or caused by lower food consumption (lower triglyceride levels).
Table 1. Survival and mean body weights of mice in the 13-week gavage studies of d-limonene
Dose (mg/kg bw/day) |
Survival (a) |
Mean body weights (grams) |
Final weight relative to vehicle controls (%) |
|||
Initial (b) |
Final |
Change (c) |
||||
Male |
0 |
10/10 |
26.6 ± 1.0 |
37.1 ± 1.0 |
+10.5 ± 1.3 |
- |
125 |
10/10 |
28.8 ± 0.7 |
37.9 ± 1.1 |
+9.1 ± 0.7 |
102.2 |
|
250 |
(d) 9/10 |
26.5 ± 0.8 |
33.9 ± 0.8 |
+7.6 ± 0.8 |
91.4 |
|
500 |
(d) 7/10 |
24.7 ± 0.9 |
34.4 ± 0.9 |
+9.7± 1.1 |
92.7 |
|
1000 |
(d) 9/10 |
28.2 ± 0.9 |
33.3 ± 0.8 |
+5.1 ± 1.1 |
89.8 |
|
2000 |
(e) 9/10 |
27.7±0.7 |
33.0 ± 0.8 |
+5.6 ± 0.8 |
88.9 |
|
Female |
0 |
10/10 |
21.3 ± 0.2 |
24.7±0.5 |
+3.4 ±0.4 |
- |
125 |
(d) 9/10 |
20.6 ± 0.3 |
25.9± 0.5 |
+5.2 ± 0.4 |
104.9 |
|
250 |
10/10 |
20.7 ± 0.3 |
25.4 ± 0.6 |
+4.7 ± 0.4 |
102.8 |
|
500 |
(f) 9/10 |
20.9 ± 0.2 |
24.9 ±0.5 |
+4.1 ± 0.4 |
100.8 |
|
1000 |
10/10 |
20.4 ±0.2 |
24.1 ± 0.7 |
+3.7 ±0.7 |
97.6 |
|
2000 |
(g) 8/10 |
21.0 ± 0.3 |
24.1 ± 0.4 |
+3.4 ± 0.3 |
97.6 |
|
(a) Number surviving/number initially in group
(b) Initial group mean body weight f standard error of the mean. Subsequent calculations are based on animals surviving to the end of the study.
(c) Mean body weight change of the survivors ± standard error of the mean
(d) Death due to gavage error
(e) Week of death: 1
(f) Week of death: 5
(g) Week of death: 3,4
Table 1. Survival and mean body weights of rats in the 13-week gavage studies of d-limonene
Dose (mg/kg bw/day) |
Survival (a) |
Mean body weights (g) |
Final weight relative to vehicle controls (%) |
|||
Initial (b) |
Final |
Change (c) |
||||
Male |
0 |
10/10 |
144 ± 2 |
333 ± 6 |
+189 ± 5 |
- |
150 |
10/10 |
145 ± 3 |
332 ± 4 |
+187 ± 3 |
100 |
|
300 |
10/10 |
149 ±2 |
330 ± 3 |
+181 ± 4 |
99 |
|
600 |
10/10 |
148 ± 2 |
314± 5 |
+166 ± 5 |
94 |
|
1200 |
10/10 |
139 ± 3 |
292 ± 5 |
+153 ± 6 |
88 |
|
2400 |
(d) 5/10 |
150 ±3 |
255 ± 10 |
+103 ± 10 |
77 |
|
Female |
0 |
10/10 |
118 ± 2 |
185 ± 2 |
+67± 4 |
- |
150 |
10/10 |
115 ± 1 |
186± 2 |
+71± 2 |
101 |
|
300 |
10/10 |
105 ± 4 |
181 ± 2 |
+76± 4 |
98 |
|
600 |
10/10 |
114 ± 1 |
184± 2 |
+70± 1 |
99 |
|
1200 |
10/10 |
116 ± 2 |
182 ± 3 |
+66± 3 |
98 |
|
2400 |
(d) 1/10 |
113 ± 1 |
164 |
+ 56 |
89 |
|
(a) Number surviving/number initially in group
(b) Initial group mean body weight ± standard error of the mean. Subsequent calculations are based on animals surviving to the end of the study.
(c) Mean body weight change of the survivors ± standard error of the mean
(d) Week of death: all 1
Table 2. Severity of kidney lesions in male rats in the 13-week gavage study of d-limonene (a)
Lesion |
Dose (mg/kg) |
|||||
Vehicle Control |
150 |
300 |
600 |
1200 |
2400 |
|
Regeneration |
(b) 0.8 |
2.4 |
2.5 |
2.5 |
3.7 |
0.9 |
Granular casts |
0 |
1.6 |
2.4 |
2.7 |
3.5 |
0.3 |
(a) Severity grades: 1 = minimal; 2 = mild; 3 = moderate; 4 = marked
(b) Average severity grade for all rats in the group
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).
Table 1. Survival and mean body weights of mice in the 13-week gavage studies of d-limonene
Dose (mg/kg bw/day) |
Survival (a) |
Mean body weights (grams) |
Final weight relative to vehicle controls (%) |
|||
Initial (b) |
Final |
Change (c) |
||||
Male |
0 |
10/10 |
26.6 ± 1.0 |
37.1 ± 1.0 |
+10.5 ± 1.3 |
- |
125 |
10/10 |
28.8 ± 0.7 |
37.9 ± 1.1 |
+9.1 ± 0.7 |
102.2 |
|
250 |
(d) 9/10 |
26.5 ± 0.8 |
33.9 ± 0.8 |
+7.6 ± 0.8 |
91.4 |
|
500 |
(d) 7/10 |
24.7 ± 0.9 |
34.4 ± 0.9 |
+9.7± 1.1 |
92.7 |
|
1000 |
(d) 9/10 |
28.2 ± 0.9 |
33.3 ± 0.8 |
+5.1 ± 1.1 |
89.8 |
|
2000 |
(e) 9/10 |
27.7±0.7 |
33.0 ± 0.8 |
+5.6 ± 0.8 |
88.9 |
|
Female |
0 |
10/10 |
21.3 ± 0.2 |
24.7±0.5 |
+3.4 ±0.4 |
- |
125 |
(d) 9/10 |
20.6 ± 0.3 |
25.9± 0.5 |
+5.2 ± 0.4 |
104.9 |
|
250 |
10/10 |
20.7 ± 0.3 |
25.4 ± 0.6 |
+4.7 ± 0.4 |
102.8 |
|
500 |
(f) 9/10 |
20.9 ± 0.2 |
24.9 ±0.5 |
+4.1 ± 0.4 |
100.8 |
|
1000 |
10/10 |
20.4 ±0.2 |
24.1 ± 0.7 |
+3.7 ±0.7 |
97.6 |
|
2000 |
(g) 8/10 |
21.0 ± 0.3 |
24.1 ± 0.4 |
+3.4 ± 0.3 |
97.6 |
|
(a) Number surviving/number initially in group
(b) Initial group mean body weight f standard error of the mean. Subsequent calculations are based on animals surviving to the end of the study.
(c) Mean body weight change of the survivors ± standard error of the mean
(d) Death due to gavage error
(e) Week of death: 1
(f) Week of death: 5
(g) Week of death: 3,4
Table 1. Survival and mean body weights of rats in the 13-week gavage studies of d-limonene
Dose (mg/kg bw/day) |
Survival (a) |
Mean body weights (g) |
Final weight relative to vehicle controls (%) |
|||
Initial (b) |
Final |
Change (c) |
||||
Male |
0 |
10/10 |
144 ± 2 |
333 ± 6 |
+189 ± 5 |
- |
150 |
10/10 |
145 ± 3 |
332 ± 4 |
+187 ± 3 |
100 |
|
300 |
10/10 |
149 ±2 |
330 ± 3 |
+181 ± 4 |
99 |
|
600 |
10/10 |
148 ± 2 |
314± 5 |
+166 ± 5 |
94 |
|
1200 |
10/10 |
139 ± 3 |
292 ± 5 |
+153 ± 6 |
88 |
|
2400 |
(d) 5/10 |
150 ±3 |
255 ± 10 |
+103 ± 10 |
77 |
|
Female |
0 |
10/10 |
118 ± 2 |
185 ± 2 |
+67± 4 |
- |
150 |
10/10 |
115 ± 1 |
186± 2 |
+71± 2 |
101 |
|
300 |
10/10 |
105 ± 4 |
181 ± 2 |
+76± 4 |
98 |
|
600 |
10/10 |
114 ± 1 |
184± 2 |
+70± 1 |
99 |
|
1200 |
10/10 |
116 ± 2 |
182 ± 3 |
+66± 3 |
98 |
|
2400 |
(d) 1/10 |
113 ± 1 |
164 |
+ 56 |
89 |
|
(a) Number surviving/number initially in group
(b) Initial group mean body weight ± standard error of the mean. Subsequent calculations are based on animals surviving to the end of the study.
(c) Mean body weight change of the survivors ± standard error of the mean
(d) Week of death: all 1
Table 2. Severity of kidney lesions in male rats in the 13-week gavage study of d-limonene (a)
Lesion |
Dose (mg/kg) |
|||||
Vehicle Control |
150 |
300 |
600 |
1200 |
2400 |
|
Regeneration |
(b) 0.8 |
2.4 |
2.5 |
2.5 |
3.7 |
0.9 |
Granular casts |
0 |
1.6 |
2.4 |
2.7 |
3.5 |
0.3 |
(a) Severity grades: 1 = minimal; 2 = mild; 3 = moderate; 4 = marked
(b) Average severity grade for all rats in the group
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
Link to relevant study records
- Endpoint:
- sub-chronic toxicity: inhalation
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 14 weeks in 2005
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Recent study conducted by NTP similarly to OECD guideline 413 with deviations: food consumption, hematology, ophthalmological examination, some organ weights were not recorded.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
- Deviations:
- yes
- Remarks:
- (food consumption, hematology, ophthalmological examination, some organ weights were not recorded)
- GLP compliance:
- not specified
- Limit test:
- no
- Species:
- mouse
- Strain:
- B6C3F1
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- After a 10- to 14-day quarantine period, animals are assigned at random to treatment groups.
- Route of administration:
- inhalation
- Type of inhalation exposure:
- not specified
- Vehicle:
- not specified
- Remarks on MMAD:
- MMAD / GSD: No data
- Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- 14 weeks; 6 hours per day
- Frequency of treatment:
- Five times per week, weekdays only
- Remarks:
- Doses / Concentrations:
25
Basis:
nominal conc. - Remarks:
- Doses / Concentrations:
50 ppm
Basis:
nominal conc. - Remarks:
- Doses / Concentrations:
100 ppm
Basis:
nominal conc. - Remarks:
- Doses / Concentrations:
200 ppm
Basis:
nominal conc. - Remarks:
- Doses / Concentrations:
400 ppm
Basis:
nominal conc. - No. of animals per sex per dose:
- 10
- Control animals:
- yes
- Dose descriptor:
- NOAEL
- Effect level:
- 50 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Presence of transitional cell hyperplasia of the urinary bladder.
- Critical effects observed:
- not specified
- Conclusions:
- The NOAEL in male and female rats is 50 ppm based on minimal to moderate hyperplasia observed in the transitional epithelium of the urinary bladder in animals treated at 100 to 400 ppm.
- Executive summary:
In a 90-day inhalation study conducted by NTP similarly to OECD guideline 413, groups of 10 animals per dose and per sex were administered for 6 hours per day, 5 weekdays per week at 0, 25, 50, 100, 200 and 400 ppm for a total of 14 weeks. The animals were observed twice per day and weighed once per week. A complete histopathologic evaluation including treatment-related gross lesions was performed on all animals including early death animals. Treatment-related lesions (target organs) were identified and these organs and gross lesions were examined to a no-effect level.
Similar effects were observed in male and female mice from the same dose level (100 ppm): minimal to moderate hyperplasia observed in the transitional epithelium of the urinary bladder.
The NOAEL in male and female rats is 50 ppm based on minimal to moderate hyperplasia observed in the transitional epithelium of the urinary bladder in animals treated at 100 to 400 ppm.
- Endpoint:
- sub-chronic toxicity: inhalation
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Recent study conducted by NTP similarly to OECD guideline 413 with deviations: food consumption, hematology, ophthalmological examination, some organ weights were not recorded.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
- Deviations:
- yes
- Remarks:
- (food consumption, hematology, ophthalmological examination, some organ weights were not recorded)
- GLP compliance:
- not specified
- Limit test:
- no
- Species:
- rat
- Strain:
- Fischer 344
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- After a 10- to 14-day quarantine period, animals are assigned at random to treatment groups.
- Route of administration:
- inhalation
- Type of inhalation exposure:
- not specified
- Vehicle:
- not specified
- Remarks on MMAD:
- MMAD / GSD: No data
- Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- 14 weeks; 6 hours per day
- Frequency of treatment:
- five times per week, weekdays only
- Remarks:
- Doses / Concentrations:
25 ppm
Basis:
nominal conc. - Remarks:
- Doses / Concentrations:
50 ppm
Basis:
nominal conc. - Remarks:
- Doses / Concentrations:
100 ppm
Basis:
nominal conc. - Remarks:
- Doses / Concentrations:
200 ppm
Basis:
nominal conc. - Remarks:
- Doses / Concentrations:
400 ppm
Basis:
nominal conc. - No. of animals per sex per dose:
- 10
- Control animals:
- yes
- Dose descriptor:
- LOAEL
- Effect level:
- 25 ppm
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: Nephropathy which is relevant only in male rats. Lower body weight gain in the high dose group. In humans, this LOAEL will not be relevant.
- Dose descriptor:
- NOAEL
- Effect level:
- 200 ppm
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- other: Based on mortality and lower body weight gain in the high dose group.
- Critical effects observed:
- not specified
- Conclusions:
- The LOAEL in male rats is the lowest dose level tested, but it is not relevant to humans as it is based on renal effects linked to alpha2μ-globulin accumulation. When considering effects other than those on
kidneys in males, a lower body weight gain was observed at 400 ppm when compared to controls.
A NOAEL could be defined in female rats at 200 ppm on the basis of mortality and a lower body weight gain at the next dose level when compared to controls.
The overall NOAEL relevant for humans is 200 ppm, equivalent to ca. 170 mg/kg bw/day. - Executive summary:
In a 90-day inhalation study conducted by NTP similarly to OECD guideline 413, groups of 10 animals per dose and per sex were administered for 6 hours per day, 5 weekdays per week at 0, 25, 50, 100,
200 and 400 ppm for a total of 14 weeks. The animals were observed twice per day and weighed once per week. A complete histopathologic evaluation including treatment-related gross lesions was
performed on all animals, including early death animals. Treatment-related lesions (target organs) were identified and these organs and gross lesions were examined to a no-effect level.
Apart from lesions including granular casts and hyaline droplets indicative of alpha2μ-globulin nephropathy observed in all treated group males, a lower body weight gain than in controls was observed in the
high dose group in both sexes. In addition, 6 females died and 3 females displayed mild tremors in the high dose group too.
The LOAEL in male rats is the lowest dose level tested, but it is not relevant to humans as it is based on renal effects linked to alpha2μ-globulin accumulation. When considering effects other than those on
kidneys in males, a lower body weight gain was observed at 400 ppm when compared to controls. A NOAEL could be defined in female rats at 200 ppm on the basis of mortality and a lower body weight gain
at the next dose level when compared to controls. The overall NOAEL relevant for humans is 200 ppm.
- Endpoint:
- sub-chronic toxicity: inhalation
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
The read across justification is presented in the document attached to this record.- Reason / purpose for cross-reference:
- read-across source
- Dose descriptor:
- LOAEL
- Effect level:
- 25 ppm
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: Nephropathy which is relevant only in male rats. Lower body weight gain in the high dose group. In humans, this LOAEL will not be relevant.
- Dose descriptor:
- NOAEL
- Effect level:
- 200 ppm
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- other: Based on mortality and lower body weight gain in the high dose group.
- Critical effects observed:
- not specified
- Conclusions:
- The repeated dose toxicity of cornmint oil was assessed using read across from the source substance alpha-pinene. The LOAEL in male rats is the lowest dose level tested, but it is not relevant to humans as it is based on renal effects linked to alpha2μ-globulin accumulation. When considering effects other than those on
kidneys in males, a lower body weight gain was observed at 400 ppm when compared to controls.
A NOAEL could be defined in female rats at 200 ppm on the basis of mortality and a lower body weight gain at the next dose level when compared to controls.
The overall NOAEL relevant for humans is 200 ppm, equivalent to ca. 170 mg/kg bw/day. - Executive summary:
The repeated dose toxicity of cornmint oil was assessed using read across from the source substance alpha-pinene. In a 90-day inhalation study conducted by NTP similarly to OECD guideline 413, groups of 10 animals per dose and per sex were administered for 6 hours per day, 5 weekdays per week at 0, 25, 50, 100,
200 and 400 ppm for a total of 14 weeks. The animals were observed twice per day and weighed once per week. A complete histopathologic evaluation including treatment-related gross lesions was
performed on all animals, including early death animals. Treatment-related lesions (target organs) were identified and these organs and gross lesions were examined to a no-effect level.
Apart from lesions including granular casts and hyaline droplets indicative of alpha2μ-globulin nephropathy observed in all treated group males, a lower body weight gain than in controls was observed in the
high dose group in both sexes. In addition, 6 females died and 3 females displayed mild tremors in the high dose group too.
The LOAEL in male rats is the lowest dose level tested, but it is not relevant to humans as it is based on renal effects linked to alpha2μ-globulin accumulation. When considering effects other than those on
kidneys in males, a lower body weight gain was observed at 400 ppm when compared to controls. A NOAEL could be defined in female rats at 200 ppm on the basis of mortality and a lower body weight gain
at the next dose level when compared to controls. The overall NOAEL relevant for humans is 200 ppm.
- Endpoint:
- sub-chronic toxicity: inhalation
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- The read across justification is presented in the document attached to this record.
- Reason / purpose for cross-reference:
- read-across source
- No. of animals per sex per dose:
- 10
- Dose descriptor:
- NOAEL
- Effect level:
- 50 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Presence of transitional cell hyperplasia of the urinary bladder.
- Critical effects observed:
- not specified
- Conclusions:
- The repeated dose toxicity of cornmint oil was assessed using read across from the source substance alpha-pinene.
The NOAEL in male and female rats is 50 ppm based on minimal to moderate hyperplasia observed in the transitional epithelium of the urinary bladder in animals treated at 100 to 400 ppm. - Executive summary:
The repeated dose toxicity of cornmint oil was assessed using read across from the source substance alpha-pinene. In a 90-day inhalation study conducted by NTP similarly to OECD guideline 413, groups of 10 animals per dose and per sex were administered for 6 hours per day, 5 weekdays per week at 0, 25, 50, 100, 200 and 400 ppm for a total of 14 weeks. The animals were observed twice per day and weighed once per week. A complete histopathologic evaluation including treatment-related gross lesions was performed on all animals including early death animals. Treatment-related lesions (target organs) were identified and these organs and gross lesions were examined to a no-effect level.
Similar effects were observed in male and female mice from the same dose level (100 ppm): minimal to moderate hyperplasia observed in the transitional epithelium of the urinary bladder.
The NOAEL in male and female rats is 50 ppm based on minimal to moderate hyperplasia observed in the transitional epithelium of the urinary bladder in animals treated at 100 to 400 ppm.
Referenceopen allclose all
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 WoE information from constituents
Overview of available Subchronic and chronic constituent data on repeated dose toxicity
In the table below the available Weight of Evidence 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.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
