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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Comet Assay
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 April 2022 to November 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian comet assay

Test material

Constituent 1
Reference substance name:
Essential oil of Spearmint obtained from the aerial part of Mentha spicata and/or Mentha cardiaca (Lamiaceae) obtained by distillation
EC Number:
946-253-9
Molecular formula:
Not applicable
IUPAC Name:
Essential oil of Spearmint obtained from the aerial part of Mentha spicata and/or Mentha cardiaca (Lamiaceae) obtained by distillation
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Purity, including information on contaminants, isomers, etc.: 100%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stable, maximum temperature: 40°C, maximum duration: shortly for transport
- Solubility in water: Not miscible or difficult to mix

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): Test material concentration of
200 mg/mL, used in the dose range finding, was treated with ultra-sonic waves until the test material was dissolved. In the main test, test material concentrations of 150 mg/mL and lower, were vortexed to obtain a clear solution.

OTHER SPECIFICS
- Specific Density: 0.9291 g/cm3 at 20°C

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han)
Details on species / strain selection:
The Wistar Han rat was the species and strain of choice because it is a readily available rodent which is commonly used for genotoxicity testing, with documented susceptibility to a wide range of toxic materials. Moreover, historical control background data has been generated with this strain. It is an accepted rodent species for nonclinical toxicity test by regulatory agencies.
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: 6 - 8 weeks
- Weight at study initiation: mean per group ranges from 154.2 gram (S.D. +/- 16.5) to 166.4 gram (S.D. +/- 15.1). The body weights of the rats at the start of the treatment were within 20% of the sex mean.
- Assigned to test groups randomly: Yes
- Fasting period before study: not specified
- Housing: Polycarbonate cages (Makrolon MIV type or 2000P Tecniplast) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. During treatment in the dose-range finding study, polycarbonate cages (Makrolon type MIII) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. Up to 5 animals of the same sex and same dosing group were housed together.
- Diet: SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany Pellets, ad libitum except during designated procedures. Results of analysis for nutritional components and environmental contaminants were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water: Municipal tap water. Freely available to each animal via water bottles. Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility. It is considered that there were no known contaminants in the water that could interfere with the outcome of the study.
- Acclimation period: The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Targeted condition 20 to 24°C. The actual daily mean temperature during the study period was 21 to 22°C
- Humidity (%): Targeted condition 40 to 70%. The actual daily mean relative humidity during the study period was 50 to 57%.
- Air changes (per hr): Ten or more
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark (except during designated procedures)

IN-LIFE DATES: From: 08 April 2022 To: 12 May 2022

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: corn oil
- Justification for choice of solvent/vehicle: not specified
- Concentration of test material in vehicle: 200 mg/mL in the dose range finding and 150 mg/mL and lower in the main test
- Amount of vehicle (if gavage or dermal): 10 mL/kg body weight
- Source: Fagron Farmaceuticals, Capelle a/d IJssel, the Netherlands
- Specific gravity: 0.92 g/mL
- Purity: not specified
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: No correction was made for the purity/composition of the test material.The test material was dissolved in corn oil. Test material concentration of 200 mg/mL, used in the dose range finding, was treated with ultra-sonic waves until the test material was dissolved. In the main test, test material concentrations of 150 mg/mL and lower, were vortexed to obtain a clear solution.

DIET PREPARATION
- Rate of preparation of diet (frequency): Test material concentrations were dosed within 3.5 hours after preparation. Any residual volumes were discarded.
- Mixing appropriate amounts with (Type of food): Pellets
- Storage temperature of food: not specified
Duration of treatment / exposure:
24-25 hours
Frequency of treatment:
Dosed twice with a 21-hour interval
Post exposure period:
3-4 hours after last treatment
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle Control (corn oil)
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
Positive control: ethyl methanesulfonate (EMS)
Dose / conc.:
375 mg/kg bw/day (nominal)
Remarks:
Spearmint oil
Dose / conc.:
750 mg/kg bw/day (nominal)
Remarks:
Spearmint oil
Dose / conc.:
1 500 mg/kg bw/day (nominal)
Remarks:
Spearmint oil
No. of animals per sex per dose:
5 males (in the highest dose group additional (3) animals may be used to compensate for possible deaths)
Control animals:
yes, concurrent vehicle
Positive control(s):
ethylmethanesulphonate
- Justification for choice of positive control(s): not specified
- Route of administration: oral gavage
- Doses / concentrations: 200 mg/kg body weight dissolved in physiological saline

Examinations

Tissues and cell types examined:
Liver
Glandular Stomach
Duodenum
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Selection of an adequate dose-range for the Comet main test was based on a dose-range finding study. The test procedure and conditions were similar to those applied in the main test. In the dose-range finding study, three dose-groups were used to define the Maximum Tolerated Dose (MTD) based on the toxic signs observed after dosing with different doses of the test material. The RF doses were: 2000, 1500 and 1000 mg/kg bw. One dose group, comprising of 3 males and 3 females, was dosed for two consecutive days (once daily) with the highest concentration of test material that was used for the main study (1500 mg/kg bw). The other two groups consisted of 1 male and 1 female animal. The observation period after dosing was one to two days. During this period mortality and physical condition were recorded at least once a day.
Based on the results of the dose-range finding study dose levels of 375, 750 and 1500 mg/kg body weight were selected as appropriate doses for the main test. Since there were no substantial differences in toxicity between sexes, only male animals were used in the main study. The highest dose was 1500 mg/kg body weight, i.e. the Maximum Tolerated Dose (MTD; described as the dose that will not kill the animals but will provoke signs of toxicity).

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The main Comet test was performed using male rats only. Five male animals were used in each treatment group. The animals were dosed twice, at 0 and 21 hours via the oral route (gavage). In addition two control groups were dosed by oral gavage at the same time points. One group of five male rats with the vehicle alone (corn oil) and a second group of three male rats with Ethyl Methane Sulfonate (EMS) as a positive control. All animals were sacrificed by abdominal aorta bleeding under isoflurane anesthesia. Tissues were sampled approximately 3-4 hours post dosing. Mortality was observed at least twice daily (morning and afternoon) beginning upon arrival through termination. Detailed clinical observations were observed at least once a day from start of treatment onwards. Individual body weights were recorded immediately prior to dosing.

DETAILS OF SLIDE PREPARATION:
Three slides per tissue per animal were prepared. The slides were marked with the study identification number, animal number and group number. Cell suspensions of liver, duodenum and glandular stomach were collected/isolated. To the cell suspension, melted low melting point agarose (LMAgarose; Trevigen, Gaithersburg, USA) was added (ratio 10:140). The cells were mixed with the LMAgarose and 50 µL was layered on a pre-coated Comet slide (Trevigen) in duplicate. The slides were incubated for approximately 20 minutes in the refrigerator in the dark until a clear ring appears at the edge of the Comet slide area. The cells on the slides were overnight (approximately 17-18 h) immersed in pre-chilled lysis solution (Trevigen) in the refrigerator. After this period the slides were immersed/rinsed in neutralization buffer (0.4 M Tris-HCl pH 7.4). The slides were then placed in freshly prepared alkaline solution for 20 or 30 (liver) minutes at room temperature in the dark. The slides were placed in the electrophoresis unit just beneath the alkaline buffer solution and the voltage was set to 0.7 Volt/cm. The electrophoresis was performed for 20 or 30 (liver) minutes under constant cooling (actual temperature 4.0°C). After electrophoresis the slides were immersed/rinsed in neutralization buffer for 5 minutes. The slides were subsequently immersed for 5 minutes in Absolut ethanol (≥99.6%, Merck) and allowed to dry at room temperature. The slides were stained for approximately 5 minutes with the fluorescent dye SYBR® Gold (Life Technologies, Bleiswijk, The Netherlands) in the refrigerator. Thereafter the slides were washed with Milli-Q water and allowed to dry at room temperature in the dark and fixed with a coverslip.
METHOD OF ANALYSIS:
To prevent bias, slides were randomly coded (per tissue) before examination of the Comets. An adhesive label with study identification number and code were placed over the marked slide. The slides were examined with a fluorescence microscope connected to a Comet Assay IV image analysis system (Perceptive instruments Ltd, Suffolk, United Kingdom). One hundred fifty Comets (50 comets of each replicate LMAgarose circle) were examined per sample.
The following criteria for scoring of Comets were used:
• Only horizontal orientated Comets were scored, with the head on the left and the tail on the right.
• Cells that showed overlap or were not sharp were not scored.
In addition, the frequency of hedgehogs was determined and documented based on the visual scoring of at least 150 cells per tissue per animal. The occurrence of hedgehogs was scored in all treatment groups and the control.

OTHER:
Histopathology:
Part of the liver, glandular stomach and duodenum from the animals (with exception of the positive control) used (after isolation of a part for the comet assay) was collected and fixed and stored in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). No histotechnology and histopathology was needed.
Evaluation criteria:
The in vivo comet is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The positive control EMS should produce at least a statistically significant increase in the percentage Tail Intensity compared to the vehicle treated animals. The response should be compatible with the data in the historical control database.
c) Adequate numbers of cells and doses have been analysed
d) The highest test dose is the MTD or 2000 mg/kg/day
Statistics:
ToxRat Professional v 3.3.0 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the comet assay data. A test material is considered positive in the Comet assay if all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided,
p < 0.05) increase in percentage Tail Intensity is detected compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test material is considered negative in the Comet assay if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in percentage Tail Intensity is detected compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are within the 95% control limits of the negative historical control data range.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Lethargy, salivation and blood crust around the mouth were observed
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

Mortality data and Clinical observations


There were no premature deaths seen in any of the test item dose groups. Clinical signs were observed in animals dosed with the test item at the MTD (1500 mg/kg), these include: lethargy, salivation and blood crust around the mouth. Lethargy was observed on day 2 predose in all the animals in the MTD/2 dose group (750 mg/kg), but was resolved by day 2 post-dose. There were no clinical observations seen in the animals of the MTD/4 dose group.


 


Evaluation of Comet Assay Slides


A summary of the results for each of the tissues of the Comet Assay is given below in Table 2, 3 and 4.


Table 2: Overview Tail Intensity in Liver Cells of Male Rats



































 

Tail Intensity (%)



S.D.



Vehicle Control



3.17



0.86



Test Material 375 mg/kg



2.46



0.71



Test Material 750 mg/kg



3.25



0.87



Test Material 1500 mg/kg



2.99



1.00



EMS 200 mg/kg



65.93



4.45



 


Table 3: Overview Tail Intensity in Duodenum Cells of Male Rats



































 

Tail Intensity (%)



S.D.



Vehicle Control



5.95



2.26



Test Material 375 mg/kg



8.79



3.01



Test Material 750 mg/kg



6.45



1.95



Test Material 1500 mg/kg



4.67



1.15



EMS 200 mg/kg



38.55



3.85



 


Table 4: Overview Tail Intensity in Glandular Stomach Cells of Male Rats



































 

Tail Intensity (%)



S.D.



Vehicle Control



4.97



0.99



Test Material 375 mg/kg



6.42



2.07



Test Material 750 mg/kg



5.60



1.25



Test Material 1500 mg/kg



5.58



2.43



EMS 200 mg/kg



48.67



2.89



No statistically significant increase in the mean Tail Intensity (%) was observed in liver, duodenum and glandular stomach cells of test material treated male treated animals compared to the vehicle treated animals. In addition, there were no Hedgehogs observed in vehicle and test material treated groups.


The mean Tail Intensity in liver, duodenum and glandular stomach cells of vehicle-treated rats was 3.17 ± 0.86% (mean ± SD), 5.95 ± 2.26% (mean ± SD) and 4.97 ± 0.99% (mean ± SD) in male animals, respectively, which is within the 95% control limits of the distribution of the historical control data for the vehicle control (Table 25). The positive control EMS induced a significant increase and showed a mean Tail Intensity of 65.93 ± 4.45% (mean ± SD; p<0.001 Students t test), 38.55 ± 3.85% (mean ± SD; p<0.001 Students t test) and  48.67 ± 2.89% (mean ± SD; p<0.001 Students t test) male animals in liver, duodenum and glandular stomach cells, respectively. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database, except in the liver cells. Since the mean Tail Intensity was just below the lower limit of the acceptability criteria, this has no effect on the validity of the results.


Adequate numbers of cells (150 cells per animal) and doses were analyzed and the highest test dose was the MTD. Hence, all criteria for an acceptable assay were met.

Applicant's summary and conclusion

Conclusions:
The test substance is not genotoxic in the Comet Assay in liver, duodenum and glandular stomach cells when sampled approximately 3-4 hours post dosing of male rats that were dosed via oral gavage for two consecutive days up to a dose of 1500 mg/kg (the maximum tolerated dose) under the conditions of the test performed according to OECD TG 489 (2016)
Executive summary:

Spearmint oil is tested in the in vivo Comet Assay test (OECD TG 489). A range-finding test was performed to find suitable dose levels of the test item and the most appopriate sex. The Comet Assay Main Test was conducted at the Maximum Tolerated Dose (MTD) 1500 mg/kg with 750 mg/kg and 375 mg/kg as the lower dose levels. Groups of male rats (five per group) were dosed at 0 hours and 21-hours and were killed 3-4 hours after the second dose administration. In addition two control groups were dosed by oral gavage at the same time points. One group of five male rats with the vehicle alone (corn oil) and a second group of three male rats with Ethyl Methane Sulfonate (EMS) as a positive control. The presence of clinical signs indicated that systemic absorption had occured. All animals were sacrificed by abdominal aorta bleeding under isoflurane anesthesia. Cells from the liver, duodenum and glandular stomache tissue were sampled approximately 3-4 hours post dosing. 


 


No statistically significant increase in the mean Tail Intensity (%) was observed in liver, duodenum and glandular stomach cells of test material treated male treated animals compared to the vehicle treated animals. In addition, there were no Hedgehogs observed in vehicle and test material treated groups.


The positive control EMS induced a significant increase in mean Tail Intensity in all examined tissues. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database, except in the liver cells. Since the mean Tail Intensity was just below the lower limit of the acceptability criteria, this has no effect on the validity of the results.