Essential oil of Spearmint obtained from the aerial part of Mentha spicata and/or Mentha cardiaca (Lamiaceae) obtained by distillation

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A0103, 2015: Ames, OECD 471, GLP: Negative

Ishidate et al., 1984: CA, non guideline, non GLP: Negative

A0104, 2017: MLA: OECD 476, GLP: Equivocal

Adam, 1998: Ames, similar to OECD 471, non-GLP: Negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 January 2015 to 09 February 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed accoring to OECD Guideline 471 and the GLP principles.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital – 5,6-Benzoflavone induced S9 from Sprague Dawley rat liver

Test concentrations with justification for top dose:

Preliminary toxicity test: 50, 158, 500, 1580, 5000 µg/plate

Exp 1 (plate incorporation)
TA1535 - S9 S9 1600, 800, 400, 200, 100 and 50.0 µg/plate
TA1535 +S9 3200, 1600, 800, 400, 200 and 100 µg/plate
TA98 ± S9 1600, 800, 400, 200, 100 and 50.0 µg/plate
TA100 ±S9 S9 1600, 800, 400, 200, 100 and 50.0 µg/plate
TA1537 ±S9 200, 100, 50.0, 25.0, 12.5, 6.25 and 3.13 µg/plate
WP2 uvrA –S9 5000, 2500, 1250, 625, 313 and 156 µg/plate
WP2 uvrA +S9 5000, 2500, 1250, 625 and 313 µg/plate

Exp. 2 (pre incubation)
WP2 uvrA –S9 5000, 2500, 1250, 625, 313, 156 and 78.1 µg/plate
WP2 uvrA + S9 5000, 2500, 1250, 625, 313 and 156 µg/plate
TA1535 + S9 3200, 1600, 800, 400, 200, 100 and 50.0 µg/plate
TA1535 − S9 1600, 800, 400, 200, 100, 50.0 and 25.0 µg/plate
TA98 ± S9 1600, 800, 400, 200, 100, 50.0 and 25.0 µg/plate
TA100 + S9 1600, 800, 400, 200, 100 and 50.0 µg/plate
TA100 − S9 800, 400, 200, 100, 50.0 and 25.0 µg/plate
TA1537 ± S9 400, 200, 100, 50.0, 25.0 and 12.5 µg/plate

Exp. 3 (pre incubation)
TA1535 −S9 25.0, 12.5, 6.25, 3.13, 1.56 and 0.781 µg/plate
TA1535 +S9 100, 50.0, 25.0, 12.5, 6.25 and 3.13 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: compatible with the survival of the bacteria and the S9 metabolic activity

Untreated negative controls:

yes

Remarks:

sterile water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation
Experiment I: Plate incorporation, Experiment II and III: preincubation
DURATION
- Preincubation period: 30 min
- Exposure duration: 72 h at 37 C

NUMBER OF REPLICATIONS: Three replicates

Solubility was determined in a preliminary test.

Evaluation criteria:

For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies
with increasing dose levels.

Statistics:

regression analysis, t test

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Precipitation: not detected

RANGE-FINDING/SCREENING STUDIES: the results of the preliminary toxicity test revealed cytotoxicity in some concentration levels, mentioned as thinning of the background lawn. Details can be found below, in section 'Any other information on results including tables'.

COMPARISON WITH HISTORICAL CONTROL DATA: some exceptions, but were considered acceptable
Sterility: confirmed based on absent clonies on additional agar plates.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: PRELIMINARY TOXICITY TEST WITHOUT METABOLIC ACTIVATION

Dose level (µg/plate)	TA-1535 Rev/pl.	TA-1537 Rev/pl.	TA-98 Rev/pl.	TA-100 Rev/pl.	WP2uvrA Rev/pl.
untreated	21	20	37	169	26
0.00	24	23	31	118	24
50.0	39	16*	35	121	24
158	25*	13*	36	108	28
500	59*	12*	26*	104	24
1580	65*	12*	40*	76*	18*
5000	50*	M	34*	M	13*

*: thinning of the background lawn
M: microcolony formation

Table 2: PRELIMINARY TOXICITY TEST WITH METABOLIC ACTIVATION

Dose level (µg/plate)	TA-1535 Rev/pl.	TA-1537 Rev/pl.	TA-98 Rev/pl.	TA-100 Rev/pl.	WP2uvrA Rev/pl.
untreated	19	21	35	169	34
0.00	22	27	32	132	31
50.0	23	15*	28	119	20
158	22	23*	46	133	27
500	28	16*	31*	123	30
1580	31*	12*	23*	75*	22
5000	13*	M*	18*	13*	17*

*: thinning of the background lawn
M: microcolony formation

**Tables of the main mutagenicity experiments 1, 2 and 3 can be found in the attached document (Tables_Results_Spearmint Spicata) below, section 'Attached background material'.

Conclusions:

negative with and without S9. Spearmint Spicata did not induce reverse mutations in S. typhimurium or E.coli, in the presence and absence of metabolic activation, under the conditions of this test.

Executive summary:

In a bacterial reverse mutation assay, Spearmint Spicata was tested in order to examine its potential to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli. The following strains were used: TA1535, TA1537, TA98, TA100 and WP2uvrA. Experiments were conducted both in the absence and presence of metabolic activation (liver S9 fraction, induced with phenobarbitone and betanaphthoflavone). A preliminary test was performed in order to determine cytotoxicity and define appropriate concentration levels for the main experiments. Both the plate incorporation and pre incubation methods were used. The study was performed according to the OECD Guideline 471.

No precipitation was seen at all concentrations tested, while some toxicity was detected. Concentrations levels were adapted based on these reults. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. The positive controls induced marked increases in the No of revertant colonies, which show the validity of this result.

It was concluded that Spearmint Spicata does not show any mutagenic activity under the conditions of this test. Therefore, the test substance does not need to be classified under the conditions of this test according to the criteria outlined in Annex I of 1272/2008/EC (CLP).

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

cells were only exposed for 24 and 48 hours without metabolic activation.

GLP compliance:

no

Type of assay:

sister chromatid exchange assay in mammalian cells

Specific details on test material used for the study:

TSCA-CAS No.: 8008-79-5

Species / strain / cell type:

mammalian cell line, other: Chinese hamster lung fibroblasts

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: originally established from the the lung of a newborn female at Cnacer Research Institute, Tokyo (Koyama, Utakoij & Ono, 1970)
- Doubling time: approximately 15 hours
- Methods for maintenance in cell culture if applicable: maintained by 4-day passages
- Modal number of chromosomes: 25

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Minimum essential medium (MEM; GIBCO) supplemented by 10% calf serum.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

without

Test concentrations with justification for top dose:

The top dose was selected in a preliminary test, estimating 50% cell growth inhibition with a cell densitometer. The cells were exposed to 3 different concentrations test substance. The highest non-cytotoxic dose used was 0.125 mg/mL

Vehicle / solvent:

- Vehicle/solvent used: ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 24 and 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hours

SPINDLE INHIBITOR: Colcemid (final concentration 0.2 µg/mL) was added 2 hours before harvesting.

STAIN: Giesma solution (1.5% at pH 6.8)

NUMBER OF REPLICATIONS: not specified

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The cells were trypsinized and suspemded in a hypotonic KCl solution (0.075M) for 13 minutes at room temperature. Cells were centrifuged and fixed with acetic acid-methanol (1:3, v/v) and applied on glass slides. Slides were air dried and stained with Giesma solution fo 12-15 minutes.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: A hundred wel spread metaphases were observed under the microscope.

DETERMINATION OF CYTOTOXICITY
- Method: cell growth inhibition was determined in a preliminary study.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

- OTHER: Incidence of polyploidy, structural chromosomal aberrations (e.g. chromatid or chomosome gaps, breaks, exchanges, ring formations, fragmentations) were recorded.

Evaluation criteria:

- The result were considered negative if the indicence of aberrations was less than 4.9%
- The result were considered equivocal if the indicence of aberrations was between 5.0 and 9.9%
- The result were considered positive if the indicence of aberrations was more than 10%
When no dose response relationship was found additional experiments with similar dose levels were carried out.

Statistics:

For a quantitative evaluation of positive samples the D20 was calculated. The dose (mg/mL) at which structural aberrations (incl. gaps) were detected in 20% of the methaphases observed. Additionaly, the TR was calculated, indicating the exchange type aberrations per unite dose (mg/mL)

Key result

Species / strain:

mammalian cell line, other: Chinese Hamster Fibroblasts

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Additional information on results:

RESULTS
- The incidence of polyploid cells after 48 hours exposure was 1%.
- The indicence of cells with structural aberrations after 24 hours exposure was 1%

CONTROL DATA
- Negative (solvent/vehicle): The indicence of aberrations was usually less than 3.0%.

Conclusions:

Under the conditions of the test, the substance was not genotoxic (without metabolic activation). The substance does not need to be classified as mutagenic according to Regulation (EC) No. 1272/2008.

Executive summary:

Ishidate et al., 1984 performed a chromosomal aberration test with Chinese hamster lung fibroblasts (non-GLP). The cells were exposed to the test substance for 24 and 48 hours without metabolic activation. The top dose was selected in a preliminary test, estimating 50% cell growth inhibition with a cell densitometer. The cells were exposed to 3 different concentrations test substance. The highest non-cytotoxic dose used was 0.125 mg/mL. A solvent control (ethanol) and untreated control experiments were performed. Incidences of polyploidy, structural chromosomal aberrations (e.g. chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations) were recorded. The incidence of polyploid cells after 48 hours exposure was 1%. The incidence of cells with structural aberrations after 24 hours exposure was 1%. Under the conditions of the test, the substance was not genotoxic. The substance does not need to be classified as mutagenic according to Regulation (EC) No. 1272/2008.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-01-28 to 2017-01-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Type of assay:

other: in vitro mammalian cell gene mutation test

Target gene:

thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: L5178Y TK+/- mouse lymphoma cells were obtained from American Type Culture Collection, Rockville, Maryland (ATCC code:CRL 9518)
- Suitability of cells: The generation time and mutation rates (spontaneous and induced) have been checked.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:Cultures of the cells are grown in RPMI 1640 minimal medium supplemented with 10% horse serum heat-inactivated at 56°C for 20 minutes before use (Complete medium 10%). The incubations are at 37°C in a 5% carbon dioxide atmosphere (100% nominal relative humidity).
- Periodically checked for Mycoplasma contamination: yes
- Periodically 'cleansed' against high spontaneous background: yes, prior to use cells were cleansed of pre-existing mutants

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat, Phenobarbital / 5,6-Benzoflavone induced S9.

Test concentrations with justification for top dose:

Assay I:
0, 99.0, 124.0, 155, 193, 242, and 302 µg/mL (-S9)
0, 193, 242, 302, 378, 472, and 590 µg/mL (+S9)
Assay II:
0, 77.2, 92.6, 111, 133, and 160 µg/mL (-S9)
0, 123, 154, 192, 240, and 300 µg/mL (+S9)
Assay III:
0, 7.5, 15, 30, 60, 120, and 240 µg/mL (-S9)
0, 104, 119, 137, 158, 181, 209, and 240 µg/mL (+S9)
Assay IV and V:
0, 84.7, 93.1, 102, 113, 124, 136, and 150 µg/mL (-S9)
0, 104, 119, 137, 158, 181, 209, and 240 µg/mL (+S9)

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding:10^6 cells/mL

DURATION
- Exposure duration:
* Cytotoxicity assay: Absence and presence of S9 metabolic activation for 3 hours and for 24 hours only in the absence of S9 metabolic activation.
* Mutation assay: In the first and second experiment, the cells were exposed to the test item for a short treatment time (3 hours). Since negative results were obtained, the third experiment in the absence of S9 metabolism was performed, using a longer treatment time (24 hours).
- Expression time: two days after treatment
- Selection time: 14 days

SELECTION AGENT: 5-trifluorothymidine (final concentration 3.0 μg/mL)

NUMBER OF REPLICATIONS: Duplicate cultures were prepared at each test point, with the exception of the positive controls which were prepared in a single culture.

NUMBER OF CELLS EVALUATED: an estimated 2 * 10^3 cells were plated in each well of four 96-well plates and supplemented with trifluorothymidine.

DETERMINATION OF CYTOTOXICITY
- Method: Relative survival
- Any supplementary information relevant to cytotoxicity: In a preliminary cytotoxicity assay, the test item was assayed, both in the absence and presence of S9 metabolic activation, at a maximum dose level of 5000 μg/mL and at a wide range of lower dose levels: 2500, 1250, 625, 313, 156, 78.1, 39.1 and 19.5 μg/mL.

Evaluation criteria:

Criteria for outcome of assay:
For a test item to be considered mutagenic in this assay, it is required that:
1. The induced mutant frequency (IMF) is higher than the global evaluation factor (GEF) suggested for the microwell method (126 x 10^-6) at one or more doses.
2. There is a significant dose-relationship as indicated by the linear trend analysis. Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological significance. Any increase in mutant frequency should lie outside the historical control range to have biological relevance

Statistics:

Calculations performed according to the guideline.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS MUTATION ASSAY
Assay I: Since the number of analysable concentrations was not sufficient for the evaluation of mutagenic effect, both in the absence and presence of S9 metabolism, a second experiment was performed using the following lower concentrations.
Assay II: No sufficient analysable concentrations were obtained even in this experiment. However, no relevant increase in mutation frequencies was noted after treatment with the test item at any dose level, thus Main Assay III was performed using the long treatment time, in the absence of S9 metabolism.
Assay III: A sufficient number of analysable concentrations were obtained in both treatment series. In order to achieve a full assessment of the mutagenic potential of the test item, two experiments with four analysable concentrations are required. Therefore, a fourth experiment was performed in the absence and presence of S9 metabolism, using a short treatment time.
Assay IV: Adequate levels of cytotoxicity were observed in both treatment series. However, cytotoxic and mutagenic effects observed at equal or similar test item concentrations were not coherent among the experiments; therefore, in agreement with the Sponsor, a fifth experiment (Main Assay V) was performed, using the same concentration/test condition combinations as for Main Assay IV. Adequate levels of cytotoxicity were observed in both treatment series also in this experiment. No precipitate was noted upon addition of the test item to the cultures and by the end of treatment incubation period in any treatment series, in any experiment.

Survival after treatment:
Cytotoxicity results are taken into consideration for the selection of dose levels to be analysed for mutation induction. At low survival levels, the mutation data are prone to a variety of artefacts (selection effects, sampling error, founder effects). Mechanisms other than direct genotoxicity per se can lead to positive results that are related to cytotoxicity and not genotoxicity (e.g. events associated with apoptosis, endonuclease release from lysosomes, etc.). For this reason, it is generally recommended that such data are treated with caution or excluded from consideration. Accordingly, mutation data obtained at concentrations which elicited RTG lower than 10% were excluded from the statistical analyses.

- In Main Assay I, in the absence of S9 metabolic activation, no cells survived after treatment at the three highest dose levels, severe toxicity was noted at 155 μg/mL, while slight to mild toxicity was observed at the next two lower concentrations. In the presence of S9 metabolism, no cells survived at the three highest dose levels, severe toxicity was noted at 302 μg/mL with a relative total growth (RTG) of 1%, while moderate toxicity was observed at 242 and 193 μg/mL. Since the number of analysable concentrations was not sufficient for the evaluation of mutagenic effect, both in the absence and presence of S9 metabolism, a second experiment was performed using a modified dose range.
- In Main Assay II, in the absence of S9 metabolic activation, no cells survived after treatment at 160 μg/mL, severe toxicity was observed at 133 μg/mL (RTG 3%), slight to moderate toxicity was noted over the remaining concentrations tested. In the presence of S9 metabolism, severe toxicity was observed at the two highest dose levels, while treatment with the test item at concentrations between 123 and 192 μg/mL yielded a slight reduction of RTG. No sufficient analysable concentrations were obtained in the absence or presence of S9 metabolism.
- In Main Assay III, in the absence of S9 metabolism using a 24 hour treatment time, no cells survived at 240 μg/mL, while treatment at the next lower concentration yielded 11% of RTG; moderate toxicity (RTG= 53%) was noted at 60.0 μg/mL, while no relevant toxicity was observed over the remaining concentrations tested. In the presence of S9 metabolism, severe toxicity (RTG=5%) was noted at 240 μg/mL, moderate toxicity was observed between 119 and 209 μg/mL, while treatment at 104 μg/mL yielded a reduction of RTG to 68% of the negative control.
- In Main Assay IV, in the absence of S9 metabolism, severe toxicity was noted at the two highest dose levels (150 and 136 μg/mL), while moderate toxicity was observed over the remaining concentrations tested. In the presence of S9 metabolic activation, severe toxicity was noted at 240 μg/mL; dose related toxicity, slight to moderate, was observed over the remaining concentrations tested.
- In Main Assay V, in the absence of S9 metabolism, severe toxicity was observed at 150 μg/mL, while treatment at the next lower concentration yielded 18% of RTG; moderate toxicity was noted at 124 μg/mL, while no relevant toxicity was noted over the remaining concentrations tested. In the presence of S9 metabolic activation, dose related toxicity, mild to moderate (RTG ranging from 19 to 56), was observed at all concentrations tested

No relevant increases in mutant frequencies were observed in Main Assays I and II. However, neither sufficient analysable dose levels were available nor adequate toxicity to evaluate mutation induction was obtained at any dose level. No increase in mutation frequency was observed in the absence of S9 metabolic activation using a 24-hour treatment time, despite test item treatment at 120 μg/mL showed an adequate RTG value (11%). Dose related and statistically significant increases in mutant frequencies, which did not reach the Global Evaluation Factor (GEF), were noted in Main Assay III in the presence of S9 metabolic activation. In Main Assay IV, increases in mutant frequencies were noted, both in the absence and presence of S9 metabolism, at concentrations which elicited a moderate toxicity (RTG between 14 and 28%). Mutation frequency at these concentrations was statistically significant and fell out of the historical control range. A statistically significant linear trend was reported in the absence of S9 metabolism. The observed increases were higher than GEF and thus could be considered a clear evidence of positive results. In Main Assay V, dose-related and statistically significant increases in mutation frequency were noted in the presence of S9 metabolism at the two highest dose levels. Mutation frequency at the highest dose level was higher than the upper confidence limit (p<5%) of the historical control range and can be therefore considered biologically relevant. However, the observed increases did not reach the Global Evaluation Factor (GEF) and therefore can not be considered a clear evidence of mutagenic effect. In the absence of S9 metabolic activation, no increases were observed at any dose level. For the negative and positive controls, the number of wells containing small colonies and those containing large colonies were reported. The small and large colony mutant frequencies were estimated and the proportion of small mutant colonies was calculated. An adequate recovery of small colony mutants was observed following treatment with the positive controls.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Both in the absence and presence of S9 metabolic activation, the test item was assayed at a maximum dose level of 5000 μg/mL and at a wide range of lower dose levels: 2500, 1250, 625, 313, 156, 78.1, 39.1 and 19.5 μg/mL. Upon addition of the test item to the cultures, a cloudy appearance of the treatment medium was noted at the two highest dose levels; particles in suspension were observed at the highest concentration. At the end of the 3 hour treatment period, cloudy appearance was observed at the two highest concentrations; precipitation was noted at 5000 μg/mL both in the absence and presence of S9 metabolism and at 2500 μg/mL only in its presence. By the end of the 24 hour treatment period, opacity and precipitation were observed at the two highest dose levels. In the absence of S9 metabolic activation, using the 3 hour treatment time, no cells survived at the five highest dose levels. No relevant toxicity was noted over the remaining concentrations tested. Using the 24 hour treatment time, no cells survived to treatment at 5000, 2500, 1250 and 625 μg/mL, while test item treatments at 313 and 156 μg/mL yielded moderate toxicity reducing relative survival (RS) to 22% and 33%, respectively. Mild toxicity (51-57% RS) was noted at the next two lower concentrations, while slight toxicity was observed at 19.5 μg/mL (RS=71%). Following treatment in the presence of S9 metabolic activation, using the short treatment time (3 hours), no cells survived to treatment at the four highest concentrations. Slight to mild toxicity was seen between 78.1 and 313 μg/mL, while no relevant toxicity was observed over the remaining dose levels.

TEST-SPECIFIC CONFOUNDING FACTORS
The pH values and osmolality of the post-treatment media were determined. The addition of the test item solution did not have any obvious effect on the osmolality or pH of the treatment medium.

Conclusions:

Although several experiments were performed, the data set obtained precludes making a conclusion of positive or negative results, therefore results obtained after treatment with the test substance should be considered equivocal.

Executive summary:

An in vitro gene mutation study in mammalian cells was performed according to OECD 476 and in compliance with GLP. The induction of 5 trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation was examined, using a fluctuation method. Based on the results obtained in a preliminary cytotoxicity assay the dose levels were determined for the first mutation assay (I): 0, 99.0, 124.0, 155, 193, 242, and 302 µg/mL (-S9) and 0, 193, 242, 302, 378, 472, and 590 µg/mL (+S9). Due to toxicity the number of analysable concentrations was not sufficient for the evaluation of mutagenic effect. Multiple experiments with different doses were performed to obtain sufficient analysable concentrations. Assay II: 0, 77.2, 92.6, 111, 133, and 160 µg/mL (-S9) and 0, 123, 154, 192, 240, and 300 µg/mL (+S9). Assay III: 0, 7.5, 15, 30, 60, 120, and 240 µg/mL (-S9) and 0, 104, 119, 137, 158, 181, 209, and 240 µg/mL (+S9). Assay IV and V: 0, 84.7, 93.1, 102, 113, 124, 136, and 150 µg/mL (-S9) 0, 104, 119, 137, 158, 181, 209, and 240 µg/mL (+S9). In the first and second experiment, the cells were exposed to the test item for a short treatment time (3 hours). Since negative results were obtained, the third experiment in the absence of S9 metabolism was performed, using a longer treatment time (24 hours). After the expression time (Two days after treatment) the cells were incubated for 14 days with the selection agent, 5-trifluorothymidine (final concentration 3.0 μg/mL). Although several experiments were performed, the data set obtained precludes making a conclusion of positive or negative results, therefore results obtained after treatment with the test substance should be considered equivocal.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Test performed according to the Ames test as described by Maron and Ames in 1983 (OECD 471 guidelines), not according to CLP.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Only four strains of salmonella used: S. typhimurium TA 97, TA 98, TA 100, and TA102.

Principles of method if other than guideline:

Not relevant

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

His-gene

Species / strain / cell type:

S. typhimurium TA 97

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

not specified

Test concentrations with justification for top dose:

250, 500, 1000, and 2000 ppm

Vehicle / solvent:

No data available

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Details on test system and experimental conditions:

No data available

Evaluation criteria:

No data available

Statistics:

No data available

Key result

Species / strain:

S. typhimurium TA 97

Genotoxicity:

negative

Remarks:

no mutagenic activity.

Key result

Species / strain:

S. typhimurium TA 98

Genotoxicity:

negative

Remarks:

no mutagenic acitivity.

Key result

Species / strain:

S. typhimurium TA 100

Genotoxicity:

negative

Remarks:

no mutagenic activity.

Key result

Species / strain:

S. typhimurium TA 102

Genotoxicity:

negative

Remarks:

no mutagenic activity.

Additional information on results:

No mutagenicity was observed at any of the concentrations.

not relevant

Conclusions:

Interpretation of result: negative No mutagenic activity observed.
M. Spicata dit not show any mutagenic activity for concentrations of 250, 500, 1000 and 2000 ppm in the Salmonella Typhimurium strains TA97, TA98, TA100, and TA102 under the conditions of this test.

Executive summary:

In this publication the ability of M. Spicata to induce mutations in the Salmonella Typhimurium strains TA97, TA98, TA100, and TA102 was investigated. The study procedures used in this study were based on the Ames Test as described by Maron and Ames (1983). M. Spicata was tested at the concentrations of 250, 500, 1000, and 2000 ppm. No mutagenicity was observed at any of these test concentrations in any of the 4 Salmonella Typhimurium strains. M. Spicata was not mutagenic under the conditions of this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

OECD 489 Comet assay: Negative

Somatic Mutation And Recombination Tests (SMART) in Drosophila melanogaster, non-GLP: Negative

Similar to OECD 474, non-GLP: Negative

Link to relevant study records

Reference

Reference 1

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

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

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

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

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

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

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.

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

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.

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.

Reference 2

Endpoint:

in vivo mammalian germ cell study: gene mutation

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

The Somatic Mutation And Recombination Tests (SMART) in Drosophila melanogaster is a well known test, in which the genotoxicity of substances can be screened.
Drosophila larvae, obtained from a cross between the multiple wing hair strain and the flare strain, were exposed to spearmint oil. After 18 hours, the larvae were washed and placed in individual vials until adult flies emerged. All experiments were performed in triplicate. The number of surviving flies were counted in both control and test cultures. Surviving female flies were scored for the presence of mosaic spots on their wings.

GLP compliance:

no

Type of assay:

somatic mutation and recombination test in Drosophila

Species:

Drosophila melanogaster

Strain:

other: multiple wing hair strain with genetic mutation mwh elmwh e, and flair strain with genetic mutation Y wcoly wco; flr3 sel TM2 Ubx130 se e

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dr. Marec
- Age at study initiation: eggs were collected during an 8h period and after 72 ± 4h larvae were removed from food to be used in the experiment.
- Weight at study initiation: not relevant
- Fasting period before study: no food available during experiment
- Diet (e.g. ad libitum): yeast-glucose medium

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 1
- Humidity (%): 60
- Photoperiod (hrs dark / hrs light): 16h ligth / 8h dark cycle

Route of administration:

other: Larvae individually housed on Petri dishes were exposed to Spearmint Oil applied to a small filter paper disk.

Vehicle:

Not applicable

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Different amounts of test substance were applied to a small filter paper disk (4 mm diameter) placed at the center of the Petri dish.
Doses tested were slightly above LD50 concentration.

Duration of treatment / exposure:

Dishes were kept at 24 ± 1°C and 60% humidity for 18h.

Frequency of treatment:

Single treatment

Post exposure period:

After the exposure period, the larvae were washed with Ringer solution and transferred to new individual vial with food until emergence of adult flies.

Remarks:

Doses / Concentrations:
1.2 µL (slightly above LD50 concentration)
Basis:
no data

No. of animals per sex per dose:

Groups of 50 larvae were used per experiment (experiment conducted in triplicate).

Control animals:

yes, concurrent no treatment

Positive control(s):

None

Tissues and cell types examined:

- Wings of female flies were mounted in Euparal solution and scored at 400x magnification for the presence of mosaic spots.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: the dose level (1.2 µL) was chosen slightly above the LD50 (1.12 µL) as determined in the range-finding study.

DETAILS OF SLIDE PREPARATION: Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): small single cell spots indicating the possible ability of M. Spicata to induce point mutations and deletions.

Evaluation criteria:

Size, number and type of cells showing malformed wing hairs were recorded. The result was corrected for the average frequency of spontaneous spots in the control experiments. A statistical differnce was noted at a 5% significance level.

Statistics:

Mortality was corrected using the following equation: (a - b)100/a, (a = the number of surviving adults in the control and b the number of surviving adults in the test experiments).
Tthe spots were grouped into four categories:
a) small single spots (with one or two affected cells, either mwh or flr).
b) large single spots (with three or more affected cells, either mwh or flr).
c) twin spots (consisting of both mwh and flr subclones).
d) total single spots.

The multiple-decision procedure (Selby and Olson, 1981; details are given in Frei and Wuergler (1988)) was used to determine statistical significance of the test results.

Sex:

male/female

Genotoxicity:

positive

Remarks:

M. Spicata shows strong mutagenic activity, but no recombination activity

Toxicity:

yes

Remarks:

very effective insecticide

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Additional information on results:

RESULTS OF MORTALITY STUDY
- Dose range: five different amounts were determined, which caused death to Drosophila lower than 100%.
- Solubility: not applicable, undiluted substance.
- Rationale for exposure: doses caused death to Drosophila lower than 100% in order to determine a LD50-value.
- Harvest times:
- High dose with and without activation:
- Other: LD50 = 1.12 µL

RESULTS OF GENOTOXICITY STUDY
- Appropriateness of dose levels and route: dose level chosen (1.2 µL) was slightly above the LD50 determined in the range-finding study.
- Statistical evaluation: a significant increase in the frequency of single spots indicating mutagenicity but no significant increase in the frequency of twin spots (indicating recombinogenic activity).

not relevant

Conclusions:

Interpretation of results: positive mutagenic but not recombinogenic.
In a SMART test in which Drosophila melanogaster was exposed to Spearmint Oil (Mentha spicata), an LD50 of 1.12 µL was observed. A concentration slightly above this LD50 (1.2 µL) induced a significantly higher frequency of small single cell spots in treated Drosophila Melanogaster larvae, which could mean that M. spicata is able to induce point mutations and deletions. Results for the statistical analysis of twin spots indicated no increased frequency, meaning that M. spicata does not exhibit recombination activity. This leads to the conclusion that M. spicata is a toxic and genotoxic (mutagenic but not recombinogenic) oil.

Executive summary:

In this report the ability of M. spicata to cause toxic and genetoxic effects in Drosophila melanogaster larvae was investigated in the following strains: mwh strain (genetic mutation: mwh elmwh e) and flr strain (genetic mutation Y wcoly wco; flr3 sel TM2 Ubx130 se e). The study procedures described in this report were based on the Somatic Mutation And Recombination Tests (SMART).

A range-finding study was performed prior to the Somatic Mutation And Recombination Tests (SMART), in order to determine the LD50 -value for M. spicata. The control group was treated with Ringer solution. The LD50 was found to be 1.12 µL. The selected dose for the main test was chosen to be slighly above the LD50, and was 1.2 µL.

Spots per wing were diagnosed once the larvae were turned into flies to determine the genotoxic potential of M. spicata. A concentration of 1.2 µL induced a significantly higher frequency of small single cell spots in treated Drosophila melanogaster larvae, indicating that M. spicata is able to induce point mutations and deletions. The statistical analysis of twin spots indicated no increased frequency, therefore, it can be concluded that M. spicata does not exhibit recombination activity. The results show that M. spicata is a toxic and genotoxic (mutagenic but not recombinogenic) oil.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Principles of method if other than guideline:

not applicable.

GLP compliance:

no

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: ddY

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Shizuoka Agricultural Cooperative Association for Laboratory Animals, Shizuoka)
- Age at study initiation: 8 weeks
- Diet (e.g. ad libitum): ad libitum (CE-2 pellets (Japan, Clea, Tokyo)
- Water (e.g. ad libitum): ad libitum

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: Olive Oil
- Concentration of test material in vehicle: Dose levels: 0, 200, 400, and 800 mg/kg

Details on exposure:

Route of exposure = intraperitoneal.
The compound was administered by one ip injection or 4 injections

Duration of treatment / exposure:

One ip injection, sampling time after 24 hours.
4 ip injections, 24 hours between each dose. Sampling after 24 hours.

Frequency of treatment:

1 ip injection (0, 200, 400, and 800 mg/kg)
4 ip injections (400 mg/kg)

Post exposure period:

Sampling was performed 24 hours after dosing.

Dose / conc.:

200 mg/kg bw (total dose)

Remarks:

Single ip injection

Dose / conc.:

400 mg/kg bw (total dose)

Remarks:

Single ip injection

Dose / conc.:

800 mg/kg bw (total dose)

Remarks:

Single ip injection

Dose / conc.:

400 other: mg/kg

Remarks:

4 ip injections

No. of animals per sex per dose:

Only male mice were used:
Pilot study: 2 male mice
Full-scale tests: 6 male mice

Control animals:

yes

Positive control(s):

Mitomycin C (Kyowa Hakka Ltd, Tokyo)
- Route of administration: intraperitoneal
- Doses / concentrations: 2.0 mg/kg

Tissues and cell types examined:

- Femoral bonemarrow cells
- Erythrocytes
- polychromatic erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Doses were selected based on a pilot study (results not reported in this study).

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Sampling took place 24 hours after dosing.

DETAILS OF SLIDE PREPARATION:
Femoral bone marrow cells were flushed with foetal bovine serum and smeared on clean glass slides. Cells were fixed with methanol for 5 minutes, and stained with Acridine Orange (for pilot experiment) or Giemsa (for full-scale test).

METHOD OF ANALYSIS:
One thousand polychromatic erythrocytes per mouse were scored using a light microscope, with a high power objective (x100), and the number of micronucleated polychromatic erythrocytes (MNPCEs) was recorded. The proportion of polychromatic erythrocytes (PCEs) among the total erythrocytes was also evaluated by observing 1000 erythrocytes on the same slide.

Statistics:

A two-stage statistical procedure was used. In the first step, the frequency of MNPCEs in each treatment group was compared with the binominal distribution specified by historical control data from the laboratory where the test was performed.
In the second step, the dose-response relationship was tested by the Cochran-Armitage trend test. A positive result was recorded only when one or more treatment group(s) showed a statistically significant difference (P < 0.01) from the sponataneous level of MNPCEs and the trend test indicated a positive dose response (P < 0.05).

For the test consisted of multiple treatments and contained one dose group, only the frequency of MNPCEs in each treatment group was compared with the binominal distribution specified by historical control data from the laboratory where the test was performed.

Sex:

male

Genotoxicity:

negative

Remarks:

For the single as well as multiple treatment tests

Toxicity:

not specified

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- Mortality: non mortaility was observed after exposure with a single injection (0, 200, 400, 800 mg/kg bw). In the dose group exposed to 4 ip injections (400 mg/kg bw) mortality was observed in 1 out of 6 animals.
- Micronucleated polychromatic erythrocytes (%):
Single injection: 0 mg/kg bw: 0.30 ± 0.25, 200 mg/kg bw: 0.17 ± 0.20, 400 mg/kg bw: 0.25 ± 023, and 800 mg/kg bw: 0.18 ± 0.12.
4 injections: 400 mg/kg bw: 0.18 ± 0.16
- Polychromatic erythrocytes (%):
Single injection: 0 mg/kg bw: 50.6 ± 6.6 , 200 mg/kg bw: 59.9 ± 1.5, 400 mg/kg bw: 53.6 ± 7.3, and 800 mg/kg bw: 57.6 ± 4.0
4 injections: 400 mg/kg bw: 48.9 ± 8.3

Conclusions:

Interpretation of results: negative No effects observed.
Spearmint oil dit not show a significant induction of micronulei for the concentrations of 200, 400, and 800 mg/kg in the male ddY mice when intraperiteally injected as either a single or multiple (4) doses. Therefore, it is not necesarry to classify Spearmint oil as mutagenic according to the criteria outlined in Annex I of 1272/2008/EC.

Executive summary:

This publication describes the ability of Spearmint oil to induce micronuclei in ddY male mice when injected peritoneally as either a single or multiple doses. The study procedures used in this study were based on the Mammalian Erythrocyte Micronucleus Test (OECD 474). Spearmint oil was tested at the concentrations of 200, 400, and 800 mg/kg for the single dose test. A dose of 400 mg/kg was selected for the multiple dose test, including 4 intraperitoneal injections. No statiscally significant induction of micronuclei was observed at any of these test concentrations in erythrocytes isolated from the bonemarrow of the ddY male mice when compared to historical control data. Therefore, it can be concluded that it is not necesarry to classify Spearmint Oil as mutagenic according to the criteria outlined in Annex I of 1272/2008/EC.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro:

A0103, 2015: In a bacterial reverse mutation assay, Spearmint Spicata was tested in order to examine its potentialto induce gene mutations in tester strains ofSalmonella typhimuriumandEscherichia coli. The following strains were used: TA1535, TA1537, TA98, TA100 and WP2uvrA. Experiments were conducted both in the absence and presence of metabolic activation (liver S9 fraction, induced with phenobarbitone and betanaphthoflavone). A preliminary test was performed in order to determine cytotoxicity and define appropriate concentration levels for the main experiments. Both the plate incorporation and pre incubation methods were used. The study was performed according to the OECD Guideline 471. No precipitation was seen at all concentrations tested, while some toxicity was detected. Concentrations levels were adapted based on these reults. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. The positive controls induced marked increases in the No of revertant colonies, which show the validity of this result. It was concluded that Spearmint Spicata does not show any mutagenic activity under the conditions of this test. Therefore, the test substance does not need to be classified under the conditions of this test according to the criteria outlined in Annex I of 1272/2008/EC (CLP).

Ishidate et al., 1984 performed a chromosomal aberration test with Chinese hamster lung fibroblasts (non-GLP). The cells were exposed to the test substance for 24 and 48 hours without metabolic activation. The top dose was selected in a preliminary test, estimating 50% cell growth inhibition with a cell densitometer. The cells were exposed to 3 different concentrations test substance. The highest non-cytotoxic dose used was 0.125 mg/mL. A solvent control (ethanol) and untreated control experiments were performed. Incidences of polyploidy, structural chromosomal aberrations (e.g. chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations) were recorded. The incidence of polyploid cells after 48 hours exposure was 1%. The incidence of cells with structural aberrations after 24 hours exposure was 1%. Under the conditions of the test, the substance was not genotoxic. The substance does not need to be classified as mutagenic according to Regulation (EC) No. 1272/2008.

A0104, 2017: An in vitro gene mutation study in mammalian cells was performed according to OECD 476 and in compliance with GLP. The induction of 5 trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation was examined, using a fluctuation method. Based on the results obtained in a preliminary cytotoxicity assay the dose levels were determined for the first mutation assay (I): 0, 99.0, 124.0, 155, 193, 242, and 302 µg/mL (-S9) and 0, 193, 242, 302, 378, 472, and 590 µg/mL (+S9). Due to toxicity the number of analysable concentrations was not sufficient for the evaluation of mutagenic effect. Multiple experiments with different doses were performed to obtain sufficient analysable concentrations. Assay II: 0, 77.2, 92.6, 111, 133, and 160 µg/mL (-S9) and 0, 123, 154, 192, 240, and 300 µg/mL (+S9). Assay III: 0, 7.5, 15, 30, 60, 120, and 240 µg/mL (-S9) and 0, 104, 119, 137, 158, 181, 209, and 240 µg/mL (+S9). Assay IV and V: 0, 84.7, 93.1, 102, 113, 124, 136, and 150 µg/mL (-S9) 0, 104, 119, 137, 158, 181, 209, and 240 µg/mL (+S9). In the first and second experiment, the cells were exposed to the test item for a short treatment time (3 hours). Since negative results were obtained, the third experiment in the absence of S9 metabolism was performed, using a longer treatment time (24 hours). After the expression time (Two days after treatment) the cells were incubated for 14 days with the selection agent, 5-trifluorothymidine (final concentration 3.0 μg/mL). Although several experiments were performed, the data set obtained precludes making a conclusion of positive or negative results, therefore results obtained after treatment with the test substance should be considered equivocal.

Adam et al., 1998: In this publication the ability of M. Spicata to induce mutations in the Salmonella Typhimurium strains TA97, TA98, TA100, and TA102 was investigated. The study procedures used in this study were based on the Ames Test as described by Maron and Ames (1983). M. Spicata was tested at the concentrations of 250, 500, 1000, and 2000 ppm. No mutagenicity was observed at any of these test concentrations in any of the 4 Salmonella Typhimurium strains. M. Spicata was not mutagenic under the conditions of this test.

In vivo

Franzios et al., 1997: In this report the ability of M. spicata to cause toxic and genetoxic effects in Drosophila melanogaster larvae was investigated in the following strains: mwh strain (genetic mutation: mwh elmwh e) and flr strain (genetic mutation Y wcoly wco; flr3 sel TM2 Ubx130 se e). The study procedures described in this report were based on the Somatic Mutation And Recombination Tests (SMART). A range-finding study was performed prior to the Somatic Mutation And Recombination Tests (SMART), in order to determine the LD50 -value for M. spicata. The control group was treated with Ringer solution. The LD50 was found to be 1.12µL. The selected dose for the main test was chosen to be slighly above the LD50, and was 1.2µL.Spots per wing were diagnosed once the larvae were turned into flies to determine the genotoxic potential of M. spicata. A concentration of 1.2 µL induced a significantly higher frequency of small single cell spots in treated Drosophila melanogaster larvae, indicating that M. spicata is able to induce point mutations and deletions. The statistical analysis of twin spots indicated no increased frequency, therefore, it can be concluded that M. spicata does not exhibit recombination activity. The results show that M. spicata is a toxic and genotoxic (mutagenic bot not recombinogenic) oil.

Hayashi et al., 1988: This publication describes the ability of Spearmint oil to induce micronuclei in ddY male mice when injected peritoneally as either a single or multiple doses. The study procedures used in this study were based on the Mammalian Erythrocyte Micronucleus Test (OECD 474). Spearmint oil was tested at the concentrations of 200, 400, and 800 mg/kg for the single dose test. A dose of 400 mg/kg was selected for the multiple dose test, including 4 intraperitoneal injections. No statiscally significant induction of micronuclei was observed at any of these test concentrations in erythrocytes isolated from the bonemarrow of the ddY male mice when compared to historical control data. Therefore, it can be concluded that it is not necesarry to classify Spearmint Oil as mutagenic according to the criteria outlined in Annex I of 1272/2008/EC and Annex VI of 67/548/EEC.

Justification for classification or non-classification

The substance is not classified for genetic toxicity, according to Regulation 1272/2008/EC (CLP).