Mentha arvensis, ext.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Gene mutation in bacteria (Bacterial reverse mutation test/Ames test): not mutagenic with and without metabolic activation (OECD471)

- Cytogenicity (Chromosome aberration test): not clastogenic (OECD 473)

- Gene mutation in mammalian cells (Mouse lymphoma assay): not mutagenic with and without metabolic activation (OECD 476)

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:

12-Jul-2010 to 22-Jul-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was conducted according to OECD guideline 471 and under GLP conditions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and B-naphthoflavone

Test concentrations with justification for top dose:

Experiment 1:
Preliminary test (without and with S9) TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 ug/plate
Main study: TA1535, TA1537 and TA98:
Without S9-mix: 3, 10, 33, 100, 333 and 1000 ug/plate
With S9-mix: 10, 33, 100, 333, 1000 and 2000 ug/plate

Experiment 2:
TA1535, TA1537, TA98 and TA100:
Without S9-mix: 3, 10, 33, 100, 333 and 1000 ug/plate
With S9-mix: 10, 33, 100, 333, 1000 and 2000 ug/plate
WP2uvrA:
Without S9-mix: 10, 33, 100, 333, 1000 and 2000 ug/plate
With S9-mix: 10, 33, 100, 333, 1000 and 3330 ug/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Test compound was stable and soluble in ethanol and ethanol has been accepted and approved by authorities and international guidelines

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9: sodium azide (TA1535); 9-aminoacridine (TA1537); 2-nitrofluorene (TA98); methylmethanesulfonate (TA100); 4-nitroquinoline-N-oxide (WP2uvrA). With S9: 2-aminoanthracene (all test strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Selection time (if incubation with a selection agent): 48 hours

SELECTION AGENT (mutation assays): agar containing Histidine or Tryptophan

NUMBER OF REPLICATIONS: Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies

OTHER EXAMINATIONS:
- Other: The presence of precipitation of the test compound on the plates was determined

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive if:
a) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of Cornmint oil (mentha arvensis) on the plates was only observed at the start of the incubation period at concentrations of 3330 and 5000 ug/plate. This precipitate manifested as small fatty particles.

RANGE-FINDING/SCREENING STUDIES: In test strain TA100, toxicity was observed at dose levels of 333 and 1000 ug/plate and above in the absence and presence of S9-mix, resp. In test strain WP2uvrA, toxicity was observed at dose levels of 1000 and 3330 ug/plate and above in the absence and presence of S9-mix, resp.

COMPARISON WITH HISTORICAL CONTROL DATA: The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
TA1535 and TA1537: without S9: 1000 ug/plate, and with S9: 2000 ug/plate
TA98: without S9: 333 ug/plate and above, and with S9: 2000 ug/plate
TA100: without S9: 333 ug/plate and above, and with S9: 1000 ug/plate and above
WP2uvrA: without S9: 1000 ug/plate and above, and with S9: 1000 ug/plate and above.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Cornmint oil (mentha arvensis) did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the 4 test strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in test strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Based on the results of this study it is concluded that Cornmint oil (mentha arvensis) is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

Cornmint oil (mentha arvensis) was tested in the Salmonella typhimurium reverse mutation assay with 4 histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and B-naphthoflavone). The study procedures described in this report were based on OECD guideline 471. In the dose range finding test, Cornmint oil (mentha arvensis) was tested up to concentrations of 5000 ug/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. Cornmint oil (mentha arvensis) did not precipitate on the plates at this dose level. Cytotoxicity was observed in both test strains. Results of this dose range finding test were reported as part of the first experiment of the mutation assay. Based on the results of the dose range finding test, Cornmint oil (mentha arvensis) was tested in the first mutation assay up to 1000 and 2000 ug/plate in the absence and presence of 5% (v/v) S9-mix, resp. in test strains TA1535, TA1537 and TA98. In an independent repeat of the assay with additional parameters, Cornmint oil (mentha arvensis) was tested at the same concentration range as the first assay in the absence and presence of 10% (v/v) S9-mix in test strains TA1535, TA1537, TA98 and TA100 and up to 2000 and 3330 ug/plate in the absence and presence of 10% (v/v) S9-mix, resp. in test strain WP2uvrA. Appropriate cytotoxicity was observed in all test strains in both experiments. Cornmint oil (mentha arvensis) did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the 4 test strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in test strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Based on the results of this study it is concluded that Cornmint oil (mentha arvensis) is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15-jul-2010 to 17-sep-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: human peripheral blood

Details on mammalian cell type (if applicable):

- Type and identity of media:
Blood samples
Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.

- Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and 30 U/mL heparin.

- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin was added.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose range finding test:
Without and with S9-mix, 3hr exposure; 24 hr fixation: 10, 33, 100, 333 and 666 µg/mL
Without S9-mix, 24/48hr exposure; 24/48 hr fixation: 10, 33, 100, 333, 666 and 1000 µg/mL
First cytogenetic test:
Without S9-mix, 3 h exposure time, 24 h fixation time: 200, 250 and 300 µg/mL
With S9-mix, 3 h exposure, 24 h fixation time: 200, 300 and 350 µg/ mL
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 100, 150 and 200 µg/mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 100, 150 and 250 µg mL
With S9-mix, 3 hr exposure; 48 hr fixation: 200, 350 and 375 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Test compound was stable and soluble in ethanol. Culture medium has been accepted and approved by authorities and international guidelines

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9

Migrated to IUCLID6: in Hank's Balanced Salt Solution: 0.5 µg/mL for a 3 h exposure period, 0.2 µg/mL for a 24 h exposure period and 0.1 µg/mL for a 48 h exposure period

Positive control substance:

cyclophosphamide

Remarks:

with S9

Migrated to IUCLID6: in Hank's Balanced Salt Solution: 10 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration: 3 hr (with and without S9-mix), 24 and 48 hr (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hr

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of each culture was determined by counting the number of metaphases per 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 666 µg/ml and above

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 333 µg/ml and above in the absence of S9, 3 hr treatment/24 hr fixation; at dose levels of 100 and 333 µg/ml and above in the absence of S9 for the continuous treatment of 24 and 48 hr, respectively and at dose levels of 333 µg/ml and above in the presence of S9, 3 hours treatment, 24 hours fixation

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Appropriate toxicity was reached at the dose levels selected for scoring.

No effects of Cornmint oil (mentha arvensis) on the number of cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. It was noted that Cornmint oil (mentha arvensis) increased the number of polyploid cells both in the absence and presence of S9-mix in the first cytogenetic assay and in the absence of S9-mix in the second cytogenetic assay.

Conclusions:

Interpretation of results (migrated information):
negative

The ability of cornmint oil to induce chromosome aberrations in human peropheral lymphocytes was investigated in two independent experiments, in accordance with OECD 473. Both in the absence and presence of metabolic activation, cornmint oil did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations. It was therefore concluded that this test is valid and that cornmint oil is not clastogenic in human lymphocytes under the conditions of this test.

Executive summary:

The ability of cornmint oil to induce chromosome aberrations in human peropheral lymphocytes was investigated in two independent experiments, in accordance with OECD 473.

Cornmint oil (mentha arvensis) did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments. No effects of Cornmint oil (mentha arvensis) on the number of cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. It was noted that Cornmint oil (mentha arvensis) increased the number of polyploid cells both in the absence and presence of S9-mix in the first cytogenetic assay and in the absence of S9-mix in the second cytogenetic assay. However, the increases were within the historical control data range. It was therefore concluded to be not biologically relevant.

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Furthermore, positive control chemicals mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

It was concluded that this test is valid and that cornmint oil is not clastogenic in human lymphocytes under the conditions of this test.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26-aug-2010 to 12-oct-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
- RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose range finding test:
Without and with S9-mix, 3 hours treatment: 33, 100, 333, 666 and 1000 µg/mL
Without S9-mix, 24 hours treatment: 33, 100, 333, 666 and 1000 µg/ml
Experiment 1:
Without S9-mix, 3 hours treatment: 3, 10, 33, 80, 100, 125, 160 and 180 µg/mL
With S9-mix, 3 hours treatment: 33, 100, 200, 250, 270, 285, 300 and 350 µg/mL
Experiment 2
Without S9-mix, 24 hours treatment: 33, 100, 125, 140, 155, 170, 185 and 200 µg/mL
With S9-mix, 3 hours treatment: 10, 33, 100, 300, 325, 350, 365 and 400 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Test compound was stable and soluble in ethanol and ethanol has been accepted and approved by authorities and international guidelines

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9

Migrated to IUCLID6: 15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period

Positive control substance:

cyclophosphamide

Remarks:

with S9

Migrated to IUCLID6: 7.5 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 3 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells plated/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)

Evaluation criteria:

The global evaluation factor (GEF) has been defined by the IWTG as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126 .

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.

Statistics:

No addtional statistical procedure

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 666 µg/mL and above

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 333 µg/mL in the absence and presence of S9, 3 hours treatment; at dose levels of 100 µg/mL in the absence of S9, 24 hours treatment

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 76 and 87% compared to the total growth of the solvent controls after the 3 and 24 hours treatment period, respectively.

In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 78 and 80% compared to the total growth of the solvent controls after the 3 hours treatment period in the first and second experiment, respectively.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: L5178Y/TK+/-3.7.2C

Conclusions:

Interpretation of results (migrated information):
negative

The mutagenic activity of cornmint oil was evaluated in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells, with independent repeat, in accordance with OECD 476. In the absence and presence of metabolic activation, cornmint oil did not induce a significant increase in the mutation frequency. It is concluded that cornmint oil is not mutagenic in the TK mutation test system under the conditions of this test.

Executive summary:

The mutagenic activity of cornmint oil was evaluated in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells, with independent repeat, in accordance with OECD 476.

In the absence of metabolic activation, cornmint oil did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modifications in the duration of treatment time.

In the presence of metabolic activation, cornmint oil did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modifications in the composition of the S9 concentration for metabolic activation.

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay. Furthermore, the positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses. Therefore, the test conditions were appropriate and the metabolic activation system (S9-mix) functioned properly.

It is concluded that cornmint oil is not mutagenic in the TK mutation test system under the conditions of this test.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

1982

Reliability:

other: See 'Remark'

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study was not performed according to GLP and with deviations to the guideline. The results are acceptable as basic supporting data. However, as the test material identity is significantly different from the substance for which a dossier is submitted (water and methanol extracts of cornmint), the study is disregarded for use in the cornmint oil dossier.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only 2 strains tested

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine gene

Species / strain / cell type:

S. typhimurium, other: TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not applicable

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix derived from the livers of rats induced with polychlorobiphenyl

Test concentrations with justification for top dose:

1, 2, 5, and 10 mg/plate

Untreated negative controls:

yes

Remarks:

Untreated

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: AF-2 (-S9 mix) and benzo(a)pyrene (+S9 mix)

Species / strain:

S. typhimurium, other: TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in methanol extracts

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Detection of mutagenicity was sometimes difficult owing to the cytotoxicity of the extracts, especially those made with methanol. The efficiency in the mutagenicity screening of crude drugs containing toxic materials may be limited.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Cornmint (mentha arvensis) did not induce a significant dose-related increase in the number of revertant (His+) colonies in the 2 test strains (TA98 and TA100), both in the absence and presence of S9-metabolic activation. Based on the results of this study it is concluded that extracts of Cornmint (mentha arvensis) are not mutagenic in the Salmonella typhimurium reverse mutation assay, under the conditions of the current study.

Executive summary:

Water extracts and methanol extracts of cornmint (mentha arvensis) were tested for mutagenicity in the Ames test with Salmonella typhimurium TA98 and TA100, with and without metabolic activation. The doses were 1, 2, 5, and 10 mg/plate. Samples giving over 100 or 300 revertant colonies per plate with strains TA98 or TA100, resp., were concluded to be positive. Within the concentration ranges tested, neither the water extracts nor the methanol extracts of cornmint (mentha arvensis), with or without S-9 activation, affected the mutagenicity rate of the test strains in this assay. Detection of mutagenicity was sometimes difficult owing to the cytotoxicity of the extracts, especially those made with methanol. The efficiency in the mutagenicity screening of crude drugs containing toxic materials may be limited. Based on the results of this study it is concluded that extracts of Cornmint (mentha arvensis) is not mutagenic in the Salmonella typhimurium reverse mutation assay.

Reference 5

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

1982

Reliability:

other: see 'Remark'

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study was not performed according to GLP. The results are acceptable as basic supporting data. However, as the test material identity is significantly different from the substance for which a dossier is submitted (water and methanol extracts of cornmint), the study is disregarded for use in the cornmint oil dossier.

Qualifier:

no guideline followed

Principles of method if other than guideline:

The mutagenicity of cornmint oil was investigated in the recombination-assay, which was carried out with spores of B. subtilis strains (Kada, T. (1980) Screening of environmental chemical mutagens by the rec-assay system with Bacillus subtilis, in: F.J. de Serres and A. Hollaender (Eds.), Chemical Mutagens, Vol. 6, Plenum, New York, pp. 149-173).

GLP compliance:

no

Type of assay:

Bacillus subtilis recombination assay

Target gene:

DNA repair-recombination of B. subtilis H17 [rec (+)] and M45 [rec (-)]

Species / strain / cell type:

bacteria, other: Bacillus subtilis H17 [rec (+)] and M45 [rec (-)]

Details on mammalian cell type (if applicable):

Not applicable

Metabolic activation:

without

Test concentrations with justification for top dose:

100 mg/ml

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Mitomycin C and AF-2

Species / strain:

bacteria, other: Bacillus subtilis H17 [rec (+)] and M45 [rec (-)]

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

bacteria, other: Bacillus subtilis H17 [rec (+)] and M45 [rec (-)]

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: other: water extraction

Remarks:

Migrated from field 'Test system'.

The methanol extraction showed a weak positive reaction: the difference in diameter between the inhibition zones for H17 and M45 strains being 2 - 5 mm. Detection of mutagenicity was sometimes difficult owing to the cytotoxicity of the extracts, especially those made with methanol. The efficiency in the mutagenicity screening of crude drugs containing toxic materials may be limited.

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation water extraction
positive without metabolic activation methanol extraction

The water extracts of cornmint (mentha arvensis) did not affect the mutagenicity rate of the test strains in this assay. The methanol extraction showed a weak positive reaction: the difference in diameter between the inhibition zones for H17 and M45 strains being 2 - 5 mm. Detection of mutagenicity was sometimes difficult owing to the cytotoxicity of the extracts, especially those made with methanol. Thus, the efficiency in the mutagenicity screening of crude drugs containing toxic materials may be limited.

Executive summary:

Water extracts and methanol extracts of cornmint (Mentha arvensis L. var. Piperascens Malinvaud) were tested for mutagenicity in the Bacillus subtilis recombination assay (Kada, T. (1980) Screening of environmental chemical mutagens by the rec-assay system with Bacillus subtilis, in: F.J. de Serres and A. Hollaender (Eds.), Chemical Mutagens, Vol. 6, Plenum, New York, pp. 149-173). On the basis of screening criteria for mutagenicity already described (Kada, 1980), lengths of inhibition zones produced by the samples on Rec + and Rec - strains were plotted, and the samples producing plots that deviated distinctly from those of negative samples were taken to be positive. Difference in diameter between the inhibition zones for H17 and M45 strains: + +: > 5 mm; +: 2 - 5 mm; -: < 2 mm. The water extracts of cornmint oil (mentha arvensis) did not affect the mutagenicity rate of the test strains in this rec-assay. The methanol extraction showed a weak positive reaction: the difference in diameter between the inhibition zones for H17 and M45 strains being 2 - 5 mm. Detection of mutagenicity was sometimes difficult owing to the cytotoxicity of the extracts, especially those made with methanol. Therefore, the efficiency in the mutagenicity screening of crude drugs containing toxic materials may be limited.

Additional information

The genotoxic potential of cornmint oil was determined in three in vitro assays.

In the bacterial reverse mutation test (Ames test), which was performed in accordance with OECD 471, cornmint oil did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the 4 test strains (Salmonella typhimurium

TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in test strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. It is concluded that cornmint oil (mentha arvensis) is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Two disregarded studies are included in which water and methanol extracts of cornmint were investigated in the bacterial reverse mutation assay and in the Bacillus subtilis recombination assay. A weak positive extraction was observed in the tested methanol extracts. However, as the extracts were cytotoxic, detection of mutagenicity was difficult. As the test material identity is significantly different from the substance for which a dossier is submitted (water and methanol extracts of cornmint), the studies are disregarded for use in the cornmint oil dossier.

In the in vitro chromosome aberration assay, which was performed in accordance with OECD 473, cornmint oil did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome abberations (cultures peripheral human lymphocytes) with or without metabolic activation, in either or the two independently repeated experiments. It is concluded that cornmint oil is not clastogenic under the conditions of the test.

In the in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells, which was performed in accordance with OECD 476, cornmint oil did not induce a significant increase in the mutation frequency in two independently repeated experiments, with and without metabolic activation. It is concluded that cornmint oil is not mutagenic under the conditions of the test.

Justification for classification or non-classification

In all 3 key genetic toxicity studies (3 in vitro tests) cornmint oil did not show any genotoxic potential. Therefore, it can be concluded that the substance is not mutagenic, and does not need to be classified for mutagenicity according to the criteria outlined in Annex I of 1272/2008/EC (CLP/EU-GHS) and Annex VI of 67/548/EEC.