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In vitro gene mutation in bacteria

Peppermint oil was examined for the mutagenic activity in bacteria in a WoE Bacterial Reverse Mutation Assay, using five S. typhimurium strains: TA1535, TA1537, TA1538, TA98, and TA100. The test was performed similar to OECD 471. Assays were conducted at 7 doses of 0.005 ul to 10 ul/plate, with and without metabolic activation. No evidence of mutagenic activity was seen at any dose level of peppermint oil, with and without metabolic activation (DeGraff, 1983, report nr HLA 642, Mutagenicity evaluation of peppermint oil in the Ames Salmonella/microsome plate test).

Two other WoE studies are available with Peppermint oil and one WoE study with Cornmint oit (Read Across). All three studies were Bacterial Reverse Mutation Assays (Ames tests). In one study (Andersen and Jensen, 1984), the mutagenic potential of peppermint oil (containing 38.1 % menthol, 33.7% menthone, and 1.7% pulegone) was tested in S. typhimurium strains TA1535, TA1537, TA98 and TA100, in the presence and absence of metabolic activation (similar to OECD 471). Dose levels used were 6.4, 32, 160, and 800 µg/plate. In addition to peppermint oil, menthol (99%), menthone (97%), and pulegone (99%) were also tested. No evidence of mutagenic activity was seen at any dose level of peppermint oil, menthol and pulegone. Cytotoxicity was noted at the maximum dose of 800 µg/plate. Addition of S9 mix seemed to make these compounds less toxic to the bacteria. In contrast, menthone (97%) induced a statistically significant increased number of revertants in the strain TA1537 without S9 at dose levels of 6.4 and 32 µg per plate, and also in the more sensitive strain TA97 without S9 at all dose levels. As the menthone content of peppermint oil was analyzed to be 33.7%, the peppermint oil was expected to be mutagenic on Salmonella strain TA1537 at dose levels 160 and 32 µg. However, no mutagenicity was observed. The explanation of this lack of mutagenicity might be due to several different types of interaction, but is not further elucidated in the study. It is concluded that peppermint oil was not mutagenic in this bacterial system (Andersen, P. H., and N. J. Jensen, 1984, Mutagenic investigation of peppermint oil in the salmonella/mammalian-microsome test. Mutat. Res. 138:17-20).

In a study by Heck et al. (1989), peppermint oil was tested in a genetic toxicity screening battery consisting of a S. typhimurium plate incorporation assay, a rat hepatocyte unscheduled DNA synthesis (UDS) assay, and a mouse lymphoma L5178Y TK+/- cell mutagenesis (MLA) assay). Dose levels reported were the highest inactive concentration tested or, where appropriate, the lowest active concentration. The S. typhimurium plate incorporation assay was performed similar to OECD 471, using strains TA1535, TA1537, TA1538, TA98 and TA100, with and without metabolic activation. Peppermint oil was inactive at 10000 µg/plate in all strains tested with or without S-9 mix. (J. D. Heck et al., 1989, An evaluation of food flavoring ingredients in a genetic toxicity screening battery, The Toxicologist 9 (1): 257).

In order to cover in vitro gene mutagenicity with a tryptophan-requiring strain of Escherichia coli (WP2uvrA), Read Across to Cornmint oil was applied. 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.

One Ames test, in which peppermint was tested, was considered insufficient for an assessment since this test does not meet important criteria of today’s standard methods according to OECD 471. The test was performed only without metabolic activation, in 4 S. typhimurium strains (TA98, TA1535, TA1537 and TA1538), at 2 concentrations. No replicates were included, and no data on cytotoxicity were reported. Although peppermint exhibited some (weak) mutagenicity in this Ames test without metabolic activation, the results were ambiguous and insufficient for an assessment (Sivaswamy, S N, et al., Indian Journal of Experimental Biology, Aug; 29(8): 730-737, 1991).

The results from the Weight of Evidence approach lead to the conclusion that peppermint oil does not cause in vitro gene mutations in bacteria.

 

In vitro cytogenicity

Two studies in which the in vitro cytogenicity of peppermint oil is investigated are considered together with read across to an in vitro cytogenicity study with cornmint oil in a Weight of Evidence approach. The first WoE study is an in vitro chromosomal aberration test using a Chinese hamster fibroblast cell line (CHL). The test was performed comparable to OECD 473. Peppermint oil induced 7.0 % chromosomal aberrations in the absence of metabolic activation, which was considered to be an equivocal result in this test. D/L Menthol (99.8%) was also tested at 0.2 mg/ml and showed a negative result without metabolic activation. The authors suggest that it is possible that the osmotic pressure of a culture medium may affect the total incidence of chromosomal aberrations in an indirect way by physiological alterations in the cells at very high doses under some experimental conditions. So a possible artifactual nature of the positive response observed might be the case in this test (Ishidate, M. L. J., et al., 1984. Primary mutagenicity screening of food additives currently used in. Food Chem. Toxicol. Vol 22, Nr. 8, p.:623-636.).

A second in vitro chromosomal aberration testusing human lymphocytes (whole peripheral blood cultures) was carried out on the essential oils extracted from peppermint. The test was performed similar to OECD Guideline 473. As whole peripheral blood cultures display many properties common with the liver microsomal cytochrome P450 system, no external metabolising enzymes were added. A series of 7 pilot experiments was carried out before 2 representative experiments were done. The cells were continuously exposed to peppermint oil at 0.10, 0.15, 0.20, 0.25, and 0.30 µl/ml for 24 hours.

Essential oil from peppermint herb was very cytotoxic: the lowest tested concentration (0.1 µl/ml) inhibited mitotic activity of the culture for more than 2 times. Peppermint essential oil induced chromosome aberrations only when inhibition of mitotic activity was 70% or higher. It seems that peppermint essential oil may be classified as "high toxicity clastogen", which induces chromosome aberrations by secondary mechanism associated with cytotoxicity. The authors suggest that it might be menthol (a typical "high toxicity clastogen") which makes up 60% of peppermint essential oil used in this work, that can be responsible for the clastogenicity of peppermint essential oil. (J.R. Lazutka et al., 2001, Genotoxicity of dill (Anethum graveolens L.), peppermint (Menthaxpiperita L.) and pine (Pinus sylvestris L.) essential oils in human lymphocytes and Drosophila melanogaster, Food and Chemical Toxicology 39, 485-492). Menthol has been shown to be a non-DNA-reactive clastogen, producing cytotoxic effects that interfere with DNA synthesis, and may not constitute a risk of mutagenicity at low exposure levels (Galloway et al., 1998, DNA synthesis as an indirect mechanism of chromosome aberrations: comparison of DNA-reactive and non-DNA-reactive clastogens, Mutation Research 400:169-186).

The third study is read across to category member cornmint oil. In an in vitro chromosome aberration assay in cultured peripheral human lymphocytes, an increase in the number of polyploidy cells was also reported. Cornmint oil (mentha arvensis) contains similar constituents as peppermint oil (a. o. menthol and menthone).The observed increases in the study with cornmint oil were within the laboratory’s historical control data and therefore not biologically relevant. Both in the absence and presence of S9 mix cornmint oil did not induce a statistically or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments (Buskens, 2010, report nr 494738 Evaluation of the ability of Cornmint oil (mentha arvensis) to induce chromosome aberrations in cultured peripheral human lymphocytes (with repeat experiment)).

Although the available in vitro chromosome aberration studies with peppermint show ambiguous or positive response. These effects are attributed to artefacts at high doses, and cytotoxicity. The same kind of effects have been observed for major constituent menthol. It is expected that under non-cytotoxic conditions, no cytogenicity will be observed. This is confirmed by the in vitro chromosome aberration study with category member cornmint oil, which contains similar constituents. In this study, no significant increase of chromosomal aberrations were observed at non-cytotoxic concentrations. Therefore, in a Weight of Evidence Approach, peppermint oil is considered not to be cytogenic.

 

In vitro DNA damage and/or repair

A Sister Chromatid Exchange (SCE) test in vitro using human lymphocytes (whole peripheral blood cultures) was carried out on the essential oils extracted from peppermint (Menthaxpiperita L.). The test was performed similar to OECD 479. As whole peripheral blood cultures display many properties common with the liver microsomal cytochrome P450 system, no external metabolising enzymes were added. The cells were continuously exposed to peppermint oil at 0.10, 0.15, 0.20, 0.25, and 0.30 µl/ml for 24 hours. Cell replicative kinetics was determined by means of Replicative Index (RI).

Peppermint oil (Menthaxpiperita L.) clearly inhibited cell replicative kinetics. At concentrations of 0.20 ul/ml and above statistically significant inhibition of cell replicative kinetics was evident. Peppermint essential oil was a relative weak SCE inducer, it induced SCE in a dose-independent manner at cytotoxic concentrations. It seems that at high doses of Peppermint essential oil "saturation" of SCE-inducing capacity occurs.. (J.R. Lazutka et al., 2001, Genotoxicity of dill (Anethum graveolens L.), peppermint (Menthaxpiperita L.) and pine (Pinus sylvestris L.) essential oils in human lymphocytes and Drosophila melanogaster, Food and Chemical Toxicology 39, 485-492).

On supporting study is available. In this study, peppermint oil was tested in a genetic toxicity screening battery consisting of a S. typhimurium plate incorporation assay (), a rat hepatocyte unscheduled DNA synthesis (UDS) assay, and a mouse lymphoma L5178Y TK+/- cell mutagenesis (MLA) assay). All assays except UDS were performed with and without metabolic activation. Dose levels reported were the highest inactive concentration tested or, where appropriate, the lowest active concentration. The UDS assay was performed similar to OECD 482. At a dose level of 155 ug/ml peppermint oil no genotoxic effect was observed without metabolic activation. (J. D. Heck et al., 1989, An evaluation of food flavoring ingredients in a genetic toxicity screening battery, The Toxicologist 9 (1): 257).

Based on the available information from the SCE and UDS test, it can be concluded that peppermint oil is not mutagenic at non-cytotoxic concentrations.

 

In vitro gene mutation in mammalian cells

In vitro gene mutation in mammalian cells was investigated in a mouse lymphoma forward mutation assay (MLA), performed similar to OECD 476. The dose range tested for 4 hours under activation conditions was 25 to 150 nl/ml, and under non-activation conditions 6.25 to 200 nl/ml. Peppermint oil did not induce reliable increases in the mutant frequency at the TK locus in these cells and is therefore considered inactive in this Mouse Lymphoma Forward Mutation Assay with and without metabolic activation (Lorillard, 1982, report nr 20989, Mutagenicity evaluation of peppermint oil in the mouse lymphoma forward mutation assay).

In a supporting study, peppermint oil was tested in a genetic toxicity screening battery consisting of a S. typhimurium plate incorporation assay (), a rat hepatocyte unscheduled DNA synthesis (UDS) assay, and a mouse lymphoma L5178Y TK+/- cell mutagenesis (MLA) assay). All assays except UDS were performed with and without metabolic activation. Dose levels reported were the highest inactive concentration tested or, where appropriate, the lowest active concentration. The MLA assay was performed similar to OECD 476. The L5178Y/TK cells were exposed to peppermint oil for 4 hours. Peppermint oil was inactive at 150 ug/ml with or without S-9. The authors discuss the conflicting positive result in the MLA assay with the negative findings of the corresponding other 2 assays. As 13 of the 17 positive responses in the MLA assay occurred in the presence of S9, the authors mention the possibility of false positives due to either an effect of the acidic test materials on the medium pH and/or osmolality, or a possible effect of S9 degradation products in the MLA test (J. D. Heck et al., 1989, An evaluation of food flavoring ingredients in a genetic toxicity screening battery, The Toxicologist 9 (1): 257).

Based on the available information from the MLA tests, it can be concluded that peppermint oil is not mutagenic in mammalian cells.


Justification for selection of genetic toxicity endpoint
No selection is made as a Weight of Evidence approach was followed which is described below.

Short description of key information:
In vitro:
- Gene mutation in bacteria (Bacterial reverse mutation test/Ames test): not mutagenic (WoE including read across; equivalent or similar to OECD 471)
- Cytogenicity (Chromosome aberration test): ambigious/cytotoxic/not clastogenic (WoE including read across; according / equivalent or similar to OECD 473)
- DNA damage and/or repair (Sister Chromatid Exchange Assay): not mutagenic without metabolic activation (similar to OECD 479)
- Gene mutation in mammalian cells (Mouse Lymphoma Assay): not mutagenic with and without metabolic activation (similar to OECD 476)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available information, it can be concluded that peppermint oil does not have genotoxic properties at non-cytotoxic concentrations. Therefore, peppermint oil 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.

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