Genotoxicity studies on some natural fragrances

Fragrances and flavouring substances are defined by their action on the human senses. They are used in a wide variety of consumer products, ranging from the “fine fragrances”, applied directly to the skin, to perfumes used in creams, lotions, detergents, cleaning agents and many other personal and household products; furthermore they are added to neutralize unpleasant odours and as food ingredients. Because of their widespread use, it is important to examine the effects on humans and environment of the exposure to fragrance materials. In this study, the effects on DNA of some fragrance materials, lavender essential oil, linalyl acetate, linalool and β-caryophyllene, were evaluated by using a battery of two in vitro tests: the bacterial reverse mutation assay (Ames test) on Salmonella typhimurium TA98 and TA100 and Escherichia coli WP2uvrA strains, both in absence and in presence of a metabolic activation system (S9); the Micronucleus assay (MN assay) on peripheral human lymphocytes. Test substances were studied, at non toxic concentrations, for their potential genotoxicity, evaluated as increase in the number of revertant colonies in the Ames test, and as increase in the frequency of micronuclei, in the MN assay; those resulted devoid of genotoxic effects were hence investigated for their antimutagenicity, i.e. as the capability to inhibit the genotoxicity of some known mutagens: 2-nitrofluorene (2NF), sodium azide (SA), methyl methanesulfonate (MMS) and 2-aminoanthracene (2AA) in the Ames test, while ethyl methanesulfonate (EMS) and demecolchicine or colcemid (COL) in the MN assay. In the MN assay, to evaluate the mechanisms involved in the antimutagenic activity, three different protocols were used: pre- and co-treatment to identify antimutagens acting as desmutagens, and post-treatment for substances acting as bioantimutagens. In the Ames test, lavender essential oil showed to be devoid of mutagenic activity in all strains tested, both with and without the S9 metabolic activator; on the contrary, a strong antimutagenic effect against the mutagenicity of 2NF was registered. Lavender oil showed also to weakly inhibit the mutagenicity of SA and 2AA. In the MN assay, the highest concentrations of lavender oil increased, in statistically significant way, the frequency of micronuclei (genotoxic effect). The mutagenic activity of lavender oil could be due to the presence of some components acting as mutagens; the different sensitivity of two tests used and the different genotoxic events investigated can explain the conflicting results obtained. To better assess the hazard or the usefulness of the lavender oil, other studies, both in vitro and in vivo, are necessary. Linalyl acetate showed to be devoid of both mutagenic and antimutagenic effects in TA98 and TA100, while a mutagenic activity, as a statistically significant increase in the number of revertant colonies, was registered in WP2uvrA, both with and without S9 mixture. In the MN assay, the monoterpene showed to induce genotoxic effects as increase in the frequency of micronuclei. The results of the mutagenic and genotoxic effects of linaly acetate, observed in WP2uvrA and in MN assay, are of great interest because never described up to now and highlight a new profile of linalyl acetate which requires to be better investigated, taking into account its use as flavouring agent. Linalool did not induce any mutagenic and genotoxic effect in both systems used; considering the widespread use of this monoterpene in numerous products, our results reassure on the safety of the exposition to linalool and contribute to better define the profile of this natural substance. Furthermore, in the Ames test the monoterpene did not interfere with the mutagens used, hence it is devoid of antimutagenic properties. β-Caryophyllene resulted devoid of mutagenic effects both in the Ames test and in the MN assay. Furthermore, a strong antimutagenic activity against 2NF in TA98 was registered; the sesquiterpene also induced a moderate inhibition in the mutagenicity of MMS in WP2uvrA and 2AA in all strains. In the MN assay, β-caryophyllene not only was devoid of genotoxic effects but showed also to significantly reduce the frequency of micronuclei induced by EMS in pre- and co-treatment. The antimutagenic activity of β-caryophyllene observed in both assays seems to be related to a desmutagenic mechanism. Results obtained in this study highlight the importance to evaluate the genotoxic profile of a substance by using multiple systems, due to the differences in the sensitivity of each test and the different genotoxic events evaluated. Furthermore, a better characterization of the genotoxic profile of lavender oil and linalyl acetate in in vivo systems is necessary to better define their activity and their safety for human and environment. Finally, data on β-caryophyllene encourage further studies to investigate the potential role of this substance as chemopreventive agent.