Evaluation of β-myrcene, α-terpinene and (+)- and (−)-α-pinene in the Salmonella/microsome assay

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Abstract

This study was undertaken to investigate the genotoxicity of β-myrcene, α-terpinene and (+) and (−)-α-pinene, monoterpenes found in a variety of plant volatile oils. β-myrcene, α-terpinene and α-pinene as well as plant oils containing these hydrocarbon monoterpenes have been used as flavoring additives in foods and beverages, as fragrances in cosmetics, and as scent in household products. Mutagenicity was evaluated by the Salmonella/microsome assay (TA100, TA98, TA97a and TA1535 tester strains), without and with addition of an extrinsic metabolic activation system (rat liver S9 fraction induced by Aroclor 1254). Two dose-complementary assays were performed so that a broad range of doses, including a number of regularly-spaced doses in the non-toxic dose interval, were tested. No increase in the number of his+ revertant colonies over the negative control values was observed in any of the four S. typhimurium tester strains. Results from the present study therefore indicated that β-myrcene, α-terpinene, and (+) and (−)-α-pinene are not mutagenic in the Ames test.

Introduction

Monoterpenes are natural ten-carbon (C10) compounds constructed from two isoprene molecules (C5H8, or hemiterpene), the five-carbon building-block of all terpenes. At least 450 structurally-different monoterpenes have been identified so far (Sticher, 1977). They are found in edible, medicinal and aromatic plants and are the main chemical constituents of their essential oils (Sticher, 1977; Erikson, 1976). Plant volatile oils as well as their monoterpenoid constituents have been widely used as flavorings additives in foods and beverages, as fragrances in cosmetics, and as intermediates in the manufacture of perfume chemicals. They have also been employed as scent in household products (e.g., detergents, soaps, room air-fresheners and insect repellents) and as active ingredients in some old drugs (Sticher, 1977; Leung, 1980). Pinene, for instance, is one of the main constituents of a mixture of six monoterpenes (Rowachol®) used to dissolve gallstones (Ellis et al., 1984), and α-terpinene is one of the putative active ingredients of tea tree (Melaleuca alternifolia) oil, an antibacterial and antifungal remedy employed in both veterinary and human medicine (Dryden et al., 2004).

Although monoterpenes are generally regarded as safe substances, some monoterpenoid constituents of plant essential oils have been found to possess hepatotoxic (e.g. pulegone) and genotoxic and carcinogenic properties (e.g. safrole). Not withstand the importance of human exposure to a variety of monoterpenoid compounds, only a few of them have been submitted to a comprehensive toxicological evaluation so far.

This study was undertaken to provide data on the genotoxic activity of three hydrocarbon monoterpenes (C10H16) exhibiting acyclic (β-myrcene), monocyclic (α-terpinene) and bicyclic (α-pinene) structures.

Section snippets

Chemicals

β-Myrcene, α-terpinene, (−)-α-pinene, (+)-α-pinene, 4-nitroquinoline-N-oxide (4-NQNO), 2-aminofluorene (2-AF), and 2-aminoanthracene (2AA) were all purchased from Sigma Chemical Co. (St. Louis, MO, USA). Sodium azide (SA) and 2-nitrofluorene (2-NF) were bought from Aldrich Chemical Co. Purity of all tested substances was 95% or higher.

Metabolic activation system (S9 mixture)

Lyophilized rat liver S9 fraction induced by Aroclor 1254 was purchased from Moltox® (Molecular Toxicology Inc, Boone, NC, USA). The S9 mixture was prepared as

Results and discussion

Toxicity of β-myrcene, α-terpinene and (+) and (−)-pinene to S. typhimurium strains TA97a, TA98, TA100 and TA1535 was investigated in a first set of experiments shown in the upper part of Table 1, Table 2, Table 3, Table 4. Toxicity was apparent either as a reduction in the number of his+ revertant bacteria colonies and or as a change of auxotrophic background growth (i.e. the background lawn). Doses at which toxicity appeared as an alteration of the background lawn are marked with an asterisk

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