Acute, reproductive, and developmental toxicity of essential oils assessed with alternative in vitro and in vivo systems

Peter Lanzerstorfer,Georg Sandner,Tobias Aumiller,Johannes Pitsch,Julian Weghuber,Bianca Mascher

doi:10.1007/s00204-020-02945-6

Peter Lanzerstorfer, Georg Sandner + Show 4 more

Open Access

https://doi.org/10.1007/s00204-020-02945-6

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Abstract

Essential oils (EOs) have attracted increased interest for different applications such as food preservatives, feed additives and ingredients in cosmetics. Due to their reported variable composition of components, they might be acutely toxic to humans and animals in small amounts. Despite the necessity, rigorous toxicity testing in terms of safety evaluation has not been reported so far, especially using alternatives to animal models. Here, we provide a strategy by use of alternative in vitro (cell cultures) and in vivo (Caenorhabditis elegans, hen’s egg test) approaches for detailed investigation of the impact of commonly used rosemary, citrus and eucalyptus essential oil on acute, developmental and reproductive toxicity as well as on mucous membrane irritation. In general, all EOs under study exhibited a comparable impact on measured parameters, with a slightly increased toxic potential of rosemary oil. In vitro cell culture results indicated a concentration-dependent decrease of cell viability for all EOs, with mean IC50 values ranging from 0.08 to 0.17% [v/v]. Similar results were obtained for the C. elegans model when using a sensitized bus-5 mutant strain, with a mean LC50 value of 0.42% [v/v]. In wild-type nematodes, approximately tenfold higher LC50 values were detected. C. elegans development and reproduction was already significantly inhibited at concentrations of 0.5% (wild-type) and 0.1% (bus-5) [v/v] of EO, respectively. Gene expression analysis revealed a significant upregulation of xenobiotic and oxidative stress genes such as cyp-14a3, gst-4, gpx-6 and sod-3. Furthermore, all three EOs under study showed an increased short-time mucous membrane irritation potential, already at 0.5% [v/v] of EO. Finally, GC–MS analysis was performed to quantitate the relative concentration of the most prominent EO compounds. In conclusion, our results demonstrate that EOs can exhibit severe toxic properties, already at low concentrations. Therefore, a detailed toxicological assessment is highly recommended for each EO and single intended application.

Highlights

Natural compounds have been a good source for new bioactive drugs for a long time and provide unique structural diversity (Cragg and Newman 2013; Lautie et al 2020)
The Regulation on cosmetic products (1223/2009), REACH (2007/2006) and Classification, Labelling and Packaging (CLP) (1272/2008) are examples of EU legislation that require the replacement of animal testing
Cell and tissue culture models, the hen’s egg test on the chorioallantoic membrane (HET-CAM) as well as the Caenorhabditis elegans (C. elegans) model have gained considerable attention as alternative screening platforms to evaluate the toxicity of bioactive compounds in various context (Haselgrubler et al 2017; Hunt 2017; Piersma 2004; Scheel et al 2011)

Summary

Introduction

Natural compounds (phytochemicals) have been a good source for new bioactive drugs for a long time and provide unique structural diversity (Cragg and Newman 2013; Lautie et al 2020). Detailed information about possible negative effects of phytochemical addition on animal and human health is often lacking Within this regard, the toxic potential of phytochemicals might be underestimated, as natural compounds are not necessarily safer than other products. There is no mandatory use of alternative methods in the field of novel food and feed additives, which have to undergo a scientificbased safety assessment. Such supplements, before being placed on the market, are currently evaluated by scientific panels, which deliver opinions on the safety and toxicity, mainly based on the available literature data (von Holst et al 2016). Cell and tissue culture models, the hen’s egg test on the chorioallantoic membrane (HET-CAM) as well as the Caenorhabditis elegans (C. elegans) model have gained considerable attention as alternative screening platforms to evaluate the toxicity of bioactive compounds in various context (Haselgrubler et al 2017; Hunt 2017; Piersma 2004; Scheel et al 2011)

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