Subacute toxicity assessment of biobased ionic liquids in rats

Highlights

• Fatty acid based ILs may be a strategy to produce low or non-toxic additives.

• No changes in lipid peroxidation were observed.

• Triglycerides was a dependent response on the IL concentration and structure.

Abstract

Ionic liquids (ILs) derived from compounds obtained from natural sources, such as fatty acids (FAs) have attracted the interest of the scientific and industrial communities because of their sustainable appeal and possible low toxic effects or nontoxicity. These aspects open new perspective of applications in other fields, which demands a better comprehension of their toxicity. This work evaluated the subacute toxicity of bis(2-hydroxyethyl)ammonium carboxylates in Wistar rats, considering the alkyl chain length of FAs (capric and oleic acids), and the concentration (0.16%, 1.6% or 3.2%, w_IL/w_Oil) of ILs added in diets. The blood serum of the rats was evaluated in relation to total cholesterol, triglycerides, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and γ-glutamyl transferase. Lipid peroxidation was determined in plasma, liver and kidney tissues by determining the level of thiobarbituric acid reactive substances. Histological analyses of the liver and kidney tissues were performed in order to evaluate morphological changes. No signal of toxicity was observed according to lipid peroxidation. Triglycerides increased with the increasing of the concentration and alkyl chain length of the IL, but no difference in serum level of lipid peroxidation was observed. This behavior may be attributed to the amphiphilic nature of FAs based ILs, which might facilitate lipid digestion. However, more studies are necessary in order to understand such behavior. Therefore, the synthesis of ILs from FAs, has been evaluated as a strategy to produce compounds with low or without toxicity for the agro-food, pharmaceutical or cosmetic industries.

Introduction

Ionic liquids (ILs) are salts with a melting temperature below 373.15 K (Rogers & Seddon, 2003). They are promising compounds and widely applied in the chemical industry, especially as alternative sustainable solvents to replace volatile organic solvents in extraction processes (Ostadjoo et al., 2018, Rogers and Seddon, 2003). The IL technology is also used in the pharmaceutical industry as a strategy to produce active pharmaceutical ingredients (APIs) (Stoimenovski, MacFarlane, Bica, & Rogers, 2010). This approach consists of combining bioactive compounds to produce drugs in the IL form with improved physical properties, such as lower melting point (e.g. liquid drugs at body temperature), and higher aqueous solubility, stability and permeability (Shamshina et al., 2017, Wang et al., 2014). The improvement of these physical properties may be related to better biological properties and bioavailability (Shamshina, Kelley, Gurau, & Rogers, 2015). This strategy also allows the delivery of two or more bioactive compounds used as IL precursors, with different or complementary functional properties at once in the body (Shamshina et al., 2015).

In the last decade, the promising properties of ILs as solvents have attracted the attention of the food industry. Literature (Toledo Hijo et al., 2016, Martins et al., 2017, Passos et al., 2014) has shown their application in extraction processes and food analysis. The main target of extraction processes using ILs are bioactive compounds from food matrices, such as phenolic compounds, essential oils, carotenoids and caffeine. ILs have been used in food analysis for the determination of preservatives, heavy metals, dyes, herbicides, pesticides and vitamins. These reported applications in the food industry are mainly focused on the commonly used ILs, such as imidazolium, phosphonium and pyridinium based ILs, which are associated with toxicity concerns or derived from compounds considered as toxic (Toledo Hijo et al., 2016). In contrast, a new concept of biobased ILs has emerged in recent years, which is based on the use of compounds obtained from natural sources for their synthesis. Based on this approach, novel promising applications of ILs in the food industry have been recently proposed (Toledo Hijo et al., 2016), especially on the production of food lubricants (Naumov Vladimir Nikolaevich, 2010, Oulego et al., 2019), surfactants (Klein et al., 2008, Toledo Hijo et al., 2017) and preservatives (Pendleton & Gilmore, 2015). These technological developments indicate that the IL strategy might be a trend in such a field. The most important feature of ILs of interest to food industry is the possibility of producing them from natural sources, bioactive compounds, or even from food ingredients (e.g. fatty acids). This is remarkable considering that the search of healthier, functional, sustainable and natural derived additives and coadjuvants is one of the major challenges facing the food industry nowadays.

In order to establish the use of ILs in the food industry, studies on their safety are already demanded, since new applications of ILs obtained from natural and bioactive sources have been recently reported and gained importance (Martins et al., 2017, Passos et al., 2014, Toledo Hijo et al., 2016). In the food industry field, toxicological studies of ILs play an important role in order to evaluate the use of natural and bioactive compounds as a feasible approach to obtain new, safe and functional additives in the IL form as alternative to replace common food additives, especially those associated to toxicity concerns, such as several preservatives (Sharma, 2015). Toxicity studies might also provide a significant contribution to the IL field considering the demanding of ILs with low or non-toxic effects in comparison to the common ILs. Thus, such studies are necessary taking into account that ILs are already considered as coadjuvants in extraction processes and food analysis and might have potential use as new food additives. However, toxicological studies of biobased ILs for food use are still scarce. The Food and Drug Administration (FDA) recommends a set of toxicological studies (e.g. genetic toxicity tests, toxicity studies with rodents and nonrodents, developmental and reproductive toxicity studies), which must be performed before considering a substance as safe in the conditions of intended use and a potential food additive (Toledo Hijo et al., 2016). In fact, the toxicity aspect of ILs has been the major drawback of using them in the food industry, despite the divergence in the literature on such a topic (Hough and Rogers, 2007, Jodynis-Liebert et al., 2009, Jodynis-Liebert et al., 2010, Landry et al., 2005, Zhao et al., 2007). Literature still presents a significant lack of works on developmental (Bailey et al., 2008, Bailey et al., 2010) and oral toxicity of ILs (Dumitrescu et al., 2014, Jodynis-Liebert et al., 2009, Jodynis-Liebert et al., 2010, Landry et al., 2005, Leitch et al., 2020, Pernak et al., 2011, Xu et al., 2011, Xu et al., 2013, Yu et al., 2008) and most of the works have been focused on the common ILs, such as those with imidazolium group (Bailey et al., 2010, Landry et al., 2005, Leitch et al., 2020, Xu et al., 2011, Xu et al., 2013, Yu et al., 2008), which have presented certain toxicity levels. On the other hand, works on the application of biobased ILs, including food related applications, are increasing and may demand studies of their oral toxicity.

In this context, a new concept of biobased ILs has gained attraction for food use due to the use of natural and bioactive compounds for their synthesis, such as fatty acids (Fan et al., 2018, Klein et al., 2013), amino acids (Hou, Li, & Zong, 2013), choline (Klein et al., 2013, Mena et al., 2020, Nockemann et al., 2007) and other natural acids (Billeci et al., 2020, Peric et al., 2013). The toxicity of ILs derived from organic acids and 2-hydroxyethylamine was firstly studied by Peric et al. (2013). The authors reported interesting results in terms of sustainability and safety, considering that they did not present ecotoxicity toward aquatic systems in comparison to the imidazolium based ILs. These studies on the toxicity of biobased ILs have been considered a big step towards the obtainment of more sustainable ILs with higher biodegradability and environmentally friendly. However, most of them have been performed using microorganisms or aquatics systems and further effort is necessary to assess their cytotoxicity and oral toxicity, taking into account their impact in animal and human health, and food safety. To the best of our knowledge, only few studies on the subacute toxicity of ILs using mammals (Jodynis-Liebert et al., 2009; Jadwiga Jodynis-Liebert et al., 2010) have been published to date and there are no data available on the subacute or acute oral toxicity of biobased ILs in mammals, especially ILs synthesized with FAs.

Fatty acids (FAs) are naturally present in vegetable oils, such as soy and sunflower oils and can be obtained through the hydrolysis of triacylglycerol molecules. They are also responsible for the acidity and off-flavor of crude vegetable oils. This is the reason why they have to be removed from crude vegetable oils, representing so a significant byproduct of the vegetable oil industry. They are Generally Recognized as Safe (GRAS) substances and food additives, and some of them are essential for the human organism, such as oleic and linoleic acids, also known as omega 9 and omega 6, for example, which have been reported to have benefits to human health, such as the prevention of cardiovascular diseases (Simopoulos, 2008). ILs derived from FAs have potential applications as soaps or surfactants (Klein et al., 2008, Klein et al., 2013, Toledo Hijo et al., 2017) and in the formulation of food lubricants (Nikolaevich, 2010).

In this context, FAs might be interesting precursors to produce low toxic or possibly edible ILs for the food industry, since they are obtained from natural sources, are food additives with functional properties and represent a renewable feedstock. Thus, oral toxicological tests are demanded to evaluate them as safe food additives or coadjuvants. Therefore, this work was aimed at evaluating the subacute toxicity of ILs derived from bis(2-hydroxyethyl)amine and FAs in Wistar rats, considering the alkyl chain length of the FAs, used as anion, and the concentration of ILs added in diets.