Effects of co-formulants on the absorption and secretion of active substances in plant protection products in vitro

Abstract

Currently, the authorisation process for plant protection products (PPPs) relies on the testing of acute and topological toxicity only. Contrastingly, the evaluation of active substances includes a more comprehensive set of toxicity studies. Nevertheless, mixture effects of active ingredients and co-formulants may result in increased toxicity. Therefore, we investigated effects of surface active co-formulants on the toxicity of two PPPs focussing on qualitative and quantitative toxicokinetic effects on absorption and secretion. The respective products are based on the active substances abamectin and fluroxypyr-meptyl and were tested for cytotoxicity in the presence or absence of the corresponding surfactants and co-formulants using Caco-2 cells. In addition, the effect of co-formulants on increased cellular permeation was quantified using LC–MS/MS, while potential kinetic mixture effects were addressed by fluorescence anisotropy measurements and ATPase assays. The results show that surface active co-formulants significantly increase the cytotoxicity of the investigated PPPs, leading to more than additive mixture effects. Moreover, analytical investigations show higher efflux ratios of both active substances and the metabolite fluroxypyr upon combination with certain concentrations of the surfactants. The results further point to a significant and concentration-dependent inhibition of Pgp transporters by most of the surfactants as well as to increased membrane fluidity. Altogether, these findings strongly support the hypothesis that surfactants contribute to increased cytotoxicity of PPPs and do so by increasing the bioavailability of the respective active substances.

Characterization of the composition of plant protection products in different formulation types employing suspect screening and unknown approaches

“…10,23 Toxicity of co-formulants The increasing use of PPPs has led to widespread concerns about their adverse effect on the environment and especially on human health, particularly those surfactants with non-ionic properties. 7 According to the harmonized classification and labeling approved by the European Union, 1,2-benzisothiazol-3(2H)-one is very toxic to aquatic life and harmful if swallowed. Moreover, this compound causes serious eye damage, skin irritation and might even cause an allergic skin reaction.…”

“…12 However, no further toxicological evaluation or authorization is required for co-formulants, because they are commonly subject to the REACH (Registration, Evaluation, Authorization and restriction of Chemicals) regulation and hence toxicologically tested and assessed depending on their annual production volume. 7 Nevertheless, some studies have demonstrated that some co-formulants, which might not be toxic themselves, can influence the toxicity of PPPs via toxicodynamic and toxicokinetic interactions. 8 Consequently, the Commission Regulation (EU) 2021/383 sets a list of co-formulants that are not accepted for inclusion in PPPs.…”

“…Co‐formulants are usually defined as substances that are added to PPPs but are neither active substances nor safeners or synergists, 3 and they provide specific properties for their application, 7 such as enhancing formulation stability or even optimizing the distribution of formulations on plant surfaces, among others. Co‐formulants perform specific functions in the formulation, such as solvents, surfactants, diluents, thickeners, dispersing agents, binding agents, stabilizing agents, wetting agents, antifoaming agents, preservatives or antifreeze agents 8‐10 …”

“…However, as generally active substances assumed to dominate toxicity, they are analyzed thoroughly for acute, chronic and subchronic effects in short‐ and long‐term in vivo studies 12 . However, no further toxicological evaluation or authorization is required for co‐formulants, because they are commonly subject to the REACH (Registration, Evaluation, Authorization and restriction of Chemicals) regulation and hence toxicologically tested and assessed depending on their annual production volume 7 . Nevertheless, some studies have demonstrated that some co‐formulants, which might not be toxic themselves, can influence the toxicity of PPPs via toxicodynamic and toxicokinetic interactions 8 .…”

Assessment of Co-Formulants in Marketed Plant Protection Products by LC-Q-Orbitrap-MS: Application of a Hybrid Data Treatment Strategy Combining Suspect Screening and Unknown Analysis

“…4 Additionally, other authors have pointed at synergistic effects between the active substances and coformulants, which could result in increased toxicity. 5,6 Concerning EU Regulations, Regulation EC No 2021/383 lists the forbidden co-formulants for use in PPPs or marketed adjuvants. 7 However, this regulation generally allows concentrations less than 0.1% (w/w) of these unacceptable coformulants unless otherwise noted, as these could be unintentional impurities.…”