Frauke Stock

Biological effects of imidazolium ionic liquids with varying chain lengths in acute Vibrio fischeri and WST-1 cell viability assays

Detailed biological studies of methyl- and some ethylimidazolium ionic liquids in luminescent bacteria as well as in the IPC-81 (leukemia cells) and C6 (glioma cells) rat cell lines are presented. Effective concentrations in these test systems are generally some orders of magnitude lower than effective concentrations [corrected] of the conventional solvents acetone, acetonitrile, methanol, and methyl t-butyl ether. No general influence of the anionic compound in the ionic liquids on toxicity could be found, although they seem to modulate toxicity in some cases. The clear influence of the alkyl chain length on toxicity was quantified by linear regression analysis. Alkyl chain length of the longer alkyl chain was varied from 3 to 10 carbon atoms. Consequences for a design of sustainable alternative solvents are briefly sketched.

How hazardous are ionic liquids? Structure–activity relationships and biological testing as important elements for sustainability evaluationThis work was presented at the Green Solvents for Catalysis Meeting held in Bruchsal, Germany, 13–16th October 2002.

For ionic liquids only few toxicological and/or ecotoxicological data are available until now. A strategy is presented which aims at an environmental risk assessment of chemicals, using a combination of structure–activity relationships (SAR), toxicological and ecotoxicological tests and modelling. The parts “test-kit-concept” and “multidimensional risk analysis” are described in detail by means of selected imidazolium ionic liquids. The iterative process of this strategy offers a tool for sustainable product design.

Anion effects on the cytotoxicity of ionic liquids

Most recent investigations concerning the toxicological and ecotoxicological risk potentials of ionic liquids are predominantly focusing on the cation moieties. In this study we elucidate, whether the anion species commonly used in ionic liquids are exhibiting intrinsic cytotoxic effects and if these effects can be rationalised by thinking in terms of structure–activity relationships (T-SAR). As test system to measure the cell viability as toxicologically relevant endpoint the IPC-81 rat leukemia cell line and the WST-1 assay were employed. Our results show an anion effect in ionic liquids on cytotoxicity for 10 of 27 tested anions. For the remaining 17 anions from our test kit no significant effect was found. With respect to structure–activity relationships, lipophilicity and/or vulnerability to hydrolytic cleavage seem to be the key structural features leading to the observed anion cytotoxicity. We also conclude that the model of concentration addition may be useful to estimate the EC50 values of ionic liquids that have not been tested or even synthesised yet. This can help to design not only task specific but also inherently safer ionic liquids.

Progress in evaluation of risk potential of ionic liquids—basis for an eco-design of sustainable products

Motivated by the prevailing need for a sustainable development and taking the principles of Green Chemistry as a starting point, the present paper describes new and updated findings regarding a sustainable product design for ionic liquids. The focus is on environmental risk. Nevertheless, cytotoxicity testing and first indicative results from a genotoxicity study extend present knowledge also with regard to possible effects on humans. The structural variability of commercially available ionic liquids as well as the abundance of theoretically accessible ionic liquids is illustrated and the consequences for an integrated risk assessment accompanying the development process are discussed. The side chain effect on toxicity for imidazolium type ionic liquids was confounded by more complex biological testing. Also, an influence of an anion on cytotoxicity is shown for the first time. Testing of presumed metabolites of the imidazolium type cations showed a significantly lower biological activity in cytotoxicity studies than their parent compounds. The importance of a purity assessment for ionic liquids is pointed out and a collection of methods that is believed to be adequate is presented. In addition to risk analysis, the use of life cycle analysis for the multi-objective problem of designing ionic liquids is sketched and an eco-design scheme for ionic liquids is proposed. In conclusion, the paper illustrates the complex nature of the development processes ionic liquids are currently undergoing and provides guidance on which aspects have to be kept in mind.

Effects of ionic liquids on the acetylcholinesterase – a structure–activity relationship consideration

Ionic liquids are discussed as sustainable green solvents, but toxicity and ecotoxicity data are rare. In this paper we present our results for different ionic liquids with the acetylcholinesterase inhibition assay. The results show that the acetylcholinesterase can be inhibited by ionic liquids containing a cation with a positively charged nitrogen and a certain lipophilicity. We tested imidazolium ionic liquids with different alkyl chains at R1 and R2 as well as with different anions and compared these results with our findings for other cation structures such as pyridinium ionic liquids and phosphonium ionic liquids. According to our results imidazolium and pyridinium ionic liquids inhibit the purified enzyme with EC50 values as low as 13 μM. The bulky phosphonium ionic liquids were less inhibitory. These results can be rationalized by structure–activity relationship considerations.

Qualitative and quantitative structure activity relationships for the inhibitory effects of cationic head groups, functionalised side chains and anions of ionic liquids on acetylcholinesterase

To contribute to a deeper insight into the hazard potential of ionic liquids to humans and the environment, an acetylcholinesterase (AchE) inhibition screening assay was used to identify toxicophore substructures and interaction potentials mediating enzyme inhibition.The positively charged nitrogen atom, a widely delocalised aromatic system, and the lipophilicity of the side chains connected to the cationic head groups can be identified as the key structural elements in binding to the enzymes active site. With respect to this, the dimethylaminopyridinium, the quinolinium and the pyridinium head groups exhibit a very strong inhibitory potential to the enzyme with IC50 values around 10 µM. In contrast, the polar and non-aromatic morpholinium head group is found to be only weakly inhibiting to the enzyme activity, with IC50 values > 500 µM.The introduction of polar hydroxy, ether or nitrile functions into the alkyl side chain is shown to be a potent structural alteration to shift the corresponding ionic liquids to a lower inhibitory potential. Supporting this fact, for a series of imidazolium cations, a QSAR correlation was set up by the linear regression of the log IC50versus the logarithm of the HPLC-derived lipophilicity parameter k0.Additionally, a broad set of anion species (inorganic, organic and complex borate anions), commonly used as ionic liquid counterions, was tested and the vast majority exhibited no effect on AchE. Only the fluoride and fluoride containing anion species which readily undergo hydrolytic cleavage can be identified to act as AchE inhibitors.

Integrated testing and intelligent assessment-new challenges under REACH.

Background, aim and scopeDue to a number of drawbacks associated with the previous regime for the assessment of new and existing chemicals, the European Union established a new regulation concerning the registration, evaluation, authorisation and restriction of chemicals (REACH). All relevant industrial chemicals must now be assessed. Instead of the authorities, industry itself is responsible for the risk assessment. To achieve better and more efficient assessments while reducing animal testing, all information—standard, non-standard and non-testing—has to be used in an integrated manner. To meet these challenges, the current technical guidance documents for risk assessment of new and existing chemicals had to be updated and extended considerably. This was done by experts in a number of REACH Implementation Projects. This paper presents the most relevant results of the expert Endpoint Working Group on Aquatic Toxicity in order to illustrate the change of paradigm in the future assessment of hazards to the aquatic environment by chemical substances.Main features and challengesREACH sets certain minimum data requirements in order to achieve a high level of protection for human health and the environment. It encourages the assessor to use alternative information instead of or in addition to standard one. This information has to be equivalent to the standard information requirement and adequate to draw overall conclusions with respect to the regulatory endpoints classification and labelling, persistent, bioaccumulative and toxic (PBT) assessment and predicted no-effect concentrations (PNEC) derivation. The main task of the expert working group was to develop guidance on how to evaluate the toxicity of a substance based on integration of information from different sources and of various degrees of uncertainty in a weight of evidence approach.Integrated testing and intelligent assessmentIn order to verify the equivalence and adequacy of different types of information, a flexible sequence of steps was proposed, covering characterisation of the substance, analysis of modes of action, identification of possible analogues, evaluation of existing in vivo and in vitro testing data as well as of QSAR results. Finally, all available data from the different steps have to be integrated to come to an overall conclusion on the toxicity of the substance. This weight of evidence approach is the basis for the development of integrated testing strategies (ITS), in that the available evidence can help to determine subsequent testing steps and is essential for an optimal assessment. Its flexibility helps to meet the different requirements for drawing conclusions on the endpoints classification and labelling, PNEC derivation as well as PBT assessment. The integration of all kinds of additional information in a multi-criteria assessment reduces the uncertainties involved with extrapolation to the ecosystem level. The weight of evidence approach is illustrated by practical examples.Conclusions and perspectivesREACH leads to higher challenges in order to make sound decisions with fewer resources, i.e. to move away from extensive standard testing to an intelligent substance-tailored approach. Expert judgement and integrated thinking are key elements of the weight of evidence concept and ITS, potentially leading to better risk assessments. Important sub-lethal effects such as endocrine disruption, which are not covered by the current procedure, can be considered. Conclusions have to be fully substantiated: Risk communication will be an important aspect of future assessments.

Structure–activity relationships of pyrithiones – IPC-81 toxicity tests with the antifouling biocide zinc pyrithione and structural analogs

Zinc pyrithione (1-hydroxypyridine-2-thione, zinc complex; ZnPT2) is currently viewed as the top prospect for replacing tributyltin antifoulants in ship paints. Thus, the risk assessment of a high scale release of ZnPT2 to the natural environment is of increasing importance. The knowledge of the molecular mechanisms related to biological effects of ZnPT2 and its transformation products is crucial for this assessment and thus for the decision whether pyrithiones are sound or “green” alternatives to organotin antifoulants. A multitude of biological effects of pyrithiones is already known while the underlying molecular mechanisms of action remain obscure. This study presents toxicological data of zinc pyrithione and several structural analogs in rat leukemic cells (IPC-81). The N-hydroxythioamide functional group proved to play a significant role in the molecular mechanisms related to the biological action. Structural analogs, which are deprived of one or more molecular interaction or chemical reaction potentials given by this group (namely pyridine, pyridine 1-oxide and pyridine 2-thione, bis(2-pyridinyl)disulfide, and three methylated metabolites), exhibit far less toxic potential in IPC-81 cells than pyrithiones (i.e., 2-pyridinethione-1-oxides). In particular the trans-metallization products of ZnPT2, iron (FePT3) and copper (CuPT2) pyrithione, and the oxidation product bis(2-pyridinyl)disulfide 1,1′-dioxide (pyrithione disulfide, (PT2)) have been proven to be as toxic as ZnPT2 and tributyltin chloride in IPC-81 cells. CuPT2, FePT3 and (PT)2 need to be considered as environmental transformations products of ZnPT2.

Thresholds of toxicological concern for endocrine active substances in the aquatic environment

The threshold of toxicological concern (TTC) concept proposes that an exposure threshold value can be derived for chemicals, below which no significant risk to human health or the environment is expected. This concept goes further than setting acceptable exposure levels for individual chemicals, because it attempts to set a de minimis value for chemicals, including those of unknown toxicity, by taking the chemicals structure or mode of action (MOA) into consideration. This study examines the use of the TTC concern concept for endocrine active substances (EAS) with an estrogenic MOA. A case study formed the basis for a workshop of regulatory, industry and academic scientists held to discuss the use of the TTC in aquatic environmental risk assessment. The feasibility and acceptability, general advantages and disadvantages, and the specific issues that need to be considered when applying the TTC concept for EAS in risk assessment were addressed. Issues surrounding the statistical approaches used to derive TTCs were also discussed. This study presents discussion points and consensus findings of the workshop.

Identification, assessment and management of "endocrine disruptors" in wildlife in the EU substance legislation—Discussion paper from the German Federal Environment Agency (UBA)

A discussion paper was developed by a panel of experts of the German Federal Environment Agency (UBA) contributing to the on-going debate on the identification, assessment and management of endocrine disruptors with a view to protect wildlife according to the EU substance legislation (plant protection products, biocides, industrial chemicals). Based on a critical synthesis of the state-of-the-art regarding regulatory requirements, testing methods, assessment schemes, decision-making criteria and risk management options, we advise an appropriate and consistent implementation of this important subject into existing chemicals legislation in Europe. Our proposal for a balanced risk management of endocrine disruptors essentially advocates transparent regulatory decision making based on a scientifically robust weight of evidence approach and an adequate risk management consistent across different legislations. With respect to the latter, a more explicit consideration of the principle of proportionality of regulatory decision making and socio-economic benefits in the on-going debate is further encouraged.