New approach methods to assess developmental and adult neurotoxicity for regulatory use: a PARC work package 5 project.

In the European regulatory context, rodent in vivo studies are the predominant source of neurotoxicity information. Although they form a cornerstone of neurotoxicological assessments, they are costly and the topic of ethical debate. While the public expects chemicals and products to be safe for the developing and mature nervous systems, considerable numbers of chemicals in commerce have not, or only to a limited extent, been assessed for their potential to cause neurotoxicity. As such, there is a societal push toward the replacement of animal models with in vitro or alternative methods. New approach methods (NAMs) can contribute to the regulatory knowledge base, increase chemical safety, and modernize chemical hazard and risk assessment. Provided they reach an acceptable level of regulatory relevance and reliability, NAMs may be considered as replacements for specific in vivo studies. The European Partnership for the Assessment of Risks from Chemicals (PARC) addresses challenges to the development and implementation of NAMs in chemical risk assessment. In collaboration with regulatory agencies, Project 5.2.1e (Neurotoxicity) aims to develop and evaluate NAMs for developmental neurotoxicity (DNT) and adult neurotoxicity (ANT) and to understand the applicability domain of specific NAMs for the detection of endocrine disruption and epigenetic perturbation. To speed up assay time and reduce costs, we identify early indicators of later-onset effects. Ultimately, we will assemble second-generation developmental neurotoxicity and first-generation adult neurotoxicity test batteries, both of which aim to provide regulatory hazard and risk assessors and industry stakeholders with robust, speedy, lower-cost, and informative next-generation hazard and risk assessment tools.

Curated mode-of-action data and effect concentrations for chemicals relevant for the aquatic environment.

Chemicals in the aquatic environment can be harmful to organisms and ecosystems. Knowledge on effect concentrations as well as on mechanisms and modes of interaction with biological molecules and signaling pathways is necessary to perform chemical risk assessment and identify toxic compounds. To this end, we developed criteria and a pipeline for harvesting and summarizing effect concentrations from the US ECOTOX database for the three aquatic species groups algae, crustaceans, and fish and researched the modes of action of more than 3,300 environmentally relevant chemicals in literature and databases. We provide a curated dataset ready to be used for risk assessment based on monitoring data and the first comprehensive collection and categorization of modes of action of environmental chemicals. Authorities, regulators, and scientists can use this data for the grouping of chemicals, the establishment of meaningful assessment groups, and the development of in vitro and in silico approaches for chemical testing and assessment.

A mechanistic understanding of the effects of polyethylene terephthalate nanoplastics in the zebrafish (Danio rerio) embryo.

Plastic pollution, especially by nanoplastics (NPs), has become an emerging topic due to the widespread existence and accumulation in the environment. The research on bioaccumulation and toxicity mechanism of NPs from polyethylene terephthalate (PET), which is widely used for packaging material, have been poorly investigated. Herein, we report the first use of high-resolution magic-angle spinning (HRMAS) NMR based metabolomics in combination with toxicity assay and behavioural end points to get systems-level understanding of toxicity mechanism of PET NPs in intact zebrafish embryos. PET NPs exhibited significant alterations on hatching and survival rate. Accumulation of PET NPs in larvae were observed in liver, intestine, and kidney, which coincide with localization of reactive oxygen species in these areas. HRMAS NMR data reveal that PET NPs cause: (1) significant alteration of metabolites related to targeting of the liver and pathways associated with detoxification and oxidative stress; (2) impairment of mitochondrial membrane integrity as reflected by elevated levels of polar head groups of phospholipids; (3) cellular bioenergetics as evidenced by changes in numerous metabolites associated with interrelated pathways of energy metabolism. Taken together, this work provides for the first time a comprehensive system level understanding of toxicity mechanism of PET NPs exposure in intact larvae.

Implementation of Zebrafish Ontologies for Toxicology Screening.

Toxicological evaluation of chemicals using early-life stage zebrafish (Danio rerio) involves the observation and recording of altered phenotypes. Substantial variability has been observed among researchers in phenotypes reported from similar studies, as well as a lack of consistent data annotation, indicating a need for both terminological and data harmonization. When examined from a data science perspective, many of these apparent differences can be parsed into the same or similar endpoints whose measurements differ only in time, methodology, or nomenclature. Ontological knowledge structures can be leveraged to integrate diverse data sets across terminologies, scales, and modalities. Building on this premise, the National Toxicology Program's Systematic Evaluation of the Application of Zebrafish in Toxicology undertook a collaborative exercise to evaluate how the application of standardized phenotype terminology improved data consistency. To accomplish this, zebrafish researchers were asked to assess images of zebrafish larvae for morphological malformations in two surveys. In the first survey, researchers were asked to annotate observed malformations using their own terminology. In the second survey, researchers were asked to annotate the images from a list of terms and definitions from the Zebrafish Phenotype Ontology. Analysis of the results suggested that the use of ontology terms increased consistency and decreased ambiguity, but a larger study is needed to confirm. We conclude that utilizing a common data standard will not only reduce the heterogeneity of reported terms but increases agreement and repeatability between different laboratories. Thus, we advocate for the development of a zebrafish phenotype atlas to help laboratories create interoperable, computable data.

Trout and Human Plasma Protein Binding of Selected Pharmaceuticals Informs the Fish Plasma Model.

Concerns are increasing that pharmaceuticals released into the environment pose a risk to nontarget organism such as fish. The fish plasma model is a read-across approach that uses human therapeutic blood plasma concentrations for estimating likely effects in fish. However, the fish plasma model neglects differences in plasma protein binding between fish and humans. Because binding data for fish plasma are scarce, the binding of 12 active pharmaceutical ingredients (APIs; acidic, basic, and neutral) to rainbow trout (Oncorhynchus mykiss) and human plasma was measured using solid-phase microextraction (SPME). The plasma/water distribution ratios (D plasma/w ) of neutral and basic APIs were similar for trout and human plasma, differing by no more than a factor of 2.7 for a given API. For the acidic APIs, the D plasma/w values of trout plasma were much lower than for human plasma, by up to a factor of 71 for naproxen. The lower affinity of the acidic APIs to trout plasma compared with human plasma suggests that the bioavailability of these APIs is higher in trout. Read-across approaches like the fish plasma model should account for differences in plasma protein binding to avoid over- or underestimation of effects in fish. For the acidic APIs, the effect ratio of the fish plasma model would increase by a factor of 5 to 60 if the unbound plasma concentrations were used to calculate the effect ratio. Environ Toxicol Chem 2022;41:559-568. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

The Role of Behavioral Ecotoxicology in Environmental Protection.

For decades, we have known that chemicals affect human and wildlife behavior. Moreover, due to recent technological and computational advances, scientists are now increasingly aware that a wide variety of contaminants and other environmental stressors adversely affect organismal behavior and subsequent ecological outcomes in terrestrial and aquatic ecosystems. There is also a groundswell of concern that regulatory ecotoxicology does not adequately consider behavior, primarily due to a lack of standardized toxicity methods. This has, in turn, led to the exclusion of many behavioral ecotoxicology studies from chemical risk assessments. To improve understanding of the challenges and opportunities for behavioral ecotoxicology within regulatory toxicology/risk assessment, a unique workshop with international representatives from the fields of behavioral ecology, ecotoxicology, regulatory (eco)toxicology, neurotoxicology, test standardization, and risk assessment resulted in the formation of consensus perspectives and recommendations, which promise to serve as a roadmap to advance interfaces among the basic and translational sciences, and regulatory practices.

Cellular Metabolism in High-Throughput In Vitro Reporter Gene Assays and Implications for the Quantitative In Vitro-In Vivo Extrapolation.

High-throughput in vitro reporter gene assays are increasingly applied to assess the potency of chemicals to alter specific cellular signaling pathways. Genetically modified reporter gene cell lines provide stable readouts of the activation of cellular receptors or transcription factors of interest, but such reporter gene assays have been criticized for not capturing cellular metabolism. We characterized the metabolic activity of the widely applied AREc32 (human breast cancer MCF-7), ARE-bla (human liver cancer HepG2), and GR-bla (human embryonic kidney HEK293) reporter gene cells in the absence and in the presence of benzo[a]pyrene (BaP), an AhR ligand known to upregulate cytochrome P450 in vitro and in vivo. We combined fluorescence microscopy with chemical analysis, real-time PCR, and ethoxyresorufin-O-deethylase activity measurements to track temporal changes in BaP and its metabolites in the cells and surrounding medium over time in relation to the expression and activity of metabolic enzymes. Decreasing BaP concentrations and formation of metabolites agreed with the high basal CYP1 activity of ARE-bla and the strong CYP1A1 mRNA induction in AREc32, whereas BaP concentrations were constant in GR-bla, in which neither metabolites nor CYP1 induction was detected. The study emphasizes that differences in sensitivity between reporter gene assays may be caused not only by different reporter constructs but also by a varying biotransformation rate of the evaluated parent chemical. The basal metabolic capacity of reporter gene cells in the absence of chemicals is not a clear indication because we demonstrated that the metabolic activity can be upregulated by AhR ligands during the assay. The combination of methods presented here is suitable to characterize the metabolic activity of cells in vitro and can improve the interpretation of in vitro reporter gene effect data and extrapolation to in vivo human exposure.

Recommendations for Improving Methods and Models for Aquatic Hazard Assessment of Ionizable Organic Chemicals.

Ionizable organic chemicals (IOCs) such as organic acids and bases are an important substance class requiring aquatic hazard evaluation. Although the aquatic toxicity of IOCs is highly dependent on the water pH, many toxicity studies in the literature cannot be interpreted because pH was not reported or not kept constant during the experiment, calling for an adaptation and improvement of testing guidelines. The modulating influence of pH on toxicity is mainly caused by pH-dependent uptake and bioaccumulation of IOCs, which can be described by ion-trapping and toxicokinetic models. The internal effect concentrations of IOCs were found to be independent of the external pH because of organisms' and cells' ability to maintain a stable internal pH milieu. If the external pH is close to the internal pH, existing quantitative structure-activity relationships (QSARs) for neutral organics can be adapted by substituting the octanol-water partition coefficient by the ionization-corrected liposome-water distribution ratio as the hydrophobicity descriptor, demonstrated by modification of the target lipid model. Charged, zwitterionic and neutral species of an IOC can all contribute to observed toxicity, either through concentration-additive mixture effects or by interaction of different species, as is the case for uncoupling of mitochondrial respiration. For specifically acting IOCs, we recommend a 2-step screening procedure with ion-trapping/QSAR models used to predict the baseline toxicity, followed by adjustment using the toxic ratio derived from in vitro systems. Receptor- or plasma-binding models also show promise for elucidating IOC toxicity. The present review is intended to help demystify the ecotoxicity of IOCs and provide recommendations for their hazard and risk assessment. Environ Toxicol Chem 2020;39:269-286. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Combined Ion-Trapping and Mass Balance Models To Describe the pH-Dependent Uptake and Toxicity of Acidic and Basic Pharmaceuticals in Zebrafish Embryos ( Danio rerio).

The aim of the current study was to understand and develop models to predict the pH-dependent toxicity of ionizable pharmaceuticals in embryos of the zebrafish Danio rerio. We found a higher uptake and toxicity with increasing neutral fraction of acids (diclofenac, genistein, naproxen, torasemide, and warfarin) and bases (metoprolol and propranolol). Simple mass balance models accounting for the partitioning to lipids and proteins in the zebrafish embryo were found to be suitable to predict the bioconcentration after 96 h of exposure if pH values did not differ much from the internal pH of 7.55. For other pH values, a kinetic ion-trap model for the zebrafish embryo explained the pH dependence of biouptake and toxicity. The total internal lethal concentrations killing 50% of the zebrafish embryos (ILC50) were calculated from the measured BCF and LC50. The resulting ILC50 were independent of external pH. Critical membrane concentrations were deduced by an internal mass balance model, and apart from diclofenac, whose specific toxicity in fish had already been established, all pharmaceuticals were confirmed to act as baseline toxicants in zebrafish.

pH-Dependent Uptake and Sublethal Effects of Antihistamines in Zebrafish (Danio rerio) Embryos.

Reported off-target effects of antihistamines in humans draw interest in ecotoxicity testing of first- and second-generation antihistamines, the latter of which have fewer reported side effects in humans. Because antihistamines are ionizable compounds, the pH influences uptake and toxicity and thus is highly relevant when conducting toxicity experiments. Zebrafish embryo toxicity tests were performed with the 3 first-generation antihistamines ketotifen, doxylamine, and dimethindene and the 2 second-generation antihistamines cetirizine and levocabastine at pH 5.5, 7.0, and 8.0. We detected effects on survival, phenotype, swimming activity, and heart rate for 4 antihistamines with the exception of levocabastine, which did not show any lethal or sublethal effects. When compared to lethal concentrations, effect concentrations neither of phenotype malformation nor of swimming activity or heart rate deviated by more than a factor of 10 from lethal concentrations, indicating that all sublethal effects were fairly nonspecific. First-generation antihistamines are weak bases and showed decreasing external effect concentrations with increasing neutral fraction, accompanied by increased uptake in the fish embryo. As a result, internal effect concentrations were independent from external pH. The pH-dependent toxicity originates from speciation-dependent uptake, with neutral species taken up in higher amounts than the corresponding ionic species. Cetirizine, which shifts from a zwitterionic to an anionic state in the measured pH range, did not show any pH-dependent uptake or toxicity. Environ Toxicol Chem 2019;00:1-11. © 2019 SETAC.

Can Environmentally Relevant Neuroactive Chemicals Specifically Be Detected with the Locomotor Response Test in Zebrafish Embryos?

Chemicals considered as neuroactive (such as certain pesticides, pharmaceuticals, and industrial chemicals) are among the largest groups of bioactive substances recently detected in European rivers. However, the determination of nervous-system-specific effects has been limited using in vitro tests or conventional end points including lethality. Thus, neurobehavioral tests using in vivo models (e.g., zebrafish embryo) have been proposed as complementary approaches. To investigate the specificity and sensitivity of a light-dark transition locomotor response (LMR) test in 4 to 5 days post fertilization zebrafish with respect to different modes of action (MoAs), we analyzed a set of 18 environmentally relevant compounds with various anticipated MoAs. We found that exposure-induced behavioral alterations were reproducible and dependent on concentration and time. Comparative and quantitative analyses of the obtained locomotor patterns revealed that behavioral effects were not restricted to compounds primarily known to target the nervous system. A clear distinction of MoAs based on locomotor patterns was not possible for most compounds. Furthermore, chemicals with an anticipated same MoA did not necessarily provoke similar behavioral phenotypes. Finally, we determined an increased sensitivity (≥10-fold) compared to observed mortality in the LMR assay for five of eight neuroactive chemicals as opposed to non-neuroactive compounds.

QSAR for baseline toxicity and classification of specific modes of action of ionizable organic chemicals in the zebrafish embryo toxicity test.

The fish embryo toxicity (FET) test with the zebrafish Danio rerio is widely used to assess the acute toxicity of chemicals thereby serving as animal alternative to the acute fish toxicity test. The minimal toxicity of neutral chemicals in the FET can be predicted with a previously published Quantitative Structure-Activity Relationship (QSAR) based on the liposome-water partition coefficient Klipw. Such a QSAR may serve to plan toxicity testing and to evaluate whether an observed effect is caused by a specific mode of action (MoA). The applicability domain of this QSAR was extended to ionizable organic chemicals (IOC) without any modification of slope and intercept simply by replacing the Klipw with the speciation-corrected liposome-water distribution ratio (Dlipw(pH)) as descriptor for the uptake into the embryo. FET LC50 values of IOCs were extracted from an existing FET database and published literature. IOCs were selected that are present concomitantly as neutral and charged, species, i.e., acids with an acidity constant pKa <10 and bases with pKa>5. IOCs were grouped according to their putative MoA of acute aquatic toxicity. The toxic ratios (TR) in the FET were derived by of the experimental FET-LC50 in comparison with the baseline toxicity QSAR. Baseline toxicants were confirmed to align well with the FET baseline toxicity QSAR (TR < 10). Chemicals identified to act as specific or reactive chemicals with the toxic ratio analysis in the FET test (TR > 10) were generally consistent with MoA classification for acute fish toxicity with a few exceptions that were suspected to have had issues with the stability of the pH during testing. One critical aspect for the effect analysis of ionizable chemicals is the pH, since the difference between pH and pKa determines the speciation and thereby the Dlipw(pH).

Influence of pH on the uptake and toxicity of ß-blockers in embryos of zebrafish, Danio rerio.

ß-Blockers are weak bases with acidity constants related to their secondary amine group. At environmental pH they are protonated with the tendency to shift to their neutral species at more alkaline pH. Here we studied the influence of pH from 5.5 to 8.6 on the toxicity of the four ß-blockers atenolol, metoprolol, labetalol and propranolol in zebrafish embryos, relating toxicity not only in a conventional way to external aqueous concentrations but also to measured internal concentrations. Besides lethality, we evaluated changes in swimming activity and heartbeat, using the Locomotor Response (LMR) method and the Vertebrate Automated Screening Technology (VAST) for high throughput imaging. Effects of metoprolol, labetalol and propranolol were detected on phenotype, heart rate and swimming activity. External effect concentrations decreased with increasing neutral fraction for all three pharmaceuticals, attributed by an enhanced uptake of the neutral species in comparison to the corresponding charged form. The LC50 of metoprolol decreased by a factor of 35 from 1.91 mM with almost complete cationic state at pH 7.0 to 0.054 mM with 8% neutral fraction at pH 8.6. For propranolol the LC50 of 2.42 mM at pH 5.5 was even 100 fold higher than the LC50 at pH 8 with 0.023 mM where 3% were neutral fraction. No effects were detected in the zebrafish embryo exposed to atenolol. The internal concentrations for metoprolol and propranolol were quantified at non-toxic concentrations and at the LC10. Apparent bioconcentration factors (BCF) ranged from 1.96 at pH 7.0 to 32.0 at pH 8.6 for metoprolol and from 1.86 at pH 5.5 to 169 at pH 8.0 for propranolol. The BCFs served to predict the internal effect concentrations from the measured external effect concentrations. Internal effect concentrations of metoprolol and propranolol were in a similar range for all pH-values and for all endpoints. Interestingly, the internal effect concentrations were in the internal concentration range of baseline toxicity, which suggests that the effects of the ß-blockers are rather unspecific, even for sublethal effects on heart rate. In summary, our data confirm that the pH-dependent toxicity related to external concentrations can be explained by toxicokinetic effects and that the internal effect concentrations are pH-independent.

Applicability of the fish embryo acute toxicity (FET) test (OECD 236) in the regulatory context of Registration, Evaluation, Authorisation, and Restriction of Chemicals (REACH).

In 2013 the Organisation for Economic Co-operation and Development (OECD) test guideline (236) for fish embryo acute toxicity (FET) was adopted. It determines the acute toxicity of chemicals to embryonic fish. Previous studies show a good correlation of FET with the standard acute fish toxicity (AFT) test; however, the potential of the FET test to predict AFT, which is required by the Registration, Evaluation, Authorisation, and Restriction of Chemicals (REACH) regulation (EC 1907/2006) and the Classification, Labelling and Packaging (CLP) Regulation (EC 1272/2008), has not yet been fully clarified. In 2015 the European Chemicals Agency (ECHA) requested that a consultant perform a scientific analysis of the applicability of FET to predict AFT. The purpose was to compare the toxicity of substances to fish embryos and to adult fish, and to investigate whether certain factors (e.g., physicochemical properties, modes of action, or chemical structures) could be used to define the applicability boundaries of the FET test. Given the limited data availability, the analysis focused on organic substances. The present critical review summarizes the main findings and discusses regulatory application of the FET test under REACH. Given some limitations (e.g., neurotoxic mode of action) and/or remaining uncertainties (e.g., deviation of some narcotic substances), it has been found that the FET test alone is currently not sufficient to meet the essential information on AFT as required by the REACH regulation. However, the test may be used within weight-of-evidence approaches together with other independent, relevant, and reliable sources of information. The present review also discusses further research needs that may overcome the remaining uncertainties and help to increase acceptance of FET as a replacement for AFT in the future. For example, an increase in the availability of data generated according to OECD test guideline 236 may provide evidence of a higher predictive power of the test. Environ Toxicol Chem 2018;37:657-670. © 2017 SETAC.

Baseline toxicity and ion-trapping models to describe the pH-dependence of bacterial toxicity of pharmaceuticals.

In numerous studies on the toxicity of ionisable organic chemicals, it has been shown that the toxicity was typically higher, when larger fractions of the neutral species were present. This observation was explained in some cases by slower uptake of charged species. In other cases it was suggested that the neutral species has intrinsically higher toxicity than the charged species or is alone responsible for the toxicity. However, even permanently charged and organic chemicals with multiple acid and base functional groups and zwitterions are toxic. We set out to reconcile the divergent views and to compare the various existing models for describing the pH-dependence of toxicity with the goal to derive one model that is valid independent of the type and number of charges on the molecule. To achieve this goal we measured the cytotoxicity of 18 acidic, 15 basic and 9 multiprotic/zwitterionic pharmaceuticals at pH 5.5 to pH 9 with the bioluminescence inhibition test using Aliivibrio fischeri (Microtox assay). This assay is useful for an evaluation of various models to describe pH-dependent toxicity because the majority of chemicals act as baseline toxicants in this 30 min cytotoxicity assay. Therefore baseline toxicity with constant membrane concentrations of the sum of all chemical species of approximately 200 mmol kglip-1 served for the validation of the suitability of the various tested models. We confirmed that most tested pharmaceuticals acted as baseline toxicants in this assay at all examined pH values, when toxicity was modeled with a mixture model of concentration addition between the neutral species and all charged species. An ion trapping model, that assumes that the membrane permeability of charged species is kinetically limited, improved model predictions for some pharmaceuticals and pH values. However, neither unhindered uptake nor no uptake of the charged species were ideal models; the reality lies presumably between the two limiting cases with a slower uptake of the charged species than the neutral species. For practical applications a previously developed QSAR model with the ionisation-corrected liposome-water distribution ratio as the sole physicochemical descriptor proved to be generally applicable for all ionisable organic chemicals including those with multiple charges and zwitterions.

General baseline toxicity QSAR for nonpolar, polar and ionisable chemicals and their mixtures in the bioluminescence inhibition assay with Aliivibrio fischeri.

The Microtox assay, a bioluminescence inhibition assay with the marine bacterium Aliivibrio fischeri, is one of the most popular bioassays for assessing the cytotoxicity of organic chemicals, mixtures and environmental samples. Most environmental chemicals act as baseline toxicants in this short-term screening assay, which is typically run with only 30 min of exposure duration. Numerous Quantitative Structure-Activity Relationships (QSARs) exist for the Microtox assay for nonpolar and polar narcosis. However, typical water pollutants, which have highly diverse structures covering a wide range of hydrophobicity and speciation from neutral to anionic and cationic, are often outside the applicability domain of these QSARs. To include all types of environmentally relevant organic pollutants we developed a general baseline toxicity QSAR using liposome-water distribution ratios as descriptors. Previous limitations in availability of experimental liposome-water partition constants were overcome by reliable prediction models based on polyparameter linear free energy relationships for neutral chemicals and the COSMOmic model for charged chemicals. With this QSAR and targeted mixture experiments we could demonstrate that ionisable chemicals fall in the applicability domain. Most investigated water pollutants acted as baseline toxicants in this bioassay, with the few outliers identified as uncouplers or reactive toxicants. The main limitation of the Microtox assay is that chemicals with a high melting point and/or high hydrophobicity were outside of the applicability domain because of their low water solubility. We quantitatively derived a solubility cut-off but also demonstrated with mixture experiments that chemicals inactive on their own can contribute to mixture toxicity, which is highly relevant for complex environmental mixtures, where these chemicals may be present at concentrations below the solubility cut-off.

From the exposome to mechanistic understanding of chemical-induced adverse effects.

The exposome encompasses an individual's exposure to exogenous chemicals, as well as endogenous chemicals that are produced or altered in response to external stressors. While the exposome concept has been established for human health, its principles can be extended to include broader ecological issues. The assessment of exposure is tightly interlinked with hazard assessment. Here, we explore if mechanistic understanding of the causal links between exposure and adverse effects on human health and the environment can be improved by integrating the exposome approach with the adverse outcome pathway (AOP) concept that structures and organizes the sequence of biological events from an initial molecular interaction of a chemical with a biological target to an adverse outcome. Complementing exposome research with the AOP concept may facilitate a mechanistic understanding of stress-induced adverse effects, examine the relative contributions from various components of the exposome, determine the primary risk drivers in complex mixtures, and promote an integrative assessment of chemical risks for both human and environmental health.

Development of a general baseline toxicity QSAR model for the fish embryo acute toxicity test.

Fish embryos have become a popular model in ecotoxicology and toxicology. The fish embryo acute toxicity test (FET) with the zebrafish embryo was recently adopted by the OECD as technical guideline TG 236 and a large database of concentrations causing 50% lethality (LC50) is available in the literature. Quantitative Structure-Activity Relationships (QSARs) of baseline toxicity (also called narcosis) are helpful to estimate the minimum toxicity of chemicals to be tested and to identify excess toxicity in existing data sets. Here, we analyzed an existing fish embryo toxicity database and established a QSAR for fish embryo LC50 using chemicals that were independently classified to act according to the non-specific mode of action of baseline toxicity. The octanol-water partition coefficient Kow is commonly applied to discriminate between non-polar and polar narcotics. Replacing the Kow by the liposome-water partition coefficient Klipw yielded a common QSAR for polar and non-polar baseline toxicants. This developed baseline toxicity QSAR was applied to compare the final mode of action (MOA) assignment of 132 chemicals. Further, we included the analysis of internal lethal concentration (ILC50) and chemical activity (La50) as complementary approaches to evaluate the robustness of the FET baseline toxicity. The analysis of the FET dataset revealed that specifically acting and reactive chemicals converged towards the baseline toxicity QSAR with increasing hydrophobicity. The developed FET baseline toxicity QSAR can be used to identify specifically acting or reactive compounds by determination of the toxic ratio and in combination with appropriate endpoints to infer the MOA for chemicals.

Zebrafish biosensor for toxicant induced muscle hyperactivity.

Robust and sensitive detection systems are a crucial asset for risk management of chemicals, which are produced in increasing number and diversity. To establish an in vivo biosensor system with quantitative readout for potential toxicant effects on motor function, we generated a transgenic zebrafish line TgBAC(hspb11:GFP) which expresses a GFP reporter under the control of regulatory elements of the small heat shock protein hspb11. Spatiotemporal hspb11 transgene expression in the musculature and the notochord matched closely that of endogenous hspb11 expression. Exposure to substances that interfere with motor function induced a dose-dependent increase of GFP intensity beginning at sub-micromolar concentrations, while washout of the chemicals reduced the level of hspb11 transgene expression. Simultaneously, these toxicants induced muscle hyperactivity with increased calcium spike height and frequency. The hspb11 transgene up-regulation induced by either chemicals or heat shock was eliminated after co-application of the anaesthetic MS-222. TgBAC(hspb11:GFP) zebrafish embryos provide a quantitative measure of muscle hyperactivity and represent a robust whole organism system for detecting chemicals that affect motor function.

Fish embryo toxicity test: identification of compounds with weak toxicity and analysis of behavioral effects to improve prediction of acute toxicity for neurotoxic compounds.

The fish embryo toxicity test has been proposed as an alternative for the acute fish toxicity test, but concerns have been raised for its predictivity given that a few compounds have been shown to exhibit a weak acute toxicity in the fish embryo. In order to better define the applicability domain and improve the predictive capacity of the fish embryo test, we performed a systematic analysis of existing fish embryo and acute fish toxicity data. A correlation analysis of a total of 153 compounds identified 28 compounds with a weaker or no toxicity in the fish embryo test. Eleven of these compounds exhibited a neurotoxic mode of action. We selected a subset of eight compounds with weaker or no embryo toxicity (cyanazine, picloram, aldicarb, azinphos-methyl, dieldrin, diquat dibromide, endosulfan, and esfenvalerate) to study toxicokinetics and a neurotoxic mode of action as potential reasons for the deviating fish embryo toxicity. Published fish embryo LC50 values were confirmed by experimental analysis of zebrafish embryo LC50 according to OECD guideline 236. Except for diquat dibromide, internal concentration analysis did not indicate a potential relation of the low sensitivity of fish embryos to a limited uptake of the compounds. Analysis of locomotor activity of diquat dibromide and the neurotoxic compounds in 98 hpf embryos (exposed for 96 h) indicated a specific effect on behavior (embryonic movement) for the neurotoxic compounds. The EC50s of behavior for neurotoxic compounds were close to the acute fish toxicity LC50. Our data provided the first evidence that the applicability domain of the fish embryo test (LC50s determination) may exclude neurotoxic compounds. However, neurotoxic compounds could be identified by changes in embryonic locomotion. Although a quantitative prediction of acute fish toxicity LC50 using behavioral assays in fish embryos may not yet be possible, the identification of neurotoxicity could trigger the conduction of a conventional fish acute toxicity test or application of assessment factors while considering the very good fish embryo-acute fish toxicity correlation for other compounds.