Do we still need animals? Surveying the role of animal-free models in Alzheimer's and Parkinson's disease research.

The use of animals in neuroscience and biomedical research remains controversial. Policy is built around the "3R" principle of "Refining, Reducing and Replacing" animal experiments, and across the globe, different initiatives stimulate the use of animal-free methods. Based on an extensive literature screen to map the development and adoption of animal-free methods in Alzheimer's and Parkinson's disease research, we find that at least two in three examined studies rely on animals or on animal-derived models. Among the animal-free studies, the relative contribution of innovative models that may replace animal experiments is limited. We argue that the distinction between animal research and alternative models presents a false dichotomy, as the role and scientific value of both animal and animal-free approaches are intertwined. Calls to halt all animal experiments appear premature, as insufficient non-animal-based alternatives are available and their development lags behind. In light of this, we highlight the need for objective, unprejudiced monitoring, and more robust performance indicators of animal-free approaches.

Minimum reporting standards based on a comprehensive review of the zebrafish embryo teratogenicity assay.

Reproductive toxicity chemical safety assessment involves extensive use of vertebrate animals for regulatory testing purposes. Although alternative methods such as the zebrafish embryo teratogenicity assay (identified in the present manuscript by the acronym ZETA) are promising for replacing tests with mammals, challenges to regulatory application involve lack of standardization and incomplete validation. To identify key protocol aspects and ultimately support improving this situation, a comprehensive review of the literature on the level of harmonization/standardization and validation status of the ZETA has been conducted. The gaps and needed advances of the available ZETA protocols were evaluated and discussed with respect to its applicability as an alternative approach for teratogenicity assessment. Based on the review outcomes, a set of minimum reporting standards for the experimental protocol is proposed. Together with other initiatives towards implementation of alternative approaches at the screening and regulatory levels, the application of minimum reporting requirements is anticipated to support future method standardization and validation, as well as identifying potential improvement aspects. Present findings are expected to ultimately support advancing the ongoing validation initiatives towards the regulatory acceptance of the ZETA.

Xenopus laevis as a Bioindicator of Endocrine Disruptors in the Region of Central Chile.

One of the direct causes of biodiversity loss is environmental pollution resulting from the use of chemicals. Different kinds of chemicals, such as persistent organic pollutants and some heavy metals, can be endocrine disruptors, which act at low doses over a long period of time and have a negative effect on the reproductive and thyroid system in vertebrates worldwide. Research on the effects of endocrine disruptors and the use of bioindicators in neotropical ecosystems where pressure on biodiversity is high is scarce. In Chile, although endocrine disruptors have been detected at different concentrations in the environments of some ecosystems, few studies have been performed on their biological effects in the field. In this work, Xenopus laevis (African clawed frog), an introduced species, is used as a bioindicator for the presence of endocrine disruptors in aquatic systems with different degrees of contamination in a Mediterranean zone in central Chile. For the first time for Chile, alterations are described that can be linked to exposure to endocrine disruptors, such as vitellogenin induction, decreased testosterone in male frogs, and histological changes in gonads. Dioxin-like and oestrogenic activity was detected in sediments at locations where it seem to be related to alterations found in the frogs. In addition, an analysis of land use/cover use revealed that urban soil was the best model to explain the variations in frog health indicators. This study points to the usefulness of an invasive species as a bioindicator for the presence of endocrine-disruptive chemicals.

Consensus statement on the need for innovation, transition and implementation of developmental neurotoxicity (DNT) testing for regulatory purposes.

This consensus statement voices the agreement of scientific stakeholders from regulatory agencies, academia and industry that a new framework needs adopting for assessment of chemicals with the potential to disrupt brain development. An increased prevalence of neurodevelopmental disorders in children has been observed that cannot solely be explained by genetics and recently pre- and postnatal exposure to environmental chemicals has been suspected as a causal factor. There is only very limited information on neurodevelopmental toxicity, leaving thousands of chemicals, that are present in the environment, with high uncertainty concerning their developmental neurotoxicity (DNT) potential. Closing this data gap with the current test guideline approach is not feasible, because the in vivo bioassays are far too resource-intensive concerning time, money and number of animals. A variety of in vitro methods are now available, that have the potential to close this data gap by permitting mode-of-action-based DNT testing employing human stem cells-derived neuronal/glial models. In vitro DNT data together with in silico approaches will in the future allow development of predictive models for DNT effects. The ultimate application goals of these new approach methods for DNT testing are their usage for different regulatory purposes.

Phenotypic and biomarker evaluation of zebrafish larvae as an alternative model to predict mammalian hepatotoxicity.

Zebrafish phenotypic assays have shown promise to assess human hepatotoxicity, though scoring of liver morphology remains subjective and difficult to standardize. Liver toxicity in zebrafish larvae at 5 days was assessed using gene expression as the biomarker approach, complementary to phenotypic analysis and analytical data on compound uptake. This approach aimed to contribute to improved hepatotoxicity prediction, with the goal of identifying biomarker(s) as a step towards the development of transgenic models for prioritization. Morphological effects of hepatotoxic compounds (acetaminophen, amiodarone, coumarin, methapyrilene and myclobutanil) and saccharin as the negative control were assessed after exposure in zebrafish larvae. The hepatotoxic compounds induced the expected zebrafish liver degeneration or changes in size, whereas saccharin did not have any phenotypic adverse effect. Analytical methods based on liquid chromatography-mass spectrometry were optimized to measure stability of selected compounds in exposure medium and internal concentration in larvae. All compounds were stable, except amiodarone for which precipitation was observed. There was a wide variation between the levels of compound in the zebrafish larvae with a higher uptake of amiodarone, methapyrilene and myclobutanil. Detection of hepatocyte markers (CP, CYP3A65, GC and TF) was accomplished by in situ hybridization of larvae to coumarin and myclobutanil and confirmed by real-time reverse transcription-quantitative polymerase chain reaction. Experiments showed decreased expression of all markers. Next, other liver-specific biomarkers (i.e. FABP10a and NR1H4) and apoptosis (i.e. CASP-3 A and TP53) or cytochrome P450-related (CYP2K19) and oxidoreductase activity-related (ZGC163022) genes, were screened. Links between basic mechanisms of liver injury and results of biomarker responses are described. Copyright © 2016 John Wiley & Sons, Ltd.

OECD validation study to assess intra- and inter-laboratory reproducibility of the zebrafish embryo toxicity test for acute aquatic toxicity testing.

The OECD validation study of the zebrafish embryo acute toxicity test (ZFET) for acute aquatic toxicity testing evaluated the ZFET reproducibility by testing 20 chemicals at 5 different concentrations in 3 independent runs in at least 3 laboratories. Stock solutions and test concentrations were analytically confirmed for 11 chemicals. Newly fertilised zebrafish eggs (20/concentration and control) were exposed for 96h to chemicals. Four apical endpoints were recorded daily as indicators of acute lethality: coagulation of the embryo, lack of somite formation, non-detachment of the tail bud from the yolk sac and lack of heartbeat. Results (LC50 values for 48/96h exposure) show that the ZFET is a robust method with a good intra- and inter-laboratory reproducibility (CV<30%) for most chemicals and laboratories. The reproducibility was lower (CV>30%) for some very toxic or volatile chemicals, and chemicals tested close to their limit of solubility. The ZFET is now available as OECD Test Guideline 236. Considering the high predictive capacity of the ZFET demonstrated by Belanger et al. (2013) in their retrospective analysis of acute fish toxicity and fish embryo acute toxicity data, the ZFET is ready to be considered for acute fish toxicity for regulatory purposes.

A European perspective on alternatives to animal testing for environmental hazard identification and risk assessment.

Tests with vertebrates are an integral part of environmental hazard identification and risk assessment of chemicals, plant protection products, pharmaceuticals, biocides, feed additives and effluents. These tests raise ethical and economic concerns and are considered as inappropriate for assessing all of the substances and effluents that require regulatory testing. Hence, there is a strong demand for replacement, reduction and refinement strategies and methods. However, until now alternative approaches have only rarely been used in regulatory settings. This review provides an overview on current regulations of chemicals and the requirements for animal tests in environmental hazard and risk assessment. It aims to highlight the potential areas for alternative approaches in environmental hazard identification and risk assessment. Perspectives and limitations of alternative approaches to animal tests using vertebrates in environmental toxicology, i.e. mainly fish and amphibians, are discussed. Free access to existing (proprietary) animal test data, availability of validated alternative methods and a practical implementation of conceptual approaches such as the Adverse Outcome Pathways and Integrated Testing Strategies were identified as major requirements towards the successful development and implementation of alternative approaches. Although this article focusses on European regulations, its considerations and conclusions are of global relevance.

An in vitro air-liquid interface inhalation platform for petroleum substances and constituents.

The goal is to optimize and show the validity of an in vitro method for inhalation testing of petroleum substances and their constituents at the air-liquid interface (ALI). The approach is demonstrated in a pilot study with ethylbenzene (EB), a mono-constituent petroleum substance, using a human alveolar epithelial cell line model. This included the development and validation of a generation facility to obtain EB vapors and the optimization of an exposure system for a negative control (clean air, CA), positive control (nitrogen dioxide), and EB vapors. The optimal settings for the VITROCELL® 24/48 system were defined. Cytotoxicity, cell viability, inflammation, and oxidative stress were assessed in A549 after exposure to EB vapors. A concentration-dependent significant decrease in mean cell viability was observed after exposure, which was confirmed by a cytotoxicity test. The oxidative stress marker superoxide dismutase 2 was sig­nificantly increased, but no concentration-response was observed. A concentration-dependent significant increase in pro-inflammatory markers C-C motif chemokine ligand 2, interleukin (IL)6, and IL8 was observed for EB-exposed A549 cells compared to CA. The data demonstrated consistency between in vivo air concentrations at which adverse respi­ratory effects were observed and ALI-concentrations affecting cell viability, provided that the actual measured in vitro delivery efficiency of the compound was considered. It can be concluded that extrapolating in vitro air concentrations (adjusted for delivery efficiency and absorption characteristics and applied for testing cell viability) to simulate in vivo air concentrations may be a promising method to screen for acute inhalation toxicity.

A Systematic Review to Compare Chemical Hazard Predictions of the Zebrafish Embryotoxicity Test With Mammalian Prenatal Developmental Toxicity.

Originally developed to inform the acute toxicity of chemicals on fish, the zebrafish embryotoxicity test (ZET) has also been proposed for assessing the prenatal developmental toxicity of chemicals, potentially replacing mammalian studies. Although extensively evaluated in primary studies, a comprehensive review summarizing the available evidence for the ZET's capacity is lacking. Therefore, we conducted a systematic review of how well the presence or absence of exposure-related findings in the ZET predicts prenatal development toxicity in studies with rats and rabbits. A two-tiered systematic review of the developmental toxicity literature was performed, a review of the ZET literature was followed by one of the mammalian literature. Data were extracted using DistillerSR, and study validity was assessed with an amended SYRCLE's risk-of-bias tool. Extracted data were analyzed for each species and substance, which provided the basis for comparing the 2 test methods. Although limited by the number of 24 included chemicals, our results suggest that the ZET has potential to identify chemicals that are mammalian prenatal developmental toxicants, with a tendency for overprediction. Furthermore, our analysis confirmed the need for further standardization of the ZET. In addition, we identified contextual and methodological challenges in the application of systematic review approaches to toxicological questions. One key to overcoming these challenges is a transition to more comprehensive and transparent planning, conduct and reporting of toxicological studies. The first step toward bringing about this change is to create broad awareness in the toxicological community of the need for and benefits of more evidence-based approaches.

Blueprint for a self-sustained European Centre for service provision in safe and sustainable innovation for nanotechnology.

The coming years are expected to bring rapid changes in the nanotechnology regulatory landscape, with the establishment of a new framework for nano-risk governance, in silico approaches for characterisation and risk assessment of nanomaterials, and novel procedures for the early identification and management of nanomaterial risks. In this context, Safe(r)-by-Design (SbD) emerges as a powerful preventive approach to support the development of safe and sustainable (SSbD) nanotechnology-based products and processes throughout the life cycle. This paper summarises the work undertaken to develop a blueprint for the deployment and operation of a permanent European Centre of collaborating laboratories and research organisations supporting safe innovation in nanotechnologies. The proposed entity, referred to as "the Centre", will establish a 'one-stop shop' for nanosafety-related services and a central contact point for addressing stakeholder questions about nanosafety. Its operation will rely on significant business, legal and market knowledge, as well as other tools developed and acquired through the EU-funded EC4SafeNano project and subsequent ongoing activities. The proposed blueprint adopts a demand-driven service update scheme to allow the necessary vigilance and flexibility to identify opportunities and adjust its activities and services in the rapidly evolving regulatory and nano risk governance landscape. The proposed Centre will play a major role as a conduit to transfer scientific knowledge between the research and commercial laboratories or consultants able to provide high quality nanosafety services, and the end-users of such services (e.g., industry, SMEs, consultancy firms, and regulatory authorities). The Centre will harmonise service provision, and bring novel risk assessment and management approaches, e.g. in silico methodologies, closer to practice, notably through SbD/SSbD, and decisively support safe and sustainable innovation of industrial production in the nanotechnology industry according to the European Chemicals Strategy for Sustainability.

Alternative air-liquid interface method for inhalation toxicity testing of a petroleum-derived substance.

In vitro-based new approach methodologies (NAMs) provide a pragmatic solution to animal testing of petroleum substances and their constituents. A previous study exposed an in vitro model (A549 cells) at the air-liquid interface (ALI) to assess inhalation toxicity of a single compound, ethylbenzene. Experimental conditions using VITROCELLⓇ 24/48 exposure system were optimized to achieve a deposition efficiency that resulted in dose-dependent biological changes. The feasibility of this set-up was evaluated for testing the complex substance gasoline, which, at only high concentrations, can induce mild respiratory irritation in animals and cough in humans.•Results showed that perpendicular ALI exposure flow systems (VITROCELL® 6/4 and 24/48) may not be appropriate for testing gasoline because it was not possible to achieve enough deposition onto the cells and in the culture medium to measure dose and to determine dose-dependent biological changes (more information can be found in 'Supplementary material and/or Additional information' section).•Structural features (e.g. aromatic or saturated hydrocarbon structure) and high hydrophobicity, together with the low concentrations of individual components in gasoline, may have caused the low deposition.•To achieve a higher deposition on the cells, A549 cells were exposed to gasoline at the ALI by passive dosing.The results demonstrate that the presented methodology is a promising NAM for inhalation toxicity testing of (semi-)volatile complex substances with low aqueous solubility.

THP-1 monocytes but not macrophages as a potential alternative for CD34+ dendritic cells to identify chemical skin sensitizers.

Early detection of the sensitizing potential of chemicals is an emerging issue for chemical, pharmaceutical and cosmetic industries. In our institute, an in vitro classification model for prediction of chemical-induced skin sensitization based on gene expression signatures in human CD34+ progenitor-derived dendritic cells (DC) has been developed. This primary cell model is able to closely mimic the induction phase of sensitization by Langerhans cells in the skin, but it has drawbacks, such as the availability of cord blood. The aim of this study was to investigate whether human in vitro cultured THP-1 monocytes or macrophages display a similar expression profile for 13 predictive gene markers previously identified in DC and whether they also possess a discriminating capacity towards skin sensitizers and non-sensitizers based on these marker genes. To this end, the cell models were exposed to 5 skin sensitizers (ammonium hexachloroplatinate IV, 1-chloro-2,4-dinitrobenzene, eugenol, para-phenylenediamine, and tetramethylthiuram disulfide) and 5 non-sensitizers (l-glutamic acid, methyl salicylate, sodium dodecyl sulfate, tributyltin chloride, and zinc sulfate) for 6, 10, and 24 h, and mRNA expression of the 13 genes was analyzed using real-time RT-PCR. The transcriptional response of 7 out of 13 genes in THP-1 monocytes was significantly correlated with DC, whereas only 2 out of 13 genes in THP-1 macrophages. After a cross-validation of a discriminant analysis of the gene expression profiles in the THP-1 monocytes, this cell model demonstrated to also have a capacity to distinguish skin sensitizers from non-sensitizers. However, the DC model was superior to the monocyte model for discrimination of (non-)sensitizing chemicals.

Gene profiles of a human bronchial epithelial cell line after in vitro exposure to respiratory (non-)sensitizing chemicals: identification of discriminating genetic markers and pathway analysis.

Respiratory sensitization is a concern for occupational and environmental health in consumer product development. Despite international regulatory requirements there is no established protocol for the identification of chemical respiratory sensitizers. New tests should be based on mechanistic understanding and should be preferentially restricted to in vitro assays. The major goal of this study was to investigate the alterations in gene expression of human bronchial epithelial (BEAS-2B) cells after exposure to respiratory sensitizers and respiratory non-sensitizing chemicals, and to identify genes that are able to discriminate between both groups of chemicals. BEAS-2B cells were exposed during 6, 10, and 24h to the respiratory sensitizers ammonium hexachloroplatinate IV, hexamethylene diisocyanate, and trimellitic anhydride, the irritants acrolein and methyl salicylate, and the skin sensitizer 1-chloro-2,4-dinitrobenzene. Overall changes in gene expression were evaluated using Agilent Whole Human Genome 4x 44K oligonucleotide arrays. Fisher Linear Discriminant Analysis was used to obtain a ranking of genes that reflects their potential to discriminate between respiratory sensitizing and respiratory non-sensitizing chemicals. The 10 most discriminative genes were BC042064, A_24_P229834, DOCK11, THC2544911, DLGAP4, NINJ1, PFKM, FLJ10986, IL28RA, and CASP9. Based on the differentially expressed genes, pathway analysis was used to identify possible underlying mechanisms of respiratory sensitization. We demonstrated that in bronchial epithelial cells the canonical PTEN signaling pathway is probably the most specific pathway in the context of respiratory sensitization. Results are indicative that the BEAS-2B cell line can be used as an alternative cell model to screen chemical compounds for their respiratory sensitizing potential.

Gene profiles of a human alveolar epithelial cell line after in vitro exposure to respiratory (non-)sensitizing chemicals: identification of discriminating genetic markers and pathway analysis.

There are currently no accepted biological prediction models for assessing the potential of a substance to cause respiratory sensitization. New tests should be based on mechanistic understanding and should be preferentially restricted to in vitro assays. The major goal of this study was to investigate the alterations in gene expression of human alveolar epithelial (A549) cells after exposure to respiratory sensitizing and non-respiratory sensitizing chemicals, and to identify genes that are able to discriminate between both groups of chemicals. A549 cells were exposed during 6, 10, and 24 h to the respiratory sensitizers ammonium hexachloroplatinate IV, hexamethylene diisocyanate, and trimellitic anhydride, the irritants acrolein and methyl salicylate, and the skin sensitizer 1-chloro-2,4-dinitrobenzene. Overall changes in gene expression were evaluated using Agilent Whole Human Genome 4x44K oligonucleotide arrays. A Fisher linear discriminant analysis was used to obtain a ranking of genes that reflects their potential to discriminate between respiratory sensitizing and respiratory non-sensitizing chemicals. Among the 20 most discriminating genes, which were categorized into molecular and biological gene ontology (GO) terms, CTLA4 could be associated with asthma and/or respiratory sensitization. When categorizing the top-1000 genes into biological GO terms, 22 genes were associated with immune function. Using a pathway analysis tool to identify possible underlying mechanisms of respiratory sensitization, no known canonical signaling pathway was observed to be activated in the A549 cell line.

Adaptation of the Systematic Review Framework to the Assessment of Toxicological Test Methods: Challenges and Lessons Learned With the Zebrafish Embryotoxicity Test.

Systematic review methodology is a means of addressing specific questions through structured, consistent, and transparent examinations of the relevant scientific evidence. This methodology has been used to advantage in clinical medicine, and is being adapted for use in other disciplines. Although some applications to toxicology have been explored, especially for hazard identification, the present preparatory study is, to our knowledge, the first attempt to adapt it to the assessment of toxicological test methods. As our test case, we chose the zebrafish embryotoxicity test (ZET) for developmental toxicity and its mammalian counterpart, the standard mammalian prenatal development toxicity study, focusing the review on how well the ZET predicts the presence or absence of chemical-induced prenatal developmental toxicity observed in mammalian studies. An interdisciplinary team prepared a systematic review protocol and adjusted it throughout this piloting phase, where needed. The final protocol was registered and will guide the main study (systematic review), which will execute the protocol to comprehensively answer the review question. The goal of this preparatory study was to translate systematic review methodology to the assessment of toxicological test method performance. Consequently, it focused on the methodological issues encountered, whereas the main study will report substantive findings. These relate to numerous systematic review steps, but primarily to searching and selecting the evidence. Applying the lessons learned to these challenges can improve not only our main study, but may also be helpful to others seeking to use systematic review methodology to compare toxicological test methods. We conclude with a series of recommendations that, if adopted, would help improve the quality of the published literature, and make conducting systematic reviews of toxicological studies faster and easier over time.

Corrigendum to Recommendation on test readiness criteria for new approach methods in toxicology: exemplified for developmental neurotoxicity.

In this manuscript, which appeared in ALTEX 35 , 306-352 ( doi:10.14573/altex.1712081 ), the Acknowledgements should read: This work was supported by the Doerenkamp-Zbinden Foundation, EFSA, the BMBF, JPI-NutriCog-Selenius, and it has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No. 681002 (EU-ToxRisk).

Repeatability and Reproducibility of the RTgill-W1 Cell Line Assay for Predicting Fish Acute Toxicity.

Predicting fish acute toxicity of chemicals in vitro is an attractive alternative method to the conventional approach using juvenile and adult fish. The rainbow trout (Oncorhynchus mykiss) cell line assay with RTgill-W1 cells has been designed for this purpose. It quantifies cell viability using fluorescent measurements for metabolic activity, cell- and lysosomal-membrane integrity on the same set of cells. Results from over 70 organic chemicals attest to the high predictive capacity of this test. We here report on the repeatability (intralaboratory variability) and reproducibility (interlaboratory variability) of the RTgill-W1 cell line assay in a round-robin study focusing on 6 test chemicals involving 6 laboratories from the industrial and academic sector. All participating laboratories were able to establish the assay according to preset quality criteria even though, apart from the lead laboratory, none had previously worked with the RTgill-W1 cell line. Concentration-response modeling, based on either nominal or geometric mean-derived measured concentrations, yielded effect concentrations (EC50) that spanned approximately 4 orders of magnitude over the chemical range, covering all fish acute toxicity categories. Coefficients of variation for intralaboratory and interlaboratory variability for the average of the 3 fluorescent cell viability measurements were 15.5% and 30.8%, respectively, which is comparable to other fish-derived, small-scale bioassays. This study therefore underlines the robustness of the RTgill-W1 cell line assay and its accurate performance when carried out by operators in different laboratory settings.

Molecular recognition of endocrine disruptors by synthetic and natural 17beta-estradiol receptors: a comparative study.

A beta-estradiol receptor binding mimic was synthesised using molecular imprinting. Bulk polymers and spherical polymer nanoparticles based on methacrylic acid and ethylene glycol dimethacrylate as the functional monomer and crosslinker, respectively, were prepared in acetonitrile. The selectivity was evaluated by radioligand binding assays. The imprinted polymers were very specific to beta-estradiol since the control polymers bound virtually none of the radioligand. The bulk polymer was then employed to screen endocrine disrupting chemicals. Structurally related steroids like alpha-estradiol, estrone and ethynylestradiol showed, respectively, 14.0, 5.0 and 0.7% of relative binding to the beta-estradiol polymer, whereas most unrelated chemicals did not bind at all. These results are compared to those obtained with a bioassay using stably transfected yeast cells in culture bearing the human estrogen receptor. The receptor was activated by several estrogen-like chemicals and to a lesser extent by some structurally related chemicals. [figure: see text]

Microarray analyses in dendritic cells reveal potential biomarkers for chemical-induced skin sensitization.

The assessment of the skin sensitising capacity of chemicals is up to now investigated using in vivo animal tests. However there has been an increasing public and governmental concern regarding the use of animals for chemical screening. This has raised the need for the development of validated in vitro alternatives. Langerhans cells are potent antigen-presenting cells that play a crucial role in the development of allergic contact dermatitis. We used CD34(+) progenitor-derived dendritic cells from cord blood as an in vitro alternative for Langerhans cells. The cells were exposed to four contact allergens (nickel sulphate, dinitrochlorobenzene, oxazolone and eugenol) and two irritants (sodium dodecyl sulphate and benzalkonium chloride) for 3, 6, 12 and 24h. Using microarray analyses we revealed a set of 25 genes with an altered gene expression pattern after exposure to allergens and not to irritants. Five out of these 25 genes were selected and their gene expression changes were confirmed with real-time reverse transcriptase polymerase chain reaction. The list of 25 genes represent valuable candidates to be further evaluated for their capacity to predict the sensitizing potential of different classes of chemicals in studies using a more extended set of (non) allergic substances.

An AOP-based alternative testing strategy to predict the impact of thyroid hormone disruption on swim bladder inflation in zebrafish.

The adverse outcome pathway (AOP) framework can be used to help support the development of alternative testing strategies aimed at predicting adverse outcomes caused by triggering specific toxicity pathways. In this paper, we present a case-study demonstrating the selection of alternative in chemico assays targeting the molecular initiating events of established AOPs, and evaluate use of the resulting data to predict higher level biological endpoints. Based on two AOPs linking inhibition of the deiodinase (DIO) enzymes to impaired posterior swim bladder inflation in fish, we used in chemico enzyme inhibition assays to measure the molecular initiating events for an array of 51 chemicals. Zebrafish embryos were then exposed to 14 compounds with different measured inhibition potentials. Effects on posterior swim bladder inflation, predicted based on the information captured by the AOPs, were evaluated. By linking the two datasets and setting thresholds, we were able to demonstrate that the in chemico dataset can be used to predict biological effects on posterior chamber inflation, with only two outliers out of the 14 tested compounds. Our results show how information organized using the AOP framework can be employed to develop or select alternative assays, and successfully forecast downstream key events along the AOP. In general, such in chemico assays could serve as a first-tier high-throughput system to screen and prioritize chemicals for subsequent acute and chronic fish testing, potentially reducing the need for long-term and costly toxicity tests requiring large numbers of animals.