Prioritization of chemicals in food for risk assessment by integrating exposure estimates and new approach methodologies: A next generation risk assessment case study.

Next generation risk assessment is defined as a knowledge-driven system that allows for cost-efficient assessment of human health risk related to chemical exposure, without animal experimentation. One of the key features of next generation risk assessment is to facilitate prioritization of chemical substances that need a more extensive toxicological evaluation, in order to address the need to assess an increasing number of substances. In this case study focusing on chemicals in food, we explored how exposure data combined with the Threshold of Toxicological Concern (TTC) concept could be used to prioritize chemicals, both for existing substances and new substances entering the market. Using a database of existing chemicals relevant for dietary exposure we calculated exposure estimates, followed by application of the TTC concept to identify substances of higher concern. Subsequently, a selected set of these priority substances was screened for toxicological potential using high-throughput screening (HTS) approaches. Remarkably, this approach resulted in alerts for a selection of substances that are already on the market and represent relevant exposure in consumers. Taken together, the case study provides proof-of-principle for the approach taken to identify substances of concern, and this approach can therefore be considered a supportive element to a next generation risk assessment strategy.

Identification of proteome markers for drug-induced liver injury in zebrafish embryos.

The zebrafish embryo (ZFE) is a promising alternative non-rodent model in toxicology, and initial studies suggested its applicability in detecting hepatic responses related to drug-induced liver injury (DILI). Here, we hypothesize that detailed analysis of underlying mechanisms of hepatotoxicity in ZFE contributes to the improved identification of hepatotoxic properties of compounds and to the reduction of rodents used for hepatotoxicity assessment. ZFEs were exposed to nine reference hepatotoxicants, targeted at induction of steatosis, cholestasis, and necrosis, and effects compared with negative controls. Protein profiles of the individual compounds were generated using LC-MS/MS. We identified differentially expressed proteins and pathways, but as these showed considerable overlap, phenotype-specific responses could not be distinguished. This led us to identify a set of common hepatotoxicity marker proteins. At the pathway level, these were mainly associated with cellular adaptive stress-responses, whereas single proteins could be linked to common hepatotoxicity-associated processes. Applying several stringency criteria to our proteomics data as well as information from other data sources resulted in a set of potential robust protein markers, notably Igf2bp1, Cox5ba, Ahnak, Itih3b.2, Psma6b, Srsf3a, Ces2b, Ces2a, Tdo2b, and Anxa1c, for the detection of adverse responses.

Dose Addition in the Induction of Craniofacial Malformations in Zebrafish Embryos Exposed to a Complex Mixture of Food-Relevant Chemicals with Dissimilar Modes of Action.

BACKGROUND: Humans are exposed to combinations of chemicals. In cumulative risk assessment (CRA), regulatory bodies such as the European Food Safety Authority consider dose addition as a default and sufficiently conservative approach. The principle of dose addition was confirmed previously for inducing craniofacial malformations in zebrafish embryos in binary mixtures of chemicals with either similar or dissimilar modes of action (MOAs). OBJECTIVES: In this study, we explored a workflow to select and experimentally test multiple compounds as a complex mixture with each of the compounds at or below its no observed adverse effect level (NOAEL), in the same zebrafish embryo model. METHODS: Selection of candidate compounds that potentially induce craniofacial malformations was done using in silico methods-structural similarity, molecular docking, and quantitative structure-activity relationships-applied to a database of chemicals relevant for oral exposure in humans via food (EuroMix inventory, n=1,598). A final subselection was made manually to represent different regulatory fields (e.g., food additives, industrial chemicals, plant protection products), different chemical families, and different MOAs. RESULTS: A final selection of eight compounds was examined in the zebrafish embryo model, and craniofacial malformations were observed in embryos exposed to each of the compounds, thus confirming the developmental toxicity as predicted by the in silico methods. When exposed to a mixture of the eight compounds, each at its NOAEL, substantial craniofacial malformations were observed; according to a dose-response analysis, even embryos exposed to a 7-fold dilution of this mixture still exhibited a slight abnormal phenotype. The cumulative effect of the compounds in the mixture was in accordance with dose addition (added doses of the individual compounds after adjustment for relative potencies), despite different MOAs of the compounds involved. DISCUSSION: This case study of a complex mixture inducing craniofacial malformations in zebrafish embryos shows that dose addition can adequately predicted the cumulative effect of a mixture of multiple substances at low doses, irrespective of the (expected) MOA. The applied workflow may be useful as an approach for CRA in general. https://doi.org/10.1289/EHP9888.

Exploring Neurobehaviour in Zebrafish Embryos as a Screening Model for Addictiveness of Substances.

Tobacco use is the leading cause of preventable death worldwide and is highly addictive. Nicotine is the main addictive compound in tobacco, but less is known about other components and additives that may contribute to tobacco addiction. The zebrafish embryo (ZFE) has been shown to be a good model to study the toxic effects of chemicals on the neurological system and thus may be a promising model to study behavioral markers of nicotine effects, which may be predictive for addictiveness. We aimed to develop a testing protocol to study nicotine tolerance in ZFE using a locomotion test with light-dark transitions as behavioral trigger. Behavioral experiments were conducted using three exposure paradigms: (1) Acute exposure to determine nicotine's effect and potency. (2) Pre-treatment with nicotine dose range followed by a single dose of nicotine, to determine which pre-treatment dose is sufficient to affect the potency of acute nicotine. (3) Pre-treatment with a single dose combined with acute exposure to a dose range to confirm the hypothesized decreased potency of the acute nicotine exposure. These exposure paradigms showed that (1) acute nicotine exposure decreased ZFE activity in response to dark conditions in a dose-dependent fashion; (2) pre-treatment with increasing concentrations dose-dependently reversed the effect of acute nicotine exposure; and (3) a fixed pre-treatment dose of nicotine induced a decreased potency of the acute nicotine exposure. This effect supported the induction of tolerance to nicotine by the pre-treatment, likely through neuroadaptation. The interpretation of these effects, particularly in view of prediction of dependence and addictiveness, and suitability of the ZFE model to test for such effects of other compounds than nicotine, are discussed.

Developmental Neurotoxicity of Environmentally Relevant Pharmaceuticals and Mixtures Thereof in a Zebrafish Embryo Behavioural Test.

Humans are exposed daily to complex mixtures of chemical substances via food intake, inhalation, and dermal contact. Developmental neurotoxicity is an understudied area and entails one of the most complex areas in toxicology. Animal studies for developmental neurotoxicity (DNT) are hardly performed in the context of regular hazard studies, as they are costly and time consuming and provide only limited information as to human relevance. There is a need for a combination of in vitro and in silico tests for the assessment of chemically induced DNT in humans. The zebrafish (Danio rerio) embryo (ZFE) provides a powerful model to study DNT because it shows fast neurodevelopment with a large resemblance to the higher vertebrate, including the human system. One of the suitable readouts for DNT testing in the zebrafish is neurobehaviour (stimulus-provoked locomotion) since this provides integrated information on the functionality and status of the entire nervous system of the embryo. In the current study, environmentally relevant pharmaceuticals and their mixtures were investigated using the zebrafish light-dark transition test. Zebrafish embryos were exposed to three neuroactive compounds of concern, carbamazepine (CBZ), fluoxetine (FLX), and venlafaxine (VNX), as well as their main metabolites, carbamazepine 10,11-epoxide (CBZ 10,11E), norfluoxetine (norFLX), and desvenlafaxine (desVNX). All the studied compounds, except CBZ 10,11E, dose-dependently inhibited zebrafish locomotor activity, providing a distinct behavioural phenotype. Mixture experiments with these pharmaceuticals identified that dose addition was confirmed for all the studied binary mixtures (CBZ-FLX, CBZ-VNX, and VNX-FLX), thereby supporting the zebrafish embryo as a model for studying the cumulative effect of chemical mixtures in DNT. This study shows that pharmaceuticals and a mixture thereof affect locomotor activity in zebrafish. The test is directly applicable in environmental risk assessment; however, further studies are required to assess the relevance of these findings for developmental neurotoxicity in humans.

An adverse outcome pathway on the disruption of retinoic acid metabolism leading to developmental craniofacial defects.

Adverse outcome pathway (AOP) is a conceptual framework that links a molecular initiating event (MIE) via intermediate key events (KEs) with adverse effects (adverse outcomes, AO) relevant for risk assessment, through defined KE relationships (KERs). The aim of the present work is to describe a linear AOP, supported by experimental data, for skeletal craniofacial defects as the AO. This AO was selected in view of its relative high incidence in humans and the suspected relation to chemical exposure. We focused on inhibition of CYP26, a retinoic acid (RA) metabolizing enzyme, as MIE, based on robust previously published data. Conazoles were selected as representative stressors. Intermediate KEs are RA disbalance, aberrant HOX gene expression, disrupted specification, migration, and differentiation of neural crest cells, and branchial arch dysmorphology. We described the biological basis of the postulated events and conducted weight of evidence (WoE) assessments. The biological plausibility and the overall empirical evidence were assessed as high and moderate, respectively, the latter taking into consideration the moderate evidence for concordance of dose-response and temporal relationships. Finally, the essentiality assessment of the KEs, considered as high, supported the robustness of the presented AOP. This AOP, which appears of relevance to humans, thus contributes to mechanistic underpinning of selected test methods, thereby supporting their application in integrated new approach test methodologies and strategies and application in a regulatory context.

The effects of aliphatic alcohols and related acid metabolites in zebrafish embryos - correlations with rat developmental toxicity and with effects in advanced life stages in fish.

The zebrafish embryo toxicity test (ZFET) is a simple medium-throughput test to inform about (sub)acute lethal effects in embryos. Enhanced analysis through morphological and teratological scoring, and through gene expression analysis, detects developmental effects and the underlying toxicological pathways. Altogether, the ZFET may inform about hazard of chemical exposure for embryonal development in humans, as well as for lethal effects in juvenile and adult fish. In this study, we compared the effects within a series of 12 aliphatic alcohols and related carboxylic acid derivatives (ethanol, acetic acid, 2-methoxyethanol, 2-methoxyacetic acid, 2-butoxyethanol, 2-butoxyacetic acid, 2-hydroxyacetic acid, 2-ethylhexan-1-ol, 2-ethylhexanoic acid, valproic acid, 2-aminoethanol, 2-(2-hydroxyethylamino)ethanol) in ZFET and early life stage (ELS, 28d) exposures, and compared ZFET results with existing results of rat developmental studies and LC50s in adult fish. High correlation scores were observed between compound potencies in ZFET with either ELS, LC50 in fish and developmental toxicity in rats, indicating similar potency ranking among the models. Compounds could be mapped to specific pathways in an adverse outcome pathway (AOP) network through morphological scoring and gene expression analysis in ZFET. Similarity of morphological effects and gene expression profiles in pairs of alcohols with their acid metabolites suggested metabolic activation of the parent alcohols, although with additional, metabolite-independent activity independent for ethanol and 2-ethylhexanol. Overall, phenotypical and gene expression analysis with these compounds indicates that the ZFET can potentially contribute to the AOP for developmental effects in rodents, and to predict toxicity of acute and chronic exposure in advanced life stages in fish.

Distinguishing mode of action of compounds inducing craniofacial malformations in zebrafish embryos to support dose-response modeling in combined exposures.

Knowledge on mode-of-action (MOA) is required to understand toxicological effects of compounds, notably in the context of risk assessment of mixtures. Such information is generally scarce, and often complicated by the existence of multiple MOAs per compound. Here, MOAs related to developmental craniofacial malformations were derived from literature, and assembled in a MOA network. A selection of gene expression markers was based on these MOAs. Next, these markers were verified by qPCR in zebrafish embryos, after exposure to reference compounds. These were: triazoles for inhibition of retinoic acid (RA) metabolism, AM580 and CD3254 for selective activation of respectively RA-receptor (RAR) and retinoid-X-receptor (RXR), dithiocarbamates for inhibition of lysyl oxidase, TCDD for activation of the aryl-hydrocarbon-receptor (AhR), VPA for inhibition of histone deacetylase (HDAC), and PFOS for activation of peroxisome proliferator-activated receptor-alpha (PPARα). Next, marker gene profiles for these reference compounds were used to map the profiles of test compounds to known MOAs. In this way, 2,4-dinitrophenol matched with the TCDD and RAR profiles, boric acid with RAR, endosulfan with PFOS, fenpropimorph with dithiocarbamates, PCB126 with AhR, and RA with triazoles and RAR profiles. Prochloraz showed no match. Activities of these compounds in ToxCast assays, and in silico analysis of binding affinity to the respective targets showed limited concordance with the marker gene expression profiles, but still confirmed the complex MOA profiles of reference and test compounds. Ultimately, this approach could be used to support modeling of mixture effects based on upfront knowledge of (dis)similarity of MOAs.

A Case Study with Triazole Fungicides to Explore Practical Application of Next-Generation Hazard Assessment Methods for Human Health.

The ongoing developments in chemical risk assessment have led to new concepts building on integration of sophisticated nonanimal models for hazard characterization. Here we explore a pragmatic approach for implementing such concepts, using a case study of three triazole fungicides, namely, flusilazole, propiconazole, and cyproconazole. The strategy applied starts with evaluating the overall level of concern by comparing exposure estimates to toxicological potential, followed by a combination of in silico tools and literature-derived high-throughput screening assays and computational elaborations to obtain insight into potential toxicological mechanisms and targets in the organism. Additionally, some targeted in vitro tests were evaluated for their utility to confirm suspected mechanisms of toxicity and to generate points of departure. Toxicological mechanisms instead of the current "end point-by-end point" approach should guide the selection of methods and assays that constitute a toolbox for next-generation risk assessment. Comparison of the obtained in silico and in vitro results with data from traditional in vivo testing revealed that, overall, nonanimal methods for hazard identification can produce adequate qualitative hazard information for risk assessment. Follow-up studies are needed to further refine the proposed approach, including the composition of the toolbox, toxicokinetics models, and models for exposure assessment.

Dose addition in chemical mixtures inducing craniofacial malformations in zebrafish (Danio rerio) embryos.

A challenge in cumulative risk assessment is to model hazard of mixtures. EFSA proposed to only combine chemicals linked to a defined endpoint, in so-called cumulative assessment groups, and use the dose-addition model as a default to predict combined effects. We investigated the effect of binary mixtures of compounds known to cause craniofacial malformations, by assessing the effect in the head skeleton (M-PQ angle) in 120hpf zebrafish embryos. We combined chemicals with similar mode of action (MOA), i.e. the triazoles cyproconazole, triadimefon and flusilazole; next, reference compounds cyproconazole or triadimefon were combined with dissimilar acting compounds, TCDD, thiram, VPA, prochloraz, fenpropimorph, PFOS, or endosulfan. These mixtures were designed as (near) equipotent combinations of the contributing compounds, in a range of cumulative concentrations. Dose-addition was assessed by evaluation of the overlap of responses of each of the 14 tested binary mixtures with those of the single compounds. All 10 test compounds induced an increase of the M-PQ angle, with varying potency and specificity. Mixture responses as predicted by dose-addition did not deviate from the observed responses, supporting dose-addition as a valid assumption for mixture risk assessment. Importantly, dose-addition was found irrespective of MOA of contributing chemicals.

Analysis of Lipid Metabolism, Immune Function, and Neurobehavior in Adult C57BL/6JxFVB Mice After Developmental Exposure to di (2-ethylhexyl) Phthalate.

Background: Developmental exposure to di (2-ethylhexyl) phthalate (DEHP) has been implicated in the onset of metabolic syndrome later in life. Alterations in neurobehavior and immune functions are also affected by phthalate exposure and may be linked to the metabolic changes caused by developmental exposure to DEHP. Objectives: Our goal was to study the effects of developmental exposure to DEHP in the context of metabolic syndrome by integrating different parameters to assess metabolic, neurobehavioral, and immune functions in one model. Methods: Female C57BL/6J mice were exposed to DEHP through the diet during gestation and lactation at doses ranging from 3.3 to 100,000 µg/kg body weight/day (µkd). During a 1-year follow-up period, a wide set of metabolic parameters was assessed in the F1 offspring, including weekly body weight measurements, food consumption, physical activity, glucose homeostasis, serum lipids, and endocrine profile. In addition, neurobehavioral and immune functions were assessed by sweet preference test, object recognition test, acute phase protein, and cytokines production. Animals were challenged with a high fat diet (HFD) in the last 9 weeks of the study. Results: Increased free fatty acids (FFA) and, high density lipoprotein (HDL-C) were observed in serum, together with a decrease in glycated hemoglobin levels in blood of 1-year old male DEHP-exposed offspring after HFD challenge. For the most sensitive endpoint measured (FFA), a lower bound of the 90%-confidence interval for benchmark dose (BMD) at a critical effect size of 5% (BMDL) of 2,160 µkd was calculated. No persistent changes in body weight or fat mass were observed. At 33,000 µkd altered performance was found in the object recognition test in males and changes in interferon (IFN)γ production were observed in females. Conclusions: Developmental exposure to DEHP combined with HFD in adulthood led to changes in lipid metabolism and neurobehavior in male offspring and cytokine production in female offspring. Our findings contribute to the evidence that DEHP is a developmental dyslipidemic chemical, however, more research is needed to further characterize adverse health outcomes and the mechanisms of action associated with the observed sex-specific effects.

Head skeleton malformations in zebrafish (Danio rerio) to assess adverse effects of mixtures of compounds.

The EU-EuroMix project adopted the strategy of the European Food Safety Authority (EFSA) for cumulative risk assessment, which limits the number of chemicals to consider in a mixture to those that induce a specific toxicological phenotype. These so-called cumulative assessment groups (CAGs) are refined at several levels, including the target organ and specific phenotype. Here, we explore the zebrafish embryo as a test model for quantitative evaluation in one such CAG, skeletal malformations, through exposure to test compounds 0-120 hpf and alcian blue cartilage staining at 120 hpf, focusing on the head skeleton. Reference compounds cyproconazole, flusilazole, metam, and thiram induced distinctive phenotypes in the head skeleton between the triazoles and dithiocarbamates. Of many evaluated parameters, the Meckel's-palatoquadrate (M-PQ) angle was selected for further assessment, based on the best combination of a small confidence interval, an intermediate maximal effect size and a gentle slope of the dose-response curve with cyproconazole and metam. Additional test compounds included in the CAG skeletal malformations database were tested for M-PQ effects, and this set was supplemented with compounds associated with craniofacial malformations or cleft palate to accommodate otherwise organized databases. This additional set included hexaconazole, all-trans-retinoic acid, AM580, CD3254, maneb, pyrimethanil, imidacloprid, pirimiphos-methyl, 2,4-dinitrophenol, 5-fluorouracil, 17alpha-ethynylestradiol (EE2), ethanol, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), PCB 126, methylmercury, boric acid, and MEHP. Most of these compounds produced a dose-response for M-PQ effects. Application of the assay in mixture testing was provided by combined exposure to cyproconazole and TCDD through the isobole method, supporting that in this case the combined effect can be modeled through concentration addition.

Fish embryo toxicity test, threshold approach, and moribund as approaches to implement 3R principles to the acute fish toxicity test.

The acute fish toxicity test (AFT) is requested by EU legal frameworks for hazard classification and risk assessment. AFT is one of the few regulatory required tests using death as an endpoint. This paper reviews efforts made to reduce, refine and replace (3Rs) AFT. We make an inventory of information requirements for AFT, summarize studies on 3Rs of AFT and give recommendations. The fish embryo toxicity test (FET) is proposed as a replacement of AFT and analyses have focused on two aspects: assessing the capacity of FET in predicting AFT and defining the applicability domain of FET. Six comparison studies have consistently shown a strong correlation of FET and AFT. In contrast, the applicability domain of FET has not yet been fully defined. FET has not yet been accepted as a replacement of AFT by any EU legal frameworks to fulfill information requirements because FET is insensitive to some chemicals. It is recommended that the outlier chemicals that do not correlate between FET and AFT should be further investigated. When necessary, additional FET data should be generated. Another effort to reduce and refine AFT is incorporation of FET into the threshold approach. Furthermore, moribund as an endpoint of fish death has been introduced in revising AFT guideline to reduce the duration of suffering for refinement. This endpoint, however, needs further work on the link of moribund and death. Global regulatory acceptance of the moribund endpoint would be critical for this development.

Programmed Effects in Neurobehavior and Antioxidative Physiology in Zebrafish Embryonically Exposed to Cadmium: Observations and Hypothesized Adverse Outcome Pathway Framework.

Non-communicable diseases (NCDs) are a major cause of premature mortality. Recent studies show that predispositions for NCDs may arise from early-life exposure to low concentrations of environmental contaminants. This developmental origins of health and disease (DOHaD) paradigm suggests that programming of an embryo can be disrupted, changing the homeostatic set point of biological functions. Epigenetic alterations are a possible underlying mechanism. Here, we investigated the DOHaD paradigm by exposing zebrafish to subtoxic concentrations of the ubiquitous contaminant cadmium during embryogenesis, followed by growth under normal conditions. Prolonged behavioral responses to physical stress and altered antioxidative physiology were observed approximately ten weeks after termination of embryonal exposure, at concentrations that were 50-3200-fold below the direct embryotoxic concentration, and interpreted as altered developmental programming. Literature was explored for possible mechanistic pathways that link embryonic subtoxic cadmium to the observed apical phenotypes, more specifically, the probability of molecular mechanisms induced by cadmium exposure leading to altered DNA methylation and subsequently to the observed apical phenotypes. This was done using the adverse outcome pathway model framework, and assessing key event relationship plausibility by tailored Bradford-Hill analysis. Thus, cadmium interaction with thiols appeared to be the major contributor to late-life effects. Cadmium-thiol interactions may lead to depletion of the methyl donor S-adenosyl-methionine, resulting in methylome alterations, and may, additionally, result in oxidative stress, which may lead to DNA oxidation, and subsequently altered DNA methyltransferase activity. In this way, DNA methylation may be affected at a critical developmental stage, causing the observed apical phenotypes.

Zebrafish embryos as a screen for DNA methylation modifications after compound exposure.

Modified epigenetic programming early in life is proposed to underlie the development of an adverse adult phenotype, known as the Developmental Origins of Health and Disease (DOHaD) concept. Several environmental contaminants have been implicated as modifying factors of the developing epigenome. This underlines the need to investigate this newly recognized toxicological risk and systematically screen for the epigenome modifying potential of compounds. In this study, we examined the applicability of the zebrafish embryo as a screening model for DNA methylation modifications. Embryos were exposed from 0 to 72 h post fertilization (hpf) to bisphenol-A (BPA), diethylstilbestrol, 17α-ethynylestradiol, nickel, cadmium, tributyltin, arsenite, perfluoroctanoic acid, valproic acid, flusilazole, 5-azacytidine (5AC) in subtoxic concentrations. Both global and site-specific methylation was examined. Global methylation was only affected by 5AC. Genome wide locus-specific analysis was performed for BPA exposed embryos using Digital Restriction Enzyme Analysis of Methylation (DREAM), which showed minimal wide scale effects on the genome, whereas potential informative markers were not confirmed by pyrosequencing. Site-specific methylation was examined in the promoter regions of three selected genes vasa, vtg1 and cyp19a2, of which vasa (ddx4) was the most responsive. This analysis distinguished estrogenic compounds from metals by direction and sensitivity of the effect compared to embryotoxicity. In conclusion, the zebrafish embryo is a potential screening tool to examine DNA methylation modifications after xenobiotic exposure. The next step is to examine the adult phenotype of exposed embryos and to analyze molecular mechanisms that potentially link epigenetic effects and altered phenotypes, to support the DOHaD hypothesis.

A transcriptomics-based hepatotoxicity comparison between the zebrafish embryo and established human and rodent in vitro and in vivo models using cyclosporine A, amiodarone and acetaminophen.

The zebrafish embryo (ZFE) is a promising alternative, non-rodent model in toxicology, which has an advantage over the traditionally used models as it contains complete biological complexity and provides a medium to high-throughput setting. Here, we assess how the ZFE compares to the traditionally used models for liver toxicity testing, i.e., in vivo mouse and rat liver, in vitro mouse and rat hepatocytes, and primary human hepatocytes. For this comparison, we analyzed gene expression changes induced by three model compounds for cholestasis, steatosis, and necrosis. The three compounds, cyclosporine A, amiodarone, and acetaminophen, were chosen because of their relevance to human toxicity and these compounds displayed hepatotoxic-specific changes in the mouse in vivo data. Compound induced expression changes in the ZFE model shared similarity with both in vivo and in vitro. Comparison on single gene level revealed the presence of model specific changes and no clear concordance across models. However, concordance was identified on the pathway level. Specifically, the pathway "regulation of metabolism - bile acids regulation of glucose and lipid metabolism via FXR" was affected across all models and compounds. In conclusion, our study with three hepatotoxic model compounds shows that the ZFE model is at least as comparable to traditional models in identifying hepatotoxic activity and has the potential for use as a pre-screen to determine the hepatotoxic potential of compounds.

Toxicological profile of ultrapure 2,2',3,4,4',5,5'-heptachlorbiphenyl (PCB 180) in adult rats.

PCB 180 is a persistent non-dioxin-like polychlorinated biphenyl (NDL-PCB) abundantly present in food and the environment. Risk characterization of NDL-PCBs is confounded by the presence of highly potent dioxin-like impurities. We used ultrapure PCB 180 to characterize its toxicity profile in a 28-day repeat dose toxicity study in young adult rats extended to cover endocrine and behavioral effects. Using a loading dose/maintenance dose regimen, groups of 5 males and 5 females were given total doses of 0, 3, 10, 30, 100, 300, 1000 or 1700 mg PCB 180/kg body weight by gavage. Dose-responses were analyzed using benchmark dose modeling based on dose and adipose tissue PCB concentrations. Body weight gain was retarded at 1700 mg/kg during loading dosing, but recovered thereafter. The most sensitive endpoint of toxicity that was used for risk characterization was altered open field behavior in females; i.e. increased activity and distance moved in the inner zone of an open field suggesting altered emotional responses to unfamiliar environment and impaired behavioral inhibition. Other dose-dependent changes included decreased serum thyroid hormones with associated histopathological changes, altered tissue retinoid levels, decreased hematocrit and hemoglobin, decreased follicle stimulating hormone and luteinizing hormone levels in males and increased expression of DNA damage markers in liver of females. Dose-dependent hypertrophy of zona fasciculata cells was observed in adrenals suggesting activation of cortex. There were gender differences in sensitivity and toxicity profiles were partly different in males and females. PCB 180 adipose tissue concentrations were clearly above the general human population levels, but close to the levels in highly exposed populations. The results demonstrate a distinct toxicological profile of PCB 180 with lack of dioxin-like properties required for assignment of WHO toxic equivalency factor. However, PCB 180 shares several toxicological targets with dioxin-like compounds emphasizing the potential for interactions.

Gene expression markers in the zebrafish embryo reflect a hepatotoxic response in animal models and humans.

The zebrafish embryo (ZFE) is a promising non-rodent model in toxicology, and initial studies suggested its applicability in detecting hepatotoxic responses. Here, we hypothesize that the detailed analysis of underlying mechanisms of hepatotoxicity in ZFE contributes to the improved identification of hepatotoxic properties of new compounds and to the reduction of rodents used for screening. ZFEs were exposed to nine reference hepatotoxicants, targeted at induction of cholestasis, steatosis and necrosis, and two non-hepatotoxic controls. Histopathology revealed various specific morphological changes in the ZFE hepatocytes indicative of cell injury. Gene expression profiles of the individual compounds were generated using microarrays. Regulation of single genes and of pathways could be linked to hepatotoxic responses in general, but phenotype-specific responses could not be distinguished. Hepatotoxicity-associated pathways included xenobiotic metabolism and oxidoreduction related pathways. Overall analysis of gene expression identified a limited set of potential biomarkers specific for a common hepatotoxicity response. This set included several cytochrome P450 genes (cyp2k19, cyp4v7, cyp2aa3), genes related to liver development (pklr) and genes important in oxidoreduction processes (zgc:163022, zgc:158614, zgc:101858 and sqrdl). In conclusion, the ZFE model allows for identification of hepatotoxicants, without discrimination into specific phenotypes.

A critical appraisal of the process of regulatory implementation of novel in vivo and in vitro methods for chemical hazard and risk assessment.

Regulatory toxicology urgently needs applicable alternative test systems that reduce animal use, testing time, and cost. European regulation on cosmetic ingredients has already banned animal experimentation for hazard identification, and public awareness drives toward additional restrictions in other regulatory frameworks as well. In addition, scientific progress stimulates a more mechanistic approach of hazard identification. Nevertheless, the implementation of alternative methods is lagging far behind their development. In search for general bottlenecks for the implementation of alternative methods, this manuscript reviews the state of the art as to the development and implementation of 10 diverse test systems in various areas of toxicological hazard assessment. They vary widely in complexity and regulatory acceptance status. The assays are reviewed as to parameters assessed, biological system involved, standardization, interpretation of results, extrapolation to human hazard, position in testing strategies, and current regulatory acceptance status. Given the diversity of alternative methods in many aspects, no common bottlenecks could be identified that hamper implementation of individual alternative assays in general. However, specific issues for the regulatory acceptance and application were identified for each assay. Acceptance of one-in-one replacement of complex in vivo tests by relatively simple in vitro assays is not feasible. Rather, innovative approaches using test batteries are required together with metabolic information and in vitro to in vivo dose extrapolation to convincingly provide the same level of information of current in vivo tests. A mechanistically based alternative approach using the Adverse Outcome Pathway concept could stimulate further (regulatory) acceptance of non-animal tests.