Nynke I. Kramer

Predicting Adult Fish Acute Lethality with the Zebrafish Embryo: Relevance of Test Duration, Endpoints, Compound Properties, and Exposure Concentration Analysis

The zebrafish embryo toxicity test has been proposed as an alternative for the acute fish toxicity test, which is required by various regulations for environmental risk assessment of chemicals. We investigated the reliability of the embryo test by probing organic industrial chemicals with a wide range of physicochemical properties, toxicities, and modes of toxic action. Moreover, the relevance of using measured versus nominal (intended) exposure concentrations, inclusion of sublethal endpoints, and different exposure durations for the comparability with reported fish acute toxicity was explored. Our results confirm a very strong correlation of zebrafish embryo to fish acute toxicity. When toxicity values were calculated based on measured exposure concentrations, the slope of the type II regression line was 1 and nearly passed through the origin (1 to 1 correlation). Measured concentrations also explained several apparent outliers. Neither prolonged exposure (up to 120 h) nor consideration of sublethal effects led to a reduced number of outliers. Yet, two types of compounds were less lethal to embryos than to adult fish: a neurotoxic compound acting via sodium channels (permethrin) and a compound requiring metabolic activation (allyl alcohol).

Dose metric considerations in in vitro assays to improve quantitative in vitro-in vivo dose extrapolations

Challenges to improve toxicological risk assessment to meet the demands of the EU chemicals legislation, REACH, and the EU 7th Amendment of the Cosmetics Directive have accelerated the development of non-animal based methods. Unfortunately, uncertainties remain surrounding the power of alternative methods such as in vitro assays to predict in vivo dose-response relationships, which impedes their use in regulatory toxicology. One issue reviewed here, is the lack of a well-defined dose metric for use in concentration-effect relationships obtained from in vitro cell assays. Traditionally, the nominal concentration has been used to define in vitro concentration-effect relationships. However, chemicals may differentially and non-specifically bind to medium constituents, well plate plastic and cells. They may also evaporate, degrade or be metabolized over the exposure period at different rates. Studies have shown that these processes may reduce the bioavailable and biologically effective dose of test chemicals in in vitro assays to levels far below their nominal concentration. This subsequently hampers the interpretation of in vitro data to predict and compare the true toxic potency of test chemicals. Therefore, this review discusses a number of dose metrics and their dependency on in vitro assay setup. Recommendations are given on when to consider alternative dose metrics instead of nominal concentrations, in order to reduce effect concentration variability between in vitro assays and between in vitro and in vivo assays in toxicology.

The Use of Biomarkers of Toxicity for Integrating In Vitro Hazard Estimates Into Risk Assessment for Humans

The role that in vitro systems can play in toxicological risk assessment is determined by the appropriateness of the chosen methods, with respect to the way in which in vitro data can be extrapolated to the in vivo situation. This report presents the results of a workshop aimed at better defining the use of in vitro-derived biomarkers of toxicity (BoT) and determining the place these data can have in human risk assessment. As a result, a conceptual framework is presented for the incorporation of in vitro-derived toxicity data into the risk assessment process. The selection of BoT takes into account that they need to distinguish adverse and adaptive changes in cells. The framework defines the place of in vitro systems in the context of data on exposure, structural and physico-chemical properties, and toxicodynamic and biokinetic modeling. It outlines the determination of a proper point-of-departure (PoD) for in vitro-in vivo extrapolation, allowing implementation in risk assessment procedures. A BoT will need to take into account both the dynamics and the kinetics of the compound in the in vitro systems. For the implementation of the proposed framework it will be necessary to collect and collate data from existing literature and new in vitro test systems, as well as to categorize biomarkers of toxicity and their relation to pathways-of-toxicity. Moreover, data selection and integration need to be driven by their usefulness in a quantitative in vitro-in vivo extrapolation (QIVIVE).

Quantifying Processes Determining the Free Concentration of Phenanthrene in Basal Cytotoxicity Assays

Difficulties may arise when extrapolating in vitro derived toxicity data to in vivo toxicity data because of the high variability and occasional low sensitivity of in vitro results. Differences in the free concentration of a test compound between in vitro and in vivo systems and between different in vitro systems may in part explain this variability and sensitivity difference. The aim of this study was to determine what assay components influence the free concentration of phenanthrene in a Balb/c 3T3 and RTgill-W1 MTT assay. Partition coefficients of phenanthrene to serum, well plate plastic, cells, and headspace were measured and subsequently used to model the free concentration of the compound in vitro. The estimated free concentration was compared to the free concentration measured in the assays using solid phase microextraction (SPME). Results indicate that the free concentration of phenanthrene, a relatively volatile and hydrophobic compound, is significantly reduced in a typical in vitro setup as it binds to matrices such as serum protein and well plate plastic. A reduction in free concentration due to increasing serum protein levels is accompanied by an increase in the median effect concentration (EC(50)) and can be modeled, with the exception of evaporation, using the partition coefficients of the compound to assay components.

Development of a partition-controlled dosing system for cell assays.

Hydrophobic and volatile chemicals have proven to be difficult to dose in cell assays. Cosolvents are often needed to dissolve these chemicals in cell culture medium. Moreover, the free concentration of these chemicals in culture medium may diminish over time due to metabolism, evaporation, and nonspecific binding to well plate surfaces and serum constituents. The aim of this study was to develop a partition-controlled dosing system to maintain constant concentrations of benzo(a)pyrene, 1,2-dichlorobenzene, and 1,2,4-trichlorobenzene in an ethoxyresorufin-O-deethylase (EROD) assay and a cytotoxicity assay with the rainbow trout (Oncorhynchus mykiss) cell lines RTL-W1 and RTgill-W1. Polydimethylsiloxane (PDMS) sheets were loaded with test chemicals in a spiked methanol/water solution and placed in the wells, filled with culture medium, of a 24-well culture plate. Cells were grown on inserts and were subsequently added to the wells with the PDMS sheets. The system reached equilibrium within 24 h, even for the very hydrophobic chemical benzo(a)pyrene. The reservoir of test chemical in PDMS was large enough to compensate for the loss of >95% of the test chemical from the culture medium. The PDMS sheets maintained medium concentrations constant for >72 h. Nominal median effect concentrations (EC(50)) were 1.3-7.0 times lower in the partition-controlled dosing systems than in conventional assays spiked using dimethyl sulfoxide (DMSO) as a carrier solvent, thus indicating that the apparent sensitivity of the bioassay increased when controlled and constant exposure conditions could be assured. The EC(50) values of the test chemicals based on free concentrations were estimated in the partition-controlled dosing systems using measured PDMS-bare culture medium partition coefficients. Results indicated that 61, 70, and 99.8% of 1,2-diclorobenzene, 1,2,4-trichlorobenzene, and benzo(a)pyrene were bound to serum constituents in the culture medium.

PBTK modelling platforms and parameter estimation tools to enable animal-free risk assessment: recommendations from a joint EPAA--EURL ECVAM ADME workshop.

Information on toxicokinetics is critical for animal-free human risk assessment. Human external exposure must be translated into human tissue doses and compared with in vitro actual cell exposure associated to effects (in vitro-in vivo comparison). Data on absorption, distribution, metabolism and excretion in humans (ADME) could be generated using in vitro and QSAR tools. Physiologically-based toxicokinetic (PBTK) computer modelling could serve to integrate disparate in vitro and in silico findings. However, there are only few freely-available PBTK platforms currently available. And although some ADME parameters can be reasonably estimated in vitro or in silico, important gaps exist. Examples include unknown or limited applicability domains and lack of (high-throughput) tools to measure penetration of barriers, partitioning between blood and tissues and metabolic clearance. This paper is based on a joint EPAA--EURL ECVAM expert meeting. It provides a state-of-the-art overview of the availability of PBTK platforms as well as the in vitro and in silico methods to parameterise basic (Tier 1) PBTK models. Five high-priority issues are presented that provide the prerequisites for wider use of non-animal based PBTK modelling for animal-free chemical risk assessment.

Effects of solvents and dosing procedure on chemical toxicity in cell-based in vitro assays.

Due to the implementation of new legislation, such as REACh, a dramatic increase of animal use for toxicity testing is expected and the search for alternatives is timely. Cell-based in vitro assays are promising alternatives. However, the behavior of chemicals in these assays is still poorly understood. We set out to quantify the exposure and associated toxicity of chemicals with different physicochemical properties toward a fish gill cell line when different solvents and procedural steps are used to introduce test chemicals to cells. Three chemicals with a range of hydrophobicity and volatility were selected and delivered in three different solvents using two common dosing procedures. Toxicity tests were coupled with chemical analysis to quantify the chemical concentrations within culture wells. The impact of solvents and dosing procedure was greatest for the most volatile and hydrophobic test chemical. We show that certain combinations of the test chemical, solvent, and procedural steps can lead to inhomogeneous distribution of the test chemical and thus differing degrees of bioavailability, resulting in quantitative differences in apparent toxicity.

Developing a list of reference chemicals for testing alternatives to whole fish toxicity tests

This paper details the derivation of a list of 60 reference chemicals for the development of alternatives to animal testing in ecotoxicology with a particular focus on fish. The chemicals were selected as a prerequisite to gather mechanistic information on the performance of alternative testing systems, namely vertebrate cell lines and fish embryos, in comparison to the fish acute lethality test. To avoid the need for additional experiments with fish, the U.S. EPA fathead minnow database was consulted as reference for whole organism responses. This database was compared to the Halle Registry of Cytotoxicity and a collation of data by the German EPA (UBA) on acute toxicity data derived from zebrafish embryos. Chemicals that were present in the fathead minnow database and in at least one of the other two databases were subject to selection. Criteria included the coverage of a wide range of toxicity and physico-chemical parameters as well as the determination of outliers of the in vivo/in vitro correlations. While the reference list of chemicals now guides our research for improving cell line and fish embryo assays to make them widely applicable, the list could be of benefit to search for alternatives in ecotoxicology in general. One example would be the use of this list to validate structure-activity prediction models, which in turn would benefit from a continuous extension of this list with regard to physico-chemical and toxicological data.

A novel hypothesis for an alkaline phosphatase 'rescue' mechanism in the hepatic acute phase immune response.

The liver isoform of the enzyme alkaline phosphatase (AP) has been used classically as a serum biomarker for hepatic disease states such as hepatitis, steatosis, cirrhosis, drug-induced liver injury, and hepatocellular carcinoma. Recent studies have demonstrated a more general anti-inflammatory role for AP, as it is capable of dephosphorylating potentially deleterious molecules such as nucleotide phosphates, the pathogenic endotoxin lipopolysaccharide (LPS), and the contact clotting pathway activator polyphosphate (polyP), thereby reducing inflammation and coagulopathy systemically. Yet the mechanism underlying the observed increase in liver AP levels in circulation during inflammatory insults is largely unknown. This paper hypothesizes an immunological role for AP in the liver and the potential of this system for damping generalized inflammation along with a wide range of ancillary pathologies. Based on the provided framework, a mechanism is proposed in which AP undergoes transcytosis in hepatocytes from the canalicular membrane to the sinusoidal membrane during inflammation and the enzymes expression is upregulated as a result. Through a tightly controlled, nucleotide-stimulated negative feedback process, AP is transported in this model as an immune complex with immunoglobulin G by the asialoglycoprotein receptor through the cell and secreted into the serum, likely using the receptors State 1 pathway. The subsequent dephosphorylation of inflammatory stimuli by AP and uptake of the circulating immune complex by endothelial cells and macrophages may lead to decreased inflammation and coagulopathy while providing an early upstream signal for the induction of a number of anti-inflammatory gene products, including AP itself.

The influence of modes of action and physicochemical properties of chemicals on the correlation between in vitro and acute fish toxicity data.

New EU legislation is providing an impetus for research aimed at replacing acute fish toxicity testing with in vitro alternatives. In line with such research, the objective of this study was to determine what factors influence the correlation between in vitro and fish toxicity data. Basal cytotoxicity (IC(50)) and acute toxicity data from fathead minnow (LC(50)) of 82 industrial organic chemicals were obtained from the Halle Registry of Cytotoxicity and the US EPA Fathead Minnow Database. A good correlation between IC(50) with LC(50) data was found (r 0.84). Yet, IC(50) data were less sensitive than LC(50) data by an order of magnitude. Using multiple regression analysis, the octanol-water partition coefficient (K(OW)) and the Henrys Law Constant (H) were found to significantly explain the low absolute sensitivity. The mode of action (MOA) of the chemical was found to significantly explain the general variation in the logIC(50)/log LC(50) regression line. These results support the notion that (a) the bioavailability of hydrophobic (high K(OW)) and volatile (high H) chemicals is significantly lower in in vitro assays than in the fish bioassay and (b) multiple cell types and endpoints should be included to mimic the modes of action possible in the whole organism.