New approach methodologies (NAMs) for human-relevant biokinetics predictions. Meeting the paradigm shift in toxicology towards an animal-free chemical risk assessment.

For almost fifteen years, the availability and regulatory acceptance of new approach methodologies (NAMs) to assess the absorption, distribution, metabolism and excretion (ADME/biokinetics) in chemical risk evaluations are a bottleneck. To enhance the field, a team of 24 experts from science, industry, and regulatory bodies, including new generation toxicologists, met at the Lorentz Centre in Leiden, The Netherlands. A range of possibilities for the use of NAMs for biokinetics in risk evaluations were formulated (for example to define species differences and human variation or to perform quantitative in vitro-in vivo extrapolations).

Use of the kinetically-derived maximum dose concept in selection of top doses for toxicity studies hampers proper hazard assessment and risk management.

The KMD (kinetically-derived maximum dose) is an increasingly advocated concept that uses toxicokinetic data in the top dose selection for toxicity testing. Application of this concept may have serious regulatory implications though, especially in the European Union. The basic assumption is that the relationship between internal and external dose (IED) shows an inflection point where linearity transits into non-linearity due to saturation of underlying processes; top doses in toxicity tests should not be above the inflection point, provided human exposures are well below this point.

Mechanism of Action of TiO: Recommendations to Reduce Uncertainties Related to Carcinogenic Potential.

The Risk Assessment Committee of the European Chemicals Agency issued an opinion on classifying titanium dioxide (TiO) as a suspected human carcinogen upon inhalation. Recent animal studies indicate that TiO may be carcinogenic through the oral route. There is considerable uncertainty on the carcinogenicity of TiO, which may be decreased if its mechanism of action becomes clearer.

The value of organs-on-chip for regulatory safety assessment.

Organs-on-chip (OC) have gained much interest as animal-free toxicity testing methods due to their closer resemblance to human tissues and longer culture viability than conventional in vitro methods. The current paper discusses where and how OCs may take a role in the transition to a more predictive, animal-free safety assessment for regulatory purposes. From a preliminary analysis of a repeated dose toxicity database, ten organs of priority for OC development for regu­latory use have been identified.

Towards harmonization of test methods for in vitro hepatic clearance studies.

Non-animal methods for toxicokinetics, such as in vitro hepatic metabolic clearance studies, play an important role in chemical risk evaluations. To gain regulatory acceptance of such clearance data, the development of a test guideline for performing in vitro hepatic clearance studies is crucial. The aim of the present study was to obtain insight in the experimental conditions of clearance studies that influence obtained intrinsic clearance (CLint) values.

Innovative organotypic in vitro models for safety assessment: aligning with regulatory requirements and understanding models of the heart, skin, and liver as paradigms.

The development of improved, innovative models for the detection of toxicity of drugs, chemicals, or chemicals in cosmetics is crucial to efficiently bring new products safely to market in a cost-effective and timely manner. In addition, improvement in models to detect toxicity may reduce the incidence of unexpected post-marketing toxicity and reduce or eliminate the need for animal testing. The safety of novel products of the pharmaceutical, chemical, or cosmetics industry must be assured; therefore, toxicological properties need to be assessed.

Development and Validation of an On-Line Water Toxicity Sensor with Immobilized Luminescent Bacteria for On-Line Surface Water Monitoring.

Surface water used for drinking water production is frequently monitored in The Netherlands using whole organism biomonitors, with for example or mussels, which respond to changes in the water quality. However, not all human-relevant toxic compounds can be detected by these biomonitors. Therefore, a new on-line biosensor has been developed, containing immobilized genetically modified bacteria, which respond to genotoxicity in the water by emitting luminescence.

Risk assessment of titanium dioxide nanoparticles via oral exposure, including toxicokinetic considerations.

Titanium dioxide white pigment consists of particles of various sizes, from which a fraction is in the nano range (<100 nm). It is applied in food as additive E 171 as well as in other products, such as food supplements and toothpaste. Here, we assessed whether a human health risk can be expected from oral ingestion of these titanium dioxide nanoparticles (TiO NPs), based on currently available information.

Oral intake of added titanium dioxide and its nanofraction from food products, food supplements and toothpaste by the Dutch population.

Titanium dioxide (TiO) is commonly applied to enhance the white colour and brightness of food products. TiO is also used as white pigment in other products such as toothpaste. A small fraction of the pigment is known to be present as nanoparticles (NPs).

Biology-inspired microphysiological system approaches to solve the prediction dilemma of substance testing.

The recent advent of microphysiological systems - microfluidic biomimetic devices that aspire to emulate the biology of human tissues, organs and circulation in vitro - is envisaged to enable a global paradigm shift in drug development. An extraordinary US governmental initiative and various dedicated research programs in Europe and Asia have led recently to the first cutting-edge achievements of human single-organ and multi-organ engineering based on microphysiological systems. The expectation is that test systems established on this basis would model various disease stages, and predict toxicity, immunogenicity, ADME profiles and treatment efficacy prior to clinical testing.

Sample preparation for combined chemical analysis and in vitro bioassay application in water quality assessment.

The combination of in vitro bioassays and chemical screening can provide a powerful toolbox to determine biologically relevant compounds in water extracts. In this study, a sample preparation method is evaluated for the suitability for both chemical analysis and in vitro bioassays. A set of 39 chemicals were spiked to surface water, which were extracted using Oasis MCX cartridges.

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 chemical's 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.

Occurrence of glucocorticogenic activity in various surface waters in The Netherlands.

Considering the important role that surface waters serve for drinking water production, it is important to know if these resources are under the impact of contaminants. Apart from environmental pollutants such as pesticides, compounds such as (xeno)estrogens have received al lot of research attention and several large monitoring campaigns have been carried out to assess estrogenic contamination in the aquatic environment. The introduction of novel in vitro bioassays enables researchers to study if - and to what extent - water bodies are under the impact of less-studied (synthetic) hormone active compounds.

Trigger values for investigation of hormonal activity in drinking water and its sources using CALUX bioassays.

To screen for hormonal activity in water samples, highly sensitive in vitro CALUX bioassays are available which allow detection of estrogenic (ERα), androgenic (AR), progestagenic (PR), and glucocorticoid (GR) activities. This paper presents trigger values for the ERα, AR, PR, and GR CALUX bioassays for agonistic hormonal activities in (drinking) water, which define a level above which human health risk cannot be waived a priori and additional examination of specific endocrine activity may be warranted. The trigger values are based on 1) acceptable or tolerable daily intake (ADI/TDI) values of specific compounds, 2) pharmacokinetic factors defining their bioavailability, 3) estimations of the bioavailability of unknown compounds with equivalent hormonal activity, 4) relative endocrine potencies, and 5) physiological, and drinking water allocation factors.

Effect of combining in vitro estrogenicity data with kinetic characteristics of estrogenic compounds on the in vivo predictive value.

With the ultimate aim of increasing the utility of in vitro assays for toxicological risk assessment, a method was developed to calculate in vivo estrogenic potencies from in vitro estrogenic potencies of compounds by taking into account systemic availability. In vitro estrogenic potencies of three model compounds (bisphenol A, genistein, and 4-nonylphenol) relative to ethinylestradiol (EE2), determined with the estrogen receptor alpha (ERα) transcriptional activation assay using hER-HeLa-9903 cells, were taken from literature and used to calculate the EE2 equivalent (EE2EQ) effect doses in the predominantly ERα-dependent rat uterotrophic assay. Compound-specific differences in hepatic clearance relative to the reference compound EE2 were determined in vitro to examine whether in vivo estrogenic potencies reported in literature could be more accurately estimated.

The dosing determines mutagenicity of hydrophobic compounds in the Ames II assay with metabolic transformation: passive dosing versus solvent spiking.

The Ames II bacterial mutagenicity assay is a new version of the standard Ames test for screening chemicals for genotoxic activity. However, the use of plastic micro-titer plates has drawbacks in the case of testing hydrophobic mutagens, since sorptive and other losses make it difficult to control and define the exposure concentrations, and they reduce availability for bacterial uptake or to the S9 enzymes. With passive dosing, a biocompatible polymer such as silicone is loaded with the test compound and acts as a partitioning source.

Are luminescent bacteria suitable for online detection and monitoring of toxic compounds in drinking water and its sources?

Biosensors based on luminescent bacteria may be valuable tools to monitor the chemical quality and safety of surface and drinking water. In this review, an overview is presented of the recombinant strains available that harbour the bacterial luciferase genes luxCDABE, and which may be used in an online biosensor for water quality monitoring. Many bacterial strains have been described for the detection of a broad range of toxicity parameters, including DNA damage, protein damage, membrane damage, oxidative stress, organic pollutants, and heavy metals.

Toxicological relevance of emerging contaminants for drinking water quality.

The detection of many new compounds in surface water, groundwater and drinking water raises considerable public concern, especially when human health based guideline values are not available it is questioned if detected concentrations affect human health. In an attempt to address this question, we derived provisional drinking water guideline values for a selection of 50 emerging contaminants relevant for drinking water and the water cycle. For only 10 contaminants, statutory guideline values were available.

Detection of multiple hormonal activities in wastewater effluents and surface water, using a panel of steroid receptor CALUX bioassays.

It is generally known that there are compounds present in the aquatic environment that can disturb endocrine processes, for example via interaction with the endogenous hormone receptors. Most research so far has focused on compounds that bind to the estrogen and/or androgen receptor, but ligands for other hormone receptors might also be present. In this study, a newly completed panel of human cell derived CALUX reporter gene bioassays was utilized to test water extracts for estrogen (ER), as well as androgen (AR), progesterone (PR), and glucocorticoid (GR) receptor mediated transactivation activity.

Bioavailability in dose and exposure assessment of organic contaminants in (eco)toxicology.

The dose is an essential element in toxicology and risk assessment. In most cases, the dose is expressed as a concentration in the external environment. The internal dose is a more direct measure for the exposure in toxicological assays, because it takes differences in bioavailability into account.

Measurement of the free concentration of octylphenol in biological samples with negligible depletion-solid phase microextraction (nd-SPME): analysis of matrix effects.

A negligible depletion-solid phase microextraction (nd-SPME) method is presented to measure free concentrations of octylphenol in biological samples. Potential confounding factors, such as matrix effects, are studied as well. Fouling of the fibre appears to occur, but it does not seem to reduce or enhance the measured uptake of octylphenol.

Toward more useful in vitro toxicity data with measured free concentrations.

In vitro assays and computer models are promising alternatives for in vivo animal testing, but the power of these alternative methods to predict in vivo risk is still very limited. One step forward is to make the outcome of in vitro assays (such as median effect concentrations (EC50 values)) independent of assay conditions such as protein content. Here we show that measured free concentrations of chemicals in the in vitro assay medium result in system-independent EC50 values.