Location: University of Utrecht (the Netherlands)

Date of establishment: 2008

Duration of contract: 6 years

End of furtherance: 2014

Chairholder: Prof. dr. Bas Blaauboer

By establishing and funding an endowed professorship on alternative methods to animal experimentation at IRAS (the Institute for Risk Assessment Sciences) the Utrecht University, the Doerenkamp-Zbinden Foundation and the Faculty of Veterinary Medicine, Utrecht University pursue the common goal to promote the development of alternative methods exclusively in the field of toxicology and their implementation into regulations through interdisciplinary research and education in an international context.

The work of the chair is focusing on the need to improve the toxicological risk assessment process, thereby acknowledging the need to reduce and replace the use of animal-based models. This requires a shift in the paradigm of toxicity testing from a system that is based on determining apical endpoints (clinical, clinical-chemical and histopathological parameters) in animal studies towards the interpretation of mechanisms of toxicity linked to changes in biological pathways in cellular systems. Using these data as the basis for a quantitative extrapolation from in vitro dose metrics to in vivo exposure scenarios in a transparent, robust and reliable system will then give improved predictions of toxicological risk. When cellular systems derived from human tissues are employed, thus avoiding the problems with interspecies extrapolations, such systems can be better used for predicting human health risks.

The chair focuses on the following lines of research:

1. Biotransformation of compounds in human in vitro systems.

2. Use of in vitro models to study mechanisms of toxicity.

3. Development of QSAR models for the estimation of relevant toxic endpoints.

4. Development and implementation of PBBK models for the interpretation of in vitro toxicity data for their relevance of exposure scenarios.

5. Research in the factors and actors involved in the implementation of alternative methods in regulatory processes.

Information about the chair at the University of Utrecht can be found at the following link:

→ http://www.uu.nl/staff/BJBlaauboer/0

Highlighted projects:

• Factors stimulating or obstructing the acceptance and implementation of the 3rs in the regulatory process.
  

Approximately 30% of the animal experiments within the European Union is done to meet regulatory requirements. Regulatory animal testing is part of the safety and quality testing done prior to the release of a product or compound onto the market to ensure the safety of humans, animals or the environment. The requirements prescribe which experiments must be carried out in order to license and release a compound or product onto the market. Over the last decades the heavy reliance on animal experimentation in this area has encountered serious objections for ethical, scientific and economical reasons. The new Directive 2011/63/EU, which regulates the protection of animals used for experimental and other scientific purposes at a European level, stipulates that alternatives, if available, should be used. The requirements dealing with the registration and release of products often leave room for regulators to choose the testing method they perceive as most suitable for the job. But even though the number of 3R methods has risen sharply over the last decades, regulatory acceptance and the implementation of alternative methods has not kept pace with the development of these tests. This means that, even if safety and quality requirements allow the assessors and industry to use available alternative methods, they often show a preference for the conventional methods. The acceptance and implementation of alternative methods proves to be a difficult process that meets with a whole range of obstacles.

This raises the question how regulators and industry use the discretionary space available to them and which factors influence the decision-making process preceding regulatory acceptance and implementation of 3R methods models.

Although a general study has been conducted to identify possible factors influencing regulatory and industries decision making whether or not to accept/implement 3R methods (Schiffelers et al, 2005), it has become clear that the actual situation differs per sector/ per product and maybe even per industry. To identify the actual importance and relative force of these different factors on the acceptance and implementation of 3R method for regulatory purposes, the examination of case-studies is needed.

Update: Withing this project a PhD thesis was published in 2016. The entire work is available for download:

>>„ANIMAL TESTING, 3R MODELS AND REGULATORY ACCEPTANCE - Technology Transition in a Risk-averse Context“<<

• Improving quantitative in vitro-in vivo extrapolation by using alternative dose metrics in vitro
  

This project aims to develop an understanding of the dose metric that is required from in vitro toxicity assays to accurately extrapolate in vitro toxicity data to in vivo relevant doses. Currently, in vitro toxicity data are expressed as nominal concentrations in culture medium. However, the use of such concentrations for quantitative in vitro-in vivo toxic dose extrapolations may introduce serious uncertainties as these concentrations are highly dependent on the in vitro set-up and may not accurately reflect the true toxic potency of the test chemical. By measuring the nominal, free and internal cell concentration of chemicals spanning a range of physicochemical properties in various in vitro (carcinogenicity) assays over time, the dependence of the observed effect concentration on non-specific binding, exposure and recovery time, cell concentration and type, metabolism, evaporation, degradation, and the chemical’s mechanism of action are be deduced. Such dependence sheds light onto whether physiologically based biokinetic (PBBK) models translating toxic potencies determined in in vitro assays to equivalent acute and chronic toxic doses in humans are best parameterized using in vitro effect concentrations expressed as nominal concentrations, initial free concentrations, free concentrations at the end of the exposure period, the geometric mean of free concentrations over time, maximum or peak concentrations in the cell, ‘critical cell burdens’ (analogous to critical body residues, CBR), or the area under a curve (AUC, concentration in cells over time).

• The role of facilitated transport by serum protein in in vitro intrinsic clearance
  

When a chemical is exposed to an in vitro cell assay in culture medium with serum protein, the effect (e.g. clearance) observed may be lower than when the chemical is exposed in culture medium without serum protein. This is because the chemical can bind to serum protein, which reduces the unbound free concentration of the chemical available for uptake into cells. Therefore, it is better to determine in vitro intrinsic clearance (CLint) based on unbound fractions of chemicals. However, serum protein may also facilitate the transport of chemicals through aqueous media (facilitated transport). Thus the presence of serum protein may increase the uptake rate of chemicals into cells and solid phase microextraction (SPME) fibers. If the uptake rate determines clearance, then the presence of serum protein may increase clearance, thus hampering the extrapolation of in vitro CLint to in vivo clearance when clearance assays use varying concentrations of serum. Therefore, the uptake rate and clearance of chemicals strongly and weakly bound to albumin, and chemicals slowly and quickly cleared in vitro is measured in a number of in vitro clearance assays (including HepaRG, HepG2 and H4IIE hepatoma cell lines) at varying concentrations of bovine serum albumin using the substrate depletion approach. To measure the free fraction, mimic uptake of these chemicals in cells, and facilitate the modeling of the transport into cells, SPME is used to extract the unbound chemical from the exposure medium.

• The development of in vitro assays to test for sex steroid hormone production interference
  

Sex steroidogenesis (i.e. CYP17 and CYP19/aromatase enzyme activity) is a target for endocrine disrupting compounds (EDCs) which by their action can disrupt the hormonal balance in humans and other organisms thus resulting in reproductive toxicity. This project aims at developing in vitro alternative (screening) methods for EDCs, more specifically directed towards identifying effects on steroidogenesis and spermatogenesis. At first, a comparison of a new model using porcine adrenal cortex microsomes (PACMs) for assessing effects on CYP17, the enzyme responsible for formation of DHEA, the most abundant circulating steroid in the human body, was evaluated and compared with the H295R cell steroidogenesis assay and the human placental microsomes (HPMs) assay for CYP19 activity (Roelofs et al, 2013). Next, our research focused at the topic of male fertility. We are currently investigating the influence of the conazoles fungicides on their ability to activate the androgen receptor (AR) via the T47D-ARE cell line and their effects on steroidogenesis in the MA-10 and TM3 mouse Leydig cell lines. Furthermore, a collaboration with the department of Pharmacology and Toxicology of the Radboud University Nijmegen Medical Centre (RUNMC) was established. This resulted in an in vitro study using MA-10 cells and HEK293 cells overexpressing specific ABC transporters to study the effect of six suggested EDCs (i.e. BPA, TBBPA, DEHP, MEHP, PFOA, and PFOS) on androgen secretion by Leydig cells and the influence of ABC transporters on this process. In the near future several suggested EDCs (e.g. conazoles fungicides) will be examined with the different in vitro models to get more insight in the mechanism of their endocrine disrupting properties.

• The development of in vitro models to study the hypothalamus pituitary gonadal (HPG) axis
  

The project aims to investigate the available in vitro models of hypothalamus pituitary gonadal (HPG) axis to study the mechanisms of action and the effects of environmental endocrine disrupting compounds on this system. The main focus was put on female HPG axis. The in vitro models include: available rat and human cell lines as well as  rat and porcine primary cultures (purchased from slaughterhouse). Testing compounds include: dioxins and non-dioxin like compounds, estrogenic / antiestrogenic  like compounds (phthalates, phytoestrogens). Achievements: new rat hypothalamic GnV-3 cell line was evaluated in the scope of the involvement of Aryl hydrocarbon receptor (Ahr) in the regulation of important neuroendocrinological processes in the hypothalamus (circadian rhythm, GnRH release, food intake). Additionally, the adverse effects of dioxin and non-dioxin like compounds was evaluated at hypothalamus, pituitary and ovarian level using rat primary cultures. Moreover, human granulosa like tumor cell line KGN was used to study the effects of  several groups of phytoestrogens (8-prenylnaringenin, quercetin, resveratrol, coumestrol, genistein) on the microenviromental modulation of granulose type tumor. Finally, the effects of estrogenic like compounds (phytoestrogens and phthalates) are investigated on the in vitro maturation of cumulus cells and oocytes using primary porcine oocytes. Furthermore, the possible effects of those compounds on embryo development will be elucidated. 

• An in vitro model to study the regulation of alkaline phosphatase induction in the liver
  

The enzyme alkaline phosphatase (AP) regulates inflammatory immune responses by dephosphorylating extracellular nucleotides and lipopolysaccharides (LPS). Several isoenzymes of AP exist of which the liver type AP is the most abundant tissue non-specific type. Upon a pro-inflammatory stimulus, AP is released from liver into the circulation and quickly cleared by Kupffer cells after dephosphorylating LPS. Clinically, it has been shown that endogenous AP was induced in open-heart surgery patients treated with a bolus and a subsequent infusion of supplemental AP during surgery, thus reducing the risk of systemic inflammatory response syndrome (SIRS). The aim of this study is to develop an in vitro assay to study the mechanism of AP induction in the liver and its release from the cells after an inflammatory insult. An insight into the mechanism should improve the search for drugs and techniques to prolong the residence time of AP in the bloodstream of patients with, for example, rheumatism and a high risk of SIRS. The mRNA expression and activity of AP in the human hepatocarcinoma cell lines, HepaRG and HepG2, as well as the osteoblast-like cell line, Saos-2, are measured after LPS stimulation under various (co-)culture conditions.

• The effect of the displacement of chemicals from serum constituents on in vitro mixture toxicity
  

In vitro assays may be used to estimate toxicity of mixtures of chemicals. The concentrations of chemicals in these assays are normally expressed as nominal concentrations. However, the freely available concentration may be much lower than the nominal concentration because the chemical may bind to serum constituents in the culture medium. When chemicals are exposed to an in vitro assay in a mixture, one chemical that is normally bound to serum protein (and thus has a low free concentration) may be displaced from serum protein by another chemical that binds more strongly to protein, thus increasing the free concentration and response of the first chemical in the assay. When nominal concentrations are used, one could falsely attribute this increase in response as being a direct effect of the second chemical. Therefore, the aim of this study is to measure the free concentration of a number of organic chemicals individually and in a mixture, in a CAFLUX and a basal cytotoxicity assay, using solid phase microextraction (SPME).

• Data on in vitro metabolism and mechanisms of action in combination with kinetic modeling: integrating in risk assessment
  

This project is being carried out in the framework of a CEFIC LRI project, which is conducted in cooperation with The Hamner Institutes in Research Triangle Park (NC, USA), the Dutch National Institute of Health and the Environment (RIVM, Bilthoven, NL) and KWR Water Research Institute (Nieuwegein, NL).

In this study an approach was developed for the integration of in silico and in vitro derived toxicity data in human risk assessment. The first part of the project was focusing on selecting a set of compounds for which there are reliable in vivo toxicity data available. For these compounds we used a number of in silico systems (DEREK, OECD-Toolbox, TOPKAT) to predict toxicity endpoints and targets in vivo. In silico systems to predict the formation of metabolites (METEOR, OECD-Toolbox) were used and added to the prediction for toxicity endpoints and targets. The outcomes were compared with known in vivo toxicity endpoints and targets. A main conclusion was that a generally good qualitative prediction was possible, with some drawbacks that can be related to the underrepresentation of certain endpoints in the in silico systems (e.g. neurotoxicity). The addition of biotransformation predictions further improved the qualitative predictions.

Part 2 used the qualitative predictions made in part 1 in selecting appropriate in vitro systems that could be used to obtain toxic concentrations for the endpoints and targets. This was mainly done on the basis of date derived from studies found in the literature, and preference was given to data derived from those studies that used well-established in vitro systems.

In part 3, kinetic modelling was used to evaluate steady-state blood concentrations of the  compounds in this study, related to no-effect levels in vivo. This was done based on in vivo and on in vitro data. The results were very comparable in the majority of the cases.

In the final part 4 of the project we used the in vitro-obtained toxicity data (from part 2) as points of departure to predict in vivo toxic doses, making use of reverse dosimetry based on the kinetic modelling done in part 3. The outcome was then compared with known in vivo human safe doses. The result showed that in general the in vitro-derived evaluations for human risk underestimated the in vivo toxicity. In over half the cases studied here, however, the ratio between the in vitro and the in vivo derived risk estimates differed no more than two orders of magnitude.

Important reasons for the underestimations are the apparent lack of relevant biotransformation processes in the in vitro systems as well as the lack of detailed data on in vitro biokinetic behaviour of the compounds under study.

Overall we conclude that the QIVIVE approach proposed here to integrate in silico- and in vitro-derived toxicity data needs refinements, mainly by improving our knowledge of the relevant biotransformation processes and how to incorporate them in in vitro systems. In general, however, qualitative predictions of endpoints was good; the quantification very much depends on the quality of the in vitro data for their relevance to qualitatively predict in vivo toxicity more precisely.

• Profiling the toxicity of new drugs: a non animal-based approach integrating toxico-dynamics and biokinetics
  

Improvement of the applicability of in vitro methods by better understanding the biokinetics of a compound within the in vitro systems, i.e. “biokinetics in vitro”.  This is the major component of the work at IRAS in relation to the EU-framework FP7programme Predict-IV. By studying the processes that determine the real (free) concentration (evaporation, binding to plastic and to medium proteins) we were able to improve the possibilities to interpret the toxicity of compounds. This was done by developing and implementing methods to determine the free concentration of compounds by means of solid-phase microextraction (SPME) methods. This was done in 3T3 cells, fish cell lines (as part of other programmes) as well as in the Caco-2 system for measuring transport over a gastrointestinal epithelium layer.