Chemical safety assessment encompasses the qualitative description of the toxic properties and also a quantification of exposure and toxic response.
The assessment of dose-effect relationship includes evaluation of exposure at the site of action.
Toxicokinetic evaluations help to relate the chemical concentration/dose to the observed toxicity effect and to understand the mode of action of the chemical and/or its metabolites.
Understanding the toxicokinetic processes that lead to the formation or distribution of the active chemical entity at the target tissue(s) is essential for estimating the dose at the toxicological target site(s).
Toxicokinetics and ADME
Toxicokinetics describes how the body handles a chemical, as a function of dose and time, in terms of the concept of ADME (absorption, distribution, metabolism and excretion):
- The rate of chemical absorption from the site of application into the blood stream
- The rate and extend of chemical movement out of blood into the tissue (distribution)
- The rate and extend of chemical biotransformation into metabolites (metabolism)
- The rate of chemical removal from the body (excretion)
The dose at target tissue(s) is the net result of the rate and magnitude of the ADME processes. Knowledge of the ADME processes is critical, especially in a century where safety assessment has to be based more and more on alternative (in silico and in vitro) methods. Human external exposure has to be translated into a human target tissue dose and compared with in vitro effect levels.
Basic toxicokinetic parameters determined from in vitro and in silico studies will also provide information on the potential for accumulation of the chemical in tissues and/or organs and the potential for inhibition or induction of biotransformation as a result of exposure to the chemical.
Toxicodynamics refers to the molecular, biochemical, and physiological effects of chemicals or their metabolites in biological systems.
These effects are the result of the interaction of the biologically effective dose of the active chemical with a molecular target. The in vitro results generated at tissue/cell or sub-cellular level have to be converted into dose-response information for the entire organism again using toxicokinetic considerations.
Since it is not always feasible or possible to measure target tissue concentration of the chemical and/or its metabolites, toxicokinetic models are increasingly being sought as valuable tools in human health safety assessment.
Physiologically-Based ToxicoKinetics (PBTK) models are mathematical descriptions of ADME processes. These models facilitate quantitative descriptions of the temporal change in the concentration of chemical and/or its metabolites in biological matrices (e.g., blood, tissue, urine, alveolar air) of the exposed organism.
PBTK models describe the organism as a set of compartments that are characterized physiologically or empirically. Two categories of parameters are needed in order to simulate the toxicokinetics of a chemical in a PBTK model:
- Physiological parameters that are chemical-independent, such as cardiac output and organ blood flow (species-, sex- and age-specific and largely available in public literature) blood flow (species-, sex- and age-specific and largely available in public literature)
- Chemical-specific parameters that have to be determined e.g. by in vitro test methods or predicted by in silico(data-based) methods for each chemical.
Regulatory acceptance and legislation
According to OECD TG 417 (OECD TG 417, 2008), toxicokinetics should be evaluated in vivo using the rat as test system.
Such toxicokinetic evaluation is carried out in a variety of sectors for regulatory purposes (mainly for drugs and food additives but also in cases for pesticides and biocides, cosmetics, environmental pollutants, occupational and industrial chemicals).
Although in vitro metabolically competent sources often provide a qualitatively and quantitative good picture of the metabolites formation in the body, comprehensive in vivo studies for metabolite profiling and identification are often still required by regulatory authorities to confirm the metabolic fate of the compound.
Metabolite profiling studies in vivo are naturally conducted in animals; however, the FDA guidelines(FDA 2008) state “we strongly recommend in vivo metabolic evaluation in humans be performed as early as feasible”.
The first aim is to compare metabolites’ profiles between human and animals used in toxicity studies, and further to identify metabolites being disproportionate in humans, i.e. present in human only or present at higher levels in human with respect to animals.
Indeed, it becomes more and more evident that animal derived toxicokinetic data are not always reliable for extrapolation to human safety assessment, due to inter-species differences in physiology, biochemical and metabolic pathways.
For these reasons, and due to requirements in EU Directive 2010/63/EU on the protection of animals used for scientific purposes, the EU Cosmetic Regulation (EC 1223/2009) and the REACH Regulation (EC 1907/2006), there is an increasing pressure to develop alternative (non-animal) toxicokinetic methods to reliably determine the necessary ADME parameters.
The European Food Safety Authority (EFSA) described in a recent guidance (EFSA, 2012) that toxicokinetic data can be derived from a suite of studies covering ADME, including in vitro, in silico and in vivo studies, and single and repeated dose kinetics.
Also in the recent Guidance on information requirements and chemical safety assessment (ECHA, 2012) it is mentioned that “Even though toxicokinetics is not a toxicological endpoint and is not specifically required by REACH, the generation of toxicokinetic information can be encouraged as a means to interpret data, assist testing strategy and study design, as well as category development, thus helping to optimise test designs”.
When moving from the classical toxicological safety assessment based on the whole animal methods to approaches based on alternative in vitro and in silico methods, toxicokinetics is perceived as a key element to assess systemic effects. This will be the case now for the cosmetic sector, where animal testing is completely banned.
For this sector the availability of a human metabolically competent test system is the prerequisite for reliable data on clearance of the chemical and/or its metabolites and for the quantitative and qualitative determination of metabolite formation once the compound is absorbed from the site of application into the blood stream.
EURL ECVAM intends to further elaborate its strategy in the toxicokinetic area with the aim to direct research and development efforts and to identify needs and today’s priorities for formal validation.
Validated toxicokinetics test method
This test method has been used as a stand-alone test method for several regulatory requirements. However, to adequately measure the flux across the dermal barrier for the purpose of integration into PBTK modeling approaches, some additional work needs to be carried out.
In addition to the officially accepted test methods, other in vitro and in silico methods for measuring ADME processes are available and are routinely used for specific in-house interests or used as supporting toxicokinetic data for the regulatory dossiers based on in vivo testing data (e.g. drug and food sector).
Extensive sets of data are available for some of them, demonstrating their importance in building up a picture of specific qualitative and quantitative toxicokinetic information.
Test methods under validation by EURL ECVAM
At experts meetings in the field of toxicokinetics and metabolism, it was agreed that a validation study providing a standard for human hepatic metabolism and toxicity would have been very beneficial.
As a follow up, EURL ECVAM coordinates the “Multi-study Validation Trial for cytochrome P450 (CYP) induction providing a reliable human-metabolic competent standard model or method using the human cryopreserved HepaRG cell line and cryopreserved human hepatocytes”.
The assay is based on cryopreserved human hepatocytes and cryopreserved human metabolically competent cell line (HepaRG) and is standardised using coded test items. The coded test items are selected on the basis evidence from in vivo human data on their CYP induction potential.
Furthermore, the chemical have been selected in order to be sure that all the three main nuclear receptors (CAR, AhR, PXR) involved in CYP induction are covered.
The endpoint “induction of CYPs” is measured following treatment with test compounds and two reference compounds (β-naphthoflavone and rifampicin), using a cocktail of prototypical substrates (Kanebratt et al., 2008) for different CYP isoforms incubated directly with the two test systems.
The results of this study are envisaged to be the starting point for a novel in vitro platform for assessing metabolism and toxicity. In addition in moving towards a mode of action based approach to safety assessment, up-regulation of CYP iso-enzymes has been identified as a key event potentially leading to a number of adverse events and, as such, this assay may contribute to the elucidation of a number of adverse outcome pathways.
Development and optimisation of alternative toxicokinetics methods
The main aim of all hazard and risk assessment strategies is to assess human health effects. Ideally, one would say that in silico and in vitro methods should model human toxicokinetic processes and should use human cells and/or tissues.
However, due to the limited availability of human cells and tissues, and the ethical concerns which are often raised in obtaining and using them, other approaches are being developed.
In order to avoid any need for species extrapolation, it is strongly recommended that the in vitro or in silico models used should provide information relevant for human hazard assessment.
In some cases, animal derived cells and tissues can comply with this objective, but in other cases, such as when Phase I and Phase II biotransformation pathways are involved, species differences are well described. In these latter cases, it is essential to incorporate strategies and approaches that take this aspect into account and will ultimately be relevant to what is happening in the human body.