Given the scenario of the development of cosmetic products based on non-animal testing strategies non-in vivo animal testing strategies (1R)Footnote 8, toxicokinetics becomes the essential and central body of information.
Information on toxicokinetics under 1R is indispensable to address three major issues:
Development and design of more efficient testing strategies: As a key starting point for any toxicological testing, it is essential to know whether a compound and/or its metabolites will be bioavailable by one of the relevant uptake routes. Only in cases where a cosmetic ingredient is bioavailable following dermal, oral or inhalation exposure, further tests on systemic and not just local toxicity would be necessary.
In vitro–in vivo extrapolation: To relate toxicodynamic information from non-in vivo animal testing (1R) to real-life situation relevant for humans, i.e. to transform an in vitro concentration–effect relationship into an in vivo dose–effect relationship. In this respect, the role of in vitro biokinetics is crucial to translate a nominal in vitro concentration to the actual level of cell exposure producing the observed effects. For the proper design and performance of in vitro studies, it is important to include kinetic and analytical aspects in the in vitro test protocols.
Identification of clearance rates and the role of metabolites: For the in vitro dynamics experiments, it is essential to know whether the cell or tissue under human exposure conditions is exposed to the parent compound and/or its metabolites. This information is required upfront and can be obtained from toxicokinetic alternative methods that identify the main metabolites and the clearance rates of the parent compound and/or its metabolites.
Under 1R, toxicokinetic studies can make use of the updated OECD 417 (July 2010) which also comprises in vitro (e.g. use of microsomal fractions or cell lines to address metabolism) and in silico (toxicokinetic modelling for the prediction of systemic exposure and internal tissue dose) methods (OECD 2010a).
Physiologically based toxicokinetic (PBTK) models are ideally suited for the integration of data produced from in vitro/in silico methods into a biologically meaningful framework and for the extrapolation to in vivo conditions.
Sensitive, specific and validated analytical methods for a new substance and its potential metabolites will be an indispensable step in gathering data for quantitative risk assessment.
A whole array of in vitro/in silico methods at various levels of development is available for most of the steps and mechanisms which govern the toxicokinetics of cosmetic substances. One exception is excretion, for which until now no in vitro/in silico methods are available; thus, there is an urgent need for further developments in this area. Also there is a lack of experience for absorption through the lung alveoli, which would also make this a priority item for research and development given the fact that this route of exposure is important for cosmetics.
For the generation of most kinetic data, non-animal methods are available or at an advanced stage of development. Given best working conditions, including resources in money and in manpower, alternative methods to predict renal and biliary excretion, as well as absorption in the lungs, need at least 5–7 years of development. However, the development of an integrated approach linking the results from in vitro/in silico methods with toxicokinetics modelling towards the full replacement of animals will take even more time.
However, it cannot be excluded that with the use of new exposure-driven risk assessment approaches, such as the TTC (threshold of toxicological concern), the need to replace at least some steps may become less relevant for regulatory decisions.
Given the scenario of non-in vivo animal testing (1R) which has to be envisaged to be in place from 2013 on, the risk assessment of cosmetics is faced with a radically altered situation as compared with the 2005 report (see Coecke et al. 2005 in Eskes and Zuang 2005). In this new framework, exposure assessment is the important first step to decide on the necessity of further testing. Only in cases where a cosmetic ingredient is bioavailable following dermal, oral or inhalation exposure further tests on systemic and not just local toxicity would be necessary. The extent of exposure is compared with a dose which has a low probability to exert a toxic effect. This dose—also referred to as the threshold of toxicological concern (TTC)—is derived from the existing knowledge and could be used for chemicals for which little or no toxicological data are available.
Toxicokinetics is characterising the absorption, distribution, metabolism and excretion of a compound (ADME). ADME and biotransformation or metabolism encompasses all aspects of a pharmacokinetic/toxicokinetic evaluation. Studies to characterise steps in the toxicokinetic processes provide information about metabolite formation, metabolic induction/inhibition and other information which might be helpful for the study design of the downstream toxicological tests (the so-called toxico-dynamics). Metabolite/toxicokinetic data may also contribute to explaining possible toxicities and modes of action and their relation to dose level and route of exposure. Physiologically based toxicokinetic (PBTK)Footnote 9 models are important to integrate the processes of absorption, distribution, metabolism and excretion (ADME) and are the tools to convert external exposure doses into internal concentrations and vice versa, thus enabling also for converting in vitro concentration–response into in vivo dose–response relationships. This chapter will illustrate that toxicokinetic data form a prerequisite for the conduct of other toxicological tests and are necessary to understand and interpret toxicological data. They are essential to extrapolate in vitro data to the human in vivo situation for the respective relevant toxicological endpoints.
It is seen necessary to maintain a dialogue between experts working on toxicokinetics and on toxicodynamics to ensure the interaction between the toxicokinetic and toxicodynamic processes are understood. The toxicity endpoints covered in other chapters deal with repeated dose exposures to xenobiotics, assessing chronic toxicities including target organ toxicities and target system toxicities, carcinogenicity, reproductive and developmental toxicity and sensitisation. It is necessary for toxicodynamic testing to take into consideration toxicokinetic processes of importance for the proper design and performance of in vitro toxicodynamic studies. Apart from ADME processes and their integration in PBTK models, in vitro biokinetics measurements are further elements which characterise the concentration–time course during in vitro toxicity testing relevant for the concentration–effect relationship, the so-called actual concentration.
The TTC concept
The TTC concept is an approach that aims to establish a human exposure threshold value below which there is a very low probability of an appreciable risk to human health, applicable to chemicals for which toxicological data are not available, based on chemical structure and toxicity data of structurally related chemicals.Footnote 10 The TTC concept is currently used in relation to oral exposure to food contact materials, to food flavourings and to genotoxic impurities in pharmaceuticals and to metabolites of plant protection products in ground water (e.g. Kroes et al. 2004; Barlow 2005). Recently, The European Cosmetic Toiletry and Perfumery Association (COLIPA) sponsored work by a group of experts to examine the potential use of the TTC concept in the safety evaluation of cosmetic ingredients (Kroes et al. 2007). As the application of the TTC principle strongly depends on the quality, completeness and relevance of the databases, which are mostly based on toxicity data after oral exposure, its applicability to the dermal or inhalation uptake routes is limited, although it has been reported that the oral TTC values could be of some use for dermal exposures (Kroes et al. 2007); further, the use of TTC for the inhalation uptake route has recently been published (Escher et al. 2010; Westmoreland et al. 2010). However, only systemic effects are considered in the databases, and no local toxicity can be evaluated with this approach. Therefore, an improvement of the currently available databases is certainly needed. In addition, the TTC principle requires sound and reliable data on exposure (which might not always be available for cosmetics such as complex plant-derived mixtures) and the possibility to apply this principle in the field of cosmetics is still an ongoing discussion at an international level. Currently, the three Scientific Committees of DG SANCO have received a mandate to prepare an opinion on this topic.
If external exposure is above the external TTC, the toxicokinetic behaviour of a substance becomes important because it provides relevant information to derive the internal concentration related to the external exposure. The internal concentration, which might be different for different target organs, constitutes the basis for deciding on the necessity to perform further toxicity studies. The decision is made by comparing the internal concentration with a concentration which has a low probability to exert a toxic effect at organism level—the internal threshold of toxicological concern (TTCint)—derived from existing knowledge. The usefulness of the TTCint concept is not yet widely discussed, but the concept is under development.
In vitro toxicokinetics as a key for 1R replacement strategies
Given the scenario of the development of compounds/products based on non-in vivo animal testing strategies (1R), toxicokinetics provides essential data for (1) establishing tools for PBTK modelling, (2) designing tests for toxicodynamic endpoints and (3) permitting a proper risk assessment. The implication for the 1R replacement paradigm is that toxicokinetic data would become the first data set to be produced using alternative methods.
Information on toxicokinetics under 1R is essential to address three major issues:
Development and design of more efficient testing strategies: As a key starting point for any toxicological testing, it is essential to know whether a substance will be bioavailable by one of the relevant uptake routes: only in cases where a cosmetic ingredient is bioavailable following dermal, oral or inhalation exposure, further tests on systemic and not just local toxicity will be necessary.
In vitro–in vivo extrapolation: To relate toxicodynamic information from non-animal-testing (1R) to real-life situation relevant for humans, i.e. to transform in vitro concentration–effect relationship into an in vivo dose–effect relationship. The most sophisticated challenge under 1R is to make in vitro data (from any type of toxicological endpoint) usable for risk assessment, i.e. to properly relate toxicodynamic information from in vitro studies to the in vivo situation, because test results under 1R will be presented as an in vitro concentration–effect relationship instead of an in vivo dose–effect relationship.
Identification of clearance rates and the role of metabolites: For the in vitro dynamics experiments, it is essential to know whether the cell or tissues are exposed to the parent compound and/or its metabolites. This information has to be known upfront based on toxicokinetic alternative methods identifying the main metabolites and the clearance rates of the parent compound and/or its metabolites.
Furthermore, nominal applied concentrations in in vitro media may greatly differ from the actual intracellular concentration due to altered bioavailability (interactions with the medium, the plate, the cell itself) or to physiological cellular processes (mechanism of transport across the membranes, biotransformation, bioaccumulation). In repeated treatments for prolonged times of exposure, to mimic exposure to cosmetic products, the uncertainty about the actual level of exposure of cells in vitro is greatly enhanced. For this reason, in vitro biokinetics should be also considered in the experimental design for the in vitro dynamics experiments in order to correlate in vitro results to in vivo actual situations.
As amply justified earlier, it is without question that under 1R scenario of full animal replacement, toxicokinetic studies have to be performed differently from the study design and execution described in the OECD test guideline (OECD 417, the version adopted 1984; effective until September 2010). The newly effective and updated OECD 417 also comprises in vitro (e.g. use of microsomal fractions to address metabolism) and in silico (toxicokinetic modelling for the prediction of systemic exposure and internal tissue dose) methods (OECD 2010a). Essentially, instead of in vivo experiments, in vitro/in silico methods have to be used to derive the relevant information. This paradigm shift is illustrated schematically in Figs. 1, 2a and b.
The relation between kinetics and dynamics for a 1R replacement strategy
Ideally, and as a general goal, predictions of tissue exposure and subsequently toxicities should be based on human in vitro/in silico data combined with proper physiologically based toxicokinetic modelling, thereby replacing animal experiments. However, there are two questions that must be resolved in order to make the in vitro results usable for risk assessment. Firstly, the relationship between the effect of the parent compound and/or the metabolites on the in vitro test system and the health effect of interest must be clearly defined in order to derive a relevant in vitro (no)effect concentration or, better, benchmark concentration (BMC). In this respect, the actual rather than the nominal in vitro concentration tested, as stated previously, is a crucial starting point, to derive relevant parameters. Already at this stage, it is essential to consider what compound [e.g. the parent compound and/or metabolite(s)] the cells, tissues or organs will be exposed to, e.g. these data would be obtained by the kineticists. Secondly, an additional task of kineticists is to convert in vitro BMC to a predicted in vivo benchmark dose (BMD). For risk assessment purposes, the predicted in vivo BMD is to be compared to human exposure data (Rotroff et al. 2010).
Importance of analytical methods in the 1R scenario
It is obvious that in this “alternative” scenario (1R), concentration measurements of the parent compound and/or the metabolites in the in vitro test system and the behaviour of a studied ingredient in the test system (in vitro kinetics or biokinetics) in general become an important part of the test design (Pelkonen et al. 2008a).
Hence, a sensitive, specific and validated analytical and quantitative method for a new substance and its potential metabolites (Tolonen et al. 2009; Pelkonen et al. 2009a) will be pre-requisite in gathering data for quantitative risk assessment.
Measurement of the actual rather than the nominal or ‘applied’ in vitro concentrationFootnote 11 in the media or in cells is fundamental to performing in vitro kinetic modelling and in vitro studies on metabolism, preferably in human-derived systems.
In vitro studies on distribution between blood/plasma and different tissues, in vitro absorption (gut, skin, lung) as well as studies on protein binding rely on the availability of appropriate analytical methods, although for this purpose in silico methods may also be available in the future, once the database containing chemical-specific toxicokinetic parameters evolves to an extent that QSAR models can be built based on these parameters.
Other fields of application for an analytical method are experiments to derive physico-chemical data, which are important as an input into QSAR for predicting the fate of substances.
Chemical-specific measurements are also important as inputs into tissue composition-dependent algorithms to estimate the partitioning of chemicals into tissues.
Importance of actual, rather than nominal concentration in the 1R scenario
Kinetics has often been evoked to explain the differences between in vivo toxicity and results obtained in vitro, limiting the possibility to use the in vitro–derived data for an in vitro–in vivo extrapolation in the risk assessment of a chemical (Pelkonen et al. 2008a). Nevertheless, surprisingly few studies have addressed the issue of in vitro kinetics (Blaauboer 2010). One of the major problems of in vitro methods is the difficulty in the extrapolation of the dose–response relationship of toxicity data obtained in vitro to the in vivo situation if the nominal concentration of a chemical applied to cells is the basis for that extrapolation. PBTK models could help, allowing estimating tissues concentrations starting from a specific exposure scenario, or vice versa, to calculate from an effective dose resulting in vivo, the concentration resulting in a toxicologically relevant effect in an in vitro system (Mielke et al. 2010).
In any case, to use the nominal concentration in the in vitro system is a bad predictor of the free concentration and therefore a prerequisite for these extrapolations is the knowledge of the actual concentrations of the chemical exerting a toxic effect in the in vitro system. The nominal concentration, even when applied as a single dose, can to a great extent deviate from the actual concentration of the chemical in the system over time, due to altered bioavailability (interactions with the medium, adsorption to the disposable plastics, binding to proteins, evaporation) or to physiological cellular processes (mechanism of transport across the membranes, bio-transformation, bioaccumulation). In repeated treatments for prolonged time of exposure, the uncertainty about the actual level of exposure of cells in vitro is greatly enhanced also due to the metabolic capacity of the in vitro system. These processes have been shown to influence the free concentration and thus the effect (Gülden et al. 2001; Gülden and Seibert 2003; Heringa, et al. 2004; Kramer et al. 2009), clearly indicating the need to estimate or measure the free concentration in the medium or the actual concentration in the cells (Zaldívar et al. 2010). One technique used to measure the free concentration in the medium is the solid-phase micro-extraction (SPME), the application of which showed that for some compounds the free concentration could differ up to two orders of magnitude from the nominal concentration (Kramer 2010).
The identification of in vitro relevant kinetic parameters, the elaboration of a tiered strategy to measure/estimate the real exposure of cells to xenobiotics and/or their metabolites in in vitro systems as key elements for IVIV extrapolation are among the major aims of PredictIV, a EU funded project, particularly of WP3: Non-animal-based models for in vitro kinetics and human kinetic prediction (see the Project website: http://www.predict-iv.toxi.uni-wuerzburg.de/). This is the first attempt in an EU Project to combine biological effects (toxicodynamics) with toxicokinetics and modelling ensuring the generation of real exposure data linked to effects. In strict cooperation with the work package (WP) of the project dealing with the identification of effects (dynamic), the studies have been designed to determine the no observed effect concentration (NOEC) in model systems based on human cells representative of in vivo target organs. Data obtained will be modelled, in close cooperation with WP partners dealing with in silico methods, by using advanced PBPK modelling, so that starting from the NOECs, it will be possible to extrapolate the corresponding in vivo dose.
This approach is in line with the recommendation coming from an ECVAM-sponsored workshop on in vitro kinetics held in ISPRA in 2007, stating: In biologically relevant in vitro systems, good experimental design should always consider the impact of relevant in vitro factors, in particular kinetic factors, on the results. In order to achieve this, close cooperation between experimenters, modellers, biostatisticians and analytical chemists is necessary, particularly beyond the stage of prototype development.
Strategic considerations of risk assessment of cosmetic ingredients
Kinetics and dynamics are inherently linked to each other in the non-animal testing era, maybe even more than under the current situation, when animal bioassays are still allowed for several toxicological endpoints. This is illustrated in Fig. 3 (Bessems 2009). The left column starts on top with exposure and ends at the bottom with target tissue dose/concentration. The second column starts with the estimated/predicted target tissue dose/concentration which would indicate the range of concentrations to be tested in vitro with as sensitive techniques as possible (including omics). If in vitro effects are not measurable that are predicted using the left column, any in vivo effects are quite unlikely and possible health risks would not be indicated.
With respect to prevalidation and the time required to prevalidation, it is worthwhile to spend a few sentences on the actual need to perform standard validation processes, such as by ECVAM. It is not uncommon that companies perform in-house validation, using often (historical) animal in vivo data. If the companies have a standard operating procedure for ADME testing, there may be chances to accept these kinds of methods via an independent expert opinion consultation under confidentiality agreements. This might be an alternative way to safeguarding consumer safety, by circumventing long-term validation programmes. Alternatively, if companies would be willing to cooperate, these in-house methods might be provided to others, as e.g. within COLIPA, and have independent experts reviewing the performance of the method including public consultation in a kind of small-scale prevalidation which could be sufficient for the purpose of ADME testing. Important in this respect is the inclusion of well-known reference compounds. In addition, for ADME testing, very high precision of a method covering one of many aspects determining blood concentration–time curves might not always be key, especially not during pre-screening when validation of a PBPK modelling prediction would always be a case-by-case validation, not a method validation. Here, robustness may be a much more important criterion than accuracy.
Under the new, non-whole-animal testing paradigm, the first step of a screening process of chemicals for possible use as a cosmetic ingredient should be to find out whether absorption is likely or not under the foreseen use scenario. To this end, the following decision tree (Fig. 4) might be very helpful before starting any effects testing (both in vivo or in vitro). Ultimately, companies could decide to stop further development, if a compound appears to be absorbed under foreseen circumstances.
One step further, if a chemical appears to be absorbed to a limited extent, enough not to block further R&D, is to assess its systemic exposure quantitatively (Fig. 5). This will deliver essential information for the in vitro effects testing.
Available non-animal methods to derive values for absorption, distribution, metabolism and excretion
In this section, a general survey on the current status of non-animal methods for deriving input parameters for PBTK modelling is presented. For details regarding several mostly in vitro and in silico methods that are under development at various stages, the reader is referred to the Supplementary Online Information, which includes several tables.
Absorption and bioavailability after dermal, inhalatory or oral exposure
Absorption is the transport across an epithelial layer. Bioavailability is more complex and is defined as the fraction of a chemical in a certain matrix that reaches the systemic circulation unchanged. In that way, it is a complex parameter, describing several processes. Although it is difficult to study in isolation in such a way that the outcome is easily applicable in PBTK models, it is important in risk assessment and possibly in intelligent testing strategies.
Release of the compound from its matrix is required for transport across the dermal, lung or intestinal epithelium and bioavailability of a compound to the body. Release depends mainly on the matrix–compound interaction. In vitro models to assess bioaccessibility are best developed for the oral route (Brandon et al. 2006). Dermal exposure and exposure via inhalation are more problematic. Some methods for these types of exposure are under development for other areas than cosmetics. They will have to be developed further for application to cosmetic ingredients in the next years.
Absorption depends on the compound-specific properties and physiology (and pathology) of the epithelial tissue. Some of the properties of importance are physico-chemical properties of a compound and the availability of specific influx and efflux transporters in the tissue.
Dermal exposure: Various in silico QSAR models are available for the skin permeability prediction of compounds although none of them has been developed for a broad applicability, i.e. for a broad range of physicochemical properties. Although some of them have been set up according to the OECD Principles for the validation of QSAR (OECD 2004a) and may be useful for specific chemicals, none of them has been widely validated up to our knowledge (Bouwman et al. 2008). Regarding in vitro absorption models, OECD Technical Guideline 428 is available (OECD 2004b) and guidance is presented as well by OECD (2004c), US EPA (2004), EFSA (2009) and SCCNFP (2003).
Inhalatory exposure: The lung can be anatomically divided into several parts: trachea, bronchi, bronchioles and alveoli. In the upper respiratory airways, the absorption is low, and it mostly occurs in the lower part. No QSAR models predicting lung absorption are known in the public literature. In vitro models to study the translocation of compounds in the lung are in various stages of development. Several more years of intensive research will be needed to provide suitable models that can enter prevalidation.
Oral exposure: In silico QSAR-like models can predict specific parameters for an unknown chemical based on structural and physicochemical similarities to various known chemicals. In vitro models such as the Caco-2 cell line can predict the absorption over a single barrier as well and are rather standard. They could be incorporated in a medium-throughput test strategy. Importantly however, and as with in silico models, the validity of these in vitro model predictions for cosmetic ingredients remains to be established, because most, if not all, of these models were developed for pharmaceutically active ingredients. While in the pharmaceutical R&D reliable prediction between 50 and 100% absorption is important, in the cosmetics arena the crucial range will be the lower absorption range, i.e. much further less than 10% or even 1% absorption. Many more years of intensive research seem necessary for cosmetic ingredients before prevalidation of in vitro or other methods that are suitable to assess the potential pneumonal toxicity of cosmetic ingredients comes in sight.
Three processes (partially linear, partially in parallel) can be distinguished that determine bioavailability: (1) release of the compound from its matrix (bioaccessibility), (2) absorption of the released fraction and (3) metabolism before reaching the systemic circulation (Oomen et al. 2003).
In order to predict the precise bioavailability of a cosmetic ingredient from a cosmetic product, it is therefore important to determine the three different processes involved. However, the bioaccessibility could be used as a measure for the maximal bioavailability. If the parent compound would be expected to cause toxicity, complete absence of bioaccessibility would indicate absence of systemic effects. There are in vitro models to measure absorption through various portals of entry, although they are at various stages of development. In vitro models for measuring metabolism are described in section “Metabolism (biotransformation)”.
In silico models to estimate oral bioavailability (Bois et al. 2010) have been developed for the use in conjunction with PBTK models. The use of these organ-level in silico models is currently the best way to integrate the inputs from tests of bioaccessibility, absorption and metabolism, including hepatic clearance in the first-pass situation, because of the complex nature of bioavailability. However clearly, relevance and reliability of these in silico models outside the pharmaceutical R&D will need quite some years of extra investigations before prevalidation, the capacity to demonstrate reliability, would be reachable.
After absorption, the distribution of a compound and its metabolites inside the body is governed by three main factors: (1) the partition of the substance with plasma proteins, (2) between blood and specific tissues and (3) the permeability of the substance to cross specialised membranes, so-called barriers (e.g. blood–brain barrier/BBB, blood–placental barrier/BPB, blood–testis barrier/BTB).
Estimation of plasma protein binding (PPB)
Only the free (unbound) fraction of a compound is available for diffusion and transport across cell membranes. Therefore, it is essential to determine the binding of a compound to plasma or serum proteins. The easy availability of human plasma has made it possible to determine the unbound fraction of compounds by performing in vitro incubations directly in human plasma.
In vitro approaches: There are three methods generally used for PPB determination: (1) equilibrium dialysis (ED) (Waters et al. 2008), (2) ultrafiltration (UF) (Zhang and Musson 2006) and (3) ultracentifugation (Nakai et al. 2004). All methods can be automated for high throughput, are easy to perform and have good precision and reproducibility. The use of combined LC/MS/MS allows high selectivity and sensitivity. Equilibrium dialysis is regarded as the “gold standard” approach (Waters et al. 2008).
In silico approaches: Two recent reviews on the in silico approaches for the estimation of PPB have been carried out by Wang and Hou (2009) and Mostrag-Szlichtyng and Worth (2010). A general correlation based on the octanol–water partition coefficient was proposed by de Bruyn and Gobas (2007) after a compilation of literature data and using a broad variety of chemicals, i.e. pesticides, polar organics, polychlorinated biphenyls, dioxins, furans, etc.
Estimation of blood–tissue partitioning
The fate of a compound in the body is determined by partitioning into the human tissues. Therefore, the knowledge of this partitioning is of fundamental importance for the understanding of a compound’s kinetic behaviour and toxic potential. The measurement of tissue storage and a molecular understanding of tissue affinity have, historically, not been studied to the same extent as plasma protein binding; however, the knowledge of these partitioning coefficients is essential for the development of PBTK models. Fortunately, quite a large number of approaches have been developed over the last years.
In vitro approaches: The available system is the vial-equilibration technique. A spiked sample of organ tissue–buffer homogenate is equilibrated and subsequently, the free (unbound) concentration of the test chemical is determined. The tissue–blood partition coefficient is calculated using results from pure buffer, tissue–buffer and blood–buffer incubations. Tissues can be mixed to obtain average values for example richly perfused tissue groups. Olive oil or octanol are often used instead of adipose tissue. The free (unbound) concentration is typically assessed by one of the following techniques: equilibrium dialysis, ultracentrifugation, headspace analysis (for volatiles) or solid-phase (micro-) extraction followed by a classical analysis such as HLPC UV or MS. The purpose of this technique is the prediction of the in vivo tissue blood partitioning and the prediction of an in vivo volume of distribution (Gargas et al. 1989; Artola-Garicano et al. 2000).
In silico approaches: Different methodologies have been developed, starting from QSAR, correlations with physicochemical properties, up to mechanistic approaches. The main problem for the generalisation of QSAR correlations has been the poor results obtained for charged molecules under physiological conditions and with charged phospholipids. However, there are mixed and mechanistic approaches with tolerable error ranges (Poulin and Theil 2002, 2009; Schmitt 2008).
Estimation of substance permeability through specialised barriers
Blood–Brain Barrier (BBB): The BBB is a regulatory interface that separates the central nervous system (CNS) from systemic blood circulation and may limit or impair the delivery of certain compounds, which makes the brain different from other tissues. There are several passive and active mechanisms of transport through the BBB (Mehdipour and Hamidi 2009).
Several in vitro BBB models are under development which integrate various cell of vascular and neural origin. Also single cell lines containing transfected transporters have been proposed as models to study BBB permeability. However, all available models are in early stages of development. Several in silico models exist to predict BBB penetration, although the vast majority of these approaches do not consider transport mechanisms taking place (Mostrag-Szlichtyng and Worth 2010). Recently, some molecular models have been developed to consider the BBB transporters (Allen and Geldenhuys 2006).
Blood–Placenta Barrier (BPB): The BPB serves to transport nutrients and waste, and other compounds such as hormones. However, the placenta does not provide a true barrier protection to the foetus from exposure to compounds present in the mother’s systemic circulation, although it might reduce the transport of certain molecules. The transfer across the placenta can occur by several active or passive processes (Myren et al. 2007).
Experimental methods to study human transplacental exposure to toxic compounds have been reviewed by Vähäkangas and Myllynen (2006). There are both primary and permanent trophoblast-derived cell models available. An ex vivo model, human perfused placenta cotyledon, offers information about transplacental transfer, placental metabolism, storage, acute toxicity and the role of transporters, as well as an estimation of foetal exposure. There are also few QSAR models, although active transport mechanisms and potential metabolism are not addressed.
Blood–Testis Barrier (BTB): In the testis, the BTB is a physical and physiological barrier which assures functions in hormonal regulation and spermatogenesis (Fawcett et al. 1970). Many systems have been tested as organ cultures, co-cultures or single cell cultures, but none has really developed for considering toxicokinetic processes.
Metabolism or biotransformation is the principal elimination route of organic chemicals; roughly 70–80% of pharmaceuticals are partially or practically completely eliminated by metabolism (Zanger et al. 2008). Due to a multitude of xenobiotic-metabolising enzymes possibly acting on a chemical with different metabolic pathways, the first screen should preferably be as comprehensive as possible. Because liver is the principal site of xenobiotic metabolism, the enzyme component in in vitro systems should preferably be liver-derived (Coecke et al. 2006; Pelkonen et al. 2005, 2008b) and of human origin, to avoid species differences (see e.g. Turpeinen et al. 2007). There is a generally accepted consensus that metabolically competent human hepatocytes or hepatocyte-like cell lines are the best enzyme source to perform the first primary screening of metabolism (Gómez-Lechón et al. 2003, 2008; Houston and Galetin 2008; Riley and Kenna 2004). The two most important endpoints measured are (1) intrinsic clearance which can be extrapolated into hepatic metabolic clearance and (2) the identification of metabolites (stable, inactive, active or reactive metabolites of concern).
In silico approaches
The available systems are of three types: (1) expert systems based on structure-metabolism rules, (2) SAR and QSAR modelling and (3) systems based on pharmacophore (ligand) or target protein modelling. There are a large number of commercial softwares available for predicting biotransformation, in various phases of development. Although in silico approaches are developing rapidly, they are still inadequate for the production of results which are accepted by regulators, and new approaches are needed to predict the major metabolic routes when there are a number of potential metabolic pathways. Reliable and good-quality databases (not limited to pharmaceuticals) are of the utmost importance for the development of reliable software for application to a wider assortment of chemicals including cosmetic ingredients and they are still in great need. Discussions of various approaches can be found in recent reviews (Testa et al. 2004; de Graaf et al. 2005; Kulkarni et al. 2005; Crivori and Poggesi 2006; Lewis and Ito 2008; Muster et al. 2008; Mostrag-Szlichtyng and Worth 2010).
The metabolic stability test is a relatively simple, fast-to-perform, but specialised analytical equipment-based MS study, to find out whether a compound is metabolically stable or labile. It is based on the disappearance of the parent compound over time (with the appropriate analytical technique) when incubated with a metabolically competent tissue preparation (e.g. a human liver preparation, preferably human hepatocytes). The rate of parent compound disappearance gives a measure of its metabolic stability and allows for the calculation of intrinsic clearance and extrapolation to hepatic (metabolic) clearance. The use of liver-based experimental systems should give a fairly reliable view of hepatic intrinsic clearance. However, to be able to predict in vivo clearance, a number of assumptions concerning the substance under study must be made, so an extrapolation model is needed (see e.g. Pelkonen and Turpeinen 2007; Rostami-Hodjegan and Tucker 2007; Webborn et al. 2007). Although no formal validation studies are known, the screening test for metabolic clearance should be relatively ready for validation after the availability of common procedures and related SOPs.
To cover also extrahepatic biotransformation, the above-described method for metabolic stability can be combined with the use of other tissues. For cosmetic substances, dermal uptake is the most prominent intake pathway and consequently methodologies for skin metabolism would be of considerable significance. Likewise, inhalation (spray applications) is also an important uptake route, and metabolism should be taken into consideration in in vitro pulmonary tests. Some efforts would be needed to standardise metabolic stability in skin and pulmonary tissues.
Metabolite profile and bioactivation
With the advent of modern MS techniques, it is possible and feasible to study both the detailed qualitative and quantitative metabolic profiles of a compound (Pelkonen et al. 2009b; Tolonen et al. 2009). The use of recombinant enzymes, transfected cells and metabolically competent human (liver-derived) cell lines or subcellular fractions is being very actively employed in pharmaceutical industry and academia. In this way, it is possible to have indication about the enzymes participating in the metabolism which allows a number of predictions about physiological, pathological and environmental factors affecting the kinetics of a compound of interest.
The formation of reactive metabolites by biotransformation seems to be the cause of deleterious effects for a large number of compounds (Park et al. 2006; Williams 2006). Even though mechanistic details of relationships between toxicities and reactive metabolites are still somewhat unclear, there is ample indirect evidence for their associations (Baillie 2006, 2008; Tang and Lu 2010).
There are direct and indirect methods to test the potential formation of reactive metabolites. Most direct assays use trapping agents (i.e. glutathione or its derivatives, semicarbazide, methoxylamine or potassium cyanide) that are able to trap both soft and hard electrophiles: conjugates are then analytically measured. The Ames test is a prime example of an indirect method for the bioactivation assay making use of metabolically competent enzyme system (which could be human-derived, if needed) and properly engineered bacteria to detect reactive, DNA-bound metabolites.
Since induction has a complex underlying mechanism, it is a good indicator for high-quality metabolic competent systems that can be used for long-term purposes (Coecke et al. 1999; Pelkonen et al. 2008b): that is why developments are ongoing to assess CYP induction in bioreactor-based systems. Obviously, the most relevant intake routes for cosmetics (dermal, inhalation) should be considered when in vitro test systems are developed.
A large number of test systems ranging from nuclear receptor binding assays to induction-competent cell lines and cryopreserved human hepatocytes are currently available. The reliability of 2 hepatic metabolically competent test systems, e.g. cryopreserved hepatocytes and cryopreserved HepaRG systems, is currently assessed by ECVAM (International Validation Trial) by using CYP induction at the enzyme level as the endpoint detection method. These test systems are widely used in pharmaceutical industry to help early drug development and are designed to detect induction of CYP enzymes relevant for the pharmaceutical area. This can represent a potential limitation since, for cosmetics, other CYP forms might play an additional or more prominent role. Thus, further progress is needed to cover this potential gap.
Due to the broad substrate specificity of metabolising enzymes, there is always a possibility that compounds would interfere with each other’s biotransformation. Inhibition of biotransformation leads to higher concentrations and delayed clearance and may cause adverse effects. At the site of entry (i.e. GI tract, skin, lung), inhibition of the first-pass metabolism would increase the blood concentration of the parent compound.
There are currently available a large number of test systems ranging from recombinant expressed enzymes (principally CYP and UGT enzymes, but increasingly also other xenobiotic-metabolising enzymes) to primary cells (hepatocytes) and permanent cell lines (Li 2008; Farkas et al. 2008). All these test systems are widely used in pharmaceutical industry and can be judged to be validated at least for pharmaceuticals. This can represent a potential limitation since, for cosmetics, other CYP forms might be relevant. In addition, some cosmetics contain complex plant-derived mixtures, and it is not elucidated to what extent current inhibition assays would be applicable. Thus, further progress should cover chemical and compositional peculiarities characteristic for the cosmetics field.
Predicting major excretion pathways of compounds is important in relation to their kinetic behaviour and the relationship to pharmacological/toxicological effects. The kidneys and the hepatobiliary system have the capacity to excrete either as the parent compound or as metabolites and are important routes for elimination of xenobiotics and their metabolites. Unfortunately, excretory processes seem to be the least developed area in the context of in vitro toxicokinetic methods probably because renal and biliary excretion, the major excretory routes, are complex processes with a number of passive components and active processes involved.
Renal excretion: Excretion by the kidney encompasses three different mechanisms and they all include the interplay of both passive movement of drugs and the participation of a number of active transporters. Even if there are examples how the involved transporters can be identified, it is difficult to use the findings to feed into a physiological model of renal excretion which includes tubular secretion and tubular reabsorption.
Biliary excretion: In humans, biliary excretion does not seem to play an important role for most of the substances. However, in cases where it matters, the process is rather complex, first preceded with the entry of the substance to the hepatocyte and its possible metabolism by the hepatic metabolic machinery. With most substances ultimately excreted into the bile, phase II metabolising enzymes produce conjugates, which are then transported across the canalicular membrane to be excreted into the bile.
Current approaches and future efforts needed. There have been few attempts for developing expert systems or computational approaches to predict renal excretion from some basic molecular and physicochemical properties. Likewise, in silico modelling attempts are being made to evaluate the molecular weight dependence of biliary excretion (well established in rats) and to develop quantitative structure–pharmacokinetic relationships to predict biliary excretion. Efforts have been undertaken to use collagen-sandwich cultures of hepatocytes as an in vitro test system for testing biliary excretion. Due to the fact that progress in the field is very recent, no systematic efforts have been undertaken to standardise the above mentioned approaches. Some pharmaceutical companies as well as academic groups have published reports on their experiences. No formal validation studies are known.
An understanding of mechanisms that determine these processes is required for the prediction of renal and biliary excretion. Physiologically based in vitro/in-silico/in vivo approaches could potentially be useful for predicting renal and biliary clearance. Whereas for biliary excretion some advances have been made with in vitro models (i.e. sandwich-cultured hepatocytes), no reports could be identified in the literature on in vitro models of renal excretion nor were reports available on in silico methods.
Integrating in vitro and in silico approaches using PBTK modelling
After a chemical compound penetrates into a living mammalian organism (following intentional administration or unintentional exposure), it is usually distributed to various tissues and organs by blood flow (Nestorov 2007). The substance can then bind to various receptors or target molecules, undergo metabolism or can be eliminated unchanged. The four processes of absorption, distribution, metabolism and elimination (ADME) constitute the pharmacokinetics of the substance studied. The term toxicokinetics is used if the substance is considered from a toxicity view point.
In general, the toxicokinetics of a compound are the function of two sets of determinants: physiological characteristics of the body (which are compound independent) and compound-specific properties. It is possible to quantify some compound-specific structural properties and to relate them to biological activity. That is the basis of the so-called Quantitative Structure–Activity Relationships (QSARs). Likewise, structural properties can be used to estimate other properties, such as lipophilicity (logKow) and blood over tissue partition coefficients. In that case, the term Quantitative Structure–Property Relationship (QSPR) is used. To quantitatively predict the toxicokinetics of a substance, it is necessary to model jointly their physiological determinants and the compound-specific properties. Physiologically based toxicokinetic (PBTK)Footnote 12 modelling is currently the most advanced tool for that task.
PBTK models are necessary tools to integrate in vitro and in silico study results
The concentration versus time profiles of a xenobiotic in tissues, or the amount of its metabolites formed, is often used as surrogate markers of internal dose or biological activity (Andersen 1995). When in vivo studies cannot be performed or when inadequate in vivo data are available, the toxicokinetics of a substance can be predicted on the basis of in vitro or in silico studies. For risk assessment purposes, in vitro systems should be mechanism based and able to generate dose/concentration–response data. The greatest obstacles to the use of in vitro systems are the integration of their data into a biologically meaningful framework and their extrapolation to in vivo conditions. PBTK models are ideally suited for this, because they can predict the biologically effective dose of an administered chemical at the target organ, tissue and even cell level (Barratt et al. 1995; Blaauboer et al. 1999; Blaauboer et al. 1996; Combes et al. 2006; DeJongh et al. 1999a; Dr. Hadwen Trust Science Review 2006). Indeed, PBTK models are increasingly used in drug development and regulatory toxicology to simulate the kinetics and metabolism of substances for a more data informed, biologically based and quantitative risk assessment (Barton et al. 2007; Boobis et al. 2008; Bouvier d’Yvoire et al. 2007; Loizou et al. 2008; Meek 2004). As such, they should be able to significantly reduce or even replace animals in many research and toxicity studies.
General description of PBTK models
A PBTK model is a mechanistic ADME model, comprising compartments that correspond directly to the organs and tissues of the body (e.g. liver, lung, muscle), connected by the cardiovascular system (see Fig. 6). The main application of PBTK models is the prediction of an appropriate target tissue dose, for the parent chemical or its active metabolites. Using an appropriate dose-metric provides a better basis for risk assessment (Barton 2009; Conolly and Butterworth 1995). The estimation of dose-metrics is regarded as the ‘linchpin’ of quantitative risk assessment (Yang et al. 1998). In the 1R approach, the question may be to predict the external dose leading to an internal dose equivalent to that of a given in vitro treatment.
PBTK models’ parameter values can be determined on the basis of:
Published models range from simple compartmental (Gibaldi and Perrier 1982; see also Pelkonen and Turpeinen 2007 for current practical solutions) to very sophisticated types (Jamei et al. 2009). Between compartments, the transport of substances is dictated by various physiological flows (blood, bile, pulmonary ventilation, etc.) or by diffusions (Gerlowski and Jain 1983; Bois and Paxman 1992). Perfusion-rate-limited kinetics applies when the tissue membrane presents no barrier to distribution. Generally, this condition is likely to be met by small lipophilic substances. In contrast, permeability-rate kinetics applies when the distribution of the substance to a tissue is rate-limited by the permeability of a compound across the tissue membrane. That condition is more common with polar compounds and large molecular structures. Consequently, PBTK models may exhibit different degrees of complexity. In the simplest and most commonly applied form (Fig. 6), each tissue is considered to be a well-stirred compartment, in which the substance distribution is limited by blood flow. In such a model, any of the tissues can be a site of elimination. However, in Fig. 6, it is assumed that the liver is the only metabolising organ and that excretion only happens in the kidney.
Building a PBTK model requires gathering a considerable amount of data which can be categorised into three groups: (1) system data (physiological, anatomical, biochemical data), (2) compound-specific data and (3) the model structure, which refers to the arrangement of tissues and organs included in the model (Rowland et al. 2004). In a sense, PBTK modelling is an integrated systems approach to both understanding the kinetic behaviour of compounds and predicting concentration–time profiles in plasma and tissues. Additional details of PBTK modelling and applications can be found elsewhere (Gerlowski and Jain 1983; Nestorov 2003; Rowland et al. 2004; Jones et al. 2009; Edginton et al. 2008; Pelkonen et al. 2008a; Kapitulnik et al. 2009; Dahl et al. 2010). Indeed, such descriptions of the body are approximate, if not rough, but a balance has to be found between precision (which implies complexity) and simplicity (for ease of use). Yet, the generic structure of a PBTK model facilitates its application to any mammalian species as long as the related system data are used. Therefore, the same structural model can approximately be used for a human, a rat or a mouse (De Buck et al. 2007).
Generic applications of PBTK modelling
Inter-individual or intra-individual extrapolations: These refer to the fact that a given exposure may induce different effects in the individuals of a population and that the same individual may respond differently to the same exposure at different times in his/her lifetime. These extrapolations are performed by setting parameter values to those of the sub-population or individual of interest and are mainly used to predict the differential effects of chemicals on sensitive populations such as children, pregnant women, the elderly, the obese, and the sick, taking into account genetic variation of key metabolic enzymes, etc. (Jamei et al. 2009). The toxicokinetic behaviour of a compound can also be studied under special conditions, such as physical activity.
Inter-dose extrapolations: These extrapolations are achieved by capturing both the linear and non-linear steps of the biological processes known to govern the kinetics of the chemical of interest, e.g. in the transport and metabolism.
Inter-route of exposure extrapolations: Any route of exposure can be described either in isolation or in combination. For example, systemic toxicity may be studied following intravenous infusion, uptake via the gastrointestinal tract, dermal absorption and inhalation via the lungs. For example, coumarin hepatotoxicity is dependent on the route of administration and can be rationalised on the basis of physiologically based modelling (Kapitulnik et al. 2009).
Specific applications of PBTK modelling in the case of the 1R for cosmetics
A tiered approach for pure predictions of toxicity: PBTK models can be used in a step by step or tiered approach. They can be first coupled to in silico quantitative structure-pharmacokinetics properties relationships (QSPR) models for partition coefficients, absorption or excretion rate constants and computer models of metabolism (assuming that such models are available for the chemical class of interest). Using expected exposure patterns, estimates of internal exposures, bioavailability, half-life, etc. can be obtained. Such results could either be sufficient to answer the question of interest or would provide at least estimates of concentration levels to be assayed in vitro. In further steps, leading to increased refinement and predictive accuracy, PBTK models can incorporate the results of specific in vitro estimates of pharmacokinetic parameters (such as absorption rates, metabolic rate constants, etc.). At any point of that approach, the PBTK model provides estimates of internal dose levels attained in predefined exposures scenarios, enabling a prediction of the most sensitive toxic endpoint, of exposure–response relationships, of no-effect levels (if the dynamic models provide toxicity thresholds), etc. (Fig. 7).
Forward dosimetry: in vitro–in vivo correlation: Historically, in chemical risk assessment, PBTK modelling has been used primarily for ‘forward dosimetry’, that is, the estimation of internal exposures in the studies characterising the toxicity of a chemical. The human chemical risk assessment arena may be described as ‘data poor’ as opposed to the ‘data-rich’ pharmaceutical arena, hence, the need to estimate internal exposure through modelling, in the absence of specific measurements. When in vitro systems, such as human cell lines, will replace animals in toxicological and safety evaluation of cosmetics, we will also need to estimate in vivo internal doses. This will require PBTK modelling, as illustrated in Fig. 7.
Reverse dosimetry: exposure reconstruction from in vitro alternatives: Recently, a number of studies have attempted to ‘reconstruct dose’ or ‘estimate external exposure’ consistent with human biological monitoring data. That exercise has been described as ‘reverse dosimetry’ (Clewell et al. 2008; Georgopoulos et al. 1994; Liao et al. 2007; Lyons et al. 2008; Roy and Georgopoulos 1998; Tan et al. 2006a, b). A similar procedure could be applied to estimate the external exposure levels leading to acute and chronic systemic toxicity, including repeated dose systemic toxicity, as estimated from in vitro alternatives methods.
Exposure reconstruction can, and should, be addressed at both the individual and population level. Population-based estimates of exposure should account for human inter-individual variability, both in the modelling of chemical disposition in the body and in the description of plausible exposure conditions.
The reconstruction of dose or exposure using Bayesian inference is recommended, even for systems where tissue dose is not linearly related to external exposure (Allen et al. 2007; Lyons et al. 2008; Sohn et al. 2004). Gelman et al. (1996) presented a general method of parameter estimation in PBTK models, and reverse dosimetry is a type of PBTK model calibration problem.
Current limitations: All the limitations of in vitro toxicokinetic assays have an impact on the predictive accuracy of PBTK models. Difficulties in predicting metabolism, renal excretion and active transport are foremost in that respect, and improvements will proceed at the pace adopted to solve these problems. More intrinsic to PBTK modelling itself is the difficulty to accurately model dermal exposure (e.g. surface area exposed, dose applied wearing and washout) and absorption (e.g. saturation of the skin layers), at least for some important chemical classes (PCBs, etc.). The most precise solutions involve partial differential equation models, even though various approximations are available (Krüse et al. 2007). This goes beyond the capabilities of commonly used PBTK modelling software, and a particular effort would need to be devoted to resolving that problem.
Checking the validity of PBTK models is much easier when they have a stable and well-documented physiological structure. That is a particular advantage of the generic PBTK models developed by Simcyp (http://www.simcyp.com), Bayer Technology Services (http://www.pk-sim.com), Cyprotex (https://www.cloegateway.com) or Simulation Plus (http://www.simulations-plus.com), etc. The need remains to validate the QSAR sub-models or the in vitro assays used to assign a PBTK model’s parameter values. Obviously, the quality of those inputs conditions the validity of the PBTK model which uses them. The validation of those sub-models and in vitro assays should be made following the relevant procedures, in the context of cosmetic ingredients. Sensitivity and uncertainty analyses can also be performed to understand which are the critical aspects of the model that might require particular attention (Bernillon and Bois 2000). Experimental or observational data are not always available to convincingly validate such complex models. ‘Virtual’ experiments simulated by varying parameters, as in sensitivity analysis, can point to important areas of future research needed to build confidence in in silico predictions. Formal optimal design techniques can also be used to that effect (Bois et al. 1999).
In any case, the major challenge will probably be the coupling of PBTK models to predictive toxicity models, at the cellular and at the organ level. Liver models are being developed (Yan et al. 2008; Orman et al. 2010), but their predictive power is far from established for chronic repeated dose toxicity.
Inventory of in vivo methods currently available
Several specific tests for studying the toxicokinetics of substances in vivo are described in Annex V to Directive 67/548. The OECD guideline 417 describes the procedure in more detail. The OECD guideline also states: “Flexibility taking into consideration the characteristics of the substance being investigated is needed in the design of toxicokinetic studies.” It should be mentioned that the OECD guideline 417 has been recently updated and adopted (July 2010) with the inclusion of in vitro and in silico methods. With the exception of dermal absorption, detailed data on the toxicokinetics including the metabolism of cosmetic ingredients is currently of limited importance and not requested. Such additional information is only required for cases where specific effects, seen in standard in vivo animal tests, have to be clarified and their relevance to humans must be proven.
For example, toxicokinetics (TK) could aid in relating concentration or dose to the observed toxicity, and to aid in understanding mechanism of toxicity. Important goals are the estimation of systemic exposure to the test substance, identification of the circulating moieties (parent substance/metabolites), the potential for accumulation of the test substance in tissues and/or organs and the potential for induction of biotransformation as a result of exposure to the test substance. Additionally, toxicokinetic studies may provide useful information for determining dose levels for toxicity studies (linear vs. non-linear kinetics), route of administration effects, bioavailability and issues related to study design.
As described in more detail in sections “Available non-animal methods to derive values for absorption, distribution, metabolism and excretion (ADME)” and “Inventory of alternative methods”, there exists a number of in vitro and/or in silico methods to study many of these TK processes. For example, in vitro biotransformation models available (e.g. hepatocytes in suspension or culture) are used to provide results considered relevant for risk assessment. The same holds true for in vitro/in silico results for oral absorption where information on chemical structure (e.g. QSAR) and physical and chemical properties (e.g. logPow) may also provide an indication of the absorption characteristics. Data on in vitro protein binding may also be considered if relevant for risk assessment.
Inventory of alternative methods
Currently used in vitro guideline
To date, only one in vitro test addressing toxicokinetics is covered by an OECD test guideline. This is the guideline on in vitro dermal absorption (OECD 428, adopted on February 2004) where the principles of this method are described (OECD 2004b). The guideline is accepted by the SCCS (SCCNFP/0750/03, Final). A guidance document of the SCCS is complementing this guideline (SCCS/1358/10).
Non-validated human in vitro/in silico approaches
Test systems to measure bioavailability and in vitro biotransformation are available (as described in the specific subchapters) and routinely used for specific in-house purposes, mainly in pharmaceutical companies. For some of them, extensive sets of data are available, demonstrating their importance to produce specific qualitative and quantitative information on various pharmacokinetic characteristics. Regulatory authorities have recognised that in vitro systems are helpful in addressing especially potential biotransformation-related issues during drug development. The application of in vitro systems for biotransformation (e.g. in microsomal preparations or isolated hepatocytes) has been described in guidance documents on studies of drug–drug interactions by US (US FDA-CDER 1997, a revised draft guideline has been published in 2006) and European authorities (EMEA 1997, a revision of the latter is currently under public consultation). The recently revised OECD guideline 417 (July 2010) foresees the use of in vitro and in silico methods.
By applying an exposure-based tiered approach, there would be no need to analyse the biotransformation of a cosmetic ingredient if the chemical had insignificant bioavailability or even if there is no toxicological relevance. The selection of the most appropriate in vitro models for determining absorption is therefore crucial for cosmetic ingredients. Once the absorption and the potential toxicological relevance is demonstrated, then further testing and toxicokinetic information would be necessary.
Ideally, in silico and in vitro methods should use metabolically competent human cells and/or tissues to model human toxicokinetic processes to avoid any need for species extrapolation, as recommended by the ECVAM Toxicokinetics Working Group (e.g. Coecke et al. 2006). However, the limited availability of human cells and tissues, and ethical concerns which are often raised, should be taken into account, although the use of human recombinant enzymes, transgenic cells in vitro and the possibility to cryopreserve human heptocytes are of great help.
In this respect, it should be noted that human genetic polymorphisms of biotransformation enzymes and transporters are not covered in conventional toxicological animal approaches. The use of human cells (or subcellular fraction), recombinant enzymes and transgenic cells in vitro are the first step in trying to pick up some well-known genetic polymorphisms. This information might be useful for the risk assessor and needs to be incorporated into a tiered strategy for toxicokinetics. The issue is of importance to drug development and therapy, and for other chemicals, but with respect to cosmetics, data could be less relevant. Similarly, “barriers”, such as the BBB, BTB and BPB, have been considered to be of minor importance in the context of cosmetics, although in individual cases their role may need clarification.
Non-validated human in vivo approaches
The microdosing approach, which makes use of extremely sensitive detection techniques such as accelerator mass spectrometry and LC–MS/MS, has been employed as a first-to-man experiment to elucidate the pharmacokinetics of pharmaceuticals (Coecke et al. 2006; Hah 2009; Lappin and Garner 2005; Oosterhuis 2010; Wilding and Bell 2005). However, the need to conduct short-term animal toxicity studies before employing microdosing would block application in the 1R situation. Interestingly, a possible approach could be to combine it with the TTC concept. In principle, human microdosing could possibly obtain ethical approval by keeping the total dose below the relevant threshold in TTC terms, although a clear difference in the cost/benefit ratio between pharmaceuticals and cosmetics should be taken into account. Usually, an amount somewhere in between 1 and 100 μg is administered (http://www.nc3rs.org.uk/downloaddoc.asp?id=339&page=193&skin=0). If the chemical is not a genotoxic compound (sufficient in vitro methods available) and not an organophosphate, the lowest threshold for exposure below which adverse effects are unlikely is 90 μg/day. Acknowledging that this threshold was based on lifelong exposure, it can be argued that this might be a promising approach for further consideration. In this evaluation, the issue of exposure route should be included, because the current TTC concept is completely based on oral toxicity studies.
Imaging techniques to study both the kinetic and dynamic behaviour of pharmaceuticals or pharmaceutical-associated materials in in vivo conditions in humans are advancing rapidly (Agdeppa and Spilker 2009; Péry et al. 2010), but similarly as with the microdosing concept, at present it is difficult to see whether imaging techniques would become tools for cosmetics risk assessment related research.
Current developments in model systems
Organotypic culture models applicable to the intestinal, pulmonary barriers and the blood–brain barrier are being actively developed. However, these are laborious experimental systems, which are not easy to handle, and are currently restricted to mechanistic investigations or specific questions facilitating in vitro–in vivo comparisons (Garberg, et al. 2005; Prieto et al. 2010; Hallier-Vanuxeem et al. 2009).
Recent developments on microfabrication technologies coupled with cell cultures techniques have allowed for the development of “cells on a chip” (El-Ali et al. 2006; Hwan Sung et al. 2010) that have been used to mimic biological systems and even as a physical representation of a PBTK model. For example, Viravaidya et al. (2004) and Viravaidya and Shuler (2004) developed a four-chamber microscale cell culture analogue (μCCA) containing “lung”, “liver”, “fat” and “other tissue” used to study the role of the naphthalene metabolism in its toxicity and bioaccumulation using cultures of L2, HepG2/C3A and differentiated 3T3-L1 adipocytes. Tatosian and Shuler (2009) studied the combined effect of several drugs for cancer treatment using HepG2/C3A as liver cells, MEG-01 as bone narrow cells and MES-SA as uterine cancer cells and MES-SA/DX-5 as a multidrug resistant variant of uterine cancer. They showed that a certain combination could inhibit MES-SA/DX-5 cell proliferation and using a PBTK model of their device they were able to scale-up to calculate doses for in vivo trials. Finally, Chao et al. (2009) using a similar approach and human hepatocytes showed that they could predict the in vivo human hepatic clearances for six compounds.
Steps or tests with novel or improved alternative methods needed
Since only in cases where a cosmetic ingredient is bioavailable following dermal, oral or inhalation exposure, further tests on systemic and not just local toxicity will be necessary, the priority for additional efforts has to be given in providing reliable alternative methods to assess the bioavailability after oral and inhalation exposure. Several efforts have been undertaken to improve the reliability of alternative test methods available assessing absorption via gut, but still more work would be required. At present, extensive experience does not exist with in vitro systems suited to measure absorption through the lung alveoli. The systems used in house have some disadvantages, and the performance of a three-dimensional in vitro culture with pulmonary cells has not yet shown for this purpose. Hence, in the light that this route of exposure is important for cosmetics, efforts are necessary to develop systems for the purpose of measuring pulmonary absorption.
At the same time, it will be essential to develop toxicodynamic experimental design including all toxicokinetic consideration necessary to transform in vitro nominal concentration–effect relationship into an in vivo dose–effect relationship and allow the extrapolation of in vitro/in silico data to in vivo dose–effect relationship. If the actual applied concentration in vitro could be determined by using appropriate biokinetic measures, the relevance for the extrapolation could be improved.
In order to do so, more investments should be done to have access to high-throughput validated analytical methods for compound and metabolite identification, as the indispensable first step before any toxicodynamic experimental design is planned allowing a quantitative risk assessment.
Several in vitro/in silico building blocks are available, but a wide variety of standard operating procedures (SOP) is used by different industries and CROs. Alternative methods are available, but no effort up to now has been made in order to get the most reliable version of such SOP accepted by regulators.
The development of in vitro/in silico methods dealing with biliary excretion and renal excretion is felt to be essential to progress to a full 1R replacement strategy based using as integrative tool the PBPK models.
Given the scenario of the development of compounds/products based on non-in vivo animal testing strategies, toxicokinetics becomes the cornerstone in the risk assessment under 1R conditions. Toxicokinetic information has to be available upfront for assessing the need for further testing dependent on the bioavailability, to plan in vitro toxicodynamic testing, and together with biokinetic in vitro data will allow to relate the in vitro information of the concentration–effect(s) relationship of the substance to an in vivo dose–effect relationship.
The following recommendations are given to pave the way forward in the field of toxicokinetics under the 1R scenario.
Firstly, absorption through the lung and excretion (via the kidneys and the biliary route) are the two processes within the ADME processes (absorption, distribution, metabolism, excretion), which have been identified as knowledge gaps.
Secondly, physiologically based toxicokinetic (PBTK) models are ideally suited for the integration of data produced from in vitro testing systems/in silico models into a biologically meaningful framework, and for the extrapolation to in vivo condition. However, even if proof of concept has been provided for the strategy how to proceed there is presently not much experience, and hence, further development and refinement is necessary. One could envisage building a publicly available user friendly tool for PBTK modelling with a repository of examples to support the use of the tool. This would promote not only PBTK as a tool for risk assessment but also the concept of risk assessment under 1R scenario as a whole. Furthermore, the working group on toxicokinetics restricted itself to kinetic aspects because it felt not charged also to explore aspects of toxicodynamics. However, it should be emphasised that the link between the results of in vitro effect testing, corrected on the basis of biokinetic measurements, and the PBTK modelling is by modelling the in vitro responses by toxicodynamic modelling (Dahl et al. 2010). Hence, the crosstalk between toxicologists measuring effects and toxicologists/scientists performing kinetic or kinetic/dynamic modelling is fundamental (see Figs. 1, 2).
Thirdly, in the areas where in vitro/in silico methods are available, it should be considered whether the conventional validation procedure is the most efficient way forward. Recognising that only one in vitro toxicokinetic method is accepted at the OECD level (OECD guideline 428 for in vitro dermal absorption), it should be considered whether and to what extent alternative methods could be utilised. It could be envisaged to work in an expert consensus procedure by collecting methods, assessing them according to test quality criteria and ranking them. Finally, by consensus a standard operating procedure (SOP) could be derived and the reliability of the method could be tested on a small sample of compounds with properties relevant for cosmetics. In vitro protein binding, in vitro metabolism and clearance, and in vitro oral absorption may be valid examples for this approach. Currently, ECVAM is carrying out a formal reliability check in the context of an international validation trial of 2 metabolic competent human hepatic systems (cryopreserved human hepatocytes and the Human HepaRG cell line) for several phase 1 biotransformation CYP isoforms.
Concerning the available in silico methods (e.g. tissue distribution), it seems necessary to explore whether substances used in cosmetics are in the chemical space of the substances which have been used to develop and validate the algorithms. If not, adjustment or even new development of algorithms has to be undertaken.
Finally, it must be clarified that all the exercises for the process of validating and of finding acceptance needs financial resources other than research, because this activity cannot be seen as a research activity but is rather a standardising activity. There will be the necessity to involve institutions which have experience and work on standardising issues (e.g. experience from pharmaceutical companies which already use a variety of alternative methodologies).
There are some fields which have not yet been considered in depth. The most important is the field of nanoparticles. We are aware of the fact that nanoparticles are currently used in cosmetic products applied to the skin. As far as we know, the presently available data show that from the products on the market absorption to the general circulation (i.e. internal exposure) does not take place. We however know that absorption through the lung alveoli may occur. We would recommend that a special working group should be set up to deal with the issues of nanoparticles in the field of cosmetics taking into account the regulations in other fields (e.g. industrial chemicals) and what has been already considered by other institutions (e.g. OECD).
Under the 1R scenario which has to be envisaged to be in place from 2013 on, the risk assessment of cosmetics is faced with a radically altered situation. In the current paradigm of risk assessment, the external exposure (in mg/kg/day) is compared to the dose for an observable effect (at the no-effect-level in mg/kg/day adjusted with appropriate assessment factors). In the old paradigm, kinetic and dynamic considerations help to understand the mode of action/interspecies differences. In the new framework, knowledge on the toxicokinetic behaviour of a substance becomes the first important piece of information.
Information on toxicokinetics under the 1R is essential to address the following three major issues:
It is essential to know, whether a substance will be bioavailable by one of the relevant uptake routes: only in cases where a cosmetic ingredient is bioavailable following dermal, oral, or inhalation exposure, further tests on systemic and not just local toxicity will be necessary.
In order to relate toxicodynamic information from non-animal-testing (1R) to real-life situation relevant for humans, it is necessary to transform the in vitro actual concentration–effect relationship into an in vivo dose–effect relationship. Physiologically based toxicokinetic modelling is the indispensable tool to enable the transformation.
In order to plan the experimental design for the in vitro dynamics experiments, it is essential to know whether the cells or tissues are exposed to the parent compound and/or its metabolites. In vitro data on metabolism support this decision.
In addition, in vitro biokinetic data recorded during in vitro toxicity testing will be crucial to derive the actual in vitro concentrations: indeed, nominal applied concentrations may greatly differ from the intracellular concentration due to altered bioavailability or to physiological cellular processes. In repeated treatments for prolonged time of exposure, to mimic exposure to cosmetic products, the uncertainty about the actual level of exposure of cells in vitro is greatly enhanced.
The development of specific and sensitive analytical methods will be the first step to obtain the necessary toxicokinetic information. Currently, there exists a number of in vitro and in silico methods to cover different aspect of the toxicokinetics processes. Although for some of them a further development/improvement is still necessary, some others of the existing methods are already well developed, but, with the notable exemption of the in vitro dermal absorption for which an OECD guideline exists, they are non-validated.
Regarding the validation of the non-validated testing methods, it should be considered whether the conventional validation procedure is the most efficient way forward and whether alternative methods could be utilised. It could be envisaged to work in an expert consensus procedure to set up Standard operating procedures by consensus and validation could be performed in testing the reliability of the methods with compounds possessing properties relevant for cosmetics. The appropriateness of available in silico methods (e.g. tissue distribution) has to be explored for substances used in cosmetics with respect to their location in the chemical space. The non-availability of methods to produce in vitro data on the absorption after inhalation exposure and on excretion have been identified as the major data gaps.
We underscore the importance of PBTK (PBPK) modelling as a necessary tool to organise and integrate the input from in vitro and in silico studies. In addition, we would like to recommend including also toxicodynamic modelling in the chain from in vitro test results to the in vivo dose–effect relationship. We also recommend supporting the development of building a publicly available user friendly tool for PBPK modelling with a repository of examples.
A special working group, probably in collaboration with other concerned agencies such as European Food Safety Authority (EFSA), should be set up to deal with the issues of nanoparticles in the field of cosmetics taking into account the regulations in other fields (e.g. industrial chemicals) and what has been already considered by other institutions (e.g. OECD).
Given the best working conditions, including resources in money and manpower, it could be predicted that the improvement of the existing methods and the development of in vitro methods for renal excretion and absorption via the inhalation route would take 5–7 years; an integrated approach linking the results from in vitro/in silico methods with physiologically based toxicokinetics in order to characterise different steps involved in toxicokinetics would take a considerably longer time.
Although animal toxicokinetic models are already rarely used in the context of cosmetics and consequently the impact of the 2013 deadline will be greatly diminished, the WG emphasises that toxicokinetics is the first step in the non-animal testing strategy for cosmetics, considering a decision tree based on systemic bioavailability and on the need to integrate biokinetics into toxicity testing.