Toxicokinetics

  • Alternative test methods and approaches

    1. ECVAM validated test methods

    2. Test methods under validation by ECVAM

    3. Development and optimisation of alternative methods

 

Background


Chemical (cosmetics, occupational and industrial chemicals, pesticides, biocides, drugs, environmental pollutants, food additives) safety assessment encompasses not only qualitative description of the toxic properties but 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 describes how the body handles a chemical, as a function of dose and time, in terms of ADME:

  • 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 (i.e. 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

There are multiple types of chemical-specific parameters. A first set are those describing the flux across barriers (penetration or permeability). These processes can be passive and active (carrier-mediated) and be related to external (gut lining, skin, lung and nasal epithelium barriers) or internal (blood-brain, testis, placenta barriers) barriers. Others describe transport between phases such as air, blood and tissue. An important set of biochemical parameters necessary for the toxicokinetic evaluation of a chemical are those describing chemical modification of the parent chemical, e.g. the metabolic parameters Km and Vmax and the parameter indicating the rate of metabolic elimination. Additionally, parameters that mathematically describe processes such as renal excretion and the fraction of the active chemical that is unbound are important in any kinetic evaluation. All these chemical-specific parameters have to be determined by in vitro test methods or predicted by in silico (data-based) methods for each chemical entity. It is therefore essential to have available in vitro or in silico methods which reliably determine such parameters. 



Regulatory framework

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 (Pelkonen et al., 2009; Tonnelier et al., 2012), 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. Several regulatory guidelines have set their thresholds for metabolites to be identified, e.g. 5% of the administered dose by OECD TG 417 toxicokinetic guidelines (OECD TG 417, 2008), 10% of the parent compound´s area under the curve in steady state by FDA’s MIST (Metabolites in Safety Testing) guideline (FDA 2008), or 10% of the parent related material by the EMA ICH M3 (R2) guideline (EMA 2009).

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 (Adler et al., 2011). 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 (Schroeder et al., 2011). 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 (Coecke et al., 2005; Coecke et al., 2012).

Important aspects to focus on in the immediate future are:

  • Respond to today’s specific regulatory demands for sector-specific regulations (e.g. start using toxicokinetic in vitro and in silico methods for read-across purposes, route-to-route extrapolation, to calculate bio-accumulative potential, waiving, obtain data on clearance of the chemical and/or its metabolites, identification of metabolites, etc..)
  • Use available toxicokinetic in vitro and in silico methods in high priority areas (e.g. skin sensitisation) for information needs on bioavailability (Aeby et al., 2010)
  • Use PBTK models to integrate rather isolated information on ADME to estimate the internal/tissue dose and use this information in designing the in vitro experiments
  • Use PBTK models for converting in vitro results (generated at tissue/cell or sub-cellular level) into dose-response information for the entire organism

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.


References
  • Adler S., Basketter D., Creton S., Pelkonen O., van Benthem J. et al. (2011) Alternative (non-animal) methods for cosmetics testing: current status and future prospects-. Arch Toxicol. 85, 367-485.
  • Aeby P, Ashikaga T, Bessou-Touya S, Schepky A, Gerberick F, Kern P, Marrec-Fairley M, Maxwell G, Ovigne JM, Sakaguchi H, Reisinger K, Tailhardat M, Martinozzi-Teissier S, Winkler P. (2010) Identifying and characterizing chemical skin sensitizers without animal testing: Colipa's research and method development program. Toxicol In Vitro 24:1465-1473.
  • Coecke S, Blaauboer BJ, Elaut G, Freeman S, Freidig A, Gensmantel N, Hoet P, Kapoulas VM, Ladstetter B, Langley G, Leahy D, Mannens G, Meneguz A, Monshouwer M, Nemery B, Pelkonen O, Pfaller W, Prieto P, Proctor N, Rogiers V, Rostami-Hodjegan A, Sabbioni E, Steiling W, van de Sandt JJ. (2005) Toxicokinetics and metabolism. Altern Lab Anim. 33: 147-175.
  • Coecke S., Pelkonen O., Leite S.B., Bernauer U., Bessems J.G., Bois F.Y., Gundert-Remy U., Loizou G., Testai E. and Zaldívar J.M. (2012) Toxicokinetics as a key to the integrated toxicity risk assessment based primarily on non-animal approaches. Toxicol In Vitro 4. In press.
  • European Chemical Agency (ECHA), Helsinki, Finland (2012) Guidance on information requirements and chemical safety assessment Chapter R.7c: Endpoint specific guidance, 1-239.
  • European Food Safety Authority (EFSA), Parma, Italy (2012). SCIENTIFIC OPINION, Guidance for submission for food additive evaluations, EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS)2, 3, EFSA Journal 10(7)2760, 1-65.
  • European Medicines Agency (EMA) (2009) ICH M3 (R2) —Guideline on Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals.
  • EC Directive 2010/63/EU on the protection of animals used for scientific purposes.
  • EC Regulation 1907/2006 on  the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).
  • EC Regulation No 1223/2009 on cosmetic products.
  • FDA Food and Drug Administration (2008) Guidance for industry – Safety Testing of drug metabolites (MIST).
  • OECD (2010) OECD Test No 417: Toxicokinetics. Guideline for the testing of chemicals. OECD, Paris, No. 417.
  • Pelkonen O, Tolonen A, Rousu T, Tursas L, Turpeinen M, Hokkanen J, Uusitalo J, Bouvier d'Yvoire M, Coecke S. (2009) Comparison of metabolic stability and metabolite identification of 55 ECVAM/ICCVAM validation compounds between human and rat liver homogenates and microsomes - a preliminary analysis. ALTEX 26:214-222.
  • Tonnelier A, Coecke S, Zaldívar JM. (2012) Screening of chemicals for human bioaccumulative potential with a physiologically based toxicokinetic model. Arch. Toxicol. 86:393-403.
  • Schroeder K, Bremm KD, Alépée N, Bessems JG, Blaauboer B, Boehn SN, Burek C, Coecke S, Gombau L, Hewitt NJ, Heylings J, Huwyler J, Jaeger M, Jagelavicius M, Jarrett N, Ketelslegers H, Kocina I, Koester J, Kreysa J, Note R, Poth A, Radtke  M, Rogiers V, Scheel J, Schulz T, Steinkellner H, Toeroek M, Whelan M, Winkler P, Diembeck W. Report from the EPAA workshop: in vitro ADME in safety testing used by EPAA industry sectors. Toxicol In Vitro. 2011 Apr;25(3):589-604. 
 

Alternative test methods and approaches


1. ECVAM validated tests methods


Only one ADME in vitro procedure has reached the level of an OECD test guideline, e.g. dermal absorption in vitro (OECD TG 428, 2004). This test method is 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 need 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.

References

  • OECD (2004) OECD Test No 428: Skin Absorption: In Vitro Method. OECD, Paris, No. 428.

 

2. Test methods under validation by ECVAM

 

Metabolism (biotransformation), which is one of the main difference inter and intra-species, play a key role in the toxicokinetic and toxicodynamic processes.

Important biochemical parameters necessary for the toxicokinetic evaluation of a chemical entity are those describing chemical modification of the parent compound, e.g. the metabolic parameters Km and Vmax and the parameter indicating the rate of metabolic elimination which relies entirely on the availability of a reliable metabolic competent source.

With regard to toxicodynamic processes, toxic effects can be increased through processes which impact the metabolic capacity of the system. Therefore, it is important to know if a chemical has critical effects on the overall metabolism of a cellular system by inducing it, inhibiting it, or by direct metabolism-mediated cytotoxic effects (OECD, 2008).

Most of the in vitro systems used for toxicodynamic evaluations lack metabolic capacities and this has been recognized as being a bottle-neck in in vitro test development (Coecke et al. 2006).

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 (Coecke et al., 1999; Coecke et al., 2005). 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 (Guillouzo et al., 2007; Richert et al. 2010; Gerin at al. 2012; Andersson et al., 2012). The coded test items are selected on the basis evidence from in vivo human data on their CYP induction potential (Abadie-Viollon et al., 2010; Abass et al., 2009). 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 (Tolson et al., 2010).  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 main objectives of the validation study are:

  • To provide a reliable test system for toxicokinetics and toxicodynamics applications. The metabolic competence of the two test systems is assessed by measuring the potential of prototypical inducers to induce CYP enzymes. CYP induction, indeed, having a complex underlying mechanisms (gene activation by xenobiotic-sensing nuclear receptors, followed by de novo protein synthesis) is a good endpoint to assess high quality metabolically competence.
  • To assess the longer term (compared to previous available test systems) metabolic competence of two test systems. HepaRG® cells could be used as a long term cellular system for metabolism of drugs and other xenobiotics with a low turnover. These substances are notorious difficult to study in present systems using primary human hepatocytes, because of short viability and stability.
  • To assess a preliminary predictive capacity of the test systems for the potential of selected test items to induce CYP enzymes. The four CYP isoforms have been selected as the main CYP enzymes involved in the metabolisms of drugs and xenobiotic and covering all the three main receptors (CAR, AhR, PXR) involved in the xenobiotic-receptor binding which triggers CYP induction (Tolson et al., 2010). Both FDA (FDA 2012; EMA 2012) and EMA Guidelines recommend CYP1A2, CYP2B6, CYP2C9 and CYP3A4 for CYP induction studies of new drug candidates to understand the impact on kinetics of both the new compound itself and on possible co-medications.
 

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.

 
References
  • Abadie-Viollon C., Martin H., Blanchard N., Pekthong D., Bachellier P., Mantion G., Heyd B., Schuler F., Coassolo P., Alexandre E. and Richert L. (2010) Follow-up to the pre-validation of a harmonised protocol for assessment of CYP induction responses in freshly isolated and cryopreserved human hepatocytes with respect to culture format, treatment, positive reference inducers and incubation conditions. Toxicol In Vitro 24, 346-356.
  • Abass K., Turpeinen M. and Pelkonen O. (2009) An evaluation of the cytochrome P450 inhibition potential of selected pesticides in human hepatic microsomes. J Environ Sci Health B. 44, 553-563.
  • Andersson T.B., Kanebratt K.P. and Kenna J.G. (2012) The HepaRG cell line: a unique in vitro tool for understanding drug metabolism and toxicology in human. Expert Opin Drug Metab Toxicol. 8, 909-920.
  • Coecke S., Rogiers V., Bayliss M., Castell J., Doehmer J., Fabre G., Fry J., Kern A. and Westmoreland C. (1999) The Use of Long-term Hepatocyte Cultures for Detecting Induction of Drug Metabolising Enzymes: The Current Status. ATLA 27, 579-638.
  • Coecke S, Blaauboer BJ, Elaut G, Freeman S, Freidig A, Gensmantel N, Hoet P, Kapoulas VM, Ladstetter B, Langley G, Leahy D, Mannens G, Meneguz A, Monshouwer M, Nemery B, Pelkonen O, Pfaller W, Prieto P, Proctor N, Rogiers V, Rostami-Hodjegan A, Sabbioni E, Steiling W, van de Sandt JJ. (2005) Toxicokinetics and metabolism. Altern Lab Anim. 33: 147-175.
  • Coecke S., Ahr H., Blaauboer B.J., Bremer S., Casati S., Castell J., Combes R., Corvi R., Crespi C.L., Cunningham M.L., Elaut G., Eletti B., Freidig A., Gennari A., Ghersi-Egea J.F., Guillouzo A., Hartung T., Hoet P., Ingelman-Sundberg M., Munn S., Janssens W., Ladstetter B., Leahy D., Long A., Meneguz A., Monshouwer M., Morath S., Nagelkerke F., Pelkonen O., Ponti J., Prieto P., Richert L., Sabbioni E., Schaack B., Steiling W., Testai E., Vericat J.A. and Worth A. (2006) Metabolism: a bottleneck in in vitro toxicological test development. The report and recommendations of ECVAM workshop 54. Altern Lab Anim. 34: 49-84.
  • European Medicine Agency (EMA), Guideline on the Investigation of Drug Interactions, CPMP/EWP/560/95/Rev. 1, 21 June 2012.
  • FDA Food and Drug Administration (2012) draft Guidance for Industry, Drug Interaction Studies —Study Design, Data Analysis, Implications for Dosing, and Labeling Recommendations, February 2012.
  • Gerin B., Dell'aiera S., Richert L., Smith S. and Chanteux H. (2012) Assessment of cytochrome P450 (1A2, 2B6, 2C9 and 3A4) induction in cryopreserved human hepatocytes cultured in 48-well plates using the cocktail strategy. Xenobiotica. 15: 1-6.
  • Guillouzo A., Corlu A., Aninat C., Glaise D., Morel F. and Guguen-Guillouzo C. (2007) The human hepatoma HepaRG cells: A highly differentiated model for studies of liver metabolism and toxicity of xenobiotics. Chemico-Biological Interactions 168, 66–73.
  • Kanebratt K.P., Diczfalusy U., Bäckström T., Sparve E., Bredberg E., Böttiger Y., Andersson T.B. and Bertilsson L. (2008) Cytochrome P450 induction by rifampicin in healthy subjects: determination using the Karolinska cocktail and the endogenous CYP3A4 marker 4beta-hydroxycholesterol. Clin Pharmacol Ther. 84: 589-594.
  • OECD (2008) Detailed review paper on the use of metabolising systems for in vitro testing of endocrine disruptors. OECD Series on testing and assessment: Testing for endocrine disrupters, OECD, Paris, No. 973D
  • Richert L., Abadie C., Bonet A., Heyd B., Mantion G., Alexandre E., Bachellier P., Kingston S., Pattenden C., Illouz S., Dennison A., Hoffmann S. and Coecke S. (2010) Inter-laboratory evaluation of the response of primary human hepatocyte cultures to model CYP inducers – A European Centre for Validation of Alternative Methods (ECVAM) – Funded pre-validation study. Toxicology in Vitro 24, 335–345.
  • Tolson AH, Wang H. Regulation of drug-metabolizing enzymes by xenobiotic receptors: PXR and CAR. Adv Drug Deliv Rev. 2010 Oct 30;62(13):1238-49.

 


3. Development and optimisation of alternative 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 (Coecke et al., 2012). 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.

References

  • Coecke S, Pelkonen O, Leite SB, Bernauer U, Bessems JG, Bois FY, Gundert-Remy U, Loizou G, Testai E, Zaldívar JM. (2012) Toxicokinetics as a key to the integrated toxicity risk assessment based primarily on non-animal approaches. Toxicol In Vitro. in press.

3.1 Barrier models

There is no need to analyse the distribution, metabolism or excretion of a chemical, unless it would become bioavailable and cross our body’s external barriers (gut lining, skin epithelial gut or nasal barriers). To come to such statement, the availability of reliable alternative models to assess the absorption of compounds through such external barriers becomes a key issue.

As indicated higher, the only validated barrier model is described in OECD TG 428 (OECD TG 428, 2004) and still some work has to be done to reliably measure the flux across the dermal barrier as a surrogate for human dermal absorption.

 

3.1.1. Gastrointestinal barrier absorption

ECVAM was involved in the studies of absorption barrier models (Le Ferrec et al., 2001). In particular, an ECVAM prevalidation study on in vitro models for the prediction of gastro-intestinal absorption was finalised in 2008 (Prieto et al., 2010). Ten test chemicals for which in vivo oral absorption data are available were tested in two laboratories. Atenolol, cimetidine and propranolol were included as reference compounds for low, medium and high intestinal absorption, respectively. Transport experiments were independently carried out in the two laboratories. Median CVs of 10.5% and 15.5% were found for each laboratory. Concerning inter-laboratory reproducibility, comparable response levels were found for the three references compounds and for paracetamol while, for the other chemicals, lower reproducibility was obtained, in particular for those actively transported. No significant differences in Papp values were reported between the two cell lines investigated. Due to the limited number of chemicals tested, a model with a real predictive value in terms of in vivo absorption is not realistic; nevertheless a preliminary prediction model has been established using two mathematical models available from the literature. Good in vitro-in vivo correlation was obtained for well absorbed compounds, while moderate and low absorbed compounds were rather overestimated. Both Caco-2 models were more reliable to identify compounds that use passive diffusion than active transport to cross the gastro-intestinal barrier (since Caco-2 cells under or overexpress only some of the transporters present in the intestinal mucosa). 

References

  • Le Ferrec E, Chesne C, Artusson P, Brayden D, Fabre G, Gires P, Guillou F, Rousset M, Rubas W, Scarino ML. (2001) In vitro models of the intestinal barrier. The report and recommendations of ECVAM Workshop 46. European Centre for the Validation of Alternative methods. Altern Lab Anim. 29:649-668.
  • Prieto P, Hoffmann S, Tirelli V, Tancredi F, González I, Bermejo M, De Angelis I. (2010). An exploratory study of two Caco-2 cell models for oral absorption: a report on their within-laboratory and between-laboratory variability, and their predictive capacity. Altern Lab Anim. 38:367-86.

 

3.1.2. Lung barrier absorption

Pulmonary absorption was identified as a high priority area to have reliable input parameters for PBTK modeling. In the priorities identified by the 2010 EURL ECVAM/DG Sanco Expert Report (Adler et al., 2011) and the joint EURL ECVAM – EPAA ADME Workshop "Potential for further integration of toxicokinetic modelling into the prediction of in vivo dose-response curves without animal experiments", held in Ispra, Italy on 13th-14th of October 2011 (Bessems et al., 2013), pulmonary absorption was underlined as a key area were more efforts are needed since no methods are ready available. In this context, the MucilAir® (Epithelix SàRL, Swiss) model was identified as a well-designed test system that with some optimization could eventually fill an important gap.

MucilAir® in vitro human 3D airway epithelium model was originally designed for assessing the toxicity of nanoparticles and chemical compounds but the potential to be used for toxicokinetic purposes as an absorption barrier has been considered as very relevant.

In a collaborative project EURL ECVAM optimised the test system MucilAir® to allow its use as an in vitro absorption barrier for toxicokinetic purposes. The absorption of chemicals through the lung barrier is analysed after apical or basolateral treatments. In order to assess the passage of the chemical that occurs due to passive diffusion and not due to the chemical toxicity and secondary barrier damage, a barrier integrity assay is performed for each test item, prior to be used in the permeability experiments. In permeability studies, the compounds are tested at known concentration and in two directions (apical to basolateral and basolateral to apical). Appropriate analytical methods (e.g. ICP-MS, HPLC, etc.) are applied in order to measure the test item concentration at specific time point established by the experimental procedure. The endpoint is represented by the permeability coefficient (Papp) that is calculated from the generated analytical raw data. Complementary parameters such as the asymmetry index or the mass balance are also calculated.

References

  • Bessems JGM, et al. PBTK modelling platforms and parameter estimation tools to enable animal-free risk assessment. Recommendations from a joint ECVAM - EPAA workshop. To be published in 2013.

 

3.1.3 Blood-brain barrier absorption

Special barriers, such as the blood–brain barrier (BBB), the blood–testis barrier and the placenta barrier, are considered to be of minor importance in the context of the cosmetic sector (Coecke et al., 2005). However, for other sectors that need to use more and more non-animal based methods these barriers are likely to gain importance. A feasibility study to evaluate the performance of five selected in vitro blood-brain barrier (BBB) models for predicting the uptake into the brain was finalised in 2008 (Prieto et al., 2008). Overall, the results of the ECVAM study point out that the new in vitro BBB model (4d/24w), suitable to automation (Culot et al., 2008), constitutes an opportunity to considerably increase the rate at which BBB permeability data can be generated. In addition, the combination of this in vitro BBB model (4d/24w) with SHSY5Y neuroblastoma cells as target cells, explored the importance of the BBB in neurotoxicity assessment (Hallier-Vanuxeem et al., 2009).

References

  • Coecke S, Blaauboer BJ, Elaut G, Freeman S, Freidig A, Gensmantel N, Hoet P, Kapoulas VM, Ladstetter B, Langley G, Leahy D, Mannens G, Meneguz A, Monshouwer M, Nemery B, Pelkonen O, Pfaller W, Prieto P, Proctor N, Rogiers V, Rostami-Hodjegan A, Sabbioni E, Steiling W, van de Sandt JJ. (2005) Toxicokinetics and metabolism. Altern Lab Anim. 33: 147-175.
  • Culot M, Lundquist S, Vanuxeem D, Nion S, Landry C, Delplace Y, Dehouck MP, Berezowski V, Fenart L, Cecchelli R.  (2008) An in vitro blood-brain barrier model for high throughput (HTS) toxicological screening. Toxicol In Vitro 22:799-811.
  • Hallier-Vanuxeem D, Prieto P, Culot M, Diallo H, Landry C, Tähti H, Cecchelli R. (2009) New strategy for alerting central nervous system toxicity: Integration of blood-brain barrier toxicity and permeability in neurotoxicity assessment. Toxicol In Vitro 23:447-453.
  • Prieto P, Blaauboer BJ, de Boer AG, Boveri M, Cecchelli R, Clemedson C, Coecke S, Forsby A, Galla HJ, Garberg P, Greenwood J, Price A, Tähti H (2004) European Centre  for the Validation of Alternative Methods. Blood-brain barrier in vitro models and their application in toxicology. The report and recommendations of ECVAM Workshop 49. Altern Lab Anim32:37-50.

 

3.2 Metabolism

A comparative study of rat and human in vitro metabolising systems (microsomes versus homogenates) on a set of chemical compounds has been carried out  in a collaborative effort between EURL ECVAM and the University of Oulu (Finland) and has been published (Olavi et al., 2009). The goals of the project were to characterise, with human and rat liver homogenate or microsomal preparations, the metabolic stability (i.e. disappearance of the parent substance from incubations with human liver preparations and appropriate cofactors for metabolism) and the appearance of metabolic products and their tentative identification. Using this approach important background information was gained for characterising the studied substances in order to predict, in the end, their behavior in the in vivo situation. Equally important is that the data obtained illustrate similarities, but also quantitative and qualitative differences between homogenate and microsomes, as well as between human and rat. The basic approach suits equally well for the characterization of metabolic capability of any tissues or cell cultures from various animals. The results of this study have served in the process of drafting the OECD Extended One-Generation Reproductive Toxicity Study (OECD TG 443, 2012) by introducing in the guideline different paragraphs related to toxicokinetics and metabolism including a specific mentioning of the need to assess in vitro metabolic processes to assess species differences. Furthermore, the guideline indicates that preliminary information on the ADME processes and bioaccumulation may be derived from chemical structure, physico-chemical data and extend of plasma protein binding or toxicokinetic studies.

Some aspects, such as human genetic polymorphisms for biotransformation enzymes, are not covered in conventional toxicological animal studies. Research and development efforts based on transgenic cells in vitro are a first step in trying to pick up some well-known genetic polymorphisms (Bogni et al., 2005). This information might be useful for a risk assessor, but needs more effort in order to be ready for incorporation in toxicokinetic evaluations. This issue is of importance for drug development and therapy, but less information is available concerning its importance with respect to other sectors.

References

  • Bogni A, Monshouwer M, Moscone A, Hidestrand M, Ingelman-Sundberg M, Hartung T, Coecke S. (2005) Substrate specific metabolism by polymorphic cytochrome P450 2D6 alleles. Toxicol In Vitro 19:621-629
  • OECD (2012) OECD Test Guideline Test No. 443: Extended One-Generation Reproductive Toxicity Study. OECD, Paris, No. 443
  • Pelkonen O, Tolonen A, Rousu T, Tursas L, Turpeinen M, Hokkanen J, Uusitalo J, Bouvier d'Yvoire M, Coecke S. (2009) Comparison of metabolic stability and metabolite identification of 55 ECVAM/ICCVAM validation compounds between human and rat liver homogenates and microsomes - a preliminary analysis. ALTEX 26:214-222.

 

 

3.2.1 Provision of a three dimensional metabolic test system for toxicokinetic and toxicodynamic applications

A pilot study used cultured HepaRG® cells as three dimensional metabolic competent structures in a spinner-bioreactor. The use of a cost-effective commercially available bioreactor, which is compatible with high-throughput cell analysis, constitutes an attractive approach for routine application. In order to assess specific aspects of the biotransformation capacity of the bioreactor-based HepaRG® system, the induction of the cytochrome P450 (CYP) 1A2, CYP 2B6, CYP 2C9, and CYP 3A4 enzymes and the activity of the phase II enzyme, uridine diphosphate glucuronoltransferase, were tested. The long-term functionality of the system was demonstrated by 7-week stable profiles of albumin secretion, CYP3A4 induction, and uridine diphosphate glucuronoltransferase activities. Immunofluorescence-based staining showed formation of tissue-like arrangements including bile canaliculi-like structures and polar distribution of transporters... The approach is a good strategy to reduce the time necessary to obtain fully differentiated cell cultures and HepaRG® cells cultured in three dimensional spinner-bioreactors are an attractive tool for toxicological studies, showing a liver-like performance and demonstrating a practical applicability for toxicokinetic and toxicodynamic approaches.

References

  • Leite SB, Wilk-Zasadna I, Zaldivar JM, Airola E, Reis-Fernandes MA, Mennecozzi M, Guguen-Guillouzo C, Chesne C, Guillou C, Alves PM, Coecke S.  (2012) Three-dimensional HepaRG model as an attractive tool for toxicity testing. Toxicol Sci. 130:106-116.

 

3.3 Physiologically-based pharmacokinetic modelling (PBPK)

An ECVAM workshop report on Physiologically-Based Kinetic Modelling (PBK) (Bouvier d’Yvoire et al., 2007) describes the strategy proposed by the participants to:

  • to better define the potential role of PBK modelling, as a set of techniques capable of contributing to the reduction, refinement and replacement of the use of laboratory animals in the risk assessment process of potentially toxic chemicals;
  • to discuss the need for technical improvement in PBK modelling and its applications;
  • to identify the need to increase understanding and, potentially, acceptance by the regulatory authorities, of the capabilities and limitations of PBK modelling techniques in toxicological risk assessment.


References

  • Bouvier d'Yvoire M, Prieto P, Blaauboer BJ, Bois FY, Boobis A, Brochot C, Coecke S, Freidig A, Gundert-Remy U, Hartung T, Jacobs MN, Lavé T, Leahy DE, Lennernäs H, Loizou GD, Meek B, Pease C, Rowland M, Spendiff M, Yang J, Zeilmaker M. (2007) Physiologically-based Kinetic Modelling (PBK Modelling): meeting the 3Rs agenda. The report and recommendations of ECVAM Workshop 63. Altern Lab Anim 35:661-671.


3.3.1 Integration of toxicokinetic modelling into the prediction of in vivo dose-response curves without animal experiments

In October 2011 the “EPAA/DG JRC ADME Workshop: Potential for further integration of toxicokinetic modelling into the prediction of in vivo dose-response curves without animal experiments” brought together 50 experts in the field.

The full report and a detailed publication are under final discussion by the Scientific Committee responsible for the publication. A synopsis document will also be published subsequently. The main aspects that will be detailed in the report are related to:

  • Identification of gaps in non-animal test methodology for the assessment of ADME.
  • Collection of models allocated to three stages of development where stage 1 is regarded as assay protocols that are suitable as input for PBTK modelling and ready for validation.
  • Addressing user-friendly PBTK software tools and free-to-use web applications.
  • Understanding the requirements for wider and increased take up and use of PBTK modelling by regulators, risk assessors and toxicologists in general.
  • Tackling the aspect of obtaining in vivo human toxicokinetic reference data via microdosing following the increased interest in by the research community, regulators and politicians.

The next step in the area of toxicokinetics is the follow-up of specific recommendations and to consolidate efforts amongst all stakeholders. Some important aspects to focus on in the immediate future can be listed based on current ongoing toxicokinetic discussions at the European leveL:

  • To respond to today’s specific regulatory demands for sector-specific regulations (e.g. to start using toxicokinetic in vitro and in silico methods for route to route extrapolation, to calculate bio-accumulative potential, waiving, read-across etc..).
  • To use available toxicokinetic in vitro and in silico methods to use in high priority area’s (e.g. skin sensitisation) for information needs on bioavailability.
  • To use PBTK models to integrate rather isolated information on ADME to estimate the internal/tissue dose and use this information in design of the in vitro experiments.
  • To use PBTK models for converting in vitro results (generated at tissue/ cell or sub-cellular level) into dose-response information for the entire organism.
  • To extend the first pilot version of the database ECVAM KinParDB containing human and rat kinetic parameters (mainly based on intravenous and oral administration) for 100 chemical substances following assessment of their reliability which are required to build PBTK models.


References

  • Bessems JGM, et al. PBTK modelling platforms and parameter estimation tools to enable animal-free risk assessment. Recommendations from a joint ECVAM - EPAA workshop. To be published in 2013.

3.3.2 Development of a predictive tool for human bioaccumulation assessment

Potential bioaccumulation of a chemical in humans is another key element in risk assessment but no models are nowadays available for bioaccumulation taking into consideration the biotransformation potential of a chemical in the human organism.

A generic PBTK model has been developed which, based on human in vitro liver metabolism data, minimal renal excretion and a chronic exposure, is able to assess the bioaccumulative potential of a chemical. The approach has been analysed using literature data on well-known bioaccumulative compounds (PCBs, DDT, PFOS), liver metabolism data from EURL ECVAM database and a subset of the ToxCast Phase I chemical library - in total 94 compounds including pharmaceuticals, plant protection products and industrial chemicals., The results suggest that human potential bioaccumulation can be assessed with two in vitro tests: one aimed at calculating the chemical binding to plasma proteins and the other at estimating the liver clearance by in vitro measuring the metabolite formation and/or the substrate depletion of the compound using human hepatocytes.

Both tests are suited for high-throughput analysis and thus can be used for prioritisations of chemical safety assessment.

References

  • Tonnelier A. Coecke S. and Zaldívar JM. (2012) Screening of chemicals for human bioaccumulative potential with a physiologically based toxicokinetic model , Arch Toxicology, 86:393-403.


3.3.3
Open-source models for the prediction of repeated dose toxicity (SEURAT-1 consortium)

Within the SEURAT-1 consortium, the COSMOS project is developing publicly available computational workflows based on the integrated use of open-access and open-source models for the prediction of repeated dose toxicity. This includes: 

  • a) the establishment of an inventory of cosmetic substances (including identifiers and chemical structures) and a repeat dose toxicity database (including oral and dermal data); 
  • b) the development of novel ways of establishing thresholds of toxicological concern (TTC), based on innovative chemistry based prediction approaches and PBB K/PBTD modelling. 

The applicability of the current TTC approach to cosmetics has also been demonstrated.

Cross-project efforts are ongoing within the SEURAT-1 cluster to capture in a systematic manner our understanding of the biological mechanisms of repeated dose toxicity. The predictive framework being developed is based on a series of key events, each of which can be modelled separately by in silico or in vitro methods, with a view to integrating the resulting information into Mode of Action (MoA) pathways and Adverse Outcome Pathways (AOPs).

References

  • Anzali S, Berthold MR, Fioravanzo E, Neagu D, Péry A, Worth AP, Yang C, Cronin MTD & Richarz A-N (2012) Development of computational models for the risk assessment of cosmetic ingredients. IFSCC Magazine 15: 249-255.
  • Worth A, Cronin M, Enoch S, Fioravanzo E, Fuart-Gatnik M, Pavan M & Yang C (2012). Applicability of the Threshold of Toxicological Concern (TTC) approach to cosmetics – preliminary analysis. JRC report EUR 25162 EN. Publications Office of the European Union, Luxembourg.


3.3.4 In vitro and in silico methods can be combined to predict target organ effects on humans under repeated dose exposure.

Within the COSMOS project, research is ongoing to provide case studies for selected chemicals that illustrate how in vitro and in silico methods can be combined to predict target organ effects on humans under repeated dose exposure. This includes the development of QSARs for skin penetration, as well as mathematical models for route-to-route extrapolation and in vitro-to-in vivo extrapolation (IVIVE). A virtual cell-based assay model has been developed to simulate the kinetics and dynamics of chemical compounds in cell-based assays. In parallel, models have been developed to simulate kinetics and dynamics at the organ level (virtual liver) and whole organism level (PBTK) models for the rat and the human). Furthermore, the models are being integrated in a multi-scale modelling approach, thereby coupling hepatocytes, via the virtual liver, to the whole organism.

References

  • Zaldívar JM, Wambaugh J & Judson R (2012). Modeling In Vitro Cell-Based Assays Experiments: Cell Population Dynamics. In Models of the ecological hierarchy: from molecules to the ecosphere, F Jørgensen & SE Jordán (Eds), pp 51-71. Developments in Environmental Modelling Vol 25. Elsevier.
  • Diaz Ochoa JG, Bucher J, Péry ARR, Zaldivar Comenges JM, Niklas J & Mauch K (2013). A multi-scale modeling framework for individualized, spatiotemporal prediction of drug effects and toxicological risk. Frontiers in Pharmoacology 3: 204.

 

3.4. ECVAM KinParDB (ECVAM Kinetic Parameters DataBase) and ECVAM KinCalTool (ECVAM Kinetics Calculation Tool)

ECVAM commissioned to RIVM the development of a pilot database with kinetic parameters of compounds used as reference substances in various in vitro toxicity testing (pre)validation programmes. The toxicokinetic properties of compounds can form valuable information in human risk assessment. In vivo as well as in vitro, biological targets are exposed to concentrations of the compounds or their metabolites. Concentrations and their time course, mostly determined in blood or plasma, provide the most direct link between the observed or predicted in vivo effects and the effects observed in vitro. Accurate quantitative knowledge of the in vivo concentration-time relationship is therefore a prerequisite for the correct interpretation of in vitro toxicity testing results. Classical compartmental modelling parameters were chosen to describe the in vivo kinetic properties as they fulfill the needs for prediction of in vivo concentration time profiles under linear conditions. Protein binding parameters were added to facilitate calculation such as unbound substance concentrations. Beside an input module (storage template) for the database, a retrieval template was developed to facilitate further use of kinetic data. The database is filled with human and rat kinetic parameters (mainly based on intravenous and oral administration) for 100 substances following assessment of their reliability. Kinetic data were collected on classical compartmental modeling parameters, which describe the absorption, distribution and elimination (metabolism and excretion) phase. Typical classical compartmental modeling parameters are systemic bioavailability (F), absorption rate constant (ka), volume of distribution (Vd) and elimination rate constant (ke). This pilot database contains kinetic parameters for internal exposure estimation, which may facilitate quantitative in vitro - in vivo extrapolation.

The ECVAM KinCalTool is a self-explaining calculation tool for the construction of a C,t-curve, using a 1- or 2-compartment kinetic model and the kinetic parameters as present in KinParDB. Kinetic parameters can be filled in the yellow cells and a C,t-curve will be presented automatically.

The KinParDB and the KinCalTool are now publicly available for use and we invite the scientific and toxicological community to make use of this application.

  

Copyright Notice

The KinParDB and the KinCalTool are the outcome of a project sponsored by the European Commission (CCR.IHCP.C432921.XO). The KinParDB and the KinCalTool are property of the European Commission: © European Communities. All rights reserved.

All uses of the KinParDB and the KinCalTool in their original version without introduction of changes are authorised, except for commercial purposes, on the condition that the source is fully acknowledged in the form "Source: European Commission, JRC, IHCP" and written notification must be given to JRC-ECVAM-CONTACT@ec.europa.eu. Changes made to the KinParDB and the KinCalTool are not authorised.

All general Copyright & Disclaimer provisions do apply as stated in the Legal Notice.