Emanuela Testai Istituto Superiore di Sanit Department of - - PowerPoint PPT Presentation

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The role of biokinetics in in vitro tests and the interpretation of results Emanuela Testai

Istituto Superiore di Sanità Department of Environment and Primary Prevention Mechanisms of Toxicity Unit Rome-Italy emanuela.testai@iss.it

Rome 17.12.2012

International Symposium on Alternative in vitro methods to characterize the role of EAS in hormone-targeted tissues

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Alternative in vitro methods to characterize the role of EAS

The International Programme for Chemical Safety (2002) has established the following definition for endocrine disrupters: Endocrine disrupters are exogenous substances or mixtures that alter function(s) of the endocrine system and consequently cause adverse health effects in an intact organism,

• r its progeny, or (sub)populations.

US EPA and IPCS do not consider endocrine disruption to be an adverse effect per se, but rather to be a mode or mechanism of action potentially leading to other outcomes, i.e. carcinogenic, reproductive/developmental effects, routinely considered in reaching regulatory decisions.

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There are at least two clear requirements for a substance to be defined as an ED: the demonstration of an adverse effect and of an endocrine disruption mode-of-action (biological plausibility).

Alternative in vitro methods to characterize the role of EAS

Difference between endocrine modulation and endocrine disruption. Many adaptive, compensatory, and physiologically normal/necessary processes result in measurable endocrine changes. It is only when these natural mechanisms are affected to such a degree that adverse effects are induced that ED occurs. Exogenous chemicals in order to affect the endocrine system must act against the background of circulating levels of endogenous hormones, which are usually much more potent than any ED (the potency issue).

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In vitro studies can be used to study the MoA and for priority testing based on hazard. For the time being their use for risk assessment purposes is limited due to difficulties in carrying out quantitative in vitro to in vivo extrapolation. Need of translating information from the cell level, to organs and subsequently to organisms and to distinguish between adaption vs. adversity, likely identifying actual in vitro marker of adversity. ‘Omics’ techniques are producing a bulk of information, but before we can quantitatively use it more knowledge is need for a correct interpretation.

Alternative in vitro methods to characterize the role of EAS

 Lack of information on actual exposure of cell: in this respect in vitro biokinetics data providing the actual level of cell exposure producing an in vitro observed effects can improve the in vitro-in vivo extrapolation.

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The knowledge of the bioavailability of a given compound by the relevant uptake routes should represent the starting point for any toxicological testing. In vivo the actual internal dose reaching the target is the more relevant parameter in evaluating exposure and in the quantitative risk assessment. When developing testing strategies, kinetics is considered the crucial body of information for the design and performance of toxicological tests and for toxicity data interpretation.

This consideration applies also to alternative/non animal testing strategy

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Alternative in vitro methods to characterize the role of EAS

Biokinetics processes have been evoked to explain the in vitro/in vivodifferences

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In vitro biokinetics

Identification of actual cellular exposure (peak concentrations, AUC, parent vs metabolites) The actual intracellular concentration may greatly differ from the nominal applied concentrations due to altered bioavailability : interactions with the medium components, the plate, the cell itself, evaporation, chemical instability (abiotic processes). physiological cellular processes : mechanism of transport across the membranes, biotransformation, bioaccumulation. In vitro the nominal applied concentration rather than the actual level

• f cell exposure is usually associated to the observed effects.

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Alternative in vitro methods to characterize the role of EAS

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Cells

Test Item

Plastic binding Evaporation Protein binding Uptake Free Concentration in the medium Target Metabolism Free Concentration Characterization

• f the cell model

Passive/Active (Transporters)

Alternative in vitro methods to characterize the role of EAS

In vitro biokinetics

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WP3:

Non animal-based models for in vitro kinetics and human kinetic prediction Predict-IV—Profiling the toxicity of new drugs: a non animal-based approach integrating toxicodynamics and biokinetics

The ultimate goal is to contribute to the derivation of NOEC values (relatively to drug safety) in model systems based on human cells representative of in vivo target

• rgans, from which it would be possible to extrapolate the

corresponding in vivo dose.

In repeated treatments for prolonged time of exposure the uncertainty about the actual level of exposure of cells in vitro is greatly enhanced (possibility of induction/inhibition phenomena, cumulative toxicity).

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Alternative in vitro methods to characterize the role of EAS

9 D0 D13

comparison Daily treatment

5 time points 5 time points

Cellule

Tempo Concentrazione 1 2 3 4 10 20 30 40 50 20 24 Cellule

Seeding

Cellule

Tempo Concentrazione 1 2 3 4 10 20 30 40 50 20 24 Cellule

First day/first dose) Last dose

 Sample collection and transport according to specific SOP  Extration from biological matrix according to specific SOP  Quantitative analysis

Alternative in vitro methods to characterize the role of EAS

Concentrations

Primary rat hepatocytes (PRH) Primary human hepatocytes(PHH) HepaRG

HD= TC10 LD= 1/10 di TC10

Human renal cells (RPTEC/TERT1)

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Human kidney cells (RPTEC/TERT1): CSA LD - HD: 5-15 µM

cells medium

LD

cells

HD

Alternative in vitro methods to characterize the role of EAS

Adsorption to the plastic in this system not relevant High potential for bioaccumulation Kinetic of intracellular conc and in the medium during 24 hrs  very low metabolic competence

Wilmes A., et al. J. Proteomics 79, 180-194 (2013)

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Wilmes A., et al. J. Proteomics 79, 180-194 (2013)

⃝  LD ∆  HD CsA

Biokinetic model: (A) CsA supernatant concentration (B)CSA Intracellular concentration

Alternative in vitro methods to characterize the role of EAS

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In the case of endocrine active substances a strong debate is going on about the ‘low dose hypothesis’, according to which “low dose effects”, which are not present at higher doses may display a non-monotonic dose-response (NMDR). Therefore for those given effect, a simple monotonic extrapolation from high to low doses during risk assessment of those substances is no more valid. environmentally-relevant doses doses in the range of typical human exposure doses below those used in traditional toxicological studies doses below the presumed NO(A)EL or BMDL derived by testing Which is the actual definition of low doses?

Can kinetics information help in explaining controversial aspects in the area of ED ?

Alternative in vitro methods to characterize the role of EAS

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Starting from Paracelsus statement All substances are poisons. It’s the dose that makes the poison the paradigm in toxicology and risk assessment is that the individual response of an organism to a chemical increases/decreases proportionally to the exposure (dose). It is generally accepted that for most chemicals (with no genotoxic potential) there is a threshold dose below which there is no adverse effect. This gives rise to a monotonic dose-response relationship In monotonic responses the effect either increases or decreases

• ver the full dose range tested.

Alternative in vitro methods to characterize the role of EAS

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The possibility exists that non monotonic dose-response relationship

• ccurs (NMDR) with U-shaped or inverted U-shaped profile.

The case of ETE is well known. As an example the effects due to copper deficiency are much more severe than the ones due to the its excess. A dose-response curve is non-monotonic when the slope of the curve changes sign somewhere within the range of doses examined. Non monotonicity is not synonymous with low dose, because there are low dose effects that follow monotonic dose-response curves.

Alternative in vitro methods to characterize the role of EAS

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Non-linearities in the toxicokinetics may be the cause of non monotonic dose-response relationship NMDR if the MoA is concentration dependent :  two receptors with different actions and different KDs.  two enzymes involved in the biotransformation with different affinity (Km) producing different metabolites with different effects.  saturation, induction/inhibition of metabolizing enzymes of the unique metabolic pathway

0.0 1.0 2.0 3.0 4.0 0.0 0.5 1.0 1.5 2.0 2.5

2C19 1A2 2B6

Low doses: the effect due to metabolites increases with the dose High doses: saturation of metabolites formation- accumulation of the parent –possible different effect or counteracting effects due to metabolites Alternative in vitro methods to characterize the role of EAS

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NMDR can be observed in studies where high-doses alters the experimental model (cell, organ or animal), thus decreasing the observed response. This could occur when the formation of aggregates, colloids or micelles at high concentrations can reduce bioavailability and therefore decrease the toxicity that appeared at lower concentrations. The same could happen when cytotoxic doses are tested in in vitro studies or in vivo, when using doses that are excessively toxic to animals (doses exceeding the maximum tolerable dose) can reduce the

• nset of an effect.

It is likely that these phenomena could contribute to generate

• nly apparent NMDR.

Alternative in vitro methods to characterize the role of EAS

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 The presence of a response at one dose level only is not sufficient to demonstrate a causal relationship.  A wide dose range and reasonably closely spaced dose intervals (<10-fold within the same study) is necessary to demonstrate U-shaped dose- responses.  Poorly described experiments in non-validated models should not be used.

Up to now no scientific consensus has been reached as to the validity of the studies supporting the ‘low dose hypothesis ‘

Alternative in vitro methods to characterize the role of EAS

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Extrapolating the windows of exposure in development in animal models to windows of exposure in human development could be problematic, due to differences in endocrine signaling across animal species. Pregnancy: Circulating estrogen concentrations during pregnancy are ≤100 times lower in mice than in women pregnant mice may be more susceptible than pregnant women to the adverse effects of estrogenic compounds Foetal life: male rat fetuses are at least an order of magnitude more sensitive than humans to in utero effects of diethylstilbestrol (DES) An additional issue is critical windows of exposure, because of which it may not be possible to identify a health-based reference value appropriate for the lifetime of the exposed population. However, critical time windows are usually covered by the existing animal testing.

Alternative in vitro methods to characterize the role of EAS

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Difference between endocrine modulation and endocrine disruption. Many adaptive, compensatory, and physiologically normal/necessary processes result in measurable endocrine changes. It is only when these natural mechanisms are affected to such a degree that adverse effects are induced that ED occurs. Exogenous chemicals in order to affect the endocrine system must act against the background of circulating levels of endogenous hormones, which are usually much more potent than any ED. The potency issue has to be taken into account in order to understand the relevance for humans. Based on estrogenic potency, human exposure to the most potent environmental estrogens would need to be at least 1000-fold higher than this level, for adverse effects relevant to the human male to be induced, and such levels of exposure are remote (Sharpe 2003)

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The most important question to be answered when the ‘low dose effect’ or NMDR are discussed is : what is the relevance of low dose effects

• bserved in animals for the human population?

Biological plausibility must be given and knowing mode of action is a prerequisite for using the information in risk assessment. Adversity vs adaptation need to be considered when defining potential impact on human health Potency of the exogenous chemical vs endogenous hormones Statistical plausibility should be also to demonstrate the non-monotonic nature of each identified dose-response relationship, which is not always an easy task due to the limited raw data available in the studies published in the scientific Literature Levels of human exposure should also be considered in order to determine in which part of a NMDR exposure occurs.

Alternative in vitro methods to characterize the role of EAS

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To test ED-induced affects postulating a NMDR would imply a change in the testing strategy: more doses to be tested in order to identify such effects, especially in the low dose area. To detect small effects at low doses, an increased number of animals in these dose groups are needed, to strengthen the statistical power. An optimum of seven doses has been proposed in a recent EFSA meeting.

Consequences for the RA

This goes in the opposite direction of the EU policy to reduce animal testing

Alternative in vitro methods to characterize the role of EAS

Studies on kinetics to check internal dose measurements (or cell exposure) have to be carried out; they would help in producing hypotheses on mode of action (MoA) and through the use of PBPK modelling in selecting the dose levels relevant for human exposure. Proposal for changes in the OECD TG to increase the n° of doses tested while decreasing the n° of animal/group to derive BMDL rather than NOAEL? Or to improve study designs incorporating endpoints beyond current OECD methods.

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Questions?

Alternative in vitro methods to characterize the role of EAS

Special thanks to: the PredictIV team Emma Di Consiglio Giuliana Pomponio To you all for your attention