The use of AOPs for

in vitro prediction of liver toxicity

Mathieu VINKEN

Application of non-animal approaches for decision-making in chemical safety assessment

10-11 December 2018

London-United Kingdom

Key events (KE)

Key event relationships (KER)

Vinken et al. (2017) Arch. Toxicol. 91: 3697-3707.

STRUCTURE

A conceptual construct that portrays existing knowledge concerning the linkage

between a direct molecular initiating event and an adverse outcome at a biological

level relevant to risk assessment

Definition

Building blocks

Villeneuve et al. (2014) Toxicol. Sci. 142: 312-320.

AOPs are not chemical-specificAny chemical or stressor can trigger the molecular initiating event (MIE)

Chemical interaction at the MIE is specific

AOPs are modularAOPs are flexible, clear, transparent and easy to use

KEs and KERs are not unique to a single AOP

An individual AOP is a pragmatic unit of development/evaluationEach AOP has a single MIE and adverse outcome (AO)

AOPs are linear

AOP networks are the functional units of predictionFor real-world applications

Interactions between AOPs triggered by single toxicants or chemical mixtures

AOPs are living documentsIterative nature

Fit-for-purpose

PRINCIPLES

ASSESSMENT

Tailored Bradford-Hill criteria

Biological plausibility of KERs

► KE level

► Mechanistic relationship between an upstream KE and a downstream KE?

Essentiality of KEs

► AOP level

► Prevention of downstream KEs or the AO by blocking an upstream KE?

Empirical support of KERs

► KER level

► Dose-response relationships?

► Temporality?

► Concordance?

Becker et al. (2015) Regul. Toxicol. Pharmacol. 72: 514-537.

Weight-of-evidenceHigh/strong confidence

Moderate confidence

Low/weak confidence

AOP KNOWLEDGE BASE

5 modules

http://aopkb.org

e.AOP. Portal

AOP Xplorer

Effectopedia

Intermediate Effects Database

AOP Wiki

Gijbels and Vinken (2017) Appl. In Vitro Toxicol. 3: 283-285.

Cholestatic liver injury induced by inhibition of the bile salt export pump

Inhibition of inducible nitric oxide synthase, hepatotoxicity and

regenerative proliferation leading to liver tumors

Peroxisome proliferator-activated receptor alpha-dependent liver

cancer

Peroxisomal fatty acid beta-oxidation inhibition leading to steatosis

Liver X receptor activation leading to hepatic steatosis

Sustained aryl hydrocarbon receptor activation leading to rodent

liver tumors

Constitutive androstane receptor suppression leading to hepatic

steatosis

Protein alkylation leading to liver fibrosis

Aryl hydrocarbon receptor activation leading to hepatic steatosis

Liver toxicity AOPs

Hepatocyte nuclear factor alpha suppression leading to hepatic steatosis

Deoxyribonucleic acid adducts leading to liver hemangiosarcoma

Pregnane X receptor activation leading to hepatic steatosis

Chronic cytochrome P450 2E1 activation leading to liver cancer

Nuclear erythroid 2-related factor repression to steatosis

Farnesoid X receptor activation leading to hepatic steatosis

Serine/threonine protein kinase 2 activation leading to hepatic

steatosis

Molecular events lead to nonalcoholic hepatic steatosis

Lysosomal damage leading to liver inflammation

Gijbels and Vinken (2017) Appl. In Vitro Toxicol. 3: 283-285.

Horvat et al. (2017) Arch. Toxicol. 91: 1523-1543.

Examples of liver toxicity AOPsDrug-induced liver fibrosis

Mellor et al. (2016) Crit. Rev. Toxicol. 46: 138-152.

Drug-induced liver steatosis

Drug-induced cholestasis

Vinken et al. (2013) Toxicol. Sci. 136: 97-106.

VALIDATION

Safety Evaluation Ultimately Replacing Animal Testing (SEURAT)

Raised in response to European Regulation (EC) No. 1223/2009

► Cosmetic products and their ingredients

► Testing and marketing ban

Public - private research initiative

► European Commission/FP7

► Cosmetics Europe

Organization

► 1 January 2011-31 December 2015

► More than 70 research institutions

► 6 projects and 1 coordinating action

www.seurat-1.eu

SCR&Tox: stem cell differentiation for human organ-specific target cells

HeMiBio: development of a hepatic microfluidic bioreactor

DETECTIVE: identification and investigation of human biomarkers

COSMOS: delivery of in silico tools to predict adverse effects of chemicals

NOTOX: development of systems biology tools for organotypic cell cultures

ToxBank: supporting integrated data analysis and servicing

COACH: coordinating action

www.seurat-1.eu

► 253 safety evaluation reports covering 220 cosmetic substances

► Focus on repeated dose toxicity testing

► SCCS safety evaluations of candidate cosmetic compounds to be included in the

annexes of European Regulation (EC) No. 1223/2009

► SCCS publishes the safety evaluation reports on open website

AOP selection

Outcome

Screening of cosmetic ingredient safety evaluation reports published by the Scientific

Committee for Consumer Safety (SCCS) between 2000 and 2009

► Steatosis and cholestasis are the most prominent forms of liver toxicity induced by

cosmetic ingredients

► Liver, kidney and spleen are the most frequently targeted organs by cosmetic

ingredients

Vinken et al. (2012) Arch. Toxicol. 86: 405-412.

In vitro experimentation

► Human hepatoma HepaRG cells

► Bosentan

AOP validation

► 3 concentrations (IC10, IC10/4, IC10/10)

► 3 exposure regimes (1 hour, 24 hours, 24 hours + 72 hours wash-out)

► Proteomics

► Metabonomics

Analyses

► Transcriptomics

Rodrigues et al. (2018) Arch. Toxicol. 6: 1939-1952.

BSEP expression and functionality

► Expression

► Functionality

Rodrigues et al. (2018) Arch. Toxicol. 6: 1939-1952.

Transcriptomics analysis

► Global gene changes

Rodrigues et al. (2018) Arch. Toxicol. 6: 1939-1952.

► Gene clustering

► Detection of established and new biomarkers

Rodrigues et al. (2018) Arch. Toxicol. 6: 1939-1952.

Rodrigues et al. (2018) Arch. Toxicol. 6: 1939-1952.

Upregulated

Downregulated

15

77

24h

Wash-out

Proteomics analysis

Rodrigues et al. (2018) Arch. Toxicol. 6: 1939-1952.

Metabonomics analysis

Rodrigues et al. (2018) Arch. Toxicol. 6: 1939-1952.

Functional and in silico testing

Nuclear receptor activation

Structural alerts and descriptors

Robustness and applicability testing

Primary human hepatocytes

Drugs and cosmetic ingredients

Quantitative optimisation: KERs

Quantitative structure-activity relationships

Concentration-response relationships

Risk assessment optimisation

Kinetic data

Exposure data

OPTIMISATION

Chemical grouping

APPLICATIONS

Test development

Vinken et al. (2017) Arch. Toxicol. 91: 3697-3707.

Integrated approaches to testing and assessment

Prioritisation/de-risking of chemicals

Classification and labelling of chemicals

Angrish et al. (2017) Toxicol. Sci. 159: 159-169.