ChemScreen update 2011

Bart van der Burg

AXLR8 meeting

Chemical substance in vitro/in silico screening syst em to predict human- and ecotoxicological effects

• EU framework program 7 (FP7; Environment programme) collaborative project

• 9 partners from 5 countries• January 1, 2010- December 31, 2013• Collaboration with US-EPA NCCT (Toxcast, Tox21)• Sister project of the NCCT/ Texas-Indiana Virtual S TAR

Centre (TIVCS)

Overview

Aims

• Generation of a simple, rapid screening system for reprotoxiceffects of chemicalso widespread implementation o suitable for regulatory purposeso within the tight time schedule of the REACH program

Why reprotox?

• Prioritised in REACH• Reproductive toxicity is important to assess both h uman

and environmental toxicity• Uses the most animals in toxicity testing• Very little alternative methods

EDUKON reprotox in aquatic species

Effect Frequency of

occurrence

Development 1702

Growth 5643

Mortality 73826

Population 10423

Reproduction 2715

• Identify sensitive parameters for reproductive toxi city and critical mechanisms involved in perturbation of these parameters

• Build medium/high throughput system using relevant tests

• Expand step-wise • Integrate with bioinformatics/data interpretation• Build integrated testing strategies, including non-

testing methods (QSAR, grouping, read-across, etc) • Keep close to application (REACH); experienced risk

assessors in the consortium

Approach

In silico prescreen

Chemical

Prioritisation

Potentiallyreprotoxic

Inte

grat

ive

tool

dev

elop

men

t

Minimal essentialscreen

Repro Screen HTP

Other toxic properties

No testing/dedicated in vivo testing

EstabishedScreeningmethods

Prioritisation

wp1

wp5

wp3

wp4

wp6

In silico reprotoxprescreen

wp2

Work package structure

1. Establish in silico prescreening and toxicity prediction methods prioritizing in vitro toxicity testing (WP1, leading partner; DTU)

2. Establish a database and an in silico prescreen to identify potential reproductive toxicants (WP2, FhG)

3. Establishment of sensitive parameters and a medium throughput ‘minimal essential’ in vitro assay panel (WP3, RIVM)

4. Establish a high throughput mechanistic pathway s creen, Repro Screen HTP, for reproductive toxicants (WP4, EKUT)

5. Integrative methods to predict in vivo reprotoxicity allowing informed decisions on prioritization for eventual fu rther testing (WP5, TNO)

6. Integration into one user-friendly tool (WP6, P&G EN)

Research program

In silico toolbox

In silico tools

• Exposure modules (TNO; human, University of Konstan z; environmental)

• Toxicity screening tool DTU (>70 QSARs)• In vivo reprotoxicity database (FeDTex, RepDose, UKO N

ecotox database and link to ToxRefDB)• Bayesian Network approach and automated decision t ool

Proctor & Gamble Eurocor and Simpple

Database human risk

• Expansion RepDose (repeated dose) and FeDTex (fertility and developmental toxicity; Fraunhofer Institute for Toxi cology and Experimental Medicine)

No of Chemicals RepDose FeDTex

before Chemscreen 661 (2217) 139 (225)

Added by Chemscreen 17 (76) 45 (68)

Status 682 (2310) 184 (293)

Further 2 138

Overall 684 322

Ecotox database

• Expansion and restructuring of EDUKON ecotox database (University of Konstanz)

• More detailed information on compounds, reprotoxic ef fects (endpoints population effects, etc)

• Extrapolation in vitro effect-based screening to eco tox effects?

Data type Data Counts

Toxicity Aquatic 133352

Toxicity Terrestrial 54184

Literature 11715

Compound Codes (listed substances) 7157

Species 3602

Choice in vitro assays for reprotoxicity

Assays feasibility study/ChemScreen selection

• 10 compounds

• 14 tests

Schenk et al. 2010 Reproductive Toxicology 30, 200- 218

Predictions ca 90% correct

Starting point ChemScreen

• screening the effects of chemicals on (in vitro) oocytematuration and fertilization

• evaluation of the consequences of those effects on the preimplantation embryo development (planned)

An in vitro assay for :

Bovine oocyte in vitro maturation assay

CALUX® human selective and responsive reporter gene assays

• Low background, high selectivity and inducibility• High sensitivity• Excellent quantification

• Single mechanism, avoid cross-talk and artifacts• Straight-forward interpretation and risk assessment • Better extrapolation to other species• Suitable to measure bioactivity in complex mixtures• Low rate of false positives greatly reduces error i n

panel of assays

CALUX® human selective and responsive reporter gene assays

CALUX/ReProGlostable

CALUX/ReProGlotransient

Expansion cell panels

Hanahan and Weinberg 2000, Cell 100, 57-70

CALUX assay panel

Nuclear receptors Signaling pathways Controls

name status cell name status cell name status cell

DR CALUX �. H4IIE kappaB CALUX �. U2OS Cytox CALUX �. U2OS

PAH CALUX �. H4IIE P21 CALUX �. U2OS MTT �. all

ER CALUX �. T47D Nrf2 CALUX �. U2OS LDH leakage �. all

ERalpha CALUX �. U2OS P53 CALUX �. U2OS Visual �. all

ERbeta CALUX �. U2OS P53 CALUX �. HepG2

ERalpha CALUX �. HEK293 TCF CALUX �. U2OS

ERbeta CALUX �. HEK293 AP1 CALUX �. U2OS

AR CALUX �. U2OS HIF1alpha CALUX �. U2OS

PR CALUX �. U2OS ER stress CALUX �. U2OS

GR CALUX �. U2OS CRE CALUX �. U2OS

TR CALUX �. U2OS ETS CALUX �. U2OS

RAR CALUX �. U2OS GLI CALUX �. U2OS

PPARγ1 CALUX �. U2OS NOTCH CALUX �. U2OS

PPARγ2 CALUX �. U2OS E2F CALUX �. U2OS

PPARα CALUX �. U2OS STAT CALUX �. U2OS

PPARδ CALUX �. U2OS Myc CALUX �. U2OS

PXR CALUX �. U2OS TGFbeta CALUX �. U2OS

LXR CALUX �. U2OS Metal CALUX �. T47D

VDR CALUX �. U2OS

MR CALUX �. U2OS

Embryonic stem cell test

Potential marker genes to simplify/enhance embryoni c stem cell test scoring

R eP roG lo Hydroxyurea

0

5

10

15

20

25

30

35

C o 0.005 0.01 0.05 0.1 0.25 0.5 0.75 1

mM Hydrox yurea

0

0.25

0.5

0.75

1

1.25

1.5

1.75

Luci human

Luci R obo

Vitality human

Vitality R obo

Automation

Ca 400 compounds per week

Automation

Tools

Screening systems

• Panel (20-50) mechanistic assays in human cells (nu clear receptors, dioxin receptor, signaling/stress /devel opmental pathways): agonism and antagonism tested

• Reporter gene assays in (mouse) embryonal stem cells (ReProGlow; developmental pathways) EKUT

• Wildtype ES/transcriptomics• Metabolising cell systems: steroidogenesis (H295R)• Zebrafish/transcriptomics (ZET; RIVM). Added from

complementary resources• Oocyte maturation (TNO) Addition from complementary

resources.

ChemScreen feasibility study selection

Adapted from:Schenk et al. 2010 Reproductive Toxicol ogy 30, 200-218

Zebrafish embryo test

CALUX

panel

H295R

Structures FS

1. Cyclosporin A (CSA)2. Monoethylhexylphthalate (MEHP)3. Valproic acid (VPA)4. D-mannitol (DML)5. Flusilazole (FLU)6. Glufosinate ammonium (GPA)7. Methoxyacetic acid (MAA)8. Retinoic acid (RA)9. Dioctyltinchloride (DOTC)10. Endosulfan (ESF) 11.Diethylstilbestrol (DES)12.Methylmercury chloride (MMC)

Sonneveld et al.2006 Toxicol. Sci., 89:173-87 ; TIV in press

r2 p range

ER 0.87 <0.0001 104

AR 0.46 <0.0001 103

PR 0.84 <0.0001 104

(Pre)Validation reporter gene assays

- In vitro -

-In

viv

o -

C

Lab1

Lab 2

Van der Burg et al 2010a,b Reproductive Toxicology 30: 18-24 & 73-80

(Pre)Validation reporter gene assays

Validation III

Tests submitted (ER and AR)

ChemScreen and validation

• EDC test submission ECVAM of individual tests; “va lidation anchors” for integrated battery approach?

• Collaboration Maurice Whelan/JRC on HTS of reporter gene assays

• Link to validation of alternative genomics-based te sts Netherlands Toxicogenomics Consortium

• Quality control (ISO/GLP)• Link to OECD

Progress

• Extension of 3 reprotox (rodent, environment) data bases (FhG,UKON)

• Identification of critical endpoints reprotox (RIVM )• Position paper current status reprotox testing (BDS , RIVM, TNO)• Set-up HTS screening (EKUT, BDS)• New assay development : CALUX panel, ReProGlo panel, ES,

steroid metabolism (RIVM, BDS, EKUT)• Assay validation (BDS)• Chemical selection (all)• QSAR tools (DTU)• Setup software tools for ITS and database analysis (P&GEN,

Simpple)• Feasibility study

Supervisory board

Dr. David Dix -Chair NCCT, US-EPA

Dr. Maria Bondesson Texas-Indiana STAR Centre

Dr Neil Carmichael ECETOC

Dr. Robert Chapin Pfizer

Prof. dr. Mark Cronin John Moores University Liverpool

Bob Diderich OECD

Dr. Andrew Worth Joint Research Centre

Thanks to

Partner

BioDetection Systems (BDS) Bart van der Burg

Fraunhofer Institute for Toxicology and

Experimental Medicine (FhG)

Inge Mangelsdorf

Netherlands Organization for Applied

Scientific Research (TNO)

Dinant Kroese

Simpple (SIM) Eduard Pauné

National Institute for Public Health and the

Environment (RIVM)

Aldert Piersma

Danish Technical University Food Institute

(DTU)

Jay Niemalä

Procter & Gamble Eurocor (P&GEN) Joanna Jaworska

Eberhard Karls University of Tübingen

(EKUT)

Michael Schwarz

University of Konstanz (UKON) Daniel Dietrich

Thanks to