ChemScreen update 2011 Bart van der Burgaxlr8.eu/workshops/van-der-burg.pdf · 1.Establish in...
Transcript of ChemScreen update 2011 Bart van der Burgaxlr8.eu/workshops/van-der-burg.pdf · 1.Establish in...
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