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learning algorithm called Support Vector Machine (SVM) (Cortes & Vapnik, 1995). In this report, we summarise the development of the GARD test. We will discuss the finding of the predictive genes, their function and relevance in the immunologic mechanisms leading to skin sensitisation, estimate the predictive power of the assay and relate it all to the deliverables and scientific goals that were set up in 2010. Deliverables Identify Predictive Genomic Signatures for MUTZ-3 Progenitors Related to Key Sensitisers The GARD assay was developed using 40 reference chemicals, including 20 sensitisers and 20 non-sensitisers (Table 1). The toxic effect of these chemicals on MUTZ-3 was assessed using a standardised Propidium Iodide protocol. In order to measure immunological effects, rather than effects induced by necrosis, all future stimulations were done in the concentrations yielding 90% relative viability (rv90). For the microarray experiments, MUTZ-3 was seeded in wells at a cell concentration of 200,000 cells/ml. Stocks of each of the reference chemicals were added, so that the final in-well concentration corresponded to the rv90 values. Following an incubation of 24h, RNA was isolated and cDNA prepared. Hybridisation, washing and scanning of Affymetrix Human Gene 1.0 ST Arrays were performed according to standardised protocols. All stimulations were performed in triplicates and vehicle controls were included. After quality control of the arrays, the data set ready for analysis consisted of 137 arrays, each with data from measurements of 29,141 transcripts. This entire procedure, which is also the suggested protocol for the assay, is summarised in Figure 1. Statistical analysis of the high dimensional Figure 1. The GARD test principle. The MUTZ-3 cell line are seeded in wells, and incubated with test substances for 24h. RNA is isolated and hybridised to arrays. Following data acquisition, the transcription levels of the predictive signature (200 genes) are analyzed, and each sample is given a decision value as either sensitiser or non-sensitiser, based on SVM classifications. 260 AXLR8-2 WORKSHOP REPORT Progress Report 2011 & AXLR8-2 Workshop Report
Table 1. List of reference chemicals and their respective vehicle and concentration used in assay development. 1) Kathon CG is a mixture of MC/MCI. The concentration of this mixture is given in %. Compound Potency Vehicle Concentration in Culture (µM) Sensitisers 2,4-Dinitrochlorobenzene Extreme DMSO 4 Oxazolone Extreme DMSO 250 Potassium dichromate Extreme Water 1.5 Kathon CG (MC/MCI) Extreme Water 0.0035%1 Formaldehyde Strong Water 80 2-Aminophenol Strong DMSO 100 2-nitro-1,4-Phenylendiamine Strong DMSO 300 p-Phenylendiamine Strong DMSO 75 Hexylcinnamic aldehyde Moderate DMSO 32.24 2-Hydroxyethyl acrylate Moderate Water 100 2-Mercaptobenzothiazole Moderate DMSO 250 Glyoxal Moderate Water 300 Cinnamaldehyde Moderate Water 120 Isoeugenol Moderate DMSO 300 Ethylendiamine Moderate Water 500 Resorcinol Moderate Water 500 Cinnamic alcohol Weak DMSO 500 Eugenol Weak DMSO 300 Penicillin G Weak Water 500 Geraniol Weak DMSO 500 Non-sensitisers 1-Butanol DMSO 500 4-Aminobenzoic acid DMSO 500 Benzaldehyde DMSO 250 Chlorobenzene DMSO 98 Diethyl phthalate DMSO 50 Dimethyl formamide Water 500 Ethyl vanillin DMSO 500 Glycerol Water 500 Isopropanol Water 500 Lactic acid Water 500 Methyl salicylate DMSO 500 AXLR8-2 WORKSHOP REPORT Progress Report 2011 & AXLR8-2 Workshop Report 261
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ALTERNATIVE TESTING STRATEGIES PROG
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ALTERNATIVE TESTING STRATEGIES PROG
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TABLE OF CONTENTS 1. 2. EXECUTIVE S
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Update on EPA’s ToxCast Programme
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challenges, needs, and priorities f
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1INTRODUCTION It is the aim of the
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and progress of these multidiscipli
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Table 2. Members of the AXLR8 Scien
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• OECD Joint Meeting Special Sess
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ACuteTox Optimisation & Pre-Validat
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to robotic screening platforms; and
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ased on functional parameters inclu
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Table 1. Outliers identified by com
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showed that for some compounds the
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Computer-based prediction of metabo
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three reference compounds revealed
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a calibration curve constructed. Th
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cell lines) or 48 hours (A704 cells
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multivariate CART results did not c
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probability) ~50% of the substances
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Publications 2010-11 Hoffmann S, Ki
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Per Artursson Uppsala University Up
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their genotoxic and carcinogenic po
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Table 1. Overview of carcinoGENOMIC
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D12.23 3 monthly Project M42 √ Bo
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annual carcinoGENOMICS meeting in N
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M3.5 Decision of most M40 Postponed
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M3.4 Identification of most suitabl
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WP1 with input from other partners
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Partners Project Co-ordinator Jos K
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combine knowledge of critical proce
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defined and published in the form o
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WP6 is devoted to the setup of a so
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Partners Co-ordinator Bart van der
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obustness, comparing results obtain
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6. Candéias S, Pons B, Viau M, et
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ESNATS Embryonic Stem Cell-Based No
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Table 1. List of deliverables due i
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D3.3.1 Toxicity gene expression sig
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Table 2. List of milestones due in
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Table 3. Test systems corresponding
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Publications 2010-11 1. Peters SJ,
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Marcel Leist Universität Konstanz
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The most significant achievements o
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Figure 3. The Sensor Dish Reader (S
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LIINTOP Optimisation of Liver & Int
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cells express most of the functions
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Figure 3. Scheme of lentivirus gene
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Figure 5. Co-culture set-up. Functi
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Figure 9. Phalloidin-TRITC staining
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Table 1. Values are expressed as pe
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formed with mannitol, atenolol, pro
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Figure 14. Comparison of Digoxin an
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By using the new technology of HCA
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Figure 17. (Left panel) Phalloidin-
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limited amount of data could be pro
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André Guillouzo Institut National
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Objectives The overall aim of this
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Table 2: Milestones achieved in 201
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detected iron (µg/ml) 80 70 6
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in the following order: uncoated ma
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analysis of leukocytes, expression
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2011 the automated protocols will b
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OpenTox Promotion, Development, Acc
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Results The OpenTox Framework The O
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Figure 1. ToxPredict: OpenTox Appli
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scientifically sound manner, so as
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Figure 4. Creating a QPRF report wi
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Figure 7. MaxTox model-building app
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Figure 8. Bioclipse interaction wit
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and model predictions, which they m
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Publications 2010-11 1. Hardy B, Do
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In vitro toxicology using isolated
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(2C-Glo-CYP2C, 3A-Glo-CYP3A) while
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neuronal models 2D (primary culture
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10 and 14 days. The liver-specific
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Frédéric Bois Institut National d
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Figure 1. The Sens-it-iv sphere. 2.
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Figure 2. The updated modular struc
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Technology Module The aim of techno
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Table 1: The final compound list. R
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Table 2. The Sens-it-iv Toolbox. N
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Figure 4. A gene profile for identi
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Figure 7. In vitro T cell priming a
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sessions at congress and meetings (
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7. Gibbs S, van Montfrans C, Kroeze
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vitro assessment of allergens. Eds:
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S.F. Martin Universitätsklinikum F
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idging the gap to human volunteer s
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combinations of transcriptomics, me
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normal animals who usually exhibit
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Workshop proposals Concepts of data
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VITROCELLOMICS Reducing Animal Expe
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esulting of the project was the abi
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Figure 3. Four-compartment artifici
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embryonic stem cells differentiatin
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196 3AXLR8-2 WORKSHOP REPORT
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Workshop participants were divided
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08.45 - 09.15 ACuteTox Annette Kopp
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3.3 Workshop Presentations 202
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Table 1. A sampling of current toxi
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Table 2. EPA activities: a timeline
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Why is a Consortium Needed While th
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Page 214 and 215: compared to controls. Most interest
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Page 218 and 219: The Virtual Liver Spatial-Temporal
Page 220 and 221: A B C D E Figure 2. Tissue reconstr
Page 222 and 223: Figure 4. Mechanisms of how hepatoc
Page 224 and 225: in rat liver in vivo (Figure 5A). H
Page 226 and 227: The IMI eTOX Project Efforts to Dev
Page 228 and 229: deliverables, which can only be ach
Page 230 and 231: A series of publications related to
Page 232 and 233: Update on EPA’s ToxCast Programme
Page 234 and 235: Phase I of ToxCast involved the eva
Page 236 and 237: Endpoint Brief Description of Bioac
Page 238 and 239: includes approximately 150 chemical
Page 240 and 241: information. For each slice, distan
Page 242 and 243: Judson RS, Houck KA, Kavlock RJ, et
Page 244 and 245: Embryonic Stem Cell Approaches Towa
Page 246 and 247: perhaps be tested efficiently in al
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Page 254 and 255: functions, establish relationships
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Page 258 and 259: assay. PLoS One. 2011; 6, e18540. 7
Page 262 and 263: Compound Potency Vehicle Concentrat
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Page 266 and 267: References Basketter DA, Evans P, F
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Page 284 and 285: Figure 3. Scatter plot of robust li
Page 286 and 287: 7. De Smedt A, Van Den Heuvel R, Va
Page 288 and 289: The OECD Adverse Outcome Pathway Ap
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Page 292 and 293: Table 1. Summary of the experimenta
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Page 306 and 307: Table 1. Established reporter cell
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Publications Carcinogenicity Tanaka
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Case Study Approaches for Implement
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assay results together with CSBP mo
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them to support multi-day testing i
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CSBP models for both receptor-media
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Hamner Presentations in 2011 • Ch
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human health risk assessment. Risk
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There is a significant scientific c
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phototoxicity and eye irritation, f
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allergy risk assessment rather than
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‘toolbox’ of non-animal methods
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332 3.4 Breakout Group Reports
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Figure 1. An illustration of the me
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methods, and the generally poor und
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3.4.3. Break-Out Group 2: Reproduct
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- Deep-sequencing of birth defect p
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Figure 2. Specific stages of the ma
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3.4.4 Break-Out Group 3: Skin Sensi
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for further progress. Better tools
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sure doses is not straightforward.
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3.5 AXLR8 Scientific Panel Recommen
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Workshop participants underlined th
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could be co-ordinated in a focused
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focusing on scientific excellence a
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Directory of Projects & Co-ordinato
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Glossary of Terms 2D/3D 3Rs CARDAM/
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