title: Inter-laboratory study of human in vitro toxicogenomics-based tests as alternative methods for evaluating chemical carcinogenicity: a bioinformatics perspective.
abstract: The assessment of the carcinogenic potential of chemicals with alternative, human-based in vitro systems has become a major goal of toxicogenomics. The central read-out of these assays is the transcriptome, and while many studies exist that explored the gene expression responses of such systems, reports on robustness and reproducibility, when testing them independently in different laboratories, are still uncommon. Furthermore, there is limited knowledge about variability induced by the data analysis protocols. We have conducted an inter-laboratory study for testing chemical carcinogenicity evaluating two human in vitro assays: hepatoma-derived cells and hTERT-immortalized renal proximal tubule epithelial cells, representing liver and kidney as major target organs. Cellular systems were initially challenged with thirty compounds, genome-wide gene expression was measured with microarrays, and hazard classifiers were built from this training set. Subsequently, each system was independently established in three different laboratories, and gene expression measurements were conducted using anonymized compounds. Data analysis was performed independently by two separate groups applying different protocols for the assessment of inter-laboratory reproducibility and for the prediction of carcinogenic hazard. As a result, both workflows came to very similar conclusions with respect to (1) identification of experimental outliers, (2) overall assessment of robustness and inter-laboratory reproducibility and (3) re-classification of the unknown compounds to the respective toxicity classes. In summary, the developed bioinformatics workflows deliver accurate measures for inter-laboratory comparison studies, and the study can be used as guidance for validation of future carcinogenicity assays in order to implement testing of human in vitro alternatives to animal testing.
authors.affiliation: Department Computational Molecular Biology, Max-Planck-Institute for Molecular Genetics, Ihnestr. 73, 14195, Berlin, Germany. herwig@molgen.mpg.de.
authors.forename: R
authors.initials: R
authors.lastname: Herwig
authors.affiliation: Genedata AG, Margarethenstrasse 38, 4053, Basel, Switzerland.
authors.forename: H
authors.initials: H
authors.lastname: Gmuender
authors.affiliation: European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM), Institute for Health and Consumer Protection (IHCP), European Commission Joint Research Centre, TP 126, Via E. Fermi 2749, 21027, Ispra, Italy.
authors.forename: R
authors.initials: R
authors.lastname: Corvi
authors.affiliation: School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Byrom Street, Liverpool, L3 3AF, UK.
authors.forename: K M
authors.initials: KM
authors.lastname: Bloch
authors.affiliation: Genedata AG, Margarethenstrasse 38, 4053, Basel, Switzerland.
authors.forename: A
authors.initials: A
authors.lastname: Brandenburg
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Valencia, Av. Blasco Ibanez 15, 46010, Valencia, Spain.
authors.forename: J
authors.initials: J
authors.lastname: Castell
authors.affiliation: Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090, Brussels, Belgium.
authors.forename: L
authors.initials: L
authors.lastname: Ceelen
authors.affiliation: Biopredic International, Parc d'affaires de la Bretèche, Bldg. A4, 35760, St Gregoire, France.
authors.forename: C
authors.initials: C
authors.lastname: Chesne
authors.affiliation: Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090, Brussels, Belgium.
authors.forename: T Y
authors.initials: TY
authors.lastname: Doktorova
authors.affiliation: Department of Toxicogenomics, Maastricht University, Maastricht, The Netherlands.
authors.forename: D
authors.initials: D
authors.lastname: Jennen
authors.affiliation: Division of Physiology, Department of Physiology and Medical Physics, Medical University of Innsbruck, Innsbruck, Austria.
authors.forename: P
authors.initials: P
authors.lastname: Jennings
authors.affiliation: Division of Physiology, Department of Physiology and Medical Physics, Medical University of Innsbruck, Innsbruck, Austria.
authors.forename: A
authors.initials: A
authors.lastname: Limonciel
authors.affiliation: School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Byrom Street, Liverpool, L3 3AF, UK.
authors.forename: E A
authors.initials: EA
authors.lastname: Lock
authors.affiliation: Conway Institute, School of Biomolecular and Biomedical Science, University College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: T
authors.initials: T
authors.lastname: McMorrow
authors.affiliation: European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM), Institute for Health and Consumer Protection (IHCP), European Commission Joint Research Centre, TP 126, Via E. Fermi 2749, 21027, Ispra, Italy.
authors.forename: P
authors.initials: P
authors.lastname: Phrakonkham
authors.affiliation: Conway Institute, School of Biomolecular and Biomedical Science, University College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: R
authors.initials: R
authors.lastname: Radford
authors.affiliation: Conway Institute, School of Biomolecular and Biomedical Science, University College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: C
authors.initials: C
authors.lastname: Slattery
authors.affiliation: Department of Risk Analysis for Products in Development, Netherlands Organisation for Applied Scientific Research (TNO), Utrechtseweg 48, 3704 HE, Zeist, The Netherlands.
authors.forename: R
authors.initials: R
authors.lastname: Stierum
authors.affiliation: Department Computational Molecular Biology, Max-Planck-Institute for Molecular Genetics, Ihnestr. 73, 14195, Berlin, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Vilardell
authors.affiliation: Genedata AG, Margarethenstrasse 38, 4053, Basel, Switzerland.
authors.forename: T
authors.initials: T
authors.lastname: Wittenberger
authors.affiliation: Department Computational Molecular Biology, Max-Planck-Institute for Molecular Genetics, Ihnestr. 73, 14195, Berlin, Germany.
authors.forename: R
authors.initials: R
authors.lastname: Yildirimman
authors.affiliation: Conway Institute, School of Biomolecular and Biomedical Science, University College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: M
authors.initials: M
authors.lastname: Ryan
authors.affiliation: Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090, Brussels, Belgium.
authors.forename: V
authors.initials: V
authors.lastname: Rogiers
authors.affiliation: Department of Toxicogenomics, Maastricht University, Maastricht, The Netherlands.
authors.forename: J
authors.initials: J
authors.lastname: Kleinjans
mesh_terms: D002273:Carcinogens / Q000145:classification / Q000633:toxicity*; D045744:Cell Line, Tumor; D019295:Computational Biology; D004305:Dose-Response Relationship, Drug; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects; D055106:Genome-Wide Association Study; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000187:drug effects* / Q000378:metabolism; D059021:Laboratory Proficiency Testing; D008099:Liver / Q000187:drug effects* / Q000378:metabolism; D015588:Observer Variation; D020411:Oligonucleotide Array Sequence Analysis; D015203:Reproducibility of Results; D018570:Risk Assessment; D013997:Time Factors; D043922:Toxicogenetics / Q000379:methods*; D059467:Transcriptome / Q000187:drug effects*; D057188:Workflow
chemical_list: D002273:Carcinogens
keywords: Bioinformatics; Carcinogenicity; In vitro assays; Inter-laboratory assessment; Pre-validation; Toxicogenomics