Introduction

The aim of the SEURAT-1 (Safety Evaluation Ultimately Replacing Animal Testing-1) research cluster, comprised of seven EU FP7 Health projects co-financed by Cosmetics Europe, is to generate a proof-of-concept to show how the latest technologies, systems toxicology and toxicogenomics can be combined to deliver a test replacement for repeated dose systemic toxicity testing on animals.

The SEURAT-1 strategy is to adopt a mode-of-action framework to describe repeated dose toxicity, combining in vitro and in silico methods to derive predictions of in vivo toxicity responses. ToxBank is the cross-cluster infrastructure project whose activities include the development of a data warehouse to provide a web-accessible shared repository of research data and protocols, a physical compounds repository, reference or “gold compounds” for use across the cluster (available via wiki.toxbank.net), and a reference resource for biomaterials.

Core technologies used in the data warehouse include the ISA-Tab universal data exchange format, Representational State Transfer (REST) web services, the W3C Resource Description Framework (RDF) and the OpenTox standards. We describe the design of the data warehouse based on cluster requirements, the implementation based on open standards, and finally the underlying concepts and initial results of a data analysis utilizing public data related to the gold compounds.

The currently proposed “21st Century toxicity testing paradigm” is based on a mode-of-action framework that relies on the understanding of biological pathways and mechanisms of action that underlie the toxicity of chemicals in vivo.[7] The SEURAT-1 program is developing a MOA-based strategy for animal-free replacements of repeated-dose toxicity testing. This paper has presented on-going work from the ToxBank infrastructure project which is supporting the research activities of the SEURAT-1 cluster, including the selection of standard reference compounds that stratify different MOAs associated with repeated dose toxicity, that are potentially relevant across multiple endpoints and organs, such as liver, kidney, heart and the brain. The standard reference chemicals will be used within the other consortia to ensure the experimental results from the different research activities can be compared.

The ToxBank data warehouse will house SEURAT-1 generated results and protocols as well as relevant data from outside the cluster. The warehouse has been developed to enable any future integrated data analysis through the use of RDF and REST-based web services. The warehouse was designed to support research scientists working on the development of replacements to the current repeated dose toxicity tests; however, as the project develops more emphasis will be placed on the use of these approaches to support stakeholders from industry and regulatory agencies for risk assessment purposes.

A goal of the SEURAT-1 project is to investigate the applicability of model systems for uncovering chemical-MOA associations and the robustness of the associations across several model systems from 2D cultures of cell line models to primary cell cultures to highly developed bioreactors. In order to achieve this both the descriptions of experimental metadata and the most relevant results need to be standardized with the use of ontologies and the SEURAT-1 keyword hierarchy. Semantic web technologies can enable flexible and on-going data mining of the entire dataset, as new data is generated and submitted to the ToxBank data warehouse, and can facilitate creating connections to external data such as the CTD and the processed data from the DrugMatrix and TG-GATEs repositories.

Similarly, while repeated dose toxicity is a focus for SEURAT-1, in many cases the biological rationale behind repeated dose toxicity is not fully understood. In the context of the MOA for toxicity, however, there are only two possibilities: either the MOA leading to repeated dose toxicity is the same as that for acute toxicity or it is different. To illustrate, carbon tetrachloride at high doses causes acute wide-spread hepatic necrosis while low repeated doses lead to fibrosis, which is still a response to necrosis, just a more limited and localized necrosis. This is an example where the primary MOA is the same for both acute and repeated dose toxicity. The repeated dose toxicity of phenobarbital, in contrast, is proposed to result from changes in locus-specific DNA methylation patterns, an MOA distinct from acute biological responses.

The compound selection strategy must therefore be based on an understanding of MOAs that underlie repeated dose toxicity so that these MOAs are adequately represented in the in vitro assays. The difficulty behind this statement is again illustrated by carbon tetrachloride. While carbon tetrachloride-induced fibrosis is presumably a response to cell death, many compounds cause hepatic cell death, but not all of them cause fibrosis upon repeated low exposures. The challenge is to understand and derive from cellular model system MOAs at a level of granularity sufficient to distinguish acute versus chronic effects in vivo.

SEURAT-1 gold compounds with diverse chemical structures (Supporting Information SI Figure S2) and MOAs (Table 5, Figure 5) were clustered by chemical-gene and chemical-GO association from the CTD. Clustering of the compounds by GO association grouped together compounds with a similar MOA. Associations of genes to the SEURAT-1 gold compounds from the CTD come mainly from microarray studies, illustrating the value of gene expression studies in generating a large number of chemical-gene associations. Microarray studies also generate more unbiased chemical-gene associations, since typically the expression levels of all the protein-coding genes in the genome are measured. For validation purposes associations uncovered by the toxicogenomics analysis would need to be experimentally verified.

Since the SEURAT-1 gold compounds have fairly well established MOAs (Table 5) the most significant result of the CTD-based data analysis is that unbiased high-throughput data also reflects the MOAs obtained from literature despite the relatively small number of compounds in the analysis, the diverse studies submitted to the CTD, and despite the compounds with the same MOA having different chemical structural features. Results of the SEURAT-1 high-throughput ‘omics profiling experiments obtained from treatments of the cellular model systems with the gold compounds and submitted to the TBDW can be analyzed in the same fashion.

The use of the CTD to analyze MOAs relevant to SEURAT-1 gold compounds also illustrates how statistically significant chemical-gene associations can be mined at the gene-level and connected to MOAs via GO categories. Such associations, if derived using a sufficiently large set of compounds to ensure specificity, can be tentatively considered biomarkers for the detection of toxicologically relevant MOAs. MOA-specific genes and pathways that are detected across several model systems or in the highest quality ones would then form a basis for designing in vitro reporter assays, such as those that are envisioned for further developments related to SEURAT-1 and its extensions.

The ultimate goal would be to fully recreate human in vivo conditions in culture but the more realistic goal of SEURAT-1 is to work towards animal-free repeated-dose toxicity testing of chemical entities overall. Establishing cellular models that enable determination of toxicity relevant modes-of-action in a reproducible manner represents an important step on the way.