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10 years of metabolomics research: The importance of quality control
Abstracts / Toxicology Letters 238S (2015) S32–S55
W03-4 10 years of metabolomics research: The importance of quality control H. Kamp 1,∗ , E. Fabian 1 , M. Frericks 1 , M. Herold 2 , G. Krennrich 1 , R. Looser 2 , W. Mellert 1 , G. Montoya 1 , E. Peter 2 , T. Ramirez 1 , M. Spitzer 2 , V. Strauss 1 , A. Strigun 2 , T. Walk 2 , B. van Ravenzwaay 1 1 2
BASF SE, Ludwigshafen, Germany metanomics GmbH, Berlin, Germany
Over the last 10 years, BASF has developed the database MetaMap® Tox containing toxicity and metabolome profiles of more than 750 compounds. The metabolome data was obtained from plasma samples taken in 4-week rat studies. In order to ensure reliability of these data generated over time, maintenance and treatment of animals, sampling and work-up of plasma, measurement of the metabolome as well as data interpretation and storage were standardized. This includes thorough documentation, the compliance with SOPs and safe data storage. These high quality standards should allow for the use of such data, beyond internal screening purposes, to reach a level of transparency sufficient to gain regulatory acceptance. Data from more than 80 control groups with each 10 males and females were analyzed to assess variability. An in depth analysis of this showed a high stability and robustness of the metabolome over a period of ten years. After artificially splitting the groups of 10 control groups into groups of five animals and comparing the number of statistically significantly regulated (false positive) metabolites, the peak of the distribution curve was left of the exact (gausian) centre, but tailed off more to the right than expected under normal distribution. From this analysis we were able to calculate density distributions (relative ratio and standard deviation) for the control values of each metabolite, which can serve as a historical control displaying the range of changes which can be expected as normal. During the course of our project we have used more than ten exact repeats to show reproducibility and reliability of the metabolome analysis (Kamp et al., 2012). Comparing these exact repeats at different levels of statistical significance, we noted that at a level of statistical significance of approximately p = 0.1, provides the best balance between matches (metabolites regulated in the same direction) and mismatches (metabolites regulated in opposite directions) was obtained. Taken together, we show that high quality standards and the analysis of control data help to improve the quality of the data and conclusions derived thereof. Comprehensive documentation and compliance with SOPs ensure highest data for internal toxicity assessment and should allow for the inclusion of such information in regulatory decision making. http://dx.doi.org/10.1016/j.toxlet.2015.08.099
W03-5 Metabolomics: An opportunity for systemic toxicity assessment in vitro T. Hartung The Johns Hopkins University, Center for Alternatives to Animal Testing, Baltimore, United States The NRC report from 2007 “Toxicity Testing in the 21st Century: A vision and a strategy” (Tox-21c) has created an atmosphere of departure suggesting to move to a new resolution, i.e. pathways of toxicity (PoT). We need for this a Human Toxome as the comprehensive pathway list, an annotation of cell types, species, toxicant classes and hazards to these pathways, an integration in
S37
systems toxicology approaches, the in-vitro-in-vivo-extrapolation by reversed dosimetry and finally making sense of the data, most probably in a probabilistic way. The NIH is funding since 2011 by a transformative research grant The Human Toxome project, which involves US EPA ToxCast, the Hamner Institute, Agilent and several members of the Tox-21c panel. The new approach is shaped around pro-estrogenic endocrine disruption as a test case. PoT identification is at the initial stage based on untargeted mass-spectrometry-based metabolomics and gene-arraybased transcriptomics. Proteomics, next-generation-sequencing and miRNomics are currently explored. Key deliverables are the quality assurance of models and analytics, bioinformatic tools for PoT identification and the design of a process for PoT annotation and validation as well as the Human Toxome knowledge database with its governance. Quality assurance (QA) starts with the model systems based on Good Cell Culture Practice guidance and in vitro reporting standards. The field of transcriptomics pioneered QA for omics technologies. For metabolomics, we recently held a series of workshop and have suggested ring trials to explore reproducibility, also across platforms. Early on, the need for QA for the new approaches as a sparring partner for their development and implementation has been noted. The Evidence-based Toxicology Collaboration (EBTC) was created in the US and Europe in 2011 and 2012, respectively. This collaboration of representatives from all stakeholder groups aims to develop tools of Evidence-based Medicine for toxicology, with the secretariat run by CAAT. Beside translating tools such as systematic reviews, meta-analysis, riskof-bias and quality-scoring tools, the EBTC developed a concept of mechanistic validation. All together, Tox-21c and its implementation activities promise a credible approach to revamp regulatory toxicology. http://dx.doi.org/10.1016/j.toxlet.2015.08.100 Workshop W04: New approaches to repeated dose toxicity assessment – Are we ready to replace animal testing? W04-1 Safety evaluation ultimately replacing animal testing: The SEURAT-1 approach? M. Whelan European Commission Joint Research Centre, Institute for Health and Consumer Protection, Ispra, Italy Launched in January 2011, SEURAT-1 is a 50 million Euro 5-year research programme, co-funded equally by the European Commission’s Directorate General for Research and Innovation and Cosmetics Europe, the industry personal care association. Forming a cluster of six research projects and one coordination project, SEURAT-1 represents a community of over 70 research partners from 16 EU Member States and includes the European Commission’s Joint Research Centre. Its aim has been to make a decisive step forward towards the replacement of in vivo repeated dose systemic toxicity testing by demonstrating how data obtained with state-of-the-art in vitro methods and computational models can be rationally integrated and exploited in the safety assessment process. The SEURAT-1 research strategy is based on the premise that new predictive approaches to toxicity assessment have to be knowledge driven, where mechanistic understanding is made explicit upfront to guide the development and combination of complementary of methods that can be selectively incorporated into a flexible decision-making framework. An overarching goal of SEURAT-1 has been the conception and implementation of Proof-
W03-4 10 years of metabolomics research: The importance of quality control H. Kamp 1,∗ , E. Fabian 1 , M. Frericks 1 , M. Herold 2 , G. Krennrich 1 , R. Looser 2 , W. Mellert 1 , G. Montoya 1 , E. Peter 2 , T. Ramirez 1 , M. Spitzer 2 , V. Strauss 1 , A. Strigun 2 , T. Walk 2 , B. van Ravenzwaay 1 1 2
BASF SE, Ludwigshafen, Germany metanomics GmbH, Berlin, Germany
Over the last 10 years, BASF has developed the database MetaMap® Tox containing toxicity and metabolome profiles of more than 750 compounds. The metabolome data was obtained from plasma samples taken in 4-week rat studies. In order to ensure reliability of these data generated over time, maintenance and treatment of animals, sampling and work-up of plasma, measurement of the metabolome as well as data interpretation and storage were standardized. This includes thorough documentation, the compliance with SOPs and safe data storage. These high quality standards should allow for the use of such data, beyond internal screening purposes, to reach a level of transparency sufficient to gain regulatory acceptance. Data from more than 80 control groups with each 10 males and females were analyzed to assess variability. An in depth analysis of this showed a high stability and robustness of the metabolome over a period of ten years. After artificially splitting the groups of 10 control groups into groups of five animals and comparing the number of statistically significantly regulated (false positive) metabolites, the peak of the distribution curve was left of the exact (gausian) centre, but tailed off more to the right than expected under normal distribution. From this analysis we were able to calculate density distributions (relative ratio and standard deviation) for the control values of each metabolite, which can serve as a historical control displaying the range of changes which can be expected as normal. During the course of our project we have used more than ten exact repeats to show reproducibility and reliability of the metabolome analysis (Kamp et al., 2012). Comparing these exact repeats at different levels of statistical significance, we noted that at a level of statistical significance of approximately p = 0.1, provides the best balance between matches (metabolites regulated in the same direction) and mismatches (metabolites regulated in opposite directions) was obtained. Taken together, we show that high quality standards and the analysis of control data help to improve the quality of the data and conclusions derived thereof. Comprehensive documentation and compliance with SOPs ensure highest data for internal toxicity assessment and should allow for the inclusion of such information in regulatory decision making. http://dx.doi.org/10.1016/j.toxlet.2015.08.099
W03-5 Metabolomics: An opportunity for systemic toxicity assessment in vitro T. Hartung The Johns Hopkins University, Center for Alternatives to Animal Testing, Baltimore, United States The NRC report from 2007 “Toxicity Testing in the 21st Century: A vision and a strategy” (Tox-21c) has created an atmosphere of departure suggesting to move to a new resolution, i.e. pathways of toxicity (PoT). We need for this a Human Toxome as the comprehensive pathway list, an annotation of cell types, species, toxicant classes and hazards to these pathways, an integration in
S37
systems toxicology approaches, the in-vitro-in-vivo-extrapolation by reversed dosimetry and finally making sense of the data, most probably in a probabilistic way. The NIH is funding since 2011 by a transformative research grant The Human Toxome project, which involves US EPA ToxCast, the Hamner Institute, Agilent and several members of the Tox-21c panel. The new approach is shaped around pro-estrogenic endocrine disruption as a test case. PoT identification is at the initial stage based on untargeted mass-spectrometry-based metabolomics and gene-arraybased transcriptomics. Proteomics, next-generation-sequencing and miRNomics are currently explored. Key deliverables are the quality assurance of models and analytics, bioinformatic tools for PoT identification and the design of a process for PoT annotation and validation as well as the Human Toxome knowledge database with its governance. Quality assurance (QA) starts with the model systems based on Good Cell Culture Practice guidance and in vitro reporting standards. The field of transcriptomics pioneered QA for omics technologies. For metabolomics, we recently held a series of workshop and have suggested ring trials to explore reproducibility, also across platforms. Early on, the need for QA for the new approaches as a sparring partner for their development and implementation has been noted. The Evidence-based Toxicology Collaboration (EBTC) was created in the US and Europe in 2011 and 2012, respectively. This collaboration of representatives from all stakeholder groups aims to develop tools of Evidence-based Medicine for toxicology, with the secretariat run by CAAT. Beside translating tools such as systematic reviews, meta-analysis, riskof-bias and quality-scoring tools, the EBTC developed a concept of mechanistic validation. All together, Tox-21c and its implementation activities promise a credible approach to revamp regulatory toxicology. http://dx.doi.org/10.1016/j.toxlet.2015.08.100 Workshop W04: New approaches to repeated dose toxicity assessment – Are we ready to replace animal testing? W04-1 Safety evaluation ultimately replacing animal testing: The SEURAT-1 approach? M. Whelan European Commission Joint Research Centre, Institute for Health and Consumer Protection, Ispra, Italy Launched in January 2011, SEURAT-1 is a 50 million Euro 5-year research programme, co-funded equally by the European Commission’s Directorate General for Research and Innovation and Cosmetics Europe, the industry personal care association. Forming a cluster of six research projects and one coordination project, SEURAT-1 represents a community of over 70 research partners from 16 EU Member States and includes the European Commission’s Joint Research Centre. Its aim has been to make a decisive step forward towards the replacement of in vivo repeated dose systemic toxicity testing by demonstrating how data obtained with state-of-the-art in vitro methods and computational models can be rationally integrated and exploited in the safety assessment process. The SEURAT-1 research strategy is based on the premise that new predictive approaches to toxicity assessment have to be knowledge driven, where mechanistic understanding is made explicit upfront to guide the development and combination of complementary of methods that can be selectively incorporated into a flexible decision-making framework. An overarching goal of SEURAT-1 has been the conception and implementation of Proof-