The EU-ToxRisk method documentation, data processing and chemical testing pipeline for the regulatory use of new approach methods.

Hazard assessment, based on new approach methods (NAM), requires the use of batteries of assays, where individual tests may be contributed by different laboratories. A unified strategy for such collaborative testing is presented. It details all procedures required to allow test information to be usable for integrated hazard assessment, strategic project decisions and/or for regulatory purposes. The EU-ToxRisk project developed a strategy to provide regulatorily valid data, and exemplified this using a panel of > 20 assays (with > 50 individual endpoints), each exposed to 19 well-known test compounds (e.g. rotenone, colchicine, mercury, paracetamol, rifampicine, paraquat, taxol). Examples of strategy implementation are provided for all aspects required to ensure data validity: (i) documentation of test methods in a publicly accessible database; (ii) deposition of standard operating procedures (SOP) at the European Union DB-ALM repository; (iii) test readiness scoring accoding to defined criteria; (iv) disclosure of the pipeline for data processing; (v) link of uncertainty measures and metadata to the data; (vi) definition of test chemicals, their handling and their behavior in test media; (vii) specification of the test purpose and overall evaluation plans. Moreover, data generation was exemplified by providing results from 25 reporter assays. A complete evaluation of the entire test battery will be described elsewhere. A major learning from the retrospective analysis of this large testing project was the need for thorough definitions of the above strategy aspects, ideally in form of a study pre-registration, to allow adequate interpretation of the data and to ensure overall scientific/toxicological validity.

Elements and development processes for test methods in toxicology and human health-relevant life science research.

Many laboratory procedures generate data on properties of chemicals, but they cannot be equated with toxicological "test methods". This apparent discrepancy is not limited to in vitro testing, using animal-free new approach methods (NAM), but also applies to animal-based testing approaches. Here, we give a brief overview of the differences between data generation and the setup or use of a complete test method. While there is excellent literature available on this topic for specialists (GIVIMP guidance; ToxTemp overview), a brief overview and easily-accessible entry point may be useful for a broader community. We provide a single figure to summarize all test method elements and processes required in the development (setup and adaptation) of a test method. The exposure scheme, the endpoint, and the test system are briefly outlined as fundamental elements of any test method. A rationale is provided, why they are not sufficient. We then explain the importance and role of purpose definition (including some information on what is modelled) and the prediction model, aka data interpretation procedure, which depends on the purpose definition, as further essential elements. This connection exemplifies that all fundamental elements are interdependent, and none can be omitted. Finally, discussion is provided on validation as a measure to provide confidence in the reliability, performance, and relevance of a test method. In this sense, validation may be considered a sixth fundamental element for practical use of test methods.

Many laboratory procedures generate data on chemicals, but they cannot be considered complete toxicological "test methods". Here, we give a brief explanation of the fundamental elements of a toxicological test method. We provide an illustration that gives a complete overview of the devel­opment of a test method for non-specialists. We introduce the six fundamental elements, i.e., the exposure scheme, the test endpoint, the test system, the purpose definition and the prediction model and describe how they work together. Finally, we discuss the concept of validation. An understanding of these concepts is important for good-quality scientific research and especially for the development and acceptance of alternatives to animal experiments.

Acceptance criteria for new approach methods in toxicology and human health-relevant life science research - part I.

Every test procedure, scientific and non-scientific, has inherent uncertainties, even when performed according to a standard operating procedure (SOP). In addition, it is prone to errors, defects, and mistakes introduced by operators, laboratory equipment, or materials used. Adherence to an SOP and comprehensive validation of the test method cannot guarantee that each test run produces data within the acceptable range of variability and with the precision and accuracy determined during the method validation. We illustrate here (part I) why controlling the validity of each test run is an important element of experimental design. The definition and application of acceptance criteria (AC) for the validity of test runs is important for the setup and use of test methods, particularly for the use of new approach methods (NAM) in toxicity testing. AC can be used for decision rules on how to handle data, e.g., to accept the data for further use (AC fulfilled) or to reject the data (AC not fulfilled). The adherence to AC has important requirements and consequences that may seem surprising at first sight: (i) AC depend on a test method's objectives, e.g., on the types/concentrations of chemicals tested, the regulatory context, the desired throughput; (ii) AC are applied and documented at each test run, while validation of a method (including the definition of AC) is only performed once; (iii) if AC are altered, then the set of data produced by a method can change. AC, if missing, are the blind spot of quality assurance: Test results may not be reliable and comparable. The establishment and uses of AC will be further detailed in part II of this series.

Standardization of Cell Culture Conditions and Routine Genomic Screening under a Quality Management System Leads to Reduced Genomic Instability in hPSCs.

Human pluripotent stem cells (hPSCs) have generated unprecedented interest in the scientific community, given their potential applications in regenerative medicine, disease modeling, toxicology and drug screening. However, hPSCs are prone to acquire genomic alterations in vitro, mainly due to suboptimal culture conditions and inappropriate routines to monitor genome integrity. This poses a challenge to both the safety of clinical applications and the reliability of basic and translational hPSC research. In this study, we aim to investigate if the implementation of a Quality Management System (QMS) such as ISO9001:2015 to ensure reproducible and standardized cell culture conditions and genomic screening strategies can decrease the prevalence of genomic alterations affecting hPSCs used for research applications. To this aim, we performed a retrospective analysis of G-banding karyotype and Comparative Genomic Hybridization array (aCGH) data generated by our group over a 5-year span of different hESC and hiPSC cultures. This work demonstrates that application of a QMS to standardize cell culture conditions and genomic monitoring routines leads to a striking improvement of genomic stability in hPSCs cultured in vitro, as evidenced by a reduced probability of potentially pathogenic chromosomal aberrations and subchromosomal genomic alterations. These results support the need to implement QMS in academic laboratories performing hPSC research.

A worldwide survey on the use of animal-derived materials and reagents in scientific experimentation.

The use of cell and tissue-based methods in basic, applied and regulatory science has been increasing exponentially. Animal-derived components, including serum, coating materials, growth factors and antibodies are routinely used in cell/tissue cultures and in general laboratory practices. In addition to ethical issues, the use and production of animal-derived materials and reagents raises many scientific concerns, generally associated with presence of undefined components and batch-to-batch variability, which may compromise experimental reproducibility. On the other hand, non-animal materials and reagents, such as human cells, alternatives to animal sera or non-animal recombinant antibodies, are becoming increasingly available, and their use is encouraged by the EU Directive 2010/63 and the Guidance Document on Good In vitro Method Practices (GIVIMP), published by the Organization for Economic Cooperation and Development (OECD). In an effort to map the current state of use of animal-derived reagents across different sectors and to identify the obstacles possibly hampering the implementation of non-animal derived alternatives, a global online survey addressed to scientists working on in vivo, in vitro, in silico methods, in academia as well as pharmaceutical or cosmetic companies, was conducted with the goal to understand: 1) the most commonly used animal-derived materials and reagents, 2) the main issues associated with the production and use of animal-derived materials and reagents, 3) the current level of knowledge on available non-animal alternative materials and reagents, and 4) what educational and information sources could be most useful or impactful to disseminate knowledge on non-animal alternatives. This paper provides an overview of the survey replies and discusses possible proposals to increase awareness, acceptance and use of non-animal ingredients.

Identifying, naming and documenting of test and tool compound stocks.

Handling of chemicals is an often-neglected area of test descriptions. Some important aspects are highlighted here, using methyl-phenyl-tetrahydropyridine (MPTP), ferrous sulfate (FeSO4·xH2O) and ciguatoxin as example compounds. These are used to provide some background on aspects of acid-base equilibria, redox state, crystal water, natural compound mixtures, and chemical naming systems. Also, solvents and impurities are addressed, for instance concerning their often high (millimolar range) concentrations in assay buffers and cell culture media. The discussion of these aspects calls for a more standardized preparation of test solutions and a more extensive disclosure of the procedure in publications; it also suggests more flexibility in data mining, as compounds with clearly different identifiers may have been used to produce highly similar or fully identical test conditions. While this short overview is not intended as definitive guidance, it does demand more active involvement of all test developers and performers with these issues, and it calls for more transparent information disclosure concerning the preparation and use of test and control chemical solutions.