Avoiding a reproducibility crisis in regulatory toxicology-on the fundamental role of ring trials.

The ongoing transition from chemical hazard and risk assessment based on animal studies to assessment relying mostly on non-animal data, requires a multitude of novel experimental methods, and this means that guidance on the validation and standardisation of test methods intended for international applicability and acceptance, needs to be updated. These so-called new approach methodologies (NAMs) must be applicable to the chemical regulatory domain and provide reliable data which are relevant to hazard and risk assessment. Confidence in and use of NAMs will depend on their reliability and relevance, and both are thoroughly assessed by validation. Validation is, however, a time- and resource-demanding process. As updates on validation guidance are conducted, the valuable components must be kept: Reliable data are and will remain fundamental. In 2016, the scientific community was made aware of the general crisis in scientific reproducibility-validated methods must not fall into this. In this commentary, we emphasize the central importance of ring trials in the validation of experimental methods. Ring trials are sometimes considered to be a major hold-up with little value added to the validation. Here, we clarify that ring trials are indispensable to demonstrate the robustness and reproducibility of a new method. Further, that methods do fail in method transfer and ring trials due to different stumbling blocks, but these provide learnings to ensure the robustness of new methods. At the same time, we identify what it would take to perform ring trials more efficiently, and how ring trials fit into the much-needed update to the guidance on the validation of NAMs.

Hazard identification, classification, and risk assessment of carcinogens: too much or too little? - Report of an ECETOC workshop.

The European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) organized a workshop "Hazard Identification, Classification and Risk Assessment of Carcinogens: Too Much or Too Little?" to explore the scientific limitations of the current binary carcinogenicity classification scheme that classifies substances as either carcinogenic or not. Classification is often based upon the rodent 2-year bioassay, which has scientific limitations and is not necessary to predict whether substances are likely human carcinogens. By contrast, tiered testing strategies founded on new approach methodologies (NAMs) followed by subchronic toxicity testing, as necessary, are useful to determine if a substance is likely carcinogenic, by which mode-of-action effects would occur and, for non-genotoxic carcinogens, the dose levels below which the key events leading to carcinogenicity are not affected. Importantly, the objective is not for NAMs to mimic high-dose effects recorded in vivo, as these are not relevant to human risk assessment. Carcinogenicity testing at the "maximum tolerated dose" does not reflect human exposure conditions, but causes major disturbances of homeostasis, which are very unlikely to occur at relevant human exposure levels. The evaluation of findings should consider biological relevance and not just statistical significance. Using this approach, safe exposures to non-genotoxic substances can be established.

21st Century Approaches for Evaluating Exposures, Biological Activity, and Risks of Complex Substances: Workshop highlights.

The June 2019 workshop 21st Century Approaches for Evaluating Exposures, Biological Activity, and Risks of Complex Substances, co-organised by the International Council of Chemical Association's Long-Range Research Initiative and the European Commission's Joint Research Centre, is summarised. Focus was the need for improved approaches to evaluate the safety of complex substances. Approximately 10% and 20% of substances registered under the EU chemicals legislation are 'multi-constituent substances' and 'substances of unknown or variable compositions, complex reaction products and biological substances' (UVCBs), respectively, and UVCBs comprise approximately 25% of the U.S. Toxic Substances Control Act Inventory. Workshop participants were asked to consider how the full promise of new approach methodologies (NAMs) could be brought to bear to evaluate complex substances. Sessions focused on using NAMs for screening, biological profiling, and in complex risk evaluations; improving read-across approaches employing new data streams; and methods to evaluate exposure and dosimetry. The workshop concluded with facilitated discussions to explore actionable steps forward. Given the diversity of complex substances, no single 'correct' approach was seen as workable. The path forward should focus on 'learning by doing' by developing and openly sharing NAM-based fit-for-purpose case examples for evaluating biological activity, exposures and risks of complex substances.

Challenges in working towards an internal threshold of toxicological concern (iTTC) for use in the safety assessment of cosmetics: Discussions from the Cosmetics Europe iTTC Working Group workshop.

The Threshold of Toxicological Concern (TTC) is an important risk assessment tool which establishes acceptable low-level exposure values to be applied to chemicals with limited toxicological data. One of the logical next steps in the continued evolution of TTC is to develop this concept further so that it is representative of internal exposures (TTC based on plasma concentration). An internal TTC (iTTC) would provide threshold values that could be utilized in exposure-based safety assessments. As part of a Cosmetics Europe (CosEu) research program, CosEu has initiated a project that is working towards the development of iTTCs that can be used for the human safety assessment. Knowing that the development of an iTTC is an ambitious and broad-spanning topic, CosEu organized a Working Group comprised a balance of multiple stakeholders (cosmetics and chemical industries, the EPA and JRC and academia) with relevant experience and expertise and workshop to critically evaluate the requirements to establish an iTTC. Outcomes from the workshop included an evaluation on the current state of the science for iTTC, the overall iTTC strategy, selection of chemical databases, capture and curation of chemical information, ADME and repeat dose data, expected challenges, as well as next steps and ongoing work.

Thresholds of Toxicological Concern for cosmetics-related substances: New database, thresholds, and enrichment of chemical space.

A new dataset of cosmetics-related chemicals for the Threshold of Toxicological Concern (TTC) approach has been compiled, comprising 552 chemicals with 219, 40, and 293 chemicals in Cramer Classes I, II, and III, respectively. Data were integrated and curated to create a database of No-/Lowest-Observed-Adverse-Effect Level (NOAEL/LOAEL) values, from which the final COSMOS TTC dataset was developed. Criteria for study inclusion and NOAEL decisions were defined, and rigorous quality control was performed for study details and assignment of Cramer classes. From the final COSMOS TTC dataset, human exposure thresholds of 42 and 7.9 µg/kg-bw/day were derived for Cramer Classes I and III, respectively. The size of Cramer Class II was insufficient for derivation of a TTC value. The COSMOS TTC dataset was then federated with the dataset of Munro and colleagues, previously published in 1996, after updating the latter using the quality control processes for this project. This federated dataset expands the chemical space and provides more robust thresholds. The 966 substances in the federated database comprise 245, 49 and 672 chemicals in Cramer Classes I, II and III, respectively. The corresponding TTC values of 46, 6.2 and 2.3 µg/kg-bw/day are broadly similar to those of the original Munro dataset.

Use of genetic toxicity data in GHS mutagenicity classification and labeling of substances.

One of the key outcomes of testing the potential genotoxicity or mutagenicity of a substance is the conclusion on whether the substance should be classified as a germ cell mutagen and the significance of this for other endpoints such as carcinogenicity. The basis for this conclusion are the criteria presented in classification and labelling systems such as the Globally Harmonized System for classification and labeling (GHS). This article reviews the classification criteria for germ cell mutagenicity and carcinogenicity and how they are applied to substances with evidence of mutagenicity. The implications and suitability of such a classification for hazard communication, risk assessment, and risk management are discussed. It is proposed that genotoxicity assessments should not focus on specifically identifying germ cell mutagens, particularly given the challenges associated with communicating this information in a meaningful way. Rather the focus should be on deriving data to characterize the mode of action and for use in the risk assessment of mutagens, which could then feed into a more robust, risk based management of mutagenic substances versus the current more hazard based approaches. Environ. Mol. Mutagen. 58:354-360, 2017. © 2017 Wiley Periodicals, Inc.

Thresholds in chemical respiratory sensitisation.

There is a continuing interest in determining whether it is possible to identify thresholds for chemical allergy. Here allergic sensitisation of the respiratory tract by chemicals is considered in this context. This is an important occupational health problem, being associated with rhinitis and asthma, and in addition provides toxicologists and risk assessors with a number of challenges. In common with all forms of allergic disease chemical respiratory allergy develops in two phases. In the first (induction) phase exposure to a chemical allergen (by an appropriate route of exposure) causes immunological priming and sensitisation of the respiratory tract. The second (elicitation) phase is triggered if a sensitised subject is exposed subsequently to the same chemical allergen via inhalation. A secondary immune response will be provoked in the respiratory tract resulting in inflammation and the signs and symptoms of a respiratory hypersensitivity reaction. In this article attention has focused on the identification of threshold values during the acquisition of sensitisation. Current mechanistic understanding of allergy is such that it can be assumed that the development of sensitisation (and also the elicitation of an allergic reaction) is a threshold phenomenon; there will be levels of exposure below which sensitisation will not be acquired. That is, all immune responses, including allergic sensitisation, have threshold requirement for the availability of antigen/allergen, below which a response will fail to develop. The issue addressed here is whether there are methods available or clinical/epidemiological data that permit the identification of such thresholds. This document reviews briefly relevant human studies of occupational asthma, and experimental models that have been developed (or are being developed) for the identification and characterisation of chemical respiratory allergens. The main conclusion drawn is that although there is evidence that the acquisition of sensitisation to chemical respiratory allergens is a dose-related phenomenon, and that thresholds exist, it is frequently difficult to define accurate numerical values for threshold exposure levels. Nevertheless, based on occupational exposure data it may sometimes be possible to derive levels of exposure in the workplace, which are safe. An additional observation is the lack currently of suitable experimental methods for both routine hazard characterisation and the measurement of thresholds, and that such methods are still some way off. Given the current trajectory of toxicology, and the move towards the use of non-animal in vitro and/or in silico) methods, there is a need to consider the development of alternative approaches for the identification and characterisation of respiratory sensitisation hazards, and for risk assessment.

Application of the TTC concept to unknown substances found in analysis of foods.

Unknown substances, not previously observed, are frequently detected in foods by quality control laboratories. In many cases, the assessment of these 'new' substances requires additional chemical analysis for their identification prior to assessing risk. This identification procedure can be time-consuming, expensive and in some instances difficult. Furthermore, in many cases, no toxicological information will be available for the substance. Therefore, there is a need to develop pragmatic tools for the assessment of the potential toxicity of substances with unknown identity to avoid delays in their risk assessment. Hence, the 'ILSI Europe expert group on the application of the threshold of toxicological concern (TTC) to unexpected peaks found in food' was established to explore whether the TTC concept may enable a more pragmatic risk assessment of unknown substances that were not previously detected in food. A step-wise approach is introduced that uses expert judgement on the source of the food, information on the analytical techniques, the dietary consumption of food sources containing the unknown substance and quantitative information of the unknown substance to assess the safety to the consumer using the TTC. By following this step-wise approach, it may be possible to apply a TTC threshold of 90 µg/day for an unknown substance in food.

Epigenetics and chemical safety assessment.

Epigenetics, as it pertains to biology and toxicology, can be defined as heritable changes in gene expression that do not involve mutations and are propagated without continued stimulus. Although potentially reversible, these heritable changes may be classified as mitotic, meiotic, or transgenerational, implicating the wide-ranging impact of epigenetic control in cellular function. A number of biological responses have been classified as being caused by an "epigenetic alteration," sometimes based on sound scientific evidence and often in lieu of an identified genetic mutation. Complicating the understanding and interpretation of perceived epigenetic alterations is an incomplete understanding of the normal state and dynamic variation of the epigenome, which can differ widely between cell and tissue types and stage of development or age. This emerging field is likely to have a profound impact on the study and practice of toxicology in coming years. This document reviews the current state of the science in epigenetic modifications, techniques used to measure these changes, and evaluates the current toxicology testing battery with respect to strengths and potential weaknesses in the identification of epigenetics changes. In addition, case studies implicating transgenerational effects induced by diethylstilbestrol, vinclozolin, and bisphenol A were reviewed to illustrate the application of epigenetics in safety assessment and the strengths and limitations of the study designs. An assessment of toxicology tests currently used in safety evaluation revealed that these tests are expected to identify any potential adverse outcomes resulting from epigenetic changes. Furthermore, in order to increase our understanding of the science of epigenetics in toxicology, this review has revealed that a solid understanding of the biology and variation in the epigenome is essential to contextualize concerns about possible adverse health effects related to epigenetic changes. Finally, the fundamental principles guiding toxicology studies, including relevant doses, dose-rates, routes of exposure, and experimental models, need to be taken into consideration in the design and interpretation of studies within this emerging area of science.

Characterizing the noncancer toxicity of mixtures using concepts from the TTC and quantitative models of uncertainty in mixture toxicity.

This article explores the use of an approach for setting default values for the noncancer toxicity, developed as part of the Threshold of Toxicological Concern (TTC), for the evaluation of the chronic noncarcinogenic effects of certain chemical mixtures. Individuals are exposed to many mixtures where there are little or no toxicological data on some or all of the mixture components. The approach developed in the TTC can provide a basis for conservative estimates of the toxicity of the mixture components when compound-specific data are not available. The application of this approach to multiple chemicals in a mixture, however, has implications for the statistical assumptions made in developing component-based estimates of mixtures. Specifically, conservative assumptions that are appropriate for one compound may become overly conservative when applied to all components of a mixture. This overestimation can be investigated by modeling the uncertainty in toxicity standards. In this article the approach is applied to both hypothetical and actual examples of chemical mixtures and the potential for overestimation is investigated. The results indicate that the use of the approach leads to conservative estimates of mixture toxicity and therefore its use is most appropriate for screening assessments of mixtures.