Towards a science-based testing strategy to identify maternal thyroid hormone imbalance and neurodevelopmental effects in the progeny - Part IV: the ECETOC and CLE Proposal for a Thyroid Function-Related Neurodevelopmental Toxicity Testing and Assessment Scheme (Thyroid-NDT-TAS).

Following the European Commission Endocrine Disruptor Criteria, substances shall be considered as having endocrine disrupting properties if they (a) elicit adverse effects, (b) have endocrine activity, and (c) the two are linked by an endocrine mode-of-action (MoA) unless the MoA is not relevant for humans. A comprehensive, structured approach to assess whether substances meet the Endocrine Disruptor Criteria for the thyroid modality (EDC-T) is currently unavailable. Here, the European Centre for Ecotoxicology and Toxicology of Chemicals Thyroxine Task Force and CropLife Europe propose a Thyroid Function-Related Neurodevelopmental Toxicity Testing and Assessment Scheme (Thyroid-NDT-TAS). In Tier 0, before entering the Thyroid-NDT-TAS, all available in vivo, in vitro and in silico data are submitted to weight-of-evidence (WoE) evaluations to determine whether the substance of interest poses a concern for thyroid disruption. If so, Tier 1 of the Thyroid-NDT-TAS includes an initial MoA and human relevance assessment (structured by the key events of possibly relevant adverse outcome pathways) and the generation of supportive in vitro/in silico data, if relevant. Only if Tier 1 is inconclusive, Tier 2 involves higher-tier testing to generate further thyroid- and/or neurodevelopment-related data. Tier 3 includes the final MoA and human relevance assessment and an overarching WoE evaluation to draw a conclusion on whether, or not, the substance meets the EDC-T. The Thyroid-NDT-TAS is based on the state-of-the-science, and it has been developed to minimise animal testing. To make human safety assessments more accurate, it is recommended to apply the Thyroid-NDT-TAS during future regulatory assessments.

Evidential requirements for the regulatory hazard and risk assessment of respiratory sensitisers: methyl methacrylate as an example.

This review addresses the need for a framework to increase the consistency, objectivity and transparency in the regulatory assessment of respiratory sensitisers and associated uncertainties. Principal issues are considered and illustrated through a case study (with methyl methacrylate). In the absence of test methods validated for regulatory use, formal documentation of the weight-of-evidence for hazard classification both at the level of integration of individual studies within lines of evidence and across a broad range of data streams was agreed to be critical for such a framework. An integrated approach is proposed to include not only occupational studies and clinical evidence for the regulatory assessment of respiratory sensitisers, but also information on structure and physical and chemical factors, predictive approaches such as structure activity analysis and in vitro and in vivo mechanistic and toxicokinetic findings. A weight-of-evidence protocol, incorporating integration of these sources of data based on predefined considerations, would contribute to transparency and consistency in the outcome of the assessment. In those cases where a decision may need to be taken on the basis of occupational findings alone, conclusions should be based on transparent weighting of relevant data on the observed prevalence of occupational asthma in various studies taking into account all relevant information including the range and nature of workplace exposures to the substance of interest, co-exposure to other chemicals and study quality.

Safety assessment scheme for menstrual cups and application for the evaluation of a menstrual cup comprised of medical grade silicone.

BACKGROUND: Ensuring menstrual cup safety is paramount, yet a menstrual cup safety assessment scheme is lacking. This paper presents a quadripartite scheme, showing how it can be applied. METHODS: The Tampax Menstrual Cup was evaluated in the safety assessment scheme: (1) Biocompatibility and chemical safety of cup constituents. Extractables were obtained under different use condition; exposure-based risk assessments (EBRA) were conducted for extractables exceeding thresholds of toxicological concern. (2) Physical impact to vaginal mucosa. After physical evaluations, the Tampax Cup and another cup were assessed in a randomised double-blinded, two-product, two-period cross-over clinical trial (65 women, mean age 34.2 years). (3) Impact to vaginal microbiota (in vitro mixed microflora assay and evaluation of vaginal swabs). (4) In vitro growth of Staphylococcus aureus and toxic shock syndrome toxin-1 (TSST-1) production. FINDINGS: Biocompatibility assessments and EBRA of cup constituents showed no safety concerns. In the randomised clinical trial, all potentially product-related adverse effects were mild, vaginal exams were unremarkable, no clinically relevant pH changes occurred, post-void residual urine volume with and without cup were similar, and self-reported measures of comfort along with reports of burning, itching and stinging between cups were comparable. Cup use had no effect on microbial growth in vitro or in the 62 subjects who completed the trial or on in vitro TSST-1 production. INTERPRETATION: The quadripartite safety assessment scheme allows evaluation of menstrual cup safety. The Tampax Cup is safe and well-tolerated upon intended use. As with all feminine hygiene products, post-market safety surveillance confirmed this conclusion. FUNDING: By Procter & Gamble.

Assessing chemical carcinogenicity: hazard identification, classification, and risk assessment. Insight from a Toxicology Forum state-of-the-science workshop.

The Toxicology Forum convened an international state-of-the-science workshop Assessing Chemical Carcinogenicity: Hazard Identification, Classification, and Risk Assessment in December 2020. Challenges related to assessing chemical carcinogenicity were organized under the topics of (1) problem formulation; (2) modes-of-action; (3) dose-response assessment; and (4) the use of new approach methodologies (NAMs). Key topics included the mechanisms of genotoxic and non-genotoxic carcinogenicity and how these in conjunction with consideration of exposure conditions might inform dose-response assessments and an overall risk assessment; approaches to evaluate the human relevance of modes-of-action observed in rodent studies; and the characterization of uncertainties. While the scientific limitations of the traditional rodent chronic bioassay were widely acknowledged, knowledge gaps that need to be overcome to facilitate the further development and uptake of NAMs were also identified. Since one single NAM is unlikely to replace the bioassay, activities to combine NAMs into integrated approaches for testing and assessment, or preferably into defined approaches for testing and assessment that include data interpretation procedures, were identified as urgent research needs. In addition, adverse outcome pathway networks can provide a framework for organizing the available evidence/data for assessing chemical carcinogenicity. Since a formally accepted decision tree to guide use of the best and most current science to advance carcinogenicity risk assessment is currently unavailable, a Decision Matrix for carcinogenicity assessment could be useful. The workshop organizers developed and presented a decision matrix to be considered within a carcinogenicity hazard and risk assessment that is offered in tabular form.

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.

The Grouping and Assessment Strategy for Organic Pigments (GRAPE): Scientific evidence to facilitate regulatory decision-making.

This article presents the Grouping and Assessment Strategy for Organic Pigments (GRAPE). GRAPE is driven by the hypotheses that low (bio)dissolution and low permeability indicate absence of systemic bioavailability and hence no systemic toxicity potential upon oral exposure, and, for inhalation exposure, that low (bio)dissolution (and absence of surface reactivity, dispersibility and in vitro effects) indicate that the organic pigment is a 'poorly soluble particle without intrinsic toxicity potential'. In GRAPE Tier 1, (bio)solubility and (bio)dissolution are assessed, and in Tier 2, in vitro Caco-2 permeability and in vitro alveolar macrophage activation. Thereafter, organic pigments are grouped by common properties (further considering structural similarity depending on the regulatory requirements). In Tier 3, absence of systemic bioavailability is verified by limited in vivo screening (rat 28-day oral and 5-day inhalation toxicity studies). If Tier 3 confirms no (or only very low) systemic bioavailability, all higher-tier endpoint-specific animal testing is scientifically not-relevant. Application of the GRAPE can serve to reduce animal testing needs for all but few representative organic pigments within a group. GRAPE stands in line with the EU REACH Regulation (Registration, Evaluation, Authorisation and Restriction of Chemicals). An ongoing research project aims at establishing a proof-of-concept of the GRAPE.

Applying 'omics technologies in chemicals risk assessment: Report of an ECETOC workshop.

Prevailing knowledge gaps in linking specific molecular changes to apical outcomes and methodological uncertainties in the generation, storage, processing, and interpretation of 'omics data limit the application of 'omics technologies in regulatory toxicology. Against this background, the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) convened a workshop Applying 'omics technologies in chemicals risk assessment that is reported herein. Ahead of the workshop, multi-expert teams drafted frameworks on best practices for (i) a Good-Laboratory Practice-like context for collecting, storing and curating 'omics data; (ii) the processing of 'omics data; and (iii) weight-of-evidence approaches for integrating 'omics data. The workshop participants confirmed the relevance of these Frameworks to facilitate the regulatory applicability and use of 'omics data, and the workshop discussions provided input for their further elaboration. Additionally, the key objective (iv) to establish approaches to connect 'omics perturbations to phenotypic alterations was addressed. Generally, it was considered promising to strive to link gene expression changes and pathway perturbations to the phenotype by mapping them to specific adverse outcome pathways. While further work is necessary before gene expression changes can be used to establish safe levels of substance exposure, the ECETOC workshop provided important incentives towards achieving this goal.

The challenge of the application of 'omics technologies in chemicals risk assessment: Background and outlook.

This survey by the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) highlights that 'omics technologies are generally not yet applied to meet standard information requirements during regulatory hazard assessment. While they are used within weight-of-evidence approaches to investigate substances' modes-of-action, consistent approaches for the generation, processing and interpretation of 'omics data are not applied. To date, no 'omics technology has been standardised or validated. Best practices for performing 'omics studies for regulatory purposes (e.g., microarrays for transcriptome profiling) remain to be established. Therefore, three frameworks for (i) establishing a Good-Laboratory Practice-like context for collecting, storing and curating 'omics data; (ii) 'omics data processing; and (iii) quantitative WoE approaches to interpret 'omics data have been developed, that are presented in this journal supplement. Application of the frameworks will enable between-study comparison of results, which will facilitate the regulatory applicability of 'omics data. The frameworks do not constitute prescriptive protocols precluding any other data analysis method, but provide a baseline for analysis that can be applied to all data allowing ready cross-comparison. Data analysis that does not follow the frameworks can be justified and the resulting data can be compared with the Framework-based common analysis output.

Modern science for better quality control of medicinal products "Towards global harmonization of 3Rs in biologicals": The report of an EPAA workshop.

This article summarizes the outcome of an international workshop organized by the European Partnership for Alternative Approaches to Animal Testing (EPAA) on Modern science for better quality control of medicinal products: Towards global harmonization of 3Rs in biologicals. As regards the safety testing of biologicals, the workshop participants agreed to actively encourage the deletion of abnormal toxicity tests and target animal batch safety tests from all relevant legal requirements and guidance documents (country-specific guidelines, pharmacopoeia monographs, WHO recommendations). To facilitate the global regulatory acceptance of non-animal methods for the potency testing of, e.g., human diphtheria and tetanus vaccines and veterinary swine erysipelas vaccines, international convergence on the scientific principles of the use of appropriately validated in vitro assays for replacing in vivo methods was identified as an overarching goal. The establishment of scientific requirements for new assays was recognized as a further means to unify regulatory approaches in different jurisdictions. It was recommended to include key regulators and manufacturers early in the corresponding discussions. Manufacturers and responsible expert groups, e.g. at the European Directorate for the Quality of Medicines and Health Care of the Council of Europe or the European Medicines Agency, were invited to consider leadership for international collaboration.

Safety assessment of nanomaterials using an advanced decision-making framework, the DF4nanoGrouping.

As presented at the 2016 TechConnect World Innovation Conference on 22-25 May 2016 in Washington DC, USA, the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) 'Nano Task Force' proposes a Decision-making framework for the grouping and testing of nanomaterials (DF4nanoGrouping) consisting of three tiers to assign nanomaterials to four main groups with possible further subgrouping to refine specific information needs. The DF4nanoGrouping covers all relevant aspects of a nanomaterial's life cycle and biological pathways: intrinsic material properties and system-dependent properties (that depend upon the nanomaterial's respective surroundings), biopersistence, uptake and biodistribution, and cellular and apical toxic effects. Use, release, and exposure route may be applied as 'qualifiers' to determine if, e.g., nanomaterials cannot be released from products, which may justify waiving of testing. The four main groups encompass (1) soluble, (2) biopersistent high aspect ratio, (3) passive, and (4) active nanomaterials. The DF4nanoGrouping foresees a stepwise evaluation of nanomaterial properties and effects with increasing biological complexity. In case studies covering carbonaceous nanomaterials, metal oxide, and metal sulfate nanomaterials, amorphous silica and organic pigments (all nanomaterials having primary particle sizes below 100 nm), the usefulness of the DF4nanoGrouping for nanomaterial hazard assessment was confirmed. The DF4nanoGrouping facilitates grouping and targeted testing of nanomaterials. It ensures that sufficient data for the risk assessment of a nanomaterial are available, and it fosters the use of non-animal methods. No studies are performed that do not provide crucial data. Thereby, the DF4nanoGrouping serves to save both animals and resources.

Eating nanomaterials: cruelty-free and safe? the EFSA guidance on risk assessment of nanomaterials in food and feed.

Nanomaterials are increasingly being added to food handling and packaging materials, or directly, to human food and animal feed. To ensure the safety of such engineered nanomaterials (ENMs), in May 2011, the European Food Safety Authority (EFSA) published a guidance document on Risk assessment of the application of nanoscience and nanotechnologies in the food and feed chain. It states that risk assessment should be performed by following a step-wise procedure. Whenever human or animal exposure to nanomaterials is expected, the general hazard characterisation scheme requests information from in vitro genotoxicity, toxicokinetic and repeated dose 90-day oral toxicity studies in rodents. Numerous prevailing uncertainties with regard to nanomaterial characterisation and their hazard and risk assessment are addressed in the guidance document. This article discusses the impact of these knowledge gaps on meeting the goal of ensuring human safety. The EFSA's guidance on the risk assessment of ENMs in food and animal feed is taken as an example for discussion, from the point of view of animal welfare, on what level of uncertainty should be considered acceptable for human safety assessment of products with non-medical applications, and whether animal testing should be considered ethically acceptable for such products.

Animal and non-animal experiments in nanotechnology - the results of a critical literature survey.

A literature survey funded by the Foundation Animalfree Research was performed to obtain an overview on animal experiments in nanotechnology. Scientific articles from Germany, France, the United Kingdom, Italy, the Netherlands and Switzerland published between 2004 and 2007 were collected. A total of 164 articles was retrieved covering in vivo nanotechnological research. The majority of animal experiments were conducted in "nanomedicine", i.e. nanotechnology in the health care area, to study targeted drug, vaccine or gene delivery. Further areas of research relate to nanotechnology-based imaging technologies, the toxicity of nanomaterials, tissue engineering for regenerative treatments, and magnetic tumour thermotherapy. Many experiments were classified as moderately and even severely distressful to the animals. Due to the significance of the scientific topics pursued, the possible scientific benefit of the research depicted in the articles is also assigned to be moderate to high. Nevertheless, it has to be asked whether such animal experiments are truly the only means to answer the scientific questions addressed in nanotechnology. An overview on non-animal test methods used in nanotechnological research revealed a broad spectrum of methodologies applied in a broad spectrum of scientific areas, including those for which animal experiments are being performed. Explicit incentives to avoid animal experiments in nanotechnology currently can only be found in the area of nanotoxicology, but not in the area of nanomedicine. From the point of view of animal welfare, not least because of the new technologies that arise due to nanotechnology, it is time for a paradigm change both in fundamental and applied biomedical research to found research strategies on non-animal test methods.

The new EU Chemicals Policy: challenges and chances for animal-free test methods.

As a first step toward preparing new chemicals legislation, in February 2001, the European Commission adopted a White Paper on a Strategy for a Future Chemicals Policy. Its main goals are to better protect humans and the environment from unknown risks through chemicals. The "promotion of non-animal testing" is one of the key elements of the proposed strategy. For low production volume chemicals, only data from in vitro tests are to be requested. The data requirements for higher production volume chemicals will be designed flexibly, so that only data relevant for the respective chemical are collected. From the point of view of animal welfare risk management, strategies should be defined before test batteries are put together. The test catalogues currently listed in the Dangerous Substances Directive, Council Directive 67/548/EEC, are to be replaced by flexible, tiered-testing strategies, and concrete waiving strategies are to be designed. The avoidance of duplicate testing should become mandatory. In order to fill prevailing knowledge gaps, the funding of alternative method research should be given priority by the EU Commission, and thus should receive a concrete budget. It should have been formulated as a key action in the European Sixth Research Framework Programme.