EURL ECVAM Recommendation on the Direct Peptide Reactivity Assay (DPRA) for Skin Sensitisation Testing

Identification of the skin sensitisation hazard of chemicals has traditionally relied on the use of animals. Progress in the development of alternative methods has been prompted by the increasing knowledge of the key biological mechanisms underlying this human health effect, as documented by the OECD's recent report summarising the key biological events leading to skin sensitisation ("Adverse Outcome Pathway" (AOP) for skin sensitisation). The molecular initiating event defined within this AOP is the covalent binding of chemicals with skin proteins. Thus peptide reactivity assays may provide valuable information in the context of integrated approaches such as Weight of Evidence (WoE) or Integrated Testing Strategies (ITS) for skin sensitisation hazard and safety assessment. Based on these considerations, EURL ECVAM coordinated a validation study on the Direct Peptide Reactivity Assay (DPRA) addressing mainly the test method’s transferability and within- and between-laboratory reproducibility. Following independent scientific peer review by the EURL ECVAM’s Scientific Advisory Committee (ESAC) and having considered the input from regulators, stakeholders, international partners and the general public, EURL ECVAM concluded that the DPRA may prove a valuable component of a WoE or ITS for skin sensitisation hazard assessment. In addition to this, the DPRA may also be able to contribute to the assessment of sensitising potency, e.g. by supporting sub-categorisation of sensitisers according to UN GHS. However it is recognised that further efforts are required to explore how DPRA data may contribute to potency assessmentJRC.I.5-Systems Toxicolog

EURL ECVAM Recommendation on the Cell Transformation Assay based on the Bhas 42 cell line

The carcinogenic potential of compounds is a crucial aspect in human hazard and risk assessment of substances. Among the various alternatives developed for carcinogenicity prediction, the cell transformation assays (CTAs) have been shown to closely model some key stages of the in vivo carcinogenesis process. Similar to previously validated in vitro CTAs, the CTA in Bhas 42 cells aims at predicting carcinogenic potential. Based on the results of a validation study coordinated by Hadano Research Institute (HRI) Food and Drug Safety Center (FDSC) and other published data, the Bhas 42 CTA protocol (including the 6-well and 96-well plate versions) was considered to be sufficiently standardised, transferable, reproducible between laboratories and relevant to support the identification of potential carcinogenicity of substances. Following independent scientific peer review by the EURL ECVAM’s Scientific Advisory Committee (ESAC) and having considered the input from regulators, stakeholders, international partners and the general public, EURL ECVAM concluded that the CTA in Bhas 42 cells shows promise for inclusion within weight of evidence or integrated testing strategy approaches to assess carcinogenic potential or to support chemical category formation and read-across. Thus EURL ECVAM recommends that an OECD test Guideline be developed. In addition, further investigations on the capability of the assay to detect tumour promoters would provide useful information on mode of action of carcinogens for risk assessment purposes.JRC.I.5-Systems Toxicolog

EURL ECVAM Recommendation on the 3T3 Neutral Red Uptake Cytotoxicity Assay for Acute Oral Toxicity Testing

Acute oral toxicity is currently being assessed by a suite of refinement test methods based on the traditional LD50 lethality test and is, besides skin sensitisation, the only remaining animal test required under REACH Annex VII. In view of assessing the use of alternatives for this health endpoint, EURL ECVAM conducted a study on the 3T3 Neutral Red Uptake cytotoxicity test method addressing the method's capacity to support specifically the identification substances not requiring classification as acute toxicants. Following independent scientific peer review of this study by EURL ECVAM's scientific advisory committee (ESAC) and having considered input from regulators, stakeholders, international partners and the general public, EURL ECVAM concludes that the 3T3 NRU test method may prove a valuable component of a WoE or ITS approach for supporting hazard identification and safety assessment in agreement with the EU CLP Regulation and international regulatory schemes implementing the upper threshold of UN GHS Category 4 as the cut-off for non-classification of substances. In particular, data from the 3T3 NRU assay may constitute an information source within a WoE approach under the provisions of the REACH regulation (Annex XI, 1.2) potentially supporting conclusions on absence of acute oral toxicity of industrial chemicals.JRC.I.5-Systems Toxicolog

EURL ECVAM Status Report on the Development, Validation and Regulatory Acceptance of Alternative Methods and Approaches (2013-April 2014)

The EURL ECVAM status report provides an update on the progress made in the development, validation and regulatory acceptance of alternative methods and approaches since the last report published in April 2013. It is informing on ongoing research and development activities, validation studies, peer reviews, recommendations, strategies and international acceptance of alternative methods and approaches. R&D activities are ongoing for the complex endpoints where the toxicological processes and the mechanistic understanding have not been sufficiently elucidated yet and for which 3Rs solutions are more difficult to find. On the other hand, good progress In the validation and regulatory acceptance is made in areas where non-animal alternative methods have been developed and validated and where the focus lies in an intelligent combination/ integration of the various non-animal approaches.JRC.I.5-Systems Toxicolog

EURL ECVAM Status Report on the Development, Validation and Regulatory Acceptance of Alternative Methods and Approaches (2015)

The EURL ECVAM status report provides an update on the progress made in the development, validation and regulatory acceptance of alternative methods and approaches and their dissemination since the last report published in June 2014. It is informing on ongoing research and development activities, validation studies, peer reviews, recommendations, strategies and regulatory/international acceptance of alternative methods and approaches and dissemination activities. R&D activities within large European or International consortia continued in toxicity areas where 3Rs solutions are more difficult to find due to the underlying complexity of the area. On the other hand, toxicity areas where promising non-animal approaches have been developed, their validation and regulatory acceptance/international adoption could be progressed. Particular emphasis was given to the best and most intelligent combination and integration of these different non-animal approaches to ultimately obtain the required information without resorting to animal testing.JRC.I.5-Systems Toxicolog

Alternative methods for regulatory toxicology – a state-of-the-art review

This state-of-the art review is based on the final report of a project carried out by the European Commission’s Joint Research Centre (JRC) for the European Chemicals Agency (ECHA). The aim of the project was to review the state of the science of non-standard methods that are available for assessing the toxicological and ecotoxicological properties of chemicals. Non-standard methods refer to alternatives to animal experiments, such as in vitro tests and computational models, as well as animal methods that are not covered by current regulatory guidelines. This report therefore reviews the current scientific status of non-standard methods for a range of human health and ecotoxicological endpoints, and provides a commentary on the mechanistic basis and regulatory applicability of these methods. For completeness, and to provide context, currently accepted (standard) methods are also summarised. In particular, the following human health endpoints are covered: a) skin irritation and corrosion; b) serious eye damage and eye irritation; c) skin sensitisation; d) acute systemic toxicity; e) repeat dose toxicity; f) genotoxicity and mutagenicity; g) carcinogenicity; h) reproductive toxicity (including effects on development and fertility); i) endocrine disruption relevant to human health; and j) toxicokinetics. In relation to ecotoxicological endpoints, the report focuses on non-standard methods for acute and chronic fish toxicity. While specific reference is made to the information needs of REACH, the Biocidal Products Regulation and the Classification, Labelling and Packaging Regulation, this review is also expected to be informative in relation to the possible use of alternative and non-standard methods in other sectors, such as cosmetics and plant protection products.JRC.I.5-Systems Toxicolog

Current information sources for hazard identification

The types of information available differ for existing and new chemicals. For established chemicals, exposure and adverse effect data may be readily available, e.g., from scientific literature, or poison control centres. Such information is often unavailable for unapproved new substances for which no practical experience exists. Toxicological information may be gained prospectively or retrospectively. Prospective information may be gained from in vivo (including human), in vitro, or in silico studies. Data may be obtained using standardized test protocols or novel mechanistic techniques, e.g., toxicogenomics. Retrospective information includes epidemiological, post-marketing surveillance, and occupational health data, but also information from non-human sentinel species studies, e.g., fish, wildlife, or livestock. Before collecting and/or evaluating toxicological information, it is important to assess its source. The quality of the data and the quality control measures used for generating the data should be assessed. An assay should be validated for its reliability and (predictive) relevance, and its mechanistic basis (relevance) should be understood. Finally, the appropriateness of the statistical methods used should be evaluated. Sources of information should also be evaluated for their relevance to the question being addressed, e.g, taking into account interspecies variability. For example, weaknesses of in vitro systems include the inability to predict pharmacokinetic parameters, whereas in silico models have this capability, but do not on the other hand provide empirical information. Epidemiological studies should be assessed for population variability, and uncertainties over dose and exposure. Other factors to consider include whether the original data are available for examination and audit. All information should be evaluated for variability and inconsistencies, and their potential causes. Laboratory testing (e.g., toxicogenomics) should adhere to validated, explicit test protocols. It is important also to consider the demonstrated applicability domain limitations of test methods based on physical/chemical properties. Lastly, publication bias should be considered. Recommendations for evidence-based toxicological assessments include increasing the availability of standardized databases of toxicological information. Integrated testing strategies, including infomatic methods to integrate and analyse data from different methodologies, are also needed. Finally, the scientific community should develop and disseminate test methods and strategies for assessing the safety of novel materials, e.g., nanomaterials.JRC.I.2-Public Health Policy Suppor

Current schemes for decision-making in toxicology

In an evaluation of current schemes for decision-making, several areas were identified for improvement. Firstly, regulatory requirements are often inconsistent (e.g., there is no human exposure data requirement for the assessment of agrochemicals). There should be a drive towards standardization so that substances or materials are assessed to a uniform and comparable standard. Secondly, decision-making could be improved by increasing the usage of reported data. Formal voluntary data submission processes should be set up, together with schemes for incorporating the data into the decision-making process. Pharmacovigilance schemes from the pharmaceutical industry may serve as models. Thirdly, the ways in which data are evaluated and integrated may be improved. Klimisch scores provide a good basis for assessing data quality, but additional information is required on the adequacy of studies with regard to specific questions (1). Despite the importance of improved standardization regarding data evaluation (e.g., by more elaborate standards or scoring systems), it is important to allow for a certain flexibility in the way data are interpreted. Fourthly, there is no current link between advances in scientific knowledge and the processes of decision-making. Processes, such as formalized decision analyses, are needed. Finally, uncertainty is not addressed explicitly in decision-making schemes, and uncertainty factors need to be less arbitrary and more evidence-based. There were specific recommendations regarding ecotoxicology. Ecosystem health is a growing concern, and decision-making is increasingly based on in situ diagnostics of low ecological levels of exposure using biomarkers of pollution. A strength of this approach is that biomarkers are measurable in individual organisms, providing early indications of ecotoxicity. Weaknesses include methods of extrapolating mechanistic evidence to the levels of populations and ecosystems. Current decision-making schemes such those of the International Agency for Research on Cancer (IARC), the United States Environmental Protection Agency (EPA), and the Organization for Economic Cooperation and Development (OECD), and the European Union Regulation on Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) legislation are science-based but do to a large extent depend on authority-led judgement. A comparison of the strengths and limitations of individual decision-making schemes may provide the basis for developing an evidence-based toxicological approach. References 1 Klimisch HJ, Andreae M, Tillmann U. A systematic approach for evaluating the quality of experimental toxicological and ecotoxicological data. Regul Toxicol Pharmacol 1997; 25: 1-5.JRC.DDG.I.3-In-vitro method