A modular strategy for the testing and assessment of non-genotoxic carcinogens.

A modular strategy is described for the testing and assessment (MoSt) of non-genotoxic carcinogenicity (NGTxC) that is suitable for regulatory applications. It utilizes and builds upon work conducted by the OECD expert group on NGTxC. The approach integrates relevant test methods from the molecular- to cellular- and further to tissue level, many of which have been recently reviewed. Six progressive modules are included in the strategy. Advice is provided for the iterative selection of the next appropriate test method within each step of the strategy. Assessment is completed by a weight of evidence conclusion, which integrates the different streams of modular information. The assessment method gives higher weight to findings that are mechanistically linked with biological relevance to carcinogenesis. With a focus on EU-REACH, and pending upon successful test method validation and acceptance, this will also enable the MoSt for NGTxC to be applied for regulatory purposes across different regulatory jurisdictions.

Skin sensitisation--moving forward with non-animal testing strategies for regulatory purposes in the EU.

In a previous EPAA-Cefic LRI workshop in 2011, issues surrounding the use and interpretation of results from the local lymph node assay were addressed. At the beginning of 2013 a second joint workshop focused greater attention on the opportunities to make use of non-animal test data, not least since a number of in vitro assays have progressed to an advanced position in terms of their formal validation. It is already recognised that information produced from non-animal assays can be used in regulatory decision-making, notably in terms of classifying a substance as a skin sensitiser. The evolution into a full replacement for hazard identification, where the decision is not to classify, requires the generation of confidence in the in vitro alternative, e.g. via formal validation, the existence of peer reviewed publications and the knowledge that the assay(s) are founded on key elements of the Adverse Outcome Pathway for skin sensitisation. It is foreseen that the validated in vitro assays and relevant QSAR models can be organised into formal testing strategies to be applied for regulatory purposes by the industry. To facilitate progress, the European Partnership for Alternative Approaches to animal testing (EPAA) provided the platform for cross-industry and regulatory dialogue, enabling an essential and open debate on the acceptability of an in vitro based integrated strategy. Based on these considerations, a follow up activity was agreed upon to explore an example of an Integrated Testing Strategy for skin sensitisation hazard identification purposes in the context of REACH submissions.

Alternative acute oral toxicity assessment under REACH based on sub-acute toxicity values.

The REACH Regulation requires information on acute oral toxicity for substances produced or imported in quantities greater than one ton per year. When registering, animal testing should be used as last resort. The standard acute oral toxicity test requires use of animals. Therefore, the European Chemicals Agency examined whether alternative ways exist to generate information on acute oral toxicity. The starting hypothesis was that low acute oral toxicity can be predicted from the results of low toxicity in oral sub-acute toxicity studies. Proving this hypothesis would allow avoiding acute toxicity oral testing whenever a sub-acute oral toxicity study is required or available and indicates low toxicity. ECHA conducted an analysis of the REACH database and found suitable studies on both acute oral and sub-acute oral toxicities for 1,256 substances. 415 of these substances had low toxicity in the sub-acute toxicity study (i.e., NO(A)EL at or above the limit test threshold of 1,000 mg/kg). For 98% of these substances, low acute oral toxicity was also reported (i.e., LD50 above the classification threshold of 2,000 mg/kg). On the other hand, no correlation was found between lower NO(A)ELs and LD50. According to the REACH Regulation, this approach for predicting acute oral toxicity needs to be considered as part of a weight of evidence analysis. Therefore, additional sources of information to support this approach are presented. Ahead of the last REACH registration deadline, in 2018, ECHA estimates that registrants of about 550 substances can omit the in vivo acute oral toxicity study by using this adaptation.

In vitro tests within the REACH information strategies.

Tonnage-based information requirements are specified in the proposal on the regulation on the Registration, Evaluation and Authorisation of Chemicals (REACH) in the European Union. The hazard assessment for toxic endpoints should be performed by using a tiered approach, i.e. as an information strategy (IS), starting with an evaluation of all of the data already available, including animal in vivo and in vitro data, and human evidence and case reports, as well as data from (Quantitative)-Structure Activity Relationships ([Q]SARs) or read-across, before any further testing is suggested. To contribute to the implementation of the REACH system, the Nordic countries launched two projects: 1) a review of currently used testing strategies, including a comparison with the REACH requirements; and 2) the development of detailed ISs for skin and eye irritation/corrosion. The review showed that the ISs and classification criteria for the selected endpoints are inconsistent in many cases. In the classification criteria, human data and in vivo test results are usually the prerequisites. Other types of information, such as data from in vitro studies, can sometimes be used, but usually as supportive evidence only. This differs from the REACH ISs, where QSARs, read-across and in vitro testing are important elements. In the other part of the project, an IS for skin and eye irritation/corrosion was proposed. The strategy was "tested" by using four high production volume (HPV) chemicals: hydrogen peroxide, methyl tertiary-butyl ether (MTBE), trivalent chromium, and diantimony trioxide, but only MTBE and trivalent chromium are dealt with in this paper. The "test" revealed that in vivo data, human case reports and physical-chemical data were available and could be used in the evaluation. Classification could be based on the proposed IS and the existing data in all cases, except for the eye irritation/corrosion of trivalent chromium. Weight-of-evidence analysis appeared to be a useful step in the ISs proposed, and including it in the REACH strategies should be considered. For these chemicals, few in vitro and (Q)SAR data were available--more of these data would be generated, if the relevant guidance and legislation on classification were updated.

International regulatory needs for development of an IATA for non-genotoxic carcinogenic chemical substances.

Although regulatory requirements for carcinogenicity testing of chemicals vary according to product sector and regulatory jurisdiction, the standard approach starts with a battery of genotoxicity tests. If any of the in vivo genotoxicity tests are positive, a lifetime rodent cancer bioassay may be requested, which allows the detection of non-genotoxic carcinogens (NGTxC). However, under most chemical regulations the cancer bioassay is rarely requested, specific requests to obtain information on non-genotoxic mechanisms of carcinogenicity are few, and there are no OECD approved screening methods. When the in vitro genotoxicity battery is negative, usually no further carcinogenicity testing is requested. Consequently NGTxC might remain unidentified and therefore the risks they may pose to human health will not be managed. In contrast to genotoxic carcinogens NGTxCact through a large variety of specific mechanisms, and a panel of tests covering multiple biological traits will be needed. The development of an Integrated Approach to Testing and Assessment (IATA) of NGTxC could assist regulatory decision makers. We examine what NGTxC are and discuss chemical regulatory requirements and limitations. With a strong drive to reduce animal testing and costs in mind, it is essential that proper and robust alternatives for animal testing (3Rs) methods for addressing non-genotoxic modes of action are developed and used. Therefore relevant in vitro mechanisms and assays are described and tentatively organized in levels of information, indicating both a possible structure of the future IATA for NGTxC and associated OECD Test Guideline development priorities.

Status and prospects of in vitro tests in risk assessment.

According to the new chemicals policy of the European Union (EU), most chemicals, i.e. the 20,000 chemicals manufactured or imported at 1-10 tons annually, should be tested primarily by using in vitro methods. Also, for other chemicals, the use of in vitro methods is encouraged in the testing strategies given in the draft EU legislation. However, the validation and international acceptance of in vitro tests has been slow. Only recently has the OECD approved four new in vitro test methods, validated by the European Centre for the Validation of Alternative Methods. An analysis of ten randomly selected risk assessment reports of the EU Existing Chemicals Risk Assessment Programme showed that in vitro studies, for example, on cytotoxicity to different cell cultures, cell transformation, metabolism and skin penetration (a total of 115 studies) were used for the assessments. Key metabolic pathways and mechanisms of toxicity have been elucidated, for some chemicals, by using in vitro methods. On the other hand, the results of in vitro studies were regarded as secondary or unreliable in some cases. For several toxic endpoints, in vitro methods will probably serve as screening tools and for mechanistic studies, while target organ toxicity or physiologically regulated adverse effects caused by long-term exposure are difficult to observe without the use of animal models.