Proposed Integrated Decision-tree Testing Strategies for Mutagenicity and Carcinogenicity in Relation to the EU REACH Legislation

Abstract

Liverpool John Moores University and FRAME recently conducted a research project sponsored by Defra, on the status of alternatives to animal testing with regard to the European Union REACH (Registration, Evaluation and Authorisation of Chemicals) system for the safety testing and risk assessment of chemicals. The project covered all the main toxicity endpoints associated with the REACH system. This paper focuses on the prospects for using alternative methods (both in vitro and in silico) for mutagenicity (genotoxicity) and carcinogenicity testing — two toxicity endpoints, which, together with reproductive toxicity, are of pivotal importance for the REACH system. The manuscript critically discusses well-established testing approaches, and in particular, the requirement for short-term in vivo tests for confirming positive mutagenicity, and the need for the rodent bioassay for detecting non-genotoxic carcinogens. Recently-proposed testing strategies focusing on non-animal approaches are also considered, and our own testing scheme is presented and supported with background information. This scheme makes maximum use of pre-existing data, computer (in silico) and in vitro methods, with weight-of-evidence assessments at each major stage. The need for the improvement of in vitro methods, to reduce the generation of false-positive results, is also discussed. Lastly, ways in which reduction and refinement measures can be used are also considered, and some recommendations are made for future research to facilitate the implementation of the proposed testing scheme.

Antimutagenic compounds and their possible mechanisms of action

“…Noteworthy, both in vitro and in vivo testing methods are used to identify the same endpoints. The European Union has already implemented this strategy; additionally, guidelines have been recommended internationally (Combes et al 2007). …”

“…Common Phase 1 in vitro mammalian tests include: the mouse lymphoma thymidine kinase (TK) gene mutation assay, which detects compounds that induce forward gene mutations in the tk gene of the L5178Y mouse lymphoma cell line, and the hypoxanthine guanine phosphorybosyl transferase (HPRT) gene mutation assay, which identifies agents that cause gene mutations in the hprt gene of a suitable cell line, such as Chinese hamster cells (Combes et al 2007; Eastmond et al 2009; Johnson 2012). …”

“…In the former assay, mammalian metaphase cells are analyzed for the presence of structural chromosome aberrations, and in the latter, micronuclei in the cytoplasm of cultured mammalian cells during interphase is detected. The micronucleus test is a procedure for the detection of both aneuploidy and clastogenicity in cultured mammalian cells (Combes et al 2007; Eastmond et al 2009). …”

Regulatory toxicology in the twenty-first century: challenges, perspectives and possible solutions

“…In addition, endocrine disruptors, low-dose effects, epigenetically active substances and the influence of background exposures are some of the currently discussed issues which might require the establishment of additional tests or endpoints. Several large projects have been initiated in Europe and North America with the aim to develop the necessary techniques and testing strategies for such complex toxicological endpoints, respectively Aeby et al 2010;Benigni et al 2010;Combes et al 2007Combes et al , 2008Kinsner-Ovaskainen et al 2009;Schenk et al 2010;Vanhaecke et al 2009). In this context, two conceptionally distinct approaches have to be distinguished:…”

A feasibility study: Can information collected to classify for mutagenicity be informative in predicting carcinogenicity?

“…In an effort to improve predictivity, strategic testing has taken the form of Integrated Testing Strategies (ITSs) (Grindon et al, 2006;Combes et al, 2007;Kirkland et al, 2007a,b;Kirkland et al, 2011). The aim of an ITS is to maximize the use of all scientific relevant information and where possible, avoid the use of animal testing.…”