ALTEX Online first published February 23, 2017 https://doi.org/10.14573/altex.1606271 1 Research article The borderline range of toxicological methods: Quantification and implications for evaluating precision Maria Leontaridou 1,2 , Daniel Urbisch 2 , Susanne N. Kolle 2 , Katharina Ott 2 , Denis S. Mulliner 3 , Silke Gabbert 1 and Robert Landsiedel 2* 1 Wageningen University, Environmental Economics and Natural Resources Group, Wageningen, the Netherlands; 2 BASF SE, Experimental Toxicology and Ecology, Ludwigshafen, Germany; 3 BASF SE, Computational Chemistry and Biology, Ludwigshafen, Germany Summary Testing methods to assess the skin sensitisation potential of a substance usually use threshold criteria to dichotomise continuous experimental read-outs into yes/no conclusions. The threshold criteria are prescribed in the respective OECD test guidelines and the conclusion is used for regulatory hazard assessment, i.e. classification and labelling of the substance. We can identify a borderline range (BR) around the classification threshold within which test results are non-conclusive due to a testing method’s biological and technical variability. We quantify BRs in the prediction models of the non-animal testing methods DPRA, LuSens and h-CLAT, and of the animal test LLNA, respectively. Depending on the size of the BR we find that between 6% and 28% of the substances in the sets tested with these methods were considered borderline. If the results of individual non- animal test methods are combined into integrated testing strategies (ITS), borderline test results of individual tests can also affect the overall assessment of the skin sensitisation potential of the testing strategy. This was analysed for the ‘2-out-of-3’ ITS: Four out of 40 substances (10%) were considered borderline. Based on our findings we propose expanding the standard binary classification of substances into ‘positive’/’negative’ or ‘hazardous’/’non - hazardous’ by adding a ‘borderline’ or ‘non-conclusive’ alert for cases where test results fall within the borderline range. Keywords: Non-animal methods, variability, borderline range, skin sensitisation. 1 Introduction Skin sensitisers are substances that can lead to an allergic response following skin contact (UNECE, 2011). An individual may be sensitised upon first contact. Subsequent contact can then provoke allergic contact dermatitis (ACD). It is estimated that ACD affects about 20% of the European and North American population at least once in their lifetime, although there is considerable variation of skin sensitisation prevalence between different age-sex groups (Thyssen et al., 2007). Data on skin sensitisation potential have to be provided for all substances produced or manufactured above one tonne per year under the European chemicals legislation REACH, and for classification and labelling of substances under the European CLP regulation (ECHA 2016). The assessment of a substance’s skin sensitisation potential has been traditionally based on data derived from animal tests such as the guinea pig based tests described in OECD TG no. 406 (OECD, 1992) or the murine local lymph node assay (LLNA) described in OECD TG no. 429 (OECD, 2002 and 2010). However, animal welfare concerns, and the regulatory enforcement e.g. by the Cosmetics Regulation (European Commission, 2009) and the REACH legislation (European Commission, 2006) have driven efforts to move away from animal to non-animal testing. A number of non-animal testing methods have been developed (Mehling et al., 2012; Reisinger et al., 2015), two of which, namely the direct peptide reactivity assay (DPRA) (Gerberick et al., 2004; Gerberick et al., 2007) and the antioxidant response element - nuclear factor erythroid 2 (ARE-Nrf2) luciferase testing methods covered by KeratinoSens TM (Natsch et al., 2011), have been validated by the European Centre for Validation of Alternative Methods (ECVAM; Italy) and are described in the OECD TG no. 442C and no. 442D (OECD, 2015a, b). LuSens (Ramirez et al., 2014; Ramirez et al., 2016) also covers the ARE-Nrf2 luciferase testing method and is currently undergoing validation. Another non-animal testing method, the human cell line activation test (h-CLAT) (Ashikaga et al., 2010; Ashikaga et al., 2006; Sakaguchi et al., 2006; Sakaguchi et al., 2010) has recently been validated by ECVAM and is described in OECD TG no. 442E (OECD, 2016). The sequential structure of molecular and cellular mechanisms causing ACD is represented by the “adverse outcome pathway” (AOP) for skin sensitisation, consisting of eleven causally linked steps, four of which were defined to be essential and specific (“key events”) (OECD, 2012a, b). The DPRA, the ARE-Nrf2 testing methods and the h-CLAT cover the first three key events of the skin sensitisation AOP. For hazard classification purposes, i.e. for assessing skin sensitisation potential, continuous data obtained from animal tests or from non-