Regarding the references for reference chemicals of alternative methods.

The selection of reference and proficiency chemicals is an important component of method validation and proficiency evaluations. Reference chemicals are a set of test substances used by a method developer to evaluate the reliability and relevance of a new method, in comparison to reference data (usually to a validated reference method). Proficiency chemicals, as defined in OECD Guidance Document on Good In Vitro Method Practices, are defined post validation as a subset of the reference chemicals or other chemicals with sufficient supporting data that are used by naïve laboratories to demonstrate technical competence with a validated test method. Proficiency chemicals should cover different physical states, several chemical classes within the applicability domain of the method and yield the full range of responses (in the validated reference method and in vivo), they shall be commercially available (at non-prohibitive costs) and have high quality reference data. If reference and subsequent proficiency chemicals are chosen without sufficient evidence for their inclusion, both test method evaluation and demonstration of technical proficiency can be hampered. In this report we present cases in which the selection of reference chemicals led to problems in the reproduction of the reference results and demonstration of technical proficiency: The variability of results was not always taken into account in selection of several reference substances of the LLNA (OECD TG 429). Based on the available reference data one proficiency chemical for the Corrositex skin corrosion test (OECD TG 435) should be replaced. Likewise, the expected in vitro result for one of the proficiency chemicals for the BCOP (OECD TG 437) was difficult to reproduce in several labs. Furthermore, it was not possible to obtain one of the proficiency chemicals for the Steroidogenesis Assay (OECD TG 456) at non-prohibitive costs at a reasonable purity. Based on these, we recommend changes of current proficiency chemicals lists with established OECD Test Guidelines and provide recommendations for developing future sets of reference chemicals.