Validation requirements for alternative methods in inhalation toxicology - case study of a German two phase prevalidation project

OECD Guidance Document 34 will be briefly recapitulated in context of special requirements for validation of alternative methods to predict inhalative toxicity. Selection of promising endpoints, standardization, optimization, assessment of variability within and between laboratories, and over time, as well as the development of a prediction model (PM) can be performed according to GD 34. However, final testing under blind conditions is often hampered by necessary analytics. Moreover, technically challenging analytics may have to be shared between labs in a rotating principle. Since each in vitro exposure experiment only provides one data point of a concentration-response, the number of test items employed has to be significantly lower than in other validation studies. We are reporting about a BMBF-funded study in four laboratories aiming at prevalidation of a methodology exposing human lung epithelial cells (A549) with toxic gases at the air/liquid interface. Using four reference gases in phase1 as a training set, both endpoints (cell viability and Comet genotoxicity) showed promising robustness; and a linear prediction model for acute in vivo toxicity could be developed. In phase2, the data set was enlarged by six gases (one overlap), and both, reproducibility and the PM of the training set could be independently confirmed.