The borderline range of toxicological methods : Quantification and implications for evaluating precision

Test methods to assess the skin sensitization potential of a substance usually use threshold criteria to dichotomize 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 inconclusive due to a test method's biological and technical variability. We quantified BRs in the prediction models of the non-animal test methods DPRA, LuSens and h-CLAT, and of the animal test LLNA, respectively. Depending on the size of the BR, we found that between 6% and 28% of the substances in the sets tested with these methods were considered borderline. When the results of individual non-animal test methods were combined into integrated testing strategies (ITS), borderline test results of individual tests also affected the overall assessment of the skin sensitization potential of the testing strategy. This was analyzed 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 "inconclusive" alert for cases where test results fall within the borderline range

Assessment of Pre- and Pro-haptens Using Nonanimal Test Methods for Skin Sensitization

Because of ethical and regulatory reasons, several nonanimal test methods to assess the skin sensitization potential of chemicals have been developed and validated. In contrast to <i>in vivo</i> methods, they lack or provide limited metabolic capacity. For this reason, identification of pro-haptens but also pre-haptens, which require molecular transformations to gain peptide reactivity, is a challenge for these methods. In this study, 27 pre- and pro-haptens were tested using nonanimal test methods. Of these, 18 provided true positive results in the direct peptide reactivity assay (DPRA; sensitivity of 67%), although lacking structural alerts for direct peptide reactivity. The reaction mechanisms leading to peptide depletion in the DPRA were therefore elucidated using mass spectrometry. Hapten–peptide adducts were identified for 13 of the 18 chemicals indicating that these pre-haptens were activated and that peptide binding occurred. Positive results for five of the 18 chemicals can be explained by dipeptide formations or the oxidation of the sulfhydryl group of the peptide. Nine of the 27 chemicals were tested negative in the DPRA. Of these, four yielded true positive results in the keratinocyte and dendritic cell based assays. Likewise, 16 of the 18 chemicals tested positive in the DPRA were also positive in either one or both of the cell-based assays. A combination of DPRA, KeratinoSens, and h-CLAT used in a 2 out of 3 weight of evidence (WoE) approach identified 22 of the 27 pre- and pro-haptens correctly (sensitivity of 81%), exhibiting a similar sensitivity as for directly acting haptens. This analysis shows that the combination of <i>in chemico</i> and <i>in vitro</i> test methods is suitable to identify pre-haptens and the majority of pro-haptens

Towards a science-based testing strategy to identify maternal thyroid hormone imbalance and neurodevelopmental effects in the progeny—part III: how is substance-mediated thyroid hormone imbalance in pregnant/lactating rats or their progeny related to neurodevelopmental effects?

This review investigated which patterns of thyroid- and brain-related effects are seen in rats upon gestational/lactational exposure to 14 substances causing thyroid hormone imbalance by four different modes-of-action (inhibition of thyroid peroxidase, sodium-iodide symporter and deiodinase activities, enhancement of thyroid hormone clearance) or to dietary iodine deficiency. Brain-related parameters included motor activity, cognitive function, acoustic startle response, hearing function, periventricular heterotopia, electrophysiology and brain gene expression. Specific modes-of-action were not related to specific patterns of brain-related effects. Based upon the rat data reviewed, maternal serum thyroid hormone levels do not show a causal relationship with statistically significant neurodevelopmental effects. Offspring serum thyroxine together with offspring serum triiodothyronine and thyroid stimulating hormone appear relevant to predict the likelihood for neurodevelopmental effects. Based upon the collated database, thresholds of ≥60%/≥50% offspring serum thyroxine reduction and ≥20% and statistically significant offspring serum triiodothyronine reduction indicate an increased likelihood for statistically significant neurodevelopmental effects; accuracies: 83% and 67% when excluding electrophysiology (and gene expression). Measurements of brain thyroid hormone levels are likely relevant, too. The extent of substance-mediated thyroid hormone imbalance appears more important than substance mode-of-action to predict neurodevelopmental impairment in rats. Pertinent research needs were identified, e.g. to determine whether the phenomenological offspring thyroid hormone thresholds are relevant for regulatory toxicity testing. The insight from this review shall be used to suggest a tiered testing strategy to determine whether gestational/lactational substance exposure may elicit thyroid hormone imbalance and potentially also neurodevelopmental effects.</p