Regarding the references for reference chemicals of alternative methods.

The selection of reference and proficiency chemicals is an important basis for 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.

Peptide reactivity associated with skin sensitization: The QSAR Toolbox and TIMES compared to the DPRA.

The molecular initiating event (MIE) of skin sensitization is the binding of a hapten to dermal proteins. This can be assessed using the in chemico direct peptide reactivity assay (DPRA) or in silico tools such as the QSAR Toolbox and TIMES SS. In this study, the suitability of these methods was analyzed by comparing their results to in vivo sensitization data of LLNA and human studies. Compared to human data, 84% of non-sensitizers and sensitizers yielded consistent results in the DPRA. In silico tools resulted in 'no alert' for 83%-100% of the non-sensitizers, but alerted only 55%-61% of the sensitizers. The inclusion of biotic and abiotic transformation simulations yielded more alerts for sensitizers, but simultaneously dropped the number of non-alerted non-sensitizers. In contrast to the DPRA, in silico tools were more consistent with results of the LLNA than human data. Interestingly, the new "DPRA profilers" (QSAR Toolbox) provided unsatisfactory results. Additionally, the results were combined in the '2 out of 3' prediction model with in vitro data derived from LuSens and h-CLAT. Using DPRA results, the model identified 90% of human sensitizers and non-sensitizers; using in silico results (including abiotic and biotic activations) instead of DPRA results led to a comparable high predictivity.

Prevalidation of the ex-vivo model PCLS for prediction of respiratory toxicity.

In acute inhalation toxicity studies, animals inhale substances at given concentrations. Without additional information, however, appropriate starting concentrations for in-vivo inhalation studies are difficult to estimate. The goal of this project was the prevalidation of precision-cut lung slices (PCLS) as an ex-vivo alternative to reduce the number of animals used in inhalation toxicity studies. According to internationally agreed principles for Prevalidation Studies, the project was conducted in three independent laboratories. The German BfR provided consultancy in validation principles and independent support with biostatistics. In all laboratories, rat PCLS were prepared and exposed to 5 concentrations of 20 industrial chemicals under submerged culture conditions for 1h. After 23 h post-incubation, toxicity was assessed by measurement of released lactate dehydrogenase and mitochondrial activity. In addition, protein content and pro-inflammatory cytokine IL-1α were measured. For all endpoints IC50 values were calculated if feasible. For each endpoint test acceptance criteria were established. This report provides the final results for all 20 chemicals. More than 900 concentration-response curves were analyzed. Log10[IC50 (µM)], obtained for all assay endpoints, showed best intra- and inter-laboratory consistency for the data obtained by WST-1 and BCA assays. While WST-1 and LDH indicated toxic effects for the majority of substances, only some of the substances induced an increase in extracellular IL-1α. Two prediction models (two-group classification model, prediction of LC50 by IC50) were developed and showed promising results.

Linking energy metabolism to dysfunctions in mitochondrial respiration--a metabolomics in vitro approach.

The study presented here describes the application of metabolite profiling of highly polar, intracellular metabolites after incubation of a mammalian fibroblast cell line with inhibitors of mitochondrial function. A metabolomics approach was used to assess the complex response of the cellular energy metabolism. Metabolic profiles of phosphorylated and carboxylated intracellular metabolites were assessed by UPLC-MS/MS and used to predict the mode of mitochondrial toxicity. Based on distinct metabolic patterns, multivariate data analysis allowed for the discrimination of two groups of toxins: inhibitors of the electron transport in mitochondrial membranes (complex IV inhibitors) and uncouplers of oxidative phosphorylation. Beyond these known interferences, metabolic profiling was able to reveal additional inhibitory effects on the cellular energy metabolism. Most prominently, for three of the toxins, metabolic patterns also disclosed an enhanced activity of the glycerol phosphate shuttle inferring the inhibition of NADH dehydrogenase at complex I. Secondly, inhibition of the electron transport was accompanied by a limiting availability of citric acid cycle intermediates and aspartate. Concomitantly, specific perturbations of the purine nucleotide cycle were observed. We have shown here that metabolomic approaches may assist to predict complex modes of action of toxic compounds on cellular level as well as to unravel specific dysfunctions in the energy metabolism.

In house validation of recombinant yeast estrogen and androgen receptor agonist and antagonist screening assays.

Besides other modes of action, endocrine disruptors may interact with hormone receptors thereby modifying the physiological function of endogenous hormones. In the present study, we report the results obtained with yeast based assays to detect the (anti-)estrogenic potential (YES) and the (anti-)androgenic potential (YAS) of 105 substances. The results show very high reproducibility and good concordance with literature data of in vivo and/or in vitro studies: the overall true positive rate, true negative rate and accuracy of the assays were 78%, 95%, and 87% (estrogen agonism), and 70%, 97%, and 90% (estrogen antagonism), 88%, 96%, and 95% (androgen agonism) and 81%, 88%, and 85% (androgen antagonism). Furthermore, the performance of the YES assay has been compared to the HeLa based transcriptional activation assay using 20 compounds. The overall true positive rate, true negative rate, and accuracy obtained for the 20 compounds were 100%, 88%, and 95% (mammalian cell based HeLa assay) and 92%, 86%, and 90% (yeast based YES assay). Taken together, the YES and YAS are robust systems, easy to handle and satisfying the requirements for screening systems that can be applied in programs including the US Environmental Protection Agency's Endocrine Disruptor Screening Program.