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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., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

While the skin sensitization hazard of substances can already be identified using non-animal methods, the classification of potency sub-categories GHS-1A and 1B is still challenging. Potency can be measured by the dose at which an effect is observed; since the protein-adduct formation is determining the dose of the allergen in the skin, peptide reactivity was used to assess the potency. The Direct Peptide Reactivity Assay (DPRA; one concentration and reaction-time) did not sufficiently discriminate between sub-categories 1A and 1B (56% accuracy compared to LLNA data, n=124). An extended protocol termed 'quantitative DPRA' (three concentrations and one reaction-time), discriminated sub-categories GHS 1A and 1B with an accuracy of 81% or 57% compared to LLNA (n=36) or human (n=14) data, respectively. The analysis of the Cys-adducts was already sufficient; additional analysis of Lys-adducts did not improve the predictivity. An additional modification, the 'kinetic DPRA' (several concentrations and reaction-times) was used to approximate the rate constant of Cys-peptide-adduct formation. 35 of 38 substances were correctly assigned to the potency sub-categories (LLNA data), and the predictivity for 14 human data was equally high. These results warrant the kinetic DPRA for further validation in order to fully replace in vivo testing for assessing skin sensitization including potency sub-classification., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Allergic contact dermatitis can develop following repeated exposure to allergenic substances. To date, hazard identification is still based on animal studies as non-animal alternatives have not yet gained global regulatory acceptance. Several non-animal methods addressing key-steps of the adverse outcome pathway (OECD, 2012) will most likely be needed to fully address this effect. Among the initial cellular events is the activation of keratinocytes and currently only one method, the KeratinoSens™, has been formally validated to address this event. In this study, a further method, the LuSens assay, that uses a human keratinocyte cell line harbouring a reporter gene construct composed of the antioxidant response element (ARE) of the rat NADPH:quinone oxidoreductase 1 gene and the luciferase gene. The assay was validated in house using a selection of 74 substances which included the LLNA performance standards. The predictivity of the LuSens assay for skin sensitization hazard identification was comparable to other non-animal methods, in particular to the KeratinoSens™. When used as part of a testing battery based on the OECD adverse outcome pathway for skin sensitization, a combination of the LuSens assay, the DPRA and a dendritic cell line activation test attained predictivities similar to that of the LLNA., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The usefulness of in vitro systems to predict acute inhalation toxicity was investigated. Nineteen substances were tested in three-dimensional human airway epithelial models, EpiAirway™ and MucilAir™, and in A549 and 3T3 monolayer cell cultures. IC(50) values were compared to rat four-hour LC(50) values classified according to EPA and GHS hazard categories. Best results were achieved with a prediction model distinguishing toxic from non-toxic substances, with satisfactory specificities and sensitivities. Using a self-made four-level prediction model to classify substances into four in vitro hazard categories, in vivo-in vitro concordance was mediocre, but could be improved by excluding substances causing pulmonary edema and emphysema in vivo. None of the test systems was outstanding, and there was no evidence that tissue or monolayer systems using respiratory tract cells provide an added value. However, the test systems only reflected bronchiole epithelia and alveolar cells and investigated cytotoxicity. Effects occurring in other cells by other mechanisms could not be recognised. Further work should optimise test protocols and expand the set of substances tested to define applicability domains. In vivo respiratory toxicity data for in vitro comparisons should distinguish different modes of action, and their relevance for human health effects should be ensured., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Allergic contact dermatitis is induced by repeated skin contact with an allergen. Assessment of the skin sensitizing potential of chemicals, agrochemicals, and especially cosmetic ingredients is currently performed with the use of animals. Animal welfare and EU legislation demand animal-free alternatives reflected in a testing and marketing ban for cosmetic ingredients beginning in 2013. The underlying mechanisms of induction and elicitation of skin sensitization are complex and a chemical needs to comply several properties being skin sensitizing. To account for the multitude of events in the induction of skin sensitization an in vitro test system will consist of a battery of various tests. Currently, we performed intralaboratory validations of four assays addressing three different events during induction of skin sensitization. (1) The Direct Peptide Reactivity Assay (DPRA) according to Gerberick and co-workers (Gerberick et al., 2004) using synthetic peptides and HPLC analysis. (2) Two dendritic cell activation assays based on the dendritic cell like cell lines U-937 and THP-1 and flow cytometric detection of the maturation markers CD54 and/or CD86 (Ashikaga et al., 2006; Python et al., 2007; Sakaguchi et al., 2006). (3) Antioxidant response element (ARE)-dependent gene activity in a HaCaT reporter gene cell line (Emter et al., 2010). We present the results of our intralaboratory validation of these assays with 23 substances of known sensitizing potential. The sensitivity, specificity, and accuracy of the individual tests were obtained by comparison to human epidemiological data as well as to data from animal tests such as the local lymph node assay., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2010 Elsevier Ltd. All rights reserved.)