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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.

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.

In order to assess whether the placental metabolism of xenobiotic compounds should be taken into consideration for physiologically-based toxicokinetic (PBTK) modelling, the activities of seven phase I and phase II enzymes have been quantified in the 18-day placenta of untreated Wistar rats. To determine their relative contribution, these activities were compared to those of untreated adult male rat liver, using commonly accepted assays. The enzymes comprised cytochrome P450 (CYP), flavin-containing monooxygenase (FMO), alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), esterase, UDP-glucuronosyltransferase (UGT), and glutathione S-transferase (GST). In contrast to liver, no activities were measurable for 7-ethylresorufin-O-dealkylase (CYP1A), 7-pentylresorufin-O-dealkylase (CYP2B), 7-benzylresorufin-O-dealkylase (CYP2B, 2C and 3 A), UGT1, UGT2 and GST in placenta, indicating that the placental activity of these enzymes was well below their hepatic activity. Low activities in placenta were determined for FMO (4%), and esterase (8%), whereas the activity of placental ADH and ALDH accounted for 35% and 40% of the hepatic activities, respectively. In support of the negligible placental CYP activity, testosterone and six model azole fungicides, which were readily metabolized by rat hepatic microsomes, failed to exhibit any metabolic turnover with rat placental microsomes. Hence, with the possible exception of ADH and ALDH, the activities of xenobiotic-metabolizing enzymes in rat placenta are too low to warrant consideration in PBTK modelling.

Several non-animal methods are now available to address the key events leading to skin sensitization as defined by the adverse outcome pathway. The KeratinoSens™ assay addresses the cellular event of keratinocyte activation and is a method accepted under OECD TG 442D. In this study, the results of an inter-laboratory evaluation of the “me-too” LuSens assay, a bioassay that uses a human keratinocyte cell line harboring a reporter gene construct composed of the rat antioxidant response element (ARE) of the NADPH:quinone oxidoreductase 1 gene and the luciferase gene, are described. Earlier in-house validation with 74 substances showed an accuracy of 82% in comparison to human data. When used in a battery of non-animal methods, even higher predictivity is achieved. To meet European validation criteria, a multicenter study was conducted in 5 laboratories. The study was divided into two phases, to assess 1) transferability of the method, and 2) reproducibility and accuracy. Phase I was performed by testing 8 non-coded test substances; the results showed a good transferability to naïve laboratories even without on-site training. Phase II was performed with 20 coded test substances (performance standards recommended by OECD, 2015). In this phase, the intra- and inter-laboratory reproducibility as well as accuracy of the method was evaluated. The data demonstrate a remarkable reproducibility of 100% and an accuracy of over 80% in identifying skin sensitizers, indicating a good concordance with in vivo data. These results demonstrate good transferability, reliability and accuracy of the method thereby achieving the standards necessary for use in a regulatory setting to detect skin sensitizers.

Skin absorption testing in vitro is a regulatory accepted alternative method (OECD Guideline 428). Different tests can be applied to evaluate the integrity of the skin samples. Here, we compared the pre- or post-run integrity tests (transepidermal electrical resistance, TEER; transepidermal water loss, TEWL; absorption of the reference compounds water, TWF, or methylene blue, BLUE) and additionally focused on co-absorption of a (3)H-labeled internal reference standard (ISTD) as integrity parameter. The results were correlated to absorption profiles of various test compounds. Limit values of 2kΩ, 10 gm(-2)h(-1) and 4.5∗10(-3)cmh(-1) for the standard methods TEER, TEWL and TWF, respectively, allowed distinguishing between impaired and intact human skin samples in general. Single skin samples did, however, not, poorly and even inversely correlate with the test-compound absorption. In contrast, results with ISTD (e.g. (3)H-testosterone) were highly correlated to the absorption of (14)C-labeled test compounds. Importantly, ISTD did not influence analytics or absorption of test compounds. Therefore, ISTD, especially when adjusted to the physico-chemical properties of test compounds, is a promising concept to assess the integrity of skin samples during the whole course of absorption experiments. However, a historical control dataset is yet necessary for a potential routine application.

The knowledge-based search engine Go3R, www.Go3R.org, has been developed to assist scientists from industry and regulatory authorities in collecting comprehensive toxicological information with a special focus on identifying available alternatives to animal testing. The semantic search paradigm of Go3R makes use of expert knowledge on 3Rs methods and regulatory toxicology, laid down in the ontology, a network of concepts, terms, and synonyms, to recognize the contents of documents. Search results are automatically sorted into a dynamic table of contents presented alongside the list of documents retrieved. This table of contents allows the user to quickly filter the set of documents by topics of interest. Documents containing hazard information are automatically assigned to a user interface following the endpoint-specific IUCLID5 categorization scheme required, e.g. for REACH registration dossiers. For this purpose, complex endpoint-specific search queries were compiled and integrated into the search engine (based upon a gold standard of 310 references that had been assigned manually to the different endpoint categories). Go3R sorts 87% of the references concordantly into the respective IUCLID5 categories. Currently, Go3R searches in the 22 million documents available in the PubMed and TOXNET databases. However, it can be customized to search in other databases including in-house databanks.

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.

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.

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.