Reconstructed human epidermis-based testing strategy of skin sensitization potential and potency classification using epidermal sensitization assay and in silico data.

The hazards and potency of skin sensitizers are traditionally determined using animal tests such as the local lymph node assay (LLNA); however, significant progress has been made in the development of non-animal test methods addressing the first three mechanistic key events of adverse outcome pathway in skin sensitization. We developed the epidermal sensitization assay (EpiSensA), which is a reconstructed human epidermis-based assay, by measuring four genes related to critical keratinocyte responses during skin sensitization. Four in vitro skin sensitization test methods (EpiSensA, direct peptide reactivity assay [DPRA], KeratinoSens™, and human cell line activation test [h-CLAT]) were systematically evaluated using 136 chemicals including lipophilic chemicals and pre/pro-haptens, which may be related to assay-specific limitations. The constructed database included existing and newly generated data. The EpiSensA showed a broader applicability domain and predicted the hazards with 82.4% and 78.8% accuracy than LLNA and human data. The EpiSensA could detect 76 out of 88 sensitizers at lower concentrations than the LLNA, indicating that the EpiSensA has higher sensitivity for the detection of minor sensitizing constituents. These results confirmed the potential use of the EpiSensA in evaluating a mixture of unknown compositions that can be evaluated by animal tests. To combine different information sources, the reconstructed human epidermis-based testing strategy (RTS) was developed based on weighted multiple information from the EpiSensA and TImes MEtabolism Simulator platform for predicting Skin Sensitization (TIMES-SS; RTSv1) or Organization for Economic Cooperation and Development (OECD) QSAR Toolbox automated workflow (RTSv2). The predictivities of the hazards and Globally Harmonized System (GHS) subcategories were equal to or better than the defined approaches (2 out of 3, integrated testing strategy [ITS]v1, and ITSv2) adopted as OECD Guideline 497.

The inter-laboratory validation study of EpiSensA for predicting skin sensitization potential.

The Epidermal Sensitization Assay (EpiSensA) is a reconstructed human epidermis (RhE)-based gene expression assay for predicting the skin sensitization potential of chemicals. Since the RhE model is covered by a stratified stratum corneum, various kinds of test chemicals, including lipophilic ones and pre-/pro-haptens, can be tested with a route of exposure akin to an in vivo assay and human exposure. This article presents the results of a formally managed validation study of the EpiSensA that was carried out by three participating laboratories. The purpose of this validation study was to assess transferability of the EpiSensA to new laboratories along with its within- (WLR) and between-laboratory reproducibility (BLR). The validation study was organized into two independent stages. As demonstrated during the first stage, where three sensitizers and one non-sensitizer were correctly predicted by all participating laboratories, the EpiSensA was successfully transferred to all three participating laboratories. For Phase I of the second stage, each participating laboratory performed three experiments with an identical set of 15 coded test chemicals resulting in WLR of 93.3%, 93.3%, and 86.7%, respectively. Furthermore, when the results from the 15 test chemicals were combined with those of the additional 12 chemicals tested in Phase II of the second stage, the BLR for 27 test chemicals was 88.9%. Moreover, the predictive capacity among the three laboratories showed 92.6% sensitivity, 63.0% specificity, 82.7% accuracy, and 77.8% balanced accuracy based on murine local lymph node assay (LLNA) results. Overall, this validation study concluded that EpiSensA is easily transferable and sufficiently robust for assessing the skin sensitization potential of chemicals.

Characterization of dermal sensitization potential for industrial or agricultural chemicals with EpiSensA.

The regulatory community is transitioning to the use of nonanimal methods for dermal sensitization assessments; however, some in vitro assays have limitations in their domain of applicability depending on the properties of chemicals being tested. This study explored the utility of epidermal sensitization assay (EpiSensA) to evaluate the sensitization potential of complex and/or "difficult to test" chemicals. Assay performance was evaluated by testing a set of 20 test chemicals including 10 methacrylate esters, 5 silicone-based compounds, 3 crop protection formulations, and 2 surfactant mixtures; each had prior in vivo data plus some in silico and in vitro data. Using the weight of evidence (WoE) assessments by REACH Lead Registrants, 14 of these chemicals were sensitizers and, six were nonsensitizers based on in vivo studies (local lymph node assay [LLNA] and/or guinea pig studies). The EpiSensA correctly predicted 16/20 materials with three test materials as false positive and one silane as false negative. This silane, classified as weak sensitizer via LLNA, also gave a "false negative" result in the KeratinoSens™ assay. Overall, consistent with prior evaluations, the EpiSensA demonstrated an accuracy level of 80% relative to available in vivo WoE assessments. In addition, potency classification based on the concentration showing positive marker gene expression of EpiSensA was performed. The EpiSensA correctly predicted the potency for all seven sensitizing methacrylates classified as weak potency via LLNA (EC3 ≥ 10%). In summary, EpiSensA could identify dermal sensitization potential of these test substances and mixtures, and continues to show promise as an in vitro alternative method for dermal sensitization.

Transferability and within- and between-laboratory reproducibilities of EpiSensA for predicting skin sensitization potential in vitro: A ring study in three laboratories.

The epidermal sensitization assay (EpiSensA) is an in vitro skin sensitization test method based on gene expression of four markers related to the induction of skin sensitization; the assay uses commercially available reconstructed human epidermis. EpiSensA has exhibited an accuracy of 90% for 72 chemicals, including lipophilic chemicals and pre-/pro-haptens, when compared with the results of the murine local lymph node assay. In this work, a ring study was performed by one lead and two naive laboratories to evaluate the transferability, as well as within- and between-laboratory reproducibilities, of EpiSensA. Three non-coded chemicals (two lipophilic sensitizers and one non-sensitizer) were tested for the assessment of transferability and 10 coded chemicals (seven sensitizers and three non-sensitizers, including four lipophilic chemicals) were tested for the assessment of reproducibility. In the transferability phase, the non-coded chemicals (two sensitizers and one non-sensitizer) were correctly classified at the two naive laboratories, indicating that the EpiSensA protocol was transferred successfully. For the within-laboratory reproducibility, the data generated with three coded chemicals tested in three independent experiments in each laboratory gave consistent predictions within laboratories. For the between-laboratory reproducibility, 9 of the 10 coded chemicals tested once in each laboratory provided consistent predictions among the three laboratories. These results suggested that EpiSensA has good transferability, as well as within- and between-laboratory reproducibility.

Binary test battery with KeratinoSens™ and h-CLAT as part of a bottom-up approach for skin sensitization hazard prediction.

Skin sensitization is one of the key safety endpoints for chemicals applied directly to the skin. Several integrated testing strategies (ITS) using multiple non-animal test methods have been developed to accurately evaluate the sensitizing potential of chemicals, but there is no regulatory-accepted ITS to classify a chemical as a non-sensitizer. In this study, the predictive performance of a binary test battery with KeratinoSens™ and h-CLAT compared to the local lymph node assay (LLNA) and human data was examined using comprehensive dataset of 203 chemicals. When two negative results indicate a non-sensitizer, the binary test battery provided sensitivity of 93.4% or 94.4% compared with the LLNA or human data. Taking into account the predictive limitations (i.e. high log Kow, pre-/pro-haptens and acyl transfer agents (or amine-reactive)), the binary test battery had extremely high sensitivity comparable to that of the 3 out of 3 ITS where three negative results of the DPRA, KeratinoSens™ and h-CLAT indicate a non-sensitizer. Therefore, the data from KeratinoSens™ or h-CLAT may provide partly redundant information on the molecular initiating event derived from DPRA. Taken together, the binary test battery of KeratinoSens™ and h-CLAT could be used as part of a bottom-up approach for skin sensitization hazard prediction.

Proof of concept testing of a positive reference material for in vivo and in vitro sensitization testing of medical devices.

In vivo skin sensitization tests are required to evaluate the biological safety of medical devices in contact with living organisms to provide safe medical care to patients. Negative and positive reference materials have been developed for biological tests of cytotoxicity, implantation, hemolysis, and in vitro skin irritation. However, skin sensitization tests are lacking. In this study, polyurethane sheets containing 1 wt/wt % 2,4-dinitrochlorobenzene (DNCB-PU) were developed and evaluated as a positive reference material for skin sensitization tests. DNCB-PU sheet extracts prepared with sesame oil elicited positive sensitization responses for in vivo sensitization potential in the guinea pig maximization test and the local lymph node assay. Furthermore, DNCB-PU sheet extracts prepared with water and acetonitrile, 10% fetal bovine serum-containing medium, or sesame oil elicited positive sensitization responses as alternatives to animal testing based on the amino acid derivative reactivity assay, human cell line activation test, and epidermal sensitization assay, respectively. These data suggest that the DNCB-PU sheet is an effective extractable positive reference material for in vivo and in vitro skin sensitization testing in medical devices. The formulation of this reference material will lead to the development of safer medical devices that contribute to patient safety.

Heparin-conjugated collagen as a potent growth factor-localizing and stabilizing scaffold for regenerative medicine.

INTRODUCTION: Growth factors are crucial bioactive molecules in vitro and in vivo. Among them, basic fibroblast growth factor (bFGF) has been used widely for various applications such as cell culture and regenerative medicine. However, bFGF has extremely poor stability in aqueous solution; thus, it is difficult to maintain its high local concentration. Heparin-conjugated materials have been studied recently as promising scaffold-immobilizing growth factors for biological and medical applications. The previous studies have focused on the local concentration maintenance and sustained release of the growth factors from the scaffold. METHODS: In this paper, we focused on the biological stability of bFGF immobilized on the heparin-conjugated collagen (hep-col) scaffold. The stability of the immobilized bFGF was quantitatively evaluated at physiological temperature (37 °C) using cell culture and ELISA. RESULTS: The immobilized bFGF had twice higher stability than the bFGF solution. Furthermore, the hep-col scaffold was able to immobilize not only bFGF but also other growth factors (i.e., vascular endothelial growth factor and hepatocyte growth factor) at high efficiency. CONCLUSIONS: The hep-col scaffold can localize several kinds of growth factors as well as stabilize bFGF under physiological temperature and is a promising potent scaffold for regenerative medicine.

Adjustment of a no expected sensitization induction level derived from Bayesian network integrated testing strategy for skin sensitization risk assessment.

Skin sensitization is a key adverse effect to be addressed during hazard identification and risk assessment of chemicals, because it is the first step in the development of allergic contact dermatitis. Multiple non-animal testing strategies incorporating in vitro tests and in silico tools have achieved good predictivities when compared with murine local lymph node assay (LLNA). The binary test battery of KeratinoSensTM and h-CLAT could be used to classify non-sensitizers as the first part of bottom-up approach. However, the quantitative risk assessment for sensitizing chemicals requires a No Expected Sensitization Induction Level (NESIL), the dose not expected to induce skin sensitization in humans. We used Bayesian network integrated testing strategy (BN ITS-3) for chemical potency classification. BN ITS-3 predictions were performed without a pre-processing step (selecting data from their physic-chemical applicability domains) or post-processing step (Michael acceptor chemistry correction), neither of which necessarily improve prediction accuracy. For chemicals within newly defined applicability domain, all under-predictions fell within one potency class when compared with LLNA results, indicating no chemicals that were incorrectly classified by more than one class. Considering the potential under-prediction by one class, a worst case value to each class from BN ITS-3 was used to derive a NESIL. When in vivo and human data from suitable analogs cannot be used to estimate the uncertainty, adjusting the NESIL derived from BN ITS-3 may help perform skin sensitization risk assessment. The overall workflow for risk assessment was demonstrated by incorporating the binary test battery of KeratinoSensTM and h-CLAT.

Sensitivity of KeratinoSensTM and h-CLAT for detecting minute amounts of sensitizers to evaluate botanical extract.

Cosmetic ingredients are often complex mixtures from natural sources such as botanical extracts that might contain minute amounts of constituents with sensitizing potential. The sensitivity of in vitro skin sensitization test methods such as KeratinoSensTM and h-CLAT for the detection of minute amounts of sensitizer in mixtures remains unclear. In this study, we assessed the detection sensitivity of the binary test battery comprising KeratinoSensTM and h-CLAT for minute amounts of sensitizers by comparing the LLNA EC3 (estimated concentration of a substance expected to produce a stimulation index of 3) values to the minimum detection concentrations (MDCs) exceeding the positive criteria for each of the two in vitro test methods. 146 sensitizers with both sets of in vitro data and LLNA data were used. MDC values for KeratinoSensTM and h-CLAT were calculated from exposure concentrations exceeding positive criteria for each in vitro test method (EC1.5 and minimum induction thresholds, respectively). The dilution rate used to expose culture medium was also considered. For 86% of analyzed sensitizers, the in vitro test methods showed MDC values lower than LLNA EC3 values, suggesting that the binary test battery with KeratinoSensTM and h-CLAT have greater sensitivity for detection of minute amounts of sensitizer than LLNA. These results suggest the high applicability of KeratinoSensTM and h-CLAT for detecting skin sensitizing constituents present in botanical extract.

The dermal sensitization threshold (DST) approach for mixtures evaluated as negative in in vitro test methods; mixture DST.

Cosmetic ingredients often comprise complex mixtures, such as botanical extracts, which may contain skin sensitizing constituents. In our previous study for the sensitivity of the evaluations of skin sensitizing constituents in mixtures using the binary in vitro test battery with KeratinoSensTM and h-CLAT, some sensitizers showed higher detection limits in in vitro test methods than in murine local lymph node assays (LLNA). Thus, to minimize the uncertainty associated with decreased sensitivity for these sensitizers, a risk assessment strategy was developed for mixtures with negative results from the binary test battery. Assuming that the no expected sensitization induction level of mixtures (mixture NESIL) can be derived for mixtures with negative in vitro test results, we assessed 146 sensitizers with in vitro and LLNA data according to the assumption of indeterminate constituents in mixtures. Finally, we calculated 95th percentile probabilities of mixture NESILs and derived dermal sensitization thresholds for mixtures (mixture DST) with negative in vitro test results of 6010 µg/cm2. Feasibility studies indicated that this approach was practical for risk assessments of products in the cosmetic industry. This approach would be a novel risk assessment strategy for incorporating the DST approach and information from in vitro test methods.

Development of an in situ evaluation system for neural cells using extracellular matrix-modeled gel culture.

Two-dimensional monolayer culture is the most popular cell culture method. However, the cells may not respond as they do in vivo because the culture conditions are different from in vivo conditions. However, hydrogel-embedding culture, which cultures cells in a biocompatible culture substrate, can produce in vivo-like cell responses, but in situ evaluation of cells in a gel is difficult. In this study, we realized an in vivo-like environment in vitro to produce cell responses similar to those in vivo and established an in situ evaluation system for hydrogel-embedded cell responses. The extracellular matrix (ECM)-modeled gel consisted of collagen and heparin (Hep-col) to mimic an in vivo-like environment. The Hep-col gel could immobilize growth factors, which is important for ECM functions. Neural stem/progenitor cells cultured in the Hep-col gel grew and differentiated more actively than in collagen, indicating an in vivo-like environment in the Hep-col gel. Second, a thin-layered gel culture system was developed to realize in situ evaluation of the gel-embedded cells. Cells in a 200-µm-thick gel could be evaluated clearly by a phase-contrast microscope and immunofluorescence staining through reduced optical and diffusional effects. Finally, we found that the neural cells cultured in this system had synaptic connections and neuronal action potentials by immunofluorescence staining and Ca2+ imaging. In conclusion, this culture method may be a valuable evaluation system for neurotoxicity testing.