LuSens: a keratinocyte based ARE reporter gene assay for use in integrated testing strategies for skin sensitization hazard identification.

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.

International ring trial of the epidermal equivalent sensitizer potency assay: reproducibility and predictive-capacity.

This study describes the international ring trial of the epidermal-equivalent (EE) sensitizer potency assay. This assay does not distinguish a sensitizer from a non-sensitizer, but may classify known skin sensitizers according to their potency. It assesses the chemical concentration resulting in 50% cytotoxicity (EE-EC50) or the 2-fold increase in IL-1α (IL-1α2x). Four laboratories received 13 coded sensitizers. Reproducible results were obtained in each laboratory. A binary prediction model, EC50≥7 mg/ml=weak to moderate sensitizer and EC50<7 mg/ml=strong to extreme sensitizer had an accuracy of 77%. A superior EE (EC50 and IL-1α2x) correlation was observed with human in vivo DSA05 data compared to LLNA-EC3 data. Human in vivo NOEL and LLNA-EC3 data correlated to a similar extent to in vitro EE data. Our results indicate that this easily transferable EE potency assay is suitable for testing chemical allergens of unknown potencies and may now be ready for further validation, providing complementary potency information to other assays already undergoing validation for assessing skin sensitization potential.

Transfer of a two-tiered keratinocyte assay: IL-18 production by NCTC2544 to determine the skin sensitizing capacity and epidermal equivalent assay to determine sensitizer potency.

At present, the identification of potentially sensitizing chemicals is carried out using animal models. However, it is very important from ethical, safety and economic point of view to have biological markers to discriminate allergy and irritation events, and to be able to classify sensitizers according to their potency, without the use of animals. Within the Sens-it-iv EU Frame Programme 6 funded Integrated Project (LSHB-CT-2005-018681), a number of in vitro, human cell based assays were developed which, when optimized and used in an integrated testing strategy, may be able to distinguish sensitizers from non-sensitizers. This study describes two of these assays, which when used in a tiered strategy, may be able to identify contact sensitizers and also to quantify sensitizer potency. Tier 1 is the human keratinocyte NCTC2544 IL-18 assay and tier 2 is the Epidermal Equivalent potency assay. The aim of this study is to show the transferability of the two-tiered approach with training chemicals: 3 sensitizers (DNCB, resorcinol, pPD) and 1 non sensitizer (lactic acid) in tier 1 and 2 sensitizers with different potency in tier 2 (DNCB; extreme and resorcinol; moderate). The chemicals were tested in a non-coded fashion. Here we describe the transferability to naïve laboratories, the establishment of the standard operating procedure, critical points, acceptance criteria and project management. Both assays were successfully transferred to laboratories that had not performed the assays previously. The two tiered approach may offer an unique opportunity to provide an alternative method to the Local Lymph Node Assay (LLNA). These assays are both based on the use of human keratinocytes, which have been shown over the last two decades, to play a key role in all phases of skin sensitization.

Non-animal test methods for predicting skin sensitization potentials.

Contact allergies are complex diseases, and it is estimated that 15-20 % of the general population suffers from contact allergy, with increasing prevalence. Evaluation of the sensitization potential of a substance is usually carried out in animal models. Nowadays, there is much interest in reducing and ultimately replacing current animal tests. Furthermore, as of 2013, the EU has posed a ban on animal testing of cosmetic ingredients that includes skin sensitization. Therefore, predictive and robust in vitro tests are urgently needed. In order to establish alternatives to animal testing, the in vitro tests must mimic the very complex interactions between the sensitizing chemical and the different parts of the immune system. This review article summarizes recent efforts to develop in vitro tests for predicting skin sensitizers. Cell-based assays, in chemico methods and, to a lesser extent, in silico methods are presented together with a discussion of their current status. With considerable progress having been achieved during the last years, the rationale today is that data from different non-animal test methods will have to be combined in order to obtain reliable hazard and potency information on potential skin sensitizers.

Putting the parts together: combining in vitro methods to test for skin sensitizing potentials.

Allergic contact dermatitis is a common skin disease and is elicited by repeated skin contact with an allergen. In the regulatory context, currently only data from animal experiments are acceptable to assess the skin sensitizing potential of substances. Animal welfare and EU Cosmetic Directive/Regulation call for the implementation of animal-free alternatives for safety assessments. The mechanisms that trigger skin sensitization are complex and various steps are involved. Therefore, a single in vitro method may not be able to accurately assess this endpoint. Non-animal methods are being developed and validated and can be used for testing strategies that ensure a reliable prediction of skin sensitization potentials. In this study, the predictivities of four in vitro assays, one in chemico and one in silico method addressing three different steps in the development of skin sensitization were assessed using 54 test substances of known sensitizing potential. The predictivity of single tests and combinations of these assays were compared. These data were used to develop an in vitro testing scheme and prediction model for the detection of skin sensitizers based on protein reactivity, activation of the Keap-1/Nrf2 signaling pathway and dendritic cell activation.

The automated FADU-assay, a potential high-throughput in vitro method for early screening of DNA breakage.

Genotoxicity tests are essential to identify compounds that have a potential to compromise not only the environment but also human and animal health, including compounds that increase the risk of cancer. At present, no single test is capable of detecting all types of genotoxic effects; therefore a battery of in vitro and, if positive, in vivo tests is necessary to determine the genotoxicity of a substance. However, the respective specificities of current assays have been criticized for their high percentage of false positive results. We recently published an automated version of the "Fluorimetric detection of Alkaline DNA Unwinding" (FADU) assay for measuring DNA strand breaks in human peripheral blood mononuclear cells or in cell lines. Using this new technology we show detection of DNA strand breaks in cells treated with several compounds known to induce DNA strand breaks by various mechanisms. We also tested toxic compounds that were not expected to induce DNA strand breaks; these were negative in the assay as expected. Finally, we included zinc oxide nanoparticles of high production volume to explore further fields of potential FADU applications. The main advantages of this assay are high reproducibility, easy handling, lack of operator bias, high-throughput, speed, and low cost.

Imidazoquinolinone, imidazopyridine, and isoquinolindione derivatives as novel and potent inhibitors of the poly(ADP-ribose) polymerase (PARP): a comparison with standard PARP inhibitors.

We have identified three novel structures for inhibitors of the poly(ADP-ribose) polymerase (PARP), a nuclear enzyme activated by strand breaks in DNA and implicated in DNA repair, apoptosis, organ dysfunction or necrosis. 2-[4-(5-Methyl-1H-imidazol-4-yl)-piperidin-1-yl]-4,5-dihydro-imidazo[4,5,1-i,j]quinolin-6-one (BYK49187), 2-(4-pyridin-2-yl-phenyl)-4,5-dihydro-imidazo[4,5,1-i,j]quinolin-6-one (BYK236864), 6-chloro-8-hydroxy-2,3-dimethyl-imidazo-[1,2-alpha]-pyridine (BYK20370), and 4-(1-methyl-1H-pyrrol-2-ylmethylene)-4H-isoquinolin-1,3-dione (BYK204165) inhibited cell-free recombinant human PARP-1 with pIC(50) values of 8.36, 7.81, 6.40, and 7.35 (pK(i) 7.97, 7.43, 5.90, and 7.05), and murine PARP-2 with pIC(50) values of 7.50, 7.55, 5.71, and 5.38, respectively. BYK49187, BYK236864, and BYK20370 displayed no selectivity for PARP-1/2, whereas BYK204165 displayed 100-fold selectivity for PARP-1. The IC(50) values for inhibition of poly(ADP-ribose) synthesis in human lung epithelial A549 and cervical carcinoma C4I cells as well in rat cardiac myoblast H9c2 cells after PARP activation by H(2)O(2) were highly significantly correlated with those at cell-free PARP-1 (r(2) = 0.89-0.96, P < 0.001) but less with those at PARP-2 (r(2) = 0.78-0.84, P < 0.01). The infarct size caused by coronary artery occlusion and reperfusion in the anesthetized rat was reduced by 22% (P < 0.05) by treatment with BYK49187 (3 mg/kg i.v. bolus and 3 mg/kg/h i.v. during 2-h reperfusion), whereas the weaker PARP inhibitors, BYK236864 and BYK20370, were not cardioprotective. In conclusion, the imidazoquinolinone BYK49187 is a potent inhibitor of human PARP-1 activity in cell-free and cellular assays in vitro and reduces myocardial infarct size in vivo. The isoquinolindione BYK204165 was found to be 100-fold more selective for PARP-1. Thus, both compounds might be novel and valuable tools for investigating PARP-1-mediated effects.