Application of Spectro-DPRA,KeratinoSens™ and h-CLAT to estimation of the skin sensitization potential of cosmetics ingredients

Ethical issues in animal toxicity testing have led to the search for alternative methods to determine the skin sensitization potential of cosmetic products. The emergence of ethical testing issues has led to the development of many alternative methods that can reliably estimate skin sensitization potentials. However, a single alternative method may not be able to achieve high predictivity due to the complexity of the skin sensitization mechanism. Therefore, several prediction assays, including both in chemico and in vitro test methods, were investigated and integrated based on the skin sensitization adverse outcome pathway. In this study, we evaluated three different integrated approaches to predict a human skin sensitization hazard using data from in vitro assays (KeratinoSens™ and human cell line activation test [h-CLAT]), and a newly developed in chemico assay (spectrophotometric direct peptide reactivity assay [Spectro-DPRA]). When the results of the in chemico and in vitro assays were combined, the predictivity of human data increased compared with that of a single assay. The highest predictivity was obtained for the approach in which sensitization potential was determined by Spectro-DPRA followed by final determination using the result of KeratinoSens™ and h-CLAT assays (96.3% sensitivity, 87.1% specificity, 86.7% positive predictive value, 96.4% negative predictive value and 91.4% accuracy compared with human data). While further optimization is needed, we believe this integrated approach may provide useful predictive data when determining the human skin sensitization potential of chemicals.     

A predictive mouse ear-swelling model for investigating topical photoallergy

The photoallergic potential of various compounds was assessed using a mouse ear-swelling model, which offers the advantage of being quantifiable and more objective than models based on subjective evaluation of erythematous skin reactions. Cyclophosphamide pretreated BALB/c mice were induced by topical treatment of the dorsal skin surface on 3 consecutive days and challenged on the ears 5 days after the last induction. For each induction and challenge treatment, mice were consecutively irradiated with ultraviolet (UV) A (10 J/cm2) and UVB (45 mJ/cm2) radiation 30 min to 1 hr after test material application. The photoallergic response to musk ambrette, a known human photoallergen, was significantly augmented when three consecutive induction exposures were used as compared with one or two inductions. The photoallergic potential of nine other known human photoallergens (tetrachlorosalicylanilide, bithionol, 6-methylcoumarin, chlorpromazine, sodium omadine, bisphenol A, sulphanilamide, fentichlor and p-aminobenzoic acid) was successfully detected using the mouse model. In each experiment, the ear thickness changes observed in the photoallergy test mice were significantly greater than the changes observed in the contact allergy, vehicle/radiation and phototoxicity control mice. Coumarin and homosalate, two agents not traditionally associated with causing photoallergy in humans or animals, did not demonstrate contact photoallergy using this model. With three of the photoallergens, musk ambrette, bithionol and tetrachlorosalicylanilide, the ear swelling response obtained was due to photoallergy alone and not due to the co-existence of both contact photoallergy and contact allergy. In addition, irradiating mice 24 hr, rather than 1 hr, following application of the test material during the induction phase resulted in a significantly reduced photoallergic response with both musk ambrette and tetrachlorosalicylanilide. These results indicate that the mouse ear-swelling model is a potentially useful model for investigative and predictive photoallergy testing.     

New approach to predict photoallergic potentials of chemicals based on murine local lymph node assay

Photoallergic dermatitis, caused by pharmaceuticals and other consumer products, is a very important issue in human health. However, S10 guidelines of the International Conference on Harmonization do not recommend the existing prediction methods for photoallergy because of their low predictability in human cases. We applied local lymph node assay (LLNA), a reliable, quantitative skin sensitization prediction test, to develop a new photoallergy prediction method. This method involves a three‐step approach: (1) ultraviolet (UV) absorption analysis; (2) determination of no observed adverse effect level for skin phototoxicity based on LLNA; and (3) photoallergy evaluation based on LLNA. Photoallergic potential of chemicals was evaluated by comparing lymph node cell proliferation among groups treated with chemicals with minimal effect levels of skin sensitization and skin phototoxicity under UV irradiation (UV+) or non‐UV irradiation (UV?). A case showing significant difference (P < .05) in lymph node cell proliferation rates between UV? and UV+ groups was considered positive for photoallergic reaction. After testing 13 chemicals, seven human photoallergens tested positive and the other six, with no evidence of causing photoallergic dermatitis or UV absorption, tested negative. Among these chemicals, both doxycycline hydrochloride and minocycline hydrochloride were tetracycline antibiotics with different photoallergic properties, and the new method clearly distinguished between the photoallergic properties of these chemicals. These findings suggested high predictability of our method; therefore, it is promising and effective in predicting human photoallergens.     

Phototoxicity testing in guinea-pigs.

The photoirritant potential of topically applied chemicals was studied using guinea-pigs. Solutions of test chemicals were applied to the skin, and after 30 min the animals were irradiated with near-ultraviolet radiation. Skin reactions were assessed subjectively between 3 and 72 hr after the start of treatment. Acridine and anthracene caused immediate photoirritation, whereas reaction to 8-methoxy-psoralen (8-MOP) was delayed; acridine was weakly active compared with the strong photoirritancy of anthracene and 8-MOP. Ethanol and a mixture of dimethylacetamide-acetone-ethanol (DAE) were satisfactory solvents, and a time interval of 15 to 30 min between application and irradiation was optimal. It is concluded that animal tests should not be recommended if ingredients have negligible absorption of sunlight. The safety hazard of ingredients absorbing near-ultraviolet and visible radiation may be assessed by laboratory animal procedures to satisfy governmental regulations. The use of guinea-pigs allows the study to act as a preliminary test for the selection of non-photoirritant concentrations for photoallergy testing and reduction of animal numbers. The risk to consumers of photoactive products may be properly assessed by human studies.     

Cause of and countermeasures for oxidation of the cysteine‐derived reagent used in the amino acid derivative reactivity assay

The amino acid derivative reactivity assay (ADRA) is an in chemico alternative to animal testing for skin sensitization that solves certain problems found in the use of the direct peptide reactivity assay (DPRA). During a recent validation study conducted at multiple laboratories as part of the process to include ADRA in an existing OECD test guideline, one of the nucleophilic reagents used in ADRA—N‐(2‐(1‐naphthyl)acetyl)‐l ‐cysteine (NAC)—was found to be susceptible to oxidation in much the same manner that the cysteine peptide used in DPRA was. Owing to this, we undertook a study to clarify the cause of the promotion of NAC oxidation. In general, cysteine and other chemicals that have thiol groups are known to oxidize in the presence of even minute quantities of metal ions. When metal ions were added to the ADRA reaction solution, Cu²⁺ promoted NAC oxidation significantly. When 0.25 μm of EDTA was added in the presence of Cu²⁺, NAC oxidation was suppressed. Based on this, we predicted that the addition of EDTA to the NAC stock solution would suppress NAC oxidation. Next, we tested 82 chemicals used in developing ADRA to determine whether EDTA affects ADRA's ability to predict sensitization. The results showed that the addition of EDTA has virtually no effect on the reactivity of NAC with a test chemical, yielding an accuracy of 87% for predictions of skin sensitization, which was roughly the same as ADRA.     

In chemico assessment of potential sensitizers: Stability and direct peptide reactivity of 24 fragrance ingredients

Twenty‐four pure fragrance ingredients of concern as potential skin sensitizers were previously subjected to degradation studies and evaluated using the high throughput with dansyl cysteamine (HTS‐DCYA) method. The experimental results showed that two‐thirds of the 24 fragrance ingredients underwent chemical degradation. In some cases, such degradation was accompanied by an increase in thio‐reactivity. These results prompted us to investigate the reactivity of the same ingredients using the direct peptide reactivity assay (DPRA). In the present work, the 24 chemicals were subjected to forced degradation for 150 days, and evaluated with both DPRA and HTS‐DCYA methods. At the end of the study, four and eight compounds remained non‐reactive in the DPRA and DCYA assay, respectively. Coumarin, benzyl salicylate, benzyl cinnamate and hexyl cinnamal were found unreactive in both assays, while cinnamal, cinnamyl alcohol, hydroxycitronellal and lilial were found negative in the DCYA but positive in the DPRA method. The incongruity in reactivity of these four compounds was attributed to a possible role of pro‐oxidants formed upon degradation, resulting in depletion of peptide without formation of apparent covalent adducts with the test chemical. To validate this hypothesis, the effect of hydrogen peroxide as model pro‐oxidant on both lysine‐ and cysteine‐heptapeptide depletion in the DPRA method was thus investigated. The obtained results showed little effect of oxidative conditions on lysine depletion, while cysteine depletion was significantly affected by concentrations above 1.1 mg/L of hydrogen peroxide. Overall, both in chemico methods confirmed chemical instability should be considered when assessing the skin sensitization potential of (un)known chemicals with alternative methods.     

Development of an in vitro photosensitization assay using human monocyte-derived cells

In this study, with the aim of developing a cell-based in vitro photosensitization assay, we examined whether changes of CD86 and CD54 expression on cells of a human monocytic cell line, THP-1, could be used to assess the photosensitizing potential of chemicals. First, we identified suitable conditions of UV-irradiation (irradiation dose; 5.0 J/cm², irradiation intensity; 1.7 mW/cm²) by investigating the effect of UV-irradiation on CD86 and CD54 expression on untreated or 6-methylcoumarin (a representative photoallergen)-treated THP-1 cells (irradiation method). However, acridine, a representative photo-irritant, augmented CD86 and CD54 expression on THP-1 cells, apparently via induction of reactive oxygen species (ROS). In order to abolish the effect of ROS, we examined CD86 and CD54 expression on THP-1 cells treated with pre-irradiated chemicals (pre-irradiation method). We found that UV-irradiated photoallergens, but not photo-irritants, enhanced CD86 and/or CD54 expression on the THP-1 cells. Finally, based on the results of irradiation, non-irradiation, and pre-irradiation with 18 test chemicals, we built a decision tree, which allows us to distinguish between photoallergens and photo-irritants. We suggest that this system may be useful for in vitro evaluation of the photoallergic potential of chemicals.     

Use of the occlusive patch to evaluate the photosensitive properties of chemicals in guinea-pigs

Guinea-pig tests were conducted on a known photocontact allergen, tetrachlorosalicylanilide (TCSA), a known phototoxin, 8-methoxypsoralen, two reportedly weak photoallergens, musk ambrette and 6-methylcoumarin, and a negative control, octylphenoxy polyethoxyethanol (Triton X-15). The data show that under the test conditions used, photosensitivity responses can be produced, and combinations of these as well as the other biological responses can be readily defined. The results indicate that musk ambrette is photoallergenic, that 8-methoxypsoralen is phototoxic and that Triton X-15 is only a slight irritant. On the other hand, results with TCSA suggest that it is a strong contact allergen and photoallergen, while 6-methylcoumarin would be considered to be a weak contact allergen with weak phototoxic properties. Previous reports that barrier destruction or adjuvanticity is necessary to produce photoallergy to musk ambrette were not confirmed; by ensuring occlusion using standard methods, the photoallergic nature of the response to this material was clearly demonstrated. A device described elsewhere (Newmann & Parker, Fd Chem. Toxic. 1985, 23, 683) has made it possible to develop methods that can be used to differentiate clearly among the possible biological responses that can occur in guinea-pigs when photoreactive materials are applied to their skin and irradiated. The probable biological responses that need to be defined, under the above conditions, are primary irritation, delayed contact hypersensitivity, phototoxicity and/or photoallergenicity.     

An in vitro model for detecting skin irritants: methyl green-pyronine staining of human skin explant cultures.

We evaluated the potential of human organotypic skin explant cultures (hOSECs) for screening skin irritants. Test chemicals were applied to the epidermis of the skin explants which were incubated for 4, 24 or 48 h in tissue culture medium. A decrease in epidermal RNA staining, visualised in frozen sections using a modified methyl-green pyronine (MGP) staining procedure, was used as a marker of irritancy. A decrease in epidermal RNA after a 4-, 24- or 48-h exposure to a certain concentration of a test chemical equated to a MGP score of 3, 2 or 1, respectively. The MGP score was 0 if there was no keratinocyte cytotoxicity after a 48-h exposure. A minimum of three donors were used per chemical and the average MGP score was used to classify the chemical as irritant or not. Chemicals with an average MGP score > or =1.5 were classified as irritants (R38), at that concentration. Chemicals with a MGP score <1.5 were not classified (NC), at that concentration. The results obtained using human skin in vitro were compared with published data obtained using cultured porcine skin, the cutaneous Draize test (from this point referred to as the "rabbit skin irritation test") and volunteer studies. There was an excellent correlation between the classification of a chemical, as R38 or NC, based on hOSEC and results of volunteer studies. The hOSEC model predicted perfectly the irritation hazard of the 22 chemicals for which volunteer data were available. The porcine OSEC correctly predicted the classification of 21 of 22 (95%) chemicals and the rabbit skin irritation test correctly predicted the classification of 14 of 15 chemicals (93%) for which data were available. In conclusion, MGP staining of human skin explant cultures can be used to predicted human skin irritancy in vivo. In addition, the data validate the use of porcine skin as an alternative to human skin for screening for dermal irritants in vitro.     

僧帽牡蛎碱性磷酸酶在变性剂作用下构象与催化活力变化的研究

从僧帽牡蛎（Ostrea Cucullata）整体中提取一种经聚丙烯酸胺凝胶电泳鉴定为均一纯的碱性磷酸酶。经不同浓度的胍和脲处理,以荧光光谱为监测手段。研究其变性和大活动力学过程,发现酶的荧光强度随着变性剂浓度的增大而逐渐下降,330.2nm处的吸收峰峰值位置也出现了一定程度的“红移”和“蓝”。测定该酶的失活和变性速度常数发现,不论该酶在胍或脲溶液中变性,其失活速度常数都较其变性速度常数大。呈现出“快失活慢构象变化”的过程。     

A novel in chemico method to detect skin sensitizers in highly diluted reaction conditions

The direct peptide reactivity assay (DPRA) is a simple and versatile alternative method for the evaluation of skin sensitization that involves the reaction of test chemicals with two peptides. However, this method requires concentrated solutions of test chemicals, and hydrophobic substances may not dissolve at the concentrations required. Furthermore, hydrophobic test chemicals may precipitate when added to the reaction solution. We previously established a high‐sensitivity method, the amino acid derivative reactivity assay (ADRA). This method uses novel cysteine (NAC) and novel lysine derivatives (NAL), which were synthesized by introducing a naphthalene ring to the amine group of cysteine and lysine residues. In this study, we modified the ADRA method by reducing the concentration of the test chemicals 100‐fold. We investigated the accuracy of skin sensitization predictions made using the modified method, which was designated the ADRA‐dilutional method (ADRA‐DM). The predictive accuracy of the ADRA‐DM for skin sensitization was 90% for 82 test chemicals which were also evaluated via the ADRA, and the predictive accuracy in the ADRA‐DM was higher than that in the ADRA and DPRA. Furthermore, no precipitation of test compounds was observed at the initiation of the ADRA‐DM reaction. These results show that the ADRA‐DM allowed the use of test chemicals at concentrations two orders of magnitude lower than that possible with the ADRA. In addition, ADRA‐DM does not have the restrictions on test compound solubility that were a major problem with the DPRA. Therefore, the ADRA‐DM is a versatile and useful method. Copyright © 2015 John Wiley & Sons, Ltd.     

Pre-validation of a Calu-3 epithelium cytotoxicity assay for predicting acute inhalation toxicity of chemicals

Although in vivo inhalation toxicity tests have been widely conducted, the testing of many chemicals is limited for economic and ethical reasons. Therefore, we previously developed an in vitro acute inhalation toxicity test method. The goal of the present pre-validation study was to evaluate the transferability, reproducibility, and predictive capacity of this method.After confirming the transferability of the Calu-3 epithelium cytotoxicity assay, reproducibility was evaluated using 20 test substances at three independent institutions. Cytotoxicity data were analyzed using statistical methods, including the intra-class correlation coefficient and Bland-Altman plots for within- and between-laboratory reproducibility. The assay for the 20 test substances showed excellent agreement within and between laboratories. To evaluate the predictive capacity, 77 test substances were analyzed for acute inhalation toxicity. Accuracy was measured using a cutoff of 40%, and the relevance was analyzed as a receiver-operating characteristic (ROC) curve. An accuracy of 72.73% was obtained, and the area under the ROC curve was 0.77, indicating moderate performance.In this study, we found that the in vitro acute inhalation toxicity test method demonstrated good reliability and relevance for predicting the acute toxicity of inhalable chemicals. Hence, this assay has potential as an alternative test for screening acutely toxic inhalants.     

Analysis of functional effects of a mixture of five pesticides using a ray design

The protection of human health from the adverse effects of cumulative environmental exposure to chemical mixtures is an important issue. Of particular interest is the detection and characterization of interaction among chemicals in complex mixtures. Response surface methodology, often supported by factorial designs, is the classical statistical experimental approach. Fixed-ratio ray designs, which may include the use of single chemical data in addition to data along mixture ray(s), have been proposed as an alternative approach. Such designs permit a reduction in the amount of experimental effort when the region of interest can be restricted to exposure-relevant mixing ratios. A 'single chemicals required' (SCR) approach and a 'single chemicals not required' (SCNR) approach are both described. The methods are illustrated with a five-chemical mixture of organophosphorus pesticides-acephate (ACE), diazinon (DIA), chlorpyrifos (CPF), malathion (MAL) and dimethoate (DIM). Their relative proportions in the mixture were based on the relative dietary human exposure estimates of each chemical as projected by the U.S. EPA Dietary Exposure Evaluation Model (DEEM). Use of the SCR and SCNR methods for binary endpoints are demonstrated using a dichotomized gait score as an indication of toxicity. For both methods, the overall hypothesis of additivity was rejected, indicating significant departure from additivity when the five pesticides were combined at the specified mixing ratio. By comparison of the predicted response under additivity to the modeled response of the experimental mixture data this departure from additivity was characterized as synergy (greater than additive toxicity). To examine the influence of malathion in the mixture, it was removed from the five-pesticide mixture (full ray) and the remaining four chemicals (reduced ray) were combined at the same relative proportions used in the full fixed-ratio ray There was not a significant departure from additivity along the ray with the four remaining pesticides omitting malathion. Thus, although malathion was not dose-responsive alone, it significantly interacted with the other pesticides.     

A new 3D reconstituted human corneal epithelium model as an alternative method for the eye irritation test

Many efforts are being made to develop new alternative in vitro test methods for the eye irritation test. Here we report a new reconstructed human corneal epithelial model (MCTT HCE model) prepared from primary-cultured human limbal epithelial cells as a new alternative in vitro eye irritation test method. In histological and immunohistochemical observation, MCTT HCE model displayed a morphology and biomarker expressions similar to intact human cornea. Moreover, the barrier function was well preserved as measured by high transepithelial electrical resistance, effective time-50 for Triton X-100, and corneal thickness. To employ the model as a new alternative method for eye irritation test, protocol refinement was performed and optimum assay condition was determined including treatment time, treatment volume, post-incubation time and rinsing method. Using the refined protocol, 25 reference chemicals with known eye irritation potentials were tested. With the viability cut-off value at 50%, chemicals were classified to irritant or non-irritant. When compared with GHS classification, the MCTT HCE model showed the accuracy of 88%, sensitivity of 100% and specificity of 77%. These results suggest that the MCTT HCE model might be useful as a new alternative eye irritation test method.     

The within‐ and between‐laboratory reproducibility and predictive capacity of the in chemico amino acid derivative reactivity assay: Results of validation study implemented in four participating laboratories

The amino acid derivative reactivity assay (ADRA) is an in chemico alternative method that focuses on protein binding as the molecular initiating event for skin sensitization. It is a simple and versatile method that has successfully solved some of the problems of the direct peptide reactivity assay (DPRA). The transferability and within‐ and between‐laboratory reproducibility of ADRA were evaluated and confirmed as part of a validation study conducted at four participating laboratories. The transfer of ADRA technology from the lead laboratory to the four participating laboratories was completed successfully during a two‐step training program, after which the skin sensitization potentials of 40 coded chemicals were predicted based on the results of ADRA testing. Within‐laboratories reproducibility was 100% (10 of 10), 100% (10 of 10), 100% (7 of 7) and 90% (9 of 10), or an average of 97.3% (36 of 37); between‐laboratory reproducibility as calculated on the results of three laboratories at the time was 91.9%. The overall predictive capacity comprised an accuracy of 86.9%, sensitivity of 81.5% and specificity of 98.1%. These results satisfied the targets set by the validation management team for demonstrating transferability, within‐ and between‐laboratory reproducibility, and predictive capacity as well as gave a clear indication that ADRA is easily transferable and sufficiently robust to be used in place of DPRA.     

The within- and between-laboratories reproducibility and predictive capacity of Amino acid Derivative Reactivity Assay using 4 mM test chemical solution: Results of ring study implemented at five participating laboratories

Amino acid derivative reactivity assay (ADRA) for skin sensitization was adopted as an alternative method in the 2019 OECD Guideline for the Testing of Chemicals (OECD TG 442C). The molar ratio of the nucleophilic reagent to the test chemicals in the reaction solution was set to 1:50. Imamura et al. reported that changing this molar ratio from 1:50 to 1:200 reduced in false negatives and improved prediction accuracy. Hence, a ring study using ADRA with 4 mM of a test chemical solution (ADRA, 4 mM) was conducted at five different laboratories to verify within- and between-laboratory reproducibilities (WLR and BLR, respectively). In this study, we investigated the WLR and BLR using 14 test chemicals grouped into three classes: (1) eight proficiency substances, (2) four test chemicals that showed false negatives in the ADRA with 1 mM test chemical solution (ADRA, 1 mM), but correctly positive in ADRA (4 mM), and (3) current positive control (phenylacetaldehyde) and a new additional positive control (squaric acid diethyl ester). The results showed 100% reproducibility and 100% accuracy for skin sensitization. Hence, it is clear that the ADRA (4 mM) is an excellent test method in contrast to the currently used ADRA (1 mM). We plan to resubmit the ADRA (4 mM) test method to the OECD Test Guideline Group in the near future so that OECD TG 442C could be revised for the convenience and benefit of many ADRA users.     

Modeling the antioxidant properties of the eye reduces the false-positive rate of a nonanimal eye irritation test (OptiSafe)

We recently identified a group of chemicals that are misclassified by most, if not all, in vitro alternative ocular irritation tests, suggesting that nonanimal tests may not fully model the ocular environment in which these chemicals interact. To address this, we evaluated the composition of tears, the first defense against foreign substances, and identified the presence of antioxidants that could detoxify reactive chemicals that otherwise may be falsely identified as irritants in alternative irritation tests. In this study, we evaluated the effects of tear antioxidants on the ocular irritation scoring of commonly overclassified chemicals (false positives) using the OptiSafe™ ocular irritation test. Six tear-related antioxidants were individually added to the OptiSafe formulation, and the effects on test outcome were determined. Ascorbic acid, the most abundant water-soluble antioxidant in tears, specifically reduced the OptiSafe false-positive rate. Titration curves showed that this reduction occurs at in vivo concentrations and is specific to chemicals identified either as producing reactive oxygen species or as crosslinkers. Importantly, the addition of tear antioxidants did not impact the detection of true negatives, true positives, or other false positives unassociated with reactive oxygen species or crosslinking. These results suggest that the addition of tear antioxidants to in vitro alternative test systems may substantially reduce the false-positive rate and improve ocular irritant detection.     

Solution behavior of IFN-beta-1a: an empirical phase diagram based approach

An empirical phase diagram approach has been developed as a practical tool to aid macromolecular preformulation/formulation studies. This method employs an eigenvector based procedure to visualize and interpret complex data sets. Human Inteferon-beta-1a, an important therapeutic protein, was used to further develop the method and test its utility. The protein was characterized in solution as a function of pH (2-8), temperature (10 degrees C-85 degrees C) and ionic strength (I = 0.1 and 1.0) using intrinsic and ANS fluorescence, Far-UV circular dichroism (Far-UV CD), Fourier Transform Infrared spectroscopy (FTIR) and derivative UV absorbance spectroscopies, as well as differential scanning calorimetry (DSC) to supplement spectroscopic thermal stability studies. Derivative UV absorbance data were initially used to construct a pH-temperature phase diagram at each ionic strength. Three distinctive phases at I = 0.1 and two major phases at I = 1.0 were identified corresponding to different conformation/aggregation states of the protein. For the first time, heterogeneous data sets (i.e., data from different techniques) including Far-UV CD, fluorescence and UV absorbance results were used to generate empirical phase diagrams. Results from different data sets are compared; precautions in applying the method and its overall utility are discussed.     

An in vitro human skin test for assessing sensitization potential

Sensitization to chemicals resulting in an allergy is an important health issue. The current gold‐standard method for identification and characterization of skin‐sensitizing chemicals was the mouse local lymph node assay (LLNA). However, for a number of reasons there has been an increasing imperative to develop alternative approaches to hazard identification that do not require the use of animals. Here we describe a human in‐vitro skin explant test for identification of sensitization hazards and the assessment of relative skin sensitizing potency. This method measures histological damage in human skin as a readout of the immune response induced by the test material. Using this approach we have measured responses to 44 chemicals including skin sensitizers, pre/pro‐haptens, respiratory sensitizers, non‐sensitizing chemicals (including skin‐irritants) and previously misclassified compounds. Based on comparisons with the LLNA, the skin explant test gave 95% specificity, 95% sensitivity, 95% concordance with a correlation coefficient of 0.9. The same specificity and sensitivity were achieved for comparison of results with published human sensitization data with a correlation coefficient of 0.91. The test also successfully identified nickel sulphate as a human skin sensitizer, which was misclassified as negative in the LLNA. In addition, sensitizers and non‐sensitizers identified as positive or negative by the skin explant test have induced high/low T cell proliferation and IFNγ production, respectively. Collectively, the data suggests the human in‐vitro skin explant test could provide the basis for a novel approach for characterization of the sensitizing activity as a first step in the risk assessment process. Copyright © 2015 John Wiley & Sons, Ltd.