The usefulness of toxicogenomics for predicting acute skin irritation on in vitro reconstructed human epidermis

In vitro models aiming at replacing the traditional animal test for determining the skin irritation potential of a test substance have been developed, evaluated in prevalidation studies and recently validated by the European Center for the Validation of Alternative Methods (ECVAM). To investigate the usefulness of toxicogenomic technologies to identify novel mechanistic endpoints for skin irritation responses, the present work challenged the human reconstituted epidermis model validated by ECVAM with four irritant chemicals and four non-classified chemicals tested at subcytotoxic concentrations. Using a specifically designed low-density DNA array, about 50 genes out of 240 were found to be significantly and differentially expressed between tissues exposed to irritant and non-irritant chemicals for at least one test chemical when compared to the seven others. These genes are involved in cell signalling, stress response, cell cycle, protein metabolism and cell structure. Among them, 16 are expressed in the same way whatever the irritant compound applied. The differential gene expressions might represent new or additional endpoints useful for the mechanistic understanding and perhaps also the hazard assessment of the skin irritation potential of chemicals and products.     

重组人皮肤模型在化妆品皮肤刺激性评价中的应用研究

目的 探讨重组人皮肤模型应用于化妆品体外皮肤刺激性评价的可行性.方法 以重组人皮肤模型(EpiSkinTM)为受试模型,在对10个已知刺激性分类的标准化学品进行方法验证的基础上,再根据化妆品的使用特性对23个产品进行皮肤刺激性分类评价,其中6个基础型化妆品和10个美容型化妆品暴露18 h、7个清洁型化妆品分别暴露18、...     

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.     

Simultaneous absorption of vitamins C and E from topical microemulsions using reconstructed human epidermis as a skin model

Antioxidants provide the mainstay for skin protection against free radical damage. The structure of microemulsions (ME), colloidal thermodynamically stable dispersions of water, oil and surfactant, allows the incorporation of both lipophilic (vitamin E) and hydrophilic (vitamin C) antioxidants in the same system. The objective of this work was to investigate the potential of non-thickened (o/w, w/o and gel-like) and thickened (with colloidal silica) ME as carriers for the two vitamins using reconstructed human epidermis (RHE). The amounts of these vitamins accumulated in and permeated across the RHE were determined, together with factors affecting skin deposition and permeation. Notable differences were observed between formulations. The absorption of vitamins C and E in RHE layers was in general enhanced by ME compared to solutions. The incorporation of vitamins in the outer phase of ME resulted in greater absorption than that when vitamins were in the inner phase. The location of the antioxidants in the ME and affinity for the vehicle appear to be crucial in the case of non-thickened ME. Addition of thickener enhanced the deposition of vitamins E and C in the RHE. By varying the composition of ME, RHE absorption of the two vitamins can be significantly modulated.     

Establishment of a new in vitro test method for evaluation of eye irritancy using a reconstructed human corneal epithelial model,LabCyte CORNEA-MODEL

Finding in vitro eye irritation testing alternatives to animal testing such as the Draize eye test, which uses rabbits, is essential from the standpoint of animal welfare. It has been developed a reconstructed human corneal epithelial model, the LabCyte CORNEA-MODEL, which has a representative corneal epithelium-like structure. Protocol optimization (pre-validation study) was examined in order to establish a new alternative method for eye irritancy evaluation with this model. From the results of the optimization experiments, the application periods for chemicals were set at 1 min for liquid chemicals or 24 h for solid chemicals, and the post-exposure incubation periods were set at 24 h for liquids or zero for solids. If the viability was less than 50%, the chemical was judged to be an eye irritant. Sixty-one chemicals were applied in the optimized protocol using the LabCyte CORNEA-MODEL and these results were evaluated in correlation with in vivo results. The predictions of the optimized LabCyte CORNEA-MODEL eye irritation test methods were highly correlated with in vivo eye irritation (sensitivity 100%, specificity 80.0%, and accuracy 91.8%). These results suggest that the LabCyte CORNEA-MODEL eye irritation test could be useful as an alternative method to the Draize eye test.     

A catch‐up validation study of an in vitro skin irritation test method using reconstructed human epidermis LabCyte EPI‐MODEL24

Three validation studies were conducted by the Japanese Society for Alternatives to Animal Experiments in order to assess the performance of a skin irritation assay using reconstructed human epidermis (RhE) LabCyte EPI‐MODEL24 (LabCyte EPI‐MODEL24 SIT) developed by the Japan Tissue Engineering Co., Ltd. (J‐TEC), and the results of these studies were submitted to the Organisation for Economic Co‐operation and Development (OECD) for the creation of a Test Guideline (TG). In the summary review report from the OECD, the peer review panel indicated the need to resolve an issue regarding the misclassification of 1‐bromohexane. To this end, a rinsing operation intended to remove exposed chemicals was reviewed and the standard operating procedure (SOP) revised by J‐TEC. Thereafter, in order to confirm general versatility of the revised SOP, a new validation management team was organized by the Japanese Center for the Validation of Alternative Methods (JaCVAM) to undertake a catch‐up validation study that would compare the revised assay with similar in vitro skin irritation assays, per OECD TG No. 439 (2010). The catch‐up validation and supplementary studies for LabCyte EPI‐MODEL24 SIT using the revised SOPs were conducted at three laboratories. These results showed that the revised SOP of LabCyte EPI‐MODEL24 SIT conformed more accurately to the classifications for skin irritation under the United Nations Globally Harmonised System of Classification and Labelling of Chemicals (UN GHS), thereby highlighting the importance of an optimized rinsing operation for the removal of exposed chemicals in obtaining consistent results from in vitro skin irritation assays. Copyright © 2013 John Wiley & Sons, Ltd.     

Prediction of skin sensitization potency using machine learning approaches

The replacement of animal use in testing for regulatory classification of skin sensitizers is a priority for US federal agencies that use data from such testing. Machine learning models that classify substances as sensitizers or non‐sensitizers without using animal data have been developed and evaluated. Because some regulatory agencies require that sensitizers be further classified into potency categories, we developed statistical models to predict skin sensitization potency for murine local lymph node assay (LLNA) and human outcomes. Input variables for our models included six physicochemical properties and data from three non‐animal test methods: direct peptide reactivity assay; human cell line activation test; and KeratinoSens™ assay. Models were built to predict three potency categories using four machine learning approaches and were validated using external test sets and leave‐one‐out cross‐validation. A one‐tiered strategy modeled all three categories of response together while a two‐tiered strategy modeled sensitizer/non‐sensitizer responses and then classified the sensitizers as strong or weak sensitizers. The two‐tiered model using the support vector machine with all assay and physicochemical data inputs provided the best performance, yielding accuracy of 88% for prediction of LLNA outcomes (120 substances) and 81% for prediction of human test outcomes (87 substances). The best one‐tiered model predicted LLNA outcomes with 78% accuracy and human outcomes with 75% accuracy. By comparison, the LLNA predicts human potency categories with 69% accuracy (60 of 87 substances correctly categorized). These results suggest that computational models using non‐animal methods may provide valuable information for assessing skin sensitization potency. Copyright © 2017 John Wiley & Sons, Ltd.     

Improved performance of the SH test as an in vitro skin sensitization test with a new predictive model and decision tree

Demands for the elimination and replacement of animal experiments for cosmetic safety assessment have increased in recent years. Evaluation of skin sensitization, however, is a critical issue in cosmetic safety assessment. The SH test is an in vitro skin sensitization test method that evaluates protein binding of chemical substances, which is an important event in skin sensitization. We previously verified the technical transferability and between-laboratory reproducibility of the SH test, a domestic test method for which no scientific research has been conducted, and improved the protocol, but also noted some unresolved issues. Therefore, in the present study, we successfully improved the operational efficiency and clarity of the final judgment of the SH test by (i) developing a new decision-making system that can make a final judgment without statistical processing, (ii) changing the statistical method, and (iii) evaluating and determining the maximum number of repetitions necessary for optimal efficiency. The improved SH test was verified by comparing it with existing test methods already adopted by the Organization for Economic Cooperation and Development. The results of this study demonstrated excellent performance of the improved SH test, with high reproducibility, reliable predictability, and good operational efficiency. The predictive performance of the improved method does not differ significantly from that of the conventional method, although it is clearer and more efficient. Therefore, the results of the present improved method are consistent with those obtained using the conventional method, with higher efficiency.     

Evaluating the sensitization potential of surfactants: integrating data from the local lymph node assay, guinea pig maximization test, and in vitro methods in a weight-of-evidence approach

An integral part of hazard and safety assessments is the estimation of a chemical's potential to cause skin sensitization. Currently, only animal tests (OECD 406 and 429) are accepted in a regulatory context. Nonanimal test methods are being developed and formally validated. In order to gain more insight into the responses induced by eight exemplary surfactants, a battery of in vivo and in vitro tests were conducted using the same batch of chemicals. In general, the surfactants were negative in the GPMT, KeratinoSens and hCLAT assays and none formed covalent adducts with test peptides. In contrast, all but one was positive in the LLNA. Most were rated as being irritants by the EpiSkin assay with the additional endpoint, IL1-alpha. The weight of evidence based on this comprehensive testing indicates that, with one exception, they are non-sensitizing skin irritants, confirming that the LLNA tends to overestimate the sensitization potential of surfactants. As results obtained from LLNAs are considered as the gold standard for the development of new nonanimal alternative test methods, results such as these highlight the necessity to carefully evaluate the applicability domains of test methods in order to develop reliable nonanimal alternative testing strategies for sensitization testing.     

Analysis of interleukin-1alpha (IL-1alpha) and interleukin-8 (IL-8) expression and release in in vitro reconstructed human epidermis for the prediction of in vivo skin irritation and/or sensitization.

In the present study, reconstructed human epidermis (RHE) was used as an in vitro model to discriminate 1-chloro-2,4-dinitrobenzene (DNCB), nickel sulfate (NiSO(4)), oxazolone (OXA), 2,4-dinitrofluorobenzene (DNFB) and 2,4,6-trinitrobenzenesulfonic acid (TNBS) as skin sensitizers from benzalkonium chloride (BC), benzoic acid (BA) and sodium lauryl sulfate (SLS) as skin irritants. Our criteria were (a) the differential IL-1alpha and IL-8 synthesis and release (b) cytotoxicity assessment by MTT assay. When the RHE are topically treated with the sensitizers, very low levels of extra- and intracellular IL-1alpha are observed although they induce significant cytotoxicity. In contrast, they exhibit a sharp maximum of IL-8 release. In the presence of the tested irritants, we observe the inverse cytokine release profile, although they induce dose-dependent cytotoxicity profiles similar to those observed with the sensitizers. Finally, IL-1alpha mRNA upregulation is observed after topical application of both sensitizers and irritants, but only the latter significantly increase extracellular IL-1alpha. In conclusion, our results suggest that the associated determination of IL-8, with IL-1alpha, and MTT conversion are at least necessary to discriminate and classify, in a single assay, irritant and sensitizing agents and represent a potential in vitro alternative to two classical in vivo assays.     

Disposition and biotransformation of C-Benzo(a)pyrene in a pig ear skin model: Ex vivo and in vitro approaches

Biotransformation of chemicals by the skin is a critical determinant of systemic exposure in humans following dermal absorption. Pig ear skin potentially represents a valuable alternative model since it closely resembles to human skin. We developed an ex vivo pig ear skin system which absorption, diffusion and metabolic capabilities were investigated using benzo(a)pyrene [B(a)P] as a model molecule. The potential of the ex vivo pig ear skin model to biotransform xenobiotics was compared with metabolic data obtained using dermal and hepatic microsomes from human and pig. ¹⁴C-B(a)P [50–800 nmol] was applied on the surface of skin models. The diffusion and the production of B(a)P metabolites were quantified by radio-HPLC, LC–MS/MS and NMR. B(a)P was extensively metabolized by pig ear skin explants, the major metabolites being B(a)P-glucuronide and sulfate conjugates. B(a)P-OHs, B(a)P-diols, B(a)P-catechols and B(a)P-diones were also identified. In the pig ear skin model developed, skin diffusion was maintained over 72 h and both phase I and phase II activities were expressed, with the formation of similar metabolites as produced in incubations with liver and skin microsomal fractions. This ex vivo model, which combines a functional skin barrier and active biotransformation capabilities, appears to represent a valuable alternative tool in transdermal exposure studies.     

Synthesis of methoxypoly(ethylene glycol) carbonate prodrugs of zidovudine and penetration through human skin in vitro

Objectives The aim of this study was to synthesise a series of novel methoxypoly (ethylene glycol) carbonate prodrugs of the antiretroviral drug zidovudine (azidothymidine, AZT) in an attempt to enhance the physicochemical properties for transdermal delivery, which may reduce the severe side‐effects and toxicity associated with high oral doses of AZT. Methods Methoxypoly(ethylene glycol) carbonates of AZT were synthesised in two steps: activation of the relevant methoxypoly(ethylene glycol) with p‐nitrophenyl chloroformate, followed by reaction with AZT. Analysis of the hydrolytic stability in phosphate buffer at pH 5.0 and 7.4 revealed that all the carbonates were markedly more stable at pH 5.0 than at pH 7.4 (0.01 m), with half‐lives ranging from 15 to 44 days at pH 5.0 and from 6 to 24 days at pH 7.4. The potential of the series to penetrate the skin was evaluated in vitro by measuring diffusion through excised abdominal female human skin at pH 5.0. Key findings Prodrugs with 1–3 or 8 oxyethylene units in the methoxypoly(ethylene glycol) moiety were found to permeate the skin whereas those with 12 or 17 units did not. The prodrug with eight oxyethylene units was the most effective penetrant, permeating the skin with a mean flux of 53.3 ± 46.5 nmol/cm² per h, which is 2.4–10.1 times that of AZT (8.55 ± 5.3 nmol/cm² per h). Conclusions The bioreversible conjugation of the methoxypoly(ethylene glycol) promoiety to AZT appears to be a promising strategy for the transdermal delivery of AZT at a therapeutic dose.     

Transfection of primary human nasal epithelial cells using a biodegradable poly (ester amine) based on polycaprolactone and polyethylenimine as a gene carrier

The purpose of this study was to prepare and characterize poly (ester amine) (PEA)/pGL3 complexes and investigate their transfection efficiency in human nasal epithelial (HNE) cells. Particle size, zeta potential, and gel retardation characteristics of PEA /pGL3 complexes were also measured. After treatment of DNase-I, protection and release assay of PEA/pGL3 complexes were performed. To assess the transfection efficiency and cytotoxicity, measurement of relative luciferase activity and MTS assay were performed. PEA/pGL3 complexes showed effective and stable DNA condensation with the particle sizes below 200 nm, implicating their potential for intracellular delivery. PEA/pGL3 complexes successfully transfected into the HNE cells with higher viability of the cells. These results suggested that, the PEA can be used as an efficient cationic polymeric vehicle which provides a versatile platform for further investigation of structure property relationship along with the controlled degradation, significant low cytotoxicity, and high transfection efficiency of the primary HNE cells.     

Kinetic-spectrophotometric method for the assay of copper(II) in human serum by catalytic oxidation of salicylic acid

A very sensitive kinetic spectrophotometric method for the determination of copper(II) concentrations as low as 0.07 ng ml⁻¹ is described. This method is based on the oxidation of salicylic acid by hydrogen peroxide in ammoniacal medium, catalysed by copper(II) ion. The figures of merit of the procedure and the results of a study of interferences are given. The method is applied to the assay of copper in human blood serum.     

Silymarin prevents the toxicity induced by benzo(a)pyrene in human erythrocytes by preserving its membrane integrity: An in vitro study

Silymarin, the purified extract from milk thistle Silybum marianum (L.) Gaertn, consists mainly of four isomeric flavonolignans: silibinin, isosilibinin, silidianin, and silichristin. The present study was carried out to evaluate the protective potential of silymarin in human erythrocytes against in vitro exposure to the carcinogen benzo(a)pyrene (B(a)P). Erythrocytes isolated from human blood were divided into four groups and treated with Vehicle [Group I], B(a)P (300 μM) [Group II], Silymarin (500 μM) + B(a)P (300 μM) [Group III], and Silymarin alone (500 μM)] [Group IV]. Silymarin treatment maintains the integrity of erythrocytes by preventing hemolysis, protein thiol oxidation and by decreasing the activity of AChE. SEM observations indicate that B(a)P induced significant alteration in the morphology of erythrocytes to echinocytes, which may be due to the interaction of B(a)P with the membrane's outer phopholipid monolayer. The light microscopic and SEM images show that silymarin treatment maintains the normal discocytic morphology of erythrocytes. The protective effect of silymarin might be attributed to its chemical structure and membranotrophic nature. The components silibinin, silydianin, and silychristin have OH in the 3rd, 5th, and 7th carbon atoms that may account for its increased antioxidant activity and removal of ROS formed during B(a)P metabolism. © 2011 Wiley Periodicals, Inc. Environ Toxicol 29: 165–175, 2014.     

Antagonism of skin conductance response (SCR) habituation during iterative photostimulation in mice: Habituation test — A new psychopharmacological method for detecting and quantifying enhancement of psychic activity

A method (habituation test) for studying habituation of the palmar skin conductance response (SCR) during iterative photostimulation in mice is described. Twenty drugs known for their CNS stimulant activity and/or beneficial action on learning were tested for their antagonism toward habituation. With most of the drugs tested, the delay in SCR extinction was dose-dependent. From the corresponding regression equations, the standard delaying doses were computed and used for classification. Fenozolone was the most active product. In descending order follow dexamphetamine and piracetam, then other amphetamines.Locomotor activity tests were run in parallel with habituation tests and the two sets of results compared.Reliability of habituation test was checked. The responsiveness of the test and the significance of the results are discussed.The applicability of the habituation test in psychopharmacological research is argued by its sensitivity to piracetam, whose nootropic activity is not detectable by classic behavioral methods.The habituation test makes it possible to test drugs for their effects on the maintenance of attention during monotonous stimulation and, more generally, for their psychic enhancement activity whatever the mechanism involved. It is thus likely to be a suitable test for nooanaleptics (in the broad meaning of the term).