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Cosmetics Europe Multi-Laboratory Pre-Validation of the SkinEthic™ Reconstituted Human Corneal Epithelium Test Method for the Prediction of Eye Irritation.
Abstract
Cosmetics Europe, The Personal Care Association, known as Colipa before 2012, conducted a program of technology transfer and assessment of Within/Between Laboratory (WLV/BLV) reproducibility of the SkinEthic™ Reconstituted Human Corneal Epithelium (HCE) as one of two human reconstructed tissue eye irritation test methods. The Skinethic™ HCE test method involves two exposure time treatment procedures - one for short time exposure (10 min - SE) and the other for long time exposure (60 min - LE) of tissues to test substance. This paper describes pre-validation studies of the Skinethic™ HCE test method (SE and LE protocols) as well as the Eye Peptide Reactivity Assay (EPRA). In the SE WLV study, 30 substances were evaluated. A consistent outcome with respect to viability measurement across all runs was observed with all substances showing an SD of less than 18%. In the LE WLV study, 44 out of 45 substances were consistently classified. These data demonstrated a high level of reproducibility within laboratory for both the SE and LE treatment procedures. For the LE BLV, 19 out of 20 substances were consistently classified between the three laboratories, again demonstrating a high level of reproducibility between laboratories. The results for EPRA WLV and BLV studies demonstrated that all substances analyzed were categorized similarly and that the method is reproducible. The SkinEthic™ HCE test method entered into the experimental phase of a formal ECVAM validation program in 2010.
... To this end, a reconstituted human corneal epithelium (HCE) tissue culture developed by SkinEthic Laboratories may be a better tool for in vitro toxicological studies. When cultivated as a multilayered and differentiated epithelium on a permeable membrane, this HCE model resembles the human cornea in vivo (Alepee et al., 2013). By using chemicals with known eye irritating potential, the use of the HCE model for eye irritation test has been validated with satisfactory specificity and reproducibility (Alepee et al., 2013;Doucet et al., 2006;Meloni et al., 2010;Seaman et al., 2010;Van Goethem et al., 2006). ...
... When cultivated as a multilayered and differentiated epithelium on a permeable membrane, this HCE model resembles the human cornea in vivo (Alepee et al., 2013). By using chemicals with known eye irritating potential, the use of the HCE model for eye irritation test has been validated with satisfactory specificity and reproducibility (Alepee et al., 2013;Doucet et al., 2006;Meloni et al., 2010;Seaman et al., 2010;Van Goethem et al., 2006). In addition, assessment of in vitro eye irritating potential of chemicals by using the HCE model showed satisfactory results compared with animal test (Pauly et al., 2012;Van Goethem et al., 2006). ...
... After exposure, each HCE tissue culture was carefully rinsed with PBS and transferred to 300 mL freshly prepared MTT solution (0.5 mg/mL in PBS; purchased from Sigma-Aldrich) in 24-well plates. MTT test has been validated as an easy and reliable method for the assessment of in vitro eye irritating potential in the HCE model based on the principle that MTT is reduced to a purple formazan by mitochondrial succinate dehydrogenase in viable cells (Alepee et al., 2013;Doucet et al., 2006;Seaman et al., 2010;Van Goethem et al., 2006). The plates were incubated for 1 h at 37 C, 5% CO 2 in a humidified incubator. ...
Context:
Eye irritation is a common complaint in indoor environment, but the causes have still not been identified among the multiple exposures in house environments. To identify the potential environmental factors responsible for eye irritation and study the possible mechanisms, an in vitro model for eye irritation is suggested.
Materials and methods:
In this study, reconstituted human corneal epithelium (HCE) tissue cultures were used to study the eye irritating and inflammatory potential of lipopolysaccharide (LPS) and dust. HCE tissue cultures were exposed to a range of concentrations of LPS for 6 h and dust for 24 h, respectively. After exposure, viability and secretion of interleukins (IL) IL-1β, IL-8, and tumor necrosis factor (TNFα) were examined. Histology was used to indicate the morphological changes after dust exposure.
Results:
Both LPS and dust affected HCE viability. There was an increased level of IL-8 after LPS exposure, while the concentrations of IL-1β and TNFα remained unaffected. Dust exposure resulted in an elevation of both IL-1β and IL-8, but not TNFα. Histology study showed increased vacuolization and reduced thickness after 24 h exposure to 5 mg/mL dust.
Discussion and conclusion:
LPS and dust showed in vitro eye irritating and inflammatory potential, and cytokines/chemokines like IL-1β and IL-8 may be involved in the mechanisms of eye irritation. The HCE tissue culture may be used as an in vitro model to study environmental exposure induced eye irritation and inflammation.
... Changes in acidity in response to an irritant are measured using a pH meter, ocular toxicity is evaluated by calculating the reduction in metabolic rate caused by the addition of a test chemical to the culture media compared to the basal metabolic state. CM has been recommended for the identification of GHS Category 1, severe irritants and GHS non irritants (Alépée et al., 2013;OECD, 2012a). The test is limited for use with test substances which do not settle or separate during analysis, primarily water soluble surfactants and surfactant-containing mixtures, but also some non-water-soluble solids, viscous chemicals or suspensions that maintain uniformity during analysis (OECD, 2012a). ...
... In vitro cultured epithelium is capable of retaining the in vivo repair mechanisms found in the native cornea (Davila et al., 1998), although these mechanisms are not always given the level of attention they deserve (Dholakiya and Barile, 2013). Epithelial models can be constructed from animal cells (commonly SIRC cells (Ubels and Clousing, 2005)) such as in the STE test, human epidermal cells, or human corneal cells, which are usually cultured in defined medium on cell culture membranes using air-lifting techniques (Alépée et al., 2013;Cotovio et al., 2007;Kaluzhny et al., 2011;Matsuda et al., 2009) to create a 3D stratified epithelium. Cytotoxicity following topical exposure is generally used as an endpoint (Curren and Harbell, 2002), and epithelial models have the potential to identify non-classified/nonirritating substances from mild irritants (Scott et al., 2010). ...
... Finding suitable, regulatory approved and validated alternatives to animal testing is a crucial aim of toxicological research (Alépée et al., 2013) with regulatory bodies keen to adopt the use of protocols that modify and reduce the number of animals used in ocular testing procedures. For alternative methods to be successfully incorporated into safety assessment procedures, they need to demonstrate that they can provide at least an equivalent or preferably superior level of protection to that obtained with current methods (Vinardell and Mitjans, 2008). ...
Given the hazardous nature of many materials and substances, ocular toxicity testing is required to evaluate the dangers associated with these substances after their exposure to the eye. Historically, animal tests such as the Draize test were exclusively used to determine the level of ocular toxicity by applying a test substance to a live rabbit’s eye and evaluating the biological response. In recent years, legislation in many developed countries has been introduced to try to reduce animal testing and promote alternative techniques. These techniques include ex vivo tests on deceased animal tissue, computational models that use algorithms to apply existing data to new chemicals and in vitro assays based on two dimensional (2D) and three dimensional (3D) cell culture models. Here we provide a comprehensive overview of the latest advances in ocular toxicity testing techniques, and discuss the regulatory framework used to evaluate their suitability.
... This model is not further discussed here since the nonocular origin of the cells represents a significant flaw. On the contrary, SkinEthic HCE™ was established with immortalized human corneal epithelial cells [60][61][62][63]. It has the advantage of using corneal cells but the inconvenience of using a cell line instead of primary cells. ...
... Validation of reconstructed human cornea-like epithelium (RhCE) models for eye irritation tests [53][54][55][56][57][58][59][60][61][62][63][64][65][66][67] Representation of corneal curvature [72] Stroma Development of models with fibroblasts that produce their own extracellular matrix [81][82][83][84][85][86] Use of 3D bioprinting to develop corneal equivalents [88] Endothelium ...
In recent decades, the establishment of complex three-dimensional (3D) models of tissues has allowed researchers to perform high-quality studies and to not only advance knowledge of the physiology of these tissues but also mimic pathological conditions to test novel therapeutic strategies. The main advantage of 3D models is that they recapitulate the spatial architecture of tissues and thereby provide more physiologically relevant information. The eye is an extremely complex organ that comprises a large variety of highly heterogeneous tissues that are divided into two asymmetrical portions: the anterior and posterior segments. The anterior segment consists of the cornea, conjunctiva, iris, ciliary body, sclera, aqueous humor, and the lens. Different diseases in these tissues can have devastating effects. To study these pathologies and develop new treatments, the use of cell culture models is instrumental, and the better the model, the more relevant the results. Thus, the development of sophisticated 3D models of ocular tissues is a significant challenge with enormous potential. In this review, we present a comprehensive overview of the latest advances in the development of 3D in vitro models of the anterior segment of the eye, with a special focus on those that use human primary cells.
... immortalized cell lines (SkinEthic HCE [7]) for the construction of these models, may represent a certain degree of difference with native human corneal tissue. To solve these issues, different 3D cornea models using primary cultures of human corneal cells have been developed [8,9]. ...
... In this study, we developed and characterized a new reconstructed human corneal model produced with primary human corneal epithelial cells that mimics the structure and the barrier integrity of a native human corneal epithelium as proven by phenotypical markers and a barrier function of nearly 1500 Ωcm 2 . Using this model, we were able to classify test substances, between irritant and non-irritant, based on cellular viability via MTT reduction as is the internationally accepted methodology for these types of studies [6,7,15,19,20]. Moreover, we also explored the changes in TEER after test substance exposure, defining a non-evaluated property of test substances: their barrier disruptive potential. ...
A new in vitro human corneal epithelial model (QobuR) obtained from normal limbal tissue has been developed to study ocular irritancy of different ophthalmic compounded drugs. Phenotypical characterization and trans-epithelial electrical resistance (TEER) of QobuR revealed essential similarities compared with a native human cornea, displaying functional markers and TEER values near 1500 Ωcm² at day 7th of cellular differentiation. Using this model, ocular irritancy and barrier integrity alterations were evaluated using MTT reaction and variations in TEER. We found that some of the Non-Irritant products evaluated still damage the corneal epithelial integrity and current protocols for ocular irritancy should therefore include a barrier integrity evaluation. Moreover, in order to comprehensively evaluate corneal permeability of the active ingredients, we propose the use of QobuR as an all-in-one alternative method for evaluating ocular irritancy, barrier disruptions and permeability rates of topically applied ocular drugs to improve current in vitro drug testing procedures.
... Here we developed a new test method for the determination of eye irritation of solid substances with MCTT HCE TM model and with the developed method, we demonstrated that MCTT HCE TM model could predict the eye irritation of 11 solid chemicals quickly and correctly in comparison with other HCE models. Solid test materials are supposed to be applied for 90 min and then post-incubated for 18 hr in the EpiOcular TM model (Kaluzhny et al., 2011; Pfannenbecker et al., 2013). And in the LabCyte CORNEA-MODEL, another recently de veloped HCE model, solid substances are treated for 24 hr without postincubation (Katoh et al., 2013), refl ecting that considerable time can be saved with MCTT HCE TM model. ...
... In addition, in MCTT HCE TM model, we could employ identical treatment time and post-incubation time for solid as used in liquid test substances through deploying separate washing schemes, which would be convenient and practical for the conduct of test. Most HCE models need separate treatment procedures for solid substances except for SkinEthic HCE TM where different treatment time is applied according to eye peptide reactivity assay (EPRA) results (Alepee et al., 2013). Generally, HCE models are provided in a 24 well format and 12 test substances can be tested in one kit of HCE model (duplicate). ...
The eye irritation potential of drug candidates or pharmaceutical ingredients should be evaluated if there is a possibility of ocular exposure. Traditionally, the ocular irritation has been evaluated by the rabbit Draize test. However, rabbit eyes are more sensitive to irritants than human eyes, therefore substantial level of false positives are unavoidable. To resolve this species difference, several three-dimensional human corneal epithelial (HCE) models have been developed as alternative eye irritation test methods. Recently, we introduced a new HCE model, MCTT HCE(TM) which is reconstructed with non-transformed human corneal cells from limbal tissues. Here, we examined if MCTT HCE(TM) can be employed to evaluate eye irritation potential of solid substances. Through optimization of washing method and exposure time, treatment time was established as 10 min and washing procedure was set up as 4 times of washing with 10 mL of PBS and shaking in 30 mL of PBS in a beaker. With the established eye irritation test protocol, 11 solid substances (5 non-irritants, 6 irritants) were evaluated which demonstrated an excellent predictive capacity (100% accuracy, 100% specificity and 100% sensitivity). We also compared the performance of our test method with rabbit Draize test results and in vitro cytotoxicity test with 2D human corneal epithelial cell lines.
... The tissue is represented by a multilayered epithelium, with five to seven cell layers and a surface area of 0.5 cm 2 . The assay was performed according to the manufacturer instructions (Alépée et al., 2013;Cotovio et al., 2010). Upon arrival, tissues were transferred to 24 well plates containing 37 • C pre-warmed maintenance media (1 mL/well) and incubated 24 h at 37 • C, 5% CO 2 and 95% relative humidity. ...
... In this field, numerous non-animal test systems have been developed over the years (OECD, 2009a,b). SkinEthic TM HCE is one of the two reconstructed human tissue that are currently available for the purpose of eye irritation and is under validation by ECVAM (Alépée et al., 2013). ...
Recent changes in regulatory requirements and social views on animal testing have incremented the development of reliable alternative tests for predicting skin and ocular irritation potential of products based on new raw materials. In this regard, botanical ingredients used in cosmetic products are among those materials, and should be carefully reviewed concerning the potential presence of irritant constituents. In particular, cosmetic products used on the face, in vicinity of the eyes or that may come in contact with mucous membranes, should avoid botanical ingredients that contain, or are suspected to contain, such ingredients. In this study, we aimed to evaluate the effect of a new cosmetic ingredient, namely, coffee silverskin (CS), with an in vitro skin and ocular irritation assay using reconstructed human epidermis, EpiSkin™, and human corneal epithelial model, SkinEthics™ HCE, and an in vivo assay. Three different extracts of CS were evaluated. The histology of the models after extracts applications was analysed. The in vitro results demonstrated that extracts were not classified as irritant and the histological analyses proved that extracts did not affect both models structure. The content of caffeine, 5-hydroxymethyl furfural and chlorogenic acid was quantified after the epidermal assay. The in vivo test carried out with the most promising extract (hydroalcoholic) showed that, with respect to irritant effects, these extracts can be regarded as safe for topical application.
... It is worth mentioning that ICCvAm recommended in particular that additional data be collected on eye irritants (Cat 2) to more adequately characterise the usefulness of the HeT-CAm. Test methods using reconstructed human tissues (rhT) modelling the corneal epithelium, like the epiOcu-lar™ eye Irritation Test (eIT) (Kaluzhny et al. 2011;Pfannenbecker et al. 2013) or the skinethic™ Human Corneal epithelium (HCe) test (van Goethem et al. 2006;Cotovio et al. 2007Cotovio et al. , 2010Alépée et al. 2013), may also be relevant for assessing conjunctiva epithelial responses using cytotoxicity as an endpoint (OeCD 2010). An eUrL eCvAm/ Cosmetics europe prospective validation study on epiO-cular™ eIT and skinethic™ HCe to evaluate their usefulness and limitations for discriminating non-classified materials versus eye irritants/chemicals inducing serious eye damage (Cat 2/Cat 1) is currently ongoing (Freeman et al. 2010). ...
... Further development and/or evaluation of alternative methods are, however, still required to fill the gaps identified in this paper, i.e. persistence/reversibility of effects and identification of Cat 2. Considering the importance of conjunctiva effects for the classification of Cat 2 chemicals (43 % in the nCD and 26 % in the rCD) and of persistence of corneal effects for the classification of Cat 1 chemicals (53 % in the nCD and 26 % in the rCD), focus and efforts may still have to be given to further development and evaluation/validation of alternative methods/strategies capable of correctly identifying these effects. several other test methods, e.g. the epiOcular™ eIT (Kaluzhny et al. 2011;Pfannenbecker et al. 2013), the skinethic™ HCe (van Goethem et al. 2006;Cotovio et al. 2007Cotovio et al. , 2010Alépée et al. 2013), the Ocular Irritection ® assay, the PorCOrA (Piehl et al. 2010, 2011 or the eveIT (spöler et al. 2007;Frentz et al. 2008), are currently undergoing validation or are in an advanced status of development/optimisation and should soon be accepted for use in a regulatory environment. With all these methods becoming available, it should now be possible to explore the development of highly predictive testing strategies capable of fully replacing the animal test. ...
For more than two decades, scientists have been trying to replace the regulatory in vivo Draize eye test by in vitro methods, but so far only partial replacement has been achieved. In order to better understand the reasons for this, historical in vivo rabbit data were analysed in detail and resampled with the purpose of (1) revealing which of the in vivo endpoints are most important in driving United Nations Globally Harmonized System/European Union Regulation on Classification, Labelling and Packaging (UN GHS/EU CLP) classification for serious eye damage/eye irritation and (2) evaluating the method’s within-test variability for proposing acceptable and justifiable target values of sensitivity and specificity for alternative methods and their combinations in testing strategies. Among the Cat 1 chemicals evaluated, 36–65 % (depending on the database) were classified based only on persistence of effects, with the remaining being classified mostly based on severe corneal effects. Iritis was found to rarely drive the classification (
... Moreover, multi-laboratory verification studies of animal substitution tests for eye irritation have been actively conducted using selected chemicals [17,18]. The predictive capacity and between-laboratory reproducibility of in vitro human corneal assays for the evaluation of eye irritation have been confirmed to sufficiently satisfy the criteria established in the OECD TG 492 performance standards. ...
The sustained growth of the market for ophthalmic medical devices has increased the demand for alternatives to animal testing for the evaluation of eye irritation. The International Organization for Standardization has acknowledged the need to develop novel in vitro tests to replace animal testing. Here, we evaluated the applicability of an alternative method based on a human corneal model to test the safety of ophthalmic medical devices. 2-Hydroxyethyl methacrylate (HEMA) and Polymethyl methacrylate (PMMA), which are used to fabricate contact lenses, were used as base materials. These materials were blended with eye irritant and non-irritant chemicals specified in the OECD Test Guideline (TG) 492 and Globally Harmonized System (GHS) classification. Then, three GLP-certified laboratories performed three replicates using the developed method using 3D reconstructed human cornea epithelium, MCTT HCETM. OECD TG 492 describes the procedure used to evaluate the eye hazard potential of the test chemical based on its ability to induce cytotoxicity in a reconstructed human cornea-like epithelium (RhCE) tissue. Results: The within-laboratory reproducibility (WLR) and between-laboratory reproducibility (BLR) were both 100%. When a polar extraction solvent was used, the sensitivity, specificity, and accuracy were all 100% in each laboratory. When a non-polar extraction solvent was used, the sensitivity was 80%, the specificity was 100%, and the accuracy was 90%. The proposed method exhibited excellent reproducibility and predictive capacity within and between laboratories. Therefore, the proposed method using the MCTT HCETM model could be used to evaluate eye irritation caused by ophthalmic medical devices.
... A few in vitro human-derived corneal-like epithelium models are commercially available as 3D human cornea equivalents [179][180][181][182][183][184][185][186][187]. The LabCyte CORNEA-MODEL24 from Japan Tissue Engineering Co. is fabricated with primary human corneal cells and has the three main corneal epithelial layers [188][189][190]. ...
In vitro cell-based models have been used for a long time since they are normally easily obtained and have an advantageous cost-benefit. Besides, they can serve a variety of ends, from studying drug absorption and metabolism to disease modeling. However, some in vitro models are too simplistic, not accurately representing the living tissues. It has been shown, mainly in the last years, that fully mimicking a tissue composition and architecture can be paramount for cellular behavior and, consequently, for the outcomes of the studies using such models. Because of this, 3D in vitro cell models have been gaining much attention, since they are able to better replicate the in vivo environment. In this review we focus on 3D models that contain mucus-producing cells, as mucus can play a pivotal role in drug absorption. Being frequently overlooked, this viscous fluid can have an impact on drug delivery. Thus, the aim of this review is to understand to which extent can mucus affect mucosal drug delivery and to provide a state-of-the-art report on the existing 3D cell-based mucus models.
... Eyes from dead rabbits and chicken had been also used to evaluate the biomaterials irritation effects on the eyes [53,54]. Scientists have developed 3D epithelial models using human skin-derived epidermal keratinocytes and human corneal epithelial cells (HCE), which are available commercially having a trademark name EpiOcular™ and SkinEthic™ respectively [55,56]. Neural red uptake assay is used to assess human keratinocyte viability [57] while, Skin-on-a-chip Skin inflammation and skin cell viability testing [59] 8 ...
Advanced biomaterials have produced a significant impact on healthcare by improving the quality of life of people with disabilities. Biomaterials are immensely used in tissue engineering, wound healing applications, and delivery of cancer targeted therapeutics. Biocompatibility and cytotoxicity screening of biomaterials on cell culture systems is the first step before their in vivo testing in animal models and subsequent clinical trials. Direct use of biomaterials on animals may create technical challenges as well as ethical concerns. In order to avoid the ethical concerns of animal use, many non-animal models such as stem cell cultures are being developed and utilized for testing their safety. However, due to several limitations including the inability to recapitulate the complex in vivo microenvironment, the application of stem cell cultures is limited. However, properties of stem cells such as their self-renewal and ability to differentiate into various cell lineages like hepatocytes, cardiomyocytes, and neural cells make them an ideal candidates for in vitro screening studies. Furthermore, the application of stem cells may overcome the challenges associated with the inability to develop a complex heterogeneous tissue using primary cells. Currently, Embryonic Stem Cells (ESCs), Adult Stem Cells (ASCs), and Induced Pluripotent Stem Cells (iPSCs) are being used as in vitro preliminary biomaterials testing models with demonstrated advantages over mature primary cell or cell line based in vitro models. This review discusses the current status and future directions of in vitro stem cell-based cultures and their derivatives such as spheroids and organoids for the screening of their safety before their application to animal models and human in translational research.
... 3D)(Araki-Sasaki et al. 1995;Zorn-Kruppa et al. 2005). The SkinEthic-HCE tissue model has been used to study ocular irritation (hazard assessment), drug discovery, and disease modeling(Meloni et al. 2011;Alepee et al. 2013; Fallacara et al. 2017;Matysik-Woźniak et al. 2017) (https:// www.episkin.com/HCE-Corneal-Epithelium). ...
Testing of all manufactured products and their ingredients for eye irritation is a regulatory requirement. In the last two decades, the development of alternatives to the in vivo Draize eye irritation test method has substantially advanced due to the improvements in primary cell isolation, cell culture techniques, and media, which have led to improved in vitro corneal tissue models and test methods. Most in vitro models for ocular toxicology attempt to reproduce the corneal epithelial tissue which consists of 4–5 layers of non-keratinized corneal epithelial cells that form tight junctions, thereby limiting the penetration of chemicals, xenobiotics, and pharmaceuticals. Also, significant efforts have been directed toward the development of more complex three-dimensional (3D) equivalents to study wound healing, drug permeation, and bioavailability. This review focuses on in vitro reconstructed 3D corneal tissue models and their utilization in ocular toxicology as well as their application to pharmacology and ophthalmic research. Current human 3D corneal epithelial cell culture models have replaced in vivo animal eye irritation tests for many applications, and substantial validation efforts are in progress to verify and approve alternative eye irritation tests for widespread use. The validation of drug absorption models and further development of models and test methods for many ophthalmic and ocular disease applications is required.
... A monolayer of Madin-Darby Canine Kidney cells has been used for Fluorescein Leakage Test (OECD TG 460) to determine ocular toxicity potential of a test substance by measuring the permeability of sodium fluorescein. Commercially available 3D epithelial models such as EpiOcular™ prepared with human skin-derived epidermal keratinocytes and SkinEthic™ with immortalized human corneal epithelial cells (HCE) (Alépée et al., 2013) have been developed for in vitro skin irritation tests (OECD TG 492). Yang et al. (2017) have reported results of a pre-validation trial for an in vitro eye irritation test using MCTT HCE™ (Biosolution Co., Seoul, South Korea) prepared from primary human corneal cells. ...
Despite a major role of experimental animals in development of biomedical research, there has been historical controversy surrounding animal research. Along with a strategy of 3Rs, various in vitro methods have been suggested to replace potentially painful animal experiments. In this review, we summarize the use of stem cells as an alternative of animal experimentation in predictive toxicology. There have been continuing researches on stem cells and stem cell-derived tissue-specific cells to develop alternative methods/biomarkers for animal toxicity testing including developmental toxicity, genotoxicity, and tissue-specific toxicity. Along with unique abilities of stem cells including self-renewal, infinite proliferation, and differentiation into multiple lineages, human stem cell-based in vitro systems have been proven valuable to increase predictive power of toxicology through providing with better scientific information related to toxic risks in humans without inter-species variability. In particular, stem cells including induced pluripotent stem cell-based system for personalized toxicological assessment could be a better option as an in vitro model system in comparison with immortalized cells with abnormal phenotype or primary cells with small quantity and batch-to-batch variation. This review will be useful for understanding the current status and future direction in using stem cells as an alternative non-animal method for predictive toxicology.
... The advantages of three dimensional methods are the tissues which are used have a capacity to withstand mechanical or chemical damage as compared to that of two dimensional methods. In order to get better results, reconstructed human corneal epithelium (RCE) is used in three-dimensional corneal epithelial models 24 . ...
The developed ophthalmic formulations should be tested for ocular toxicity testing like draize eye test. Rapid growth in the field of ocular toxicity testing replaces the usage of animals and to adopt the modified protocols. The researchers have to develop properly validated and regulatory approved options to animal testing. Alternative ocular toxicity testing is based on corneal structure and mechanism through which the specific ocular formulation causes irritation to the eyes. Several alternative assay methods have been introduced in the field of ocular toxicity testing like in vitro organotypic model for testing ocular toxicity and several other methods like Bovine Corneal Opacity and Permeability Assay (BCOP Assay), isolated chicken eye test, isolated rabbit eye test and Hen's egg-test on the chorioallantoic membrane. The ultimate aim of alternate methods of ocular toxicity testing is to develop properly validated and regulatory approved best practices. Hence this review highlights the development in the field of alternative ocular toxicity testing methods.
... The Skinethic HCE model is prepared with immortalized human corneal epithelial cells (HCE) (68). The resulting tissue forms a multilayered, stratified epithelium with an overall thickness of 60 mm, similar to normal human corneal epithelium (69). ...
Human eyes and skin are frequently exposed to chemicals accidentally or on purpose due to their external location. Therefore, chemicals are required to undergo the evaluation of the ocular and dermal irritancy for their safe handling and use before release into the market. Draize rabbit eye and skin irritation test developed in 1944, has been a gold standard test which was enlisted as OECD TG 404 and OECD TG 405 but it has been criticized with respect to animal welfare due to invasive and cruel procedure. To replace it, diverse alternatives have been developed: (i) For Draize eye irritation test, organotypic assay, in vitro cytotoxicity-based method, in chemico tests, in silico prediction model, and 3D reconstructed human cornea-like epithelium (RhCE); (ii) For Draize skin irritation test, in vitro cytotoxicity-based cell model, and 3D reconstructed human epidermis models (RhE). Of these, RhCE and RhE models are getting spotlight as a promising alternative with a wide applicability domain covering cosmetics and personal care products. In this review, we overviewed the current alternatives to Draize test with a focus on 3D human epithelium models to provide an insight into advancing and widening their utility.
... For decades, the Draize rabbit eye irritation test has been the globally accepted regulatory method for assessing eye irritation potential of chemicals 2 . However, replacing the animal test with in vitro testing remains as an urgent mission in toxicological research 3 . ...
Aim:
To evaluate eye irritation potential of palm-based methyl ester sulphonates (MES) of different chain lengths; C12, C14, C16, C16:18.
Methods:
The Bovine Corneal Opacity and Permeability test method (BCOP), OECD Test Guideline 437, was used as an initial step to study the inducing effect of palm-based MES on irreversible eye damage. The second assessment involved the use of reconstructed human corneal-like epithelium test method, OECD Test Guideline 492 using SkinEthic™ Human Corneal Epithelium to study the potential effect of palm-based MES on eye irritancy. The palm-based MES were prepared in 10% solution (w/v) in deionized water and tested as a liquid and surfactant test substances whereby both test conducted according to the liquid/surfactant treatment protocol.
Results:
The preliminary BCOP results showed that palm-based MES; C12, C14, C16, C16:18 were not classified as severe eye irritants test substances with in vitro irritancy score between 3 and the threshold level of 55. The second evaluation using SkinEthic™ HCE model showed that palm-based MES; C12, C14, C16, C16:18 and three commercial samples were potentially irritants to the eyes with mean tissue viability ? 60% and classified as Category 2 according to United Nations Globally Harmonized System of Classification and Labelling of Chemicals. However, there are some limitations of the proposed ocular irritation classification of palm-based MES due to insolubility of long chain MES in 10% solution (w/v) in deionized water.
Conclusion:
Therefore, future studies to clarify the eye irritation potential of the palm-based MES will be needed, and could include; methods to improve the test substance solubility, use of test protocol for solids, and/or inclusion of a benchmark anionic surfactant, such as sodium dodecyl sulphate within the study design.
... The outcome of this expert meeting identified that test methods using RhCE could be considered for incorporation into a testing strategy as an initial step in a Bottom-Up approach or the second step in a Top-Down approach. Currently, there are two such test methods available namely, the EpiOcular™EIT (Kaluzhny et al., 2011;Pfannenbecker et al., 2013;OECD, 2015b) and the SkinEthic™ Human Corneal Epithelium (HCE) test (Van Goethem et al., 2006;Cotovio et al., 2007Cotovio et al., , 2010; Alépée et al., 2013). Recently, the SkinEthic™ HCE model has been optimized for the evaluation of liquids (Eye Irritation Testing of Liquids, EITL) in a multicentre prospective study demonstrating an overall predictive capacity of 84.4% with 99.0% sensitivity and 68.5% specificity . ...
... Due to the numerous applications of these raw materials, their safety profile is well established. Oleth-20 and oleth-3 were ranked non-or moderately irritating by the Draize test, [46][47][48], unlike the cationic surfactant cetyltrimethylammonium chloride that may cause irreversible eye damage associated with corneal opacities and conjunctival irritation [49]. The base formulation showed weak irritant potential due to the low quaternary ammonium salt content , and the addition of quercetin did not aggravate the result. ...
... The outcome of this expert meeting identified that test methods using RhCE could be considered for incorporation into a testing strategy as an initial step in a Bottom-Up approach or the second step in a Top-Down approach. Currently, there are two such test methods available namely, the EpiOcular™EIT (Kaluzhny et al., 2011;Pfannenbecker et al., 2013;OECD, 2015b) and the SkinEthic™ Human Corneal Epithelium (HCE) test (Van Goethem et al., 2006;Cotovio et al., 2007Cotovio et al., , 2010; Alépée et al., 2013). Recently, the SkinEthic™ HCE model has been optimized for the evaluation of liquids (Eye Irritation Testing of Liquids, EITL) in a multicentre prospective study demonstrating an overall predictive capacity of 84.4% with 99.0% sensitivity and 68.5% specificity . ...
... 6,7 Some of these corneal models are being evaluated in a multicenter validation study to determine their applicability domain and potential utility in regulatory-driven safety testing. 8,9 The currently available scientifically valid tissue-engineered 3D models, however, as well as 3D models undergoing formal validation studies, are protected by patents and/or are being commercialized using proprietary tissue culture procedures owned by only a few private companies. As a consequence, their continued commercial availability is dependent upon the economic market, single-corporate strategies, and customs regulation, and so their accessibility might not be guaranteed for the long-term as well as in geographically diverse locations. ...
... The outcome of this expert meeting identified that test methods that are using RhCE could be considered for incorporation into a testing strategy as an initial step in a Bottom-Up approach or the second step in a Top-Down approach. Currently, there are two such test methods available namely, the EpiOcular™ Eye Irritation Test (EIT) (Kaluzhny et al., 2011;Pfannenbecker et al., 2013;OECD, 2015b) and the SkinEthic™ Human Corneal Epithelium (HCE) test (Van Goethem et al., 2006;Cotovio et al., 2007Cotovio et al., , 2010Alépée et al., 2013). In the past, various test protocols using the SkinEthic™ HCE model have been evaluated in order to improve the predictive capacity of this test method. ...
... 6,7 Some of these corneal models are being evaluated in a multicenter validation study to determine their applicability domain and potential utility in regulatory-driven safety testing. 8,9 The currently available scientifically valid tissue-engineered 3D models, however, as well as 3D models undergoing formal validation studies, are protected by patents and/or are being commercialized using proprietary tissue culture procedures owned by only a few private companies. As a consequence, their continued commercial availability is dependent upon the economic market, single-corporate strategies, and customs regulation, and so their accessibility might not be guaranteed for the long-term as well as in geographically diverse locations. ...
Today, engineered three-dimensional tissue models from human cells of the skin, cornea, mucosa, and respiratory tract are widely used in pharmaceutical, chemical, consumer, and cosmetic industries as alternatives to animal testing. The currently available scientifically valid 3D tissue-engineered models, however, are protected by patents and/or are being commercialized using proprietary tissue culture procedures owned by only a few private companies. As a consequence, their continued commercial availability might not be guaranteed in the long-term. In collaboration with its partners, the ALEXANDRA Association strives to ensure a long-term, sustainable, and global implementation of alternative methods based on nonprofit, patent-free, and proprietary-free development of ‘‘open source’’ (OS) human tissue engineering technologies. By doing so, the association proposes a new standard in 3D tissue reconstruction as a basis for animal-free testing method implementation by supporting the development, production, validation, and technology transfer based on a global OS concept.
... With the increasing concern over the scientific validity and ethical standards of such tests, the 7th amendment of the EU cosmetics directive has banned the use of animals for testing cosmetic ingredients for eye irritation since 2009 (EU, 2003). Also, the necessity of performing safety testing, required by the introduction of REACH (Registration, Evaluation, Authorization and Restriction of Chemicals) legislation to support the use of new and existing chemicals, demands suitable non-animal alternatives (EU, 2006;Alepee et al., 2013). ...
... Three representative RhT/MTT-based test methods covering the toxicity endpoints of serious eye damage/ eye irritation, skin irritation and skin corrosion were used for this purpose. For serious eye damage/eye irritation this was the EpiOcular™ Eye Irritation Test (EIT) (in connection with the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)/Cosmetics Europe RhT test methods Eye Irritation Validation Study (EIVS) (Freeman et al., 2010;Pfannenbecker et al., 2013;Alépée et al., 2013). For skin irritation this was EpiSkin™ as representative of OECD TG 439 (OECD, 2013b) and for skin corrosion this was SkinEthic™ RHE as representative of OECD TG 431 (OECD, 2013a). ...
A number of in vitro test methods using Reconstructed human Tissues (RhT) are regulatory accepted for evaluation of skin corrosion/irritation. In such methods, test chemical corrosion/irritation potential is determined by measuring tissue viability using the photometric MTT-reduction assay. A known limitation of this assay is possible interference of strongly coloured test chemicals with measurement of formazan by absorbance (OD). To address this, Cosmetics Europe evaluated use of HPLC/UPLC-spectrophotometry as an alternative formazan measurement system. Using the approach recommended by the FDA guidance for validation of bio-analytical methods, three independent laboratories established and qualified their HPLC/UPLC-spectrophotometry systems to reproducibly measure formazan from tissue extracts. Up to 26 chemicals were then tested in RhT test systems for eye/skin irritation and skin corrosion. Results support that: 1) HPLC/UPLC-spectrophotometry formazan measurement is highly reproducible; 2) formazan measurement by HPLC/UPLC-spectrophotometry and OD gave almost identical tissue viabilities for test chemicals not exhibiting colour interference nor direct MTT reduction; 3) independent of the test system used, HPLC/UPLC-spectrophotometry can measure formazan for strongly coloured test chemicals when this is not possible by absorbance only. It is therefore recommended that HPLC/UPLC-spectrophotometry to measure formazan be included in the procedures of in vitro RhT-based test methods, irrespective of the test system used and the toxicity endpoint evaluated to extend the applicability of these test methods to strongly coloured chemicals.
Copyright © 2015. Published by Elsevier Ltd.
The Food and Drug Administration's recent decision to eliminate mandatory animal testing for drug approval marks a significant shift to alternative methods. Similarly, the European Parliament is advocating for a faster transition, reflecting public preference for animal‐free research practices. In vitro tissue models are increasingly recognized as valuable tools for regulatory assessments before clinical trials, in line with the 3R principles (Replace, Reduce, Refine). Despite their potential, barriers such as the need for standardization, availability, and cost hinder their widespread adoption. To address these challenges, the Robotic Enabled Biological Automation (ReBiA) system is developed. This system uses a dual‐arm robot capable of standardizing laboratory processes within a closed automated environment, translating manual processes into automated ones. This reduces the need for process‐specific developments, making in vitro tissue models more consistent and cost‐effective. ReBiA's performance is demonstrated through producing human reconstructed epidermis, human airway epithelial models, and human intestinal organoids. Analyses confirm that these models match the morphology and protein expression of manually prepared and native tissues, with similar cell viability. These successes highlight ReBiA's potential to lower barriers to broader adoption of in vitro tissue models, supporting a shift toward more ethical and advanced research methods.
The accurate identification of chemicals with ocular toxicity is of paramount importance in health hazard assessment. In contemporary chemical toxicology, there is a growing emphasis on refining, reducing, and replacing animal testing in safety evaluations. Therefore, the development of robust computational tools is crucial for regulatory applications. The performance of predictive models is heavily reliant on the quality and quantity of data. In this investigation, we amalgamated the most extensive dataset (4901 compounds) sourced from governmental GHS‐compliant databases and literature to develop binary classification models of chemical ocular toxicity. We employed 12 molecular representations in conjunction with six machine learning algorithms and two deep learning algorithms to create a series of binary classification models. The findings indicated that the deep learning method GCN outperformed the machine learning models in cross‐validation, achieving an impressive AUC of 0.915. However, the top‐performing machine learning model (RF‐Descriptor) demonstrated excellent performance with an AUC of 0.869 on the test set and was therefore selected as the best model. To enhance model interpretability, we conducted the SHAP method and attention weights analysis. The two approaches offered visual depictions of the relevance of key descriptors and substructures in predicting ocular toxicity of chemicals. Thus, we successfully struck a delicate balance between data quality and model interpretability, rendering our model valuable for predicting and comprehending potential ocular‐toxic compounds in the early stages of drug discovery.
With an increase in the progression of research and development in the medical field, the experimental use of animals for the efficacy and safety testing of pharmaceuticals is on rise. Every year, millions of animals are used for experimental testing during which these suffer from pain and are then eventually sacrificed. Besides bioethical issues, animal experimentation is associated with many disadvantages like high cost, the requirement of skilled manpower, approval, and is time-consuming. Therefore, attempts have been made by researchers to design and develop a number of alternative methods that could bypass animal experiments. These methods not only give accurate results but can also save lives of millions of animals annually. Research techniques, including computer and robotics together with molecular biology techniques, are applied to discover new methods to replace animal testing. Several alternative methods are discussed in this review. Some of these methods can predict the behavior of drugs accurately and are as reliable as in-vivo animal models. Furthermore, these alternative methods offer a variety of advantages over experimental animals. However, there is still a great need to discover and develop new, accurate, and reliable methods to replace experimental animals.
The testing and classification of chemicals to determine adverse ocular effects are routinely conducted to ensure that materials are appropriately classified, labeled, and meet regulatory and safety guidelines. We have performed a same-chemical analysis using publicly available validation study results and compared the performance between tests for the same chemicals. To normalize for chemical selection, we matched chemicals tested by pairs of tests so that each matched set compared performance for the exact same chemicals. Same-chemical accuracy comparisons demonstrate a chemical selection effect that results in a wide range of overlapping false-positive (FP) rates and accuracies for all test methods. In addition, the analysis suggests that a tiered-testing strategy with specific combinations of tests can reduce the FP rate for some combinations. However, reductions in the FP rates were typically accompanied by an increase in the false-negative rates, resulting in minimal advantage in terms of accuracy. In addition, actual improvements in the FP rate after retesting positives with a second test are not as good as the theoretical improvements because some chemicals and functional groups appear to be broadly misclassified by all test methods, which, to the extent the tests make the same-chemical misclassifications, reduces the advantage of using tiered-testing strategies.
Die Risikobewertung von Chemikalien ist für die öffentliche Gesundheit von entschei-dender Bedeutung, weshalb strenge Testverfahren zu deren toxikologischer Begutach-tung angewandt werden. Die ursprünglich tierbasierten Testverfahren werden aufgrund von neuen wissenschaftlichen Erkenntnissen und wegen ökonomischer Ineffizienz sowie ethischer Fragwürdigkeit immer mehr durch alternative Methoden ohne Tiermodelle ersetzt. Für den toxikologischen Endpunkt der Augenreizung wurden bereits die ersten alternativen Testsysteme auf der Basis von ex vivo- oder in vitro-Modellen entwickelt. Jedoch ist bis dato kein alternatives Testsystem in der Lage, das gesamte Spektrum der verschiedenen Kategorien der Augenreizungen nach dem global harmonisierten System zur Einstufung und Kennzeichnung von Chemikalien (GHS) vorherzusagen und damit den tierbasierten Draize-Augenreizungstest vollends zu ersetzen. Gründe hierfür sind fehlende physiologische Merkmale im Modell sowie eine destruktive Analysemethode. Aufgrund dessen wurden in dieser Studie die Hypothesen getestet, ob ein verbessertes In-vitro-Modell oder eine zerstörungsfreie, hochsensitive Analysemethode die Vorher-sagekraft des Augenreizungstests verbessern können. Dafür wurden zunächst neue Mo-delle aus humanen Hornhaut- und Hautepithelzellen entwickelt. Die Modelle aus pri-mären cornealen Zellen zeigten eine gewebespezifische Expression der Marker Zytokera-tin 3 und 12 sowie Loricrin. In beiden Modellen konnte durch die Verkürzung der Kul-turdauer die Ausbildung einer Hornschicht verhindert werden. Die Modelle wiesen dadurch eine sensiblere Barriere vergleichbar der nativen Cornea auf. Darüber hinaus konnte durch die chemische Quervernetzung mit Polyethylenglykolsuccinimidylglutara-tester ein transparentes, nicht kontrahierendes Stroma-Äquivalent etabliert werden. Der Stroma-Ersatz konnte zur Generierung von Hemi- und Voll-Cornea-Äquivalenten einge-setzt werden und lieferte somit erste Ansatzpunkte für die Rekonstruktion der nativen Hornhaut. Parallel dazu konnte ein zerstörungsfreies Analyseverfahren basierend auf der Impe-danzspektroskopie entwickelt werden, das wiederholte Messungen der Gewebeintegri-tät zulässt. Zur verbesserten Messung der Barriere in dreidimensionalen Modelle wurde hierfür ein neuer Parameter, der transepitheliale elektrische Widerstand (TEER) bei der Frequenz von 1000 Hz, der TEER1000 Hz definiert, der eine genauere Aussage über die Integrität der Modelle zulässt. Durch die Kombination der entwickelten cornealen Epithelzellmodelle mit der TEER1000 Hz-Messung konnte die Prädikitivität des Augenrei-zungstests auf 78 - 100 % erhöht werden. Von besonderer Bedeutung ist dabei, dass die nicht destruktive Messung des TEER1000 Hz zum ersten Mal erlaubte, die Persistenz von Irritationen durch wiederholte Messungen in einem in vitro-Modell zu erkennen und somit die GHS-Kategorie 1 von GHS-Kategorie 2 zu unterscheiden. Der wissenschaftli-che Gewinn dieser Forschungsarbeit ist ein neues Testverfahren, das alle GHS-Kategorien in einem einzigen in vitro-Test nachweisen und den Draize-Augenreizungstest gänzlich ersetzen kann.
Numerous studies have attempted to develop a new in vitro eye irritation test (EIT). To obtain more reliable results from EIT, potential new biomarkers that reflect eye irritation by chemicals must be identified. We investigated candidate biomarkers for eye irritation, using a proteomics approach. Sodium lauryl sulfate (SLS) or benzalkonium chloride (BAC) was applied on a reconstructed human cornea-like epithelium model, MCTT HCETM, and corneal protein expression was examined by two-dimensional gel electrophoresis. We found that ezrin (EZR) was significantly up-regulated by SLS or BAC. In addition, up-regulation of EZR in immortalized human corneal cells treated with SLS or BAC was confirmed by quantitative reverse transcription-PCR and western blot analysis. Furthermore, other well-known eye irritants such as cetylpyridinium bromide, Triton X-100, cyclohexanol, ethanol, 2-methyl-1-pentanol, and sodium hydroxide significantly increased EZR expression in immortalized human corneal cells. Induction of EZR promoter activity in irritant-treated human corneal cells was confirmed by a luciferase gene reporter assay. In conclusion, EZR expression may be a potential biomarker for detecting eye irritation, which may substantially improve the performance of in vitro EIT.
Introduction:
A variety of in vitro tests to replace the Draize test have been developed; however, there is no available method for assessing the full spectrum of Globally Harmonized System (GHS) categories. Human cornea-like three-dimensional (3D) reconstructed tissue models are the most promising in vitro systems. The objective of this study was to evaluate the ocular toxicity of 11 test substances using the EpiOcular™ model after performing proficiency tests. We further evaluated the effectiveness of ezrin staining as a complementary marker in histological analysis to overcome the limitation of eye irritation tests using 3D reconstructed human corneal epithelium models.
Methods:
The assessment of ocular toxicity was performed by the suggested OECD TG 492 procedure. After treatment with proficiency test chemicals and 10 test substances, EpiOcular™ tissue models were stained with hematoxylin and eosin and ezrin, and the histological changes were observed by immunofluorescence microscopy.
Results:
The ocular toxicity assessment of 10 test chemicals using the EpiOcular™ eye irritation test were in accordance with the UN GHS classification of test chemicals. Histological analysis of ezrin staining showed that the cell membranes of models treated with 10 out of 11 non-irritant chemicals were maintained, whereas those of models treated with 14 eye irritant substances resulted in the apparent translocation of ezrins from the cell membrane to the cytoplasm or nucleus by destruction of cell membrane.
Discussion:
Ezrin may be used as a complementary marker to more accurately assess ocular toxicity using 3D reconstructed human cornea-like epithelium models.
Cell culture systems are essential tools used in a wide range of biomedical and clinical studies. Two dimensional cell culture models (2D) provide basic information on cytotoxicity, penetration and accumulation of drugs in cells and they are of outmost importance when selecting new compounds of the desired biopharmaceutical properties as candidates for novel drugs. The improvement over 2D growing cells are three dimensional (3D) tissue models that mimic in vivo conditions and the functions of living tissue more accurately. These models reduce the cost of drug development, enable more efficient drug screening, minimise failure rate in medicine discovery and eliminate animal use during experiments. The article provides an overview of 2D cell cultures and 3D tissue models–their properties, basic procedures, conditions of culturing and applications.
Background:
Tryptophan metabolites formed along kynurenine pathway may affect cell proliferation and tissue function. This pathway presents potential sites for drug discovery. Tryptophan and its metabolites kynurenine and kynurenic acid may be involved in the physiology and pathology of the ocular surface. The purpose of this work is to investigate the effect exerted by tryptophan, kynurenine and kynurenic acid upon corneal epithelium.
Methods:
A SkinEthic™ HCE human reconstructed corneal epithelium model was used. WST-1 test was used to examine cell proliferation and viability, and the Griess reaction for nitric oxide determination. The levels of IL-6 and IL-10 were measured by means of ELISA assay. All analyses were carried out after the cells were exposed to tryptophan, kynurenine and kynurenic acid at concentrations of 5, 50 or 100μM for 0-24h and 24-48h.
Results:
Tryptophan (100μM), kynurenine (100μM) and kynurenic acid (5-100μM) slightly increased the viability and proliferation of corneal epithelium. All of the tested compounds decreased cellular NO release. Kynurenine (50-100μM) and tryptophan (50-100μM) decreased while tryptophan (5μM) and kynurenic acid (100μM) increased the release of IL-6. All of the tested substances increased the level of IL-10 and decreased the IL-6 to IL-10 ratio.
Conclusions:
Tryptophan, kynurenine and kynurenic acid affect physiological processes in corneal epithelium and therefore may play a significant role in the physiology and pathology of the ocular surface. Our results indicate that the use of these compounds may be considered in the treatment of ocular surface diseases.
Here, we report the results of a prevalidation trial for an in vitro eye irritation test (EIT) using the reconstructed human cornea-like epithelium, MCTT HCE™. The optimal cutoff to determine irritation in the prediction model was established at 35% with the receiver operation characteristics(ROC) curve for 126 substances. Within-lab(WL) and between-lab(BL) reproducibility was tested for 20 reference substances by 3 participating laboratories. Viability data described by mean ± SD or ± 1/2 difference between duplicate wells, and scatter plots, demonstrated the WL/BL consistency. WL/BL concordance with the binary decision, whether non-irritant or irritant was estimated to be 85–95% and 95%, respectively. WL/BL reproducibility of viability data was further supported by a strong correlation(ICC, r > 0.9). WL/BL agreement of binary decisions was also examined by Fleiss' Kappa statistics, which showed a strong level of agreement (> 0.78), nevertheless weaker than the reproducibility of the viability. The EIT with MCTT HCE™ exhibited a sensitivity of 82.2% (60/73), a specificity of 81.1% (43/53), and an accuracy of 81.8% (103/126) for 126 reference substances (for liquids; a sensitivity of 100% (47/47), a specificity of 70.6% (24/34), and an accuracy of 87.7% (71/81), and for solids, a sensitivity of 50% (13/26), a specificity of 100% (19/19), and an accuracy of 71.1% (32/45), suggesting that the accuracy is satisfactory but the sensitivity needs improvement, which shall be addressed through correcting the poor sensitivity for solid substances in future full validation trials.
The assessment of ocular toxicity (i.e., eye irritation and serious eye damage) is important to ensure the safety of products and their components used in our daily life. In several EU legislations related to chemicals and products, the generation of information on eye irritation and serious eye damage represents a standard requirement.
The traditional eye irritation test, also known as the Draize eye test, is performed on albino rabbits. Ethical and scientific considerations as well as legal requirements in EU legislations have triggered the development and validation of alternative methods to the Draize test. Since no in vitro test taken individually can nowadays fully replace the Draize eye test, testing schemes combining strengths of particular in vitro assays were proposed. The conceptual framework of testing strategies based on a Bottom-Up or Top-Down approach is described in this chapter. In addition, a summary of the most promising in vitro test methods as well as their validation and regulatory acceptance status is given. Finally some strategic proposals on the way forward in this area are made.
Inhibition of hepatic efflux transporters (BSEP, MRP2) is one of the important mechanisms in drug-induced liver injury and in particular cholestasis. The inhibitory effect of compounds on these two hepatic transporters is commonly delineated using inverted membrane vesicles from Sf9 insect cells overexpressing the BSEP or MRP2 protein. However, due to lack of bioactivation and metabolism of the compound, and the lack of the expression and functionality of other sinusoidal efflux and uptake transporters in the cell-free assay system, it is difficult to fully understand the role of a given hepatic transporter in cholestasis. Therefore, our aim was to characterize and develop a competent cell-based bile canalicular imaging assay for measuring the overall inhibitory effects of compounds on biliary transport. Since species differences have been reported for some compounds, we developed the assay using both rat and human hepatocytes. Two fluorescent dyes, cholyl-lysyl-fluorescein (CLF) and carboxymethyl flourescein diacetate (CMFDA), have been reported in the literature to measure BSEP and MRP2 inhibition. We show in our study that neither CLF nor CMFDA is a specific substrate for either BSEP/Bsep or MRP2/Mrp2 as demonstrated by generating Km values for each transporter. Most of the drugs tested inhibited CLF accumulation and exhibited less potency toward CMFDA accumulation in hepatocytes. We found the highest concordance between IC50 values for inhibition of CLF accumulation in human hepatoyctes and IC50 values generated in the human BSEP vesicle assay. This suggests that the human vesicle-based assays could suffice as a primary screen to avoid future potential human liver injury.
In the last decades significant regulatory attempts were made to replace, refine and reduce animal testing to assess the risk of consumer products for the human eye. As the original in vivo Draize eye test has been criticized for limited predictivity, costs and ethical issues, several animal-free test methods have been developed to categorize substances according to the global harmonized system (GHS) for eye irritation.This review summarizes the progress of alternative test methods for the assessment of eye irritation. Based on the corneal anatomy and the current knowledge of the mechanisms causing eye irritation, different ex vivo and in vitro methods will be presented and discussed in regard of possible limitations and their status of regulatory acceptance. In addition to established in vitro models, this review will also highlight emerging, full thickness cornea models that might be applicable to predict all GHS categories.
Evaluation of the eye irritation is essential in the development of new cosmetic products. Draize rabbit eye irritation test has been widely used in which chemicals are directly applied to rabbit eye, and the symptoms and signs of eyes are scored. However, due to the invasive procedure, it causes substantial pain and discomfort to animals. Recently, we reported in vitro eye irritation test method using a 3D human corneal epithelial model (MCTT HCE™) which is reconstructed from remaining human tissues after a corneal transplantation. This model exhibited an excellent predictive capacity for 25 reference chemicals (sensitivity 100 %, specificity 77 % and accuracy 88 % vs. GHS). To improve the test performance, we explored new biomarkers for the eye irritation through transcriptomic approach. Three surfactants were selected as model eye irritants that include sodium lauryl sulfate, benzalkonium chloride and triton X-100. After test chemicals were treated, we investigated differentially expressed genes through a whole-gene microarray (Affymetrix GeneChip(®) Human Gene 2.0 ST Array, 48,000 probes). As a result, we identified that mRNAs of cornifelin (CNFN), a constituent of the insoluble cornified cell envelope of stratified squamous epithelia, and early growth response-1 (EGR1), a nuclear transcriptional regulator, were significantly up-regulated by all three irritants. Up-regulation of CNFN and EGR1 was further confirmed by Q-RT-PCR, and immunohistochemistry revealed increased level of CNFN in irritant-treated tissues, supporting the relevance of CNFN and EGR1 as new biomarkers for eye irritation.
In spite of over 20 years of effort, no single in vitro assay has been developed and validated as a full regulatory replacement for the Draize Eye Irritation test. However, companies have been using in vitro methods to screen new formulations and in some cases as their primary assessment of eye irritation potential for many years. The present report shows the outcome of an Expert Meeting convened by the European Centre for the Validation of Alternative Methods in February 2005 to identify test strategies for eye irritation. In this workshop test developers/users were requested to nominate methods to be considered as a basis for the identification of such testing strategies. Assays were evaluated and categorized based on their proposed applicability domains (e.g., categories of irritation severity, modes of action, chemical class, physicochemical compatibility). The analyses were based on the data developed from current practice and published studies, the ability to predict depth of injury (within the applicable range of severity), modes of action that could be addressed and compatibility with different physiochemical forms. The difficulty in predicting the middle category of irritancy (e.g. R36, GHS Categories 2A and 2B) was recognized. The testing scheme proposes using a Bottom-Up (begin with using test methods that can accurately identify non-irritants) or Top-Down (begin with using test methods that can accurately identify severe irritants) progression of in vitro tests (based on expected irritancy). Irrespective of the starting point, the approach would identify non-irritants and severe irritants, leaving all others to the (mild/moderate) irritant GHS 2/R36 categories.
The European Centre for the Validation of Alternative Methods (ECVAM) proposes to make the validation process more flexible, while maintaining its high standards. The various aspects of validation are broken down into independent modules, and the information necessary to complete each module is defined. The data required to assess test validity in an independent peer review, not the process, are thus emphasised. Once the information to satisfy all the modules is complete, the test can enter the peer-review process. In this way, the between-laboratory variability and predictive capacity of a test can be assessed independently. Thinking in terms of validity principles will broaden the applicability of the validation process to a variety of tests and procedures, including the generation of new tests, new technologies (for example, genomics, proteomics), computer-based models (for example, quantitative structure-activity relationship models), and expert systems. This proposal also aims to take into account existing information, defining this as retrospective validation, in contrast to a prospective validation study, which has been the predominant approach to date. This will permit the assessment of test validity by completing the missing information via the relevant validation procedure: prospective validation, retrospective validation, catch-up validation, or a combination of these procedures.
This multicentre study aimed at evaluating the reliability (reproducibility) and relevance (predictivity) of a new commercially available human corneal epithelial (HCE) model (SkinEthic Laboratories, Nice, France) to assess acute ocular irritation. A prevalidation approach (protocol optimisation, transfer and performance) was followed and at each of the four participating laboratories, 20 coded reference chemicals, covering the whole range of irritancy, were tested. The compounds were applied topically to the HCE cultures and the level of cytotoxicity (tissue viability and histological analysis) was determined. Once a standardised protocol was established, a high level of reproducibility between the laboratories was observed. In order to assess the capability of the HCE model to discriminate between irritants (I) and non-irritants (NI), a classification prediction model (PM) was defined based on a viability cut-off value of 60%. The obtained in vitro classifications were compared with different in vivo classifications (e.g. Globally Harmonised System) which were calculated from individual rabbit data described in the ECETOC data bank. Although an overall concordance of 80% was obtained (sensitivity = 100% and specificity = 56%), the predictivity of the HCE model substantially increased when other sources of in vivo and in vitro data were taken into account.
In the interest of reducing animal use, in vitro alternatives for skin sensitization testing are under development. One unifying characteristic of chemical allergens is the requirement that they react with proteins for the effective induction of skin sensitization. The majority of chemical allergens are electrophilic and react with nucleophilic amino acids. To determine whether and to what extent reactivity correlates with skin sensitization potential, 82 chemicals comprising allergens of different potencies and nonallergenic chemicals were evaluated for their ability to react with reduced glutathione (GSH) or with two synthetic peptides containing either a single cysteine or lysine. Following a 15-min reaction time with GSH, or a 24-h reaction time with the two synthetic peptides, the samples were analyzed by high-performance liquid chromatography. UV detection was used to monitor the depletion of GSH or the peptides. The peptide reactivity data were compared with existing local lymph node assay data using recursive partitioning methodology to build a classification tree that allowed a ranking of reactivity as minimal, low, moderate, and high. Generally, nonallergens and weak allergens demonstrated minimal to low peptide reactivity, whereas moderate to extremely potent allergens displayed moderate to high peptide reactivity. Classifying minimal reactivity as nonsensitizers and low, moderate, and high reactivity as sensitizers, it was determined that a model based on cysteine and lysine gave a prediction accuracy of 89%. The results of these investigations reveal that measurement of peptide reactivity has considerable potential utility as a screening approach for skin sensitization testing, and thereby for reducing reliance on animal-based test methods.
The corneal epithelium, comprising ve or six layers of differentiated ectodermal cells, protects the cornea and contributes to its optical properties (Lemp, 1987; Beuerman and Pedroza-Schmidt, 1996). Injury to the cornea presents a substantial threat to vision (Gasset et al., 1974; Clayton et al., 1985; Osgood et al., 1990). Thus, determining the risk of corneal injury from exposure to chemicals both at home and in the workplace is of critical importance.
This study investigated the potential of a new in vitro model to assess eye irritation. Three dimensional human reconstituted epithelial cultures (REC), grown from a squamous cell carcinoma line (TR146), were used to quantify the irritating potential of cosmetic formulations. A mean cytotoxicity index (MCI), reflecting the time course of toxicity for products applied to the cultures, was used as an endpoint to quantify irritation. Forty cosmetic formulated products were tested in both the in vitro cytotoxicity test and the traditional Draize test, The MCI obtained with this new model correlated (p < 0.001, Rp = 0.89) with a Draize modified maximum average score (MMAS). Across product categories tested, a simple linear correlation was found. In addition, it was possible to correctly identify 100% of the cosmetic test products into two classes: irritant or nonirritant by setting appropriate in vitro cut-off values. Despite the different types of formulae tested, high sensitivity, high specificity, and high concordance values were obtained, using this in vitro model. Its performance assessed through a Kappa test gave a value of K = 1.00. These preliminary results indicate the promise of this new three-dimensional human epithelial model for the in vitro assessment of eye irritation.
The 7 th amendment of the EU Cosmetic Directive will lead to the ban of animal testing for cosmetic ingredients in 2009 (acute tests). Many efforts have been made in order to find reliable and relevant alternatives methods to eye irritation testing. 3-D in vitro technology allows the evaluation of chemicals in conditions similar to in vivo use. In this study, we have used the SkinEthic Human Corneal Epithelial Model (HCE) to evaluate in vitro eye irritation potential of chemicals. Viability (MTT test) was used as the main endpoint to classify test-chemicals. The optimization of the in vitro protocol was based on a specific 60 min. contact time, adapted applied quantities (60 mg or 60µl/cm²) and a specific 16 hours post-treatment incubation period. A set of 102 chemicals belonging to different families, with known in vivo data was investigated in order to internally validate the protocol. The derived Predictive Model using a 50% viability cut off allowed the prediction of 2 classes: Irritants (R41 and R36 EU risk phrases) and Non Irritants (Non Classified). Performances were characterized by a good sensitivity (86%), a good specificity (83%) and good accuracy (84%). Results showed that the HCE model and the associated PM is a promising in vitro tool for the prediction of ocular irritancy.
Cosmetics Europe, The Personal Care Association (known as Colipa before 2012), conducted a program of technology transfer and within/between laboratory reproducibility of MatTek Corporation's EpiOcular(TM) Eye Irritation Test (EIT) as one of the two human reconstructed tissue test methods. This EIT EpiOcular(TM) used a single exposure period for each chemical and a prediction model based on a cut-off in relative survival [⩽ 60% = irritant (I) (GHS categories 2 and 1); > 60% = no classification (NC)]. Test substance single exposure time was 30 minutes with a 2-hour post-exposure incubation for liquids and 90 minutes with an 18-hour post-exposure incubation for solids. Tissue viability was determined by tetrazolium dye (MTT) reduction. Combinations of 20 coded chemicals were tested in 7 laboratories. Standardized laboratory documentation was used by all laboratories. Twenty liquids (11 NC/9 I) plus 5 solids (3 NC/2 I) were selected so that both exposure regimens could be assessed. Concurrent positive (methyl acetate) and negative (water) controls were tested in each trial. In all, 298 independent trials were performed and demonstrated 99.7% agreement in prediction (NC/I) across the laboratories. Coefficients of variation for the% survival for tissues from each treatment group across laboratories were generally low. This protocol has entered in 2010 the experimental phase of a formal ECVAM validation program.
The 7th amendment of the EU Cosmetics Directive led to the ban of eye irritation testing for cosmetic ingredients in animals, effective from March 11th 2009. Over the last 20years, many efforts have been made to find reliable and relevant alternative methods. The SkinEthic HCE model was used to evaluate the in vitro eye irritancy potential of substances from a cosmetic industry portfolio. An optimized protocol based on a specific 1-h treatment and a 16-h post-treatment incubation period was first assessed on a set of 102 substances. The prediction model (PM) based on a 50% viability cut-off, allowed to draw up two classes (Irritants and Non-Irritants), with good associated sensitivity (86.2%) and specificity (83.5%). To check the robustness of the method, the evaluated set was expanded up to 435 substances. Final performances maintained a high level and were characterized by an overall accuracy value > 82% when using EU or GHS classification rules. Results showed that the SkinEthic HCE test method is a promising in vitro tool for the prediction of eye irritancy. Optimization datasets were shared with the COLIPA Eye Irritation Project Team and ECVAM experts, and reviewed as part of an ongoing progression to enter an ECVAM prospective validation study for eye irritation.
A tetrazolium salt has been used to develop a quantitative colorimetric assay for mammalian cell survival and proliferation. The assay detects living, but not dead cells and the signal generated is dependent on the degree of activation of the cells. This method can therefore be used to measure cytotoxicity, proliferation or activation. The results can be read on a multiwell scanning spectrophotometer (ELISA reader) and show a high degree of precision. No washing steps are used in the assay. The main advantages of the colorimetric assay are its rapidity and precision, and the lack of any radioisotope. We have used the assay to measure proliferative lymphokines, mitogen stimulations and complement-mediated lysis.
The evaluation of eye and skin irritation potential is essential to ensuring the safety of individuals in contact with a wide variety of substances designed for industrial, pharmaceutical or cosmetic use. The Draize rabbit eye and skin irritancy tests have been used for 60 years to attempt to predict the human ocular and dermal irritation of such products. The Draize test has been the standard for ocular and dermal safety assessments for decades. However, several aspects of the test have been criticised. These include: the subjectivity of the method; the overestimation of human responses; and the method's cruelty. The inadequacies of the Draize test have led to several laboratories over the last 20 years making efforts to develop in vitro assays to replace it. Protocols that use different types of cell cultures and other methods have been devised to study eye and skin irritation. Different commercial kits have also been developed to study eye and skin irritation, based on the action of chemicals on these tissues. This article presents a review of the main alternatives developed to replace the use of animals in the study of chemical irritation. Particular attention is paid to the reproducibility of each method.
Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67
- Eu
EU, 2008. Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No. 1907/2006. Official Journal of the European Union L 353/1-1354.
Three-dimensional construct of the human corneal epithelium for in vitro toxicology Alternative Toxicological Methods
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Nguyen, D.H., Beuerman, R.W., de Wever, B., Rosdy, M., 2003. Three-dimensional construct of the human corneal epithelium for in vitro toxicology. In: Salem, H., Katz, S.A. (Eds.), Alternative Toxicological Methods. CRC Press, pp. 147–159.
the peptide reactivity was 100% concordant for the two lab-oratories. This demonstrates the effectiveness of the training
- Lysine
Lysine, the peptide reactivity was 100% concordant for the two lab-oratories. This demonstrates the effectiveness of the training.
The use of alternative methods and clinical tests in the ocular risk assessment of cosmetic formulations
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Directive 2003/15/EC of the European parliament and of the council of 27 February 2003 amending council directive 76/768/EEC on the approximation of the laws of the member states relating to cosmetic products
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EU, 2003. Directive 2003/15/EC of the European parliament and of the council of 27 February 2003 amending council directive 76/768/EEC on the approximation of the laws of the member states relating to cosmetic products. Official Journal of the European Union L66, 26–35.