Helena Kandarova

Comparison of human skin irritation patch test data with in vitro skin irritation assays and animal data

Background: Efforts to replace the rabbit skin irritation test have been underway for many years, encouraged by the EU Cosmetics Directive and REACH. Recently various in vitro tests have been developed, evaluated and validated.

An In Vitro Skin Irritation Test (SIT) using the EpiDerm Reconstructed Human Epidermal (RHE) Model

The EpiDerm Skin Irritation test (EpiDerm SIT) was developed and validated for in vitro skin irritation testing of chemicals, including cosmetic and pharmaceutical ingredients. The EpiDerm SIT utilizes the 3D in vitro reconstructed human epidermal (RHE) model EpiDerm. The procedure described in this protocol allows for discrimination between irritants of GHS category 2 and non-irritants. The test is performed over the course of a 4 day time period, consisting of pre-incubation, 60 minute exposure, 42 hour post-incubation and MTT viability assay. After tissue receipt and overnight pre-incubation (Day 0), tissues are topically exposed to the test chemicals (Day 1), which can be liquid, semisolid, solid or waxy. Three tissues are used for each test chemical, as well as for the positive control (5% aq. SDS solution), and a negative control (DPBS). Chemical exposure lasts for 60 minutes, 35 min of which the tissues are kept in an incubator at 37 degrees C. The test substances are then removed from the tissue surface by an extensive washing procedure. The tissue inserts are blotted and transferred to fresh medium. After a 24 hr incubation period (Day 2), the medium is exchanged. The medium can be saved for further analysis of cytokines or other endpoints of interest. After the medium exchange, tissues are incubated for an additional 18 hours. At the end of the entire 42 h post-incubation (day 3), the tissues are transferred into yellow MTT solution and incubated for 3 hours. The resultant purple-blue formazan salt, formed mainly by mitochondrial metabolism, is extracted for 2 hours using isopropanol. The optical density of the extracted formazan is determined using a spectrophotometer. A chemical is classified as an irritant if the tissue viability relative to the negative control treated tissues is reduced below 50%. This procedure can be used as full replacement of the in vivo rabbit skin irritation test for hazard identification and labeling of chemicals in line with EU regulations.

Review of skin irritation/corrosion Hazards on the basis of human data: A regulatory perspective.

Abstract Regulatory classification of skin irritation has historically been based on rabbit data, however current toxicology processes are transitioning to in vitro alternatives. The in vitro assays have to provide sufficient level of sensitivity as well as specificity to be accepted as replacement methods for the existing in vivo assays. This is usually achieved by comparing the in vitro results to classifications obtained in animals. Significant drawback of this approach is that neither in vivo nor in vitro methods are calibrated against human hazard data and results obtained in these assays may not correspond to situation in human. The main objective of this review was to establish an extended database of substances classified according to their human hazard to serve for further development of alternative methods relevant to human health as well as resource for improved regulatory classification. The literature has been reviewed to assemble all the available information on the testing of substances in the human 4 h human patch test, which is the only standardized protocol in humans matching the exposure conditions of the regulatory accepted in vivo rabbit skin irritation test. A total of 81 substances tested according to the defined 4 h human patch test protocol were found and collated into a dataset together with their existing in vivo classifications published in the literature. While about 50% of the substances in the database are classified as irritating based on the rabbit skin test, on using the 4 h HPT test, less than 20% were identified as acutely irritant to human skin. Based on the presented data, it can be concluded that the rabbit skin irritation test largely over-predicts human responses for the evaluated chemicals. Correct classification of the acute skin irritation hazard will only be possible if newly developed in vitro toxicology methods will be calibrated to produce results relevant to man.

Two novel prediction models improve predictions of skin corrosive sub-categories by test methods of OECD Test Guideline No. 431

Alternative test methods often use prediction models (PMs) for converting endpoint measurements into predictions. Two PMs are used for the skin corrosion tests (SCTs) of the OECD Test Guideline No. 431 (TG 431). One is specific to EpiSkin™ test method, whereas EpiDerm™, SkinEthic™ RHE and epiCS® share a common PM. These methods are based on reconstructed human epidermis models wherein cell viability values are measured. Their PMs allow translating those values into sub-categories of corrosive chemicals, Category 1A (Cat1A) and a combination of Categories 1B/1C (Cat1BC), and identifying non-corrosive (NC) chemicals. EpiSkin™s PM already results in sufficiently accurate predictions. The common PM of the three others accurately identifies all corrosive chemicals but, for sub-categorization, an important fraction of Cat1BC chemicals (40-50%) is over-predicted as Cat1A. This paper presents a post-hoc analysis of validation data on a set of n=80 chemicals. It investigates: why this common PM causes these over-predictions and how two novel PMs that we developed (PMvar1 and PMvar2) improve the predictive capacity of these methods. PMvar1 is based on a two-step approach; PMvar2 is based on a single composite indicator of cell viability. Both showed a greater capacity to predict Cat1BC, while Cat1A correct predictions remaining at least at the same level of EpiSkin™. We suggest revising TG 431, to include the novel PMs in view of improving the predictive capacity of its SCTs.

CON4EI: Selection of the reference chemicals for hazard identification and labelling of eye irritating chemicals

Assessment of the acute eye irritation potential is part of the international regulatory requirements for testing of chemicals. In the past, several prospective and retrospective validation studies have taken place in the area of serious eye damage/eye irritation testing. Success in terms of complete replacement of the regulatory in vivo Draize rabbit eye test has not yet been achieved. A very important aspect to ensure development of successful alternative test methods and/or strategies for serious eye damage/eye irritation testing is the selection of appropriate reference chemicals. A set of 80 reference chemicals was selected for the CEFIC-LRI-AIMT6-VITO CON4EI (CONsortium for in vitro Eye Irritation testing strategy) project, in collaboration with Cosmetics Europe, from the Draize Reference Database published by Cosmetics Europe based on key criteria that were set in their paper (e.g. balanced by important driver of classification and physical state). The most important goals of the CON4EI project were to identify the performance of eight in vitro alternative tests in terms of driver of classification and to identify similarities/differences between the methods in order the build a successful testing strategy that can discriminate between all UN GHS categories. This paper provides background on selection of the test chemicals.

CON4EI: Short Time Exposure (STE) test method for hazard identification and labelling of eye irritating chemicals

Assessment of ocular irritancy is an international regulatory requirement in the safety evaluation of industrial and consumer products. Although many in vitro ocular irritation assays exist, alone they are incapable of fully categorizing chemicals. Therefore, the CEFIC-LRI-AIMT6-VITO CON4EI consortium was developed to assess the reliability of eight in vitro test methods and establish an optimal tiered-testing strategy. One assay selected was the Short Time Exposure (STE) assay. This assay measures the viability of SIRC rabbit corneal cells after 5min exposure to 5% and 0.05% solutions of test material, and is capable of categorizing of Category 1 and No Category chemicals. The accuracy of the STE test method to identify Cat 1 chemicals was 61.3% with 23.7% sensitivity and 95.2% specificity. If non-soluble chemicals and unqualified results were excluded, the performance to identify Cat 1 chemicals remained similar (accuracy 62.2% with 22.7% sensitivity and 100% specificity). The accuracy of the STE test method to identify No Cat chemicals was 72.5% with 66.2% sensitivity and 100% specificity. Excluding highly volatile chemicals, non-surfactant solids and non-qualified results resulted in an important improvement of the performance of the STE test method (accuracy 96.2% with 81.8% sensitivity and 100% specificity). Furthermore, it seems that solids are more difficult to test in the STE, 71.4% of the solids resulted in unqualified results (solubility issues and/or high variation between independent runs) whereas for liquids 13.2% of the results were not qualified, supporting the restriction of the test method regarding the testing of solids.

CON4EI: SkinEthic™ Human Corneal Epithelium Eye Irritation Test (SkinEthic™ HCE EIT) for hazard identification and labelling of eye irritating chemicals

Assessment of ocular irritancy is an international regulatory requirement and a necessary step in the safety evaluation of industrial and consumer products. Although a number of in vitro ocular irritation assays exist, none are capable of fully categorizing chemicals as a stand-alone assay. Therefore, the CEFIC-LRI-AIMT6-VITO CON4EI (CONsortium for in vitro Eye Irritation testing strategy) project was developed with the goal of assessing the reliability of eight in vitro/alternative test methods as well as establishing an optimal tiered-testing strategy. One of the in vitro assays selected was the validated SkinEthic™ Human Corneal Epithelium Eye Irritation Test method (SkinEthic™ HCE EIT). The SkinEthic™ HCE EIT has already demonstrated its capacity to correctly identify chemicals (both substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage (No Category). The goal of this study was to evaluate the performance of the SkinEthic™ HCE EIT test method in terms of the important in vivo drivers of classification. For the performance with respect to the drivers all in vivo Cat 1 and No Cat chemicals were 100% correctly identified. For Cat 2 chemicals the liquids and the solids had a sensitivity of 100% and 85.7%, respectively. For the SkinEthic™ HCE EIT test method, 100% concordance in predictions (No Cat versus No prediction can be made) between the two participating laboratories was obtained. The accuracy of the SkinEthic™ HCE EIT was 97.5% with 100% sensitivity and 96.9% specificity. The SkinEthic™ HCE EIT confirms its excellent results of the validation studies.

Preparation of irritant polymer samples for an in vitro round robin study

A round robin study using reconstructed human epidermis (RhE) tissues was conducted to test medical device polymer extracts for skin irritation potential. Test samples were four irritant and three non-irritant medical device polymers. Five of these polymer samples were developed and two were obtained commercially. The three non-irritant samples were comprised of 100% 80A polyurethane, one-part silicone, and polyvinyl chloride (PVC). The polyurethane samples were made using a hot-melt process, while the silicone samples were created by mixing and casting. The PVC samples were commercially produced sheets. The four irritant samples were comprised of one-part silicone and 25% heptanoic acid (HA), two-part silicone and 15% sodium dodecyl sulfate (SDS), PVC and 4% Genapol® X-100, and PVC and 5.8% Genapol® X-080. The HA, SDS, and Genapol® X-100 samples were produced using the mixing and casting method, while the Genapol® X-080 sheet samples were obtained commercially. During development, irritant polymer samples were extracted using polar and non-polar solvents that were subsequently analyzed chemically. Samples with sufficient levels of extracted irritants were tested on RhE tissues to confirm their irritation potential. Polymers that passed this screening test were used in the round robin study described elsewhere in this special edition.

CON4EI: EpiOcular™ Eye Irritation Test (EpiOcular™ EIT) for hazard identification and labelling of eye irritating chemicals

Assessment of the acute eye irritation potential is part of the international regulatory requirements for testing of chemicals. The objective of the CON4EI project was to develop tiered testing strategies for eye irritation assessment. A set of 80 reference chemicals (38 liquids and 42 solids) was tested with eight different methods. Here, the results obtained with the EpiOcular™ Eye Irritation Test (EIT), adopted as OECD TG 492, are shown. The primary aim of this study was to evaluate of the performance of the test method to discriminate between chemicals not requiring classification for serious eye damage/eye irritancy (No Category) and chemicals requiring classification and labelling. In addition, the predictive capacity in terms of in vivo drivers of classification (i.e. corneal opacity, conjunctival redness and persistence at day 21) was investigated. EpiOcular™ EIT achieved a sensitivity of 97%, a specificity of 87% and accuracy of 95% and also confirmed its excellent reproducibility (100%) from the original validation. The assay was applicable to all chemical categories tested in this project and its performance was not limited to the particular driver of the classification. In addition to the existing prediction model for dichotomous categorization, a new prediction model for Cat 1 is suggested.

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&A) for liquids, 100.0/68.4/ and 84.6% SS&A for solids, and 97.6/68.3/ and 83.1% for overall SS&A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.