A.R. Van Rompay

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.

Multi-laboratory validation of SkinEthic HCE test method for testing serious eye damage/eye irritation using liquid chemicals.

A prospective multicentric study of the reconstructed human corneal epithelial tissue-based in vitro test method (SkinEthic™ HCE) was conducted to evaluate its usefulness to identify chemicals as either not classified for serious eye damage/eye irritation (No Cat.) or as classified (Cat. 1/Cat. 2) within UN GHS. The aim of this study was to demonstrate the transferability and reproducibility of the SkinEthic™ HCE EITL protocol for liquids and define its predictive capacity. Briefly, 60 chemicals were three times tested (double blinded) in 3 laboratories and 45 additional chemicals were tested three times in one laboratory. Good within laboratory reproducibility was achieved of at least 88.3% (53/60) and 92.4% (97/105) for the extended data set. Furthermore, the overall concordance between the laboratories was 93.3% (56/60). The accuracy of the SkinEthic™ HCE EITL for the extended dataset, based on bootstrap resampling, was 84.4% (95% CI: 81.9% to 87.6%) with a sensitivity of 99.0% (95% CI: 96.4% to 100%) and specificity of 68.5% (95% CI: 64.0% to 74.0%), thereby meeting all acceptance criteria for predictive capacity. This efficient transferable and reproducible assay is a promising tool to be integrated within a battery of assays to perform an eye irritation risk assessment.

Multi-laboratory evaluation of SkinEthic HCE test method for testing serious eye damage/eye irritation using solid chemicals and overall performance of the test method with regard to solid and liquid chemicals testing

A prospective multicentre study of the reconstructed human corneal epithelial tissue-based in vitro test method (SkinEthic™ HCE) was conducted to evaluate its usefulness to identify chemicals as either not classified for serious eye damage/eye irritation (No Cat.) or as classified (Cat. 1/Cat. 2) within UN GHS. The aim of this study was to demonstrate the transferability and reproducibility of the SkinEthic™ HCE EITS protocol for solids and define its predictive capacity. Briefly, 60 chemicals were three times tested (double blinded) in 3 laboratories and 35 additional chemicals were tested three times in one laboratory. Good within laboratory reproducibility was achieved of at least 95% (57/60) and 96.8% (92/95) for the extended data set. Furthermore, the overall concordance between the laboratories was 96.7% (58/60). The accuracy of the SkinEthic™ HCE EITS for the extended dataset, based on bootstrap resampling, was 81.0% (95% CI: 78.9% to 83.2%) with a sensitivity of 90.5% (95% CI: 88.1% to 92.9%) and specificity of 73.6% (95% CI: 71.7% to 75.5%). Overall, 200 chemicals were tested (105 liquids (EITL protocol) and 95 solids (EITS protocol)) resulting in a sensitivity of 95.2%, specificity of 72.1% and accuracy of 83.7%, thereby meeting all acceptance criteria for predictive capacity.

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.

CON4EI: Development of testing strategies for hazard identification and labelling for serious eye damage and eye irritation of chemicals

Assessment of acute eye irritation potential is part of the international regulatory requirements for safety testing of chemicals. In the last decades, many efforts have been made in the search for alternative methods to replace the regulatory in vivo Draize rabbit eye test (OECD TG 405). Success in terms of complete replacement of the regulatory in vivo Draize rabbit eye test has not yet been achieved. The main objective of the CEFIC-LRI-AIMT6-VITO CON4EI (CONsortium for in vitro Eye Irritation testing strategy) project was to develop tiered testing strategies for serious eye damage and eye irritation assessment that can lead to complete replacement of OECD TG 405. A set of 80 reference chemicals (e.g. balanced by important driver of classification and physical state), was tested with seven test methods. Based on the results of this project, three different strategies were suggested. We have provided a standalone (EpiOcular ET-50), a two-tiered and three-tiered strategy, that can be used to distinguish between Cat 1 and Cat 2 chemicals and chemicals that do not require classification (No Cat). The two-tiered and three-tiered strategies use an RhCE test method (EpiOcular EIT or SkinEthic™ EIT) at the bottom (identification No Cat) in combination with the BCOP LLBO (two-tiered strategy) or BCOP OP-KIT and SMI (three-tiered strategy) at the top (identification Cat 1). For our proposed strategies, 71.1% - 82.9% Cat 1, 64.2% - 68.5% Cat 2 and ≥80% No Cat chemicals were correctly identified. Also, similar results were obtained for the Top-Down and Bottom-Up approach.

CON4EI: Evaluation of QSAR models for hazard identification and labelling of eye irritating chemicals

Assessment of ocular irritation is a regulatory requirement in 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 stand-alone assays. Therefore, the CEFIC-LRI-AIMT6-VITO CON4EI (CONsortium for in vitro Eye Irritation testing strategy) project was developed to assess the reliability of eight in vitro test methods and computational models as well as establishing an optimal tiered-testing strategy. For three computational models (Toxtree, and Case Ultra EYE_DRAIZE and EYE_IRR) performance parameters were calculated. Coverage ranged from 15 to 58%. Coverage was 2 to 3.4 times higher for liquids than for solids. The lowest number of false positives (5%) was reached with EYE_IRR; this model however also gave a high number of false negatives (46%). The lowest number of false negatives (25%) was seen with Toxtree; for liquids Toxtree predicted the lowest number of false negatives (11%), for solids EYE_DRAIZE did (17%). It can be concluded that the training sets should be enlarged with high quality data. The tested models are not yet sufficiently powerful for stand-alone evaluations, but that they can surely become of value in an integrated weight-of-evidence approach in hazard assessment.

CON4EI: Slug Mucosal Irritation (SMI) test method for hazard identification and labelling of serious eye damaging and 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. The objective of CEFIC-LRI-AIMT6-VITO CON4EI (CONsortium for in vitro Eye Irritation testing strategy) project was to develop tiered testing strategies for eye irritation assessment that can lead to complete replacement of the in vivo Draize rabbit eye test (OECD TG 405). A set of 80 reference chemicals was tested with seven test methods, one method was the Slug Mucosal Irritation (SMI) test method. The method measures the amount of mucus produced (MP) during a single 1-hour contact with a 1% and 10% dilution of the chemical. Based on the total MP, a classification (Cat 1, Cat 2, or No Cat) is predicted. The SMI test method correctly identified 65.8% of the Cat 1 chemicals with a specificity of 90.5% (low over-prediction rate for in vivo Cat 2 and No Cat chemicals). Mispredictions were predominantly unidirectional towards lower classifications with 26.7% of the liquids and 40% of the solids being underpredicted. In general, the performance was better for liquids than for solids with respectively 76.5% vs 57.1% (Cat 1), 61.5% vs 50% (Cat 2), and 87.5% vs 85.7% (No Cat) being identified correctly.