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The focus of Cosmetics Europe's programme on serious eye damage/eye irritation is on development of testing strategies and defined approaches for identification of ocular effects of chemicals in the context of OECD's Guidance Document on an Integrated Approach on Testing and Assessment (IATA) for Serious Eye Damage and Eye Irritation. Cosmetics Europe created a comprehensive database of chemicals for which in vitro data are available with corresponding historical in vivo Draize eye data. This database allowed further exploration of the initially proposed strategies from the CON4EI project and to identify opportunities for refinement. The current analysis focused on the development of a defined approach, applicable to liquid non-surfactant chemicals, neat and in dilution, that can distinguish between the three UN GHS categories (Cat. 1, Cat. 2, and No Cat.). Combining the modified-protocol Short Time Exposure (STE) test method (OECD TG 491 with extension to highly volatile substances) with the Bovine Corneal Opacity and Permeability Laser Light-Based Opacitometer (BCOP LLBO) test method in a Bottom-Up approach identified 81.2% Cat. 1, 56.3% Cat. 2, and 85.3% No. Cat correctly, with an NPV of 96.7% and a PPV of 68.6%. Therefore, the performance of the defined approach was better than the standalone test methods., (Copyright © 2019. Published by Elsevier Ltd.)

A thorough understanding of which of the effects assessed in the in vivo Draize eye test are responsible for driving UN GHS/EU CLP classification is critical for an adequate selection of chemicals to be used in the development and/or evaluation of alternative methods/strategies and for properly assessing their predictive capacity and limitations. For this reason, Cosmetics Europe has compiled a database of Draize data (Draize eye test Reference Database, DRD) from external lists that were created to support past validation activities. This database contains 681 independent in vivo studies on 634 individual chemicals representing a wide range of chemical classes. A description of all the ocular effects observed in vivo, i.e. degree of severity and persistence of corneal opacity (CO), iritis, and/or conjunctiva effects, was added for each individual study in the database, and the studies were categorised according to their UN GHS/EU CLP classification and the main effect driving the classification. An evaluation of the various in vivo drivers of classification compiled in the database was performed to establish which of these are most important from a regulatory point of view. These analyses established that the most important drivers for Cat 1 Classification are (1) CO mean ≥ 3 (days 1-3) (severity) and (2) CO persistence on day 21 in the absence of severity, and those for Cat 2 classification are (3) CO mean ≥ 1 and (4) conjunctival redness mean ≥ 2. Moreover, it is shown that all classifiable effects (including persistence and CO = 4) should be present in ≥60 % of the animals to drive a classification. As a consequence, our analyses suggest the need for a critical revision of the UN GHS/EU CLP decision criteria for the Cat 1 classification of chemicals. Finally, a number of key criteria are identified that should be taken into consideration when selecting reference chemicals for the development, evaluation and/or validation of alternative methods and/or strategies for serious eye damage/eye irritation testing. Most important, the DRD is an invaluable tool for any future activity involving the selection of reference chemicals., Competing Interests: The authors declare that they have no conflict of interest.

A.I.S.E. investigated the suitability of the regulatory adopted ICE in vitro test method (OECD TG 438) with or without histopathology to identify detergent and cleaning formulations having extreme pH that require classification as EU CLP/UN GHS Category 1. To this aim, 18 extreme pH detergent and cleaning formulations were tested covering both alkaline and acidic extreme pHs. The ICE standard test method following OECD Test Guideline 438 showed good concordance with in vivo classification (83%) and good and balanced specificity and sensitivity values (83%) which are in line with the performances of currently adopted in vitro test guidelines, confirming its suitability to identify Category 1 extreme pH detergent and cleaning products. In contrast to previous findings obtained with non-extreme pH formulations, the use of histopathology did not improve the sensitivity of the assay whilst it strongly decreased its specificity for the extreme pH formulations. Furthermore, use of non-testing prediction rules for classification showed poor concordance values (33% for the extreme pH rule and 61% for the EU CLP additivity approach) with high rates of over-prediction (100% for the extreme pH rule and 50% for the additivity approach), indicating that these non-testing prediction rules are not suitable to predict Category 1 hazards of extreme pH detergent and cleaning formulations., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A.I.S.E. investigated the suitability of histopathological evaluations as an additional endpoint to the regulatory adopted ICE in vitro test method (OECD TG 438) to identify non-extreme pH detergent and cleaning products that require classification as EU CLP/UN GHS Category 1 (serious eye damage). To this aim, a total of 30 non-extreme pH products covering the range of in vivo classifications for eye irritation, and representing various product categories were tested. Epithelium vacuolation (mid and lower layers) and erosion (at least moderate) were found to be the most relevant histopathological effects induced by products classified in vivo as Category 1. Histopathology criteria specifically developed for non-extreme pH detergent and cleaning products were shown to correctly identify materials classified as Category 1 based on in vivo persistent effects, and to significantly increase the overall sensitivity of the standard ICE prediction model for Category 1 identification (to 75%) whilst maintaining a good concordance (73%). In contrast, use of EU CLP additivity approach for classification of mixtures was considerably less predictive, with a concordance of only 27%, and 100% over-predictions of non-Category 1 products. As such, use of histopathology as an addition to the ICE test method was found suitable to identify EU CLP/UN GHS Category 1 non-extreme pH detergent and cleaning products and to allow a better discrimination from Category 2 products., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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 (<4 % of both Cat 1 and Cat 2 chemicals). The two most important endpoints driving Cat 2 classification are conjunctiva redness (75-81 %) and corneal opacity (54-75 %). The resampling analyses demonstrated an overall probability of at least 11 % that chemicals classified as Cat 1 by the Draize eye test could be equally identified as Cat 2 and of about 12 % for Cat 2 chemicals to be equally identified as No Cat. On the other hand, the over-classification error for No Cat and Cat 2 was negligible (<1 %), which strongly suggests a high over-predictive power of the Draize eye test. Moreover, our analyses of the classification drivers suggest a critical revision of the UN GHS/EU CLP decision criteria for the classification of chemicals based on Draize eye test data, in particular Cat 1 based only on persistence of conjunctiva effects or corneal opacity scores of 4. In order to successfully replace the regulatory in vivo Draize eye test, it will be important to recognise these uncertainties and to have in vitro tools to address the most important in vivo endpoints identified in this paper.

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™ Eye Irritation Test (EIT) as one of the two human reconstructed tissue test methods. This EIT EpiOcular™ 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 min with a 2-h post-exposure incubation for liquids and 90 min with an 18-h 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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The need for alternative approaches to replace the in vivo rabbit Draize eye test for evaluation of eye irritation of cosmetic ingredients has been recognised by the cosmetics industry for many years. Extensive research has lead to the development of several assays, some of which have undergone formal validation. Even though, to date, no single in vitro assay has been validated as a full replacement for the rabbit Draize eye test, organotypic assays are accepted for specific and limited regulatory purposes. Although not formally validated, several other in vitro models have been used for over a decade by the cosmetics industry as valuable tools in a weight of evidence approach for the safety assessment of ingredients and finished products. In light of the deadlines established in the EU Cosmetics Directive for cessation of animal testing for cosmetic ingredients, a COLIPA scientific meeting was held in Brussels on 30th January, 2008 to review the use of alternative approaches and to set up a decision-tree approach for their integration into tiered testing strategies for hazard and safety assessment of cosmetic ingredients and their use in products. Furthermore, recommendations are given on how remaining data gaps and research needs can be addressed.

The focus of Cosmetics Europe's ocular toxicity programme is on development of testing strategies and defined approaches for identification of ocular effects of chemicals in the context of OECD's Guidance Document on an Integrated Approach on Testing and Assessment (IATA) for Serious Eye Damage and Eye Irritation. Cosmetics Europe created a comprehensive database of chemicals for which in vitro data are available with corresponding historical in vivo Draize eye data and physicochemical properties. This database allowed further exploration of the initially proposed strategies from the CON4EI project and to identify opportunities for refinement. One key outcome of this project is that combining in vitro test methods (RhCE and BCOP LLBO) with physicochemical properties in a two-step Bottom-Up approach applicable to neat liquids, resulted in an improvement of the specificity, without reducing the sensitivity, when compared to the combination of in vitro methods alone. The Bottom-Up approach proposed here for neat liquids correctly predicted 58.3% (EpiOcular™ EIT followed by BCOP LLBO) to 62.6% (SkinEthic™ HCE EIT followed by BCOP LLBO) of No Cat., 59.1% to 68.7% of Cat. 2, and 76.5% of Cat. 1. Incorporating specific physicochemical properties with this Bottom-Up approach increased the correct identification of No Cat. neat liquids to between 72.7% and 79.7%. • Develop defined approach (DA) for assessment of serious eye damage/eye irritation • Development of exclusion rules for physicochemical properties to identify No Cat. • Proposed DA's: 2 combinations of RhCE test methods with the BCOP LLOB • Incorporation of physicochemical property exclusion rules with DA's improve their specificity. [ABSTRACT FROM AUTHOR]

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 (10min – SE) and the other for long time exposure (60min – 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 analysed were categorised 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. [Copyright &y& Elsevier]