Local Tolerance Testing Under REACH: Accepted Non-animal Methods Are Not on Equal Footing with Animal Tests
Ursula G. Sauer, Erin H. Hill, Rodger D. Curren, Susanne N. Kolle, Wera Teubner, Annette Mehling and Robert Landsiedel
In general, no single non-animal method can cover the complexity of any given animal test. Therefore, fixed sets of in vitro (and in chemico) methods have been combined into testing strategies for skin and eye irritation and skin sensitisation testing, with pre-defined prediction models for substance classification. Many of these methods have been adopted as OECD test guidelines. Various testing strategies have been successfully validated in extensive in-house and inter-laboratory studies, but they have not yet received formal acceptance for substance classification. Therefore, under the European REACH Regulation, data from testing strategies can, in general, only be used in so-called weight-of-evidence approaches. While animal testing data generated under the specific REACH information requirements are per se sufficient, the sufficiency of weight-of-evidence approaches can be questioned under the REACH system, and further animal testing can be required. This constitutes an imbalance between the regulatory acceptance of data from approved non-animal methods and animal tests that is not justified on scientific grounds. To ensure that testing strategies for local tolerance testing truly serve to replace animal testing for the REACH registration 2018 deadline (when the majority of existing chemicals have to be registered), clarity on their regulatory acceptance as complete replacements is urgently required.
Felix Spöler, Oya Kray, Stefan Kray, Claudia Panfil, and Norbert F. Schrage
Ocular irritation testing is a common requirement for the classification, labelling and packaging of chemicals (substances and mixtures). The in vivo Draize rabbit eye test (OECD Test Guideline 405) is considered to be the regulatory reference method for the classification of chemicals according to their potential to induce eye injury. In the Draize test, chemicals are applied to rabbit eyes in vivo, and changes are monitored over time. If no damage is observed, the chemical is not categorised. Otherwise, the classification depends on the severity and reversibility of the damage. Alternative test methods have to be designed to match the classifications from the in vivo reference method. However, observation of damage reversibility is usually not possible in vitro. Within the present study, a new organotypic method based on rabbit corneas obtained from food production is demonstrated to close this gap. The Ex Vivo Eye Irritation Test (EVEIT) retains the full biochemical activity of the corneal epithelium, epithelial stem cells and endothelium. This permits the in-depth analysis of ocular chemical trauma beyond that achievable by using established in vitro methods. In particular, the EVEIT is the first test to permit the direct monitoring of recovery of all corneal layers after damage. To develop a prediction model for the EVEIT that is comparable to the GHS system, 37 reference chemicals were analysed. The experimental data were used to derive a three-level potency ranking of eye irritation and corrosion that best fits the GHS categorisation. In vivo data available in the literature were used for comparison. When compared with GHS classification predictions, the overall accuracy of the three-level potency ranking was 78%. The classification of chemicals as irritating versus non-irritating resulted in 96% sensitivity, 91% specificity and 95% accuracy.
The EpiOcular™ Eye Irritation Test is the Method of Choice for the In Vitro Eye Irritation Testing of Agrochemical Formulations:
Correlation Analysis of EpiOcular Eye Irritation Test and BCOP Test Data According to the UN GHS, US EPA and Brazil ANVISA Classification Schemes
Susanne N. Kolle, Maria Cecilia Rey Moreno, Winfried Mayer, Andrew van Cott, Bennard van Ravenzwaay and Robert Landsiede
The Bovine Corneal Opacity and Permeability (BCOP) test is commonly used for the identification of severe ocular irritants (GHS Category 1), but it is not recommended for the identification of ocular irritants (GHS Category 2). The incorporation of human reconstructed tissue model-based tests into a tiered test strategy to identify ocular non-irritants and replace the Draize rabbit eye irritation test has been suggested (OECD TG 405). The value of the EpiOcular™ Eye Irritation Test (EIT) for the prediction of ocular non-irritants (GHS No Category) has been demonstrated, and an OECD Test Guideline (TG) was drafted in 2014. The purpose of this study was to evaluate whether the BCOP test, in conjunction with corneal histopathology (as suggested for the evaluation of the depth of the injury) and/or the EpiOcular-EIT, could be used to predict the eye irritation potential of agrochemical formulations according to the UN GHS, US EPA and Brazil ANVISA classification schemes. We have assessed opacity, permeability and histopathology in the BCOP assay, and relative tissue viability in the EpiOcular-EIT, for 97 agrochemical formulations with available in vivo eye irritation data. By using the OECD TG 437 protocol for liquids, the BCOP test did not result in sufficient correct predictions of severe ocular irritants for any of the three classification schemes. The lack of sensitivity could be improved somewhat by the inclusion of corneal histopathology, but the relative viability in the EpiOcular-EIT clearly outperformed the BCOP test for all three classification schemes. The predictive capacity of the EpiOcular-EIT for ocular non-irritants (UN GHS No Category) for the 97 agrochemical formulations tested (91% sensitivity, 72% specificity and 82% accuracy for UN GHS classification) was comparable to that obtained in the formal validation exercise underlying the OECD draft TG. We therefore conclude that the EpiOcular-EIT is currently the best in vitro method for the prediction of the eye irritation potential of liquid agrochemical formulations.
The EpiOcular Eye Irritation Test (EIT) for Hazard Identification and Labelling of Eye Irritating Chemicals: Protocol Optimisation for Solid Materials and the Results after Extended Shipment
Yulia Kaluzhny, Helena Kandárová, Yuki Handa, Jane DeLuca, Thoa Truong, Amy Hunter, Paul Kearney, Laurence d’Argembeau-Thornton and Mitchell Klausner
The 7th Amendment to the EU Cosmetics Directive and the EU REACH Regulation have reinforced the need for in vitro ocular test methods. Validated in vitro ocular toxicity tests that can predict the human response to chemicals, cosmetics and other consumer products are required for the safety assessment of materials that intentionally, or inadvertently, come into contact with the eye. The EpiOcular Eye Irritation Test (EIT), which uses the normal human cell-based EpiOcular™ tissue model, was developed to address this need. The EpiOcular-EIT is able to discriminate, with high sensitivity and accuracy, between ocular irritant/corrosive materials and those that require no labelling. Although the original EpiOcular-EIT protocol was successfully pre-validated in an international, multicentre study sponsored by COLIPA (the predecessor to Cosmetics Europe), data from two larger studies (the EURL ECVAM–COLIPA validation study and an independent in-house validation at BASF SE) resulted in a sensitivity for the protocol for solids that was below the acceptance criteria set by the Validation Management Group (VMG) for eye irritation, and indicated the need for improvement of the assay’s sensitivity for solids. By increasing the exposure time for solid materials from 90 minutes to 6 hours, the optimised EpiOcular-EIT protocol achieved 100% sensitivity, 68.4% specificity and 84.6% accuracy, thereby meeting all the acceptance criteria set by the VMG. In addition, to satisfy the needs of Japan and the Pacific region, the EpiOcular-EIT method was evaluated for its performance after extended shipment and storage of the tissues (4–5 days), and it was confirmed that the assay performs with similar levels of sensitivity, specificity and reproducibility in these circumstances.
Development of Potential Alternatives to the Draize Eye Test: The CTF A Evaluation of Alternatives Program
Stephen D. Gettings
The EYTEX™ System and the Neutral Red Uptake Inhibition Assay in Cultured Hep G2 Cells as Alternative Methods for In Vivo Eye Irritation Following the EEC Protocol
Paul J. Dierickx
The Role of Structure-activity Relationships and Expert Systems in Alternative Strategies for the Determination of Skin Sensitisation, Skin Corrosivity and Eye Irritation
Martin D. Barratt
The Development and Characterisation of a Structure-activity Relationship Model of the Draize Eye Irritation Test
Herbert S. Rosenkranz, Ying Ping Zhang and Gilles Klopman2
A structure-activity relationship (SAR) model based on the results of 297 chemicals tested in the Draize eye irritation assay was developed. The SAR model displayed a predictivity of 74% for chemicals not included in the model. The SAR analysis indicated that chemical reactivity was not a requirement for eye irritation. The major structural determinants included hydrophilicity, alkalinity (i.e. primary, secondary and tertiary amines), acidity (for example, the carboxylic acid moiety), and putative lipophobic 4.5–5.4Å receptor-binding ligands. The analysis revealed that, while there were significant structural overlaps between the SAR models of ocular irritation, allergic contact dermatitis and respiratory hypersensitivity, there was much less overlap between ocular irritation and cell toxicity. This decreased overlap must be considered in developing strategies to replace the Draize test with in vitro cellular toxicity assays.
Herbert S. Rosenkranz and Albert R. Cunningham
The newly developed “chemical diversity approach” was used to determine whether or not it is likely that a panel of in vitro cell toxicity assays capable of predicting in vivo eye irritation could be assembled. The analyses, based upon available and validated structure–activity relationship models of toxicity in cultured human HeLa cells and murine Balb/c 3T3 cells, indicate that a battery of cytotoxicity tests could provide a viable alternative to the animal-based procedure.