Ocular irritation reversibility assessment of a laundry detergent using the Porcine Corneal Opacity Reversibility Assay (PorCORA): a focused study.

Consumer product manufacturers utilise a spectrum of alternative ocular irritation assays, as these tests do not require the use of live animals. Despite their usefulness, no regulatory-accepted assay assesses the reversibility of ocular damage, a key criterion of GHS ocular classification, like the rabbit eye test (i.e., Draize Rabbit Eye Test [DRET]) . The Porcine Corneal Opacity Reversibility Assay (PorCORA), an ex vivo intact corneal tissue culture model, predicts the reversibility of damage by ocular irritants. Inclusion of the damage reversibility endpoint in the PorCORA supplements other alternative test methods for ocular irritation, by assessing induced eye damage and the ability of this damage to reverse (heal) without the use of live animals to distinguish between Globally Harmonised System of Classification and Labelling of Chemicals (GHS) ocular classifications. In this focused study, results of a Bovine Corneal Opacity and Permeability (BCOP) test of a laundry detergent, neat and 10% dilution, (product mixture from S.C. Johnson & Son, Inc. [SCJ]) classified the product into GHS Category 1; however, the BCOP test cannot assess the reversibility of ocular damage. The laundry detergent was evaluated using the PorCORA, where ocular damage induced by the detergent was fully reversed within seven days. Evaluation of the reversibility of ocular damage using the PorCORA in this focused study can add strength to the weight-of-evidence (WoE) analysis approach in ocular hazard assessment. This WoE approach strengthens the argument that the PorCORA can be used to supplement BCOP data, and that this laundry detergent is not an irreversible eye irritant.

Validation of the OptiSafe™ eye irritation test.

PURPOSE: OptiSafe is an in chemico test method that identifies potential eye irritants based on macromolecular damage following test chemical exposure. The OptiSafe protocol includes a prescreen assessment that identifies test chemicals that are outside the applicability domain of the test method and thus determines the optimal procedure. We assessed the usefulness and limitations of the OptiSafe test method for identifying chemicals not requiring classification for ocular irritation (i.e. bottom-up testing strategy). MATERIALS AND METHODS: Seventeen chemicals were selected by the lead laboratory and tested as an independent study. Ninety-five unique coded chemicals were selected by a validation management team to assess the intra- and interlaboratory reproducibility and accuracy of OptiSafe in a multilaboratory, three-phased validation study. Three laboratories (lead laboratory and two naïve laboratories) evaluated 35 chemicals, with the remaining 60 chemicals evaluated by the lead laboratory only. Test method performance was assessed by comparing classifications based on OptiSafe results to classifications based on available retrospective in vivo data, using both the EPA and GHS eye irritation hazard classification systems. No prospective in vivo testing was conducted. RESULTS: Phase I testing of five chemicals showed that the method could be transferred to naïve laboratories; within-lab reproducibility ranged from 93% to 100% for both classification systems. Thirty coded chemicals were evaluated in Phase II of the validation study to demonstrate both intra- and interlaboratory reproducibility. Intralaboratory reproducibility for both EPA and GHS classification systems for Phase II of the validation study ranged from 93% to 99%, while interlaboratory reproducibility was 91% for both systems. Test method accuracy for the EPA and GHS classification systems based on results from individual laboratories ranged from 82% to 88% and from 78% to 88%, respectively, among the three laboratories; false negative rates ranged from 0% to 7% (EPA) and 0% to 15% (GHS). When results across all three laboratories were combined based on the majority classification, test method accuracy and false negative rates were 89% and 0%, respectively, for both classification systems, while false positive rates were 25% and 23% for the EPA and GHS classification systems, respectively. Validation study Phase III evaluation of an additional 60 chemicals by the lead laboratory provided a comprehensive assessment of test method accuracy and defined the applicability domain of the method. Based on chemicals tested in Phases II and III by the lead laboratory, test method accuracy was 83% and 79% for the EPA and GHS classification systems, respectively; false negative rates were 4% (EPA) and 0% (GHS); and false positive rates were 40% (EPA) and 42% (GHS). Potential causes of false positives in certain chemical (e.g. ethers and alcohols) or hazard classes are being further investigated. CONCLUSION: The OptiSafe test method is useful for identifying nonsurfactant substances not requiring classification for ocular irritancy. OptiSafe represents a new tool for the in vitro assessment of ocular toxicity in a tiered-testing strategy where chemicals can be initially tested and identified as not requiring hazard classification.

An epidermal equivalent assay for identification and ranking potency of contact sensitizers.

The purpose of this study was to explore the possibility of combining the epidermal equivalent (EE) potency assay with the assay which assesses release of interleukin-18 (IL-18) to provide a single test for identification and classification of skin sensitizing chemicals, including chemicals of low water solubility or stability. A protocol was developed using different 3D-epidermal models including in house VUMC model, epiCS® (previously EST1000™), MatTek EpiDerm™ and SkinEthic™ RHE and also the impact of different vehicles (acetone:olive oil 4:1, 1% DMSO, ethanol, water) was investigated. Following topical exposure for 24h to 17 contact allergens and 13 non-sensitizers a robust increase in IL-18 release was observed only after exposure to contact allergens. A putative prediction model is proposed from data obtained from two laboratories yielding 95% accuracy. Correlating the in vitro EE sensitizer potency data, which assesses the chemical concentration which results in 50% cytotoxicity (EE-EC50) with human and animal data showed a superior correlation with human DSA05 (µg/cm(2)) data (Spearman r=0.8500; P value (two-tailed)=0.0061) compared to LLNA data (Spearman r=0.5968; P value (two-tailed)=0.0542). DSA05=induction dose per skin area that produces a positive response in 5% of the tested population Also a good correlation was observed for release of IL-18 (SI-2) into culture supernatants with human DSA05 data (Spearman r=0.8333; P value (two-tailed)=0.0154). This easily transferable human in vitro assay appears to be very promising, but additional testing of a larger chemical set with the different EE models is required to fully evaluate the utility of this assay and to establish a definitive prediction model.

Guidance on assessing the methodological and reporting quality of toxicologically relevant studies: A scoping review.

Assessments of methodological and reporting quality are critical to adequately judging the credibility of a study's conclusions and to gauging its potential reproducibility. To aid those seeking to assess the methodological or reporting quality of studies relevant to toxicology, we conducted a scoping review of the available guidance with respect to four types of studies: in vivo and in vitro, (quantitative) structure-activity relationships ([Q]SARs), physico-chemical, and human observational studies. Our aims were to identify the available guidance in this diverse literature, briefly summarize each document, and distill the common elements of these documents for each study type. In general, we found considerable guidance for in vivo and human studies, but only one paper addressed in vitro studies exclusively. The guidance for (Q)SAR studies and physico-chemical studies was scant but authoritative. There was substantial overlap across guidance documents in the proposed criteria for both methodological and reporting quality. Some guidance documents address toxicology research directly, whereas others address preclinical research generally or clinical research and therefore may not be fully applicable to the toxicology context without some translation. Another challenge is the degree to which assessments of methodological quality in toxicology should focus on risk of bias - as in clinical medicine and healthcare - or be broadened to include other quality measures, such as confirming the identity of test substances prior to exposure. Our review is intended primarily for those in toxicology and risk assessment seeking an entry point into the extensive and diverse literature on methodological and reporting quality applicable to their work.

Porcine Corneal Ocular Reversibility Assay (PorCORA) predicts ocular damage and recovery for global regulatory agency hazard categories.

In this study, we examined the capacity of the Porcine Corneal Ocular Reversibility Assay (PorCORA) to classify the reversibility of ocular effects for 32 test compounds (20 reversible, 12 irreversible) from various chemical classes. PorCORA predicted 28 of 32 compounds correctly when compared to historical rabbit eye test data. The correlation coefficient for PorCORA versus historical rabbit test data was 0.84, based on the last day of damaged cornea reversal. These results demonstrate a high correlation between corneal irritation recovery time in the PorCORA and the rabbit eye. When compared to historical Modified Maximal Average Score (MMAS) in rabbit eyes, PorCORA yielded a correlation coefficient of 0.80, demonstrating ability to predict MMAS. PorCORA was highly predictive of regulatory agency ocular hazard classification categories, resulting in 91% accuracy for EU R41 and GHS Category 1. PorCORA was also predictive of EPA Category I (88% accuracy). Overall, the accuracy (88-91%), sensitivity (79-86%), specificity (94%), positive predictivity (94%), and negative predictivity (85-89%) for all three regulatory classifications indicate that ocular irritation hazardous effects were well predicted by the PorCORA. This study suggests that PorCORA could help discriminate between EU R36 and R41, GHS Categories 1 and 2, and EPA Categories I and II.

Novel cultured porcine corneal irritancy assay with reversibility endpoint.

Several alternative assays exist to assess ocular irritancy without the use of live animals. However, these assays cannot address ocular injury reversibility. Reversibility is an issue critical to regulatory authorities and manufactures of commercial products, as ocular irritation caused by misuse or accidental exposure to a product may cause irreversible eye damage. Here we report the development and initial characterization of a novel ocular irritation assay that addresses ocular injury reversibility. This assay, the Porcine Corneal Ocular Reversibility Assay (PorCORA), uses an air-interface porcine corneal culture system to sustain ex vivo porcine corneas as a model system. These corneas are maintained in culture for 21 days to determine if cornea injury, once inflicted, will reverse. Corneal injury reversibility is measured using Sodium Fluorescein (NaFl) stain to detect compromised epithelial barrier function. In this study, we examined the effects of five compounds on the cultured corneas: phosphate-buffered saline (PBS), 100% Ethanol (EtOH), 3% Sodium Dodecyl Sulfate (SDS), 1% Benzalkonium Chloride (BAK), and 10% Sodium Hydroxide (NaOH). Overall, the persistence of corneal effects between historical Draize rabbit eye data and PorCORA indicates a correlation coefficient of 0.98 (for the five compounds tested) and a correlation coefficient of 0.97 with the Draize modified maximal average score (MMAS). Finally, both fluorescence confocal microscopy and histopathology evidence demonstrates that the PorCORA and NaFl measurements are indicative of actual cellular and tissue damage. PorCORA shows promise as a potential non-animal replacement assay capable of predicting ocular damage reversibility.