Further optimisation of a macromolecular ocular irritation test (OptiSafeTM).

PURPOSE: OptiSafeTM (OS) is a shelf stable, nonanimal test for ocular irritation. A recent database search found that half of the OS false positive (FP) materials were associated with reactive oxygen chemistries but were not eye irritants in vivo (based on historical rabbit studies by other groups). We hypothesized that naturally occurring tear antioxidants protect the eye from reactive chemistries in vivo and that specific tear chemistries might help explain why some materials are FP for nonanimal tests but are reported as nonirritants in the live animal. To test this hypothesis, a prior study evaluated tear antioxidants and found that the tear antioxidant ascorbic acid, added at human physiological tear levels to the OS test, specifically reduced the measured values for these FPs but did not reduce the true-positive rate. Based on these findings, the OS test method was further optimized. The purpose of the current study was to comprehensively evaluate the performance of the further optimized test method for detection of ocular irritants. MATERIALS AND METHODS: The OS test measures chemically induced damage to macromolecules and relates these measured values to ocular irritancy. To improve the performance of OS, we made updates to the material to be tested physiochemical handling procedures, prediction model, and test method to include tear-level concentrations of ascorbic acid. We then retested the 78 chemicals from the prior OS-coded validation study in triplicate and compared the accuracy of the 'nonirritant versus irritant' prediction for the further optimized method with the prior results. RESULTS: We report that for the detection of 'nonirritant' versus 'irritant' (GHS NC versus categories 2B/A and 1) test substances, the further optimized OS test with ascorbic acid compared with the original version has a FP rate that is reduced from 40.0 to 22.2%, the false-negative (FN) rate remains at 0.0%, and the accuracy improved from 80.3% to 89.2%. CONCLUSION: A comparison to OECD-adopted tests demonstrates that the further optimized OS test, like the original method, has a higher accuracy and lower FN rate for the detection of 'nonirritants' versus 'irritants' (GHS Category NC versus 2B/A and 1) than the other eye irritation tests (BCOP, EpiOcularTM Eye Irritation Test, ICE, Ocular Irritection®, and STE).

Expansion of the application domain of a macromolecular ocular irritation test (OptiSafe™).

The OptiSafe (OS) test is shelf-stable, macromolecular eye irritation test that does not include any animal ingredient or component ("vegan"). The purpose of this study was to evaluate the test's accuracy for an expanded application domain for both the original and recently updated OS method. This study involved the testing of additional ocular corrosives and previously excluded foaming agents ("surfactants") using both the original and updated OS methods and then combining these data with prior validation data for a total of 147 chemicals. Predictivity was evaluated by a statistical comparison of the OptiSafe predictions with historical in vivo "Draize" rabbit eye data for the same chemicals (from public databases). We report that for the detection of chemicals not requiring classification for eye irritation [Globally Harmonized System of Classification and Labeling of Chemicals (GHS) No Category], the accuracy, specificity, and sensitivity were 92.8%, 79.6%, and 100.0%, respectively, for the updated method; for the detection of chemicals inducing extreme eye damage/corrosion (GHS Category 1), the accuracy, specificity, and sensitivity were 79.4%, 71.8%, and 91.7%, respectively, for the updated method. Results indicate that both the original and updated methods have a high accuracy for the expanded application domain that included ocular corrosives and surfactants.

Ascorbic acid specifically reduces the misclassification of nonirritating reactive chemicals in the OptiSafe™ macromolecular eye irritation test.

Recently, we showed that the addition of physiological concentrations of ascorbic acid, a tear antioxidant, to the OptiSafe™ macromolecular eye irritation test reduced the false-positive (FP) rate for chemicals that had reactive chemistries, leading to the formation of reactive oxygen species (ROS) and molecular crosslinking. The purpose of the current study was to 1) increase the number of chemicals tested to comprehensibly determine whether the antioxidant-associated reduction in OD is specific to FP chemicals associated with ROS chemistries and 2) determine whether the addition of antioxidants interferes with the detection of true positive (TP) and true negative (TN) ocular irritants. We report that when ascorbic acid is added to the test reagents, retesting of FP chemicals with reactive chemistries show significantly reduced OD values (P < 0.05). Importantly, ascorbic acid had no significant effect on the OD values of TP or TN chemicals regardless of chemical reactivity. These findings suggest that supplementation of ascorbic acid in alternative ocular irritation tests may help improve the detection of TN for those commonly misclassified reactive chemicals.

Modeling the antioxidant properties of the eye reduces the false-positive rate of a nonanimal eye irritation test (OptiSafe).

We recently identified a group of chemicals that are misclassified by most, if not all, in vitro alternative ocular irritation tests, suggesting that nonanimal tests may not fully model the ocular environment in which these chemicals interact. To address this, we evaluated the composition of tears, the first defense against foreign substances, and identified the presence of antioxidants that could detoxify reactive chemicals that otherwise may be falsely identified as irritants in alternative irritation tests. In this study, we evaluated the effects of tear antioxidants on the ocular irritation scoring of commonly overclassified chemicals (false positives) using the OptiSafe™ ocular irritation test. Six tear-related antioxidants were individually added to the OptiSafe formulation, and the effects on test outcome were determined. Ascorbic acid, the most abundant water-soluble antioxidant in tears, specifically reduced the OptiSafe false-positive rate. Titration curves showed that this reduction occurs at in vivo concentrations and is specific to chemicals identified either as producing reactive oxygen species or as crosslinkers. Importantly, the addition of tear antioxidants did not impact the detection of true negatives, true positives, or other false positives unassociated with reactive oxygen species or crosslinking. These results suggest that the addition of tear antioxidants to in vitro alternative test systems may substantially reduce the false-positive rate and improve ocular irritant detection.

Same-chemical comparison of nonanimal eye irritation test methods: Bovine corneal opacity and permeability, EpiOcular™, isolated chicken eye, ocular Irritection®, OptiSafe™, and short time exposure.

The testing and classification of chemicals to determine adverse ocular effects are routinely conducted to ensure that materials are appropriately classified, labeled, and meet regulatory and safety guidelines. We have performed a same-chemical analysis using publicly available validation study results and compared the performance between tests for the same chemicals. To normalize for chemical selection, we matched chemicals tested by pairs of tests so that each matched set compared performance for the exact same chemicals. Same-chemical accuracy comparisons demonstrate a chemical selection effect that results in a wide range of overlapping false-positive (FP) rates and accuracies for all test methods. In addition, the analysis suggests that a tiered-testing strategy with specific combinations of tests can reduce the FP rate for some combinations. However, reductions in the FP rates were typically accompanied by an increase in the false-negative rates, resulting in minimal advantage in terms of accuracy. In addition, actual improvements in the FP rate after retesting positives with a second test are not as good as the theoretical improvements because some chemicals and functional groups appear to be broadly misclassified by all test methods, which, to the extent the tests make the same-chemical misclassifications, reduces the advantage of using tiered-testing strategies.

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 in vitro depth of injury prediction model for a histopathologic classification of EPA and GHS eye irritants.

The purpose of this study was to develop Globally Harmonized System (GHS) and U.S. Environmental Protection Agency (EPA) prediction models for classifying irritant materials based on histopathologic in vitro depth of injury (DoI) measurements. Sixteen different materials were selected, representing all classes of toxicity, according to the GHS and EPA classification systems. Food-source rabbit eyes, similar to eyes used for the widely accepted Bovine Corneal Opacity and Permeability and Isolated Chicken Eye ocular irritation tests, were used. Tissues were exposed to test material for 1 min, and corneas were collected at 3- and 24-hours post-exposure. Tissues were then fixed and processed for live/dead biomarker fluorescent staining using phalloidin. DoI was then measured, and the percent DoI values for the epithelium and stroma were compared to the EPA and GHS classifications. Excluding surfactants, EPA nonclassified (category IV) materials showed no stromal and very slight epithelial damage (≤10%) to the cornea, whereas EPA corrosive (category I) materials showed significantly greater damage (P < 0.001), ranging from 39% to 100% of the stromal depth. Importantly, EPA reversible (categories II and III) materials showed significant damage to the epithelium (>10%, P < 0.005) but significantly less severe damage to the corneal stroma (P < 0.001), ranging from 1% to 38% of the stromal depth. GHS nonclassified (category NC) irritants caused damage to the epithelium but not to the stroma. All GHS class 2 materials showed damage to the stroma (1-11%), whereas GHS corrosives caused significantly greater damage to the stroma (38-100%; P < 0.001). Additionally, one corrosive material, which produced a stromal DoI of 99% at 24 h, produced no apparent damage at 3-hours post-exposure. Based on these findings, histopathologic EPA and GHS prediction models are proposed that appear to separate and identify reversible irritants from other irritant classes. Furthermore, GHS classification appears to require stromal damage, whereas NC materials may or may not damage the corneal epithelium.

Performance characteristics of colorimetric protein microarrays compared to ELISA.

Miniaturization and multiplexing are two areas that need improvement in immunoassays. However, it is unclear how associated changes in size, substrate, binding capacity, protein concentration, and other parameters affect sensitivity and quantitative range. In the study here we compared a new protein microarray substrate and system (Zeta-Grip, Miragene Inc., Anaheim, CA) with enzyme-linked immunosorbent assay (ELISA) for the application of immunoassays. We found that the protein microarray system performed better than ELISA in both sensitivity and quantitative range for use in immunoassay.

Development of a sensitive, colorometric microarray assay for allergen-responsive human IgE.

With the existence of several thousand unique human allergens, a multiplex format, such as protein microarrays, is an attractive option for allergy screening. To determine the feasibility and sensitivity of using an enzyme-based, colorimetric protein microarray assay, three common allergens (mold, dust-mite, grass) were arrayed and added sera assayed for responsive human IgE. Normal, low positive, and negative control samples were assayed to determine optimal reaction parameters. Sensitivity of the assay (in international units, IU) was determined by constructing a standard curve using World Health Organization (WHO) standards. The system described here can reliably detect allergen-specific IgE below 0.35 IU, the current WHO standard cutoff. By taking advantage of the sensitivity of enzyme-linked immunosorbent assays (ELISAs) and the multiplex format of microarrays, we have achieved a high throughput system, capable of screening patients for allergen-susceptibility with optimal sensitivity.

Development of a protein microarray library and a system for rheumatoid-arthritis disease marker screening.

Early detection and intervention can delay the onset of symptoms of RA (rheumatoid arthritis), but current diagnostic tests suffer from low sensitivity and specificity, making such early disease detection difficult. Analysis of body fluids for the identification of multiple molecular markers and the subsequent determination of modifications in their associated protein structures emerges as a possibility for early detection. The structural modifications of these markers are thought to play a pivotal role in defining the pathological state of diseased joints. Therefore a proteome library and system for RA disease screening has been developed and investigated in the present study. The large (39 S) mammalian ribosomal subunit has been identified as a potential RA marker. This protein is known as L 35, and antibodies to this protein have been found to be expressed in patients suffering from RA.

Development of an individual-specific autoantibody (ISA) protein microarray.

We have developed an ISA (individual-specific autoantibody) identification method to identify biological samples based on an individual's unique class of autoantibodies. This method involved the presentation of human proteins derived from crude lysates after SDS/PAGE separation and transferance to a solid support. In the present study, ISA strips are produced and developed on a new protein microarray. In making the ISA strips, it was found that variation in protein migration during electrophoresis and strip-manufacturing quality control limit the reliability of the assay. Therefore it was decided to semi-purify and separate proteins by column chromatography in large batches and to develop an ISA-specific protein microarray. It was found that this ISA protein microarray approximates a similar serum titre as the ISA strip and is predicted to circumvent the batch-to-batch production issues related to SDS/PAGE.