Validation of the OptiSafe™ eye irritation test

Abstract

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.

Keywords:

• Ocular irritation
• validation
• OptiSafe
• alternative
• regulatory

Acknowledgements

The authors thank Drs. S. Ferguson and M. DeVito for their thoughtful review of the manuscript and Ms. C. Sprankle for editorial review. Members of the VMT included A. Layton, D. Lowther, J. Merrill, J. Matheson, J. Barroso, and K. Yozzo.

Disclaimer

This article may be the work product of an employee or group of employees of the United States CPSC, EPA, FDA, NIEHS, or the European Union Reference Laboratory for Alternatives to Animal Testing. However, the statements, opinions, or conclusions contained therein do not necessarily represent the statements, opinions, or conclusions of these organizations or the United States government. ILS staff provides technical support for NICEATM, but do not represent NIEHS, NTP, or the official positions of any federal agency.

Disclosure statement

Stewart Lebrun owns both Lebrun Labs and the patent: Biochemistry Based Ocular Toxicity Assay, Publication number: 20160290982, granted on August 7, 2018, which covers the OptiSafe test.

Additional information

Funding

Research and work in this publication were supported by the National Institute of Environmental Health Sciences, National Institutes of Health under Contract No. HHSN273201500010C to ILS in support of NICEATM, and under Award Number R44ES025501.