Ocular Toxicity Test Method Evaluations
Regulatory agencies require testing of chemicals and products to protect workers and consumers from potential eye injury hazards. This testing, referred to as ocular toxicity testing, represents one of the four most commonly conducted product safety tests. Ocular toxicity tests are conducted to identify ocular corrosives (substances that cause permanent eye tissue damage) and ocular irritants (substances that cause temporary eye tissue damage). Evaluation of test methods that reliably identify ocular corrosives and irritants while replacing, reducing, and refining animal use is a high priority for NICEATM and ICCVAM.
ICCVAM has recommended the use of in vitro ocular toxicity test methods and testing strategies that replace animal use to
- Identify products that require eye safety hazard labeling
- Determine appropriate hazard labeling category (corrosive versus irritant)
NICEATM and ICCVAM also evaluated approaches to reduce and refine animal use for eye safety testing, including:
- Employing topical anesthetics, systemic analgesics, and humane endpoints to prevent or minimize pain and distress during in vivo ocular irritation testing
- Conducting data analyses to support proposed classification criteria that would use fewer animals and meet current U.S. eye hazard classification requirements
Summaries of these evaluations appear below. Click hyperlinked headings for more information about specific projects.
OptiSafe Test Method
OptiSafe is an in vitro test method in which a test substance is applied to a semipermeable membrane. Damage to macromolecules in the membrane is measured to assess the test substance’s potential to cause eye irritation.
NICEATM reviewed a validation study conducted by the OptiSafe test method developer, Lebrun Labs, and concluded that the study data indicated that the OptiSafe method compared favorably to other in vitro ocular toxicity testing methods. NICEATM is currently coordinating a validation study of the OptiSafe test method to demonstrate the reproducibility of the method among Lebrun Labs and two naïve laboratories. The study received support from an NIEHS Small Business Innovation Research grant. The study is ongoing and is expected to be completed in mid-2018.
The short time exposure (STE) test is an in vitro test proposed for identifying ocular irritants. NICEATM compared STE test data with data generated using the traditional rabbit eye test. The results of the comparison, including comments from external reviewers, was included in a proposal to the Organisation for Economic Co-operation and Development (OECD) for a test guideline for the STE test.
Eye safety testing procedures vary among U.S. agencies. The U.S. Code of Federal Regulations (16 CFR 1500.42) provides criteria and procedures for identifying eye injury hazards based on rabbit eye test results. However, these procedures do not provide criteria to classify results obtained from a three-animal test.
NICEATM, in collaboration with ICCVAM, conducted an analysis to determine appropriate three-animal test classification criteria that were equivalent to the current testing procedure criteria cited in 16 CFR 1500.42. In 2012, ICCVAM recommended a procedure that uses 50% to 83% fewer animals than accepted testing procedures and harmonizes the number of animals used for ocular toxicity testing across U.S. regulatory agencies and international test guidelines based on their analysis.
ICCVAM considered comments from both the Scientific Advisory Committee on Alternative Toxicological Methods (SACATM) and from the public as it finalized its recommendations. These recommendations have been forwarded to Federal agencies.
The endorsement of several alternative testing methods by Federal agencies has provided regulated industries with important new tools for assessing potential eye injury hazards. The new test methods were the topic of a 2011 workshop organized by NICEATM and ICCVAM. The workshop identified best practices for chemical and product ocular toxicity testing using methods and approaches that replace, reduce, and refine animal use.
Routine Use of Topical Anesthetics, Systemic Analgesics, and Humane Endpoints to Avoid or Minimize Pain and Distress in Eye Safety Testing
Despite progress in alternative ocular toxicity test method development, no alternative test method has been adopted by regulatory authorities as a complete replacement for the in vivo rabbit eye test. Accordingly, when the rabbit eye test is necessary, ICCVAM recommends that pain management procedures always be used. A test method protocol describing specific procedures to prevent or minimize animal pain and distress is included in the ICCVAM report evaluating the use of anesthetics, analgesics, and humane endpoints during the in vivo eye test.
Validation Status of In Vitro Test Methods Proposed for Identifying Eye Injury Hazard Potential of Chemicals and Products
ICCVAM evaluated and developed recommendations for five in vitro test methods used to identify ocular irritants:
- Bovine corneal opacity and permeability (BCOP)
- Cytosensor microphysiometer (CM)
- Hen's egg test - chorioallantoic membrane (HET-CAM)
- Isolated chicken eye (ICE)
- Isolated rabbit eye (IRE)
ICCVAM recommended that the CM test method be used as a screening tool to identify:
- Certain types of water-soluble substances that cause permanent or severe eye injuries (“top-down” screen)
- Substances that do not require any hazard labeling (“bottom-up” screen) for eye irritation
ICCVAM concluded that the performance of the other four test methods must be improved before they can be used in regulatory safety testing to identify substances that may cause nonsevere eye injuries or that do not require eye irritation hazard labeling. The 2010 report on the ICCVAM evaluation of these test methods includes recommendations for future studies that could potentially improve these methods.
ICCVAM previously recommended the use of the BCOP and ICE test methods to identify substances that may cause severe or permanent eye injuries (see “In Vitro Test Methods for Detecting Ocular Corrosives and Severe Irritants”, below).
In Vitro Testing Strategy for Ocular Hazard Classification and Labeling of Antimicrobial Cleaning Products
In 2008, NICEATM received a U.S. Environmental Protection Agency (EPA)-sponsored submission for an in vitro testing strategy to assess the eye irritation potential of antimicrobial cleaning products. This proposed testing strategy used the BCOP, CM, and EpiOcular test methods to determine the EPA hazard category and labeling requirements for this class of products. ICCVAM evaluated the proposed strategy and recommended further studies to characterize its usefulness and limitations. As part of this evaluation, ICCVAM recommended that the low volume eye test, a test method used to generate data supporting the proposed testing strategy validity, should not be used for future regulatory testing.
In 2006, ICCVAM forwarded its first recommendations for the use of in vitro test methods for eye injury hazard classification testing to Federal agencies.
ICCVAM recommended including the BCOP and ICE test methods in a weight-of-evidence approach to identify products with the potential to cause permanent or severe eye injuries, noting specific limitations for certain chemical classes and/or physical properties. Substances that test positive in these assays can be classified as ocular corrosives or severe irritants without further testing in animals. Furthermore, ICCVAM recommended that these in vitro test methods be considered for eye injury hazard classification testing before using animals, and used whenever appropriate.
Subsequent to acceptance of the ICCVAM recommendations by U.S. Federal agencies, the OECD formally adopted guidelines describing the use of the BCOP and ICE test methods. These guidelines, designated Test Guidelines 437 and 438, respectively, were based on the ICCVAM recommendations. Acceptance of Test Guidelines 437 and 438 permits the international use of these test methods to identify substances that may cause permanent or severe eye injuries without the need for animal testing.
NICEATM and ICCVAM worked with international partners to organize two 2005 scientific symposia on eye safety testing.
Symposium I (Mechanisms of Chemically Induced Ocular Injury and Recovery) identified research needed to address current knowledge gaps and to advance the development and validation of test methods for ocular toxicity testing that protect human health and replace, reduce, or refine animal use.
The participants in Symposium I recommended that:
- Data collected from objective, quantitative endpoints and biomarkers should be used to assess the severity of chemically induced eye injuries in animal safety studies and human accidental exposures.
- The routine collection of this data can be expected to:
- Provide insights into chemical-specific mechanisms of eye injury and recovery
- Support the development and validation of more predictive mechanism-based in vitro test models
- Improve the accuracy and reliability of eye injury hazard assessments
- Aid in identifying earlier predictive, mechanism-based earlier humane endpoints
Symposium II (Minimizing Pain and Distress in Ocular Toxicity Testing) reviewed the current understanding of the sources and mechanisms of pain and distress in ocular toxicity testing. Workshop presentations identified current best practices for recognizing, preventing, and alleviating ocular pain and distress and identified additional research, development, and validation studies necessary to support scientifically valid ocular toxicity testing procedures that avoid pain and distress.
The participants in Symposium II recommended that:
- Topical anesthetics and systemic analgesics should routinely be administered before ocular toxicity testing in animals to avoid or minimize pain and distress that might occur during and after the initial application of test substances
- Systemic analgesics should routinely be administered when there is evidence of potentially painful damage to the eye or when clinical signs indicate pain or distress
- Specific eye injuries that would not be expected to reverse within 21 days could be identified as humane endpoints that could be used to end a study early
Both symposia were sponsored by ICCVAM, NICEATM, the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM), and the European Cosmetic, Toiletry and Perfumery Association. The symposia were organized by ICCVAM, NICEATM, and EURL ECVAM.