Eye Irritation/Serious Eye Damage

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Toxicity Endpoints & Tests

Eye Irritation / Serious Eye Damage

Last updated: June 26, 2016

TOXICITY ENDPOINT(S)

One component in the safety assessment of many types of products is the evaluation of their potential to cause eye injury. Eye irritation is defined as “the production of changes in the eye following the application of test substance to the anterior surface of the eye, which are fully reversible within 21 days of application” (UNECE, Part 3.3, 2015). Serious eye damage (eye corrosion) is defined as “the production of tissue damage in the eye, or serious physical decay of vision, following application of a test substance to the anterior surface of the eye, which is not fully reversible within 21 days of application” (UNECE, Part 3.3, 2015).

The anterior surface of the eye is covered by the cornea and the conjunctiva. The cornea is the most exposed area and, therefore, the most likely part of the eye to be involved in a chemical exposure to the eye. Chemical injury to the cornea can result in the loss of vision. Accordingly, corneal injury in the animal test for eye irritation/corrosion accounts for about 73% of the total ocular toxicity score. For these reasons, the cornea has been the primary tissue modeled in in vitro assays for accessing eye injury.

diagram of eye structure

Structure of the eye. Source: National Eye Institute, image is unchanged and licensed under Creative Commons 4.0.

The cornea is composed of three cellular layers. The outermost layer, the corneal epithelium, is composed of 5-7 epithelial cell layers in the human cornea. The surface layers of cells are connected by tight junctions that modulate the permeation of molecules into the tissue. The stroma, beneath the corneal epithelium, is composed of keratocytes (fibroblast-like cells) interspersed in the stromal collagen matrix. A single cell layer of corneal endothelial cells forms the innermost cellular layer of the cornea. Nerve cells penetrate through the cornea into the corneal epithelium, making it one of the most highly innervated tissues in the body; these cells are readily perturbed when the corneal epithelial tight junctions are disrupted.

Many mild eye irritants act by disrupting or damaging only the surface cells of the corneal epithelium, and the cornea can repair this type of damage within a short time. The stronger the eye irritant, the deeper it penetrates into the next layer of the cornea, the stroma. Very damaging materials might penetrate deep enough to cause irreversible injury, including damage to the corneal endothelial cell layer, where tight junctions modulate the penetration of water and other substances from the cornea to the aqueous humor.

The conjunctiva covers the remaining surface of the eye and is also important in protecting the eye from environmental insults. Injury to the conjunctiva has been assigned about 18% of the in vivo eye injury score in the Draize test but has largely been ignored in in vitro assessments of chemical eye injury. Conjunctival injury may be irrelevant in moderate to severe eye injury, where the corneal effects are largely predictive of reversibility and outcome, but could be useful in assessing milder effects, especially for products used in and around the eye (Ward et al., 2000). For example, the conjunctiva can exhibit different mechanisms of injury than the cornea due to its different physiology (Smit et al., 2003). The conjunctiva contains goblet cells, which secrete the mucin layer that protects the surface of the eye, as well as immune and vascular components important in the eye irritation and ocular allergy responses.

The iris is the third ocular tissue assessed for response to an irritant in the in vivo eye test. It is generally agreed that an in vitro iris assessment for most substances is not necessary, as iris responses occur only upon significant disruption to the ocular surface barrier of the cornea (ILSI TCAAT, 2006). Therefore, the degree of corneal injury should be predictive of potential iris effects.

THE ANIMAL TEST(S)

Developed in 1944, the Draize rabbit eye irritation test remains the standard method for evaluating the ocular irritation/corrosion potential of a substance for regulatory testing purposes (Draize et al., 1944; ILSI TCAAT, 1998). In this test, a material is instilled into one eye of albino rabbits (the other eye serving as the negative control), and the response of the animals is monitored using a standardized scoring system for injury to the cornea, conjunctiva, and iris. Ocular responses are scored at 1, 24, 48, and 72 hours. The animals are observed until the full magnitude and reversibility of the ocular injury can be evaluated—for up to 21 days. Reversibility of the ocular injury is an important considedration in the classification of a substance causing eye irritation versus serious eye damage. Different modifications of the test require different numbers of animals, although no more than three animals is the typical current standard.

Draize rabbit eye data have proved to be highly variable, generally overpredictive of human eye injury, and sometimes incorrect due to species differences in the ocular response to specific substances. A variant of the traditional Draize test, the low-volume eye test (LVET), which uses one-tenth the dosing volume of the traditional test placed directly on the cornea, as opposed to the conjunctival sac, is reported to give responses closer to those observed in humans, and when used with confocal microscopy shown to correlate with the degree of corneal injury for some classes of chemicals (Maurer et al., 2002; Jester et al., 2006). The LVET was endorsed as valid by ICCVAM in 2009, but only for the use of existing data to support the validation of new alternative test methods. Since there is insufficient evidence that LVET causes less pain and suffering than the traditional Draize rabbit eye test, the development of new data was discouraged, and new LVET data should not be accepted by agencies.

TEST GUIDANCE AND REGULATIONS

Regulatory authorities in most countries require ocular safety assessments and commonly have some version of the Draize rabbit eye test as part of their testing guidelines (NTP, 2011, 2016; EURL ECVAM, n.d.)

The Organisation for Economic Co-operation and Development (OECD) Test Guideline (TG) 405, Acute Eye Irritation/Corrosion (OECD, 2012) and the Globally Harmonized System (GHS) for Classification and Labeling of Chemicals, Part 3.3 (UNECE, 2015) describe internationally accepted guidelines using animal methods for eye irritation/corrosion studies. OECD TG 405 was updated in 2012 to reflect new efforts to refine and reduce animal use in ocular irritation testing. OECD guidelines, validation authority documents, and other regulatory acceptance for non-animal approaches for assessing serious eye damage/eye irritation are described in the section below.

GHS proposes a tiered approach to the classification of substances for serious eye damage/eye irritation. First, existing human and then existing animal data are to be considered. When the data satisfy the criteria, a classification can be made (GHS, 2015, p. 137). If further information is needed, skin corrosion data/testing, pH extremes of the test substance, eye effects from structurally-related substances, and data from validated in vitro tests can be part of a weight-of-evidence approach. The totality of the information is to be considered in a weight-of-evidence decision on hazard assessment and classification, “ideally without conducting new animal tests” (GHS, 2015, p. 139).

Most guidance for serious eye damage/eye irritation indicate that a chemical found to be strongly irritating or corrosive in skin studies, or with pH extremes (pH ≤ 2 or ≥ 11.5), does not need to be tested because the response can be assumed to be severe in the eye. Relevant guidance should be consulted.

ECVAM developed a template and explanatory document for “calculating eye irritation classifications from Draize in vivo eye irritation data, according to the rules of the classification systems of the European Union Dangerous Substances Directive (EU DSD), the European Union Regulation on Classification, Labelling and Packaging (EU CLP), the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS) and of the United States Environmental Protection Agency (US EPA).” The eye irritation classifications from existing Draize data can then be used in assessing the performance of alternative methods.

VALIDATED NON-ANIMAL METHODS

Non-animal methods for assessing serious eye damage/eye irritation currently endorsed as valid by one or more international validation authority and/or accepted internationally are listed in Table 1. Some references to regional regulatory acceptance, as well as links to OECD TGs and validation study documents are also provided. The more comprehensive AltTox Table of Validated & Accepted Alternative Methods provides additional information on endorsed approaches for refinement and reduction of animal use in ocular testing.

A substantial portion of the progress in refining and validating many of the ocular alternative approaches is the result of the collaborative efforts between the US EPA’s Office of Pesticide Programs (OPP), the Institute for In Vitro Science (IIVS), and ICCVAM, resulting in the “Alternate testing framework for classification of eye irritation potential of EPA pesticide products” (EPA, 2015; ICCVAM, 2015). This policy document explains the expanded use of the Bovine Cornea and Permeability (BCOP) assay for OPP’s antimicrobial testing in a decision tree approach that uses three in vitro assays: BCOP, the EpiOcular assay, and the Cytosensor Microphysiometer assay. The EPA recommends using the proposed assays for the testing covered in this policy, but states that when departures are appropriate “proposed alternatives to the methods recommended in the document can be submitted, with supporting rationale,” to be assessed on a case-by-case basis (EPA, 2015, p. 4). Alternative tests conducted and submitted for other classes of pesticides also will be considered on a case-by-case basis; “the applicant is encouraged to consult or submit the proposed test protocol to the EPA for review before conducting the study” (EPA, 2015, p. 4).

Although there are many alternatives to the Draize rabbit eye test listed in Table 1, there are still many ocular testing needs where an alternative method is not available, or is not accepted by a particular agency. Test method limitations are identified as part of any test method validation process. One example would be limitations of the assay itself, such as methods that are not compatible with a particular physical property of some test substances. Another limitation could be the range of chemical classes or the applicability domain for which a method is considered valid, which may be limited due to the numbers and types of substances evaluated in the validation study itself, so could later be expanded. For example, the BCOP and the isolated chicken eye (ICE) test methods were originally endorsed for the assessment of corrosive and severely irritating materials, but following additional validation the OECD TGs were revised “to extend the applicability domain of the BCOP and ICE for the additional purpose of ocular irritation testing.”

The applicability domain for a particular in vitro method can also be limited, as is the case for assessing the full range of ocular irritation, by the biological limitations of the in vitro system. In ocular testing, the cell-based assays have been found more predictive for non/mild/moderate eye irritants, while the ex vivo models can better predict moderate to severe irritants. The exact cut-off between eye irritation and serious eye damage is also difficult to define with non-animal systems that lack the ability to model the reversibility of the eye tissue injury, traditionally used in regulatory classifications. When cell or ex vivo models are maintained in culture, some can model a partial degree of recovery, but it is not comparable to the in vivo system due to the lack of the full complement of biological systems participating in in vivo recovery from ocular injuries, which can include immune and neural components. There are other practical approaches to addressing the recovery issue that will be considered further when the Emerging Science & Policy section is updated.

Table 1. Alternative methods, including in vitro and ex vivo assays and combination method test strategies, for assessing eye irritation/serious eye damage for regulatory testing purposes.

Method

Test Purpose

Validation Authority*

International Acceptance*

Regional Acceptance

Bovine Corneal Opacity Permeability (BCOP) Test

Eye irritation

ICCVAM (2007)

ESAC (2007)

JaCVAM (2013)

OECD TG 437 (2009; updated 2013)a

OECD Summary Document (2013)

REACH regulation

Isolated Chicken Eye (ICE) test

Eye irritation

ICCVAM (2007)

ESAC (2007)

JaCVAM (2014)

OECD TG 438 (2009; updated 2013)a

Draft revised 2015

OECD Summary Document 188 Part 1 (2013); 188 Part 2 (2013)

Draft Position Paper on ICE for UN GHS No Category

REACH regulation

Cytosensor Microphysiometer

Eye irritation

ESAC (2009)b

ICCVAM (2010)c

Draft OECD TG (2012)

Short Time Exposure (STE) in vitro test method for identifying chemicals not requiring classification for eye irritation or serious eye damage

Eye irritation

ICCVAM (2013)

OECD TG 491 (2015)

REACH regulation

Reconstructed Human Cornea-like Epithelium (RhCE) test for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damaged

Eye irritation

EURL ECVAM

OECD TG 492 (2015)

REACH regulation

Alternate testing framework for classification of eye irritation potential of EPA pesticide productse

Eye irritation

EPA OPP (2015)

Bovine Corneal Opacity Permeability (BCOP) test

Serious eye damage/corrosion

ICCVAM (2007)

ESAC (2007)

JaCVAM (2009)

OECD TG 437 (2009; updated 2013)a

OECD Summary Document 189 (2013)

REACH regulation

Isolated Chicken Eye (ICE) test

Serious eye damage/corrosion

ICCVAM (2007)

ESAC (2007)

JaCVAM (2009)

OECD TG 438 (2009; updated 2013)a

Draft revised 2015

OECD Summary Document 188 Part 1 (2013); 188 Part 2 (2013)

REACH regulation

Cytosensor Microphysiometer

Serious eye damage/corrosion

ESAC (2009)b

ICCVAM (2010)c

Draft OECD TG (2012)

Fluorescein Leakage

Serious eye damage/corrosion

ESAC (2009)f

JaCVAM (2012)

OECD TG 460 (2012)

OECD Summary Document No. 180

REACH regulation

Hen’s Egg Test-ChorioAllantoic Membrane (HET-CAM)

Serious eye damage/corrosion

EU Competent Authorities for Dangerous Substances Directiveg

Isolated Rabbit Eye test

Serious eye damage/ corrosion

EU Competent Authorities for Dangerous Substances Directiveg

Short Time Exposure (STE) in vitro test method for identifying chemicals inducing serious eye damage

Serious eye damage/ corrosion

ICCVAM (2013)

OECD TG 491 (2015)

REACH regulation

Alternate testing framework for classification of eye irritation potential of EPA pesticide productse

Serious eye damage/ corrosion

EPA OPP (2015)

*Links for accessing documents from organizations mentioned in the table:

a) BCOP and ICE methods can be used to identify chemicals as either 1) causing “serious eye damage” (category 1 of GHS, or 2) not requiring classification for eye irritation or serious eye damage according to GHS (new to the 2013 TG updates).

b) Recommended for use as initial step within a Top-Down Approach to identify ocular corrosives and severe irritants (EU R41, GHS Category 1, and EPA Category I) for water-soluble chemicals and/or as initial step within a Bottom-Up Approach to identify non-irritants (EU:NC; GHS: NC; EPA: cat IV) for water-soluble surfactants and water-soluble surfactant-containing mixtures; does NOT correctly identify moderate and mild ocular irritants (EU: R36; GHS: Cat 2A/B; EPA: Cat II/III) so can be used for only two of the three EU and GHS classification categories for ocular irritation; cannot be used for default categorization; additional limitations on equipment availability.

c) Can be used as screening test to distinguish water-soluble surfactant chemicals and certain types of surfactant-containing formulations that are not labeled as irritants (i.e., EPA Category IV, EU Not Labeled, FHSA Not Labeled) from all other hazard categories (i.e., EPA Category I, II, III; EU R41, R36; FHSA Irritant) for hazard classification and labeling under EPA, EU and FHSA classification systems; high false negative rate (24%-40%) for non-surfactant substances and formulations; high false positive rate (50% to 69%) for substances not labeled as irritants. Can be used as a screening test to identify water soluble substances as ocular corrosives and severe irritants (i.e., EPA Category I, EU R41, GHS Category 1) in tiered-testing strategy as part of weight-of-evidence approach; negative results need to be tested in another test method.

d) The only method currently covered by this TG is the EpiOcular™ Eye Irritation Test (EIT) developed using human skin cells; available from MatTek Corp.

e) Includes explanation of expanded use of BCOP for antimicrobial testing, and decision tree approach using 3 in vitro assays (see IIVS Decision Tree Approach):

  • Bovine Corneal Opacity and Permeability test (BCOP)
  • EpiOcular assay
  • Cytosensor Microphysiometer assay

f) Recommended for use as initial step within a Top-Down Approach to identify ocular corrosives and severe irritants (EU R41, GSH Category 1, and EPA Category I) for water-soluble chemicals; further refinement with respect to variability and applicability domain recommended.

g) Although not formally endorsed as valid, positive outcomes can be used for classifying and labeling substances as severe eye irritants (R41) in the EU.

Further information on the use and/or development of in vitro methods being developed for Eye Irritation/Serious Eye Damage testing can be found at:

Author(s)/Contributor(s):
Sherry L. Ward, PhD, MBA
AltTox Contributing Editor

AltTox Editorial Board reviewer(s):
George Daston, PhD
Procter & Gamble

Disclaimer

The information provided here is intended only as an overview, and is neither guidance or a comprehensive review of the laws and regulations on testing for eye irritation/serious eye damage. Individual countries/ regions and their regulatory authorities usually provide specific guidance on hazard/toxicity testing requirements.