Regulatory agencies require testing of chemicals and products to
protect workers and consumers from potential eye injury hazards. This type of test represents one of
the four most commonly conducted product safety tests. Evaluation of test methods that reliably identify
such hazards and also replace, reduce, and refine animal use is a high priority for NICEATM and ICCVAM.
ICCVAM activities in the area of ocular toxicity include the following evaluations:
- Validation status of in vitro test methods that can assess the potential
of test substances to cause severe or permanent injury to the eye without using live animals
- Validation status of in vitro test methods and testing strategies that
can assess the potential of test substances to cause reversible and nonsevere
injury to the eye or for identifying substances as not requiring ocular hazard
labeling without using live animals
- Routine use of topical anesthetics, systemic analgesics, and
humane endpoints to avoid or minimize pain and distress during in vivo ocular irritation testing
- Data analysis in support of proposed classification criteria that will
provide the same or greater level of eye hazard classification as current U.S. regulations, while using 50% to 83% fewer animals.
Click on heading links below for more information on ICCVAM evaluations of ocular toxicity test methods,
as well as links to relevant ICCVAM documents,
Federal Register notices, and peer review information.
View summary of ICCVAM recommendations on ocular test methods transmitted to
Federal agencies - September 2010
Evaluation of the Short Time Exposure Test
The short time exposure (STE) test (described in Takahashi et al 2008:
view reference via PubMed
) measures the viability of cultured cells from an established
rabbit corneal epithelial
cell line following test substance exposure. This in vitro test is proposed for identifying
eye injury hazard potential of chemicals and products.
NICEATM prepared a summary review document (SRD) on the STE test based on data provided by Kao
Corporation, the test method sponsor. The SRD presents an evaluation of STE test method accuracy,
sensitivity, specificity, false positive rate, and false negative rate based on test substances with
corresponding in vivo data. The SRD also evaluates the performance of the STE as a screening test to
distinguish severe eye irritants and corrosives from all other substances, as well as its
performance as a screening test to distinguish substances that do not require hazard labeling or are
minimal irritants from all other substances.
The SRD was provided by the National Toxicology Program to four external reviewers. A summary of
reviewer comments is provided in the SRD. The reviewers indicated that the database of compounds
tested for the SRD was generally sufficient and the review thorough.
Read the NICEATM Summary Review Document on the Short Time Exposure Test (June 2013)
The final SRD, including a summary of reviewers’ comments, was provided to Kao Corporation. Kao
Corporation is currently preparing documents for submission of the STE test to the Organisation for Economic
Cooperation and Development (OECD) as an in vitro
alternative to current ocular hazard identification tests. The NICEATM SRD will be provided to
OECD in support of this submission.
Eye safety testing procedures vary among U.S. agencies. Current testing
procedures specified in the U.S. Code of Federal Regulations (16 CFR 1500.42) provide criteria and
procedures for identifying eye hazards based on rabbit eye test results. However, these current
testing procedures do not provide criteria to classify results obtained from a three-animal test.
Therefore, NICEATM, in collaboration with ICCVAM, conducted an analysis to determine classification
criteria based on results from a three-animal test that would maintain hazard classification
equivalent to that provided by current testing procedures (16 CFR 1500.42).
When it is determined necessary to use animals for eye hazard testing, the recommendations
provide procedures that use 50% to 83% fewer animals than some current testing procedures. The
recommendations also harmonize the number of animals used for eye safety testing across U.S.
regulatory agencies and international test guidelines.
ICCVAM considered comments from the public and from the
Scientific Advisory Committee on Alternative Toxicological Methods (SACATM)
as it finalized its recommendations. These
recommendations have been forwarded to Federal agencies, with agency responses expected in 2013.
Federal agency endorsement of several alternative testing methods has provided health
agencies and regulated industries with important new tools for assessing potential
eye injury hazards presented by chemicals
and products. The new test methods were the topic of a January 2011 workshop organized by
NICEATM and ICCVAM. The workshop provided information on current best practices for
safety testing to determine whether
chemicals and products may cause eye injuries using methods and approaches that replace, reduce,
and refine animal use.
Despite progress in development of alternative test methods that do not use live animals, nearly
all eye hazard classification testing has been conducted using the rabbit eye test. ICCVAM recommends
that pain management procedures should always be used when it is determined
necessary to conduct the rabbit eye
test. A report on the ICCVAM evaluation of the use of anesthetics, analgesics, and humane
endpoints in eye hazard classification testing includes a test method protocol
that describes specific procedures to
be used to avoid or minimize animal pain and distress.
ICCVAM has made recommendations on the use of five in vitro test methods for identifying
substances with the potential to cause reversible and nonsevere eye injuries:
- 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 recommends that the CM test method can be used as a
screening test to identify some types of water-soluble substances that may cause permanent or severe eye injuries.
ICCVAM also recommends that the CM test method can be used to determine if a limited range of
substances will not cause sufficient injury to require hazard labeling for eye irritation. ICCVAM
concluded that the performance of the other four test methods must be improved before they can
be used used in regulatory safety testing to identify substances with the potential to cause
reversible, nonsevere eye injuries or that do not require hazard labeling for eye irritation.
2010 report on the ICCVAM evaluation of these test methods includes recommendations
for future studies that could potentially
improve these test methods. These recommendations were addressed in recent studies using the IRE
test method (see “Evaluation of the Short Time Exposure Test, the Isolated Rabbit Eye Test,
and Other In Vitro Eye Safety Testing Methods and Approaches,” above).
ICCVAM previously recommended that the BCOP and ICE test methods may be used
to identify substances that may cause severe or permanent eye injuries
“In Vitro Test Methods for Detecting Ocular Corrosives and Severe Irritants”, below).
In 2008, NICEATM received a submission, sponsored by the U.S. Environmental Protection
Agency (EPA), for a testing strategy to assess the eye irritation
potential of antimicrobial cleaning products without using live animals.
This proposed testing strategy utilized the BCOP, CM, and EpiOcular
test methods to determine the EPA hazard category and labeling requirements for these substances.
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 (LVET), a test method that was used to generate data supporting the
validity of the proposed testing strategy, should not be used for future regulatory testing.
Histopathology in Ocular Safety Testing
A continuing priority for NICEATM and ICCVAM is to identify and
promote research, development, translation, and validation activities for alternative test methods
with the potential to replace the rabbit eye test for identification of substances that are
potential eye injury hazards. The current major obstacle to replacing animals for eye hazard classification testing is
the lack of in vitro methods that can accurately identify chemicals and products that are capable of
causing severe or permanent eye injuries.
To this end, one activity proposed in the 2009
Implementation Plan was a workshop on the incorporation of histopathology into in vitro ocular
safety testing protocols. The goal of such a workshop woudld be to determine if the
additional information yielded by histopathology analysis might improve the ability of
these methods to predict a test substance's potential to cause
severe and irreversible injuries to the eye. However, several recent evaluations and
publications have failed to demonstrate the usefulness of histopathology as an additional endpoint
for currently accepted in vitro methods. Thus, a workshop does not appear warranted at this
View a NICEATM summary
of the current
status of the usefulness of histopathology evaluation
as an additional endpoint in in vitro eye hazard testing methods (November 2011)
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 that the BCOP and ICE test methods
can be used as part of a weight-of-evidence approach to
identify substances that may cause permanent or severe eye injuries, with 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.
ICCVAM also recommended
that these in vitro test methods should be considered before using animals for eye injury hazard classification
testing and used when determined appropriate. The ICCVAM recommendations were accepted by Federal agencies,
and the two in vitro test methods may now be used
instead of animal tests for certain regulatory testing purposes.
The use of these tests will eliminate
eye safety testing in animals of most substances likely to
cause the most severe pain and discomfort.
Subsequent to the U.S. acceptance of the ICCVAM recommendations, 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 use of these
test methods internationally to identify substances that may cause permanent or severe eye injuries
without the need for animal testing.
Two Scientific Symposia on ocular toxicity topics were held in 2005. Both symposia were
sponsored by ICCVAM, NICEATM, the European Centre for the Validation
of Alternative Methods (ECVAM), and the European Cosmetic, Toiletry and Perfumery Association,
and were organized by ICCVAM, NICEATM, and ECVAM.
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 ocular toxicity
test methods for regulatory testing that provide for protection of
human health while replacing, reducing, or refining the use of animals.
The participants in Symposium I made the following recommendations:
- Data collected from objective, quantitative endpoints and biomarkers should be used to assess the severity of
chemically induced ocular 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 ocular injury and recovery
- Support the development and validation of more predictive mechanism-based in vitro test models
- Improve the accuracy and reliability of ocular hazard assessments
- Aid in identifying predictive, mechanism-based earlier humane endpoints
Minimizing Pain and Distress in Ocular Toxicity Testing reviewed current understanding of the sources and mechanisms
of pain and distress in ocular toxicity testing; identified current best practices for preventing, recognizing,
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 made the following recommendations:
- Topical anesthetics and systemic analgesics should routinely be administered before ocular
testing 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 ocular damage or when there are clinical signs indicative of pain or distress
- Specific ocular injuries identified by the experts that would not be expected to reverse
within 21 days could be used as humane endpoints to end a study early