Evaluations of In Vitro Test Methods

ICCVAM evaluated and produced recommendations, published in 2006 and 2010, for in vitro test methods used to identify ocular corrosives (substances that can severely injure or cause permanent damage to eyes) or ocular irritants (substances that can cause nonpermanent eye injury or discomfort).

Evaluation of In Vitro Test Methods to Identify Ocular Corrosives and Severe Irritants

In its 2006 evaluation, ICCVAM recommended the use of the bovine corneal opacity and permeability (BCOP) and the isolated chicken eye (ICE) test methods in a tiered-testing strategy to determine eye injury hazards. Substances that test positive using these test methods can be classified as ocular corrosives or severe irritants without testing in animals or additional in vitro eye irritation methods.

ICCVAM Report and Related Documents

Test Method Evaluation Report (November 2006) including ICCVAM recommendations

Expert Panel Report: Evaluation of the Current Validation Status of In Vitro Test Methods for Identifying Ocular Corrosives and Severe Irritants (March 2005)

Addendum to the Expert Panel Report (November 2005)

OECD Test Guidelines 437 and 438

The ICCVAM recommendations on the BCOP and ICE test methods were the basis for test guideline proposals submitted to OECD. Test Guideline 437, which describes the BCOP, and Test Guideline 438, which describes the ICE, were formally adopted by OECD in September 2009. Both test guidelines were revised in 2013 to allow classification of chemicals not requiring classification for eye irritation or serious eye damage. Additional revisions have been made to both test guidelines, the most recent updates being published in 2025.  

View OECD Test Guideline 437 on the OECD iLibrary website

View OECD Test Guideline 438 on the OECD iLibrary website

Recommendations and Agency Responses

The ICCVAM recommendations were communicated to Federal agencies in letters from Dr. Samuel H. Wilson, Acting Director, NIEHS, to each agency head. Links to these letters, and to the responses received from the agency heads, can be found below.

Further Evaluations of In Vitro Ocular Test Methods

In 2010, ICCVAM issued recommendations on the use of the BCOP, the ICE, and three other in vitro test methods for identifying reversible and nonsevere eye injuries:

  • The Cytosensor microphysiometer (CM) test method.
  • The hen's egg test - chorioallantoic membrane (HET-CAM) test method.
  • The isolated rabbit eye (IRE) test method.

ICCVAM recommended that the CM test method could be used as a screening test to identify some types of water-soluble substances that may cause permanent or severe eye injuries. ICCVAM also recommended that the CM test method could be used, for a limited range of substances, to identify chemicals and products that do not present sufficient potential to cause eye injuries to require eye hazard labeling. The CM test method was the first in vitro test method available in the U.S. for this purpose.

ICCVAM concluded that the performance of the other four test methods required improvement before they could be used in regulatory safety testing to classify substances as having the potential to cause reversible, nonsevere eye injuries or as not requiring hazard labeling for eye irritation. As noted above, subsequent evaluations have validated the use of the BCOP and ICE methods to identify substances not requiring hazard labeling for eye irritation.

Test method evaluation report with details of ICCVAM recommendations and test method evaluation:

Peer Review Panel Report (July 2009)

Evaluations of a Proposed Antimicrobial Cleaning Product Testing Strategy and the Low Volume Eye Test

Concurrently with their second evaluation of the in vitro ocular test methods, ICCVAM evaluated a non-animal testing strategy to assess the eye irritation potential of antimicrobial cleaning products. The testing strategy used the BCOP, CM, and EpiOcular test methods, and had been proposed by the EPA to determine hazard category and labeling requirements. ICCVAM concluded that, at that time, there were insufficient data with which to adequately demonstrate that the proposed strategy could classify test substances to all four EPA ocular hazard categories. ICCVAM recommended future studies that could further characterize the usefulness and limitations of a testing strategy for this class of products.

The ICCVAM recommendations and ICCVAM-recommended protocols for the test methods are in the ICCVAM Test Method Evaluation Report: Current Validation Status of a Proposed In Vitro Testing Strategy for U.S. Environmental Protection Agency Ocular Hazard Classification and Labeling of Antimicrobial Cleaning Products (2010).

As part of its evaluation of the antimicrobial cleaning products testing strategy, ICCVAM also reviewed the validation status of the low volume eye test (LVET). The LVET is similar to the traditional in vivo rabbit eye test but uses a smaller volume of test substance and a different application procedure. Data generated using the LVET were used as reference data to partially substantiate the validity of the in vitro test methods used in the antimicrobial cleaning products testing strategy.

ICCVAM recommended that the LVET should not be used for future regulatory testing. The ICCVAM recommendations are included in the ICCVAM Test Method Evaluation Report: Recommendation to Discontinue Use of the Low Volume Eye Test for Ocular Safety Testing.

In 2013, the EPA Office of Pesticide Programs established the use of an alternative testing approach to assess the eye irritation potential for antimicrobial cleaning products, which could also be used for other classes of pesticides and pesticide products on a case-by-case basis. This testing approach modified the 2008 proposal to consider the ICCVAM recommendations and studies submitted by product registrants. EPA updated the approach in 2015 to expand the applicability of the BCOP method. A guidance document is available on the EPA website.

Recommendations and Agency Responses

The ICCVAM recommendations on the in vitro test methods, the antimicrobial cleaning project strategy, and the LVET were communicated by NIEHS Director Linda Birnbaum to federal agencies in September 2010. These letters and responses received from federal agencies are available below.