Still Looking for a Validated In Vitro Eye Test

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Still Looking for a Validated In Vitro Eye Test

Sherry Ward, AltTox Contributing Editor

Published: June 8, 2009

The March 11 European Union animal test ban was another reminder of the long-standing need for a validated non-animal test method or test scheme that can replace the Draize rabbit eye test.

 

Recent Reviews of In Vitro Ocular Test Methods

Validation authorities in Europe, the European Centre for the Validation of Alternative Methods (ECVAM), and in the US, the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), have recently conducted scientific reviews on a number of in vitro ocular test methods. The purpose of these reviews was to identify one or more methods that could be scientifically validated and possibly recommended for acceptance by regulatory authorities.

The ECVAM Scientific Advisory Committee (ESAC) statements regarding the scientific validity of four methods reviewed late last year are expected in July. The four cell function-based test methods reviewed by an ECVAM panel were:

  • Fluorescein leakage
  • Red blood cell haemolysis
  • Neutral red release
  • Cytosensor Microphysiometer®

An ICCVAM International Scientific Peer Review Panel evaluated a number of in vitro ocular eye irritation test methods on May 19-21 in Bethesda, Maryland. The following individual test methods and a test scheme were reviewed by the ICCVAM Panel:

  • Bovine corneal opacity and permeability (BCOP)
  • Isolated chicken eye (ICE)
  • Isolated rabbit eye (IRE)
  • Hen’s egg test-chorioallantoic membrane (HET-CAM)
  • EpiOcularTM (only anti-microbial cleaning products [AMCPs] data were considered)
  • Cytosensor Microphysiometer® (Cytosensor)
  • Test scheme proposed for AMCPs based on using EpiOcularTM, Cytosensor, and BCOP

The ICCVAM Panel evaluated the validation status of each of the in vitro methods according to established Federal and international criteria, and commented on draft ICCVAM recommendations regarding the usefulness and limitations of each test method as well as proposed future studies.

Dr. A. Wallace Hayes, the Panel chairperson, will report on the recommendations of the Panel at the June 25-26 meeting of ICCVAM’s scientific advisory committee (SACATM) in Arlington, Virginia. The peer panel’s published report will be available in July.

ECVAM is still evaluating some of the tests that were reviewed at the NICEATM-ICCVAM peer panel meeting. Therefore, it may take some time before ECVAM will be able to comment on the results of the US meeting, according to Dr. Horst Spielmann, chairperson of the ECVAM Validation Management Group.

NICEATM released a brief summary of the ocular peer review panel report shortly after the public meeting. This rapid update to stakeholders illustrates their continued efforts at transparency. Outcomes made public in this report related to in vitro toxicity test methods include the following:

1) The Panel recommended that the BCOP and Cytosensor methods “could be used in limited circumstances as screening tests to identify some products and substances that would not require hazard labeling for eye irritation.”

Interpretation: For limited types of chemicals and product types, the BCOP and Cytosensor were recommended for use in a bottom-up testing approach, meaning they could be used to identify substances that are not considered to be eye irritants (not labeled) and additional testing would not be required. (An important exception is that BCOP was not recommended as a screening method for EPA Category IV substances.) This complements the top-down approach validated previously where the BCOP or ICE method can be used to identify corrosive or severe eye irritants, thereby eliminating those substances that test positive (severe/corrosive) from further eye irritation testing requirements. Regulatory authorities typically have one or two additional hazard categories for mild to moderate eye irritants that fall in between non-irritants (not labeled as an irritant) and severe/corrosives eye irritants. In vitro test methods reviewed by the Panel were not considered accurate for evaluating materials in these middle categories, meaning that the Draize rabbit eye test is still the only method for identifying these substances.

2) The Panel recommendation for the test scheme proposed for anti-microbial cleaning products (Cytosensor, EpiOcularTM, and BCOP) was that “there are insufficient data to demonstrate that the non-animal testing strategies can accurately classify antimicrobial products in all four EPA ocular hazard categories.” Additional recommendations on how the testing strategy can be used in a limited manner and recommendations for its further development can be expected in the full panel report.

These in vitro ocular test methods reviewed by ECVAM and ICCVAM may sound familiar. All of them have been around for more than a decade and most have been evaluated in previous validation efforts. The status and testing needs for validating these methods were reviewed by ECVAM in 2005. However, between that time and the present validation efforts, significant new laboratory studies were not conducted for some of the methods.

 

What’s in the Pipeline

Two human cell-based ocular models have recently been qualified for a validation study by COLIPA (the European cosmetics industry’s trade association) and ECVAM. The SkinEthic human corneal epithelial cell model (SkinEthic HCE) is based on similar in vitro ocular cell models developed in the 1990’s. The MatTek EpiOcular model, composed of non-ocular human epithelial cells, has been widely used as a research and testing model for many years. Both of these models have performed well in preliminary studies for certain product types and chemical classes.

Enucleated animal eyes (BCOP, P(porcine)COP, ICE, and IRE) have provided good screening assays and have been widely used by industry for many years. Data suggest that these methods could be improved; however, only a limited effort has been made in refining these test systems and endpoints to get sufficiently predictive methods for their broader validation.

New efforts to advance in vitro and in silico toxicity test methods claim to be based on the merits of ‘better science.’ Compelling arguments have not been made against the logic that hazard assessments based on cellular toxicity pathways in human cellular or tissue models would provide risk assessments based on ‘better science.’ The problem is that insufficient resources have been invested in developing the science (test models and endpoints). Science has always been based on incremental progress. In vitro toxicology is no exception. Many studies should simultaneously be underway for the development and validation of new eye irritation methods alone to expect significant progress to occur. As an analogy, consider where we would be in our progress for new cancer treatments if the National Cancer Institute had decided to wait five years between the funding of each new drug development study to see how the previous study had turned out.

Science and technology are no longer the primary limits to progress. We have the ability to develop biologically-relevant cell-based and organ culture models that can: 1) replicate relevant structures of the human eye; 2) be used to model endpoints relevant to human eye irritation; and 3) be assembled into test schemes for the prediction of human eye irritation for different classes of chemicals over the range of eye irritation. Challenges remain for the modeling and statistical analysis needed to define and optimize a test scheme, and for determining its scientific validity.

Among other issues, the lack of sufficient government funding for academic and small business in vitro toxicology research has been a serious hindrance to progress in the US. The lack of funding for the development of commercially viable in vitro models has been especially problematic. Even the well-funded US agencies charged with this task fall far short in providing sufficient requests for proposals for in vitro toxicology research. I will leave it up to the EU researchers to identify the problems in their efforts. The question now is whether we will overcome past deficiencies and move forward in a concerted effort for progress. The sooner this is done, the greater the “cost savings” for all.