Test Method Validation and Regulatory Acceptance 2009: ECVAM

This article provides a brief summary of 2009 activities at the European Centre for the Validation of Alternative Methods (ECVAM). It is the third and final part of our updates on validation and regulatory acceptance activities that took place in 2009. The first part covered the Japanese Center for the Validation of Alternative Methods (JaCVAM) and the second part covered the US-based National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods-Interagency Coordinating Committee on the Validation of Alternative Methods (NICEATM-ICCVAM).

To facilitate the replacement of existing toxicity tests with new ones, national and international authorities have developed processes and criteria for evaluating new test methods. The major steps for partially or fully replacing an existing regulatory test with a new method are:

• Test method development
• Protocol optimization
• Multi-laboratory validation studies
• Peer review / independent assessment
• Regulatory acceptance consideration (national and international levels)

National validation centers prefer to liaise with test method developers during all phases of development and validation. Once adequate validation studies have been completed, the new or revised method can be submitted to one of the validation centers for an assessment of its scientific validity, including its demonstrated usefulness and limitations for the specific proposed use. In addition to submissions from external organizations, the centers are often involved in a variety of ways in the validation process from test development to peer review/assessment, although the details of their processes may vary.

ECVAM 2009

ECVAM provides technical support to the following services of the European Commission:

• DG Environment: in relation to Directive 86/609/EEC (Protection of Laboratory Animals) and Directive 67/548/EEC (Dangerous substances);
• DG Enterprise: in relation to chemical products, the safety of cosmetics, medicines, and vaccines; and
• DG Health and Consumer Protection: in relation to the Scientific Committee on Cosmetics and Non-Food Products;

In addition, ECVAM collaborates with:

• DG Research: which funds research on the development of alternative test methods.

The ECVAM is located in Ispra, Italy and has been under the leadership of Dr. Joachim Kreysa since November 1, 2008. In 2009, the "Alternative Methods and ECVAM" became an "Action" within the Institute for Health & Consumer Protection (IHCP) of the European Commission Joint Research Centre (EC-JRC), and is primarily supported by two of the IHCP scientific Units, In-Vitro Methods and Systems Toxicology. This revised organizational structure provides ECVAM with access to the broad range of competencies available across the IHCP.

The priorities of ECVAM, which are determined by EU legislation, were described in a June 2009 presentation by Dr. Kreysa as: REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals), cosmetics, pesticides, and animal protection and welfare. He advocated integrated testing strategies (ITS) as "the way to go" for new testing approaches for complex endpoints such as reproductive toxicity and toxicokinetics. ECVAM has already begun to discuss the complexities of validation related to ITS, and published a report from a joint workshop with the European Partnership on Alternative Approaches to Animal Testing (EPAA) on overcoming barriers to validation for ITS approaches (Kinsner-Ovaskainen, et al., 2009a).

ECVAM will continue to participate in and support toxicology R&D, organize workshops, and participate in international initiatives such as the International Cooperation on Alternative Test Methods (ICATM). ECVAM's recently relaunched alternatives database, DB-ALM, continues to add new content and users in support of the dissemination of information on alternatives. ECVAM also provides public access to ToxRTool, a software program for assessing the reliability of in vitro and in vivo toxicological data (Schneider, et al., 2009). Plans for ECVAM include improving the transparency of decision-making during its validation process, stimulating test development and test submissions for validation, and strengthening peer review and stakeholder dialogue.

The main purpose of this article is to describe the specific progress and accomplishments of ECVAM on the review and validation of new or revised toxicity test methods during 2009. The key driver for ECVAM has always been EU legislation, including the 7^th Amendment of the EU Cosmetics Directive (2003/15/EC), which has imposed deadlines for the replacement of animals for testing cosmetic ingredients, and the EU chemicals legislation REACH (EC 1907/2006), which will require more toxicity testing. These legislative mandates have stimulated the need for developing validated alternative test methods to reduce the numbers of animals in regulatory testing.

As of December 2009, ECVAM reported that they have evaluated 34 alternative methods according to generally accepted validation principles; however, most of these methods do not yet allow full replacement of the animal tests.

Table 1 summarizes ECVAM's activities related to the evaluation of new or revised toxicity test methods that were ongoing or completed in 2009. The first six entries describe the statements issued in 2009 by ECVAM's Scientific Advisory Committee (ESAC). Table 2 describes regulatory acceptance activities during 2009 related to test methods that have been endorsed by the ESAC.

In addition to test method review and validation activities, ECVAM continues to participate in several research projects in many areas including acute toxicity, genotoxicity, carcinogenicity, eye irritation, skin sensitization, biologics testing, and reproductive toxicity. ECVAM is also participating in research projects funded by the European Commission , including Integrated Projects funded within the 6^th and 7^th Framework Programmes (FP6 and FP7) such as: ACuteTox, carcinoGENOMICS, COMICS, ESNATS, INVITROHEART, Predictomics, ReProTect, Sens-it-iv, TOXDROP, and VITROCELLOMICS. The project websites and the 2009 Progress Report on FP6 and FP7 projects on alternative testing strategies are excellent resources for detailed information on these programs.

A recent publication describes ECVAM's 3-pronged approach to "developing and validating alternative methods in the field of acute toxicity" (Kinsner-Ovaskainen, et al., 2009b):

• The ACuteTox, FP6 project aims to develop a non-animal testing strategy that can replace acute oral toxicity testing in vivo; Acutoxbase is further described in the Kinsner-Ovaskainen, et al. publication (2009c)
• A follow-up validation study on the ‘validated' BALB/3T3 Neutral Red Uptake cytotoxicity assay to assess its predictive capacity for discriminating between toxic and non-toxic substances
• Retrospective data analysis from 28-day repeated dose toxicity studies to assess value of testing by different routes of exposure

ECVAM also supported research on the use of human stem cells for toxicological testing applications. A 2009 review article by Vojnits and Bremer discusses parameters for comparing the use of induced pluripotent stem cells (iPSC) as a substitute to using human embryonic stem cells (hECS) for toxicity testing applications.

Table 1. Toxicity test methods in some stage of assessment by ECVAM in 2009.

ESAC: ECVAM's Scientific Advisory Committee

Sources of information for Table 1:

• Kreysa, J. (2009). ECVAM - update. Powerpoint presentation at June 2009 SACATM meeting. Available here.
• Munn, S. (2009). Alternative Methods and ECVAM. Powerpoint presentation at December 2009 10th Anniversary REMA. Available here.
• ECVAM and ACuteTox websites
• ¹Kandárová, H., Hayden, P., Klausner, M., et al. (2009). In vitro skin irritation testing: Improving the sensitivity of the EpiDerm Skin Irritation Test protocol. Altern. Lab. Anim. 37, 671-689.
• ²Cotovio, J., Grandidier, M.H., Lelièvre, D., et al. (2010). In vitro assessment of eye irritancy using the Reconstructed Human Corneal Epithelial SkinEthic HCE model: Application to 435 substances from consumer products industry. Toxicol. In Vitro. 24, 523-537.
• ³Richert, L., Abadie, C., Bonet, A., et al. (2010). Inter-laboratory evaluation of the response of primary human hepatocyte cultures to model CYP inducers - a European Centre for Validation of Alternative Methods (ECVAM) - funded pre-validation study. Toxicol. In Vitro. 24, 335-345.

Table 2. Regulatory acceptance in 2009 of test methods endorsed by the ESAC.

ESAC: ECVAM's Scientific Advisory Committee

Source for Table 2:
ECVAM website

Note: AltTox prepared this review article using publicly available sources of information. ECVAM declined our invitation to review the content.