Test Method Validation and Regulatory Acceptance 2009: ECVAM

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Test Method Validation and Regulatory Acceptance 2009: ECVAM

Published: May 20, 2010
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 7th 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 6th and 7th 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.

Test MethodToxicity EndpointValidation Status
EpiSkin; Modified EpiDerm SIT; SkinEthic RHESkin IrritationESAC endorses performance of three ECVAM-validated Reconstructed Human Epidermis (RHE) methods for testing under the United Nations Globally Harmonized System (UN GHS)
EST-1000 RHESkin CorrosionESAC endorses method for predicting skin corrosion potential based on meeting performance standards in OECD TG 431
EpiSkin; Modified EpiDerm SIT1; SkinEthic RHESkin IrritationESAC endorses revised ECVAM Performance Standards for validating skin irritation testing methods based on RHE methods; update was necessary due to adoption of UN GHS system by EU in December 2008
RHE skin modelsSkin CorrosionESAC Statement on reference chemicals for validating reconstructed human epidermis for skin corrosion testing; ESAC supports technical update on performance standards to OECD TG 431 and recommends substitution of two Reference Chemicals of TG 431 with other ones
Cytosensor Microphysiometer and Fluorescein Leakage assaysEye IrritationESAC peer review 2009; ESAC endorses use of Cytosensor Microphysiometer and Fluorescein Leakage assays within the conceptual framework of a testing strategy for eye irritation previously developed within an ECVAM workshop
Rabbit Low Volume Eye Test (LVET)Eye IrritationESAC Statement concludes LVET cannot be recommended as a prospective testing method for generating new data; ESAC acknowledges potential usefulness of existing LVET data for cleaning products/ingredients for making weight-of-evidence decisions on further testing
Human reconstituted tissue models: SkinEthic RCE (peptide binding and GSH) and EpiOcularEye IrritationBegin validation study in collaboration with COLIPA
SkinEthic HCE modelEye IrritationReview of test data from industry as preparation for prospective validation study2
Organotypic assays (BCOP, ICE) for non to mild irritantsEye IrritationValidation study endorsed
Irritection assayEye IrritationValidation study endorsed
PorCORA (Porcine Cornea Opacity and Reversibility Assay)Eye IrritationNew test submission endorsed
Slug Mucosal Irritation AssayEye IrritationNew test submission endorsed
3 partial replacement methods: Direct Peptide Reactivity Assay (DPRA); Human Cell Line Activation Test (dendritic cell-based assay) (hCLAT); Myeloid U937 Skin Sensitization Test (MUSST)Skin SensitizationInternational prevalidation study started in 2009; ECVAM lead
h-CLAT using THP-1 cellsSkin SensitizationValidation study beginning 2009; JaCVAM lead, ECVAM collaboration
In vivo and in vitro COMET assaysGenotoxicityInternational validation study; JaCVAM lead
Micronucleus and Comet Assays in reconstituted (3D) human skin modelsGenotoxicityPrevalidation study being coordinated by COLIPA
Two Cell Transformation Assays (CTA) based on SHE and Balb/c 3T3 cellsCarcinogenicityInternational validation study in collaboration with NICEATM-ICCVAM and JaCVAM started in 2009; ECVAM lead
Cell Transformation Assay using Bhras cellsCarcinogenicityInternational validation study in collaboration with NICEATM-ICCVAM and JaCVAM started in 2009; JaCVAM lead
Toxiline-DSP Test for detection of okadaic acid and dinophysistoxins in shellfishBiologics TestingECVAM on management team of validation study that will be submitted for ESAC review
In Vitro Trout S9 Fraction Assay for bioaccumulation (reduction method)EcotoxicityValidation study review
Zebrafish Embryo Toxicity testEcotoxicityOECD validation study being coordinated by ECVAM
Threshold approach reduction strategyEcotoxicityRevision of OECD TG 203 initiated
PANVERA Estrogen Receptor Binding TestReproductive Toxicity/ Endocrine Disruptors2009 outsourced validation study in progress
HeLa Estrogen Receptor Antagonist AssayReproductive Toxicity/ Endocrine Disruptors2009 outsourced validation study in progress
VITO/B Anti-estrogenic compounds; BAYER/DReproductive Toxicity/ Endocrine DisruptorsNew test results submitted to ECVAM
MELN (MCF-7 human breast cancer cells)Reproductive Toxicity/ Endocrine DisruptorsNew test results submitted to ECVAM
LUMI-CELL Estrogen Receptor Transcriptional Activation AssayReproductive Toxicity/ Endocrine DisruptorsOngoing international validation study in collaboration with NICEATM-ICCVAM and JaCVAM
STTA AssayReproductive Toxicity/ Endocrine DisruptorsInternational validation study of estrogen antagonists started in 2008 in collaboration with JaCVAM
Balb 3T3/NRU Cytotoxicity AssayAcute Oral Systemic ToxicityValidation study to evaluate usefulness for discriminating toxic from non-toxic substances; ECVAM lead; NICEATM-ICCVAM participating as observer during analyses after data has been collected
ACuteTox testing strategyAcute Systemic ToxicityNo new activities in 2009, ACuteTox website discontinued
Human HepaRG CYP induction test method; Human Cryohep CYP induction test methodAcute Oral Systemic Toxicity (Toxicokinetics, ADME, PBPK modelling)Prevalidation study completed3; Validation study in collaboration with NICEATM-ICCVAM planned

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.

Test MethodToxicity EndpointRegulatory Acceptance Status
Five in vitro pyrogen test methodsPyrogenicityThe general method 2.6.30 Monocyte-activation test that covers the in vitro pyrogen tests was adopted by the European Pharmacopoeia Commission in March 2009
BCOP and ICE OECD Test GuidelinesEye CorrosionOECD Test Guidelines 437 and 438 adopted September 2009
EST-1000 RHESkin CorrosionCompliant with OECD Test Guideline 431 and EU Test Method B.40 as of June 2009
EpiDerm SIT and SkinEthic RHE assaySkin IrritationMethod B.46 included into Annex to 440/2008/EC in July 2009 during its 1st adaptation to technical progress (761/2009/EC). On December 11, 2009, the Draft Guideline, In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method, was posted on the OECD website for comment.
EPISKIN and EpiDermSkin IrritationMethod B.46 included into Annex to 440/2008/EC in July 2009 during its 1st adaptation to technical progress (761/2009/EC): EPISKIN as stand-alone test for replacement of Draize Skin Irritation Test; EpiDerm as screening method or as part of a sequential testing strategy. On December 11, 2009, the Draft Guideline, In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method, was posted on the OECD website for comment.

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.

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