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EU: Assessment of cell transformation assays (CTAs) by EURL ECVAM

The European Union Reference Laboratory for Alternatives to Animal Testing (EURLECVAM) has published a recommendation addressing three in vitro cell transformation assays (CTAs), the Syrian hamster embryonic (SHE) CTA performed at pH 6.7 and pH 7.0, and the BALB/c 3T3 CTA. 

This EURLECVAM recommendation addresses the performance of the CTAs and provides advice on possible regulatory applicability, limitations, and proper scientific use of the methods. It also suggests possible follow-up activities1.

This recommendation, which un­derwent public commenting, recommends the development of an OECD Test Guideline for the Syrian SHECTA and, considering the limited differences between the protocols at pH 6.7 and pH 7.0, it suggests that both SHECTAs should be incorporated into a single Test Guideline. The BALB/c 3T3 CTA protocol should be further used to expand on the reproducibility of the assay and confirm the suitability of the new statistical approach and data interpretation procedure applied. However, the recommendation also states that, from a 3Rs perspective, this CTA should be considered more appropriate than the SHECTA since it uses a cell line instead of primary cells. In general the recommendation concludes that the CTAs have the potential for partial replacement and reduction when used in a weight of evidence approach for hazard identification and risk assessment.

The details of the ECVAM study on which the recommendation is based, together with the recommended protocols and photo catalogues developed during this effort, is electronically published ahead of print in a special issue of Mutation Research on cell transformation2 (Special issue on cell transformation, in press) and will be made available in DB-ALM3 in the form of INVITTOXprotocols.

The carcinogenic potential of compounds is a crucial aspect in human hazard and risk assessment of chemicals. Among the in vitro alternatives that have been developed for predicting carcinogenicity, CTAs have been shown to involve a multistage process that closely models important stages of in vivo carcinogenesis and have the potential to detect both genotoxic and non-genotoxic carcinogens. These assays have been in use for decades and are considered to provide useful additional information to more routinely employed tests for assessing carcinogenic potential.

The OECD finalized in 2007 a Detailed Review Paper (DRP No. 31) on three CTAs (Syrian hamster embryo [SHE] cell assay, BALB/c 3T3 and C3H101/2 established cell line assays) for the detection of chemical carcinogens4 with the aim of determining whether the available data were sufficient for developing OECD Test Guidelines for any of the three CTAs. As a follow-up to this OECD effort, the OECD Working Group of National Coordinators of the Test Guideline Programme (WNT) recommended that a formal evaluation of the assays, in particular focusing on development of standardized transferable protocols and further information on assay reproducibility, should be considered prior to development of draft OECD Test Guidelines. 

On the background of the above, and following the recommendations of two ECVAM expert meetings, ECVAM coordinated a study aiming at the standardization and subsequent evaluation of the three CTA protocols mentioned above with regard to their transferability and reproducibility. The study complemented the OECD DRP No. 31 by providing prospective data on reliability while addressing predictive capacity only to a limited extent, since an extensive body of evidence on the predictivity of CTAs was available and summarized in the DRP.

EURL ECVAM requested its Scientific Advisory Committee (ESAC), to perform a peer review and provide scientific advice on the performance of the study. Both, the report of the ESAC Working Group that carried out the peer review on behalf of ESAC and the ESAC Opinion that was based on this report, were adopted by ESAC in February 2011 [ht­tp://], and made available to the OECD in time for its annual WNTmeeting in April 2011. 

Raffaella Corvi, PhD
European Commission
DG Joint Research Centre (JRC)
Institute for Health and Consumer Protection - Validation of Alternative Methods Unit
European Union Reference Laboratory for Alternative Methods to Animal Testing
Via E. Fermi 2749 TP 580
21027 Ispra (VA) ITALY

1The ECVAM study reports, the ESAC Working Group report, the ESAC opinion and the EURL-ECVAM recommendation on cell transformation assays are available at: alt-animal-testing/
2 Special Issue on cell transformation, R. Corvi and Ph. Vanparys, Guest Editors, Mutation Research, Genetic Toxicology and Environmental Research, in press. Link to the Preface:
3 DB-ALM database:
4 OECD, Detailed review paper on cell transformation assays for detection of chemical carcinogens. officialdocuments/displaydocumentpdf?cote=env/jm/mono(2007)18&doclanguage=en

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