BALB/c 3T3 cell transformation assay for the prediction of carcinogenic potential of chemicals and environmental mixtures Maria Grazia Mascolo a , Stefania Perdichizzi a , Francesca Rotondo b , Elena Morandi a , Angela Guerrini c , Paola Silingardi a , Monica Vaccari a, * , Sandro Grilli c , Annamaria Colacci a a Environmental Carcinogenesis and Risk Assessment, Environmental Protection and Health Prevention Agency – Emilia-Romagna Region (ER-EPA), Viale Filopanti 22, 40126 Bologna, Italy b Department of Social, Cognitive and Quantitative Sciences, University of Modena and Reggio Emilia, Via Allegri 9, I-42100 Reggio Emilia, Italy c Department of Experimental Pathology-Cancer Research Section, School of Medicine, University of Bologna, Viale Filopanti 22, I-40126 Bologna, Italy article info Article history: Received 30 July 2009 Accepted 7 March 2010 Available online 11 March 2010 Keywords: BALB/c 3T3 transformation assay Alternative in vitro tests Carcinogenicity prediction abstract The prediction of the carcinogenic risk for humans is mostly based on animal experiments. For the last 20 years, however, the scientific community has paid great attention to alternative strategies in compli- ance with common moral and ethical values. The new European chemical regulation REACH (Reg. EC 1907/2006) requires the performance of new studies in vertebrates only as a last resort. REACH asks for the development of validated in vitro protocols that can replace, in the medium to the long term, ani- mal bioassays. An in vitro cell transformation assay (CTA) is proposed as an alternative to in vivo carcin- ogenicity testing. This assay is reported in the list of accepted methods for REACH (Reg. EC 440/2008). The BALB/c 3T3 model represents one of the most well-known CTAs and is regarded as a useful tool to screen single chemicals or complex mixtures for carcinogenicity prediction. In this study we used a mod- ified protocol to highlight the transforming potential of three single compounds, ethinylestradiol (EE), azathioprine (AZA-T), melphalan, and two polychlorinated biphenyls (PCBs) mixtures, which are known or suspected to be human carcinogens. We also evaluated the activity of the antioxidant alpha-lipoic acid (ALA), a promising tumor chemopreventive. A significant increase in transformation frequency was observed when the BALB/c 3T3 cells were exposed to EE, AZA-T or melphalan as well as after PCBs treat- ment. On the contrary, ALA did not induce any increase of foci occurrence. Our results confirm the suit- ability of the improved protocol to discriminate carcinogenic compounds and support the use of BALB/c 3T3 cell transformation assay as a possible alternative to predict carcinogenic risk to humans. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction In order to test chemical safety during the last two decades, many efforts have been made to develop alternative methods to animal assays. However, the replacement of animal tests is still a matter of debate. Besides the ethical aspect, the use of animal tests is time and cost consuming and extrapolation of the results to man is a challenging and often imprecise exercise. On the other hand, no real validated in vitro test is currently available as a ready-to-go alternative to the currently used in vivo tests in order to assess acute, subacute, chronic toxicity as well as carcinogenicity and reproductive toxicology. Thus, alternative methods are more re- garded as tools to refine or reduce rather than replace animal bio- assays. Recently, the new European chemical regulation, aiming at the Registration, Evaluation and Authorization of Chemicals (REACH), which strongly supports the development and use of alternative tests to replace eventually animal bioassays, gave added momentum to this process (Reg. EC 1907/2006). REACH alternative methods include non-testing, i.e. structure–activity relationships, and testing strategies (in vitro, ex vivo, reduced/re- fined methods in vivo). Among in vitro testing methods, cell trans- formation appears to be one of the most suitable tools to predict the carcinogenic properties of chemicals (Lilienblum et al., 2008). Chemical carcinogenesis is a complex, multistage and multifacto- rial process. To date, options for absolute replacement of the two-year rodent carcinogenicity bioassay do not exist, even if the use of in vitro technologies could play a valuable role for the 0887-2333/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2010.03.003 Abbreviations: A1260, Aroclor 1260; ACE, absolute clonal efficiency; ALA, alpha- lipoic acid; AP, antipain; AZA-T, azathioprine; 1,2-DBE, 1,2-dibromoethane; CTA, cell transformation assay; EE, ethinylestradiol; GSH, glutathione; 3-MCA, 3- methylcholanthrene; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; PCBs, polychlorinated biphenyls; PCB mix, an equimolar reconstituted mixture of 18 congeners; RCE, relative clonal efficiency; TF, transformation frequency. * Corresponding author. Tel.: +39 51 2094790. E-mail address: m.vaccari@istbiotech.it (M. Vaccari). Toxicology in Vitro 24 (2010) 1292–1300 Contents lists available at ScienceDirect Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit