Reproductive Toxicology 51 (2015) 106–113 Contents lists available at ScienceDirect Reproductive Toxicology j ourna l ho me pa g e: www.elsevier.com/locate/reprotox Transferability and inter-laboratory variability assessment of the in vitro bovine oocyte fertilization test Irene Tessaro a , Silvia C. Modina a,b , Gabriella Crotti c , Federica Franciosi a , Silvia Colleoni c , Valentina Lodde a , Cesare Galli c,d,e , Giovanna Lazzari c,e , Alberto M. Luciano a,b,∗ a Reproductive and Developmental Biology Laboratory, Department of Health, Animal Science and Food Safety, University of Milan, 20133 Milan, Italy b Interdepartmental Research Centre for the Study of Biological Effects of Nano-Concentrations (CREBION), University of Milan, 20133 Milan, Italy c Avantea, Laboratory of Reproductive Technologies, 26100 Cremona, Italy d Dipartimento di Scienze Mediche Veterinarie, Università di Bologna, 40064 Bologna, Italy e Fondazione Avantea, Cremona, Italy a r t i c l e i n f o Article history: Received 5 February 2014 Received in revised form 16 December 2014 Accepted 6 January 2015 Available online 25 January 2015 Keywords: Reproductive toxicity Oocyte Spermatozoa ReProTect Fertilization Bovine Alternative methods 3Rs principle a b s t r a c t The dramatic increase in the number of animals required for reproductive toxicity testing imposes the validation of alternative methods to reduce the use of laboratory animals. As we previously demon- strated for in vitro maturation test of bovine oocytes, the present study describes the transferability assessment and the inter-laboratory variability of an in vitro test able to identify chemical effects during the process of bovine oocyte fertilization. Eight chemicals with well-known toxic properties (benzo[a]pyrene, busulfan, cadmium chloride, cycloheximide, diethylstilbestrol, ketoconazole, methy- lacetoacetate, mifepristone/RU-486) were tested in two well-trained laboratories. The statistical analysis demonstrated no differences in the EC 50 values for each chemical in within (inter-runs) and in between- laboratory variability of the proposed test. We therefore conclude that the bovine in vitro fertilization test could advance toward the validation process as alternative in vitro method and become part of an integrated testing strategy in order to predict chemical hazards on mammalian fertility. © 2015 Elsevier Inc. All rights reserved. 1. Introduction Because of the complexity of the mammalian reproductive cycle and for the lack of validated alternative tests for most of the pro- cesses encompassed in this system, testing in living animals is presently the only tool available for hazard assessment of repro- ductive toxicants. The already high number of test animals is even predicted to increase because of the implementation of the Reg- ulation on Registration, Evaluation, Authorization and Restriction of Chemicals (REACH; [1,2]), in order to ensure safety for human health and the environment [3,4]. Nevertheless REACH discour- ages tests on live vertebrates, so that alternative methods for chemicals testing need to be developed and validated. Therefore, besides bringing about a decrease in the number of animal tests also ∗ Corresponding author at: Dipartimento di Scienze Veterinarie per la Salute, la Produzione Animale e la Sicurezza Alimentare, Università degli Studi di Milano, Via Celoria, 10, 20133 Milano, Italy. Tel.: +39 02 50317969; fax: +39 02 50317980. E-mail address: alberto.luciano@unimi.it (A.M. Luciano). following the 3Rs principle (refinement, reduction, replacement), a new testing strategy for reproductive toxicology should provide more detailed information on mechanisms of toxicity in the dif- ferent target processes, leading to a new risk assessment model [5]. Under the ReProTect European network (http://www. reprotect.eu; [5]) several in vitro tests that mimic different aspects and mechanisms of the reproductive cycle have been optimized following the procedure of the modular approach for test development [6] recommended by the European Centre for the Validation of Alternative Methods (ECVAM). This step-by-step process begins with the test definition consisting on the iden- tification of mechanistic basis, standard operation procedures (SOP), and relevant toxicological endpoints. Then the assay is per- formed on a board of recognized reproductive toxicants to obtain dose–response curves and to demonstrate their repeatability [5]. In the field of female fertility, the mammalian oocyte represents the source of transcripts and energy substrates that allows the ini- tiation of embryo development with the formation of female and male pronuclei after fertilization, followed by the first embryonic http://dx.doi.org/10.1016/j.reprotox.2015.01.001 0890-6238/© 2015 Elsevier Inc. All rights reserved.