To ensure that inherent toxicity of our tested chemicals and their mixtures did not induce a downturn in testosterone synthesis, thereby confounding any genuine anti-androgenic effects, we assessed the functional integrity of the testosterone-producing Leydig cell population by immunolabeling of cytochrome P450 (CYP11A1), a key enzyme of the androgen biosynthesis pathway. We also monitored cell apoptosis by immunolabeling of cleaved caspase 3. After fixation in paraformaldehyde or Bouin solution and paraffin embedding, the randomly collected exposed explants were cut, mounted into slides, and labeled with either a rabbit anti-cytochrome P450, family 11, subfamily A, polypeptide 1 (CYP11A1) antibody (1/200; HPA016436, Sigma-Aldrich) or a rabbit anticleaved capsase-3 antibody 1/200; 9661S (Cell Signaling Technology), as described by Ben Maamar et al. 2015 ). Cleaved caspase-3 immunostaining required a step of antigen retrieval performed by incubation in preheated Tris-EDTA buffer 10/1 mM (pH ) at 80°C for 40 min and cooled at room temperature ( Ben Maamar et al. 2015 ). All the microscopic observations were made at low and high magnifications, and representative photos were taken at 40× magnification. Taken alone or in combination, both the testicular histopathology, the Leydig cell labeling and the cleaved caspase-3 positive cells staining provided a qualitative overall assessment of the integrity of the explant for each chemical at any concentration.