Mechanism of the toxic effect of Carbendazim on the pubertal seminiferous epithelium: An ex vivo study

Abstracts / Reproductive Toxicology 48 (2014) 23–36

P-10 Mechanism of the toxic effect of Carbendazim on the pubertal seminiferous epithelium: An ex vivo study Perrard Marie-Hélène 1,∗ , Carette Diane 2 , Blondet Antonine 3 , Martin Guillaume 3 , Christin Emilie 3 , Pointis Georges 2 , Durand Philippe 3 1

CNRS-INSERM U 846, Lyon, France INSERM U 1065, Nice, France 3 Kallistem, Lyon, France 2

Carbendazim (CBZ) is a fungicide used to prevent and eliminate fungal plant diseases. In vivo studies reported that CBZ induces severe testicular alterations in male rats. The cellular and molecular mechanisms of CBZ are not completely understood; even its status of endocrine-disruptor is still debated. We used our validated rat seminiferous tubule culture model (Bio-AlteR® ), developed by Durand’s team, for testing concentrations of CBZ at least 60-fold lower than the serum concentrations of CBZ found in rats treated with high doses of CBZ (25 mg/kg). Cultured seminiferous tubules from 20 to 22 day old rats were treated with CBZ 50 nM, 500 nM or 5 ␮M. The integrity of the blood-testis barrier [Trans-Epithelial Electrical Resistance (TEER), immunocytochemistry of Connexine 43 (Cx43) and of Claudin11], the various cell populations (flow cytometric analyses and quantification of mRNAs specific of the different cell populations) and hormonal receptor mRNAs were studied. The TEER was slightly increased by CBZ from day 16 onward, and the expression of Cx43 and gap junction functionality were decreased, while the expression of Claudin 11 was increased. CBZ increased somatic cells and decreased germ cells in a dosedependent manner from day 14 onward. CBZ increased young spermatocytes only on days 7 and 14. Secondary spermatocytes were increased or decreased according to the day of culture and the concentration of CBZ. However, round spermatids were always decreased by all 3 concentrations of CBZ in a dose-dependent manner, indicating a blockade of the second meiotic division. These results were completed by measurement of mRNAs specific of the different germ cell types. CBZ induced an increase of estrogen receptor ␤ mRNA on days 7 and 14. Hence, by regulating the expression of estrogen receptor ␤, low concentrations of CBZ do possess endocrine-disruptor effects leading to alteration of spermatogenesis. http://dx.doi.org/10.1016/j.reprotox.2014.07.038 P-11 Propylparaben has no estrogenic activity when administered for 3 months in juvenile rats Lakshmi Sivaraman 1 , Louise Pouliot 2,∗ , Yue Zhao 1 , Guowen Liu 1 , Hongwu Shen 1 , Bonnie Wang 1 , Cecilia Parrula 1 , Thomas Brodie 1 , Michael Graziano 1 , MaryEllen McNerney 1 1 2

R&D, Bristol-Myers Squibb Co., NJ, USA Charles River Preclinical Services, Montreal, Canada

Introduction: Parabens, esters of p-hydroxybenzoic acid, are used as antimicrobial preservatives in pharmaceuticals. There has been considerable controversy regarding the potential of parabens for estrogen-mimetic activity, and widespread concern regarding their use in pediatric medicinal formulations. The scientific literature contains conflicting results from several studies. A comprehensive study was designed and conducted to deter-

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mine the potential of propylparaben (PB) to elicit estrogen-mimetic effects. Objectives: (1) Determine the potential estrogen-mimetic effects of PB in juvenile rats; (2) determine the systemic exposures to PB and metabolites; and (3) evaluate the reversibility of any observed changes. Methods: PB was administered by oral gavage once daily to groups of Crl:CD(SD) rats at doses of 0 (vehicle), 10, 100, or 1000 mg/kg/day from Postnatal Days (PNDs) 4 through 90. Evaluations included clinical observations, body weight, food consumption (after weaning on PND 21), evidence of sexual maturation (vaginal opening and preputial separation), clinical pathology, anatomic pathology of male and female reproductive tracts, and toxicokinetic profiles on PNDs 7, 21 and 83. Recovery phase evaluations additionally included estrus cyclicity, mating (PND 91) and fertility assessments. F2 generation evaluations included viability, birth weight, litter size, clinical observations, and external and visceral examinations of pups for evidence of dysmorphology. Results: PB was well tolerated by neonatal, juvenile, and adult rats when administered from PNDs 4–90 at doses ≤1000 mg/kg/day. There were no effects of PB on sexual maturation or reproductive function in male or female rats, nor changes in organ weights or microscopic pathology of reproductive tissues suggestive of estrogen mimetic effects of PB or its metabolites. Exposures to PB and metabolites were confirmed in toxicokinetic evaluations. Conclusion: Propylparaben is not an estrogen-mimetic in juvenile rats. http://dx.doi.org/10.1016/j.reprotox.2014.07.039 P-12 Reduction and refinement of rodent juvenile toxicity studies – The simple approach to cross-fostering Emily S. Richmond ∗ , Sally A. Clode Sequani Ltd, Ledbury, UK The need for juvenile rodent toxicity studies to support paediatric clinical testing of pharmaceutical products has become increasingly commonplace, with appropriately designed nonclinical juvenile studies providing safety data, predictive of toxicity in the paediatric population. Whilst study designs vary hugely due to their tailored approach, almost exclusively these are large and complex studies, with animal usage exceeding any other study in the non-clinical programme. It is vital therefore that all options to reduce and refine these studies are explored. Many publications have focused on the justification of different endpoints and questioned the utility of these studies, however, the simple process of how animals are selected for study is often overlooked, despite the significant impact this can have on total animal usage and the robustness of data. The pup selection approach which is usually adopted, only allocates to study a small number of the dam’s natural offspring per litter, due to concern over maternal genetic bias. The cross-fostering approach has often been considered a superior method, whereby the offspring are distributed to multiple litters, removing any genetic bias or culling, and allowing all offspring to be selected for use. This reduces animal usage, provides better quality data and greater study flexibility. However, due to practical feasibility, cross-fostering has been traditionally discounted as idealist. This poster aims to raise industry awareness of a unique cross-fostering approach, developed at Sequani Ltd in a joint collaboration with our animal supplier, Charles River, UK. This approach involves cross-fostering to specific requirements at the suppliers