A Combined Morphometric and Statistical Approach to Assess Nonmonotonicity in the Developing Mammary Gland of Rats in the CLARITY-BPA Study

摘要

Background: The Consortium Linking Academic and Regulatory Insights on Bisphenol-A (CLARITY-BPA) is a rare collaboration of guideline-compliant (core) studies and academic hypothesis-based studies to assess the effects of bisphenol A (BPA). Objectives: We aimed to a ) determine whether BPA showed effects on the developing rat mammary gland using new quantitative and established semiquantitative methods in two laboratories, b ) develop a software tool for automatic evaluation of quantifiable aspects of the mammary ductal tree, and c ) compare those methods. Methods: Sprague-Dawley rats were exposed to BPA, vehicle, or positive control [ethinyl estradiol (EE2)] by oral gavage beginning on gestational day (GD)6 and continuing with direct dosing of the pups after birth. There were two studies: subchronic and chronic. The latter used two exposure regimes, one stopping at postnatal day (PND)21 (stop-dose) the other continuing until tissue harvest (continuous). Glands were harvested at multiple time points; whole mounts and histological specimens were analyzed blinded to treatment. Results: The subchronic study’s semiquantitative analysis revealed no significant differences between control and BPA dose groups at PND21, whereas at PND90 there were significant differences between control and the lowest BPA dose and between control and the lowest EE2 dose in animals in estrus. Quantitative, automatized analysis of the chronic PND21 specimens displayed nonmonotonic BPA effects, with a breaking point between the 25 and 250 μ g / kg body weight (BW) per day doses. This breaking point was confirmed by a global statistical analysis of chronic study animals at PND90 and 6 months analyzed by the quantitative method. The BPA response was different from the EE2 effect for many features. Conclusions: Both the semiquantitative and the quantitative methods revealed nonmonotonic effects of BPA. The quantitative unsupervised analysis used 91 measurements and produced the most striking nonmonotonic dose–response curves. At all time points, lower doses resulted in larger effects, consistent with the core study, which revealed a significant increase of mammary adenocarcinoma incidence in the stop-dose animals at the lowest BPA dose tested.