Scientific and Regulatory Policy Committee (SRPC) Points to Consider

Abstract

The U.S. Environmental Protection Agency Endocrine Disruptor Screening Program (EDSP) is a multitiered approach to determine the potential for environmental chemicals to alter the endocrine system. The Pubertal Development and Thyroid Function in Intact Juvenile/Peripubertal Female and Male Rats (OPPTS 890.1450, 890.1500) are 2 of the 9 EDSP tier 1 test Guidelines, which assess upstream mechanistic pathways along with downstream morphological end points including histological evaluation of the kidneys, thyroid, and select male/female reproductive tissues (ovaries, uterus, testes, and epididymides). These assays are part of a battery of in vivo and in vitro screens used for initial detection of test article endocrine activity. In this Points to Consider article, we describe tissue processing, evaluation, and nomenclature to aid in standardization of assay results across laboratories. Pubertal assay end points addressed include organ weights, estrous cyclicity, clinical pathology, hormonal assays, and histological evaluation. Potential treatment-related findings that may indicate endocrine disruption are reviewed. Additional tissues that may be useful in assessment of endocrine disruption (vagina, mammary glands, and liver) are discussed. This Points to Consider article is intended to provide information for evaluating peripubertal tissues within the context of individual assay end points, the overall pubertal assay, and tier I assays of the EDSP program.

Preparation of High-quality Hematoxylin and Eosin–stained Sections from Rodent Mammary Gland Whole Mounts for Histopathologic Review

“…wean, puberty and early pregnancy) is highly recommended, due to its small tissue size and relatively low epithelial density. We anticipate that this method will 1) serve as a standardized protocol to reduce inter-lab variability, 2) increase awareness within the scientific community of the ease with which the mammary gland can be incorporated into studies at minimal added cost (Keane et al 2015), and 3) stimulate collection of the mammary gland into chemical test guideline studies, which may or may not include evaluations (OECD TG443 and NTP), fail to focus on specific mammary gland developmental endpoints (Makris 2011) or are not collected at all (OPPTS 890.1450, US EPA 2009 and OPPTS 890.1500 2009b). …”

“…The regular evaluation of mammary tissue from mice and rats during various life stages of development (i.e., wean, puberty, and early pregnancy) is highly recommended, due to its small tissue size and relatively low epithelial density. We anticipate that this method will (1) serve as a standardized protocol to reduce interlab variability, (2) increase awareness within the scientific community of the ease with which the mammary gland can be incorporated into studies at minimal added cost (Keane et al 2015) Although it is probable that other histochemical and immunohistochemical staining may be applied to sections derived from whole mounts, it was beyond the scope of this work and further testing would be necessary to make these determinations. Furthermore, it may be possible to prepare smaller sections of abnormal tissue (i.e., punch-biopsy samples) in the same fashion as the whole gland; however, those technical variables were not tested and would require further efforts.…”

Scientific and Regulatory Policy Committee Points to Consider Review

“…This is particularly important for the female reproductive tract, since the morphology is highly synchronized and extremely sensitive to hormonal perturbation. Normal cyclicity can be disrupted by environmental factors, such as lighting, social hierarchy or secondary factors such as stress and body weight changes, and these in turn will affect the morphologic appearance of the various portions of the reproductive tract (Keane et al , 2015, Bussiere et al , 2013, Everds et al , 2013, Weinbauer et al , 2008). …”

Comparison of the sensitivity of histopathological and immunohistochemical analyses and blood hormone levels for early detection of antithyroid effects in rats treated with thyroid peroxidase inhibitors

“…Although evaluation of these circulating hormones in rodent toxicity studies can be useful for detection of antithyroid chemicals, these assays are associated with several problems, including high cost and high variability depending on blood sampling conditions. Blood T3, T4, and TSH levels change rapidly in response to stress conditions (Helmreich & Tylee, 2011; Keane et al, 2015), and assay sensitivity varies not only among methods but also between experiments, even with the same measurement method (Li et al, 2019). Therefore, more appropriate and efficient detection methods need to be developed.…”