Mammary Gland Assessment in Guideline Toxicology Studies for Environmental Chemicals Susan Makris U.S. EPA, Office of Research and Development National Center for Environmental Assessment July 21, 2011 This presentation does not necessarily represent the policy or opinions of the U.S. EPA.

Background Evaluation of structural and/or functional integrity of the mammary gland across life stages is integral to assessment of developmental, reproductive, and carcinogenic risk for environmental chemicals Question: how well do standard toxicology guideline protocols assess mammary gland development, adult function, or pathology (including cancer)? Makris, S.L. (2011) Current assessment of the effects of environmental chemicals on the mammary gland in guideline EPA, OECD, and NTP rodent studies. Environmental Health Perspectives (published on-line: doi:10.1289/ehp.1002676).

Toxicology Guidelines Standard testing protocol (study design); a tool that facilitates standardized data collection Includes recommendations for: Study methods Endpoints to assess Data analysis Guidelines do not address when to conduct the study, or what substances to test Each guideline focuses on specific exposures, endpoints, life stages, test species, etc. Similar guidelines exist for various regulatory agencies

Analysis Prenatal Developmental Toxicity Screening Developmental/Reproduction 2-Generation Reproduction Reproductive Assessment by Continuous Breeding (RACB) 1-Generation Reproduction Extended 1-Generation Reproduction Developmental Neurotoxicity Uterotrophic Assay Hershberger Assay Juvenile/Pubertal Male or Female Assay Subacute or Subchronic Chronic or Carcinogenicity EPA, OECD, and NTP toxicology guidelines with: Reproduction phase(s) Development Lactation Endocrine assays Postmortem evaluation Organ weights Necropsy observations Histopathology Evaluation of study designs for MG-specific recommendations Note: study designs are described in the manuscript, and study diagrams are provided in supplemental materials on EHP website. EPA = Environmental Protection Agency OECD = Organisation for Economic Co-operation and Development NTP = National Toxicology Program

Findings In reproduction and endocrine studies, the guidelines do not clearly indicate the need to focus on evaluation of endpoints that are specific to MG development, health, or function Developmental and functional assessments are indirect or non-specific (e.g., surrogate measures of offspring growth and survival) Endpoints that can be useful in assessing MG toxicity: Some protocols include endocrine-mediated biomarkers (e.g., timing of puberty) that could be informative about MG development and function Clinical observations and postmortem examination may identify effects In subchronic and chronic studies, MG pathology (when required) is generally: Conducted on adult animals (not during development) Female-centric (males are often not evaluated) Not linked to developmental exposures (exception: NTP)

Recommendations Guidelines should be written/revised to specifically address evaluation of MG endpoints In studies that include peripubertal offspring that were treated in utero and/or postnatally, MG tissue should be evaluated using sensitive methods (e.g., whole mount technique) In subchronic and chronic rodent studies, exposure should be initiated in utero The evaluation of male MG tissue should be routinely required