Journal of Toxicology 5 The registrant develops protocols Independent peer review Meets requirements Comments The registrant submits ﬁnal reports to EPA ORD review The registrant submits revised protocols to EPA The registrant submits revised reports to EPA Comments Comments ORD review Meets requirements Comments Independent peer review The registrant submits ﬁnal protocols to EPA conducts studies then prepares draft reports Figure 1: Schematic overview of steps used to develop and review study protocols and produce ﬁnal reports. Independent peer review was carried out by the appropriate TAP. Completed ﬁnal reports for all of the manganese studies can be found in the Federal Docket Management System (FDMS) at http://www.regulations.gov identiﬁed by docket number EPA-HQ-OAR-2004-0074. EPA: Environmental Protection Agency; ORD: EPA Oﬃce of Research and Development. ﬁne structure (XAFS) spectroscopy, showed the presence of three major manganese-containing components in tailpipe emissions: a divalent manganese phosphate, sulfate, and oxides (most likely as Mn3O4) [29–31]. The percentage of each component varied somewhat depending on the driving cycle and vehicle but remained relatively constant with the sulfate and the phosphate forms being the major (∼80%) components [1]. The USEPA subsequently agreed to the use of com- mercially available manganese phosphate (as hureaulite), MnSO4, and Mn3O4 for the inhalation studies rather than rely on the use of a dynamometer system that used MMT in the fuel source to generate exhaust particulates. This decision also allowed the Hamner research team to achieve the high exposure concentrations (mg/m3) required to conduct the study. Such high concentrations were necessary to ensure tissue accumulation so the data could be used to inform the subsequent PBPK models. Similar aerosol concentrations and particle sizes were used in the Hamner inhalation studies to facilitate direct comparison of tissue manganese burdens among these experiments. Early studies used all three forms of manganese while later experiments relied upon the form of manganese (MnSO4) that produced the greatest increase in brain manganese concentrations, representing a “worse- case scenario” with regards to potential brain delivery. Another unique feature of the MMT alternative Tier 2 test program was the use of rhesus monkeys for certain stud- ies. A substantial literature base describing species diﬀerences in neurological responses following high-dose manganese exposure existed at the time the Alternative Tier 2 program was launched. Unlike rats, manganese-exposed monkeys develop distribution patterns for this metal within the brain that mimic those seen in heavily exposed people and develop similar neuropathology and behavioral responses [32–34]. Despite these pharmacodynamic diﬀerences in response between rats and humans, pharmacokinetic data obtained from rats remained valuable because rats and primates show similar overall pharmacokinetic responses to manganese exposure, including the induction of homeostatic control mechanisms. The rat data provided critical insights into the dose-response relationship for inhaled manganese, especially during diﬀerent life stages. Once the decision to use rhesus monkeys was reached, there was a concerted eﬀort to maximize the data that could be obtained from this study. Study endpoints included tissue manganese concentrations following MnSO4 exposure [35], brain magnetic resonance imaging (MRI) studies [36], respi- ratory tract pathology [37], catecholamine neurochemistry [38], biomarkers of neurotoxicity and oxidative stress [39], and metabolomic biomarkers of exposure [40]. The USEPA waived the GLP requirements for certain study endpoints, including the MRI study, neurochemistry measurements, and the assessment of biomarker endpoints. Another challenge that faced the research team was conduct of the required gestational and lactational inhala- tional studies. These studies required the coexposure of lactating rat dams and their pups. Inhalation developmental neurotoxicity and pharmacokinetic studies generally rely on maternal separation during exposure, resulting in pup stress that may alter development, thereby confounding study results. To overcome this potential confounding variable, Vitarella and coworkers [41] developed a unique single- animal exposure cylinder designed to house a rat dam and her litter. These investigators ﬁrst tested concentrations of manganese phosphate within the exposure cylinder to verify that particle concentrations within this system were equivalent to those achieved within a stainless steel 1-m3 inhalation chamber. Once developed, this system was then used to support the rat lactational exposure study required by the USEPA [42]. 5. Key Research Findings A number of signiﬁcant discoveries emerged from this testing program (Table 2). While these studies are described in detail