Journal of Toxicology dams and their oﬀspring to air or MnSO4 (0.05, 0.5, or 1 mg Mn/m3) for 6 hr/day, 7 days/week starting 28 days prior to breeding and from PND 1 through 18. The experimentally determined manganese concentration in neonatal striatum and the model-predicted AUC for this brain region did not imply signiﬁcantly higher exposures in the pups when compared to those in adults up to the inhaled dose of 1 mg/m3 [55, 56]. Despite the virtual absence of basal biliary excretion in neonatal rats, they appear to induce their biliary excretion when challenged with excess manganese through the oral route [57]. This inducible excretion in neonates was shown to be applicable to inhaled manganese as well to a level comparable to adults [56]. Because neonates have an increased requirement for manganese for optimal brain development, further elaboration of the dose-response rela- tionship between brain manganese levels and neurotoxicity may further elucidate the potential vulnerability of neonatal brain to manganese. Studies in Rhesus Monkeys. The Hamner inhalation study that exposed juvenile rhesus monkeys to MnSO4 is amongst the most critical completed to date with manganese. In this study, one group of monkeys was exposed to either air or MnSO4 (0.06, 0.3, or 1.5 mg Mn/m3) for 65 exposure days (6 hr/day, 5 days/week) before tissue analysis. Additional monkeys were exposed to MnSO4 at 1.5 mg Mn/m3 for 15 or 33 exposure days and evaluated immediately thereafter or for 65 exposure days followed by a 45- or 90-day delay before evaluation. Monkeys exposed to MnSO4 at ≥0.06 mg Mn/m3 developed increased manganese concentrations in the globus pallidus, putamen, olfactory epithelium, olfactory bulb, and cerebellum. Absolute manganese concentrations in the MnSO4-exposed monkeys demonstrated a decreasing peripheral-central concentration gradient within the olfac- tory system (i.e., olfactory epithelium olfactory bulb > olfactory tract > olfactory cortex). These data are consis- tent with direct olfactory transport of inhaled manganese. Increased pallidal manganese concentrations were evident by brain MRI and further conﬁrmed by atomic absorption spec- trometry analysis of the tissues [36]. MRI changes seen in this monkey study were similar to those reported in welders that have had high manganese exposure and subsequently developed bilateral hyperintensity on T1-weighted images in the globus pallidus and other brain regions [7]. Signal hyperintensities could not be visualized by MRI between the olfactory bulb and more distal sites, suggesting that direct translocation of manganese from the olfactory bulb to the globus pallidus did not occur in the manganese-exposed monkeys. Metabolomic analysis of serum and chemical analysis of brain tissues from the MnSO4-exposed monkeys revealed changes indicative of oxidative stress at higher exposure concentrations [39, 40]. Dorman and coworkers [37] also reported that exposure of monkeys to MnSO4 at 1.5 mg Mn/m3 for ≥15 exposure days resulted in increased lung manganese concentrations, mild subacute bronchiolitis, alveolar duct inﬂammation, and proliferation of bronchus- associated lymphoid tissue. Bronchiolitis and alveolar duct inﬂammatory changes were absent 45 days after exposure, 7 suggesting that these lesions are reversible upon cessation of subchronic high-dose manganese exposure. 6. Lessons Learned The work described herein represents the most extensive set of pharmacokinetic studies performed to date under the USEPA Alternative Tier 2 requirements. The pharmacoki- netic data that was collected through this testing program has dramatically improved our understanding of the health risks posed by manganese. These studies have led to an improved understanding of the exposure conditions that lead to increased concentrations of the metal within the adult and developing brain and other tissues. This work has also led to the development of predictive, PBPK models for inhaled manganese that relate lung, brain, and other tissue manganese concentrations to exposure concentrations [2]. These PBPK models should lead to the development of human health risk assessments for inhaled manganese that will consider both its essentiality and neurotoxicity. This testing program also represents an example of productive industry/government cooperation despite the challenging regulatory climate under which it commenced [22]. Evidence of cooperation was manifested by the sponsor’s willingness to conduct additional studies that were outside of the scope of the required testing pro- gram. Some examples included work to evaluate olfactory transport of manganese following inhalation, short-term (2 weeks) pharmacokinetic studies, and experiments designed to examine the elimination kinetics of inhaled manganese (see Table 1). Many of these studies were needed to develop the framework for subsequent development of the PBPK models. In addition, the sponsor also voluntarily developed the human PBPK models, as only the animal models were required by USEPA. All parties involved in this program shared a common desire to develop the most robust set of experiments possible. Figure 1 shows the extensive review and oversight by EPA and others that contributed to the development of robust experimental protocols. In addition, all parties involved recognized the value of the development and use of novel technologies (e.g., inhalation exposure systems for lactating rats) and incorporation of a wide array of endpoints, including MRI. There was also great value in conducting the work at a multidisciplinary indepen- dent research institute that could help facilitate discussions between the USEPA and the research sponsor. Incorporation of additional manganese TAPs composed of external experts also built on the Hamner’s rich history of independent external peer review of their research programs. This testing program also beneﬁted from multiple postdoctoral fellows and undergraduate researchers; thus, this program also played a key role in training new scientists. Finally, the MMT testing program is aligned with the National Research Council vision described in their report entitled “Toxicity Testing in the 21st Century: A Vision and a Strategy” [58]. The three main components of the NRC vision for the future of toxicity testing are chemical characterization, toxicity testing, and dose-response and