6 Journal of Toxicology Table 2: Notable scientiﬁc contributions derived from the MMT Alternative Tier 2 test program. Pharmacokinetic endpoint Chemical form of manganese Dose and duration dependences Homeostatic control Dose metrics Route of exposure Olfactory transport Species diﬀerences Key Finding(s) Lung uptake and brain (tissue) delivery is highly inﬂuenced by solubility (sulfate phosphate > tetroxide) Manganese uptake and elimination rates depend on exposure dose and exposure duration Delivery to the brain and development of pseudosteady manganese concentrations develop rapidly Biliary excretion shows similar time and concentration dependencies. Manganese concentration in brain remains controlled at low levels of exposure and accumulates at air concentrations >10–50 µg/m3 Dose rate rather than cumulative dose appears to be the appropriate dose metric at low levels of exposure The observed pharmacokinetic diﬀerences between dietary and inhaled manganese can be attributed to rates of uptake and elimination required to achieve the same target tissue doses Inhaled manganese is taken up by the nasal olfactory epithelium and transported directly via the olfactory nerve to the olfactory bulb Despite speciﬁc pharmacokinetic diﬀerences between rats and nonhuman primates, similar overall pharmacokinetic responses to and homeostatic controls of manganese were observed across species in their individual publications, several signiﬁcant ﬁndings are summarized here. Solubility. The Hamner particle solubility studies showed that hureaulite and Mn3O4 are relatively insoluble in simu- lated lung lining ﬂuids, while MnSO4 is considerably more soluble in biological ﬂuids [52]. These studies also showed that soluble manganese forms like the sulfate are more rapidly cleared from the rat lung and delivered to the rat brain following inhalation than are insoluble manganese oxide and phosphate particles. Direct Olfactory to Deep Brain Transport. One project goal was to determine whether inhaled manganese could be transported to the brain directly via the olfactory nerve. Interest in this topic was sparked by the knowledge that the olfactory system forms a direct interface between the air and the brain. The Hamner conducted studies in rats using short- term (90 min) inhalation exposure to radiolabeled (54Mn) aerosols in rats with one occluded nostril, thus restricting olfactory transport of manganese to only one side of the rat brain. These novel studies dramatically demonstrated that the olfactory route contributes the vast majority (>90%) of the 54Mn found in the olfactory pathway of the rat brain up to 8 days following acute inhalation exposure. To our knowledge, this was the ﬁrst study to demonstrate that an inhaled metal could be delivered to the olfactory brain regions directly via the olfactory nerve. Although olfactory transport rapidly delivers manganese to brain structures in the olfactory pathway, it appears to be relatively slow (and ineﬃcient) in delivering inhaled manganese to the rat striatum and other more distant brain structures [53]. Addition of Inhaled Manganese to Existing Oral Exposures. Individuals with either deﬁcient or excessive manganese tissue burdens have been postulated to be at increased risk for manganese toxicity following inhalation exposure [45]. Two related 14-day inhalation studies conducted by the Hamner demonstrated that manganese body burden does not inﬂuence brain manganese concentrations following inhalation [44, 54]. These studies placed postnatal day (PND) 10 rats on specially formulated diets that contained 2, 10, or 100 ppm manganese. The lowest and highest diets were chosen in order to provide the animals with a marginally deﬁcient or high-normal level of manganese. The 10 ppm manganese diet used in the studies met rodent dietary guidelines. Once tissue manganese concentrations stabilized (i.e., after 2 months on the special diets), rats were exposed by whole-body inhalation for 6 hr/day on 14 consecutive days to MnSO4 or Mn3O4 at concentrations equivalent to 0, 0.03, or 0.3 mg Mn/m3. Feeding the 2 ppm manganese diet was associated with a number of eﬀects, including reduced body weight gain, decreased liver manganese concentrations, and reduced whole-body manganese clearance rates. Although rats kept on this diet and then exposed to 0.3 mg Mn/m3 developed increased manganese concentrations in some tissues, the studies did not demonstrate any statistically sig- niﬁcant diet and inhalation interactions on brain manganese concentrations. Neonatal Exposures. Concerns have been raised regarding increased risk of neonates for manganese-induced neuro- toxicity [32]. The increased sensitivity of neonatal animals to manganese appears to be due in part to their ability to develop higher brain manganese levels than adults when faced with equivalent or lesser manganese exposures by high- dose oral gavage [51]. Factors inﬂuencing this increased susceptibility of neonatal animals may include enhanced manganese absorption from the gastrointestinal tract, an incompletely formed blood-brain barrier, and a greatly reduced basal biliary manganese excretory rate until wean- ing. However, information was more limited regarding the potential risks in neonates exposed to airborne manganese. To further assess this concern, the Hamner exposed rat