4 Pharmacokinetic endpoint of interest Particle solubility and dissolution kinetics Exposure-response and WB clearance Particle solubility and WB clearance Diet-inhalation interaction and WB clearance Individual susceptibility WB clearance Nasal pathology Individual susceptibility Species diﬀerences Brain imaging Respiratory tract pathology and neurochemistry Olfactory transport of Mn Voluntary Regulatory Status Voluntary Voluntary Voluntary Voluntary Required Required Required Animal Species Rat Rat Rat Rat Rat Rat Rat Rhesus monkey Manganese species and exposure conditions Phosphate, sulfate, and tetraoxide—intratracheal instillation 0, 0.04, 0.08, or 0.16 µg Mn/g Rats killed at 0, 1, 3, or 14 d Phosphate inhalation 6 h/d for either 5 d/wk or 7 d/wk at 0, 0.03, 0.3, or 3 mg Mn/m3 for up to 14 d Sulfate and tetroxide inhalation 6 h/d for 7 d/wk at 0, 0.03, 0.3, or 3 mg Mn/m3 for 14 d Sulfate or tetroxide inhalation 6 h/d for 7 d/wk at 0, 0.03, or 0.3 mg Mn/m3 for 14 d Low (2 ppm), suﬃcient (10 ppm), or high (100 ppm) Mn diets Chloride and phosphate inhalation ∼0.5 mg Mn/m3 for 90 min Occluded nostril model Inhalation Exposed 6 h/d for 5 d/wk to sulfate at 0.01, 0.1, or 0.5 mg Mn/m3 or phosphate at 0.1 mg Mn/m3 for up to 90 d Adult male, adult female, and senescent male Sulfate inhalation Exposed 6 h/d for 7 d/wk at 0, 0.05, 0.5, or 1 mg Mn/m3 throughout the majority of pregnancy or lactation. Sulfate inhalation Exposed 6 h/d for 5 d/wk at 0, 0.06, 0.3, or 1.5 mg Mn/m3 for up to 90 d. Journal of Toxicology Primary publication(s) [42] [43] [44] [45, 46] [47, 48] [49, 50] [41, 51] [34–38] Table 1: Overview of the pharmacokinetic studies conducted in response to the Alternative Tier 2 test program for MMT. The following forms of manganese (Mn) were used in these studies: Mn tetroxide, Mn sulfate, and Mn phosphate. Some studies also involved intravenous administration of radiolabeled Mn chloride (54MnCl2) to assess whole-body (WB) clearance. the USEPA Good Laboratory Practice (GLP) Standards for Inhalation Exposure Health Eﬀects Testing (40 CFR Part 79.60). All studies were performed at The Hamner Institutes for Health Sciences (Hamner) (formerly the Chemical Industry Institute of Toxicology (CIIT)). All required study protocols, protocol amendments, and draft ﬁnal reports underwent independent scientiﬁc review by project speciﬁc “technical advisory panels” (TAPs) composed of individuals with expertise in inhalation toxicology, pharmacokinetics, and neurotoxicology (Figure 1). Members of the TAPs, which changed for the diﬀerent facets of the test program, were chosen by the study sponsor with input from the testing laboratory and approved by the USEPA. All study results underwent additional independent peer review during sub- sequent publication of the work in scientiﬁc journals. Several critical decisions were reached early in the MMT test program. These concerned the form of manganese to be examined, animal species used, and endpoints of interest. The main focus of the test program was to evaluate combustion products of MMT. This decision was inﬂuenced in part by the observation that MMT undergoes rapid photolysis when exposed to light, forming methylcyclopenta- diene, cyclopentadiene, carbon monoxide, and a manganese carbonyl that is readily oxidized to trimanganese tetroxide [26]. One early dilemma facing study toxicologists was determination of these combustion products. Early studies performed by Ter Haar and coworkers [27] showed that the primary combustion product from MMT was Mn3O4. This study used fuel spiked with large concentrations of MMT, an automobile without a catalytic converter, and X- ray diﬀraction methods for chemical speciation, resulting in an artifactual apparent enrichment of Mn3O4 in the exhaust. Zayed et al. [28], using scanning electron microscopy coupled with energy dispersive X-ray spectrometry, also suggested that the chemical form of emitted manganese was as an oxide but could not rule out sulfates and other manganese species. Other studies, using lower treat rates representative of actual use patterns and more advanced analytical methods, including X-ray absorption near-edge structure (XANES) spectra and K-edge X-ray absorption