An in vitro approach to determine the human relevance of anti-spermatogenic effects of 4-methylmorpholine 4-oxide, monohydrate (NMMO) in rat reproductive toxicity studies.

Reduced sperm counts have been observed in male rats in an extended one generation reproductive toxicity study (EOGRTS, OECD 443) following repeated administration of 300 mg/kg/day N-Methylmorpholine N-oxide (NMMO). However, no adverse effects on reproductive organs have been reported in studies conducted with NMMO, and the mode of action (MOA) for the effects of NMMO on spermatogenesis is unknown, which complicates the interpretation of these data for human risk assessment. Here, a New Approach Method (NAM) strategy was used to evaluate NMMO MOA and compare interspecies susceptibility for anti-spermatogenic effects using organotypic in vitro assays combined with in vitro metabolism and in vitro to in vivo extrapolation (IVIVE) biokinetic modeling to compare predicted oral equivalent doses (OEDs) in human and rat. Dose-response data were collected in isolated germ cells and in an ex vivo seminiferous tubule model that recapitulates the interaction between the somatic environment and differentiating germ cells to account for potential direct and indirect effects on germ cells. With regard to direct spermatogenic effects, the human isolated germ cell model showed no toxicity at doses ≤300 µM (OED ≤ 86 mg/kg/day). With regard to indirect effects, the rat ex vivo model demonstrated dose-dependent decreases in secondary spermatocyte populations at OEDs ≥89 mg/kg/day, and reduced expression of RNAs specific to several stages of spermatogenesis (spermatogonia, pachytene spermatocytes, round spermatids) at OED = 267 mg/kg/day, consistent with in vivo observations. In contrast, the monkey ex vivo model did not show dose-dependent decreases in these same RNAs, and often demonstrated increased trends instead. These studies demonstrate clear quantitative and qualitative differences in the rat and primate response to NMMO. Furthermore, effects observed in the rat in vitro culture were not observed in the monkey at concentrations equivalent to in vivo doses of up to 1376 mg/kg/day, which is higher than the in vivo dose limit in the EOGRT study, indicating that the isolated findings on spermatogenesis in the rat studies are not likely to be relevant to humans.

Review of the Evidence from Epidemiology, Toxicology, and Lung Bioavailability on the Carcinogenicity of Inhaled Iron Oxide Particulates.

Since the iron-age and throughout the industrial age, humans have been exposed to iron oxides. Here, we review the evidence from epidemiology, toxicology, and lung bioavailability as to whether iron oxides are likely to act as human lung carcinogens. Current evidence suggests that observed lung tumors in rats result from a generic particle overload effect and local inflammation that is rat-specific under the dosing conditions of intratracheal instillation. This mode of action therefore, is not relevant to human exposure. However, there are emerging differences seen in vitro, in cell uptake and cell bioavailability between "bulk" iron oxides and "nano" iron oxides. "Bulk" particulates, as defined here, are those where greater than 70% are >100 nm in diameter. Similarly, "nano" iron oxides are defined in this context as particulates where the majority, usually >95% for pure engineered forms of primary particulates (not agglomerates), fall in the range 1-100 nm in diameter. From the weight of scientific evidence, "bulk" iron oxides are not genotoxic/mutagenic. Recent evidence for "nano" iron oxide is conflicting regarding genotoxic potential, albeit genotoxicity was not observed in an in vivo acute oral dose study, and "nano" iron oxides are considered safe and are being investigated for biomedical uses; there is no specific in vivo genotoxicity study on "nano" iron oxides via inhalation. Some evidence is available that suggests, hypothetically due to the larger surface area of "nano" iron oxide particulates, that toxicity could be exerted via the generation of reactive oxygen species (ROS) in the cell. However, the potential for ROS generation as a basis for explaining rodent tumorigenicity is only apparent if free iron from intracellular "nano" scale iron oxide becomes bioavailable at significant levels inside the cell. This would not be expected from "bulk" iron oxide particulates. Furthermore, human epidemiological evidence from a number of studies suggests that iron oxide is not a human carcinogen, and therefore, based upon the complete weight of evidence, we conclude that "bulk" iron oxides are not human carcinogens.