702 R. Golden leukemogenic chemicals are shared by formaldehyde. Inhaled formaldehyde, even up to 15 ppm, does not enter the blood and reach any sites distal to the nasal epithe- lium or alter normal endogenous blood levels (Lu et al., 2010a; Heck et al., 1985. Cassanova et al., 1988), and does not cause hematotoxicity, pancytopenia, or myelotoxico- ity in animals or humans (Golden et al., 2006; ATSDR, 1999). Despite the established inconsistency of inhaled formaldehyde with the biological properties of known human leukemogens, DeVoney et al. (2006a, 2006b) pro- posed a hypothetical mode of action. The most impor- tant characteristic of this hypothesis is that it avoids the necessity for formaldehyde entering the systemic circu- lation, transporting to the bone marrow, or producing myelotoxicity. Instead, DeVoney et al. (2006a, 2006b) postulate the involvement of nasal-associated lymphoid tissue (NALT) at the point of entry in the nose, with form- aldehyde adversely affecting stem cells or hematopoietic progenitor cells (HPCs) that circulate in NALT, causing those cells to be malignantly transformed. These trans- formed cells are assumed to travel to the bone marrow leading to the development of leukemia in the absence of myelotoxicity. Several similar MOAs incorporating many of the same elements have also been proposed by Zhang et al. (2009a, 2009b). Due to the central importance of NALT in the DeVoney et al. hypotheses, Kuper et al. (2009) investigated whether NALT and local lymph nodes might be affected by inhaled formaldehyde. This 28-day inhalation study was con- ducted in F344 rats and B6C3F1 mice at formaldehyde concentrations of 0, 0.5, 1, 2, 6, 10, and 15 ppm to deter- mine if local lymphoid tissues are a target. Both NALT and upper-respiratory-tract draining lymph nodes were stained with hematoxylin and eosin (H&E) or immuno- histochemically for cell proliferation via BrdU incorpo- ration. Light microscopy revealed slight-to-moderate hyperplasia of NALT lymphoepithelium and an increased epithelial cell proliferation rate in rats exposed to 15 ppm. Analysis of rat NALT and lymph nodes did not reveal effects at lower exposure levels, whereas similar tissues from mice were not affected at any dose. As concluded by the authors, “…the only distinct effect of FA vapor on NALT and local lymph nodes in Fischer 344 rats and B6C3F1 mice was simple hyperplasia of the lymphoepithelium of NALT in rats exposed to15 ppm. Therefore, the results of the present study did not support the hypothesis that FA may induce hematological malignancies by reacting with local lymphoid tissues such as NALT.” Another study directly relevant to the hypothesis proposed by DeVoney et al. (2006a, 2006b) (i.e., transfer from affected cells in NALT to circulating myeloid pro- genitor cells) was conducted by Neuss et al. (2010), in which primary human nasal epithelial cells (HNECs) and isolated lymphocytes were co-cultivated in vitro to deter- mine whether reactive formaldehyde can be passed from nasal epithelial cells (site of first contact) to lymphocytes located in close proximity and induce DNA damage in these cells. A modified comet assay was used as a sensi- tive method for the detection of formaldehyde-induced DNA-protein cross-links (DPX) because these are the most relevant type of formaldehyde-induced DNA dam- age. The results clearly indicated that co-cultivation of lymphocytes with HNECs exposed to formaldehyde for 1 hour causes a concentration-related induction of DPX in these cells when co-cultivation takes place in the exposure medium. However, when the exposure medium was changed after formaldehyde treatment of HNECs and before lymphocytes are added, no induc- tion of DPX was measured in lymphocytes even after exposure of HNECs to high formaldehyde concentra- tions (300 μM) and extended duration of co-cultivation (4 hours). Direct measurement of formaldehyde in the cell culture medium demonstrated that formaldehyde was not released even from highly exposed cells into the cell culture medium. These results suggest that formal- dehyde that comes into contact with nasal epithelial cells is not released and therefore cannot damage other cells in close proximity to the epithelial cells. Assuming these results also apply in vivo, formaldehyde would only be genotoxic to directly exposed cells with no delivery to other cells or to distant sites. These results do not support the hypothetical mechanism proposed by DeVoney et al. (2006a, 2006b) that formaldehyde-induced leukemia is initiated by damaging circulating hematopoietic stem or progenitor cells in the nasal passages, with subsequent distribution to the bone marrow leading to the develop- ment of leukemia. Rodents chronically exposed to high formaldehyde concentrations for 2 years in the numerous inhalation bioassays had NALT and other local lymphoid tissues and none of those animals were reported to have devel- oped leukemia. Interestingly, lymphoid tissues identical to NALT are also found in the stomach, i.e., gastric-as- sociated lymphoid tissue (GALT). Consequently, if the hypothesized formaldehyde-induced mutations in stem or hematopoietic progenitor cells in NALT are a proxi- mate cause of leukemia, the same must be hypothesized as occurring in GALT. However, several chronic inges- tion studies in which animals were exposed via drinking water to large concentrations of formaldehyde have not produced any cancers, including leukemia or lymphoma (e.g., Tobe et al., 1989; Til et al., 1989). Also, neither of these studies showed any effects on hematological parameters. The issue pertaining to GALT was also indirectly addressed in comments submitted by scientists from other government agencies who reviewed the draft US EPA/IRIS assessment prior to its release. Because form- aldehyde is naturally found in many foods (fruits and vegetables [3−60 mg/kg], meat and fish [6−20 mg/kg], shellfish [1−100 mg/kg], and milk and milk products [1−3.3 mg/kg]) (US EPA, 2010a), there is a potential for substantial dietary exposure. As with the formaldehyde drinking water studies, this would also result in direct contact with GALT. After first observing that “The toxicity Critical Reviews in Toxicology