676 R. Golden direct contact with the eyes or upper respiratory tract is the necessity for absorption and transport to distant sites. This issue has substantial implications for assess- ing whether inhaled formaldehyde would be capable of causing leukemia, which requires, at a minimum, that it reaches the blood or bone marrow to initiate this disease. Although there is no detectable increase in endogenous formaldehyde blood concentrations following inhalation exposure to formaldehyde, recent evaluations by both IARC (2009) and NTP (2009) have raised questions con- cerning this issue as it might pertain to distant site toxic- ity. Particularly with respect to explaining the biological basis for formaldehyde-induced leukemia, the potential for distant site toxicity (which would be obligatory for this disease) is based on a few publications (i.e., Zhang et al., 2009, 2010) hypothesizing that inhaled formaldehyde can increase endogenous free formaldehyde (i.e., gaseous) levels in the blood, with subsequent distant site toxic- ity. As a consequence, it is postulated that increased free formaldehyde can lead to adverse effects, either directly on the bone marrow or on circulating hematopoietic pro- genitor cells (HPCs) or stem cells, leading to myelotoxic- ity, decreased circulating red and white blood cells (i.e., pancytopenia), and ultimately leukemia. In conjunction with the epidemiology data, the conclusion that inhaled formaldehyde can raise endogenous levels with deleteri- ous consequences to the hematopoietic system played a role in the decisions by both IARC and NTP to elevate formaldehyde from a probable or reasonably anticipated carcinogen to a known human carcinogen. Following inhalation exposure to formaldehyde, the key hypothesis pertaining to formaldehyde-induced leu- kemia involves absorption and transport of exogenous formaldehyde to the bone marrow, with subsequent myelotoxicity, and/or direct interaction (i.e., muta- tion) in the circulation with susceptible stem cells, with the “transformed” cells traveling to the bone marrow to initiate the leukemogenic process. In support of this hypotheses, Zhang et al. (2009, 2010) describe how gas- eous formaldehyde in the presence of water (from the blood) dissolves and is converted to its hydrated form, methanediol [CH2(OH)2] (also known as methylene gly- col) and therefore could potentially reach the bone mar- row in this form, i.e., “…methandiol…which can readily penetrate into tissues, may travel to the marrow through the blood where it is in equilibrium with reactive form- aldehyde. The formaldehyde, once generated, can react with cellular macromolecules producing toxic injury.” An almost identical statement appears in the IARC’s (2009) recent deliberations, “In aqueous solution, formaldehyde is rapidly converted to its diol form, methanediol (form- aldehyde hydrate, CH2(OH)2 or methylene glycol), and a dynamic equiblirium with formaldehyde is formed. The concentration of the diol versus that of formaldehyde depends on the precise conditions (temperature, pH, formaldehyde concentration) under which the reaction occurs.... Importantly, methanediol, with a molecular weight of only 48, can readily penetrate into tissues (Fox, 1985). Thus, formaldehyde may reach the marrow through the blood as methanediol, where it equilibrates again to reactive formaldehyde. The formaldehyde, once regener- ated, can react with cellular macromolecules producing toxic injury.” In addition, NTP (2009) also describes a similar logic involving methanediol to help explain how a reactive chemical such as formaldehyde can be distrib- uted and undergo metabolism throughout the body, also citing Fox et al. (1985) as well as Matubayasi et al. (2007). The cited basis for methanediol as a way for formalde- hyde to be distributed to distant sites is a publication by Fox et al. (1985) on the use of 4% formaldehyde solutions for tissue fixation. At this concentration, formaldehyde rapidly penetrates dead tissues to denature and cross-link proteins, thereby also arresting enzymatic degradation. However, such a comparison, primarily extrapolating an event in non-living tissue at a high concentration of form- aldehyde to living tissue, in vivo, at low concentration is suspect. Hypothesizing about the biological activity of formaldehyde based on tissue fixing concentrations of 4% (i.e., 40,000 ppm) to normal endogenous concentra- tions of 2–3 ppm, which are at least 10,000 times lower, is questionable. The paper by Matubayasi et al. (2007) also has no relevance to biological systems, since it is about the formaldehyde/methanediol equilibrium at varying temperatures and concludes that in hot water (≈200°C), the equilibrium is shifted toward free formaldehyde. Because formaldehyde in its hydrated form (i.e., meth- anediol) is already present in the blood, it has penetrated every tissue in the body due to its ubiquitous presence. 1.Formaldehyde kinetics The hypothesis implying that the formaldehyde-meth- anediol equilibrium is inexplicably disrupted at distant sites, thereby leading to release of free formaldehyde, which then causes adverse effects illustrates a fundamen- tal misunderstanding of the well-established kinetics of formaldehyde. Gaseous formaldehyde, as a non-hydrated aldehyde, predominates only in the air phase. Whether in the extracellular spaces or within cells, free formaldehyde will be present at extremely low concentrations, since it first reacts reversibly with water to form an acetal (i.e., a more chemically correct designation of the hydrated form than methanediol) and then interacts with glutathi- one (GSH) to form a thioacetal. The equilibrium constant for the acetal versus free formaldehyde strongly favors the acetal by a factor of approximately 7000. In other words, at physiological temperature and pH, >99.9% of formaldehyde is present as methanediol, with <0.1% as free formaldehyde. Consequently, the assertions by Zhang et al. (2010) and IARC (2009) are not consistent with formaldehyde kinetics, as neither the acetal (meth- anediol) nor the thioacetal represents ways in which inhaled gaseous formaldehyde could travel through the circulation and reach distant tissues. Consequently, it is unknown (and not explained by Zhang et al., 2009, 2010; IARC, 2009; or NTP, 2009) how this equilibrium would be disrupted either in the circulation or at distant sites Critical Reviews in Toxicology