704 R. Golden a consequence of un-repaired or mis-repaired DNA- protein cross-links (DPX) and the preponderence of data shows that formaldehyde acts predominantly by a clasto- genic and not by an aneugenic mode of action. Molecular characterization of formaldehyde-induced micronuclei in cultured human lymphocytes and V79 cells as well as micronuclei measured in buccal and nasal cells of formaldehyde-exposed subjects showed that micronu- clei occurred as a consequence of chromosome break- age and not of aneuploidy (Schmid and Speit, 2007; Speit et al., 2007a, 2007b, 2008; Titenko-Holland et al., 1996). The only reported association between formaldehyde exposure and aneuploidy comes from a biomonitoring study with subjects exposed to formaldehyde at the work- place (Orsiere et al., 2006); however, the plausibility and reliability of this result has been questioned (Schmid and Speit, 2007). A negative in vivo micronucleus test with rats exposed to formaldehyde by inhalation for 4 weeks at concentrations up to 15 ppm showed that formaldehyde does not induce aneuploidy in bone marrow cells (Speit et al., 2009). Recent reports of no exogenously derived 13CD2-formaldehyde-DNA adducts in the bone marrow or lymphocytes (Lu et al., 2010a; Moeller et al., 2011) also challenges the likelihood that formaldehyde was the cause of the findings reported by Zhang et al. (2010). The findings on the lack of delivery of inhaled form- aldehyde to the bone marrow have now been extended to non-human primates. As described in conjunction with NPC, the study by Moeller et al. (2010) also deter- mined the presence of endogenous and exogenous formaldehyde in DNA from bone marrow of cynomolgus macaques exposed to 1.9 and 6.1 ppm of 13CD2-formal- dehyde for 6 hours a day for 2 consecutive days. In bone marrow, no exogenous DNA adducts were detected, even though ~10-fold larger amounts of DNA were analyzed. However, endogenous N2-hydroxymethyl-dG adducts were present at 17.48 ± 2.61 and 12.43 ± 3.63 adducts/107 dG in bone marrow DNA from the 1.9 and 6.1 ppm expo- sures, respectively. Using the stable isotope data in primates reported by Moeller et al. (2010), Swenberg et al. (2010) developed a cancer risk estimate approach using a unique “bottom up” methodology that extrapolates upward from back- ground (endogenous) exposure and response, rather than the typical “top down” approach that often requires downward extrapolation from exogenous exposures. Since no exogenous formaldehyde-dG DNA adducts were detected in the bone marrow and white blood cells, it was assumed that if such adducts were present their amounts would have to be less that the limit of detection (LOD) of the sensitive analytical method used. Since endogenous and exogenous formaldehyde dG adducts are chemically indistinguishable following an environmental exposure, both should be implicated to the same extent in the carcinogenicity of low exposures to formaldehyde. It was further assumed that the levels of endogenous and exogenous dG adducts in various rat and primate tissues are similar to what would be found in corresponding human tissues, after adjusting for spe- cies differences. As noted by Swenberg et al. (2010), “…no [13CD ]-OH-methyl dG adducts were detectable when 2 312 μ g of bone marrow DNA was analyzed. In contrast, high numbers of endogenous OH-methyl dG adducts were present in bone marrow DNA. The number of endog- enous adducts in the 312 μ g sample of bone marrow DNA was 13.9 adducts/107 dG. If one constructs a worst case scenario by assuming that exogenous adducts were just below the LOD in this sample, less than 1 [13CD ]-OH- 2 methyl dG adducts/1010 dG could have existed. This would mean that less than 1 exogenous DNA adduct was present for every 13,900 endogenous formaldehyde adducts. It is difficult to conceive of a mechanism by which 1/13,900 identical DNA adducts could drive the biology that leads to carcinogenesis.” 6.Potential NPC and leukemia risks based on stable isotope studies Swenberg et al. (2010) also used the results from the “bot- tom up” approach described above to calculate upper bound estimates of extra lifetime cancer risk from contin- uous exposure to 1 ppm formaldehyde with comparisons to the risks calculated in the US EPA/IRIS document. For NPC, the upper bound extra risk estimates span a near 20-fold range, from a low of 0.39 × 10−3 (using monkey data) up to 7.49 × 10−3 (using rat data), both of which were less than the corresponding US EPA estimate of 1.1 × 10−2. Even greater disparity with US EPA’s values was reported for the risk estimates for Hodgkin’s lymphoma and leukemia. At 1 ppm, the two Hodgkin’s lymphoma estimates were 81-fold (rat data) and 250-fold (monkey data), respectively, lower than the US EPA’s estimate of 1.7 × 10−3. The leukemia estimates ranged from 45-fold (rat data) to more than 19,000-fold (monkey data) lower than the US EPA estimate of 0.057. At 1 ppb, the lifetime risk estimates for these two cancers were all well below the de minimus 1 × 10−6 level. As described by Swenberg et al. (2010), the disparities between the adduct-based and epidemiology-based cancer risk estimates for distant sites strongly suggest that the excess risks of leukemia and Hodgkin’s lymphoma reported from occupational exposures to formaldehyde exposures cannot be due to formaldehyde. As concluded by Swenberg et al. (2010) based on the plausible assumption that “…formaldehyde dG adducts provide a valid molecular dosimeter for relat- ing potential human cancer risks to formaldehyde expo- sure, the far larger risks derived from adult human data are simply not credible.” 7.Formaldehyde hematotoxicity There is also the question of formaldehyde-induced hematotoxicity (i.e., decreased blood counts) and the conclusion by Zhang et al. (2010) that this was consis- tent with toxic effects on the bone marrow (i.e., myelo- toxicity). Of interest, Zhang et al. (2010) equate their reported findings with formaldehyde as being similar to those produced by benzene or therapy-related AML. Critical Reviews in Toxicology