Is inhalation exposure to formaldehyde a biologically plausible cause of lymphohematopoietic malignancies?

Regulatory Toxicology and Pharmacology 51 (2008) 119–133

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Is inhalation exposure to formaldehyde a biologically plausible cause of lymphohematopoietic malignancies? David Pyatt a,c,*,1, Ethan Natelson d, Robert Golden b,*,1 a

Summit Toxicology, L.L.P., 509 N Bermont, Lafayette, CO 80026, USA ToxLogic LLC, Potomac, MD, USA c University of Colorado Health Sciences Center, Denver, CO, USA d Weill Medical College of Cornell University, Methodist Hospital, Houston, TX, USA b

a r t i c l e

i n f o

Article history: Received 18 December 2007 Available online 18 March 2008 Keywords: Formaldehyde Leukemia Leukemogenesis Lymphoma Lymphohematopoietic Lymphomagenesis Mode of action Lymphohematopoietic malignancies

a b s t r a c t The United States Environmental Protection Agency (EPA) recently proposed a hypothetical mode of action (MOA) to explain how inhaled formaldehyde (FA) might induce leukemia, lymphoma and a variety of other lymphohematopoietic (LHP) malignancies in occupationally exposed workers. The central hypothesis requires that B lymphocytes or hematopoietic progenitor cells (HPC) present at the ‘‘portal of entry (POE)” undergo sustained mutagenic change as a result of direct FA exposure. These modified cells would then migrate back to the bone marrow or primary lymphatic tissue and subsequently develop into specific LHP disease states. Chemical interaction at the POE is an absolute requirement for the hypothesized MOA as there is no convincing evidence that inhaled FA causes distant site (e.g., bone marrow) toxicity. The purpose of this review is to critically evaluate this proposed MOA within the context of the existing data concerning the toxicokinetic and biological properties of FA, the current understanding of the induction of chemically-induced leukemias and lymphomas, as well as within EPA’s specific guidelines for evaluating the MOA of chemically-induced cancers. Specifically, we examine the scientific support for the hypothesis that FA exposure may induce carcinogenic transformation of localized lymphocytes or peripheral hematopoietic progenitor cells (HPC) in the absence of discernable systemic hematopoietic toxicity (i.e., peripheral transformation). While little or no empirical evidence exists upon which to fully evaluate the proposed hypothesis, available data does not support the proposed concept of ‘‘peripheral transformation” at the chemical entry site. Numerous animal bioassays evaluating chronic inhalation of FA clearly do not support this hypothesis since no properly conducted study as ever shown an increase in any LHP malignancy. Moreover, the notion that FA can cause any LHP malignancy is not supported with either epidemiologic data or current understanding of differing etiologies and risk factors for the various hematopoietic and lymphoproliferative malignancies. It is therefore concluded that existing science does not support the proposed MOA as a logical explanation for proposing that FA is a realistic etiological factor for any LHP malignancy. Ó 2008 Elsevier Inc. All rights reserved.

1. Introduction In March 2006, scientists from the United States Environmental Protection Agency (EPA) presented a poster ‘‘Hypothesized Mode of Action for Formaldehyde-induced Lymphoid Malignancies” at the Annual Meeting of the Society of Toxicology (SOT), in San Diego, California (DeVoney et al., 2006a). This proposal was slightly expanded and again presented at the Society for Risk Analysis (SRA) meeting in Baltimore, MD several months later in a presentation ‘‘A Hypothesized Mode of Action in Support of the Biological * Corresponding authors. Fax: +1 303 666 9109. E-mail addresses: [email protected] (D. Pyatt), rgolden124@aol. com (R. Golden). 1 Both authors contributed equally to this manuscript. 0273-2300/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.yrtph.2008.03.003

Plausibility of Formaldehyde-Induced Lymphohematopoietic Malignancies” (DeVoney et al., 2006b). These presentations were presumably intended to provide a biologically plausible mode of action (MOA) to support the unproven conclusion that there was an elevated risk of lymphoid and myeloid malignancies among formaldehyde (FA) exposed workers. The MOA hypothesis developed by EPA scientists appears to imply that inhaled FA has the ability to induce all forms of myeloid and lymphoid leukemia as well as non-Hodgkin’s lymphoma (NHL), Hodgkin’s lymphoma, multiple myeloma (MM) and other less common hematopoietic malignancies. If true, this would be an unprecedented finding, as no chemical or physical agent at any exposure level (including high dose-ionizing radiation), has been shown to induce all forms of such malignancies. Were this MOA directed exclusively at myeloid leukemia, it would be the first instance of a chemical without

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established bone marrow or systemic hematopoietic toxicity causing this disease. As many of these diseases (e.g., Hodgkin’s lymphoma, MM, etc.) have no established chemical etiology FA would also be the first chemical described as an etiological agent in their development. There is no indication that EPA intends to publish this MOA hypothesis in the peer reviewed literature. Consequently, because of the well recognized clinical and cytological diversity among lymphohematopoietic diseases, a hypothesis of this sweeping scope and possible public health implications demands critical examination from a wider scientific audience. In 2006, the International Agency for Research on Cancer (IARC) re-evaluated the carcinogenic potential of FA and concluded that two recent studies provided ‘‘strong but not sufficient evidence for a causal association between leukemia and occupational exposure to formaldehyde.” This conclusion was based primarily on the observation that ‘‘. . . the Working Group could not identify a mechanism for leukemia induction, and this tempered their interpretation of the epidemiological evidence” (IARC, 2006). It was also concluded that previously discounted excess leukemia incidence reported in seven earlier studies of embalmers, funeral–parlor workers, pathologists and anatomists, were now supported by the analysis of two studies of US industrial workers (i.e., Hauptmann et al., 2003; Pinkerton et al., 2004). It should be noted that the concern expressed by IARC was directed only at leukemia and did not extend to other LHP malignancies. It appears that the hypothesis proposed by EPA has been put forward to address IARC’s concern that a mechanism for leukemia induction by FA could not be identified. However, this has not been explicitly stated nor is there an explanation for why the issue appears to have been expanded to include all forms of LHP malignancies instead of just leukemia as stressed by IARC. Furthermore, it is also unknown if this MOA was intended only to explain disease findings in occupationally exposed workers or if EPA’s concerns extend to non-occupational exposures as well. Because FA is virtually ubiquitous in both indoor and outdoor air (albeit at low concentrations) and present in the blood as part of normal metabolic processes, the suggestion that FA poses a health risk at ‘background’ levels would clearly have substantial regulatory and public health implications. An important part of our evaluation of this hypothesized MOA requires application and adherence to a set of objective criteria by which scientific hypotheses can be critically evaluated. These are often referred to as the ‘‘Hill criteria”, which have been modified and adopted by EPA (2005) for application to a body of data in order to determine the scientific weight that should be accorded to a hypothesized MOA. The Hill criteria (in addition to other objective guidelines discussed in detail below), have been endorsed by the EPA (2005) for evaluating scientific data which support a MOA for any, regulated chemical. Unfortunately, as detailed in this review, it is difficult to systematically apply these criteria to the hypothesized MOA for FA as presented by DeVoney et al. (2006a,b) since much of the critical experimental data are lacking. In order to explore the validity of the proposed MOA this paper addresses the following interrelated topics:  Brief overview of FA absorption, metabolism and other toxicokinetic properties.  Brief review of animal toxicity data, particularly the numerous long-term bioassays that have evaluated potential carcinogenic effects of inhaled FA (Appendix A).  Brief overview of epidemiological data, focusing specifically on LHP malignancies (Appendix A).  Biological plausibility of FA as a leukemogenic substance.  Overview of EPA MOA hypothesis for FA-induced LHP malignancies.  Critique and evaluation of EPA’s hypothesized MOA.

 Overview of EPA’s guidelines for evaluating a proposed MOA and the extent to which the hypothesized MOA and supporting data for FA-induced LHP malignancies fulfill these guidelines. A comprehensive review of many of these topics is outside the scope of this (or any single) manuscript. However, we have attempted to provide sufficient background information to permit readers to more fully appreciate the relevant scientific issues. 2. Brief overview of formaldehyde biology and metabolism FA is a ubiquitous and naturally occurring substance present in virtually all living organisms. It is highly reactive and likely exerts its corrosive and cytotoxic effects due to its ability to readily combine with free, unprotonated amino groups of amino acids or DNA to yield hydroxymethyl amino acid derivatives and a proton (H+). It is believed that FA toxicity results when intracellular levels saturate FA dehydrogenase (FDH) and other highly efficient metabolic detoxification activity, thereby overwhelming the natural protection against FA (ATSDR, 1999). This permits intact FA to exert adverse effects locally. The primary metabolite of FA is formate (Fig. 1) which is not as reactive as FA itself and can either enter into the one-carbon metabolic pool for incorporation into other cellular components, be excreted as a salt in the urine, or further metabolized to carbon dioxide (ATSDR, 1999). The metabolic pathway to formate production is catalyzed by cytosolic glutathione (GSH)dependent FA dehydrogenase (FDH). The reaction of FA with GSH yields S-hydroxymethylglutathione which, in the presence of NAD+ and FDH, forms the thiol ester of formic acid via the action of S-formyl glutathione hydrolase (SFGH). There is compelling scientific evidence conclusively demonstrating that inhaled FA does not enter the systemic circulation to modify normally present endogenous levels (ATSDR, 1999; Heck and Casanova, 2004). This is likely due to the high water solubility of FA and its rapid metabolism. The lack of systemic distribution is evidenced by a variety of studies in rodents, monkeys and humans. In one inhalation experiment, systemic distribution was not reported following exposures to FA as high as 14 ppm in rats and 1.9 ppm in humans (Heck et al., 1985). In another study, rhesus monkeys were exposed to 6 ppm FA (6 h/day, 5 d/wk for 4 wks) and the FA concentration in the blood measured by GCMS. FA concentrations immediately after the final exposure in the exposed and unexposed animals were 1.84 and 2.42 lg/g blood, respectively. Additionally, 45 h after the last exposure, blood concentrations did not differ significantly, indicating that blood levels were independent of external exposure (Casanova et al., 1988). These observations are critical to the issues discussed in this review as they indicate that (1) systemic distribution of FA does not occur and (2) there are substantial levels of endogenous FA normally present in the blood. It seems clear that as long as inhaled levels of FA are below concentrations that can be rapidly metabolized by tissue FA dehydrogenase and other highly efficient detoxification enzymes, normal endogenous concentrations (0.1 mM) of FA in the blood do not increase (ATSDR, 1999; Heck and Casanova, 2004).

HCHO + GSH

FDH GSH –S-CH2O ---------NAD+

SFGH -------------GSH – SH + HCOO --------------------

GSH – S – HCO + NADH + H+

CO2

Na Formate (excreted in urine) Fig. 1. Primary metabolic pathway of formaldehyde biotransformation.

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Radio-labeled FA has also been used to explore these issues further. Following inhalation exposure of rats with 15 ppm FA, various tissues including respiratory mucosa and the bone marrow were analyzed (Casanova-Schmitz et al., 1984). The major distribution at all concentrations occurred in the respiratory mucosa (i.e., metabolism of FA with subsequent entry into the one carbon pool). Radioactivity was found in the bone marrow, but was the result of one-carbon units derived from metabolism of the labeled FA. Data obtained from this experiment demonstrated that inhaled FA did not form covalent adducts (i.e., DNA protein crosslinking) with macromolecules in the bone marrow (Casanova-Schmitz et al., 1984). It has been shown that following very high exposures (i.e., 80 mg/kg/day for 4 wks), the metabolic detoxification capacity can be partially overwhelmed (Vargova et al., 1992). Under such extreme exposure conditions, some un-metabolized FA may enter the systemic circulation (ATSDR, 1999). However, Casanova and Heck (1987) demonstrated that depletion of glutathione (GHS) which significantly inhibited the metabolism of FA still did not result in inhaled FA reaching the bone marrow. In this study, GSH depleted rats were exposed to radio-labeled FA up to10 ppm. Again, there was measurable distribution to the nasal respiratory mucosa, but not to the bone marrow. Consequently, there is sufficient metabolic capacity to handle any realistic potential exposure to FA without increases in the peripheral blood FA concentrations. Finally, the inability of exogenous FA to increase blood concentrations was also confirmed by Franks (2005) in a sophisticated mathematical model for the absorption and metabolism of FA vapor by humans. The results of this model were consistent with in vivo data and demonstrated that following inhalation exposure, the increase in FA concentration in the blood is insignificant compared to existing endogenous levels. This model demonstrated that it was unlikely that inhaled FA could cause toxicity at sites other than the initial site of contact. 3. Formaldehyde and LHP malignancies in animal studies In general terms, the hypothesized MOA involves inhaled FA reaching susceptible cells (lymphocytes or HPC) in the nasal associated lymphoid tissue (NALT) and then via an unknown mechanism causing a sustained malignant transformation of these target cells which ultimately leads to the development of various LHP malignancies. In a very real sense, this hypothesis has already been extensively tested as an unforeseen but now relevant consequence of the numerous long-term bioassays conducted with FA in rodents (i.e., rats and mice). Like humans, both rats and mice have hematopoietic tissue at the portal of entry [i.e., NALT and gastric associated lymphoid tissue (GALT) which are discussed in detail below] as well as peripheral HPC with comparable functions across different species (Haley, 2003; Kuper et al., 2003). Because both NALT and HPC are hypothesized as key targets in the MOA for FA-induced LHP malignancies, these tissues should also be vulnerable to FA-induced toxicity and malignant transformation (see below for detailed discussion). There are well documented and important differences in hematopoiesis and lymphopoiesis between humans and rodents. However, as reviewed below, all chemicals known to produce leukemia in humans have also shown this capability in rodent studies with either rats or mice. In this regard, the various hematopoietic tissues (e.g., NALT) hypothesized by EPA to be potential targets of FA toxicity have, in fact, been chronically exposed to high concentrations of inhaled FA in numerous independent rodent cancer bioassays. As part of routine carcinogenicity testing protocols, numerous tissues (including lymph nodes, blood and bone marrow) are collected and assessed microscopically for pathological changes. While some peripheral tissues, such as the NALT are not typically collected, there is no evidence

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that chronic, high dose-exposure to FA (via either oral or inhalation dosing) causes hematopoietic toxicity or an increased incidence of any type of hematopoietic malignancy. If FA were capable of inducing leukemias and lymphomas in humans, it seems likely that there would be some indication of a similar potential in rodents. As summarized in Appendix A, there have been at least 10 chronic carcinogenicity studies on FA, four in which animals were exposed via ingestion and six involving inhalation exposure. With the exception of one questionable ingestion study (i.e., Soffritti et al., 1989; Soffritti et al., 2002, discussed in detail in Appendix A) none of the nine other studies have reported FA-induced carcinogenic effects other than at the site of administration, i.e., nasal cancer in rats and mice following inhalation exposure and possibly gastric cancer (not gastric lymphoma) in rats following ingestion exposure. This body of the literature does not support the position that FA exposure is causally associated with leukemia, lymphoma, bone marrow toxicity or hematological abnormalities. One inhalation study (i.e., Kerns et al., 1983) has been cited by DeVoney et al. (2006a,b) as showing a significant increase in lymphoma in female B6C3F1 mice and leukemia in female F-344 rats. The basis for this conclusion does not come from the published study, which does not mention either lymphoma or leukemia incidence, but rather from an ad hoc re-analysis of the underlying Battelle (1981) data by DeVoney and co-workers. This re-analysis included the generation of dose-response curves for each kind of cancer.2 However, a careful review of these data does not support the claim that either leukemias or lymphomas were increased as a consequence of FA exposure and no evidence of a dose-response relationship was evident [i.e., there was no difference between controls and the most highly exposed animals (15 ppm) in the incidence of either leukemia or lymphoma] (see Appendix A for additional details). It is also important to note that in the numerous long-term carcinogenicity studies on FA there has also been no indication of adverse effects on any hematological parameters. In these studies, neither inhalation exposure (Appelman et al., 1988; Kamata et al., 1997; Kerns et al., 1983; Feron et al., 1988) nor oral exposure (Johannsen et al., 1986; Til et al., 1989; Tobe et al., 1989) to high doses of FA has produced any evidence of adverse hematological effects. As suggested by ATSDR (1999), the lack of hematopoietic toxicity in these studies is ‘‘likely related to rapid metabolism prior to the formaldehyde reaching the blood and blood-forming components (bone marrow).” Experimental data obtained with FA is thus in contrast to exposure to all other established leukemogenic agents (e.g., benzene, alkylating agents, topoisomerase inhibitors, ionizing radiation), which produce a consistent constellation of pathological effects in experimental animals (Eastmond, 1998; Golden et al., 2006). As described in greater detail below, all established leukemogenic chemicals have the following toxicological characteristics: (1) all are associated with dose-related hematotoxicity, (2) all can induce bone marrow hypoplasia and even aplasia in highly exposed animals, (3) all have the documented ability to induce dysplastic morphological changes in bone marrow cells and (4) all have been shown to reproducibly produce hematopoietic malignancies in rodents (Larson et al., 1996; Larson, 2000; Greenberger et al., 1996; Greaves, 1997; Nyandoto et al., 1998). In almost every case the hematopoietic tumors observed in rodents are lymphoid in origin, usually aggressive thymic derived T cell lymphomas without a precise pathological counterpart in humans. Nonetheless, there is no evidence that exposures to FA result in any of the changes common

2 An explanation for the basis of this interpretation was requested from EPA, but has not been provided.

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to these established leukemogens (Ward et al., 1990; Eastmond, 1998). 4. Overview of human epidemiological data There is a robust literature base evaluating cancer mortality in FA exposed workers. A number of small studies have involved embalmers and anatomists with some, but not all, reporting a potential association between exposure to FA and increased mortality from leukemia. However, due to substantial confounding variables such as exposure to other chemicals and poor disease classification, this body of the literature had been largely discounted as demonstrating a potential risk of leukemia resulting from an exposure to FA. Interest in some of these older studies was renewed following the National Cancer Institute (NCI) publication in 2003 of a large cohort mortality update study of FA exposed workers (Hauptmann et al., 2003) and the IARC (2006) reliance on this study as the primary basis for the re-evaluation of FA. While this study reported increased relative risk of leukemia associated with peak exposure to FA (an unconventional and little, if ever used, measure of exposure) there was no significant association with either cumulative exposure or with duration of exposure, the two metrics generally believed to be the most indicative of overall exposure risks among other chemical leukemogens. Due to the substantial issues involving the interpretation of the results of this study, the original data from the NCI study were reanalyzed by Marsh and Youk (2004) using US and local county rate-based standardized mortality ratios (SMRs) to recompute relative risks (RR) of leukemia and myeloid leukemia (ML) for the same categories of FA exposure as used by NCI. This re-analysis revealed statistically significant deficits in deaths from both leukemia and myeloid leukemia in the internal control group. Therefore, the elevated RR was likely the result of or at least strongly influenced by the deficits in deaths from these diseases in the internal comparison group. When an external comparison group was used with normal or standard background mortality from leukemia and myeloid leukemia, there was no significant increase in disease observed for any exposure measurement. This reanalysis, therefore, did not support the conclusions that a causal association between FA exposure and increased mortality from leukemia and ML exists. It should be noted that the NCI cohort is in the process of being updated once again with additional mortality data through 2004. While the results of this update are not yet available it is clear that if the mortality deficits reported by Hauptmann et al. (2003) in non-exposed and low exposed workers continue and mortality from LHP malignancies remains the same or even declines, relative risks will likely be artificially enhanced due to inappropriate comparisons with such deficits. While it is beyond the scope of this review to go into detail concerning the body of epidemiological literature germane to this issue, Hauptmann et al. (2003) and the re-analysis by Marsh and Youk (2004) as well as several other recent studies are briefly reviewed in Appendix A. 5. Biological plausibility of formaldehyde as a cause of any lymphohematopoietic malignancy All established leukemogenic exposures described to date have the capacity to exert bone marrow and hematopoietic toxicity as well as demonstrate positive effects in a range of in vitro tests for hematopoietic toxicity. In other words, all of these substances share a commonality of biological plausibility as support for their demonstrated leukemogenic properties (Golden et al., 2006; Eastmond, 1998). This includes high dose-exposure to myelotoxic chemicals including benzene and certain classes of chemotherapeutic agents and to ionizing radiation. Such therapeutic chemicals

include alkylating agents (e.g., cyclophosphamide, chlorambucil, Myleran) and topoisomerase II reactive drugs (e.g., etoposide, teniposide and doxorubicin) and taxanes. A comprehensive review, sponsored by the EPA, of chemical leukemogenesis confirms the necessity of a general sequence of biological events which precede the development of leukemia in either experimental animals or humans (Eastmond, 1998). These are: (1) unequivocal evidence that the particular suspect chemical leukemogen reaches the bone marrow following exposure, (2) demonstrable toxic effects on bone marrow cells as evidenced by altered cellularity, morphology and cytogenetic changes, including some believed to be directly related to the development of AML, (3) documented hematotoxicity with sufficient doses inducing peripheral blood cytopenias in both humans and experimental animals (Advani et al., 1983; Irons and Stillman, 1996; Larson et al., 1996; Pedersen-Bjergaard et al., 1984; Pedersen-Bjergaard et al., 1995; Elias et al., 2000; Vardiman et al., 1983). The above events are clearly evident in chemically-induced leukemia and likely play fundamental roles in disease initiation and/or progression. Additionally, current models of leukemogenesis indicate that the leukemogen has genotoxic properties, even though this characteristic alone is insufficient to conclude that a particular genotoxic chemical is capable of inducing leukemogenic alterations. Cigarette smoking is also an established etiological factor in the development of AML (US Department of Health and Human Services, 2004). While the exact leukemogenic substance(s) present in cigarette smoke is unknown, there is clear evidence that compounds present in cigarette smoke (e.g., nicotine) are hematotoxic and travel to the bone marrow following exposure. For example, mice exposed to cigarette smoke exhibited a reduction in the activity of bone marrow derived HPC, as measured in a colony-forming unit assay (CFU). Additionally, nicotine was shown to be inhibitory in long-term bone marrow cultures (in vitro hematopoiesis) and blocked adherent-layer formation from bone marrow stromal cells (Khadoyanidi et al., 2001). Further, in vivo studies in neonatal mice exposed to nicotine during gestation report a significant decrease in the number of bone marrow hematopoietic progenitor cells, as measured by CFU and long-term culture assays (Serobyan et al., 2005; Pandit et al., 2006). Therefore, while cigarette smoke does not exactly fit into the paradigm for the leukemogenic chemicals described above, there is still definitive evidence that it exhibits systemic hematotoxicity and can target the bone marrow. These key fundamental characteristics for leukemogenic chemicals are not fulfilled by FA. Instead, the robust database on FA demonstrates that (1) normal metabolic processes prevent FA from entering the systemic circulation even following prolonged inhalation exposure to high concentrations and (2) that neither the bone marrow nor the hematopoietic system is a target for FA-induced toxicity. Coupled with the negative leukemia/lymphoma findings in long-term animal bioassays, these data do not provide a biologically plausible basis for concluding that FA is a leukemogenic substance. As discussed below, a major problem with the hypothesized MOA is that it does not specify which ‘‘lymphohematopoietic” malignancy FA might cause. This makes it extremely difficult to address, since there are a wide variety of distinct disorders that fall under this rubric. The hematopoietic malignancy with clear evidence of a chemical etiology is AML, as described above. The only lymphoproliferative malignancy with any alleged relation to chemical exposure is non-Hodgkin lymphoma (NHL). NHL is a term used to describe a diverse grouping of diseases, with over 25 morphologically distinct subtypes and often changing subtype classification (Harris et al., 1999). As recently commented. . .”there is etiologic heterogeneity among the various subtypes of lymphoid neoplasms. However, epidemiologic analyses by disease subtype have proven challenging due to numerous clinical and pathologic schemes used

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to classify lymphomas and lymphoid leukemia over the past several decades” (Morton et al., 2007). Although the overwhelming majority of cases have no readily identifiable cause, in addition to a strong familial predisposition and conditions leading to severe immunosuppression, a variety of infectious etiological agents, both bacterial and viral have been causally linked to the development of NHL (Aisenberg, 1997; Altieri et al., 2005; Baris and Zahm, 2000; Chang et al., 2005; Ansell and Armitage, 2005). For example, transplant patients who are required to maintain long-term treatment with immunosuppressive therapy, including azothioprine, cyclosporine A or tacrolimus (FK-506) have a greatly increased risk of developing NHL syndrome which is typically viral-induced (Mueller, 1999). In addition, autoimmune diseases, including rheumatoid arthritis, Sjögren’s syndrome and others also confer an increased predisposition to the development of NHL (Hardell and Axelson, 1998a; Symmons, 2007). Additionally, the compound Remicade and other anti-tumor necrosis factor drugs also increase the risk of NHL, which can result in both B or T cell lymphoma. However, there is no reliable evidence that any environmental or occupational chemical exposure, including FA leads to the development of NHL (Blair et al., 1993; Zheng et al., 2001; Cantor et al., 1992; Eriksson et al., 1992; Parsonnet et al., 1994; Alexander et al., 2007). Confirmatory of the above, there is no increase in the incidence or mortality of NHL reported in any epidemiological study of workers occupationally exposed to FA. Although, some continue to suggest that such exposure is a possible etiology (i.e., DeVoney et al., 2006a,b), the hypothesized MOA also includes multiple myeloma and Hodgkin lymphoma. Multiple myeloma (MM) is a malignancy of plasma cells (fully differentiated B lymphocytes). The neoplastic cells preferentially reside in the bone marrow of afflicted individuals but other tissues such as lymph nodes, spleen and skin may be involved. Rarely, tumor cells will circulate in the peripheral blood, a condition sometimes referred to as plasma cell leukemia. A diverse set of morphological and molecular observations suggest that the cell of origin for MM is a mature B lymphocyte that has undergone antigenic stimulation outside the bone marrow (Bakkus et al., 1995; Cohen et al., 1987; Ho et al., 2002; Tricot, 2002; Sahota et al., 1999). Since a significant increase in the incidence and mortality of MM was first reported in 1950, there has been a suspicion that occupational or environmental factors might play a role in the etiology and progression of this disease. Potential environmental and/ or occupational exposures that have received the most attention include petroleum products containing benzene, polyaromatic hydrocarbons (PAH), diesel exhaust, ionizing radiation, pesticides, solvents and cigarette smoking. Overall, the results of studies evaluating these exposures include slight increases, slight decreases or usually no change in the relative risk of MM (Boffetta et al., 1989; La Vecchia et al., 1989; Pottern et al., 1992; Torres et al., 1970; Aksoy et al., 1984; Marsh et al., 1991). The lack of consistency in this rather large body of epidemiological data does not allow for an epidemiological link between any of these risk factors and the development of MM to be established. Hodgkin disease or Hodgkin lymphoma (HL) is a lymphoid neoplasm thought to originate in a unicentric fashion in lymph nodes and then spread in a predictable pattern via the lymphatic system. A hallmark of HL is the presence of the Reed Sternberg cell (RS) and its attendant population of reactive cells; monocytes, T lymphocytes, eosinophils and other cell types. Recent evidence seems to support that different forms of HL may actually have different etiologies and risk factors (Lee et al., 2003). There is a clear bimodal distribution in incidence observed in the US and other countries. One peak occurs between 15 and 35 years old and the other occurs in individuals over 60. From an epidemiological point of view, there is not much consistency in the etiological data. While there have

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been a few clustering episodes, no common etiological factors have been identified including any type of occupational exposure. The occupational exposures with the most consistent epidemiological data are jobs that involving exposure to wood and wood dust. However, even these are insufficient to conclude that wood or wood dust is causally associated with HL. Other chemical exposures such as benzene or herbicides have been evaluated and are fairly consistent in demonstrating a lack of a relationship with HL risk. Lastly, there is little evidence to support than even ionizing radiation increases the risk of HL. Therefore, no established occupational or environmental risk factors exist for any subtype of HL. This is important within the context of this review as there is no precedence for any chemical, including FA, to cause either MM or HL. As described, FA does not possess any of the toxicological properties that have been consistently reported for leukemogenic exposures. Therefore, if FA exposure was somehow related to an increased risk of AML, it would be acting through an undiscovered pathway or mechanism. Additionally, there is no precedent for chemical exposures (including FA) to result in the development of several lymphoproliferative malignancies (e.g., MM and HL).

6. Overview of EPA hypothesized mode of action s for formaldehyde-induced lymphohematopoietic (LHP) malignancies The hypothesized MOA for FA-induced LHP malignancies is based on the two presentations by DeVoney et al. (2006a,b) and discussions with Dr. DeVoney by one of the authors (DP). Accordingly, it is our understanding that the hypothesized MOA relies on the following postulated key assertions and/or assumptions: (1) Many lymphoid malignancies arise outside of the bone marrow, (2) lymphoid tissue is present at the portal of entry (POE) and represents a potential target cell population in NALT, (3) circulating stem cells or HPC can be exposed to FA, ostensibly in the lungs or nasal passages at the POE, (4) FA has been reported to result in leukemia or lymphomas in rats exposed via the inhalation and oral routes, (5) FA is genotoxic and (6) there are some epidemiological data suggesting an association between FA exposure and LHP malignancies. In order for the hypothesized MOA to be operational and remain consistent with the established toxicological and biological properties of FA, two critical assumptions are necessary. First, B or T lymphocytes in the peripheral lymphoid organs/tissues such as the NALT are targets for FA-induced malignant transformation, which then could potentially result in any form of lymphoid malignancy. Second, peripheral transformation of primitive stem cells or committed myeloid hematopoietic progenitor cells (HPC) also potentially occurs as a consequence of FA exposure. This necessarily must also occur through a mechanism that does not involve FA entering the peripheral circulation (see above). Peripheral exposure (however it occurs) then results in the development of myeloid leukemia, both chronic and acute forms, in the absence of any detectable bone marrow or hematopoietic toxicity (e.g., cytopenias, etc.). These steps are critically important as an underlying tenet for the proposed MOA to be operative as it circumvents the necessity for distant site toxicity. As discussed previously, distant site toxicity has been convincingly shown to not occur following FA exposure. As discussed below, neither of these critical assumptions are supported by experimental data and, therefore, remain speculative at best. 6.1. Disease classification (nosology) At the outset of any scholarly discussion on potential etiological agents and various hematopoietic malignancies, there must be

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absolute clarity on the specific types of hematopoietic or lymphopoietic malignancies under discussion. This is required because the term ‘‘lymphohematopoietic malignancies” encompasses such a widely disparate grouping of diseases. Without at least some degree of nosologic precision, it is impossible to discuss the cell of origin or etiology in a scientifically meaningful way. The tendency to ‘‘lump” various ‘‘immune system” cancers or even ‘‘leukemias” into large collective groupings has been recognized as clinically and biologically inappropriate by the IARC, WHO, OSHA and others (Levine and Bloomfield, 1992; Linet, 1985; Schottenfeld and Fraumeni, 1982). As a result, most recent epidemiological investigations attempting to elucidate etiological factors in the development of hematopoietic malignancies have segregated the various malignancies and subgroups in their analyses. In contrast, EPA’s hypothesized MOA does not even attempt to specify the hematopoietic malignancy(ies) that it is intended to explain. Consequently, in the absence of a precisely defined disease(s), it can only be assumed that EPA has adopted the position that FA exposure is associated with all known forms of myeloid and lymphoid malignancies. This substantially undermines the scientific weight that should be accorded this (or any) proposed MOA (Linet, 1985; Schottenfeld and Fraumeni, 1982). Considering the diversity of hematopoietic diseases that exist and the widely disparate etiological factors associated with these diseases, it is scientifically incomprehensible that FA is a risk factor for all of them.

7. Key sequence of proposed events In this section, the key events and assumptions that have been presented by EPA as support for the hypothesized MOA for FA-induced LHP malignancies are critically evaluated (DeVoney et al., 2006a,b). Each is systematically assessed in order to illustrate the extent of empirical support for the overall MOA hypothesis. It should be noted that the first two elements have already been discussed in other sections of this paper.  Epidemiological studies have associated FA exposure with both leukemia and lymphomas. The epidemiological data are briefly summarized in Appendix A and provide very limited evidence that occupational exposure to FA is related to any type of hematopoietic disease. Interested readers are encouraged to review the key original studies (i.e., Coggon et al., 2003; Hauptmann et al., 2003; Pinkerton et al., 2004), a critical data re-analysis (i.e., Marsh and Youk, 2004) or several published reviews on this subject (Cole and Axten, 2004; Collins, 2004; Collins, 2004).  Animal data support the hypothesized MOA. As summarized in Appendix A, the animal data clearly does not demonstrate an increase in LHP malignancies following FA exposure. In fact, these extensive data show just the opposite and provide convincing evidence that the proposed MOA is not operational. Consequently, it is unclear how this body of data could be interpreted as support for the hypothesized MOA.  Exposure to exogenous FA by inhalation leads to direct contact of immune cells present at the point of entry (POE) in the nasal mucosa, specifically mucus associated lymph tissue (MALT), i.e., nasal associated lymph tissue (NALT). This key element of the proposed MOA circumvents the need for inhaled FA to enter the blood. Active lymphocytes (i.e., immune cells) are clearly present in the NALT and MALT. However, lympho-

cytes in the NALT are not in direct contact with the airways and are believed to be exposed to antigens via the M cells (Fujimura, 2000; Claeys et al., 1996). Based on the reactivity and rapid metabolism of FA, it is not certain that inhaled FA would pass un-metabolized through the M cells or other epithelial cells to reach lymphocytes. Consequently, there is no basis for assuming that they are in direct contact with inhaled FA and DeVoney et al. (2006a,b) provide no supporting data for this speculation. Therefore, the extent to which this could even hypothetically occur, for example, at FA concentrations that exceeded the metabolic capacity of the tissues would likely vary depending on the concentration of FA in the inhaled air. Lacking any real data, this element of the proposed MOA simply cannot be further evaluated.  Human studies demonstrate increased micronuclei (MN) in nasal, oral and buccal epithelial cells of nasal origin.  Human studies indicate increased clastogenic damage in peripheral blood (PB) lymphocytes in FA exposed workers/students (MN, SCE and CA).  Increased MN and SCE in pulmonary lavage cells of FA exposed F344 rats. The above three interrelated points relate to local and systemic genotoxic effects of FA and are presumably included in the proposed MOA with the implication that there might be a relationship between these events and disease etiology. The question that must be addressed, however, is whether any of these types of data are relevant with respect to the hypothesized MOA for FA-induced HLP malignancies. As noted by DeVoney et al. (2006a,b), there are studies which report local genotoxic effects of inhaled FA on the frequency of micronuclei (MN) in nasal and buccal epithelial cells. A critical review of this data concluded that it should be interpreted with caution because of uncertainty regarding the biology of the methods (e.g., time kinetics of micronucleus formation), the lack of adequate positive control data and the high and unexplained assay variability (Speit and Schmid, 2006). The reported results lack consistency and the methodology lacks standardization. Although several studies suggest that FA can express its genotoxic potential in directly exposed tissues, they do not permit an ability to assess the local genotoxicity of FA in humans or to draw meaningful conclusions with respect to the relevance of how such data might play a role in disease etiology (Thomson et al., 1984; Vasudeva and Anand, 1996). In a recent study under strictly controlled GLP (Good Laboratory Practices)-like conditions, 21 volunteers (10 women and 11 men) were exposed to FA for 4 h/day over 10 days at levels of 0.15– 0.5 ppm with 4 peaks of 1.0 ppm for 15 min each. MN in buccal cells were determined 1 wk prior to the start of the study (control 1), at the start of the study before exposure (control 2) and the end of the exposure period of 10 days and 7, 14 and 21 days thereafter. There was no significant increase in MN frequency at any time point after the end of exposure in comparison to controls thereby demonstrating that FA does not induce MN in buccal epithelial cells in humans in the range of the occupational exposure limit (Speit et al., 2007). Furthermore, there is no evidence that any of these findings (i.e., MN) are etiologically linked to LHP malignancies. Indeed, epithelial cells are not proposed as playing any etiological role in the hypothesized MOA for FA-induced LHP malignancies. Consequently, there is no causal or even hypothetical basis for citing these data as supporting evidence for the proposed MOA. Local genotoxic effects after FA exposure have been clearly demonstrated in experimental animals. A dose-related induction of DNA protein crosslinks (DPX) in nasal cells was found in inhalation studies with rats for FA doses ranging from 0.3 to 10 ppm.

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However, the relationship between the induction of DPX (i.e., the primary DNA lesions) and the formation of stable mutations (e.g., micronuclei on the chromosomal level) in nasal epithelial cells has not yet been established. DPX can be induced in all cell layers of the nasal epithelium while micronuclei only occur after relevant exposure of the dividing basal cell layer. Dallas et al. (1992) conducted a cytogenetic analysis of lung (i.e., pulmonary lavage fluid) and bone marrow cells in rats after repeated exposure to FA. Male Sprague–Dawley rats were exposed to 0, 0.5, 3 or 15 ppm FA for 6 h/day, 5 d/wk for 1 and 8 wks. There was an increase in pulmonary lavage cells with CA after both 1 and 8 wks of exposure with the greatest effect in animals exposed at 15 ppm for 8 wks. However, there were no differences in the proportion of bone marrow cells with CA between animals exposed to FA and controls at either 1 or 8 wks at any dose-level. These data show local genotoxic effects of FA, but also confirm the fact that inhaled FA does not enter the blood to induce systemic genotoxicity. This study would also appear to suggest the possibility that bronchial lymphocytes could be affected and then reenter the circulation, although this was not mentioned. In contrast to possible local genotoxic effects at the first site of contact, systemic genotoxic effects of FA are highly unlikely (for a review see Heck and Casanova, 2004) and various experimental studies clearly demonstrate that FA levels or DPX levels are not increased in organs (e.g., blood, bone marrow) which are not in direct contact with FA. However, conflicting results have been published regarding potential systemic genotoxic effects in humans. In some studies, increased amounts of DPX, sister chromatid exchanges (SCE), chromosome aberrations (CA) and MN were measured in peripheral lymphocytes from FA exposed workers. Increased levels of DPX in blood of FA exposed subjects were only reported by one group (Shaham et al., 2003) using a standard method (K-SDS assay). However, the effects measured were not correlated with FAexposure and are in conflict with experimental studies under controlled exposure conditions (Heck et al., 1985; Casanova and Heck, 1987). An accumulation of DPX in lymphocytes cannot be assumed because continuous removal of DPX was clearly demonstrated with a half-life between 4 and 18 h (Quievyrn and Zhitkovich, 2000; Schmid and Speit, 2007). Furthermore, it is still unclear whether the effects measured by Shaham et al. (2003) are actually related to FA exposure. Shaham et al. (2003) and other authors (Yager et al., 1986; He et al., 1998; Ye et al., 2005) also measured increased SCE frequencies in peripheral blood after FA exposure. However, SCE are formed during in vitro cultivation of lymphocytes when cells with increased damage levels pass through DNA synthesis. A recent experimental approach clearly defined the conditions (i.e., DPX levels during S-phase) that are necessary to induce SCE (Schmid and Speit, 2007). On the basis of these results it can be concluded that the reported SCE frequencies in lymphocytes of FA exposed subjects may have nothing to do with the FA exposure. The same holds true for other cytogenetic endpoints such as MN and CA (Schmid and Speit, 2007). Consequently, these studies provide documentation that FA does not exert systemic genotoxic effects in humans. However, even with the caveats noted above, it is impossible to completely exclude the hypothetical possibility that single lymphocytes exposed to FA locally, could re-enter the systemic circulation. The facile ability of lymphocytes, to leave the blood and enter tissues and re-circulate back into the blood via the lymphatic system is well known (Young, 1999). This phenomenon is central to the role of the lymphatic system in immune defenses (Olszewski, 2003; Schnuda, 1978; Young, 1999). Consequently, it is plausible that inhaled FA may interact with lymphocytes in bronchial lymphoid tissues. If exposure is sufficiently high, DPX may be formed with subsequent recirculation of the affected lymphocytes back into the blood. However, there is no basis for concluding that

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this would lead to positive results in the cytogenetic assays because only a few cells would carry increased damage and the sample size in these assays is small (Speit, 2006; Speit et al., 2007). Additionally, there is also no evidence (and indeed none has been offered) that FA-affected lymphocytes would be capable of somehow triggering the development of the broad spectrum of LHP malignancies hypothesized in the proposed MOA. With respect to the issues discussed in this section, i.e., that SCE or CA in peripheral lymphocytes might be etiologically implicated in disease risk, it is worthwhile to note that the anti-metabolite methotrexate is well established as producing multiple chromosomal abnormalities in human lymphocytes both in vitro and in vivo (Monticello et al., 1996; IARC, 1987; Deng et al., 2005). However, after many years of observation on thousands of patients with rheumatoid arthritis, lupus, psoriasis and various malignancies treated with this drug, there is no evidence of an increase risk of secondary AML even following prolonged use. Other commonly used drugs with no evidence of the potential for the induction of leukemia, yet with clastogenic properties when studied in vitro by lymphocyte analysis include the antibiotics metronidazole and trimethoprin and the diuretic, hydrochlorothiazide (Andrianopoulos et al., 2006; Abou-Eisha, 2006; Mudry et al., 1994). These important observations illustrates the limited value of using cultured lymphocyte CA as somehow predictive of a particular chemical’s leukemogenic effect in humans.  DNA protein crosslinks and FA–DNA adducts are formed in susceptible cells.  Resulting DNA lesions result in mutations during cell proliferation. Due to the interrelated nature of these two points they are discussed together. DNA protein crosslinks (DPX) and FA–DNA adducts are well established as consequences of sufficient exposure to FA (Casanova and Heck, 1987) Unrepaired DPX seem to be closely related to the induction of other genotoxic and mutagenic effects in directly exposed proliferating cells in vitro (Merk and Speit, 1998). However, particularly with respect to DPX, due to their rapid repair, there is no evidence (nor is any provided by DeVoney et al. (2006a,b)) that this event leads to a permanent mutation in the relevant target cells in vivo. In a recent study, FA was tested for genotoxicity in human blood cultures in order to follow FA-induced DPX and its repair. The results clearly demonstrated that while DPX (determined by the comet assay) are induced at FA concentrations of 25 lM, the DPX induced by FA concentrations up to 100 lM are completely removed before lymphocytes begin to replicate. These results suggest that cytogenetic effects of FA are unlikely to occur in blood cultures of FA exposed subjects (Schmid and Speit, 2007). Moreover, even assuming that the hypothesized MOA were operative, the LHP malignancy most likely to arise from genotoxicity or cytogenetic changes in lymphocytes would be NHL, which has not been reported as elevated in any occupational cohort of FA exposed workers. The above elements in the DeVoney et al. (2006a,b) MOA hypothesis illustrates the unfounded assumptions required in order to make the case that FA is capable of initiating events possibly leading to the development of LHP malignancies. In the absence of any supporting data demonstrating that DPX (which is an indicator of exposure, but not an indicator for permanent mutations) in affected cells is not repaired prior to cell division it is unwarranted to hypothesize that this would actually occur. If this were an element in the sequence of events leading to the development of LHP malignancies, it would be incumbent to cite experimental data which support this. Such data are conspicuously absent in the presentations by DeVoney et al. (2006a,b) and do not appear to exist.  Leukemic stem cells (LSC) arises from myeloid stem cells in circulation.

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Secondary AML arising consequent to chemical or radiation exposure (s-AML) is believed to be a multistep process involving both genetic as well as epigenetic events. The cell of origin for secondary and de novo AML is believed to be a myeloid committed hematopoietic progenitor cell (HPC), historically found only in the bone marrow. Recent developments in experimental hematology have demonstrated that a small population of HPCs also freely circulates in the peripheral blood (Fritsch et al., 1996; Uchida et al., 1997; Prosper et al., 1997; Holm et al., 1999). The presence of HPC outside the bone marrow (extramedullary) is consistent with the hypothetical possibility that peripheral HPC could be the cell of origin in chemically-induced leukemia. Further, initial steps in leukemogenic transformation could theoretically occur in the absence of bone marrow involvement/toxicity. For this to occur, a circulating HPC would need to undergo malignant transformation in the periphery, followed by migration back into the bone marrow, where the disease becomes manifest. However, evidence accumulated over decades of study in both experimental animals and humans has consistently demonstrated that hematotoxicity and bone marrow damage almost universally accompany the development of chemically-induced leukemia, and indeed may be an absolute requirement (Park and Koeffler, 1996; Pedersen-Bjergaard et al., 1984; Levine and Bloomfield, 1992; Albitar et al., 2002). As previously discussed, all known leukemogenic agents possess a similar set of toxicological characteristics that would include bone marrow damage or at the very least, bone marrow involvement. (Pyatt, 2004; Larson et al., 1996; Larson, 2000; Greenberger et al., 1996; Greaves, 1997; Nyandoto et al., 1998; Eastmond, 1998). Additionally, there are clear examples of where alterations in the bone marrow likely play a fundamental role in the development of leukemia, e.g., in AML development in donor cells following stem cell transplantation (Lawler, 1997). Therefore, while the possibility of chemically-induced peripheral transformation of myeloid HPC cannot be completely ruled out, there is no evidence that it occurs, so at the present time it remains merely a hypothetical possibility. However, based on present knowledge, leukemic transformation by FA or any other chemical, in the absence of bone marrow toxicity or involvement is not supportable with existing scientific evidence. Further, the assumption that FA causes changes in circulating HPC would require that FA enter the circulation. Since there is no evidence that this occurs, even following high dose-exposure, the likelihood that this plays any role in disease etiology is substantially reduced. The preceding discussion is based on the assumption that AML is the myeloid disease in question. However, if the hypothesis includes CML then additional, even more speculative assumptions must be made in order to accept this as plausible. The primary obstacle is the fact that no chemical has ever been demonstrated to play an etiological role in the development of CML. Indeed, none are cited since FA would be unique in this regard and would be the first and only chemical to be associated with CML.

 Bone marrow compartment not involved in leukemia/lymphoma formation.  Immune cells vulnerable to neoplastic transformation are present in MALT and therefore targets of FA’s mutagenic effects (e.g. intraepithelial lymphocytes, and germinal center cells). Lymphoma/leukemia increased through transformation of immune cells at the POE. Because the above two elements in the proposed MOA are somewhat interconnected they are discussed together. As with many of the other assertions in this MOA approach, there is no real evidence or data presented that supports either point as playing an etiological role in the genesis of any type of LHP malignancies. Instead, these two elements appear to be based on a series of diverse

observations that while true, do not add up to the conclusion that NALT or MALT is the source of FA-induced LHP malignancies. These observations are: (1) routine clonal expansion of lymphocytes in NALT tissues, (2) B lymphocytes undergo somatic hypermutation, (3) plasticity of differentiation, (4) direct contact between FA and MALT tissue occurs and (5) NALT tissue is more prominent after exposure to antigens (DeVoney et al., 2006a,b). Although often multicentric in origin, a large percentage of lymphatic tumors, particularly non-Hodgkin lymphomas, appear to originate outside the bone marrow. As such, these tumors could be theoretically independent of bone marrow involvement. Specialized lymphoid tissue exists in the upper respiratory tract of most mammals (including humans). This tissue (i.e., NALT) is one example of a larger collection of peripheral lymphoid tissues associated with the mucosal lining. Collectively, MALT is strategically located in several anatomical positions, but is concentrated primarily in the gastrointestinal and upper respiratory tract (Addas et al., 2000; Jandl, 1987, 1997). These immune tissues represent the first line of defense again ingested or inhaled antigens and are critical components in host defense (Addas et al., 2000). Mature, naïve B lymphocytes (which have not responded to antigen) congregate into distinct regions called ‘‘follicles” which can easily be distinguished histologically in lymph nodes and other lymphoid tissues (Addas et al., 2000). Upon appropriate antigenic stimulation, naïve lymphocytes within these follicles begin a period of intense proliferation, giving rise to ‘‘germinal centers” (Addas et al., 2000). During the proliferative activity which occurs in an immune response, highly specific antibody is produced by the responding B lymphocytes. Additionally, a process called ‘‘affinity maturation” frequently takes place which results in the production of antibody with a greatly increased affinity for the initiating antigen. This refinement in antibody binding facilitates the removal and/or destruction of the antigen. In the simplest terms, the increase in antibody binding capacity (affinity maturation) results from multiple somatic mutations that spontaneously occur within the DNA encoding for the variable region of the antibody. Because of a greatly increased rate of spontaneous mutations, this process is sometimes referred to as ‘‘hypermutation’ and can result in the ‘‘maturing” antibody varying as much as 5% from the original antibody (Yang et al., 1999; Kanzler et al., 2000). A role for somatic mutations acquired during affinity maturation has been suggested to be important in the origin of MALT associated lymphomas (Dunn-Walters et al., 2001; McBlane et al., 1995). However, it must be recognized that the process of acquiring somatic mutations is in no way dependent on the chemical composition or genotoxicity of the inhaled material. This is evidenced by a variety of epidemiological studies that suggest chronic antigenic stimulation may increases the risk of NHL (Scherr et al., 1992; Armitage et al., 1993; Hardell and Axelson, 1998a; Hardell et al., 1998b; Alexander et al., 2007). Despite the inherent, regulatory safeguards in place, specific lymphoid malignancies are associated with MALT. The most common and best characterized are lymphomas located in the GI tract. GALT (gut associated lymphoid tissue) related tumors are often associated with a Helicobacter pylori infection (Stolte, 1992; Cammarota et al., 1995; Chan, 1996). Lymphomas observed in the upper respiratory tract are quite rare, but have also been reported (Shohat et al., 2004; Shikama et al., 2001; Quraishi et al., 2000). In nasal associated lymphomas, there is typically a strong association with Epstein Barr Virus (EBV) infection, again indicating a possible infectious etiology. Irrespective of the precise etiology, all lymphoid tumors arising within the MALT (including NALT) are classifiable as NHL (McDonnell et al., 1993; Hockenbery, 1994; Pittaluga et al., 1996). Probably the strongest evidence against applying this series of assumptions to support the proposed MOA is the fact that NHL is not elevated in any recent epidemiological study of FA ex-

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Not fulfilled

Not fulfilled

Not fulfilled

Not fulfilled

Fulfilled

MOA analysis based on physical, chemical, and biological information that helps to explain key events in an agent’s influence on development of tumors

Degree of consensus and general acceptance among scientists regarding interpretation of the significance and specificity of the tests

Conduct of the tests in accordance with generally accepted protocols

A statistically significant association between events and a tumor response observed in well conducted studies is generally supportive of causation. Consistent observations in a number of such studies with differing experimental designs increase that support, because different designs may reduce unknown biases. A lack of strength, consistency and specificity of association weakens the causal conclusions for a

Consistency of results in different test systems and different species. Is the hypothesized MOA sufficiently supported in the test animals? Similar dose-response relationships for tumor and mode of action-related effects

8.1. Strength, consistency, specificity of association

Not fulfilled

All known leukemogenic chemicals produce bone marrow toxicity, mutations and resulting leukemia; while FA is mutagenic, it does not enter the blood or reach bone marrow; no experimental evidence directly supports the proposed MOA for FA No studies exist which demonstrate malignant transformation of NALT/MALT cells leading to development of leukemia/lymphoma as a consequence of FA exposure Numerous high dose-bioassays in rats via inhalation and ingestion; all tested animals have NALT and GALT; with a single exception (see text), no studies show any evidence of leukemia/lymphoma No data showing involvement of NALT/GALT in leukemogenesis or lymphoma formation following FA exposure With one exception (see Appendix A) all relevant bioassays conducted in accordance with generally accepted protocols No consensus or acceptance among scientists that (1) chemical leukemogenesis (e.g., AML) occurs without bone marrow toxicity, (2) any chemical has caused CML or HL, (3) FA causes malignant transformation of immune cells in NALT or transformation of hematopoietic progenitor cells in the peripheral circulation The physical, chemical and biological properties of FA do not support the proposed MOA for FA-induced leukemia/lymphoma; all tissues continuously bathed in 2.5 ppm FA from blood; highly efficient metabolic processes prevent excursions above 2.5 ppm; inhaled FA does not enter the blood to affect normally occurring concentrations; no data on malignant transformation of immune cells in NALT or GALT tissues Mechanistic relevance of the data to cancer (leukemia/lymphoma)

DeVoney et al. (2006a) state that ‘‘A biologically plausible mode of action (MOA) is proposed for FA-induced LHP malignancies based on the framework provided in the current US EPA Guidelines for Carcinogen Risk Assessment. FA-induced mutation, in conjunction with proliferation of immune cells, are the key components for the proposed MOA.” It is important to point out that the framework referred to (i.e., EPA, 2005) is a detailed description of how empirical data in support of a hypothesized MOA should be assessed and evaluated. This framework was not designed to evaluate the biological plausibility of a hypothetical MOA, particularly in the absence of any empirical data. For example, the framework places substantial emphasis on considerations for causality pertaining to epidemiologic data as originally articulated by Hill (1965). Later, these ‘‘causation criteria” were extended to experimental studies although retaining the basic principles of Hill in a modified context. As described in EPA (2005), ‘‘The modified Hill criteria can be useful for organizing thinking about aspects of causation, and they are consistent with the scientific method of developing hypotheses and testing those hypotheses experimentally. . . a key question is whether the data to support a mode of action meet the standards generally applied in experimental biology regarding inference of causation. All pertinent studies are reviewed in analyzing a mode of action, and an overall weighing of evidence is performed, laying out the strengths, weaknesses and uncertainties of the case as well as potential alternative positions and rationales. Identifying data gaps and research needs is also part of the assessment. To evaluate whether an hypothesized mode of action is operative, an analysis starts with an outline of the scientific findings regarding the hypothesized key events leading to cancer, and then weighing information to determine whether there is a causal relationship between these events and cancer formation , i.e., that the effects are critical for induction of tumors [emphasis added]. Essentially none of the above caveats or guidelines has been met, i.e., no key elements of the proposed MOA have been tested experimentally, virtually no data or pertinent studies explicitly about key elements in the MOA exist, no strengths, weaknesses or uncertainties have been addressed, and no causal relationship between key events and cancer formation has been determined. In essence, the proposed MOA consists primarily of data gaps and research needs rather than a plausible explanation for how inhaled FA can cause LHP malignancies. As illustrated below and in Table 1, data proposed to support EPA’s hypothesized MOA are either non-existent or do not fulfill the explicit criteria in the EPA (2005) guidelines.

Table 1 EPA guidelines and criteria for judging data in support of mode of action data

8. EPA criteria for evaluation of mode of action data

Number of studies of each endpoint

Relevant MOA data for formaldehyde (FA) Criteria or guideline

Extent that criteria fulfilled

posed workers. Thus, if the proposed MOA were actually operative, NHL arising in the NALT (i.e., nasal lymphoma) would likely be the primary observed malignancy. The conspicuous absence of nasal lymphomas in any of the studies of FA exposed workers is powerful empirical evidence that this does not occur. Further, even if one were to assume that NALT tissue could hypothetically be the site of FA-induced lymphoid malignancies, this does not provide a reasonable MOA for myeloid leukemia or the range of LHP malignancies suggested by EPA.

Not fulfilled

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It is important that the hypothesized mode of action and the events that are part of it be based on contemporaneous understanding of the biology of cancer to be accepted. If the body of information under scrutiny is consistent with other examples (including structurally related agents) for which the hypothesized mode of action is accepted, the case is strengthened. EPA provides no supporting data for their position that FA exposure can potentially result in all forms of LHP diseases. As discussed in some detail, FA clearly does not fit into the pattern described for chemically-induced leukemia (i.e., AML subsequent to exposure to benzene, alkylating agents, topoisomerase inhibitors, etc.). Consequently, it is biologically implausible that a naturally occurring chemical such as FA would be capable of inducing the broad range of LHP malignancies as suggested by the hypothesized MOA.

studies of FA exposed workers. Conversely, the hypothesized MOA is even more speculative with regard to myeloid leukemias. With regard to the proposed peripheral transformation of myeloid committed HPC, it has been shown that there are no established causes of AML in humans that do not carry the potential for hematopoietic and/or bone marrow damage. FA does not fit the toxicological profile of a chemical capable of inducing leukemogenic transformation in humans. There is no biologically plausible mechanism to explain how a chemical with no documented hematotoxicity or delivery to the bone marrow (like FA) can induce AML in humans. Forth, peripheral transformation, in the absence of bone marrow toxicity, is difficult to reconcile with existing scientific evidence. Therefore, the question posed as the title of this paper—is inhalation exposure to formaldehyde a biologically plausible cause of lymphohematopoietic malignancies—can only be answered in the negative. Many of the observations and facts presented as part of the key events in the proposed MOA are true to a limited extent, but do not actually support the proposed hypothesis. This elaborate hypothesis appears to be an attempt to explain the leukemia and Hodgkin lymphoma findings reported by Hauptmann et al. (2003). However, the significant deficit of deaths in the internal comparison groups used for the risk estimations for these two diseases is a far more straightforward explanation. The bottom line with respect to this hypothesized MOA is that it is only a hypothesis. Science moves forward when data are obtained to support or refute a hypothesis. To a large extent, the numerous bioassays with FA in experimental animals have already tested this hypothesis and have provided consistently negative data. The highly inconsistent and largely negative pattern of LHP malignancies observed in studies of FA exposed workers also suggests that this MOA is not functional (e.g., lack of an increased NHL risk). Overall, the hypothesized MOA for FA-induced LHP malignancies is not based on the best available science, is not supported by available data, and has not been confirmed through appropriate experimentation. Scientifically sound public health and regulatory decisions should be based on verifiable, reproducible data. As such data are lacking, this hypothesis should play no role in regulatory decisions.

8.4. Identification of key events

Acknowledgments

In order to judge how well data support involvement of a key event in carcinogenic processes, the experimental definition of the event or events should be clear and reproducible. To support an association, experiments should define and measure an event consistently. A number of key events have been proposed as critical elements in EPA’s hypothesized MOA. However, as described above, no relevant experimental data are provided to support any of these key events.

Two of the authors (D.P. and R.G.) were compensated for this work by the Formaldehyde Council Inc. (FCI). However, the views presented in this paper are strictly those of the authors.

particular mode of action. There are no relevant experimental data involving FA-induced malignant transformation of immune cells in NALT or FA effects on circulating HPC leading to the development of any of the numerous hypothesized LHP malignancies. Consequently, none of these criteria can be satisfied. 8.2. Dose-response concordance If a key event and tumor endpoints increase with dose such that the key events forecast the appearance of tumors at a later time or higher dose, a causal association can be strengthened. Dose-response associations of the key event with other precursor events can add further strength. Difficulty arises when an event is not causal but accompanies the process generally. For example, if tumors and the hypothesized precursor both increase with dose, the two responses will be correlated regardless of whether a causal relationship exists.There are also no relevant experimental dose-response data involving FA-induced malignant transformation of immune cells in NALT or circulating HPC. In fact, the lack of any positive dose-response data for LHP malignancies from the numerous animal carcinogenicity studies is compelling evidence that the hypothesized MOA is not operational. Consequently, this criterion cannot be satisfied. 8.3. Biological plausibility and coherence

Appendix A A.1. Overview of formaldehyde and LHP malignancies in animal studies

9. Conclusions After careful review and evaluation of the proposed MOA for FA-induced LHP malignancies hypothesized by US EPA, several key conclusions and observations are apparent. First and foremost is the conspicuous lack of scientific support that the proposed MOA or any of its elements actually occurs. The hypothesized MOA, while interesting, is essentially devoid of relevant supporting data. Second, there is an inappropriate lack of clarity with regard to the specific diseases this hypothesized MOA was intended to support. The apparent speculation that inhaled FA was capable of causing all known forms of LHP malignancies is unwarranted and scientifically untenable. Third, the incidence of NHL which is the most biologically coherent disease hypothetically associated with the proposed MOA (related to NALT) is not increased in numerous

There have been at least 10 chronic carcinogenicity studies on FA, four in which animals were exposed via ingestion (Soffritti et al., 1989, 2002; Takahashi et al., 1986; Til et al., 1989; Tobe et al., 1989) and six involving inhalation exposure (Kerns et al., 1983; Tobe et al., 1989; Sellakumar et al., 1985; Feron et al., 1990; Kamata et al., 1997; Monticello et al., 1996 and Swenberg et al., 1980). None of these studies have reported FA-induced carcinogenic effects other than at the site of administration, i.e., nasal cancer in rats and mice following inhalation exposure and possibly gastric cancer (not gastric lymphoma) in rats following ingestion exposure. One ingestion study (i.e., Soffritti et al., 1989; Soffritti et al., 2002, discussed below) did report positive findings that are inconsistent with the other nine studies. This body of the literature does not support the position that FA exposure is causally associ-

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ated with leukemia, lymphoma, bone marrow toxicity or hematological abnormalities. For example, Til et al. (1989) conducted a two-year drinking water study of FA in Wistar rats. The mean FA doses administered to male and female animals were 0, 1.2, 15 or 82 mg/kg/day and 0, 1.8, 21 or 109 mg/kg/day, respectively. Treatment-related changes were only noted in the gastric mucosa; there was no evidence of carcinogenicity either in the stomach or any other site or tissue, including the hematopoietic system. Takahashi et al. (1986) and Tobe et al. (1989) also provide no evidence that oral exposure to FA resulted in increased incidence of hematopoietic malignancies or pathology. The only oral administration study which has reported a carcinogenic effect of FA at a site distant from the portal of entry (i.e., nasal passages or gastric mucosa) was by Soffritti et al. (1989). In this study, male and female Sprague–Dawley rats of different ages were exposed to FA in their drinking water at concentrations of 0, 10, 50, 100, 500, 1000, 1500 and 2500 mg/l for up to 104 wks. Histopathology examinations were conducted on most tissues including the femur. As reported by Soffritti et al., 1989 there was an increase in ‘‘lymphoblastic leukemias and lymphosarcomas” and ‘‘immunoblastic lymphosarcomas” although the anatomic location of these neoplasms was not identified. While this increased incidence of lymphoproliferative neoplasms occurred at doses >500 mg/l, the lack of any statistical analysis of the data precludes an ability to accurately assess this observation. Further, the reported increase in examples of ‘‘immunoblastic lymphosarcoma” did not appear to be dose-related and the incidence of ‘‘other leukemia” was similar in exposed and control animals. Bone marrow was one of the tissues specifically examined as part of routine histopathology, yet there was no mention of any abnormality in this tissue. None of the prior contradictory observations from other oral dosing studies (some with even higher administered doses of FA) which were available when Soffritti et al. (1989) submitted their report were mentioned or discussed. In addition, while Soffritti et al. (1989) present historical control incidence data for stomach, intestine and GI neoplasms in Sprague–Dawley rats, similar historical control data for lymphoblastic leukemia–lymphosarcoma are not included. As noted by Feron et al. (1990, 1991), the historical incidence of spontaneous leukemias in untreated Sprague–Dawley rats varies widely with the colony used. Therefore, in the absence of appropriate background rates, it is possible that the reported increases may be unrelated to FA exposure. After reviewing Soffritti et al. (1989), both the Agency for Toxic Substances and Disease Registry (ATSDR, 1999) as well as the Cancer Assessment Committee of the Center for Food Safety and Applied Nutrition, US Food and Drug Administration (FDA), expressed skepticism about the reported results. For example, the FDA concluded that the data reported were ‘‘unreliable” due to ‘‘a lack of critical detail . . . questionable histopathological conclusions, and the use of unusual nomenclature to describe the tumors.” Consequently, the FDA ‘‘determined that there is no basis to conclude that formaldehyde is a carcinogen when ingested” (USFDA, 1998). Soffritti et al. (2002) again reported the results from which appear to be taken directly from their earlier study with the exception that the reported incidence of leukemia doubled in most treatment groups, i.e., 45 vs. 91 in males and 34 vs. 60 in females. Information on historical control incidences rates of leukemia was still lacking and there was no explanation for the dramatic changes in the incidence of leukemia between the two reports. Finally, based on data from studies on other test compounds [i.e., methyl tertiary butyl ether (MTBE), methanol and aspartame] conducted in the same laboratory (i.e., Ramazzini Foundation) where the FA study was conducted, it now appears possible that the findings reported by Soffritti et al. (1989, 2002) may be unrelated to FA. Leukemias and lymphomas in exposed rats are typi-

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cally found in the blood or lymph nodes. However, in studies conducted with the above noted chemical compounds increases in ‘‘hemolymphoreticular neoplasias” were overwhelmingly reported as found in the lungs of both control and dosed animals. In reviewing the data reported by Soffritti et al. (2006) and Belpoggi et al., 2006 on aspartame, the European Food Safety Authority (EFSA, 2006) concluded that, ‘‘the increased incidence of lymphomas/leukemias reported in treated rats was unrelated to aspartame, given the high background incidence of chronic inflammatory changes in the lungs and the lack of a positive dose-response relationship. It is well known that such tumors can arise as a result of abundant lymphoid hyperplasia in the lungs of rats suffering from chronic respiratory disease. The most plausible explanation of the findings in this study with respect to lymphomas/leukemias is that they have developed in a colony suffering from chronic respiratory disease. The slight increase in incidence in rats fed aspartame is considered to be an incidental finding and should therefore be dismissed.” Given the well documented deficiencies of the Soffritti et al. (1989, 2002) study, it is impossible to reconcile this data within the context of the other ingestion studies. Consequently, this study does not provide any credible evidence in support of the hypothesized MOA. There have also been six chronic inhalation studies with FA, all yielding consistently negative results with regard to the appearance of hematopoietic malignancies (Kerns et al., 1983; Tobe et al., 1989; Sellakumar et al., 1985; Feron et al., 1988; Kamata et al., 1997; Monticello et al., 1996; Swenberg et al., 1980). Chronic inhalation exposure to FA concentrations as high as 15 ppm did not increase the incidence of distant site tumors (including hematopoietic) in any study However, one inhalation study (i.e., Kerns et al., 1983) is cited by DeVoney et al. (2006a,b) as showing a significant increase (including dose-response relationships) in lymphoma in female B6C3F1 mice and leukemia in female F-344 rats. The basis for this conclusion does not come from the published study, which does not mention either lymphoma or leukemia incidence, but rather from an ad hoc re-analysis of the underlying Battelle (1981) data. This re-analysis included the generation of dose-response curves for each kind of cancer. A careful review of these data does not support the claim that either leukemias or lymphomas were increased as a consequence of FA exposure and no evidence of a dose-response relationship. As described in the histopathology portion of the original report (Battelle, 1981), in mice, lymphomas were observed in numerous organs, with the reported incidence in the female controls and high dose-group to be 16% and 22%, respectively. The difference in lymphoma incidence was not statistically significant. Leukemias were assessed in FA treated experimental animals based on the combined incidence of mononuclear cell leukemia (MCL) and undifferentiated leukemia in male and female F-344 rats. Leukemia incidence was 9% in controls for both sexes and 4% and 6% for males and females, respectively, at the 15 ppm dose-level. This clearly does not support a positive dose-response relationship in rats between leukemia and inhalation of FA. Moreover, with respect to leukemia, it should be noted that F-344 rats are particularly susceptible to the development of mononuclear cell leukemia (MCL or aging Fisher rat leukemia) that has no human analogous hematopoietic disease. A.2. Overview of epidemiological data on formaldehyde exposed workers There is a robust literature base evaluating cancer mortality in FA exposed workers. A number of small studies have involved embalmers and anatomists with some, but not all, reporting a potential association between exposure to FA and increased mortality from leukemia. However, due to substantial confounding variables such as exposure to other chemicals and poor disease classification, this body of the literature had been largely discounted as

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demonstrating a potential risk of leukemia resulting from an exposure to FA. Interest in some of these older studies was renewed following the National Cancer Institute (NCI) publication in 2003 of a large cohort mortality update study of FA exposed workers (Hauptmann et al., 2003), studies on several other large occupational cohorts as well as the reclassification of FA by IARC in 2006. The NCI study consisted of a cohort of 25,619 industrial workers at 10 US industrial plants where FA was either produced or used in the production of other products (Hauptmann et al., 2003). FA exposures were assessed by peak, average intensity, cumulative and duration. An internal control group was selected and used in the risk analysis. Compared with workers exposed to low peak levels of FA (0.1–1.9 ppm), relative risks for myeloid leukemia were 2.43 (95% CI = 0.81–7.25) and 3.46 (95% CI = 1.27–9.43) for workers exposed to peak levels of 2.0–3.9 ppm and P4.0 ppm, respectively. Compared with workers exposed to low levels of average exposure intensity of FA (0.1–0.4 ppm), workers exposed to 0.5–0.9 ppm and P1.0 ppm average intensity had relative risks of 1.15 (95% CI = 0.41–3.23) and 2.49 (95% CI = 1.03–6.03) for myeloid leukemia, respectively. The relative risk for leukemia was not significantly associated with either cumulative exposure or with duration of exposure, the two metrics that are generally believed to be the most indicative of overall exposure risks among other chemical leukemogens. Table A.1 illustrates the reported findings with respect to all LHP malignancies associated with peak exposure (>4 ppm) to FA and average intensity of exposure (>1 ppm) to FA. There was no distinction made between the acute vs. chronic forms of myeloid leukemia and there was no evidence of an increased risk of NHL, MM or lymphatic leukemia (acute and chronic combined). In fact, there is no evidence of increased risk with any lymphoid malignancy except for Hodgkin’s lymphoma (HD). Importantly, there was a deficit reported in mortality from both myeloid leukemia and Hodgkin’s lymphoma in the control groups used for comparisons. This deficit would have clearly influenced the risk estimates and may be critical in terms of how these data should be interpreted. Using the original data from the NCI study, this cohort was reanalyzed by Marsh and Youk (2004). The US and local county rate-based standardized mortality ratios (SMRs) and relative risks (RR) of leukemia and myeloid leukemia (ML) were recomputed by the same four categories of FA exposure metrics as used by NCI. Additionally, an alternative categorization based on tertiles of deaths from all leukemia among exposed subjects was included. This re-analysis revealed statistically significant deficits in deaths from leukemias and myeloid leukemias in the internal control group. Therefore, the elevated risks reported in the NCI study are likely the result of or at least strongly influenced by the deficits in deaths from these diseases in the internal comparison group. The two disease groupings with the highest RR were myeloid leukemia and HL. As discussed above, there was a significant deficient of these malignancies in the internal control population. When an external comparison group was used with normal or standard

background mortality from myeloid leukemia and HL, there was no significant increase in disease observed for any exposure measurement. The alternative exposure categorization based on average intensity yielded SMRs for leukemia and ML that were close to 1.0 in the highest exposure category, and also demonstrated less evidence of a trend in RRs for leukemia and ML. Similar to the findings in the original NCI study, no association with leukemia or ML was reported with cumulative FA exposure or duration of FA exposure. Additionally, there was no consistent evidence that leukemia or ML risks increased with increasing duration of time spent in a given highest peak exposure. This re-analysis, therefore, did not support the conclusions that a causal association between FA exposure and increased mortality from leukemia and ML exists. It is important to note that while the use of an internal comparison group is typically the methodology employed in NCI studies, when significant deficits in the comparison group are evident, this choice should become questionable. This concern is particularly relevant when all significant findings disappear when mortality rates in the exposed population are compared with an external group with expected mortality rates. It is not known if EPA has taken this reanalysis under consideration in their MOA hypothesis. The NCI cohort is in the process of being updated once again with additional mortality data through 2004. While the results of this update are not yet available several issues are worth noting. Given the substantial problems and issues identified by Marsh and Youk (2004) in their re-analysis of the previous update (i.e., Hauptmann et al., 2003), it will be incumbent on NCI to address them in the soon to be released update of this cohort. Failure to do this would undermine any potential positive findings concerning associations between formaldehyde exposure and LHP malignancies. The first concerns the mortality deficits reported by Hauptmann et al. (2003) in non-exposed and low exposed workers. If these deficits continue and mortality from LHP malignancies remains the same or even declines, relative risks will likely be artificially enhanced due to inappropriate comparisons with such deficits. The other issue will be the exposure metrics used by NCI for the generation of relative risks. As noted by Marsh and Youk (2004) findings which rely exclusively on peak exposure with no significant findings based on more commonly used measures (e.g., average, cumulative or duration of exposure) are highly questionable. Even positive findings based on number of peaks as suggested by Marsh and Youk (2004) would be more valid than the single peak exposure metric. In another study, the mortality experience of 11,039 garment workers exposed to FA for three or more months at three plants was evaluated (Pinkerton et al., 2004). While noting that the mean time weighted average FA exposure at the three plants in the early 1980s was 0.15 ppm and that past exposures may have been substantially higher, no individual FA exposure measurements were available. Compared to standard US mortality rates, the total cohort mortality from myeloid leukemia was not significantly in-

Table A.1 Summary of relative risks for LHP malignancies reported by Hauptmann et al. (2003) Cause of death

Relative risk (95% CI) (peak exposure at highest level)

Significant trend with exposure

Relative risk (95% CI)(ave. intensity exposure at highest level)

Significant trend with exposure

All LHP malignancies NHL HDa MM Leukemiaa Lymphatic leukemia Myeloid leukemiaa

1.87 1.23 3.35 1.67 2.46 1.39 3.46

Yes No Yes No Yes No Yes

1.5 (1.01–2.24) 0.98 (0.43–2.2) 3.12 (0.91–10.74) 1.42 (0.56–3.58) 1.68 (0.91–3.08) 1.43 (0.47–4.34) 2.49 (1.03–6.03)

Yes No Yes No No No No

(1.27–2.75) (0.59–2.55) (0.97–11.59) (0.68–4.12) (1.31–4.62) (0.46–4.17) (1.27–9.43)

a Deficits observed in 0 ppm peak exposure group; for HD RR = 0.51 (0.06–4.52), for leukemia RR = 0.78 (0.25–2.43) and for myeloid leukemia RR = 0.67 (0.12–3.61). Deficits in these diseases were also observed in the average intensity and cumulative exposure analysis.

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creased (SMR = 1.44, 95% CI = 0.80–2.37). However, among workers with both 10 or more years of exposure and 20 years or more since first exposure, mortality from leukemia and myeloid leukemia were significantly increased (SMR = 1.92, 95% CI = 1.08–3.17 and SMR = 2.55, 95% CI = 1.10–5.03, respectively). Lymphocytic leukemia mortality was not statistically elevated in any worker category analyzed (SMR 2.12, 95% CI = 0.78–4.62). Pinkerton et al. (2004) note that their results were in contrast to two other studies involving more than 30,000 workers with much higher levels of FA exposure (>2 ppm) in which mortality from leukemia was not increased (i.e., Blair et al., 1986; Gardner et al., 1993). Collins and Lineker (2004) observed that the long latency for leukemia deaths observed in the two industrial studies that reported increased risk was not consistent with the effects of a leukemogenic chemical such as benzene. While latency is difficult to measure accurately, it is generally believed that all established leukemogenic chemicals induce AML in 2–10 years following exposure to sufficient amounts of the chemical to meaningfully increase the risk (La Vecchia et al., 1989; Larson, 2000; Levine and Bloomfield, 1992). In another recent updated study of a cohort of 14,014 men employed after 1937 at six British factories where FA was produced or used, there was no increased mortality from total leukemia relative to the incidence in the national population including those exposed at a time weighted average (TWA) of 2 ppm or greater (SMR = 0.71, 95% CI = 0.31–1.39) (Coggon et al., 2003). There was also no evidence of any exposure related increases in MM, HL or NHL. It should be noted that FA exposures in this study were likely substantially greater than in the studies by NCI or Pinkerton et al. (2004). A meta-analysis of 18 epidemiology studies of workers (i.e., embalmers, pathologists/anatomists and occupational manufacturing or use) exposed to FA that reported leukemia incidence rates (but not necessarily leukemia subtypes or lymphomas) was conducted (Collins and Lineker, 2004). Findings were summarized across studies to calculate the meta-relative risk values (mRR), confidence intervals and the heterogeneity of the risk estimates for several study characteristics. There was an overall increase in leukemia in embalmers (mRR = 1.6, 95% CI = 1.2–6.0) and a marginal increase observed in pathologists/anatomists (mRR = 1.4, 95% CI = 1.0–1.9). No quantitative exposure estimates were available for any these occupational groups. Industrial workers, with the highest documented FA exposures showed no increased risk of leukemia (mRR = 0.9, 95% CI = 0.8–1.0). A review of the available epidemiological data was undertaken to determine if a potential causal association between leukemia and exposure to FA exists (Cole and Axten, 2004). The available literature was critically evaluated within the context of established causation criteria, i.e., consistency, strength of association, coherence, temporality, dose-response and biological plausibility. These authors concluded that ‘‘In sum, then, the formaldehyde-leukemia hypothesis fails each of the four guidelines of general causation. This is hardly surprising in view of the weak and inconsistent findings in the most recent epidemiologic research and the consistent findings in animal studies.” Another position suggested by EPA’s hypothesized MOA is that chronic myeloid leukemia (CML) is the predominant type of myeloid leukemia associated with FA and this drives the risk estimates in the occupational cohorts. However, the empirical support for this position is not readily apparent.3 Unfortunately, many studies do not segregate leukemia subtypes, so there are relatively little data to evaluate this position one way or the other. Based on three observed cases in a small study on anatomists, Stroup et al. (1986) re3 In a discussion with one of the authors (DD) an unpublished tabulation of data from NCI was described as the basis for this claim; however, despite requesting these data they were not provided.

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