3
Toxicokinetics and Modes of Action of Formaldehyde

This chapter provides the committee’s review of the draft IRIS assessment that is relevant to formaldehyde toxicokinetics, carcinogenic modes of action, pharmacokinetic models, and biologically based dose-response (BBDR) models.1 The committee comments on the Environmental Protection Agency (EPA) analysis of the fate of inhaled formaldehyde in the respiratory tract (portal of entry) and at more distant sites reached through systemic circulation, the use of formaldehyde-induced cross-links as biomarkers, and the ability of formaldehyde to cause systemic genotoxic effects. The committee also reviews EPA’s use of the computational pharmacokinetic models and BBDR models that have been developed for formaldehyde and considers EPA’s analysis of the sensitivity of low-dose BBDR-model estimates to small changes in model design or model inputs.

The discussion provided here is not intended to be exhaustive but rather focuses on the evidence presented in the draft IRIS assessment that was used to support EPA’s key conclusions. It also dwells on the inhalation pathway rather than other exposure pathways because the inhalation pathway is the focus of the draft IRIS assessment. In conducting its review, the committee attempted to answer several central questions underlying the approach taken by EPA, including the following:

  • Is formaldehyde an endogenous chemical?

  • What is the immediate fate of inhaled formaldehyde?

  • Is inhaled formaldehyde available systemically?

1

This chapter focuses on carcinogenic modes of action. Known or hypothesized modes of action for other effects, such as airway irritation, are discussed elsewhere in this report.



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3 Toxicokinetics and Modes of Action of Formaldehyde This chapter provides the committee’s review of the draft IRIS assessment that is relevant to formaldehyde toxicokinetics, carcinogenic modes of action, pharmacokinetic models, and biologically based dose-response (BBDR) mod- els.1 The committee comments on the Environmental Protection Agency (EPA) analysis of the fate of inhaled formaldehyde in the respiratory tract (portal of entry) and at more distant sites reached through systemic circulation, the use of formaldehyde-induced cross-links as biomarkers, and the ability of formalde- hyde to cause systemic genotoxic effects. The committee also reviews EPA’s use of the computational pharmacokinetic models and BBDR models that have been developed for formaldehyde and considers EPA’s analysis of the sensitiv- ity of low-dose BBDR-model estimates to small changes in model design or model inputs. The discussion provided here is not intended to be exhaustive but rather focuses on the evidence presented in the draft IRIS assessment that was used to support EPA’s key conclusions. It also dwells on the inhalation pathway rather than other exposure pathways because the inhalation pathway is the focus of the draft IRIS assessment. In conducting its review, the committee attempted to an- swer several central questions underlying the approach taken by EPA, including the following:  Is formaldehyde an endogenous chemical?  What is the immediate fate of inhaled formaldehyde?  Is inhaled formaldehyde available systemically? 1 This chapter focuses on carcinogenic modes of action. Known or hypothesized modes of action for other effects, such as airway irritation, are discussed elsewhere in this report. 29

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30 Review of EPA’s Draft IRIS Assessment of Formaldehyde  Can formaldehyde-related effects alter its toxicokinetics?  Are formaldehyde-induced cross-links useful biomarkers of exposure?  Can inhaled formaldehyde have systemic genotoxic effects?  Are useful computational pharmacokinetic models for formaldehyde inhalation available?  Has a mode of action for formaldehyde carcinogenesis been identified?  What is the status of BBDR models for formaldehyde?  Should the BBDR models available for formaldehyde be used in EPA’s quantitative assessment? Some of those questions could be answered by weighing the evidence from re- search studies considered in the draft IRIS assessment; others could not be an- swered by the committee with high confidence. Overall, the committee found that the chapters describing the toxicokinet- ics, modes of action, and various models are well organized and that the draft IRIS assessment accurately reflects the current understanding of the toxicokinet- ics of inhaled formaldehyde and provides a thorough review of the metabolism, cytotoxicity, and genotoxicity of formaldehyde. The literature review in the draft IRIS assessment appears to be up to date and to include all major and recent studies published as of the release date. TOXICOKINETICS Is Formaldehyde an Endogenous Chemical? The committee notes that EPA satisfactorily describes the current under- standing of endogenous formaldehyde. It is well established that formaldehyde is produced endogenously by enzymatic and nonenzymatic pathways or as a detoxification product of xenobiotics during cellular metabolism (ATSDR 1999). There is also broad agreement that formaldehyde originating from meta- bolic or dietary sources is normally present at low concentrations in all tissues, cells, and bodily fluids. The concentration of endogenous formaldehyde in the blood of rats, monkeys, and humans is about 0.1 mM (Heck et al. 1985; Casa- nova et al. 1988). Background concentrations in the liver and nasal mucosa of the rat are 2-4 times those in the blood (Heck et al. 1982). Endogenous tissue formaldehyde concentrations are similar to concentrations (about 0.05 mM) that induce genotoxicity and cytolethality in vitro (Heck and Casanova 2004). Heck et al. (1985) did not observe an increase in blood formaldehyde con- centrations in rats and humans after exposure to inhaled formaldehyde at 14.4 ppm (2 hr) or 1.9 ppm (40 min), respectively. Subchronic studies conducted in rhesus monkeys have also shown that blood formaldehyde concentration was not measurably altered by exposure to airborne formaldehyde at 6 ppm for 6 hr/day 5 days/week for 4 weeks (Casanova-Schmitz et al. 1984).

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Toxicokinetics and Modes of Action of Formaldehyde 31 Formaldehyde has also been measured in exhaled breath, but the interpre- tation of some measurements made with mass spectrometry has been questioned (Spanel and Smith 2008; Schripp et al. 2010). Spanel and Smith (2008) showed that a trace contaminant (up to 1%) of the reagent gas used in real-time mass- spectrometric methods—specifically proton-transfer reaction mass spectrometry (PTR-MS) and selected ion flow tube mass spectrometry (SIFT-MS)—reacts with endogenous methanol and ethanol that is normally found in exhaled breath to produce the same main ion (mass-to-charge ratio of 31) as is used to measure formaldehyde. Thus, they concluded that up to 5 ppb of the formaldehyde con- centration determined in the exhaled breath of humans reported in earlier studies that did not account for this confounding may be due to methanol or ethanol and not formaldehyde; that is, 1% of total background concentrations of methanol or ethanol of about 500 ppb would be misclassified as formaldehyde. The commit- tee concurs with EPA’s concerns as to whether some published exhaled-breath measurements of formaldehyde are analytically valid. The committee also notes that this methodologic problem is inconsistently addressed by EPA in its re- analysis of the exhaled-breath experiments. The committee concludes, however, that regardless of the methodologic issue related to breath analysis, formalde- hyde is normally present at a few parts per billion in exhaled breath after the measurement error associated with a trace contaminant in the reagent gas used in previous mass-spectrometric methods is taken into account. The committee concludes that formaldehyde is an endogenous compound and that this finding complicates assessments of the risk posed by inhalation of formaldehyde. The committee emphasizes that the natural presence of various concentrations of formaldehyde in target tissues remains an important uncer- tainty with regard to assessment of the additional dose received by inhalation. What Is the Immediate Fate of Inhaled Formaldehyde? Formaldehyde has been the subject of multiple toxicokinetic studies in ro- dents, dogs, and monkeys and of numerous in vitro and biomonitoring studies in humans. Although there may be quantitative differences between species, the fate of formaldehyde is qualitatively similar among species. The draft IRIS as- sessment provides an extensive and thorough review of the literature on the fate of formaldehyde in the body. Formaldehyde is highly water-soluble and exists in water almost exclu- sively in a reversible hydrated form (methanediol). Thus, less than 0.1% of for- maldehyde can be considered “free” once it enters the body. Formaldehyde is highly reactive at the site of entry and reacts readily with macromolecules, in- cluding DNA to form DNA-protein (DPX) and DNA-DNA (DDX) cross-links. Formaldehyde is oxidized to formate by a low-Km (400-μM) mitochondrial al- dehyde dehydrogenase-2 (ALDH2) or via a two-enzyme system that converts nonenzymatically formed glutathione adducts (S-hydroxymethylglutathione) to the intermediate S-formylglutathione, which is then metabolized to formate and

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32 Review of EPA’s Draft IRIS Assessment of Formaldehyde glutathione by S-formylglutathione hydrolase. Those metabolic processes con- tribute to the short biologic half-life of formaldehyde. Inhaled formaldehyde is absorbed primarily in the upper airways because of its high water solubility, metabolism, and reactivity. Inhaled formaldehyde in the nasal cavity initially contacts the mucus layer lining the epithelium (Figure 3-1). Once in the mucus layer, formaldehyde undergoes a reversible reaction with water to form methanediol. Albumin in the mucus that lines the human nasal epithelium (Figure 3-1) forms an additional barrier to the systemic absorp- tion of formaldehyde (Bogdanffy et al. 1987). The solubility of formaldehyde in mucus and the ciliary movement and ingestion of mucus may account for the removal of as much as 42% of the inhaled dose in rodents (Schlosser 1999). Diffusion is the dominant transport mechanism for formaldehyde through the mucus layer. Some inhaled formaldehyde passes through the mucus layer to reach the epithelium where its transformation and removal occur by enzymatic reactions with the nasal tissue and nonenzymatic reactions with glutathione and macromolecules, including proteins and DNA. FIGURE 3-1 Schematic representation of the mammalian nasal epithelium. Inhaled for- maldehyde reacts rapidly with macromolecules in the tissue and the albumin in the mucus that lines the respiratory epithelium; these reactions result in a steep concentration gradi- ent. Formaldehyde crossing the basement membrane can react further with macromole- cules in the submucosal layer or reach the systemic circulation. The figure also shows a representation of the nasal-associated lymphoid tissue (NALT) that is generally present near the ethmoid turbinates on either side of the nasal septum and near the ventral naso- pharyngeal duct. The NALT is one putative site of formaldehyde interactions with lym- phoid tissues, but direct evidence that supports this hypothesis is lacking.

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Toxicokinetics and Modes of Action of Formaldehyde 33 Removal of formaldehyde from the air by the upper respiratory tract is ef- ficient. For example, nasal uptake of inhaled formaldehyde in dogs is nearly complete (Egle 1972). Penetration of formaldehyde to more distal airways was not observed in formaldehyde-exposed dogs even in the presence of increased breathing rates or exposure to formaldehyde at high concentrations (Egle 1972). In rats and mice, which are obligate nasal breathers with a highly complex nasal- airway geometry and large ratio of surface area to lumen volume, most inhaled formaldehyde (average, about 97%) is also taken up in the nasal cavity (Patter- son et al. 1986). Computer models predict that systemic delivery of formalde- hyde in rodents is extremely low and would not increase formaldehyde concen- trations in tissues (Franks 2005). The results of those simulations are supported by animal studies that show that formaldehyde inhalation in rodents or nonhu- man primates did not alter blood formaldehyde concentrations. Studies using DPX formation as an exposure surrogate corroborate that conclusion. For exam- ple, Casanova-Schmitz et al. (1984) found that exposure of rats to 14C- and 3H- labeled formaldehyde at 15 ppm did not result in DPX formation in bone mar- row. However, they did observe non-linear DPX formation in the nasal mucosa at concentrations of 2 ppm or greater. In humans, oral breathing bypasses the uptake in the nasal epithelium; this breathing pattern leads to increased delivery of formaldehyde to the intermediate regions of the respiratory tract (that is, from the oral cavity to the upper conduct- ing airways of the lungs) (Overton et al. 2001). Nasal uptake of formaldehyde is also reduced as ventilation rates increase. The committee concludes that the immediate fate of inhaled formaldehyde is primarily absorption at the site of first contact where it undergoes extensive local metabolism and reactions with macromolecules, despite species differ- ences in uptake resulting from different breathing patterns (for example, orona- sal breathing in humans vs nasal breathing in rodents). Although the site of ab- sorption in the respiratory tract may depend on airway anatomy (simple vs complex), breathing pattern (nasal vs oronasal), and ventilation rate, the net re- sult is that inhaled formaldehyde predominantly remains in the respiratory epi- thelium. Is Inhaled Formaldehyde Available Systemically? As mentioned earlier, several studies have demonstrated that inhaled for- maldehyde has at most little systemic bioavailability. Some experiments exam- ined the disposition of 14C-labeled formaldehyde, and others used techniques of analytic chemistry—such as gas or liquid chromatography coupled to mass spec- trometry (for example, GC-MS)—to evaluate changes in formaldehyde concen- trations in blood or tissue after inhalation. The studies have found that formal- dehyde undergoes rapid elimination in blood with virtually no increase in “free” formaldehyde in blood or systemic tissues. Although the studies demonstrate

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34 Review of EPA’s Draft IRIS Assessment of Formaldehyde relatively long retention of the 14C radiolabel, this observation most likely re- flects incorporation and turn over of the so-called 1-carbon pool. As noted by EPA, “measuring the distribution of the absorbed formaldehyde based on 14C- radiolabeling and GC-MS studies alone is problematic because it is difficult to resolve (through these studies) whether it is free, reversibly bound, irreversibly bound, formate, one-carbon pool, etc. This is of significance with regard to un- derstanding the availability of the absorbed formaldehyde” (EPA 2010, p. 3-12). The committee shares EPA’s concern. A series of studies using dual-labeled (14C/3H) formaldehyde in rats has been performed to address the analytic concern (Casanova-Schmitz and Heck 1983; Casanova-Schmitz et al. 1984). The draft IRIS assessment accurately summarizes the main conclusions reached from those experiments, namely that “labeling in the nasal mucosa was due to both covalent binding and metabolic incorporation,” that “DPX [were] formed at 2 ppm or greater in the respiratory mucosa,” and that “formaldehyde did not bind covalently to bone marrow mac- romolecules at any exposure concentration” (up to 15 ppm) (EPA 2010, p. 3- 12). The labeling of bone marrow macromolecules was found by the investiga- tors to be due entirely to metabolic incorporation of the radiolabels, not to direct covalent binding of intact formaldehyde. The committee views those findings as supporting the hypothesis that inhaled formaldehyde is not delivered systemi- cally under the exposure conditions used in the studies (0.3-15.0 ppm, 6 hr) (EPA 2010). The committee also found that the more contemporary work performed by Lu et al. (2010) that examined formaldehyde-induced DNA adducts and DDX cross-links provided no direct evidence of systemic availability of inhaled for- maldehyde. The Lu et al. (2010) study used 13CD2-labeled formaldehyde and showed that 13CD2-formaldehyde-DNA adducts and DDX were confined to the nasal cavity of exposed F344 rats, even though they examined much more DNA isolated from bone marrow, lymphocytes, and other tissues at distant sites for the adducts. The male Fischer 344 rats were exposed to [13CD2]-formaldehyde at 10 ppm for 1 or 5 days (6 hr/day) with a single nose-only unit. The strongest data cited by EPA in support of systemic delivery of inhaled formaldehyde come from several studies in which antibodies to formaldehyde- hemoglobin and formaldehyde-albumin adducts were detected in blood from exposed workers, smokers, and laboratory animals. The studies did not defini- tively demonstrate, however, whether adduct formation occurs at a site distant from the portal of entry. For example, it is not known whether the adducts could be formed in the airway submucosal capillary beds or reflect systemic delivery of formaldehyde. Moreover, the draft IRIS assessment does not evaluate the antibody work as critically as the direct chemical-analysis approaches. The committee found that the draft does not offer a sufficient basis for EPA’s reli- ance on the antibody data to support the hypothesis that formaldehyde (or its hydrated form, methanediol) may reach sites distal to the portal of entry and produce effects at those sites.

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Toxicokinetics and Modes of Action of Formaldehyde 35 Questions have arisen regarding the possibility that formaldehyde reaches distal sites as methanediol. However, although equilibrium dynamics indicate that methanediol would constitute more than 99.9% of the total free and hy- drated formaldehyde, the experimental data described above provide compelling evidence that hydration of formaldehyde to methanediol does not enhance deliv- ery of formaldehyde beyond the portal of entry to distal tissues. Furthermore, Georgieva et al. (2003) used a pharmacokinetic modeling approach that explic- itly accounted for the competing processes of hydration, dehydration, diffusion, reactivity with macromolecules, and metabolism and demonstrated that hydra- tion-dehydration reaction rates determined from equilibrium studies in water are not applicable in biologic tissues, given that their use in the model resulted in simulations that were inconsistent with the available data. For example, the cal- culated dehydration rate from equilibrium dynamics studies in water was so small relative to other competing rates that too little formaldehyde would be available to account for the measured DPX rates. Thus, the data provide a strong indication that the hydration-dehydration reaction should not be rate-limiting and can thus be ignored in modeling the disposition of inhaled formaldehyde in nasal tissues. EPA also suggested that systemic delivery of formaldehyde-glutathione adducts and latter release of free formaldehyde may result in delivery of formal- dehyde to sites distal to the respiratory tract. However, experimental data sup- porting that hypothesis are lacking, as acknowledged by the draft IRIS assess- ment. In fact, additional data based on even more sensitive analytic methods published since the draft assessment was released casts further doubt on the hy- pothesis that formaldehyde reaches the systemic distribution in a form that can react with macromolecules in tissues remote from the portal of entry (Lu et al. 2011; Moeller et al. 2011; Swenberg et al. 2011). The committee also found two divergent statements regarding systemic delivery of formaldehyde in the draft IRIS assessment. Some parts of the draft assume that the high reactivity and extensive nasal absorption of formaldehyde restrict the systemic delivery of inhaled formaldehyde to the upper respiratory tract (for example, EPA 2010, p. 4-371). Under that assumption, systemic re- sponses—including neurotoxicity, reproductive toxicity, and leukemia—are unlikely to arise from the direct delivery of formaldehyde (or methanediol) to a distant site in the body, such as the brain, the reproductive tract, and the bone marrow. Other portions of the document presume systemic delivery of formal- dehyde (or its conjugates) and use this presumption to account in part for the systemic effects (see, for example, p. 4-1, lines 16-19; p. 4-472, line 18; Section 4.5.3.1.8; and p. 6-23, line 31). The committee found the inconsistency to be troubling, and the divergent assumptions are not justified. The committee concludes that the issue of whether inhaled formaldehyde can reach the systemic circulation is extremely important in assessing any risk of adverse outcomes at nonrespiratory sites associated with inhalation of formalde- hyde. Moreover, the committee concludes that the weight of evidence suggests that it is unlikely for formaldehyde to appear in the blood as an intact molecule,

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36 Review of EPA’s Draft IRIS Assessment of Formaldehyde except perhaps after exposures at doses that are high enough to overwhelm the metabolic capability of the tissue at the site of entry. Thus, although many sensi- tive and selective investigative approaches have been used, systemic concentra- tions from inhaled formaldehyde are indistinguishable from endogenous back- ground concentrations. The committee, however, notes the importance of differentiating between systemic delivery of formaldehyde and systemic effects. The possibility remains that systemic delivery of formaldehyde is not a prereq- uisite for some of the reported systemic effects seen after formaldehyde expo- sure. Those effects may result from indirect modes of action associated with local effects, especially irritation, inflammation, and stress. Can Formaldehyde-Related Effects Alter Its Toxicokinetics? Formaldehyde is an irritant gas in humans and animals. Rodents exposed to sufficiently high formaldehyde concentrations develop reflex bradypnea, de- creased body temperature, reduced oxygen demand, and reduced nasal muco- ciliary clearance. For example, the minute volume decreased by 45% in rats and 75% in mice during formaldehyde inhalation at 15 ppm for 10 min compared with controls (Chang et al. 1983). The species difference may contribute to the difference between rats and mice in the incidence of nasal lesions. At identical exposures, mice were found to receive a lower effective dose at the target tissue in the nasal cavities than rats because mice have a greater reduction in minute ventilation in response to sensory irritation of the respiratory tract. The lower effective dose of mice was verified by the observation that mice had smaller increases in formaldehyde-induced cell proliferation in the nasal mucosa than rats. The findings could explain why higher formaldehyde exposure concentra- tions were needed to induce the same degree of toxic effects (such as nasal tu- mors) in mice as occurs in rats at lower exposures (Chang et al. 1983). The committee notes that the CFD models of Kimbell et al. (2001a,b) do not account for potential effects of sensory irritation on ventilation inasmuch as only two mass-transfer coefficients, one for mucus-coated and one for non-mucus-coated epithelial regions of the nose, were used in all simulations to derive uptake into nasal tissues. However, later models that account for DPX cross-links and cyto- toxicity (Conolly et al. 2000, 2002, 2003, 2004; Georgieva et al. 2003) relied on animal data that were obtained at concentrations that potentially caused irritation to derive parameters associated with metabolism and reactivity; thus, the poten- tial effect of altered ventilation was indirectly compensated for in those model simulations. EPA also hypothesized that airway remodeling induced by formaldehyde exposure might alter formaldehyde dosimetry. For example, squamous epithe- lium absorbs considerably less formaldehyde than other epithelial types (Kim- bell et al. 1997). Metaplasia of the anterior nasal epithelium to a squamous epithelial phenotype occurs in rats exposed repeatedly to formaldehyde at 3 ppm or higher (Kimbell et al. 1997). Although EPA identified the consequences of

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Toxicokinetics and Modes of Action of Formaldehyde 37 metaplasia for dosimetry, the committee concludes that this issue is not particu- larly important at the low exposure concentrations relevant to derivation of a reference concentration (RfC). The committee agrees with EPA’s conclusion that “certain formaldehyde- related effects have the potential to modulate its uptake and clearance” (EPA 2010, p. 3-5). Some of the effects, such as changes in mucociliary function and altered nasal epithelium, could occur in humans. However, reflex bradypnea and related modulating effects seen in rodents do not occur in phylogenetically higher animals (nonhuman primates) or humans. Thus, formaldehyde exposures at concentrations relevant for an RfC or unit risk are unlikely to alter its toxi- cokinetics. Are Formaldehyde-Induced Cross-Links Useful Biomarkers of Exposure? As noted earlier, formaldehyde readily forms DPX and DDX cross-links. A variety of analytic chemistry approaches, including radiolabeled formalde- hyde tracers, have been used to evaluate formaldehyde binding to macromole- cules and to differentiate binding from metabolic incorporation. For example, rats given intravenous injections of 14C-formaldehyde or 14C-formate, a metabo- lite of formaldehyde that contributes to the one-carbon pool, develop similar blood radioactivity profiles; this suggests that labeling of blood macromolecules is due to metabolic incorporation rather than adduct formation (Heck and Casa- nova 2004). DPX formation in human white blood cells after in vitro exposure to for- maldehyde (0-10 mM) for 1.5 hr rose linearly with increasing formaldehyde concentrations above 0.001 mM (Shaham et al. 1996). The kinetics of DPX formation in vivo (and in some in vitro systems) appear more complicated and reflect a balance between DPX formation and repair processes. Overall, DPX have been detected in upper and lower respiratory tracts of rodents and nonhu- man primates. For example, Casanova and colleagues (Casanova et al. 1989, 1994) conducted formaldehyde inhalation studies in male F344 rats to determine DPX formation. In one study, DPX concentrations were measured from the na- sal lateral meatus, medial meatus, and posterior meatus after inhalation of 14C- formaldehyde (Casanova et al. 1994). The sites were selected because they were associated with a high tumor incidence in a formaldehyde bioassay (Monticello et al. 1989, 1991). The other study measured DPX concentrations in nasal mu- cosal tissue taken from the entire nasal cavity (Casanova et al. 1989). Formation of DPX demonstrated a nonlinear dose-response relationship within the nasal epithelium; statistically significant increases occurred after exposure at concen- trations of 0.3 ppm and higher. Although the mechanisms for DPX-induced cell death have not been es- tablished, DPX formation has been used as a key event linking external expo- sures with cellular responses (mutagenicity, cytotoxicity, and compensatory cellular regeneration) in the development of pharmacokinetic and BBDR mod-

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38 Review of EPA’s Draft IRIS Assessment of Formaldehyde els. Pharmacokinetic models that describe in vivo DPX formation have included saturable pathways to describe enzymatic formaldehyde metabolism, a first- order pathway to represent formaldehyde's reaction with tissue constituents, and first-order binding to DNA (Hubal et al. 1997; Conolly et al. 2000). Yang and co-workers (2010) have suggested that a single, saturable pathway competing with DNA binding could describe the DPX data fairly well in rodents exposed to formaldehyde at 15 ppm or less. The models predict that less than 1% is cova- lently bound as DPX in the airway (Casanova et al. 1991; Heck and Casanova 1994). DPX have also been reported in circulating lymphocytes from formalde- hyde-exposed people (Shaham et al. 1996, 1997, 2003). For example, Shaham et al. (1996) measured DPX concentrations in human white blood cells from 12 workers exposed to formaldehyde and from eight controls. The authors found a statistically significant difference in the DPX concentrations in lymphocytes between the formaldehyde workers and the unexposed controls. However, only four of the 12 exposed workers had DPX concentrations above the upper range of the controls. The authors also reported a linear relationship between years of exposure and the amount of DPX and concluded that DPX may be used as a biomarker of formaldehyde exposure. Several limitations of the study have been identified by others (Casanova et al. 1996). Most notably, the control group’s exposure history, including smoking prevalence, was poorly defined. Shaham et al. (1996) presented minimal exposure-assessment data, so the study was not used quantitatively by EPA. The committee agrees with EPA’s decision. The concentrations of DPX formed by formaldehyde in nasal airways have been modeled and used by EPA as an internal dose surrogate to update its health assessment of formaldehyde (EPA 1991; Hernandez et al. 1994). On the basis of different model assumptions that are discussed in more detail later in this chap- ter, EPA changed the values of the parameters in the DPX model (Subramaniam et al. 2007), which was used to derive internal dose-related points of departure for human extrapolation. On the basis of the evidence, the draft IRIS assessment states that “DNA protein cross-links (DPXs) formed by formaldehyde (cova- lently bound in this case) have been regarded as a surrogate dose metric for the intracellular concentration of formaldehyde [Casanova et al. 1989, 1991; Her- nandez et al. 1994]. This is particularly relevant because of the nonlinear dose response for DPX formation due to saturation of enzymatic defenses at high concentrations [Casanova et al. 1989, 1991]. Thus, the ability to measure DPX is an important development” (EPA 2010, p. 3-12). The committee agrees with the use of DPX as a biomarker of exposure in the draft IRIS assessment. Can Inhaled Formaldehyde Have Systemic Genotoxic Effects? Formaldehyde is a genotoxic (DNA-reactive) chemical. Formaldehyde- induced DNA damage is postulated to lead to mutations and clastogenesis, criti- cal cytogenetic events in the carcinogenic mode of action. The evidence of for-

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Toxicokinetics and Modes of Action of Formaldehyde 39 maldehyde genotoxicity includes DPX cross-links, chromosomal aberrations, micronuclei, and sister-chromatid exchanges. A large number of in vitro tests for genotoxicity—including bacterial mutation, DNA strand breaks, chromosomal aberrations, and sister-chromatid exchange assays—are positive when formalde- hyde is used. The studies on genotoxicity of formaldehyde, particularly those involving in vivo exposures of humans and animals, have provided strong evi- dence that formaldehyde genotoxicity occurs in the nasal mucosa and peripheral (circulating) blood lymphocytes. The database on genotoxicity assessment using peripheral blood lymphocytes from exposed human cohorts is appreciable and contains studies from different countries and various exposure scenarios and spans a period of more than 20 years. Some more recent studies (Costa et al. 2008) provide evidence of clastogenicity of formaldehyde coupled with individ- ual exposure assessment. Although studies in humans showed some inconsistent results regarding the extent and form of the cytogenetic changes associated with formaldehyde exposure, the overall body of evidence suggests that inhaled for- maldehyde has an effect that may be detected in blood cells in the systemic cir- culation. The committee notes that it is unknown whether formaldehyde genotoxicity arises from interactions that occur at the site of contact—for exam- ple, in nasal-associated lymphoid tissue in the nasal mucosa (Figure 3-1)—or as the result of local circulation of lymphocytes in blood that perfuses portal-of- entry tissues. Overall, the draft IRIS assessment concludes that formaldehyde may act through a mutagenic mode of action and that this effect is not restricted to the site of entry (EPA 2010). As noted earlier, multiple lines of evidence support the conclusion that formaldehyde is mutagenic. The committee agrees with EPA and further notes that EPA’s conclusion is consistent with the current Guidelines for Carcinogen Risk Assessment (EPA 2005). The committee acknowledges that the database on the cytogenetic effects of formaldehyde in humans is supportive of EPA’s second conclusion, that the mutagenic action of formaldehyde is not restricted to tissues at the point of con- tact. However, available data are insufficient to support definitive conclusions on several key issues. First, exposure assessment in the relevant human studies was generally lacking, and the effects observed occurred in highly exposed workers. In the absence of understanding of the shape of the dose-response curve for cytogenetic changes at low doses, it is difficult to extrapolate the find- ings to environmental exposures. Second, the mechanism of cytogenetic effects in circulating blood cells is not established—an uncertainty that complicates the committee’s ability to link exposure with effects at distant sites. That data gap is especially problematic given the growing body of evidence that formaldehyde is not available systemically in any reactive form. Thus, it can only be hypothe- sized that systemic effects, such as cytogenetic effects in circulating blood lym- phocytes, originate by as yet unproven mechanisms in portal-of-entry tissues. The committee concludes that there is great uncertainty and associated controversy regarding the following issues: the ability of formaldehyde to cause DNA damage at distal (that is, other than portal-of-entry) sites, the relative con-

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In a follow-on analysis, Crump et al. (2008) made an arbitrary change in the DPX-based effect on initiated cell replication by theorizing that if an initiated cell is created by a specific mutation that impairs cell-cycle control, there may be a mitigation of cell replication that is observed in the low-dose cell proliferation of normal cells (that is, in the negative vs baseline replication portion of the J-shaped dose-response curve) and hence a shift of the cell division of an initiated cell in the model toward greater rates at low doses. They argued that this theoretical possibility provides biologic motivation to test the effect on the MVK model because of the absence of data for doing otherwise. The change disconnects the birth and death rates of initiated cells from constraints used by Conolly et al. based on normal cells. The committee concludes that this change is contrary to the explanation provided by Monticello et al. (1996), who suggested that it is not a mutation in cell-cycle check points that results in lower cell-division rates than control at low exposures but rather an increase in the time that it takes for DNA-repair processes to eliminate the DPX before the cell can resume the process of cell division that leads to a cell-division rate at low exposures that is below that seen under basal conditions. These are two fundamentally different mechanisms with different connotations for risk—the mutagenic one chosen by EPA and the DNA-repair mode of action supported by several other publications on DPX cited by Conolly et al. (2003, 2004) and Monticello et al. (1996). Because they argue that there are no data to refute these assumed and arbitrary adjustments of the Conolly et al. models, they state that the onus is on others to show that such small changes cannot occur (that is, prove a negative before the authors would accept the contention that the Conolly et al. models are at all conservative as Conolly et al. suggested). That standard cannot be met. High; the MVK model is very sensitive to the choice of controls and their effect on basal mutation rates. Basal mutation rates for normal and intermediate cells are unknown and had to There were zero squamous cell carcinomas in control rats in the two bioassays used to define the basal be optimized from sparse information using mutation rates of normal and intermediate cells in the two-stage, MVK dose-response model. Conolly et al. several assumptions (2004) used results from the full National Toxicology Program historical control database. That is a point of contention by EPA, which believes that only historical controls from inhalation bioassays (and those in the same laboratory as the formaldehyde study) can be used in a relevant comparison. Squamous cell carcinomas are so rare that some leeway in approximating basal rates may have to be accepted, even though EPA’s point is technically correct. When mutation rates, cell-division rates, and mutation intensity vary as a function of age (that is, not treated as constants over all ages), the Hoogenveen solution can lead to more errors than alternative models that respect the nonhomogeneity of the model parameters (Crump et al. 2005). Although EPA may be technically (Continued) 53

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54 TABLE 3-3 Continued Parameter Assumptions Likely Effect on Model correct, it was unclear to the committee whether there is a significant effect on the Conolly et al. (2004) prediction unless their parameter values are substantially changed, which would open the Crump et al. and Subramaniam et al. models up to an equally fair criticism of their assumptions and choices in model parameters. Estimating parameters for basal mutation rates for a normal to intermediate and intermediate to malignant transformation in humans is subject to even more uncertainty than in the rat. Crump et al. (2008) made seemingly arbitrary changes in the birth and death rates of initiated cells, claiming that there are no data to suggest that they cannot do that and that their changes were small relative to changes in this relationship at high formaldehyde flux rates. Although no changes in the predicted data range were observed, the effect of the low-dose and high-dose extrapolations was huge. Intermediate-cell parameter values were related Moderate. The proportionality constant for relating DPX to the human cell-mutation rate (KMU) was to normal-cell parameter values and constrained adjusted from the upper bounds on the rat value rather than the maximum likelihood estimate of the rat value, by relevant cell-proliferation and tumor data which was zero, by a ratio of the basal mutation rate of human to rat; this assumes that the mutation mechanism is the same in control vs formaldehyde exposure and that DPX can be viewed as a promutagenic event. Those assumptions suggest that human cells are more difficult to mutate than rat cells on the basis of their evaluation of the literature, and they force the model to include clonal growth at exposures below those known to cause cell proliferation in the rat. The authors view the assumptions as conservative. Changes in epithelial cell thickness due to Moderate. The first-order clearance of DPX could be slower than that used by Conolly et al. (2003, 2004). formaldehyde exposure had no effect on Over time, epithelial tissue in targeted regions of the nose thickens. The thickening could conceivably dilute DPX measurements used to calibrate the DPX concentrations in the measured tissues to such an extent that residual concentrations 18 hr after BBDR models exposure are not different from those in naïve animals, and this would affect the determination of DPX clearance rates. Abbreviations: DPX, DNA-protein crosslinks; BBDR, biologically based dose-response; CFD, computational fluid dynamics; EPA, Environ- mental Protection Agency; and MVK, Moolgavkar-Venzon-Knudson.

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Toxicokinetics and Modes of Action of Formaldehyde 55  Assumptions used in defining parameters associated with mutation, birth, and death rates of intermediate cells, cells that cannot be directly measured or even identified histologically (that is, intermediate cells in the MVK 2-stage model structure are a surrogate for potentially multiple stages of transformed but not yet malignant cells). Other sources of variability and uncertainty were also explored, but the three noted were the main focus of EPA’s reanalysis. The committee agrees that the sensitivity analysis added value to the interpretation of the Conolly et al. models, although it raised questions about the degree to which manipulations of the range of model parameter values can and should be performed to reflect po- tentially divergent outcomes. EPA’s reanalysis also identified a flaw in one of the numeric approaches used in the original models and corrected it to improve the reliability of the simulation—another value added. Alternative model structures fitted to the data used by Conolly et al. can yield different low-dose extrapolation results (Crump et al. 2005, 2008; Subra- maniam et al. 2007, 2008). For example, Subramaniam et al. (2007) assessed the sensitivity of model-predicted tumor response to two major Conolly et al. as- sumptions. First, Conolly et al. pooled all National Toxicology Program histori- cal controls to establish basal mutation rates. Second, data obtained by lumping animals found to have tumors at scheduled sacrifice with animals that died as a result of tumors were used to estimate formaldehyde-specific mutation rates. Subramaniam et al. also examined the choice for the first-order clearance of DPX used by Conolly et al. to match in vivo data with a slower clearance deter- mined in vitro and how this could still be consistent with the in vivo data. The purpose of the Subramaniam et al. work was to explore whether the mutagenic mode of action, as exemplified by DPX as a precursor, has an insignificant role in predicted tumor responses. Their analysis indicated that in the range of expo- sures in which tumors occur, the mutagenic mode of action could have contrib- uted up to 74% of the added tumor probability, whereas Conolly et al. (2003, 2004) showed very little contribution by this mode of action in the human when they used an upper bound on the DPX proportionality constant rather than the maximum-likelihood estimate of zero from the rat data. The committee agrees with EPA that existing data are insufficient to establish the potential biologic variability in model parameters associated with the mutagenic mode of action adequately. However, because the mutagenic mode of action is the major reason for adopting the default low-dose linear extrapolation methods over application of the BBDR models in the draft assessment, the committee recommends that the manipulations that lead to such high contributions of mutagenicity to the mode of action for nasal tumors be reconciled with the observations that formal- dehyde is endogenous, that nasal tumors are very rare in both rats and humans, and that no increases in tumor frequency have been observed in animal studies at formaldehyde exposure concentrations that do not also cause cytotoxicity. In a follow-on paper, Crump et al. (2008) evaluated the sensitivity of model output to varying such parameters as mutation, birth, and death rates of

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56 Review of EPA’s Draft IRIS Assessment of Formaldehyde initiated cells and concluded that small changes in these parameters can result in similar fits to experimental data but yield markedly different low-dose extrapo- lations. The authors argued that—inasmuch as there are no data that define mu- tation, birth, and death rates of initiated cells, let alone data that identify what an initiated cell is—the onus is on others to demonstrate that the small changes that they made in the Conolly et al. (2004) model cannot occur. However, that can never be established with certainty. Although the committee finds that testing of alternative models and the variability in model parameters is consistent with EPA’s cancer guidelines, some of the more extreme model scenarios should not have been used as a basis for rejecting the BBDR approach. In particular, ad- justments of parameter values associated with mutation, birth, and death rates of initiated cells used in EPA’s analysis of alternative models that yielded the most extreme deviations from the Conolly et al. (2004) low-dose extrapolations also produced unrealistically high added risks for humans at concentrations that have been observed in the environment of occupationally exposed workers (100% incidence at concentrations as low as about 0.1-1 ppm). Thus, the committee recommends that manipulations of model parameters that yield results that are biologically implausible or inconsistent with the available data be discarded and not used as a basis for rejecting the overall model. The committee concludes that the existing BBDR models were developed from an impressive exposure- and time-dependent database on the modes of actions of formaldehyde in rats and monkeys; exquisitely detailed dosimetry models in rats, monkeys, and humans; and sparse data on humans that required scale-up of key model parameter values from animal studies and other biologic data and epidemiologic observations to constrain the human model predictions. The scope of the research makes this one of the best-developed BBDR models to date for any chemical, even with its acknowledged uncertainties. The commit- tee also acknowledges that the draft IRIS assessment provides a thorough review of the BBDR models, the major assumptions underpinning the extrapolation to humans, and EPA’s own series of papers that evaluated the sensitivity of the BBDR models to these assumptions even though the committee may not agree with the validity of all the resulting manipulations. Should the Biologically Based Dose-Response Models Be Used in the Environmental Protection Agency Quantitative Assessment? As a result of the agency's reanalysis of the models, EPA chose not to use the full rat and human BBDR models to estimate unit risks. Instead, in a bench- mark-dose approach, EPA used the CFD-derived determinations of formalde- hyde flux to the entire surface of mucus-coated epithelium to derive a point of departure based on nasal cancers in rats. It then extrapolated to zero dose by using a default linearized multistage approach. The committee is concerned about that approach for low-dose extrapola- tion. The committee found that the evaluations of the original models and EPA’s

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Toxicokinetics and Modes of Action of Formaldehyde 57 reanalysis conflicted with respect to the intent or purpose of using the formalde- hyde BBDR models in human health assessments. The conflict is evident in the discussions in the draft IRIS assessment. For example, the reanalysis by Subra- maniam et al. is used to support the mutagenic mode of action of formaldehyde and to reduce support for using the BBDR models on the basis of the uncertain- ties in parameter estimation and assumptions in the models. In contrast, Conolly et al. (2003) focused their model parameter estimates to represent “best-fit,” using maximum likelihood estimates, whereas Subramaniam et al. and Crump et al. pushed parameter assumptions in a single direction to show that different assumptions that fit the experimental data can yield different results of low-dose extrapolation. The committee is concerned about the possibility that those ad- justments of the Conolly et al. models may not be scientifically defensible. The committee was also struck by the relative lack of transparency in the draft IRIS assessment’s description of the decision to use the peer-reviewed BBDR models minimally. The Conolly et al. (2003, 2004) rat and human dose-response models, as the authors themselves state, are biologically motivated and mechanistic. The main purpose of a mechanistic model is to predict as accurately as possible a response to a given exposure and to provide a rational framework for extrapola- tions outside the range of experimental data and between species. The biologic basis of the mechanistic models implies that parameters associated with anat- omy, physiology, physical and chemical properties, biochemical interactions, and dynamic responses are constrained within biologic and physical limits; they are unlike purely empirical, statistical, or mathematical models that are fit to a given dataset. A key feature of a biologically motivated mechanistic model is that all relevant data must be reconciled with the model and that, if they cannot be, a reasonable explanation for disagreements must be articulated. Model predictions must also be reconciled with plausible outcomes, which serve as final con- straints on model structure and parameter estimates. Finally, and perhaps most important, the integrated model structure forces one to identify and articulate the greatest uncertainties and variability that would affect model outcome (that is, model sensitivity) and the focus of future research. That last feature is perhaps the greatest value of the model. Although several assumptions and parameter estimates were adjusted in the Conolly et al. (2003, 2004) models in a stated attempt to make the models more conservative (that is, to imply greater risk for a given exposure or to open the door to alternative modes of action), the main use of the models was to provide best estimates of risk that could then be ad- justed to incorporate uncertainties and variability in a health-protective manner. In the view of the models’ developers, the adjustments of some of the model parameter values were sufficiently conservative and plausible. EPA disagreed with the contention that the Conolly et al. models were conservative and sought to evaluate and identify parameters that the models were most sensitive to and sources of uncertainty in the data and the models.

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58 Review of EPA’s Draft IRIS Assessment of Formaldehyde The committee acknowledges and agrees with EPA that the Conolly et al. (2003, 2004) models, like most models, contain weaknesses and tenuously sup- ported assumptions. Conolly and co-workers felt that they made several conser- vative assumptions in their models— use of hockey-stick rather than J-shaped models for cell proliferation, use of overall respiratory tract cancer incidence in humans to calculate basal mutation rates, and use of an upper bound on the pro- portionality parameter relating DPX to mutation. EPA pushed that concept fur- ther by making even more conservative assumptions within the models that cu- mulatively resulted in radical departures from the results of the Conolly et al. models with regard to low-dose extrapolation of tumor incidence. The commit- tee notes that EPA forced changes in the model parameter values in a direction that yielded more conservative results rather than one that yielded a best fit to the data. The committee is also concerned that EPA directed substantial effort to- ward refuting many of the assumptions and conclusions of the Conolly et al. (2003, 2004) models rather than trying to fill the data gaps that were clearly ar- ticulated by the models. One of the major strengths of the Conolly et al. models is that the developers had to reconcile all the data or specify why they could not be reconciled. The integrated model structure allows one to identify uncertain- ties clearly and how they may affect model behavior. Conolly and co-workers were clear on that point and expressed the need for new data that could anchor many of the parameter values that had to be optimized from rather sparse data sets. Regardless, the committee recommends that for completeness and transpar- ency the BBDR models published by Conolly et al. (2003, 2004), with the flaw in one numeric approach identified by EPA corrected, be used in the draft IRIS assessment and that the results be compared with those of the approach that was used in the draft assessment. CONCLUSIONS AND RECOMMENDATIONS The draft IRIS assessment of formaldehyde provides an exhaustive discus- sion of formaldehyde toxicokinetics, carcinogenic modes of action, and various models. Although the committee agrees with much of the narrative, several is- sues need to be addressed in the revision of the draft assessment. First, there is broad agreement that formaldehyde is normally present in all tissues, cells, and bodily fluids and that natural occurrence complicates any formaldehyde risk assessment. Thus, an improved understanding of when exogenous formaldehyde exposure appreciably alters normal endogenous formaldehyde concentrations is needed. One approach that EPA could use would be to complete an analysis of variability and uncertainty in measuring and predicting target-tissue formalde- hyde concentrations among species. Only with such an analysis can one begin to identify and address openly and transparently the question of how much added risk for an endogenous compound is acceptable.

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Toxicokinetics and Modes of Action of Formaldehyde 59 Second, inhaled formaldehyde, a highly reactive chemical, is absorbed primarily in the upper airways and remains predominantly in the respiratory epithelium. The weight of evidence indicates that formaldehyde probably does not appear in the blood as an intact molecule except at doses high enough to overwhelm the metabolic capability of the exposed tissue. The draft IRIS as- sessment presents divergent opinions regarding the systemic delivery of formal- dehyde that need to be resolved. Third, the committee agrees with EPA that formaldehyde is a genotoxic chemical and that it is therefore reasonable to conclude that formaldehyde may act through a mutagenic mode of action. However, cytotoxicity and compensatory cell proliferation also appear to play important roles in formaldehyde-induced nasal tumors. Although the draft IRIS assessment discusses that mode of action, it relies on the mutagenic mode of action to justify low-dose extrapolations. The commit- tee recommends that EPA provide alternative calculations that factor in nonlineari- ties associated with the cytotoxicity-compensatory cell proliferation mode of ac- tion and assess the strengths and weaknesses of each approach. Fourth, over the last decade, several models have been developed to help to evaluate the risks associated with formaldehyde exposure, and EPA exten- sively evaluated several of them. EPA did use the CFD models to derive human equivalent concentrations but restricted their application to the experimental range used in the animal studies and did not extrapolate to low exposures. The committee, however, recommends that the CFD models be used to extrapolate to low concentrations, that the results be included in the overall evaluation, and that EPA explain clearly its use of the CFD modeling approaches. Furthermore, EPA, on the basis of extreme alternative model scenarios, chose not to use the BBDR models developed by Conolly et al. (2003, 2004); however, the commit- tee questions the validity of some of these scenarios and recommends that the BBDR models developed by Conolly and co-workers be used (with the flaw in one numeric approach identified by EPA corrected), that the results be compared with those of the approach currently presented in the draft IRIS assessment, and that the strengths and weaknesses of each approach be discussed. Fifth, in rewriting the sections of the draft IRIS assessment that pertain to the topics reviewed in this chapter, EPA should consider the implications of the most recent work. References to older studies on DNA-adduct measurements may need to be reanalyzed in light of the most recent analytic techniques that achieved superior sensitivity (for example, Lu et al. 2010). In particular, the committee finds the recent study of Lu et al. (2010) to be highly informative and the first one to distinguish clearly between exogenous and endogenous formal- dehyde-induced DNA adducts. Although the study does not challenge the notion that DNA adducts play only a minor, if any, role in formaldehyde genotoxicity and carcinogenicity, compared with DNA-protein cross-links, it adds to the evi- dence of the inability of formaldehyde to reach distant sites. Likewise, the posi- tive study by Wang et al. (2009) is not adequately described in the draft IRIS assessment, nor is it clear to the committee why so much emphasis is placed on the study by Craft et al. (1987).

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Toxicokinetics and Modes of Action of Formaldehyde 61 Conolly, R.B., J.S. Kimbell, D.B. Janszen, and F.J. Miller. 2002. Dose response for for- maldehyde-induced cytotoxicity in the human respiratory tract. Regul. Toxicol. Pharmacol. 35(1):32-43. Conolly, R.B., J.S. Kimbell, D. Janszen, P.M. Schlosser, D. Kalisak, J. Preston, and F.J. Miller. 2003. Biologically motivated computational modeling of formaldehyde carcinogenicity in the F344 rat. Toxicol. Sci. 75(2):432-447. Conolly, R.B., J.S. Kimbell, D. Janszen, P.M. Schlosser, D. Kalisak, J. Preston, and F.J. Miller. 2004. Human respiratory tract cancer risks of inhaled formaldehyde: Dose-response predictions derived from biologically motivated computational modeling of a combined rodent and human dataset. Toxicol. Sci. 82(1):279- 296. Costa, S., P. Coelho, C. Costa, S. Silva, O. Mayan, L.S. Santos, J. Gaspar, and J.P. Teixeira. 2008. Genotoxic damage in pathology anatomy laboratory workers exposed to formaldehyde. Toxicology 252(1-3):40-48. Craft, T.R., E. Bermudez, and T.R. Skopek. 1987. Formaldehyde mutagenesis and forma- tion of DNA–protein crosslinks in human lymphoblasts in vitro. Mutat. Res. 176(1):147-155. Crump, K.S., R.P. Subramaniam, and C.B. Van Landingham. 2005. A numerical solution to the nonhomogeneous two-stage MVK model of cancer. Risk Anal. 25(4):921-926. Crump, K.S., C. Chen, J.F. Fox, C. Van Landingham, and R. Subramaniam. 2008. Sensi- tivity analysis of biologically motivated model for formaldehyde-induced respi- ratory cancer in humans. Ann. Occup. Hyg. 52(6):481-495. Crump, K.S., C. Chen, W.A. Chiu, T.A. Louis, C.J. Portier, R.P. Subramaniam, and P.D. White. 2010. What role for biologically based dose-response models in estimat- ing low-dose risk? Environ. Health Perspect. 118(5):585-588. Egle, Jr., J.L. 1972. Retention of inhaled formaldehyde, propionaldehyde, and acrolein in the dog. Arch. Environ. Health. 25(2):119-124. EPA (U.S. Environmental Protection Agency). 1991. Formaldehyde Risk Assessment Update, Final Draft. Office of Toxic Substances, U.S. Environmental Protec- tion Agency, Washington, DC. June 11, 1991. EPA (U.S. Environmental Protection Agency). 2005. Guidelines for Carcinogen Risk As- sessment. EPA/630/P-03/001F. Risk Assessment Forum, U.S. Environmental Protection Agency, Washington, DC. March 2005 [online]. Available: http://www.epa.gov/raf/publications/pdfs/CANCER_GUIDELINES_FINAL_3-2 5-05.PDF [accessed Nov. 23, 2010]. EPA (U.S. Environmental Protection Agency). 2010. Toxicological Review of Formal- dehyde (CAS No. 50-00-0) – Inhalation Assessment: In Support of Summary Information on the Integrated Risk Information System (IRIS). External Re- view Draft. EPA/635/R-10/002A. U.S. Environmental Protection Agency, Washington, DC [online]. Available: http://cfpub.epa.gov/ncea/iris_drafts/recor display.cfm?deid=223614 [accessed Nov. 22, 2010]. Franks, S.J. 2005. A mathematical model for the absorption and metabolism of formalde- hyde vapour by humans. Toxicol. Appl. Pharmacol. 206(3):309-320. Garcia, G.J., J.D. Schroeter, R.A. Segal, J. Stanek, G.L. Foureman, and J.S. Kimbell. 2009. Dosimetry of nasal uptake of water-soluble and reactive gases: A first study of interhuman variability. Inhal. Toxicol. 21(7):607-618. Georgieva, A.V., J.S. Kimbell, and P.M. Schlosser. 2003. A distributed parameter model for formaldehyde uptake and disposition in the rat nasal lining. Inhal. Toxicol. 15(14):1435-1463.

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