Human respiratory tract cancer risks of inhaled formaldehyde: dose-response predictions derived from biologically-motivated computational modeling of a combined rodent and human dataset.

Formaldehyde inhalation at 6 ppm and above causes nasal squamous cell carcinoma (SCC) in F344 rats. The quantitative implications of the rat tumors for human cancer risk are of interest, since epidemiological studies have provided only equivocal evidence that formaldehyde is a human carcinogen. Conolly et al. (Toxicol. Sci. 75, 432-447, 2003) analyzed the rat tumor dose-response assuming that both DNA-reactive and cytotoxic effects of formaldehyde contribute to SCC development. The key elements of their approach were: (1) use of a three-dimensional computer reconstruction of the rat nasal passages and computational fluid dynamics (CFD) modeling to predict regional dosimetry of formaldehyde; (2) association of the flux of formaldehyde into the nasal mucosa, as predicted by the CFD model, with formation of DNA-protein cross-links (DPX) and with cytolethality/regenerative cellular proliferation (CRCP); and (3) use of a two-stage clonal growth model to link DPX and CRCP with tumor formation. With this structure, the prediction of the tumor dose response was extremely sensitive to cell kinetics. The raw dose-response data for CRCP are J-shaped, and use of these data led to a predicted J-shaped dose response for tumors, notwithstanding a concurrent low-dose-linear, directly mutagenic effect of formaldehyde mediated by DPX. In the present work the modeling approach used by Conolly et al. (ibid.) was extended to humans. Regional dosimetry predictions for the entire respiratory tract were obtained by merging a three-dimensional CFD model for the human nose with a one-dimensional typical path model for the lower respiratory tract. In other respects, the human model was structurally identical to the rat model. The predicted human dose response for DPX was obtained by scale-up of a computational model for DPX calibrated against rat and rhesus monkey data. The rat dose response for CRCP was used "as is" for the human model, since no preferable alternative was identified. Three sets of baseline parameter values for the human clonal growth model were obtained through separate calibrations against respiratory tract cancer incidence data for nonsmokers, smokers, and a mixed population of nonsmokers and smokers, respectively. Additional risks of respiratory tract cancer were predicted to be negative up to about one ppm for all three cases when the raw CRCP data from the rat were used. When a hockey-stick-shaped model was fit to the rat CRCP data and used in place of the raw data, positive maximum likelihood estimates (MLE) of additional risk were obtained. These MLE estimates were lower, for some comparisons by as much as 1,000-fold, than MLE estimates from previous cancer dose-response assessments for formaldehyde. Breathing rate variations associated with different physical activity levels did not make large changes in predicted additional risks. In summary, this analysis of the human implications of the rat SCC data indicates that (1) cancer risks associated with inhaled formaldehyde are de minimis (10(-6) or less) at relevant human exposure levels, and (2) protection from the noncancer effects of formaldehyde should be sufficient to protect from its potential carcinogenic effects.     

Dose response for formaldehyde-induced cytotoxicity in the human respiratory tract

Human studies of the sensory irritant effects of formaldehyde are complicated by the subjective nature of some clinical endpoints. This limits the usefulness of such studies for quantitative noncancer risk assessment of airborne formaldehyde. Objective measures of the noncancer effects of formaldehyde, such as the rate of regenerative cellular proliferation (RCP) secondary to cytolethality, can be obtained from laboratory animals but present the challenge of interspecies extrapolation of the data. To the extent that uncertainties associated with this extrapolation can be reduced, however, dose-response data obtained in laboratory animals are a viable alternative to clinical studies. Here, we describe the extrapolation of dose-response data for RCP from F344 rats to humans. Rats inhaled formaldehyde (0, 0.7, 2.0, 6.0, 10, and 15 ppm) 6 h/day, 5 days/week for up to 2 years. The dose response for RCP was J-shaped, with the rates of RCP at 0.7 and 2.0 ppm below but not statistically different from control, while the rates at the higher concentrations were significantly greater than control. Both the raw J-shaped data and a hockey-stick-shaped curve fitted to the raw data were used for predicting the human dose response for RCP. Cells lining the nasal airways of F344 rats and rhesus monkeys are comparably sensitive to the cytolethal effects of inhaled formaldehyde, suggesting that the equivalent human cells are also likely to be comparably sensitive. Using this assumption, the challenge of rat-to-human extrapolation was reduced to accurate prediction of site-specific flux of formaldehyde from inhaled air into the tissue lining the human respiratory tract. A computational fluid dynamics model of air flow and gas transport in the human nasal airways was linked to a typical path model of the human lung to provide site-specific flux predictions throughout the respiratory tract. Since breathing rate affects formaldehyde dosimetry, cytotoxicity dose-response curves were predicted for three standard working levels. With the most vigorous working level, the lowest concentrations of formaldehyde predicted to exert any cytotoxic effects in humans were 1.0 and 0.6 ppm, for the J-shaped and hockey-stick-shaped RCP curves, respectively. The predicted levels of response at the lowest effect concentrations are smaller than can be measured clinically. Published literature showing that the cytotoxicity of inhaled formaldehyde is related to exposure level rather than to duration of exposure suggests that the present analysis is a reasonable basis for derivation of standards for continuous human exposure.     

A distributed-parameter model for formaldehyde uptake and disposition in the rat nasal lining

DNA-protein cross-links (DPX) serve as a dosimeter for inhaled formaldehyde and are associated with tumor induction in rat nasal passages after chronic exposure to 6 ppm and above. To determine the role of epithelium-specific morphometry in formaldehyde-induced patterns of injury, we developed a mathematical model that links airflow-driven formaldehyde uptake with DPX formation in regions of the rat nose with high and low tumor incidence. A three-dimensional, anatomically accurate computational fluid dynamics model of rat nasal airflow and inhaled gas uptake was integrated with a physiologically based mathematical model incorporating tissue thickness, formaldehyde diffusion, its removal by enzymatic and nonenzymatic processes, and DNA distribution in the nasal mucosa to predict DPX formation. The model implicitly incorporates the reversible conversion of formaldehyde to methylene glycol. Where possible, parameter values were taken from the literature or estimated using published correlations. The Michaelis-Menten kinetic constants Vmax and Km, as well as a first-order constant for formaldehyde removal, were left as fitted parameters. The resultant model fit to the experimentally measured DPX in the high- and low-tumor-incidence regions of the rat nasal passages was very good. Sensitivity analysis indicates that among the fitted parameters, model fits are most sensitive to Vmax and that predictions were sensitive to changes in tissue thickness when all other parameters are held constant. The model structure facilitates extrapolation to primates and humans and application to other soluble, reactive gases.     

A Distributed-Parameter Model for Formaldehyde Uptake and Disposition in the Rat Nasal Lining

DNA–protein cross-links (DPX) serve as a dosimeter for inhaled formaldehyde and are associated with tumor induction in rat nasal passages after chronic exposure to 6 ppm and above. To determine the role of epithelium-specific morphometry in formaldehyde-induced patterns of injury, we developed a mathematical model that links airflow-driven formaldehyde uptake with DPX formation in regions of the rat nose with high and low tumor incidence. A three-dimensional, anatomically accurate computational fluid dynamics model of rat nasal airflow and inhaled gas uptake was integrated with a physiologically based mathematical model incorporating tissue thickness, formaldehyde diffusion, its removal by enzymatic and nonenzymatic processes, and DNA distribution in the nasal mucosa to predict DPX formation. The model implicitly incorporates the reversible conversion of formaldehyde to methylene glycol. Where possible, parameter values were taken from the literature or estimated using published correlations. The Michaelis–Menten kinetic constants V_max and K_m, as well as a first-order constant for formaldehyde removal, were left as fitted parameters. The resultant model fit to the experimentally measured DPX in the high- and low-tumor-incidence regions of the rat nasal passages was very good. Sensitivity analysis indicates that among the fitted parameters, model fits are most sensitive to V_max and that predictions were sensitive to changes in tissue thickness when all other parameters are held constant. The model structure facilitates extrapolation to primates and humans and application to other soluble, reactive gases.     

Dosimetry modeling of inhaled formaldehyde: comparisons of local flux predictions in the rat, monkey, and human nasal passages.

Formaldehyde-induced nasal squamous cell carcinomas in rats and squamous metaplasia in rats and rhesus monkeys occur in specific regions of the nose with species-specific distribution patterns. Experimental approaches addressing local differences in formaldehyde uptake patterns and dose are limited by the resolution of dissection techniques used to obtain tissue samples and the rapid metabolism of absorbed formaldehyde in the nasal mucosa. Anatomically accurate, 3-dimensional computational fluid dynamics models of F344 rat, rhesus monkey, and human nasal passages were used to estimate and compare regional inhaled formaldehyde uptake patterns predicted among these species. Maximum flux values, averaged over a breath, in nonsquamous epithelium were estimated to be 2620, 4492, and 2082 pmol/(mm(2)-h-ppm) in the rat, monkey, and human respectively. Flux values predicted in sites where cell proliferation rates were measured as similar in rats and monkeys were also similar, as were fluxes predicted in a region of high tumor incidence in the rat nose and the anterior portion of the human nose. Regional formaldehyde flux estimates are directly applicable to clonal growth modeling of formaldehyde carcinogenesis to help reduce uncertainty in human cancer risk estimates.     

A Parallelogram Approach for Safety Evaluation of Ingested Acetaldehyde

Route-specific carcinogenicity data are often lacking for compounds of regulatory importance. Acetaldehyde (AA), for example, a natural constituent in foods, is a rodent carcinogen via the inhalation route, but oral carcinogenicity data are not available. In the absence of such data, a parallelogram approach can be used to estimate the oral carcinogenic potency of this chemical. The relative potency of AA to the structurally related compound formaldehyde (FA) in the nose and the relative potency of FA in the nose and stomach serve as the basis for estimating the potency of AA in the stomach. On a dosimetric basis, inhaled AA is 14- to 35-fold less potent than FA in producing nasal DNA–protein crosslinks (DPX), subchronic tissue injury, and tumors. Ingested AA is also considerably (∼5-fold) less potent than FA in producing gastric injury and DPX. Compared to the nose, the stomach is 10- to 60-fold less sensitive to both AA- and FA-induced DPX and subchronic tissue injury. The parallelogram approach will not supplant long-term oral carcinogenicity studies with AA; however, the consistent pattern of decreased sensitivity of acetaldehyde compared to formaldehyde, lower sensitivity of the stomach to the nose, and the lack of gastric tumorigenicity of orally ingested formaldehyde strongly suggests that ingested acetaldehyde is not likely to be carcinogenic. Successful estimation of the carcinogenic potency of ingested glutaraldehyde, for which chronic oral and inhalation data are available, provides further support that the parallelogram approach can provide a reasonable estimate of the carcinogenic potency of closely related aldehydes, such as AA.     

The implausibility of leukemia induction by formaldehyde: a critical review of the biological evidence on distant-site toxicity

Formaldehyde is a naturally occurring biological compound that is present in tissues, cells, and bodily fluids. It is also a potent nasal irritant, a cytotoxicant at high doses, and a nasal carcinogen in rats exposed to high airborne concentrations. The normal endogenous concentration of formaldehyde in the blood is approximately 0.1 mM in rats, monkeys, and humans, and it is 2- to 4-fold higher in the liver and nasal mucosa of the rat. Inhaled formaldehyde enters the one-carbon pool, and the carbon atom is rapidly incorporated into macromolecules throughout the body. Oxidation to formate catalyzed by glutathione-dependent and -independent dehydrogenases in nasal tissues is a major route of detoxication and generally precedes incorporation. The possibility that inhaled formaldehyde might induce various forms of distant-site toxicity has been proposed, but no convincing evidence for such toxicity has been obtained in experimental studies. This review summarizes the biological evidence that pertains to the issue of leukemia induction by formaldehyde, which includes: (1) the failure of inhaled formaldehyde to increase the formaldehyde concentration in the blood of rats, monkeys, or humans exposed to concentrations of 14.4, 6, or 1.9 ppm, respectively; (2) the lack of detectable protein adducts or DNA-protein cross-links (DPX) in the bone marrow of normal rats exposed to [3H]- and [14C]formaldehyde at concentrations as high as 15 ppm; (3) the lack of detectable protein adducts or DPX in the bone marrow of glutathione-depleted (metabolically inhibited) rats exposed to [3H]- and [14C]formaldehyde at concentrations as high as 10 ppm; (4) the lack of detectable DPX in the bone marrow of Rhesus monkeys exposed to [14C]formaldehyde at concentrations as high as 6 ppm; (5) the failure of formaldehyde to induce leukemia in any of seven long-term inhalation bioassays in rats, mice, or hamsters; and (6) the failure of formaldehyde to induce chromosomal aberrations in the bone marrow of rats exposed to airborne concentrations as high as 15 ppm or of mice injected intraperitoneally with formaldehyde at doses as high as 25 mg/kg. Biological evidence that might be regarded as supporting the possibility of leukemia induction by formaldehyde includes: (1) the detection of cytogenetic abnormalities in circulating lymphocytes in seven studies of human subjects exposed to ambient concentrations in the workplace (but not in seven other studies of human subjects or in rats exposed to 15 ppm); (2) the induction of leukemia in rats in a single questionable drinking water study with formaldehyde concentrations as high as 1.5 g/L (but not in three other drinking water studies with concentrations as high as 1.9 or 5 g/L); (3) the detection of chromosomal aberrations in the bone marrow of rats exposed to very low concentrations of formaldehyde (0.4 or 1.2 ppm) (but not in another study at concentrations as high as 15 ppm); and (4) an apparent increase in the fraction of protein-associated DNA (assumed to be due to DPX) in circulating lymphocytes of humans exposed to ambient concentrations in the workplace (1-3 ppm). This evidence is regarded as inconsequential for several reasons, including lack of reproducibility, inadequate reporting of experimental methods, inconsistency with other data, or insufficient analytical sensitivity, and therefore, it provides little justification for or against the possibility that inhaled formaldehyde may be a leukemogen. In contrast to these inconclusive findings, the abundance of negative evidence mentioned above is undisputed and strongly suggests that there is no delivery of inhaled formaldehyde to distant sites. Combined with the fact that formaldehyde naturally occurs throughout the body, and that multiple inhalation bioassays have not induced leukemia in animals, the negative findings provide convincing evidence that formaldehyde is not leukemogenic.     

USE OF COMPUTATIONAL FLUID DYNAMICS MODELS FOR DOSIMETRY OF INHALED GASES IN THE NASAL PASSAGES

Computational fluid dynamics (CFD) models of the nasal passages of a rat, monkey, and human are being used (1) to determine important factors affecting nasal uptake, (2) to make interspecies dosimetric comparisons, (3) to provide detailed anatomical information for the rat, monkey, and human nasal passages, and (4) to provide estimates of regional air-phase mass transport coefficients (a measure of the resistance to gas transport from inhaled air to airway walls) in the nasal passages of all three species. For many inhaled materials, lesion location in the nose follows patterns that are both site and species specific. For reactive, water-soluble (Category 1) gases, regional uptake can be a major factor in determining lesion location. Since direct measurement of airflow and uptake is experimentally difficult, CFD models are used here to predict uptake patterns quantitatively in three-dimensional reconstructions of the F344 rat, rhesus monkey, and human nasal passages. In formaldehyde uptake simulations, absorption processes were assumed to be as rapid as possible, and regional flux (transport rate) of inhaled formaldehyde to airway walls was calculated for rats, primates, and humans. For uptake of gases like vinyl acetate and acrylic acid vapors, physiologically based pharmacokinetic uptake models incorporating anatomical and physical information from the CFD models were developed to estimate nasal tissue dose in animals and humans. The use of biologically based models in risk assessment makes sources of uncertainty explicit and, in doing so, allows quantification of uncertainty through sensitivity analyses. Limited resources can then be focused on reduction of important sources of uncertainty to make risk estimates more accurate.     

Use of computational fluid dynamics models for dosimetry of inhaled gases in the nasal passages

Computational fluid dynamics (CFD) models of the nasal passages of a rat, monkey, and human are being used (1) to determine important factors affecting nasal uptake, (2) to make interspecies dosimetric comparisons, (3) to provide detailed anatomical information for the rat, monkey, and human nasal passages, and (4) to provide estimates of regional air-phase mass transport coefficients (a measure of the resistance to gas transport from inhaled air to airway walls) in the nasal passages of all three species. For many inhaled materials, lesion location in the nose follows patterns that are both site and species specific. For reactive, water-soluble (Category 1) gases, regional uptake can be a major factor in determining lesion location. Since direct measurement of airflow and uptake is experimentally difficult, CFD models are used here to predict uptake patterns quantitatively in three-dimensional reconstructions of the F344 rat, rhesus monkey, and human nasal passages. In formaldehyde uptake simulations, absorption processes were assumed to be as rapid as possible, and regional flux (transport rate) of inhaled formaldehyde to airway walls was calculated for rats, primates, and humans. For uptake of gases like vinyl acetate and acrylic acid vapors, physiologically based pharmacokinetic uptake models incorporating anatomical and physical information from the CFD models were developed to estimate nasal tissue dose in animals and humans. The use of biologically based models in risk assessment makes sources of uncertainty explicit and, in doing so, allows quantification of uncertainty through sensitivity analyses. Limited resources can then be focused on reduction of important sources of uncertainty to make risk estimates more accurate.     

The Importance of Delivered Dose in Estimating Low-Dose Cancer Risk from Inhalation Exposure to Formaldehyde

The Importance of Delivered Dose in Estimating Low-Dose CancerRisk from Inhalation Exposure to Formaldehyde. STARR, T. B.,AND BUCK, R. D. (1984). Fundam, AppL Toxicol. 4, 740–753.Data have recently been obtained on the concentration of formaldehydecovalently bound to the respiratory mucosal DNA of Fischer-344rats following two 6-hr inhalation exposures to gaseous formaldehyde.These data provide a direct short-term measure of the deliveredformaldehyde dose in target tissue as a function of the formaldehydeconcentration in ambient air. They also demonstrate that thedelivered dose/administered dose relationship is significantlynonlinear. Since chronic inhalation exposure of Fischer-344rats to high concentrations of gaseous formaldehyde inducessquamous cell carcinomas of the nasal cavity, and since widespreadconcern exists that formaldehyde exposure may also pose a cancerrisk for humans, the implications of this nonlinearity for low-doserisk extrapolation were investigated. The incidence of nasalsquamous cell carcinomas in a chronic formaldehyde inhalationbioassay was reanalyzed with several low-dose extrapolationmodels, using the estimated concentration of formaldehyde covalentlybound to respiratory mucosal DNA as the measure of exposure.For this purpose, it was assumed that the short-term observationsof covalent binding were representative of steady-state conditionsduring the course of the chronic study and further, that thecovalent binding of formaldehyde to target tissue DNA is animportant factor in nasal tumor induction. Resulting maximumlikelihood risk estimates and upper 95% confidence bounds wereunilaterally lower than the corresponding risk measures basedon administered dose, irrespective of the dose-response modelemployed. Reductions in estimated risk ranged from a factorof 2.5, for the multistage model upper 95% confidence bound,to over 10 orders of magnitude, for the probit model upper 95%confidence bound. These results indicate that the concept ofdelivered dose can have a significant impact on estimates oflow-dose risk and should therefore at least be considered asan alternative dose measure in assessments of human cancer riskfrom formaldehyde exposure     

DNA--Protein Cross-links and Cell Replication at Specific Sites in the Nose of F344 Rats Exposed Subchronically to Formaldehyde

Chronic exposures to high concentrations (>6 ppm) of formaldehyde(HCHO) induce cell proliferation, squamous metaplasia, and squamouscell carcinomas in F344 rats. To assess the cancer risk associatedwith HCHO exposure, DNA-protein cross-links (DPX) formed ina single exposure of naive (previously unexposed) rats and monkeyshave been used as a surrogate for the internal dose. Since thequantity of DPX may differ in subchronically exposed animals,the effects of preexposure to HCHO on the acute DPX yield (concentrationof DPX following a single exposure) and the cumulative DPX yield(concentration of DPX following repeated exposures) were determined.Male F344 rats were preexposed (PE) to 0.7, 2, 6, or 15 ppmof HCHO (6 hr/day, 5 days/week, 11 weeks + 4 days). Naive (N)rats were exposed to room air. On the 5th day of the 12th week,PE and N rats were simultaneously exposed (3 hr) to H¹⁴CHO atthe same concentrations used for preexposure. Acute DPX yieldsand cell replication (incorporation of ¹⁴C into DNA) were determinedin the mucosal lining of the nasal lateral meatus (LM) (hightumor site in HCHO bioassay) and the medial and posterior meatuses(M:PM) (low tumor site in bioassay). DPX yields in the LM wereapproximately sixfold higher than in the M:PM. At 0.7 and 2ppm, no differences between PE and N rats were detected in eithertissue. At 6 and 15 ppm, acute DPX yields in the LM of PE ratswere approximately half those of N rats, but no differenceswere detected in the M:PM. Cell proliferation was induced inPE rats at 6 ppm (LM only) and especially at 15 ppm (LM andM:PM). Cumulative DPX yields were measured indirectly by determiningthe decrease in extractability of DNA from proteins. PE ratswere preexposed to 6 or 10 ppm as above, while N rats were exposedto room air. Both groups (PE and N) were then exposed (3 hr)to the same concentration of unlabeled HCHO. DPX yields increasedin a concentration-dependent manner in both groups, but theyields were smaller in PE than N rats, suggesting that no accumulationof DPX occurred in PE rats. The results demonstrate that atconcentrations <2 ppm, N and PE rats are equivalent withrespect to the formation of DPX. At concentrations >6 ppm,N and PE rats are not equivalent, but the impact of this high-doseeffect on low-dose cancer risk estimates derived with the linearizedmultistage model is small.     

Deposition of inhaled nanoparticles in the rat nasal passages: Dose to the olfactory region

In vivo experiments have shown that nanoparticles depositing in the rat olfactory region can translocate to the brain via the olfactory nerve. Quantitative predictions of the dose delivered by inhalation to the olfactory region are needed to clarify this route of exposure and to evaluate the dose-response effects of exposure to toxic nanoparticles. Previous in vivo and in vitro studies quantified the percentage of inhaled nanoparticles that deposit in the rat nasal passages, but olfactory dose was not determined. The dose to specific nasal epithelium types is expected to vary with inhalation rate and particle size. The purpose of this investigation, therefore, was to develop estimates of nanoparticle deposition in the nasal and, more specifically, olfactory regions of the rat. A three-dimensional, anatomically accurate, computational fluid dynamics (CFD) model of the rat nasal passages was employed to simulate inhaled airflow and to calculate nasal deposition efficiency. Particle sizes from 1 to 100?nm and airflow rates of 288, 432, and 576 ml/min (1, 1.5, and 2 times the estimated resting minute volume) were simulated. The simulations predicted that olfactory deposition is maximum at 6–9% of inhaled material for 3- to 4-nm particles. The spatial distribution of deposited particles was predicted to change significantly with particle size, with 3-nm particles depositing mostly in the anterior nose, while 30-nm particles were more uniformly distributed throughout the nasal passages.     

Statistical analysis of nonmonotonic dose-response relationships: research design and analysis of nasal cell proliferation in rats exposed to formaldehyde.

Statistical analyses of nonmonotonic dose-response curves are proposed, experimental designs to detect low-dose effects of J-shaped curves are suggested, and sample sizes are provided. For quantal data such as cancer incidence rates, much larger numbers of animals are required than for continuous data such as biomarker measurements. For example, 155 animals per dose group are required to have at least an 80% chance of detecting a decrease from a 20% incidence in controls to an incidence of 10% at a low dose. For a continuous measurement, only 14 animals per group are required to have at least an 80% chance of detecting a change of the mean by one standard deviation of the control group. Experimental designs based on three dose groups plus controls are discussed to detect nonmonotonicity or to estimate the zero equivalent dose (ZED), i.e., the dose that produces a response equal to the average response in the controls. Cell proliferation data in the nasal respiratory epithelium of rats exposed to formaldehyde by inhalation are used to illustrate the statistical procedures. Statistically significant departures from a monotonic dose response were obtained for time-weighted average labeling indices with an estimated ZED at a formaldehyde dose of 5.4 ppm, with a lower 95% confidence limit of 2.7 ppm. It is concluded that demonstration of a statistically significant bi-phasic dose-response curve, together with estimation of the resulting ZED, could serve as a point-of departure in establishing a reference dose for low-dose risk assessment.     

Depletion of nasal mucosal glutathione by acrolein and enhancement of formaldehyde-induced DNA-protein cross-linking by simultaneous exposure to acrolein

Incubation of homogenates of rat nasal mucosa with acrolein resulted in the apparent formation of DNA-protein cross-links. However, inhalation exposure of male Fischer-344 rats to acrolein (2.0 ppm, 6 h) did not cause detectable DNA-protein cross-linking in the nasal respiratory mucosa. Simultaneous exposure of rats to both acrolein (2.0 ppm) and formaldehyde (6.0 ppm) for 6 h resulted in a significantly higher yield of DNA-protein cross-links than was obtained following exposure to formaldehyde (6.0 ppm) alone. Acrolein exposure at concentrations of 0.1, 0.5, 1.0, or 2.5 ppm resulted in a concentration-dependent depletion of nonprotein sulfhydryl groups in the nasal respiratory mucosa. A plausible explanation for the enhancement of DNA-protein cross-links by simultaneous exposure to formaldehyde and acrolein may be that depletion of glutathione by acrolein inhibited the oxidative metabolism of formaldehyde, leading to an increase of formaldehyde-induced DNA-protein cross-links.     

Evaluation of effects from repeated inhalation exposure of F344 rats to high concentrations of propylene.

Chronic exposure to propylene does not result in any increased incidence of tumors, yet does increase N7-hydroxypropylguanine (N7-HPGua) adducts in tissue DNA. To investigate any potential for genotoxicity (mutagenicity or clastogenicity), male F344 rats were exposed via inhalation to up to 10,000 ppm propylene for 1, 3, or 20 days (6 h/day, 5 days/week). The endpoints examined included gene (Hprt, splenocytes) and chromosomal (bone marrow micronucleus [MN]) mutations, hemoglobin (hydroxypropylvaline, HPVal) adducts in systemic blood, and DNA adducts (N7-HPGua) in several tissues. Similarly exposed female and male F344 rats, implanted with bromodeoxyuridine (BrdU) minipumps, were evaluated for nasal effects (irritation via histopathology and cell proliferation via BrdU). Internal dose measures provided clear evidence for propylene exposure, with HPVal increased for all exposures; N7-HPGua was increased in all tissues from rats exposed for more than 1 day (except lymphocytes). Saturation of propylene conversion to propylene oxide was apparent from the adduct dose-response curves. There were no biologically significant genotoxic effects demonstrated at any exposure level, with no increase in Hprt mutant frequency or in bone marrow MN formation. In addition, no histopathological changes were noted in rodent nasal tissues nor any induction of cell proliferation in nasal tissues. These results demonstrate that repeated exposure of rats to high concentrations of propylene (< or = 10,000 ppm) does not produce evidence of local nasal cavity toxicity or evidence of systemic genotoxicity to hematopoietic tissue, despite the formation of N7-HPGua adducts. In addition, these data indicate that formation of N7-HPGua does not correlate with any measure of genotoxic effect, neither mutagenic nor clastogenic.     

Application of physiological computational fluid dynamics models to predict interspecies nasal dosimetry of inhaled acrolein

Acrolein is a highly soluble and reactive aldehyde and is a potent upper-respiratory-tract irritant. Acrolein-induced nasal lesions in rodents include olfactory epithelial atrophy and inflammation, epithelial hyperplasia, and squamous metaplasia of the respiratory epithelium. Nasal uptake of inhaled acrolein in rats is moderate to high, and depends on inspiratory flow rate, exposure duration, and concentration. In this study, anatomically accurate three-dimensional computational fluid dynamics (CFD) models were used to simulate steady-state inspiratory airflow and to quantitatively predict acrolein tissue dose in rat and human nasal passages. A multilayered epithelial structure was included in the CFD models to incorporate clearance of inhaled acrolein by diffusion, blood flow, and first-order and saturable metabolic pathways. Kinetic parameters for these pathways were initially estimated by fitting a pharmacokinetic model with a similar epithelial structure to time-averaged acrolein nasal extraction data and were then further adjusted using the CFD model. Predicted air:tissue flux from the rat nasal CFD model compared well with the distribution of acrolein-induced nasal lesions from a subchronic acrolein inhalation study. These correlations were used to estimate a tissue dose-based no-observed-adverse-effect level (NOAEL) for inhaled acrolein. A human nasal CFD model was used to extrapolate effects in laboratory animals to human exposure conditions on the basis of localized tissue dose and tissue responses. Assuming that equivalent tissue dose will induce similar effects across species, a NOAEL human equivalent concentration for inhaled acrolein was estimated to be 8 ppb.     

Regional increases in rat nasal epithelial cell proliferation following acute and subchronic inhalation of formaldehyde.

Short-term studies (9 days) in the rat have demonstrated that formaldehyde-induced nasal epithelial lesions are associated with increases in surface epithelial cell proliferation rates. The present studies were designed, in part, to investigate cell proliferation rates in the nasal epithelium of rats exposed to formaldehyde for a longer duration in order to determine if correlations exist between (1) the concentration-response in cell proliferation rate with the previously published formaldehyde bioassay tumor response; (2) sites of increased cell proliferation and the regions of the nasal passages that exhibit formaldehyde-induced cytotoxicity; and (3) sites of increased cell proliferation and the regions of the rat nasal passages previously determined to be most susceptible to neoplasia (i.e., the lateral meatus and nasal septum of the anterior nasal passages). Another important endpoint of this study was to provide data for a comparison of formaldehyde-induced responses in rats with previous findings in rhesus monkeys. Fischer-344 rats were exposed to 0, 0.7, 2, 6, 10, or 15 ppm formaldehyde for up to 6 weeks and pulse labeled with tritiated thymidine prior to each scheduled termination. Exposure to formaldehyde at 6 ppm or higher induced site-specific lesions in the nasal respiratory epithelium and was associated with increases in cell proliferation rate which remained statistically elevated throughout the 6 weeks. While a direct correlation between sites susceptible to formaldehyde-induced nasal cancer and increased cell proliferation was not evident, results from the present studies did demonstrate a clear correlation between sites of cellular injury and increases in cell proliferation and a concentration-dependent response which correlated with the previously published formaldehyde bioassay tumor response. Furthermore, this work demonstrated that formaldehyde-induced responses in rats exposed to 6 ppm were morphologically similar to those reported in the rhesus monkey; however, the distribution of lesions between the two species differed significantly.     

Do rats comply with EPA policy on cancer risk assessment for formaldehyde?

Formaldehyde has been shown to cause nasal squamous cell carcinomas in the rat following 2-year inhalation exposure. The incidence of this tumor in a historical data base of 16,794 rats was nil, indicating that it is a rare spontaneous tumor. Five different mathematical extrapolation models were applied to the rat nasal tumor data to produce estimates at 10(-4) risk (the size of the historical data base) of between 3.232 and 0.003 ppm formaldehyde depending on the model and choice of maximum likelihood estimate or lower confidence limit values. Assuming that an ambient level of 0.07 ppm formaldehyde exists in a rat house, the multistage linear model did not predict correctly within the observed data. The EPA policy model (multistage third order) was not inconsistent with the observed data (P = 0.259). However, the unit risk, derived from this form of modeling, shows considerable inconsistency, at ambient levels of formaldehyde, when compared to observed incidences of nasal tumors in the general human population. It is proposed that the multistage models are inappropriate, and that caution should be exercised in the extrapolation of highly nonlinear animal tumor data.     

Formaldehyde and Glutaraldehyde and Nasal Cytotoxicity: Case Study Within the Context of the 2006 IPCS Human Framework for the Analysis of a Cancer Mode of Action for Humans

Formaldehyde and glutaraldehyde cause toxicity to the nasal epithelium of rats and mice upon inhalation. In addition, formaldehyde above certain concentrations induces dose-related increases in nasal tumors in rats and mice, but glutaraldehyde does not. Using the 2006 IPCS human framework for the analysis of cancer mode of action (MOA), an MOA for formaldehyde was formulated and its relevance was tested against the properties of the noncarcinogenic glutaraldehyde. These compounds produce similar patterns of response in histopathology and in genotoxicity tests (although formaldehyde has been much more extensively tested studied). The MOA is based on the induction of sustained cytotoxicity and reparative cell proliferation induced by formaldehyde at concentrations that also induce nasal tumors upon long-term exposure. Data on dose dependency and temporal relationships of key events are consistent with this MOA. While a genotoxic MOA can never be ruled out for a compound that is clearly genotoxic, at least in vitro, the nongenotoxic properties fundamental to the proposed MOA can explain the neoplastic response in the nose and may be more informative than genotoxicity in risk assessment. It is not yet fully explained why glutaraldehyde remains noncarcinogenic upon inhalation, but its greater inherent toxicity may be a key factor. The dual aldehyde functions in glutaraldehyde are likely to produce damage resulting in fewer kinetic possibilities (particularly for proteins involved in differentiation control) and lower potential for repair (nucleic acids) than would be the case for formaldehyde. While there have been few studies of possible glutaraldehyde-associated cancer, the evidence that formaldehyde is a human carcinogen is strong for nasopharyngeal cancers, although less so for sinonasal cancers. This apparent discrepancy could be due in part to the classification of human nasal tumors with tumors of the sinuses, which would receive much less exposure to inhaled formaldehyde. Evaluation of the human relevance of the proposed MOA of formaldehyde in rodents is restricted by human data limitations, although the key events are plausible. It is clear that the human relevance of the formaldehyde MOA in rodents cannot be excluded on either kinetic or dynamic grounds.     

Formaldehyde and glutaraldehyde and nasal cytotoxicity: case study within the context of the 2006 IPCS Human Framework for the Analysis of a cancer mode of action for humans

Formaldehyde and glutaraldehyde cause toxicity to the nasal epithelium of rats and mice upon inhalation. In addition, formaldehyde above certain concentrations induces dose-related increases in nasal tumors in rats and mice, but glutaraldehyde does not. Using the 2006 IPCS human framework for the analysis of cancer mode of action (MOA), an MOA for formaldehyde was formulated and its relevance was tested against the properties of the noncarcinogenic glutaraldehyde. These compounds produce similar patterns of response in histopathology and in genotoxicity tests (although formaldehyde has been much more extensively tested studied). The MOA is based on the induction of sustained cytotoxicity and reparative cell proliferation induced by formaldehyde at concentrations that also induce nasal tumors upon long-term exposure. Data on dose dependency and temporal relationships of key events are consistent with this MOA. While a genotoxic MOA can never be ruled out for a compound that is clearly genotoxic, at least in vitro, the nongenotoxic properties fundamental to the proposed MOA can explain the neoplastic response in the nose and may be more informative than genotoxicity in risk assessment. It is not yet fully explained why glutaraldehyde remains noncarcinogenic upon inhalation, but its greater inherent toxicity may be a key factor. The dual aldehyde functions in glutaraldehyde are likely to produce damage resulting in fewer kinetic possibilities (particularly for proteins involved in differentiation control) and lower potential for repair (nucleic acids) than would be the case for formaldehyde. While there have been few studies of possible glutaraldehyde-associated cancer, the evidence that formaldehyde is a human carcinogen is strong for nasopharyngeal cancers, although less so for sinonasal cancers. This apparent discrepancy could be due in part to the classification of human nasal tumors with tumors of the sinuses, which would receive much less exposure to inhaled formaldehyde. Evaluation of the human relevance of the proposed MOA of formaldehyde in rodents is restricted by human data limitations, although the key events are plausible. It is clear that the human relevance of the formaldehyde MOA in rodents cannot be excluded on either kinetic or dynamic grounds.