Mode of action and dose-response framework analysis for receptor-mediated toxicity: The aryl hydrocarbon receptor as a case study.

Dioxins and dioxin-like compounds are tumor promoters that cause liver cancer in rats and mice. The aryl hydrocarbon receptor (AHR) has been implicated as a key component in this tumor promotion response. Despite extensive knowledge of the toxicology of dioxins, no mode of action (MOA) hypothesis for their tumorigenicity has been formally documented using the Human Relevance MOA framework developed by the International Programme on Chemical Safety (IPCS). To address this information gap, an expert panel was convened as part of a workshop on receptor-mediated liver tumorigenicity. Liver tumors induced by ligands of the AHR were assessed using data for dioxins and related chemicals as a case study. The panel proposed a MOA beginning with sustained AHR activation, eventually leading to liver tumors via a number of other processes, including increased cell proliferation of previously initiated altered hepatic foci, inhibition of intrafocal apoptosis and proliferation of oval cells. These processes have been identified and grouped as three key events within the hepatocarcinogenic MOA: (1) sustained AHR activation, (2) alterations in cellular growth and homeostasis and (3) pre-neoplastic tissue changes. These key events were identified through application of the Bradford-Hill considerations in terms of both their necessity for the apical event/adverse outcome and their human relevance. The panel identified data supporting the identification and dose-response behavior of key events, alteration of the dose-response by numerous modulating factors and data gaps that potentially impact the MOA. The current effort of applying the systematic frameworks for identifying key events and assessing human relevance to the AHR activation in the tumorigenicity of dioxins and related chemicals is novel at this time. The results should help direct future regulatory efforts and research activities aimed at better understanding the potential human cancer risks associated with dioxin exposure.

Correlation of regional and nonlinear formaldehyde-induced nasal cancer with proliferating populations of cells.

Formaldehyde induces nonlinear, concentration-related increases in nasal epithelial cell proliferation and squamous cell carcinomas (SCC) in rats. A formaldehyde carcinogenicity study was conducted in which a major end point was correlation of cell proliferation indices with sites of formaldehyde-induced SCC. A poor correlation in certain sites led to incorporation of the number of cells in each site into the correlation. Rats were exposed (6h/day, 5 days/week) to formaldehyde (0, 0.7, 2, 6, 10 or 15 ppm) for up to 24 months with interim sacrifice time points at 3, 6, 12, and 18 mo. A unit length labeling index (ULLI; S-phase nuclei/mm basement membrane) was determined for specific nasal regions in addition to a population-weighted ULLI (PWULLI). The PWULLI was defined as the product of regional ULLI and total number of nasal epithelial cells in the respective site. Nasal SCC sites of origin were mapped. Formaldehyde induced SCC in a highly nonlinear fashion, with no observed effect at the level of 2 ppm, a minimal response at 6 ppm, and a sharp increase at 10 and 15 ppm. The tumor incidence was 1, 22, and 47% at 6, 10 and 15 ppm, respectively. ULLI was significantly (P<0.05) increased at 10 and 15 ppm but not at the lower concentrations. There was a good correlation between PWULLI and regional tumor incidence (R(2) = 0.88), while the correlation of regional SCC with ULLI was relatively poor (R(2) = 0.46). We conclude that target cell population size and sustained increases of cell proliferation in these populations, determined by differences in regional airflow-driven formaldehyde binding to DNA dose to these sites, coupled with the known nonlinear kinetics of formaldehyde binding to DNA, can together account for the nonlinearity and site specificity of formaldehyde-induced nasal SCC in rats.

Opportunities for improving techniques for interspecies extrapolation in the risk assessment process.

Quantitative estimates of human carcinogenic risk from chemical exposure are currently derived primarily from linearized multistage model analyses of the tumor response as observed in chronic laboratory animal bioassays versus administered dose. The numerous ad hoc assumptions that provide a rationale for this generic approach to carcinogenic risk assessment can only be evaluated critically when mechanistic data directly relevant to the low-dose and interspecies extrapolation problems are available. Clear needs exist to develop such ancillary data bases and the means for explicitly incorporating them into the risk estimation process. Target site dosimetry provides one useful organizing concept. Physiological response modeling can account systematically for interspecies variations in the distribution and disposition of chemicals in relation to external measures of exposure. Direct measurements of interactions of chemicals and their metabolites with specific target macromolecules can provide sensitive and biologically meaningful exposure indices. Alternatively, quantitation of toxic effects such as altered cell regulation and differentiation can serve the same purpose. Virus and oncogene activation, DNA damage and repair, and enhanced cell proliferation provide additional biological markers of exposure. They may also comprise critical elements of the carcinogenic process. Identification of the actual mechanisms involved should eventually lead to the development of risk assessment models that adequately reflect the unique biological and toxicological characteristics of different species-chemical combinations.

The trouble with TEFs.

Comments on Van den Berg, et al. Toxic equivalency factors (TEFs) for PCBs, PCDDs, PCDFs for humans and wildlife. Environ Health Perspect 106:775-792 (1998)

Significant shortcomings of the U.S. Environmental Protection Agency's latest draft risk characterization for dioxin-like compounds.

The United States Environmental Protection Agency (U.S. EPA) has concluded that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a human carcinogen, and it has stated that the lifetime all-cancer mortality risk attributable solely to the current background body burden of dioxin-like compounds could be as high as 1.3 per 100. U.S. EPA's most current human cancer risk estimate was obtained from a linear dose-response model fit to the data from three epidemiology studies of TCDD-exposed chemical workers. Herein it is shown that the U.S. EPA model fails to provide an adequate fit to that data, whereas an intercept-only model, having no slope whatsoever, is entirely adequate. Although the epidemiology data used by U.S. EPA are consistent with a significant elevation in all-cancer mortality, by about 32%, among TCDD-exposed workers, this elevation should not be attributed to the workers' TCDD exposure.

Reevaluation of the cancer potency factor of toxaphene: recommendations from a peer review panel.

This reevaluation of the current U.S. EPA cancer potency factor for toxaphene is based upon a review of toxaphene carcinogenesis bioassays in mice conducted by Litton Bionetics (unpublished report, 1978) and the National Cancer Institute (NCI) (Technical Report Series No. 37, conducted by Gulf South Research Institute, 1979). The mechanistic data available for toxaphene, including consideration of the potential of the compound to induce genotoxicity, was examined with an emphasis on whether this information supports a change in the cancer potency factor. If a quantitative dose-response assessment for toxaphene is to be performed, the data from both the NCI and Litton cancer bioassays should be used. Additionally, liver tumor results from female mice, rather than male mice, should be used for estimating potential human cancer risk because the background rate of liver tumors in females is lower and less variable than that exhibited by males. An ED(10) was estimated as the point of departure. The mechanistic data were not sufficient to fully support a margin of exposure approach. Therefore, we believe that applying a linear extrapolation from the ED(10) to the origin is an appropriate means to estimate risk at low doses. This is a highly conservative approach and, when it is applied, we conclude that the current EPA cancer potency factor should be reduced from 1.1 (mg/kg/day)(-1) to 0.1 (mg/kg/day)(-1).

Binding of formaldehyde to human and rat nasal mucus and bovine serum albumin.

The function of the nasal mucociliary apparatus, an important airway defense mechanism, is inhibited by inhaled formaldehyde. Nasal mucus, which contains significant concentrations of glycoprotein and soluble proteins, is an integral component of this system. This investigation addresses some reactions of formaldehyde with human and rat mucus in vitro in comparison with a model protein, bovine serum albumin. [14C]Formaldehyde was incubated with reconstituted preparations of human and rat nasal mucus or bovine serum albumin. Formaldehyde adducts, stabilized by sodium cyanoborohydride reduction to methylamines, were separated by Sepharose 2B gel filtration. [14C]Formaldehyde bound exclusively to one component of nasal mucus which had an elution volume identical to that of albumin. There was no detectable binding to the large molecular weight glycoproteins. The time course of reaction of formaldehyde to free amino groups was then measured using the fluorescamine technique. Formaldehyde binding was characterized by an initial fast phase (less than 2 min) followed by a slower phase which appeared to approach equilibrium (greater than 60 min). The rate of binding to human and rat nasal mucus was similar to albumin. Irreversible binding of formaldehyde to albumin was insignificant within the first 60 min indicating the reversibility of binding during this time. These data indicate that within the first 60 min, formaldehyde reacts rapidly and reversibly with nasal mucus and that it binds primarily to one component of nasal mucus. Gel filtration analysis suggests this component may be albumin although other low molecular weight proteins cannot be ruled out.

Quantitative cancer risk estimation for formaldehyde.

Of primary concern are irreversible effects, such as cancer induction, that formaldehyde exposure could have on human health. Dose-response data from human exposure situations would provide the most solid foundation for risk assessment, avoiding problematic extrapolations from the health effects seen in nonhuman species. However, epidemiologic studies of human formaldehyde exposure have provided little definitive information regarding dose-response. Reliance must consequently be placed on laboratory animal evidence. An impressive array of data points to significantly nonlinear relationships between rodent tumor incidence and administered dose, and between target tissue dose and administered dose (the latter for both rodents and Rhesus monkeys) following exposure to formaldehyde by inhalation. Disproportionately less formaldehyde binds covalently to the DNA of nasal respiratory epithelium at low than at high airborne concentrations. Use of this internal measure of delivered dose in analyses of rodent bioassay nasal tumor response yields multistage model estimates of low-dose risk, both point and upper bound, that are lower than equivalent estimates based upon airborne formaldehyde concentration. In addition, risk estimates obtained for Rhesus monkeys appear at least 10-fold lower than corresponding estimates for identically exposed Fischer-344 rats.

The role of mechanistic data in dose-response modeling.

The results from employing metabolized dose in the case of vinyl chloride demonstrate that the delivered dose concept can radically alter the shape of the dose-response curve in the observable response range. Simultaneously, it can eliminate the obvious bias in low-dose risk estimates that arises from use of an inappropriate dose measure, namely, the administered airborne vinyl chloride concentration. For formaldehyde, the data regarding covalent binding to respiratory mucosal DNA provide the best measure of target tissue exposure that is currently available. The results obtained with this dose measure demonstrate that incorporation of the delivered dose concept into any of the commonly used low-dose extrapolation procedures leads to a unilateral reduction in estimated cancer risk associated with the exposure of rats to low airborne formaldehyde concentrations. These findings strongly suggest that low-dose risk estimates obtained with the administered formaldehyde dose as the measure of exposure are biased conservatively, and that regulatory assessments of formaldehyde should consider the delivered formaldehyde dose to be the valid and relevant measure of exposure. Additional research is, of course, required to refine and elaborate the delivered dose concept, especially for human exposures. Nevertheless, this concept provides a convenient vehicle for incorporating mechanistic information directly into the quantitative risk assessment process in a meaningful and relevant manner. Clearly, risk estimates based on delivered dose reflect what is known of the underlying biological reality more faithfully than estimates that are based solely on unverified assumptions and the findings in chronic bioassays. Use of the delivered dose concept should be strongly encouraged, since it can help place quantitative risk assessment on a sound and scientifically defensible footing.

The importance of delivered dose in estimating low-dose cancer risk from inhalation exposure to formaldehyde.

Data have recently been obtained on the concentration of formaldehyde covalently bound to the respiratory mucosal DNA of Fischer-344 rats following two 6-hr inhalation exposures to gaseous formaldehyde. These data provide a direct short-term measure of the delivered formaldehyde dose in target tissue as a function of the formaldehyde concentration in ambient air. They also demonstrate that the delivered dose/administered dose relationship is significantly nonlinear. Since chronic inhalation exposure of Fischer-344 rats to high concentrations of gaseous formaldehyde induces squamous cell carcinomas of the nasal cavity, and sine widespread concern exists that formaldehyde exposure may also pose a cancer risk for humans, the implications of this nonlinearity for low-dose risk extrapolation were investigated. The incidence of nasal squamous cell carcinomas in a chronic formaldehyde inhalation bioassay was reanalyzed with several low-dose extrapolation models, using the estimated concentration of formaldehyde covalently bound to respiratory mucosal DNA as the measure of exposure. For this purpose, it was assumed that the short-term observations of covalent binding were representative of steady-state conditions during the course of the chronic study and further, that the covalent binding of formaldehyde to target tissue DNA is an important factor in nasal tumor induction. Resulting maximum likelihood risk estimates and upper 95% confidence bounds were unilaterally lower than the corresponding risk measures based on administered dose, irrespective of the dose-response model employed. Reductions in estimated risk ranged from a factor of 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 of delivered dose can have a significant impact on estimates of low-dose risk and should therefore at least be considered as an alternative dose measure in assessments of human cancer risk from formaldehyde exposure.

Assessing effects of occupational exposure on fertility with indirect standardization.

The use of indirect standardization in the assessment of the fertility of occupationally exposed workers is briefly reviewed and critiqued. The calculation of expected births in the method of Levine et al. (J Occup Med 1980;22:781-91) is modified to eliminate negative bias. An example is given using data from a 1977 survey of 60 male workers at a chemical manufacturing plant in Denver, Colorado, who were exposed to dibromochloropropane. The example illustrates how in-plant nonexposed reproductive experience provides a valuable supplement to US fertility tables which are specific only to race, birth cohort, age, and parity. It is also shown, however, that explicit control for potential confounding factors not included in the tables, such as marital status and surgical sterilization, can actually create rather than alleviate confounding error. This occurs when the additional factors co-vary in the reference population with the factors already included in the tables. For martial status, the control-induced error was readily minimized by restricting analysis to married experience at parity one or greater. For surgical sterilization, the corresponding error could not be reduced without severely compromising sample size, and hence control of this potential confounder in similar circumstances is not recommended.

Survival models for fertility evaluation.

"Two proportional hazards models for cohort fertility evaluation are constructed. Time-dependent covariates describe sources of heterogeneity between and within women regarding fertility characteristics. In the first model, U.S. birth rates specific to maternal age, race, parity, and birth cohort are used as underlying hazard rates. Covariate effects are estimated by maximizing the full likelihood. In the second model, covariate effects are estimated via Cox regression with stratified underlying hazard rates regarded as unknown nuisance parameters." The authors illustrate the models "with an evaluation of the fertility histories of the wives of workers at a manufacturing plant with potential for hazardous exposure. Adjustments to the U.S. birth rates for maternal age and parity zero experience are required with the first approach. Then, despite differences in the model-specific estimation procedures, the point estimates of the exposure effect and the estimated standard errors from the two models are practically equivalent."

Experimental design constraints on carcinogenic potency estimates.

The multistage model is used by U.S. regulatory agencies to calculate estimates of the carcinogenic potency (beta) of chemicals; the data for these estimates are generally obtained from chronic rodent bioassays. Three quantities characterize each group tested in the chronic bioassay: the dose level, the sample size, and the number responding to the dose. The dose levels tested are fixed by conventional protocols; the typical National Toxicology Program (NTP) experimental design calls for use of the maximum tolerated dose (MTD), one-half and one-fourth MTD, plus a control group. Only rarely are doses even one order of magnitude less than the MTD utilized in chronic bioassays. This experimental design constraint on dose selection limits the possible values of beta that can arise from multistage model analyses of chronic bioassay data. Sample size is also constrained by the experimental design of the chronic bioassay; the typical sample size in NTP studies is 50 animals. Occasionally, fewer animals are used, but only rarely are more. Thus, the multistage model which theoretically has three variable quantities with which to estimate carcinogenic potency, has in practice only one: the incidence of treatment-related response. Even this can vary within only a narrow range determined by sample size, the control incidence, and the level of statistical significance desired. The net result of these design constraints is that carcinogenic potency estimates derived from multistage-model analyses of chronic bioassay data may vary within only a narrow range surrounding the inverse maximum dose tested. We have illustrated this by calculating the largest possible finite potency estimates that could have arisen from the experimental designs used to test 82 mouse carcinogens in chronic bioassays. On average these maximum potency estimates were within one order of magnitude of the inverse maximum dose tested. We thus conclude that the chronic rodent bioassay, in and of itself, is altogether inadequate as a data source for estimating the risk to humans from exposure to carcinogenic chemicals.

Comparison of cancer risks projected from animal bioassays to epidemiologic studies of acrylonitrile-exposed workers.

Bioassay findings have demonstrated that acrylonitrile (ACN) is a rodent carcinogen, but the available epidemiologic evidence provides little support for the human carcinogenicity of ACN. This discordance between laboratory animal and human study findings is explored by determining post hoc the statistical power of 11 epidemiologic studies of ACN-exposed workers to detect the all-site and brain cancer excesses that are projected from rodent drinking water bioassay data. At reasonable estimates of the level and duration of exposures among the occupational cohorts, a majority of the human studies had sufficient power (> 80%) to detect the projected excesses, yet such responses were consistently absent. We conclude, subject to certain caveats, that the upper bound estimate of ACN's inhalation cancer potency of 1.5 x 10(-4) per ppm is too high to be consistent with the human ACN experience.

Chronic bioassays: relevance to quantitative risk assessment of carcinogens.

Conventional carcinogenic potency estimates for chemicals have been compared across rodent species. The high correlations previously demonstrated between maximum likelihood estimates (MLE) for chemicals identified as positive carcinogens in both rats and mice (r = 0.83, n = 83) are shown to occur also for chemicals that were negative in both species (r = 0.85, n = 51), for chemicals causing tumors in rats but not mice (r = 0.55, n = 15), and for those causing tumors in mice but not rats (r = 0.68, n = 25). Corresponding upper-bound estimates of carcinogenic potency are also highly correlated across rodent species. The correlations arise from (i) the strong interspecies correlation between maximum doses tested in chronic bioassays, (ii) the small group sizes utilized, and (iii) the narrow range of doses typically tested. These factors constrain conventional ML and upper-bound potency estimates to lie very close to the inverse maximum doses tested, irrespective of the bioassay's qualitative outcomes. The potency estimates are thus artifacts of experimental design and would seem to provide little information on the actual human cancer risks from chemical exposure. Risk assessment models can reveal true potency differences across species, but they must incorporate relevant mechanistic information before quantitative risk extrapolation from rodents to humans is scientifically defensible.

Differentiation between metabolic incorporation and covalent binding in the labeling of macromolecules in the rat nasal mucosa and bone marrow by inhaled [14C]- and [3H]formaldehyde.

The mechanisms of labeling of macromolecules (DNA, RNA, and protein) in the respiratory and olfactory mucosa, and in the bone marrow (femur) of male Fischer-344 rats exposed to [14C]- and [3H]formaldehyde [( 14C]- and [3H]CH2O) were investigated. Animals were exposed for 6 hr to atmospheres containing [14C]- and [3H]CH2O at concentrations of 0.3, 2, 6, 10, or 15 ppm, 1 day following a single pre-exposure to the same concentration of unlabeled CH2O. The major route of nucleic acid labeling at all concentrations and in all tissues was metabolic incorporation; protein labeling in the respiratory mucosa was mainly due to covalent binding at the higher CH2O concentrations. Incorporation of [14C]CH2O into DNA in the respiratory mucosa was maximal at 6 ppm but decreased at higher concentrations, whereas labeling of DNA in the olfactory mucosa and bone marrow increased monotonically with concentration. Evidence for covalent binding of CH2O to respiratory mucosal DNA was obtained at CH2O concentrations equal to or greater than 2 ppm. The concentration of CH2O covalently bound to DNA at 6 ppm was 10.5-fold higher than at 2 ppm, indicating significant nonlinearity of DNA binding with respect to the inhaled formaldehyde concentration under these conditions. Covalent binding to proteins increased in an essentially linear manner with increases in the airborne concentration. No evidence was obtained for the formation of covalent adducts with macromolecules in the olfactory mucosa or bone marrow. The nonlinear increase in covalent binding to respiratory mucosal DNA with increasing CH2O concentrations may be explained either by a decrease in the efficiency of defense mechanisms or by an increase in the availability of reaction sites on the DNA resulting from increased cell turnover.

Fertility of workers. A comparison of logistic regression and indirect standardization.

Estimates of the effect of occupational exposure on the fertility of men employed at three chemical plants were obtained from data stored at the Chemical Industry Institute of Toxicology using logistic regression and indirect standardization. Logistic regression was explored as a possible alternative to indirect standardization because it 1) permits consideration of potential confounding variables, such as the relative spacing between consecutive births, that are not included in the characterization of the fertility of an external reference population, and 2) may enable the study of occupational cohorts for which fertility data from an appropriate external reference population are not available. In addition to the main effects of age, parity, and birth cohort, main effects of certain lag variables which characterize the timing of birth events during the five-year period preceding each person-year were found to be significant at all three plants. Interactions of some of these lag variables with age also emerged as significant. Despite this, both methods were consistent in accepting or rejecting the null hypothesis regarding the effect of exposure at two of the plants. At the third plant, however, the logistic regression yielded a significant interaction between exposure status and one of the lag variables. The net exposure effect was significantly increased fertility in exposed person-years for which no birth had occurred 2-3 years prior, and nonsignificantly decreased fertility in exposed person-years for which such a prior birth had occurred. While this may be a spurious finding, it suggests that both methods of analysis should continue to be explored until a larger body of similar comparative studies has accumulated.

More precise localization of nasal tumors associated with chronic exposure of F-344 rats to formaldehyde gas.

Considerable interest and research have resulted from the finding that squamous cell carcinomas, polypoid adenomas, and a small number of other nasal neoplasms occurred in F-344 rats following chronic inhalation exposure to formaldehyde. These tumors were reported to originate in the anterior portion of the nasal cavity but their precise location in the nose was not determined. Histologic sections from the nasal passages of these rats have been reexamined and the location of each tumor has been recorded. The majority of squamous cell carcinomas occurred on the anterior portion of the lateral aspect of the nasoturbinate and adjacent lateral wall (57%) or the midventral nasal septum (26%). Polypoid adenomas were confined to a small region of the anterior nasal cavity and were restricted to the free margins of the naso-and maxilloturbinates and lateral wall adjacent to these margins. One neoplasm, considered to be the malignant counterpart of the polypoid adenoma, originated on the dorsal margin of the maxilloturbinate in the same region of the nose. Remaining neoplasms were generally too large or too poorly preserved for assessment of their site of origin. Mechanistic studies directed toward a better understanding of the nasal carcinogenicity of formaldehyde, or other nasal carcinogens. should take into account the anatomic sites of origin of the neoplasms whenever this can be determined.

Mode-of-action-based dosimeters for interspecies extrapolation of vinyl acetate inhalation risk.

Vinyl acetate is used in the manufacture of many polymers. The Clean Air Act Amendments of 1990 require that an inhalation risk assessment be conducted to assess risks to human health from ambient exposures. Vinyl acetate is a nasal carcinogen in rats and induces olfactory degeneration in rats and mice. Because of the many unique aspects of the rodent nasal cavity compared to that of humans, conventional means for extrapolating dosimetry between species are not appropriate. Physiologically based pharmacokinetic (PBPK) and pharmacodynamic (PD) modeling can address many of these unique aspects. A PBPK/PD model has been developed for vinyl acetate, but the choice of appropriate dosimeter(s) to use for interspecies extrapolation depends on a hypothesis regarding mode of action. This article summarizes the key studies that formulate a mode of action hypothesis for vinyl acetate. Dose-response relationships for vinyl acetate-induced nonneoplastic and neoplastic responses are highly nonlinear, suggesting complex kinetic processes. Carboxylesterase-dependent metabolism of vinyl acetate forms acetic acid, a potent cytotoxicant, and acetaldehyde, a weak clastogen. Cell death, proposed to be the result of intracellular acidification, results in restorative cell proliferation. In conjunction with sufficient genetic damage, induced by spontaneous mutation and acetaldehyde-induced DNA-protein cross-links (DPX), olfactory degeneration progresses to a state of elevated proliferation and eventually, at high vinyl acetate concentrations, to neoplastic transformation. Thus, reduction in intracellular pH (pHi) is proposed as the dosimeter most closely linked to the earliest stages of vinyl acetate toxicity. Consequently, risk assessments that are based on protection of nasal epithelium from intracellular acidification will be protective of all subsequent pathological responses related to vinyl acetate exposure. Proposing a reasonable mode of action is an important step in any risk assessment and is critical to the choice of dosimeter(s) to be used for interspecies dosimetry extrapolation.

Assessing exposure to dinitrotoluene using a biological monitor.

Exposure of workers to dinitrotoluene (DNT) was evaluated at a DNT manufacturing plant. Urine was collected over 72 hours; work diaries were prepared dialy; breathing zone air was sampled; and skin and environmental surfaces were wiped. Chemical analysis was performed using gas chromatography or gas chromatography/mass spectrometry. Proportions of urinary DNT and metabolites deviated substantially from those reported in rats exposed to 2,4-DNT; but as with rats, females appeared to excrete considerably more dinitrobenzyl glucuronide. Between persons on any one day and within persons on different days, considerable variation existed in the proportions of metabolites excreted. The peak rate of excretion was likely to occur toward the end of a work shift or shortly afterward. Most urinary metabolites related to exposure during an eight-hour shift had been excreted by the start of work the following day. Estimates of the maximum one-day exposure incurred by a participant in this study ranged from 0.24 to 1.00 mg of technical-grade DNT per kilogram of body weight. A large proportion of the DNT absorbed by DNT operators and loaders, it is suggested, may have entered through the skin or the gastrointestinal tract.