The Scientific Basis of Uncertainty Factors Used in Setting Occupational Exposure Limits.

The uncertainty factor concept is integrated into health risk assessments for all aspects of public health practice, including by most organizations that derive occupational exposure limits. The use of uncertainty factors is predicated on the assumption that a sufficient reduction in exposure from those at the boundary for the onset of adverse effects will yield a safe exposure level for at least the great majority of the exposed population, including vulnerable subgroups. There are differences in the application of the uncertainty factor approach among groups that conduct occupational assessments; however, there are common areas of uncertainty which are considered by all or nearly all occupational exposure limit-setting organizations. Five key uncertainties that are often examined include interspecies variability in response when extrapolating from animal studies to humans, response variability in humans, uncertainty in estimating a no-effect level from a dose where effects were observed, extrapolation from shorter duration studies to a full life-time exposure, and other insufficiencies in the overall health effects database indicating that the most sensitive adverse effect may not have been evaluated. In addition, a modifying factor is used by some organizations to account for other remaining uncertainties-typically related to exposure scenarios or accounting for the interplay among the five areas noted above. Consideration of uncertainties in occupational exposure limit derivation is a systematic process whereby the factors applied are not arbitrary, although they are mathematically imprecise. As the scientific basis for uncertainty factor application has improved, default uncertainty factors are now used only in the absence of chemical-specific data, and the trend is to replace them with chemical-specific adjustment factors whenever possible. The increased application of scientific data in the development of uncertainty factors for individual chemicals also has the benefit of increasing the transparency of occupational exposure limit derivation. Improved characterization of the scientific basis for uncertainty factors has led to increasing rigor and transparency in their application as part of the overall occupational exposure limit derivation process.

Spontaneous abortion, sex ratio, and paternal occupational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

There is conflicting research regarding an association between fetal death and paternal exposure to Agent Orange, a phenoxy herbicide widely used in Vietnam that was contaminated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Men who worked in the U.S. factories that produced Agent Orange were exposed to TCDD at levels hundreds of times higher than TCDD levels in the general population. Wives of TCDD-exposed chemical workers and wives of nonexposed neighborhood referents were interviewed to determine reproductive history. Paternal serum TCDD level at time of conception was estimated for each pregnancy using serum samples taken in 1987. Estimated TCDD levels of workers during or after exposure were high (median, 254 ppt; range, 3-16,340 ppt) compared to referent levels (median, 6 ppt; range, 2-19 ppt). No association between paternal TCDD level at the time of conception and spontaneous abortion was observed among pregnancies fathered by workers with TCDD levels of < 20 ppt [odds ratio (OR) = 0.77; 95% confidence interval (CI), 0.48-1.22], 20 to < 255 ppt (OR = 0.81; 95% CI, 0.40-1.63), 255 to < 1,120, (OR = 0.69; 95% CI, 0.30-1.58), and >or= 1,120 ppt (OR = 0.95; 95% CI, 0.42-2.17) compared to pregnancies fathered by referents. The sex ratio [males/(males + females)] of offspring also did not differ by TCDD exposure (0.53 and 0.54 among workers and referents, respectively). We did not find an association between paternal serum TCDD level and spontaneous abortion or sex ratio of offspring in this population. The estimated TCDD levels in this exposed worker population were much higher than in other studies, providing additional evidence that paternal TCDD exposure does not increase the risk of spontaneous abortion at levels above those observed in the general population. The study could not evaluate the effect of father's childhood or prenatal TCDD exposure on subsequent sex ratio.

Methodological issues of using observational human data in lung dosimetry models for particulates.

INTRODUCTION: The use of human data to calibrate and validate a physiologically based pharmacokinetic (PBPK) model has the clear advantage of pertaining to the species of interest, namely humans. A challenge in using these data is their often sparse, heterogeneous nature, which may require special methods. Approaches for evaluating sources of variability and uncertainty in a human lung dosimetry model are described in this study. METHODS: A multivariate optimization procedure was used to fit a dosimetry model to data of 131 U.S. coal miners. These data include workplace exposures and end-of-life particle burdens in the lungs and hilar lymph nodes. Uncertainty in model structure was investigated by fitting various model forms for particle clearance and sequestration of particles in the lung interstitium. A sensitivity analysis was performed to determine which model parameters had the most influence on model output. Distributions of clearance parameters were estimated by fitting the model to each individual's data, and this information was used to predict inter-individual differences in lung particle burdens at given exposures. The influence of smoking history, race and pulmonary fibrosis on the individual's estimated clearance parameters was also evaluated. RESULTS: The model structure that provided the best fit to these coal miner data includes a first-order interstitialization process and no dose-dependent decline in alveolar clearance. The parameter that had the largest influence on model output is fractional deposition. Race and fibrosis severity category were statistically significant predictors of individual's estimated alveolar clearance rate coefficients (P < 0.03 and P < 0.01-0.06, respectively), but smoking history (ever, never) was not (P < 0.4). Adjustments for these group differences provided some improvement in the dosimetry model fit (up to 25% reduction in the mean squared error), although unexplained inter-individual differences made up the largest source of variability. Lung burdens were inversely associated with the miners' estimated clearance parameters, e.g. individuals with slower estimated clearance had higher observed lung burdens. CONCLUSIONS: The methods described in this study were used to examine issues of uncertainty in the model structure and variability of the miners' estimated clearance parameters. Estimated individual clearance had a large influence on predicted lung burden, which would also affect disease risk. These findings are useful for risk assessment, by providing estimates of the distribution of lung burdens expected under given exposure conditions.

Human cancer risk and exposure to 1,3-butadiene--a tale of mice and men.

OBJECTIVES: The purpose of this study was to evaluate empirically the relevance of animal-bioassay-based models for predicting human risks from exposure to 1,3-butadiene (BD) using epidemiologic data. METHODS: Relative-risk results obtained with a regression model in a recent epidemiologic study were used to estimate leukemia risk for occupational and environmental exposures to BD and to compare these estimates with those previously derived from an analysis of animal bioassay data. RESULTS: The estimates of risk were found to be highly dependent on the model used when low levels of exposure were evaluated that are of environmental concern, but not at the levels of occupational concern. For example, at the level (1 part per million) of the recently revised standard of the Occupational Safety and Health Administration in the United States the estimates of lifetime excess risk ranged from 1 to 8 per 1000 workers. The range of the risk estimates derived from the epidemiologic models was remarkably similar to the range of risk estimates for occupational exposures (1 to 9 per thousand) previously developed by Dankovic et al in 1993 from an analysis of a mouse bioassay study for lymphocytic lymphoma. CONCLUSIONS: Results for BD seem to provide another example of a high degree of concordance between the risk predictions from models of toxicologic and epidemiologic data, particularly at occupational levels of exposure.

Evaluation of Particle Clearance and Retention Kinetics in the Lungs of U.S. Coal Miners.

Rodent studies are frequently used to assess risk in humans, yet it is not known whether the overloading of lung clearance, as observed in rodents, occurs in humans, or whether overloading is related to particle-related lung diseases in humans. The objective of this study is to develop a biologically based mathematical model to describe the retention and clearance of respirable coal mine dust in the lungs of humans. A human dosimetric lung model was developed that includes alveolar, interstitial, and hilar lymph-node compartments. The model describes the particle mass transfer kinetics among these compartments and clearance via the tracheobronchi. The model was calibrated using data in U.S. coal miners, including individual working lifetime exposure histories and lung and lymph-node particle burdens. The model fit to the human data was evaluated using a least-squared error criterion. The end-of-life lung dust burdens of all coal miners in this study were substantially greater than expected from a simple, linear first-order model with effective clearance, yet their lung and lymph-node dust burdens were lower than expected from the rodent-based overload model, particularly at higher exposures. The best fitting model included a predominant first-order interstitial compartment, in which the particles are essentially sequestered (with very slow clearance to the lymph nodes), and a first-order alveolar clearance compartment with either no dose-dependent decline (overloading) or much less than expected from the rodent studies. These findings are consistent with the findings from magnetopneumography studies of clearance in retired miners and from studies of particle retention patterns in rodents and primates. This human dosimetric lung model is useful for evaluating the kinetic differences of particle retention in humans and rodents, and for evaluating the lung closes in humans given different exposure scenarios.

An introduction to the use of physiologically based pharmacokinetic models in risk assessment.

Many extrapolation issues surface in quantitative risk assessments. The extrapolation from high-dose animal studies to low-dose human exposures is of particular concern. Physiologically based pharmacokinetic (PBPK) models are often proposed as tools to mitigate the problems of extrapolation. These models provide a representation of the disposition, metabolism, and excretion of xenobiotics that are believed to possess the potential of inducing adverse human health responses. Given a model of xenobiotic disposition that is applicable for multiple species and appropriate for nonlinearity of the xenobiotic biotransformation process, better extrapolation may be possible. Unfortunately, the true structure of these models (e.g. number of compartments, type of metabolism, etc.) is seldom known, and attributes of these models (tissue volumes, partition coefficients, etc.) are often experimentally determined and often only central measures of these quantities are reported. We describe the use of PBPK models in risk assessment, the structural and parameter uncertainty in these models, and provide a simple illustration of how these characteristics can be incorporated in a statistical analysis of PBPK models. Additional complexity in the analysis of variability in the models is also outlined. This discussion is illustrated using data from methylene chloride.

Occupational exposure to chrysotile asbestos and cancer risk: a review of the amphibole hypothesis.

OBJECTIVES: This article examines the credibility and policy implications of the "amphibole hypothesis," which postulates that (1) the mesotheliomas observed among workers exposed to chrysotile asbestos may be explained by confounding exposures to amphiboles, and (2) chrysotile may have lower carcinogenic potency than amphiboles. METHODS: A critical review was conducted of the lung burden, epidemiologic, toxicologic, and mechanistic studies that provide the basis for the amphibole hypothesis. RESULTS: Mechanistic and lung burden studies do not provide convincing evidence for the amphibole hypothesis. Toxicologic and epidemiologic studies provide strong evidence that chrysotile is associated with an increased risk of lung cancer and mesothelioma. Chrysotile may be less potent than some amphiboles for inducing mesotheliomas, but there is little evidence to indicate lower lung cancer risk. CONCLUSIONS: Given the evidence of a significant lung cancer risk, the lack of conclusive evidence for the amphibole hypothesis, and the fact that workers are generally exposed to a mixture of fibers, we conclude that it is prudent to treat chrysotile with virtually the same level of concern as the amphibole forms of asbestos.

The impact of exercise and intersubject variability on dose estimates for dichloromethane derived from a physiologically based pharmacokinetic model.

Andersen et al. and Reitz et al. have developed physiologically based pharmacokinetic models for the human metabolism of methylene chloride (dichloromethane; DCM) and have advanced the hypothesis that the carcinogenicity of DCM is related to target organ metabolism of DCM by glutathione S-transferase (GST). The models included physiological parameters appropriate for humans at rest and metabolic parameters based on average rates of DCM metabolism. Increasing the model parameters describing cardiac output, alveolar ventilation, and blood flows to tissues from resting values to values consistent with light work conditions, and assuming a 25 ppm exposure for an 8-hr work day, increases the estimated GST-metabolized dose for human liver by a factor of 2.9 compared to the GST-metabolized does estimated of Reitz et al. These modifications also increase the GST-metabolized dose to the lung by 2.4-fold. If the model is also modified to reflect individual variation in DCM metabolism (in addition to the modifications for light work conditions), the estimated GST-metabolized dose for human liver ranges from 0 to as much as 5.4-fold greater than the dose estimated by Reitz et al. The GST-metabolized dose to the lung ranges from 0 to as much as 3.6-fold greater than the dose estimated by Reitz et al. These results indicate that some occupationally-exposed individuals may receive GST-metabolized doses several-fold greater than the Reitz et al. human dose estimates.

Time-to-tumour risk assessment for 1,3-butadiene based on exposure of mice to low doses by inhalation.

The excess risk for cancer due to lifetime occupational exposure to 1,3-butadiene at the proposed US Occupational Safety and Health Administration standard of 2 ppm was estimated on the basis of a quantitative risk assessment. The risk assessment was based on a recent study by the US National Toxicology Program of the carcinogenicity of butadiene in B6C3F1 mice, using exposure concentrations ranging from 6.25 to 625 ppm butadiene and controls. Cancer risks were estimated using a multistage Weibull time-to-tumour model; the dose was based on the external butadiene concentration, owing to the low-dose linearity of butadiene metabolism. The parameters of the time-to-tumour model were estimated for seven tumour sites in male mice and nine in female mice. The risk estimates were extrapolated from mice to humans on the basis of body weight raised to the three-fourths power, and the median lifespan of mice was equated to a human lifespan of 74 years. Estimates of excess risk for lifetime occupational exposure (8 h/day, 5 days/week, 50 weeks/year, for 45 years) to 2 ppm butadiene ranged from 0.2 per 10,000 workers, based on female mouse heart haemangiosarcomas, to 600 per 10,000 workers, based on female mouse lung tumours. An analysis was performed to assess the effects of varying the modelling assumptions (incidental versus fatal tumours, inclusion or exclusion of the 625-ppm dose group, and basis for interspecies scaling) on the risk estimates from the female mouse lung tumour model. Depending on the assumptions, estimates of lifetime excess risk derived from the female mouse lung model ranged from 60 per 10,000 to 1600 per 10,000. These results suggest that exposures to butadiene in the work place should be reduced to the lowest feasible level.

Complex mixture effects on the dermal absorption of benzo[a]pyrene and other polycyclic aromatic hydrocarbons from mouse skin.

To study the dermal penetration of benzo[a]pyrene (BAP) in relation to other polycyclic aromatic hydrocarbons (PAHs), a complex mixture of PAHs was applied to the backs of CD-1 mice, and the dermal residence times of BAP and eleven other PAHs were determined using gas chromatography. The dermal penetration of BAP was found to be representative of the other PAHs studied, with a dermal half-life of 6.7 h. Half-lives of the other eleven PAHs ranged from 5.0 to 8.8 h. The dermal half-life of BAP applied in a volatile organic solvent, rather than a PAH mixture, was 3.0 h. The effects of five complex organic mixtures, with boiling points ranging from 300-700 degrees F to greater than 850 degrees F, on the dermal residence time of BAP was studied by adding radiolabeled BAP to the mixtures, and applying them to the backs of mice. All of the mixtures studied increased the dermal residence time of BAP by amounts ranging from 1.8-fold to 6.9-fold.

Preparation of microgram quantities of BaP-DNA adducts using isolated rat hepatocytes in vitro.

The analysis of carcinogen-DNA adducts generally requires the preparation (by chemical or biological means) of DNA adduct standards, in amounts sufficient for chemical characterization. We have established conditions for the in vitro biological preparation of microgram quantities of DNA adducts derived from benzo[a]pyrene (BaP), fluoranthene and 7,12-dimethylbenzanthracene, using isolated rat hepatocytes. The metabolic activation of 180 microM BaP by isolated rat hepatocytes in a calf-thymus-DNA (CT-DNA)-supplemented medium resulted in the formation of 2.9 micrograms of BaP adducted to 56.7 mg of DNA. The average level of binding in this experiment was 148 +/- 8 pmol BaP bound/1 mg DNA, which compares favorably to the 10-30 pmol BAP/1 mg DNA which is typical of mouse skin adducts in vivo. In another experiment, BaP-DNA adduct formation in calf-thymus DNA added to hepatocyte incubations was further increased to 327 +/- 27 pmol/mg DNA, by physical shearing of the DNA prior to the incubation. The HPLC profile of the BaP adducts produced using hepatocytes plus CT-DNA is virtually indistinguishable from that produced by tumor-initiating doses of BaP applied to mouse skin in vivo, and the major DNA adduct formed by the hepatocytes co-elutes with the (+)-anti-diol-epoxide adduct of deoxyguanosine. Similar experiments using fluoranthene and 7,12-dimethylbenzanthracene also resulted in substantial DNA adduct formation; however, incubations using dibenz[a,h]anthracene did not. These results indicate that isolated rat hepatocytes in vitro can be useful for the preparation of DNA adducts of a number of polycyclic aromatic hydrocarbons, in quantities sufficient for chemical characterization.

Inhibition of benzo[a]pyrene-7,8-diol formation in vitro by complex organic mixtures.

Coal-derived complex organic mixtures [COM] with boiling points greater than or equal to 370 degrees C (greater than or equal to 700 degrees F) are known to inhibit both mouse skin tumor initiation by benzo[a]pyrene [BAP], and BAP-induced bacterial mutagenesis. We have examined the effects of 5 COM, with boiling points of 149-370 degrees C (300-700 degrees F), 370-398 degrees C (700-750 degrees F), 398-426 degrees C (750-800 degrees F), 426-454 degrees C (800-850 degrees F), and greater than 454 degrees C (greater than 850 degrees F), on both the rate and the route of BAP metabolism by rat liver homogenates in vitro. When co-metabolized in 40:1 excess with BAP, all of the COM reduced the rate of BAP metabolism. The 149-370 degrees C (300-700 degrees F) COM reduced the initial rate of BAP metabolism to 34% of the rate for BAP alone, while the four higher-boiling COM reduced it to 6.3-9.3% of the rate for BAP alone. In addition, the 2 highest-boiling COM (426-454 degrees C and greater than 454 degrees C boiling points) were found to reduce the percentage of BAP metabolized to BAP-7,8-diol, in comparison to incubations using BAP alone. The 370-398 degrees C and 398-426 degrees C COM did not alter the percentage of BAP metabolized to BAP-7,8-diol, while the 149-370 degrees C COM increased it. Both the general inhibition of BAP metabolism (by all of the COM), and the specific inhibition of BAP-7,8-diol formation (by the highest-boiling COM) may play a role in the inhibition of formation of BAP-induced skin tumors by these materials.

Influences of complex organic mixtures on tumor-initiating activity, DNA binding and adducts of benzo[a]pyrene.

Co-incubation of benzo[a]pyrene (BaP) and coal-derived complex organic mixtures has been shown to decrease the metabolism and mutagenic activity of BaP. Because of these influences, five mixtures were co-administered dermally to mice to initiate tumor development. Results from these studies demonstrated that BaP tumor-initiating activity was decreased substantially by four of the five mixtures. When one of the mixtures was separated into chemical class fractions, the polycyclic aromatic hydrocarbon (PAH) and nitrogen-containing polycyclic aromatic compound fractions were the most effective, and the aliphatic and hydroxy-PAH fractions were the least effective as inhibitors of BaP-induced tumor initiation. Binding of [3H]BaP to epidermal DNA under conditions identical to those used for tumor initiation was decreased by co-administration of all five mixtures. Calculations of the number of tumors produced/micrograms BaP bound to DNA demonstrated that co-administration of this carcinogen with the mixtures consistently increased the effectiveness of the bound BaP at producing tumors by approximately a factor of 2. The HPLC radioactivity profiles of enzyme-hydrolyzed, adducted DNA indicated that, in the presence of the mixtures, the predominant adducts were derived from BaP-diol epoxide (BPDE); however, the mixtures decreased the ratios of the anti-BPDE-deoxyguanosine to syn-BPDE-deoxy-guanosine adducts. These data indicate that the prevailing influences of the mixtures (i.e. decreased DNA binding and adduct shifts) were similar to those observed with other bioassays following co-administration of binary mixtures. Furthermore, the data demonstrate that both DNA binding and adduct profiles are important in determining the contribution of a known carcinogen to tumor initiation by complex organic mixtures.

Induction of hepatic aryl hydrocarbon hydroxylase activity in rainbow trout (Salmo gairdneri) exposed to coal liquids.

1. The hepatotoxic response of rainbow trout (Salmo gairdneri) to a middle distillate (MD) and heavy distillate (HD) coal liquid was determined following administration by injection or in food. Hepatic aryl hydrocarbon hydroxylase (AHH) activity was compared to the known AHH inducer, benzo(a)pyrene. 2. Acute toxicities (LD50) of 1500 and 1410 mg/kg were obtained 120 hr after intraperitoneal injection (i.p.) for MD and HD coal liquids, respectively. Only the HD induced AHH activity when injected at 5% and 10% of the acute LD50. 3. Ingestion of 7 or 70 mg HD/kg body weight for 1 week resulted in AHH induction at 5-7 times that of control values. Levels of hepatic AHH activity in fish treated at 7 mg HD/kg declined to 2.6 times control values 7 days after exposure. Maximum induction was observed for the 70 mg HD/kg group that was provided control food for 1 week following 2 weeks of exposure. 4. The hepatic index (HI) or liver to body weight ratio generally increased along with increased activity in the enzyme response system.

The role of 4-bromophenol and 4-bromocatechol in bromobenzene covalent binding and toxicity in isolated rat hepatocytes.

4-Bromophenol and 4-bromocatechol are formed as metabolites of bromobenzene in vivo and in isolated rat hepatocytes. Both of these metabolites may potentially contribute to the hepatotoxicity of bromobenzene. Bromobenzene metabolism in hepatocytes isolated from phenobarbital-treated rats forms 0.12 to 0.17 mM 4-bromophenol and 4-bromocatechol in 2 hr, with 1 to 3 mM bromobenzene. The role of activated metabolites derived from 4-bromophenol and 4-bromocatechol in bromobenzene covalent binding and toxicity was investigated with isolated hepatocytes in suspension. The covalent binding of the phenol and the catechol was increased four- to eightfold by the addition of unlabeled bromobenzene. Two-hour incubations of 0.25 mM 14C-labeled 4-bromophenol or 4-bromocatechol with hepatocytes isolated from phenobarbital-treated rats resulted, under these conditions, in no significant toxicity, and approximately 4 and 25%, respectively, of the covalent binding associated with bromobenzene itself. Two- and six-hour incubations with higher 4-bromophenol and 4-bromocatechol concentrations demonstrated that 1 to 3 mM substrate concentrations were required for cytotoxicity. These results show that metabolically produced 4-bromophenol and 4-bromocatechol do not play significant roles in the production of bromobenzene cytotoxicity in isolated hepatocytes, and that they contribute only modestly to bromobenzene covalent binding.