Prediction of ozone-induced lung function responses in humans

The main purpose of this study was to evaluate the ability of a human exposure-response model, which describes ozone-induced changes in forced expiratory volume in 1 second (FEV₁) across a wide range of dynamic exposure conditions, to predict responses in independent data. We first conducted an n-fold cross-validation of the model using samples of the original EPA data from which the model was developed. We then identified seven more recently published studies with controlled exposures to a wide range of ozone exposure patterns relevant to the current ambient ozone health standard and used the model to calculate the mean predicted responses for the exposure conditions of the individual studies that we compared to the mean observed responses reported in these studies. The n-fold cross-validation indicated good internal agreement between mean predicted and mean observed responses in the original data used to develop the model. The model accurately captured the patterns of response in each of the seven independent studies with a tendency to overpredict the observed responses by about 1 percentage point of FEV₁ decrement on average. We conclude that the model is currently capable of predicting human FEV₁ responses across a wide range of dynamic exposure conditions and anticipate further improvements in predictions with the addition of low-concentration exposure data.     

Modelling of individual subject ozone exposure response kinetics

Context: A better understanding of individual subject ozone (O₃) exposure response kinetics will provide insight into how to improve models used in the risk assessment of ambient ozone exposure.

Objective: To develop a simple two compartment exposure–response model that describes individual subject decrements in forced expiratory volume in one second (FEV₁) induced by the acute inhalation of O₃ lasting up to 8 h.

Methods: FEV₁ measurements of 220 subjects who participated in 14 previously completed studies were fit to the model using both particle swarm and nonlinear least squares optimization techniques to identify three subject-specific coefficients producing minimum “global” and local errors, respectively. Observed and predicted decrements in FEV₁ of the 220 subjects were used for validation of the model. Further validation was provided by comparing the observed O₃-induced FEV₁ decrements in an additional eight studies with predicted values obtained using model coefficients estimated from the 220 subjects used in cross validation.

Results: Overall the individual subject measured and modeled FEV₁ decrements were highly correlated (mean R² of 0.69 ± 0.24). In addition, it was shown that a matrix of individual subject model coefficients can be used to predict the mean and variance of group decrements in FEV₁.

Conclusion: This modeling approach provides insight into individual subject O₃ exposure response kinetics and provides a potential starting point for improving the risk assessment of environmental O₃ exposure.     

Ozone exposure-response model for lung function changes: an alternate variability structure

Abstract

Context: A statistical model that accurately predicts human forced expiratory volume in one second (FEV₁) response to ozone exposure has been identified and proposed as the foundation for future risk assessments for ambient ozone. We believe that the assumptions about intra-subject variability in the published model can be improved and hypothesize that more realistic assumptions will improve the fit of the model and the accuracy of risk assessments based on the model.

Objective: Identify alternate assumptions about intra-subject variability and compare goodness-of-fit for models with various variability structures.

Materials and methods: Models were fit to an existing data set using a statistical program for fitting nonlinear mixed models. Goodness-of-fit was assessed using Akaike’s Information Criteria (AIC) and visual examination of graphical figures showing observed and predicted values.

Results: The AIC indicated that a model that assumed intra-subject variability was related to the magnitude of individual response fit the data better than a model that assumes intra-subject variability is constant across individuals and exposures (the original model). This finding was consistent with the variability of observed responses for filtered air exposures and for exposures predicted to be below the threshold for response.

Conclusion: An ozone exposure-response model that assumes intra-subject variability increases with individual mean FEV₁ response appears to fit the data better than one that assumes constant variability.     

Development of a large-scale computer-controlled ozone inhalation exposure system for rodents

Objective: Complete systems for laboratory-based inhalation toxicology studies are typically not commercially available; therefore, inhalation toxicologists utilize custom-made exposure systems. Here we report on the design, construction, testing, operation and maintenance of a newly developed in vivo rodent ozone inhalation exposure system.

Materials and methods: Key design requirements for the system included large-capacity exposure chambers to facilitate studies with large sample sizes, automatic and precise control of chamber ozone concentrations, as well as automated data collection on airflow and environmental conditions. The exposure system contains two Hazelton H-1000 stainless steel and glass exposure chambers, each providing capacity for up to 180 mice or 96 rats. We developed an empirically tuned proportional-integral-derivative control loop that provides stable ozone concentrations throughout the exposure period (typically 3h), after a short ramp time (～8?min), and across a tested concentration range of 0.2–2?ppm. Specific details on the combination of analog and digital input/output system for environmental data acquisition, control and safety systems are provided, and we outline the steps involved in maintenance and calibration of the system.

Results: We show that the exposure system produces consistent ozone exposures both within and across experiments, as evidenced by low coefficients of variation in chamber ozone concentration and consistent biological responses (airway inflammation) in mice, respectively.

Conclusion: Thus, we have created a large and robust ozone exposure system, facilitating future studies on the health effects of ozone in rodents.     

Wheezing and lung function measured in subjects exposed to various levels of fine particles and polycyclic aromatic hydrocarbons

The main purpose of the study was to assess the occurrence of wheezing and lung function in non-smoking women exposed to various levels of fine particulate matter(FP) and polycyclic aromatic hydrocarbons (PAH). Out of the total study group, 152 women were included in the lower exposed group (PM_2.5 ≤34.3μg/m³ or PAHs ≤ 22.9ng/ m³) and 96 persons in higher concentrations of both air pollutants (PM_2.5>34.3μg/m³ and PAHs > 22.9ng/ m³). Except for FVC and FEV1, all lung forced ventilatory flows (PEFR, FEF_25% FEF_50%, FEF_75%, FEF_25−75%) were significantly lower in the higher exposed group. The findings suggest bronchoconstriction within the respiratory tract, which may be related to the exposure under study. This was consistent with a higher prevalence of wheezing in more exposed subjects. It was shown that higher levels of both pollutants increased the risk of wheezing by factor 5.6 (95% CI: 1.77–17.8) after accounting for potential confounders such as allergic diseases and exposure to ETS. This study suggests that pollutants in question may have the capacity to promote broncho-constriction and asthmatic symptoms, possibly by bronchial inflammation resulting from the exposure.     

A critical review of the relationship between occupational exposure to diesel emissions and lung cancer risk

In 2012, a working group of the International Agency for Research on Cancer classified diesel exhaust (DE) as a human carcinogen (Group 1). This decision was primarily based on the findings of the Diesel Exhaust in Miners Study (DEMS). The disparity between the results of various methodological approaches applied to the DEMS led to several critical commentaries. An expert panel was subsequently set up by the Health Effects Institute to evaluate the DEMS results, together with a large study in the trucking industry. The panel concluded that both studies provided a useful basis for quantitative risk assessments (QRAs) of DE exposure. However, the results of both studies were non-definitive as the studies suffer from several methodological shortcomings. We conducted a critical review of the studies used by the International Agency for Research on Cancer (IARC) working group to evaluate the relationship between DE and lung cancer. The aim was to assess whether the available studies support the statement of a causal relationship and, secondarily if they could be used for QRA. Our review highlights several methodological flaws in the studies, amongst them overadjustment bias, selection bias, and confounding bias. The conclusion from our review is that the currently published studies provide little evidence for a definite causal link between DE exposure and lung cancer risk. Based on two studies in miners, the DEMS and the German Potash Miners study, QRA may be conducted. However, the DEMS data should be reanalyzed in advance to avoid bias that affects the presently published risk estimates.     

Identification of a critical dose level for risk assessment: developments in benchmark dose analysis of continuous endpoints.

The benchmark dose (BMD) method has been recommended to replace the no-observed-adverse-effect-level (NOAEL) approach in health risk assessment of chemical substances. In the present article, developments in BMD analysis from continuous experimental data are proposed. The suggested approach defines the BMD as the dose at which the slope of the S-shaped dose-response relationship changes the most in the low-dose region. This dose resides in a region where the sensitivity to chemical exposure may start to change noticeably. It is shown that the response (defined as a percent change relative to the magnitude, or size, of response) corresponding to the dose where the slope changes the most depends on the geometrical shape of the dose-response curve; the response becomes lower as the curve becomes more asymmetrical and threshold-like in the low-dose region. Given a symmetrical case, described by the Hill function, the response associated with the critical dose level becomes 21% (defined as a percent change relative to the magnitude, or size, of response). According to a limiting case of asymmetry and threshold-like characteristics, reflected by a Gompertz curve, the response corresponding to the dose of interest becomes as low as 7.3% (defined as a percent change relative to the magnitude, or size, of response). Use of a response in the range of 5-10% when estimating the BMD conservatively accounts for uncertainties associated with the proposed strategy, and may be appropriate in a risk assessment point of view. The present investigation also indicated that a BMD defined according to the suggested procedure may be estimated more precisely relative to BMDs defined under other approaches for continuous data.     

Increasing exposure levels cause an abrupt change in the absorption and metabolism of acutely inhaled benzo(a)pyrene in the isolated, ventilated, and perfused lung of the rat.

The carcinogenic polycyclic aromatic hydrocarbons (PAHs) are active primarily at the site of entry to the body. Lung cancer following inhalation of PAH-containing aerosols such as tobacco smoke is one likely example. A suggested mechanism for this site preference is a slow passage of the highly lipophilic PAHs through the thicker epithelia of the conducting airways, accompanied by substantial local metabolism in airway epithelium. However, it is likely that the airway epithelium will become saturated with PAHs at surprisingly low exposure levels. The purpose of this research was to quantify the level of saturation for inhaled benzo(a)pyrene (BaP) in the isolated, perfused lung (IPL) of the rat. BaP was coated onto carrier particles of silica 3.5 microm diameter at three different levels. The DustGun aerosol generator was then used to deliver respectively 2.2, 36, and 8400 ng of BaP to the IPL with the carrier particles in less than 1 min. For 77 min after the exposure, single-pass perfusate was collected from the lungs. Lungs were then removed and, with the perfusate, analyzed for BaP and metabolites. Results show that the absorption and metabolism of inhaled BaP in the lungs was highly dose dependent. At low exposure levels absorption of BaP in the mucosa was proportional to the concentration in the air/blood barrier and proceeded with substantial local metabolism. At higher exposure levels the capacity of the epithelium to dissolve and metabolize BaP became saturated, and the absorption rate remained constant until crystalline BaP had dissolved, and the process proceeded with much smaller fractions of BaP metabolites produced in the mucosa. This phenomenon may explain the well-known difficulties of inducing lung cancer in laboratory animals with inhalants containing carcinogenic PAHs, where similar lifespan exposures are used as humans may experience but with much higher dose rates.     

Critical review of the human data on short-term nitrogen dioxide (NO2) exposures: Evidence for NO2 no-effect levels

Nitrogen dioxide (NO₂) is a ubiquitous atmospheric pollutant due to the widespread prevalence of both natural and anthropogenic sources, and it can be a respiratory irritant when inhaled at elevated concentrations. Evidence for health effects of ambient NO₂ derives from three types of studies: observational epidemiology, human clinical exposures, and animal toxicology. Our review focuses on the human clinical studies of adverse health effects of short-term NO₂ exposures, given the substantial uncertainties and limitations in interpretation of the other lines of evidence. We examined more than 50 experimental studies of humans inhaling NO₂, finding notably that the reporting of statistically significant changes in lung function and bronchial sensitivity did not show a consistent trend with increasing NO₂ concentrations. Functional changes were generally mild and transient, the reported effects were not uniformly adverse, and they were not usually accompanied by NO₂-dependent increases in symptoms. The available human clinical results do not establish a mechanistic pathway leading to adverse health impacts for short-term NO₂ exposures at levels typical of maximum 1-h concentrations in the present-day ambient environment (i.e., below 0.2 ppm). Our review of these data indicates that a health-protective, short-term NO₂ guideline level for susceptible (and healthy) populations would reflect a policy choice between 0.2 and 0.6 ppm.

Extended abstract

Nitrogen dioxide (NO₂) is a ubiquitous atmospheric pollutant due to the widespread prevalence of both natural and anthropogenic sources, and it can be a respiratory irritant when inhaled at elevated concentrations. Natural NO₂ sources include volcanic action, forest fires, lightning, and the stratosphere; man-made NO₂ emissions derive from fossil fuel combustion and incineration.

The current National Ambient Air Quality Standard (NAAQS) for NO₂, initially established in 1971, is 0.053 ppm (annual average). Ambient concentrations monitored in urban areas in the United States are ~0.015 ppm, as an annual mean, i.e., below the current NAAQS. Short-term (1-h peak) NO₂ concentrations outdoors are not likely to exceed 0.2 ppm, and even 1-h periods exceeding 0.1 ppm are infrequent. Inside homes, 1-h NO₂ peaks, typically arising from gas cooking, can range between 0.4 and 1.5 ppm.

The health effects evidence of relevance to ambient NO₂ derives from three lines of investigation: epidemiology studies, human clinical studies, and animal toxicology studies. The NO₂ epidemiology remains inconsistent and uncertain due to the potential for exposure misclassification, residual confounding, and co-pollutant effects, whereas animal toxicology findings using high levels of NO₂ exposure require extrapolation to humans exposed at low ambient NO₂ levels. Given the limitations and uncertainties in the other lines of health effects evidence, our review thus focused on clinical studies where human volunteers (including asthmatics, children, and elderly) inhaled NO₂ at levels from 0.1 to 3.5 ppm during short-term (½–6-h) exposures, often combined with exercise, and occasionally combined with co-pollutants. We examined the reported biological effects and classified them into (a) lung immune responses and inflammation, (b) lung function changes and airway hyperresponsiveness (AHR), and (c) health effects outside the lungs (extrapulmonary).

We examined more than 50 experimental studies of humans inhaling NO₂, finding that such clinical data on short-term exposure allowed discrimination of NO₂ no-effect levels versus lowest-adverse-effects levels. Our conclusions are summarized by these six points: For lung immune responses and inflammation: (1) healthy subjects exposed to NO₂ below 1 ppm do not show pulmonary inflammation; (2) at 2 ppm for 4?h, neutrophils and cytokines in lung-lavage fluid can increase, but these changes do not necessarily correlate with significant or sustained changes in lung function; (3) there is no consistent evidence that NO₂ concentrations below 2 ppm increase susceptibility to viral infection; (4) for asthmatics and individuals having chronic obstructive pulmonary disease (COPD), NO₂-induced lung inflammation is not expected below 0.6 ppm, although one research group reported enhancement of proinflammatory processes at 0.26 ppm. With regard to NO₂-induced AHR: (5) studies of responses to specific or nonspecific airway challenges (e.g., ragweed, methacholine) suggest that asthmatic individuals were not affected by NO₂ up to about 0.6 ppm, although some sensitive subsets may respond to levels as low as 0.2 ppm. And finally, for extra-pulmonary effects: (6) such effects (e.g., changes in blood chemistry) generally required NO₂ concentrations above 1–2 ppm.

Overall, our review of data from experiments with humans indicates that a health-protective, short-term-average NO₂ guideline level for susceptible populations (and healthy populations) would reflect a policy choice between 0.2 and 0.6 ppm. The available human clinical results do not establish a mechanistic pathway leading to adverse health impacts for short-term NO₂ exposures at levels typical of maximum 1-h concentrations in the present-day ambient environment (i.e., below 0.2 ppm).     

The in vitro and in vivo investigation of a novel small chamber dry powder inhalation delivery system for preclinical dosing to rats

Abstract

Purpose: This research describes a novel “minitower” dry powder delivery system for nose-only delivery of dry powder aerosols to spontaneously breathing rats.

Methods: The minitower system forces pressurized air through pre-filled capsules to deliver aerosolized drug to four nose ports; three of which house spontaneously breathing rats, with the fourth used as a control. Within each port are vent filters which capture drug that was not inhaled for further quantitation. These vent filters along with a novel control system referred to as the “artificial rat lung”, allow for the theoretical amount of drug delivered and subsequently inhaled by each rat to be calculated.

Results: In vitro and in vivo studies have demonstrated this system’s ability to deliver aerosolized drug to rats. The in vitro study showed that ～30% of the starting dose reached the 4 ports and was available for inhalation. During in-vivo studies, rats inhaled ～34% of the delivered dose. Of the estimated inhaled dose, 12–18% was detectable in the various tissue samples, with over 30% of the recovered dose found in the rat’s lungs.

Conclusion: Results show that this system is capable of reproducibly delivering drug to the lungs of spontaneously breathing rats. Advantages over current delivery methods include being amenable to the administration of multiple doses and using less (milligram) amount of starting material. In addition, this technique avoids anesthesia which is typically required for instillation or insufflation, and thus has the potential as an efficient and noninvasive aerosol delivery method for preclinical drug development.     

Development and application of a skin cancer slope factor for exposures to benzo[a]pyrene in soil

Humans may be dermally exposed to the carcinogenic substance benzo[a]pyrene (B[a]P) via contact with soil at contaminated sites. The potential for risk is typically assessed using the proportion of dose estimated to penetrate through exposed skin for comparison with an oral route slope factor. An alternate dermal slope factor of 25 (mg/kg day)(-1) was previously developed (Knafla et al., 2006) based on skin carcinogenicity, since skin painting studies with mice suggest the formation of epidermal tumors may be a more sensitive endpoint than systemic tumors following dermal exposure. An extension of this work resulted in a skin cancer slope factor derived on a per unit skin surface area basis of 3.5 (μg/cm(2)day)(-1) that can be used to estimate risk as a function of exposed surface area. Various factors were examined for interspecies extrapolation of risks from mice to humans and for estimating skin exposures to B[a]P in soil. Using a nominal soil concentration of 1.0mg/kg, a range of cancer risk values of 29-220 in 100,000 was calculated. Soil concentrations associated with a one in 100,000 risk ranged from 0.0046 to 0.035 μg/g, which are lower than those derived using an oral slope factor. These results suggest that B[a]P-related skin cancer (point of contact) risks should be considered at contaminated sites.     

Development of a physiologically based pharmacokinetic model for estradiol in rats and humans: a biologically motivated quantitative framework for evaluating responses to estradiol and other endocrine-active compounds.

A physiologically based pharmacokinetic (PBPK) model for estradiol (E2) in rats and humans (male and female) was developed to provide a quantitative tool for evaluating the importance of physiological parameters on E2 blood and tissue concentration time-course and for predicting blood and tissue concentrations in rats and humans. A hepatic extraction model was developed to evaluate the significance of plasma protein binding on the hepatic extraction of E2 and the approach was integrated into the E2 model. Sufficient data was available to parameterize and validate oral and iv routes. The E2 model simulations of E2 blood and tissue concentrations compared well to experimental values. Estrogen receptor content strongly impacts distribution and elimination kinetics of E2 as well as tissue concentrations. The prolonged terminal elimination phase seen after iv bolus administration reflects the slow release of receptor bound E2 from tissues. E2 uptake behavior in the ovariectomized, but not intact rat uterus, was best described as diffusion-limited. Simulations with the hepatic extraction model predicted extensive binding of E2 to albumin (rat) and SHBG (sex-hormone binding globulin humans), although hepatic extraction does not appear to be restricted to the unbound fraction, implying that the total plasma E2 concentration is important when considering hepatic uptake. Important determinants of E2 disposition are tissue ER content and binding affinity, nonreceptor binding proteins, vascular permeability, partition coefficients, hepatic blood flow, and extrahepatic metabolism. As an integral part of a research program, the quantitative framework developed for E2 can be extended to other endocrine-active compounds (EACs) and used to evaluate the biological activity of EACs.     

Integration of a plasma protein binding factor to the Chemical-Specific Adjustment Factor (CSAF) for facilitating the estimation of uncertainties in interspecies extrapolations when deriving health-based exposure limits for active pharmaceutical ingredients: Investigation of recent drug datasets

The objective was to challenge cross-species extrapolation factors with which to scale animal doses to human by any route for non-carcinogenic endpoints. The conventional hypothesis of the toxicokinetics (TK)-toxicodynamics (TD) relationship was equal toxicity at equal plasma level of the total drug moiety in each species, but this should also follow the free drug assumption, which states that only the unbound drug moiety in plasma may elicit a TD effect in tissue. Therefore, a protein binding factor (PBF) was combined with the Chemical-Specific Adjustment Factor (CSAF) (i.e., CSAF x PBF). The value of PBF of each drug was set equal to the ratio between human and animals of the unbound fraction in plasma (fu_p). Recent drug datasets were investigated. Our results indicate that any CSAF value would be increased or decreased while PBF deviates to the unity, and this required more attention. Accordingly, further testing indicated that the CSAF values set equal to basic allometric uncertainty factors according to the conventional hypothesis (dog∼2, monkey∼3.1, rat∼7, mouse∼12) would increase by including PBF for 30% of the drugs tested that showed a superior fu_p value in human compared to animals. However, default uncertainty factors in the range of 10–100 were less frequently exceeded. Overall, PBF could be combined with any other uncertainty factor to get reliable estimate of CSAF for each bound drug in deriving health-based exposure limits.