Are rat results from intratracheal instillation of 19 granular dusts a reliable basis for predicting cancer risk?

We analyzed the so-called "19-dust-studies" (19-DS) that reported lifetime lung tumor occurrence in female rats following repetitive, short-term intratracheal instillation (ITI) of 19 different insoluble dusts. In the 19-DS, lung instillation of up to 120mg/rat of granular, biopersistent, low-specific-toxicity particles (GBP) caused about half of the rats to develop lung tumors, but the relevance of these data to deriving exposure limits for GBP is uncertain. Specific drawbacks to using the 19-DS for risk assessment include: (1) Delivery, via ITI, of a worker's estimated lifetime lung dose causes "lung overload" in rats, and is not equivalent to lifetime inhalation exposure; (2) The response of rats to insoluble-particle "lung overload" is stereotyped and unique to that species; (3) The 19-DS did not include low-dose studies, and the dose-response showed saturation at the high levels; (4) When the lung-overload threshold is exceeded, rats develop lung tumors from ongoing inflammation (as opposed to particle-specific toxicity); that is, the dramatically increased dose-delivery rate evokes mechanisms not relevant to gradual exposure; and (5) workers historically exposed to potentially lung-overloading burdens of inhaled dust (e.g., coal workers, underground miners using diesel equipment) do not exhibit an established lung-cancer excess. Our critical review of the data from the 19-DS suggests that the reported results for GBP are not a reliable basis for predicting human lung cancer risk, e.g., for the typical inhaled-dose conditions for which worker exposure limits to GBP are promulgated.

What is new in diesel.

We review information from the past 5 years on changes in diesel exhaust (DE) emissions and developments in the study of DE toxicity. New DE technologies have changed the composition of DE considerably, reducing emissions of many of the components of health concern. The increasing similarity of modern diesel and compressed natural-gas engine emissions needs to be reflected in any regulatory analysis. Even for historical DE emissions, considerable study of DE exposure in animals or humans has not produced data useful for risk assessment. DE inhalation exposure in most species (hamsters, guinea pigs, mice) does not produce lung tumors. Inhalation studies of DE in rats found lung tumors only with lung overload, and tumors also occurred when inert dusts were inhaled at overload concentrations. The animal data are reassuring and show that DE is not a concern at ambient exposure levels. Re-analyses of occupational epidemiology have been shown to have serious shortcomings that do not allow the derivation of a quantitative cancer risk. Studies of railroad workers found no dose response; rather cancer risk appeared to decrease for individuals with greater DE exposure. Studies of truck drivers also suffer from serious flaws because of misconceptions about the year that diesel was introduced, lack of an adequate latency period, and the realization that drivers exhibited increased lung cancer risk even prior to the diesel era. Recent industrial hygiene studies of drivers show that DE was not likely to be a primary source of particles or polycyclic aromatic hydrocarbons. Further, there is no dose response across occupations. In fact, the occupation (underground miners) with the highest exposure to DE does not exhibit increased cancer risks. This new information seriously weakens earlier risk characterizations of DE by various regulatory groups. New research and better exposure measurements are needed before a reliable risk assessment of DE can be produced.

Carbon black and soot: two different substances.

Carbon blacks are manufactured under controlled conditions for commercial use primarily in the rubber, painting, and printing industries. In contrast, soots are unwanted byproducts from the combustion of carbon-based materials for the generation of energy or heat, or for the disposal of waste. Unfortunately, the terms carbon black and soot often have been used interchangeably; however, carbon black is physically and chemically distinct from soot. Greater than 97% of carbon black consists of elemental carbon arranged as aciniform particulate. Depending on the type of soot, the relative amount of carbon (< 60% of the total particle mass), the type of particulate carbon, and particle characteristics (size, shape, and heterogeneity) can vary considerably. For both carbon black and soot, other elements and chemical compounds are associated with the particulate carbon. Total inorganics (ash) represent < 1% of the carbon black particle mass. Organic compounds can be extracted from particle surfaces (solvent extractable fraction [SOF]), and for carbon black, also are < 1% of the particle mass. Soots have much greater percentages of ash, SOF, or both, than carbon black. There has been concern about the adsorbed organic compounds because of potential biologic activity. For carbon black, the SOF is strongly adherent to carbon surfaces and is not released by biological fluids. The types of organic compounds consist primarily of unsubstituted polycyclic aromatic hydrocarbons and are not as biologically potent as those compounds present in soot. Thus, carbon black is distinctly different from soot, and when discussing potential health effects, care must be taken to differentiate between the two types of carbon-based particles.

Meta-analysis of rat lung tumors from lifetime inhalation of diesel exhaust.

Estimating the carcinogenic potential of exposure to diesel-engine exhaust particulates (DEPs) is problematic. In rats, high concentrations of DEPs (> 1,000 microg/m(3)) inhaled over a lifetime result in excess lung tumors. However, data for rats exposed to DEP at concentrations not associated with lung overload are consistent with no tumorigenic effect. Individual rat studies have only a limited number of exposure groups; therefore, we combined the tumor data from eight chronic inhalation studies in a meta-analysis. Statistical analysis identified a threshold of response between 200 and 600 microg/m(3) average continuous lifetime exposure, consistent with biological-effect thresholds reported by other investigators. Our exposure-response analysis of all rats with < 600 microg/m(3) average continuous lifetime exposure found no tumorigenic effect of DEP in these rats. When we evaluated all rat studies, accounted for a threshold and for inhomogeneity between experiments, and expressed the results in terms of human unit risk (UR), we found a negative maximum-likelihood human UR of -32 (times) 10(-6) per microgram per cubic meter (microg/m(3)), but this was not statistically significantly different from zero. Extrapolating the rat upper 95th percentile confidence limit to humans gave an upper-bound human UR of 9.3 (times) 10(-6) per microg/m(3)]. This upper-bound human UR, derived from all of the data points (including 1,087 animals below the estimated threshold and 1,433 in the control groups), falls entirely below the range of estimates derived from lung-overloaded rats or from epidemiology of railroad workers. Our meta-analysis of the low-exposure data in rats does not support a lung cancer risk for DEP exposure at nonoverload conditions. Average ambient concentrations of DEP (0-3 microg/m(3)) are < 1% of the concentration associated here with a threshold of tumor response in the rat bioassay.

Comparative mutagenic dose of ambient diesel engine exhaust.

Diesel engine exhaust contains carbon-based particles that can be inhaled and deposited on lung surfaces. Concern about the carcinogenic potential of diesel engine exhaust derives in part from the mutagenic activity of organics that can be extracted from exhaust particles. However, the lung cancer risk is controversial, and diesel exhaust is a candidate for further evaluation. A comparative potency approach can be used to rank the mutagenic risk of diesel exhaust with other combustion products. We compared the specific mutagenic activities of cigarette smoke condensate (CSC) and diesel exhaust particle extract (DEPE) and estimated "mutagenic dose" to the lungs. Although the specific mutagenic activities of CSC and DEPE are similar in magnitude, it is the dose reaching the lungs that is more relevant for comparing mutagenic potential. We calculated that, depending on the source of CSC and DEPE, a person would have to inhale approximately 63 to 181 mg of particulate from diesel engine exhaust to match the mutagenic dose of 1 cigarette. We also calculated that a person would have to breathe diesel exhaust (1.5 microg/m(3), estimated total personal exposure) for 6 to 16 yr to equal the mutagenic dose of 1 cigarette. Although instructive, comparative potency results should be used cautiously due to the need for simplifying assumptions. For example, the type of mutagenic assay and the source of cigarettes and diesel engine exhaust could affect dose estimates to some degree; however, the extent to which diesel particle mutagens are bioavailable would have an even greater effect on estimates of relative risk. For both cigarette smoke and diesel exhaust particles, we assumed that the organic mutagens are 100% bioavailable. In summary, our analysis showed a larger mutagenic dose-to-target-tissues in the smoke of one cigarette as compared to a year of exposure to diesel exhaust particulate at ambient levels.

Estimating dermal transfer from PCB-contaminated porous surfaces.

Health risks posed by dermal contact with PCB-contaminated porous surfaces have not been directly demonstrated and are difficult to estimate indirectly. Surface contamination by organic compounds is commonly assessed by collecting wipe samples with hexane as the solvent. However, for porous surfaces, hexane wipe characterization is of limited direct use when estimating potential human exposure. Particularly for porous surfaces, the relationship between the amount of organic material collected by hexane and the amount actually picked up by, for example, a person's hand touch is unknown. To better mimic PCB pickup by casual hand contact with contaminated concrete surfaces, we used alternate solvents and wipe application methods that more closely mimic casual dermal contact. Our sampling results were compared to PCB pickup using hexane-wetted wipes and the standard rubbing protocol. Dry and oil-wetted samples, applied without rubbing, picked up less than 1% of the PCBs picked up by the standard hexane procedure; with rubbing, they picked up about 2%. Without rubbing, saline-wetted wipes picked up 2.5%; with rubbing, they picked up about 12%. While the nature of dermal contact with a contaminated surface cannot be perfectly reproduced with a wipe sample, our results with alternate wiping solvents and rubbing methods more closely mimic hand contact than the standard hexane wipe protocol. The relative pickup estimates presented in this paper can be used in conjunction with site-specific PCB hexane wipe results to estimate dermal pickup rates at sites with PCB-contaminated concrete.

Issues in setting health-based cleanup levels for arsenic in soil.

Health risk assessments often do not take into account the unique aspects of evaluating exposures to arsenic in soil. For example, risks from ingestion of arsenic in soil are often based on toxicity factors derived from studies of arsenic (soluble arsenate or arsenite) in drinking water. However, the toxicity of arsenic in drinking water cannot be directly extrapolated to toxicity of soil arsenic because of differences in chemical form, bioavailability, and excretion kinetics. Because of the differences between soil arsenic and water arsenic, we conclude that risks from arsenic in soil are lower than what would be calculated using default toxicity values for arsenic in drinking water. Site-specific risk assessments for arsenic in soil can be improved by characterizing the form of arsenic in soil, by conducting animal feeding or in vitro bioavailability studies using site soils, and by conducting studies to evaluate the relationship between urinary arsenic and soil arsenic levels. Such data could be used to more accurately measure the contribution that soil arsenic makes to total intake of arsenic. Available data suggest that arsenic usually makes a small contribution to this total.

Can low-level 50/60 Hz electric and magnetic fields cause biological effects?

Some epidemiological studies have suggested that exposure to ambient, low-level 50/60 Hz electric and magnetic fields (EMFs) increases risk of disease. Whether this association has a causal basis depends in part on whether the electrical, chemical and mechanical "signals" induced within living cells by ambient EMFs are detectable in the complex milieu of voltages, currents and forces present within the living organism. Magnetic responsiveness has been found in some animals and bacteria; aquatic animals (e.g. sharks and rays) can sense weak electric fields. We outline the physics of several mechanisms by which EMFs may interact: (1) Energy transfer by acceleration of ions and charged proteins modifies cell membranes and receptor proteins; however, EMF energies are far below those typical of biomolecules in the cell. (2) Electric fields induced inside the body exert force on electric charges and electric moments; however, these forces are considerably smaller than typical biological forces. (3) The magnetic moments of ferromagnetic particles and free radical molecules interact with magnetic fields, but magnetic-moment sensory cells have not been found in humans, and modification of radical recombination rates by EMFs in a biological system is highly problematic. (4) Resonant interactions involve EMFs driving vibrational or orbital transitions in ion-biomolecule complexes; these mechanisms conflict with accepted physics, and many experimental tests have not found the predicted effects. (5) Temporal averaging or spatial summation can improve the ratio of "signal" to "noise" in any system, but this "mechanism" requires biological structures and neural processes having the necessary capabilities of EMF detection and temporal averaging that have not been found in humans. In summary, biological effects in humans due to extremely low-frequency EMFs of the order of those found in residential environments [< or = 2 microT (< or = 20 mG)] are implausible based on current understanding of physics and biology. Biological effects in humans at higher fields [> 10 microT (> 100 mG)] might reach plausibility as a result of time-averaging in combination with a magnetic-moment transduction mechanism; but even here, neither specialized EMF transduction structures nor appropriate averaging networks have been demonstrated. The bypothesis that the epidemiological associations observed between 50/60 Hz EMFs and disease reflect a causal relationship is not supported by what is known about mechanisms.

Radio frequency radiation (RFR): the nature of exposure and carcinogenic potential.

Epidemiologic evidence on the relation between radio-frequency radiation (RFR) and cancer is reviewed. Radio-wave communications are used extensively in modern society; thus, we are all subject to RFR created by radio, television, wireless telephony, emergency communications, radar, etc. Interest in the health effects of RFR has been motivated by the rapid growth in wireless communications and by media reports expressing concern that specific diseases may be caused by RFR exposure, e.g., from cellular telephone handsets. Due to the ubiquitous presence of RFR, the public health implication of any connection between RFR and cancer risk is potentially significant. (It is important to keep RFR distinct from power-line electromagnetic fields.) Comparison of potential risks from RFR exposure with other occupational and environmental health risks requires evaluating the level of support from available epidemiology, from studies with laboratory animals, and from mechanistic or biophysical information about the interaction of RFR with living tissues. A large number of studies have been done with laboratory animals and with in vitro systems; a more limited set of epidemiologic studies is available. Effects from RFR exposure that lead to temperature increases have been consistently reported, but 'non-thermal' effects have not been substantiated. Also, there are no mechanistic theories that support 'non-thermal' interactions with biology. Evidence to support a causal relationship between exposure to RFR and human cancers is scant. Our present state of knowledge about exposure, mechanisms, epidemiology, and animal studies does not identify significant cancer risks.

Lung cancer rates in carbon-black workers are discordant with predictions from rat bioassay data.

The recent demonstration of lung tumors in rats chronically exposed to airborne carbon-black particles has prompted reevaluation of the carcinogenicity of carbon black. However, accumulating evidence suggests that rat responses to inhaled particles are unique and are mediated by the sequelae to lung overload conditions. To test the predictive value of the rat inhalation bioassay for human lung cancer risk, we examined epidemiologic studies of workers exposed to carbon black during its manufacture or use. Industries in which significant airborne carbon-black exposure has occurred were not associated with increased lung cancer risk. We also predicted the number of occupational lung cancer cases that one would expect in exposed workers, based on the cancer potency of carbon black derived from rat studies. Our quantitative comparison of the tumorigenicity of carbon black predicted from rat studies to the lung cancer rate in carbon-black workers showed a marked discrepancy between the lung cancers predicted and those actually observed. We found that far more lung cancers are predicted from the rat bioassay than can be demonstrated in workers. We determined that it is highly unlikely that this discrepancy is due to chance. We conclude that extrapolating the incidence of lung tumors in rats inhaling inert, insoluble particles, such as carbon black, to humans must be seriously questioned. Using rat inhalation bioassay data for carbon black to estimate lung tumor risk in humans must be seriously questioned unless the mechanisms of the rat's unique response are shown to be relevant to humans.

Analysis of diesel-exhaust unit-risk estimates derived from animal bioassays.

Chronic inhalation of diesel exhaust (DE) causes lung tumors in rats; epidemiologic studies suggest that DE may be a potential human carcinogen. We compared the DE unit risks for human lung cancer as derived by Smith and Stayner (1991), Hattis and Silver (1992), Pepelko and Chen (1993), California Environmental Protection Agency, Office of Environmental Health Hazard Assessment (1994), and the U.S. Environmental Protection Agency (USEPA) (1994). All five sets of authors used identical rat bioassay data (Mauderly et al., 1987). Although different in detail, the dose-response models were uniformly linear and nonthreshold. However, each set of authors chose a different approach in relating the rat data to DE unit risk in humans. The predicted unit risks for continuous lifetime exposure to 1 microgram/m3 DE differed by an 80-fold factor between the highest [8 x 10(-4), Hattis and Silver] and the lowest [0.1 x 10(-4), Pepelko and Chen]. The choice of dose-input parameters and how each group treated particle overload were the major factors affecting the different risk estimates. Several unanswered questions undermine the current use of the rat bioassay for DE risk assessment: (1) Differences in emission products and exposure scenarios between laboratory studies and human exposure, (2) occurrence of an apparent threshold in the rat-lung-tumor response, (3) uncertainty as to the appropriate lung-dose metric and its low-dose extrapolation, (4) the role of lung overload, and (5) the cause of species-specific biological susceptibility.

Electric and magnetic fields (EMF): what do we know about the health effects?

Because we use electric power throughout our homes and workplaces, we are all subject to electric and magnetic fields (EMF) that are created by the voltages and currents present in electrical conductors and electrical equipment. Interest in the health effects of electric-power EMF has been motivated by several epidemiologic studies showing weak associations between surrogate measures of low-level EMF exposure and both childhood cancers and adult cancers. Due to the ubiquitous presence of electric power, the public health implications of this association are potentially very significant; however, good evidence for a causal relationship between exposure to EMF and any health effect has not been found. Yet, uncertainty, plus the suggestive epidemiologic findings, have fueled public anxiety and media attention. To put this potential health threat into perspective, it is important to consider the strength of the epidemiology, the availability of supporting animal studies, and mechanistic or biophysical information about the interaction of EMF with matter. Any discussion of steps that might be taken to avoid potential EMF health-effects needs to incorporate a perspective on how hypothetical EMF risks relate to other hazards to life and health.

Designing EMF experiments: what is required to characterize "exposure"?

Anyone who has attempted to organize and synthesize the results of research on biological effects of electric and magnetic fields (EMF) has experienced frustration when trying to evaluate the comparability of EMF exposures among separate studies. Reporting of exposure characteristics is often incomplete, and some investigators focus on particular nuances of exposure, which in other laboratories go unrecorded because they are not regarded as important. The obstacles encountered when comparing studies, when designing replication studies, and when evaluating research proposals could be reduced were a more standardized approach taken in describing "EMF exposure." To this end, a numerical listing of 18 separate parameters important to EMF exposure characterization is proposed. Although the goal of this list is primarily to expedite the description of EMF exposure, references are provided to examples of EMF exposures and to detailed discussions of EMF exposure systems.

Reactive oxygen in skeletal muscle. I. Intracellular oxidant kinetics and fatigue in vitro.

We hypothesized that muscle fiber bundles produce reactive oxygen intermediates and that reactive oxidant species contribute to muscular fatigue in vitro. Fiber bundles from rat diaphragm were mounted in chambers containing Krebs-Ringer solution. In studies of intracellular oxidant kinetics, bundles were loaded with 2',7'-dichlorofluorescin, a fluorochrome that emits at 520 nm when oxidized; emissions were quantified using a fluorescence microscope. Emissions from unstimulated muscles increased over time (P < 0.001). Accumulation of fluorescence was slowed by addition of catalase (P < 0.001) or superoxide dismutase (P < 0.001) and was accelerated by repetitive muscular contraction (P < 0.05). To determine effects of reactive oxygen intermediates on fatigue, curarized bundles were stimulated to contract isometrically; force was measured. Catalase, superoxide dismutase, and dimethyl sulfoxide were screened for effects on low- and high-frequency fatigue. Antioxidants inhibited low-frequency fatigue [after 5 min of repetitive contractions, force at 30 Hz was 20% greater than control (P < 0.015)] and increased the variability of fatigue at 30 Hz (P < 0.03). Antioxidants did not alter high-frequency (200-Hz) fatigue. We conclude that 1) diaphragm fiber bundles produce reactive oxygen intermediates, including O2-. and H2O2; 2) muscular contraction increases intracellular oxidant levels; and 3) reactive oxygen intermediates promote low-frequency fatigue in this preparation.

Heterogeneity of phagocytosis for inhaled versus instilled material.

Animal experiments involving lung exposure to particles and toxins often use intratracheal instillation, although inhalation is more physiologically relevant to human respiratory tract exposures. Using nontoxic magnetic particles, we examined the particle content distribution within the macrophage population lavaged from the lungs of Syrian golden hamsters exposed either by inhalation or by intratracheal instillation. One to 3 days after iron oxide particle delivery to the lungs, lung macrophages were harvested from animals in pairs, one exposed by inhalation and one exposed by instillation. The macrophages were then magnetically fractionated according to magnetic iron oxide content by flowing the cell suspension through tubing placed within the magnetic field gradient of an electromagnet. Macrophages that had ingested the largest quantity of magnetic iron oxide preferentially collected adjacent to the tubing wall when the electromagnet was at its lowest current; those with fewer particulates collected at correspondingly higher amperages; those cells that were never pulled out of the flowing stream had little or no magnetic iron oxide content. Our results showed that 80% of the lavaged cell population from animals exposed by inhalation had measurable particle content, whereas by instillation, many cells (70%) received no dose at all. Exposure by inhalation produced a more even distribution of dose among lung macrophages. Macrophage intracellular motions showed a decrease when cell content exceeded 7 to 8% of cell volume. We conclude that the method of particle delivery to lungs can influence the dose distribution among lung macrophages.

Kinetics of phagocytosis and phagosome-lysosome fusion in hamster lung and peritoneal macrophages.

The time course of phagocytosis and phagosome-lysosome fusion (PLF) in lung and peritoneal macrophages (LMs and PMs) was measured. Lysosomes in unelicited hamster LMs and PMs were labeled with lucifer yellow. Macrophages then phagocytized heat-killed Saccharomyces cerevisiae (yeast) and were evaluated at several time points for the degree to which yeast particles were adherent vs. internalized and for the presence or absence of PLF as based on the presence or absence of lucifer yellow in yeast-containing phagosomes. A three-compartment model (adherent, ingested, fused) of independent phagocytosis and PLF was developed; the number of yeast particles in each compartment was counted, and rate constants for ingestion and fusion were determined. Comparison of rate constants showed that ingestion was significantly faster in PMs (0.047 +/- 0.005 min-1) than in LMs (0.016 +/- 0.005 min-1) (mean +/- pooled SEM; P less than 0.001). Similarly, PLF was significantly faster in PMs (0.109 +/- 0.013 min-1) than in LMs (0.046 +/- 0.013 min-1) (P less than 0.003).

Viscoelastic and motile properties of hamster lung and peritoneal macrophages.

The motile and rheologic properties of hamster lung and peritoneal macrophages (LMs and PMs) were examined by following the motions of magnetizable iron oxide (gamma-Fe2O3) particles contained within phagolysosomes of these cells. As a measure of intracellular motility, gamma-Fe2O3 particles in cells were magnetically aligned and the decay rate of the remanent magnetic field (RMF) in the direction of initial magnetization was monitored over time. Cytoplasmic rheology was measured by twisting the intracellular particles with a magnetic field (Btw) applied perpendicularly to the direction of initial magnetization. We measured changes in the RMF associated with application and removal of Btw. Intracellular motility in LMs and PMs was not significantly different (P greater than 0.20); similarly, cytoplasmic viscosity was not significantly different in LMs and PMs (P greater than 0.12); deformation on application of torque was significantly greater (P less than 0.0001) and elastic recoil on removal of torque was significantly smaller (P less than 0.0001) in PMs than in LMs; and by qualitative observation, the yield stress of cytoplasm (associated with a plastic, nonrecoverable deformation) was lower in PMs than in LMs. These results show that although cytoplasmic motion and viscosity are similar in the two cell types, PM cytoplasm is less stiff than LM cytoplasm as determined by yield stress.

Cell organelle motions in bronchoalveolar lavage macrophages from smokers and nonsmokers.

The migratory and phagocytic capabilities of pulmonary macrophages are important elements in lung defense against particles and pathogens deposited on alveolar surfaces. Both functions rely on macrophage cytoplasmic movements. We examined how a common respiratory exposure, cigarette smoking, affects intracellular motions in human pulmonary macrophages (HPM phi). We observed that HPM phi isolated by bronchoalveolar lavage from human volunteers and cultured in vitro were capable of ingesting unopsonized magnetic iron oxide particles localized within phagosomes and phagolysosomes. Upon magnetization, the particles collectively produced a remanent magnetic field (RMF). Motions of particle-containing organelles caused a decay of the RMF, or "relaxation." We applied this technique, which is an alternative to optical microscopy, for evaluating both movement and viscosity of macrophage cytoplasm. In our in vitro studies, we found that HPM phi isolated from smokers exhibited more rapid RMF decay than did HPM phi isolated from nonsmokers. Rotating the intracellular magnetic particles with external fields showed that the HPM phi cytosol was highly viscous in both smoker and nonsmoker cells. In both cell groups, resistance to particle rotation was increased by 2.6 ng/ml phorbol myristate acetate. Our in vitro magnetometric quantification of intracellular particle movement in isolated HPM phi suggests that lung macrophage cell organelle motions are increased in smokers; this may be the mechanism for the enhanced in vivo magnetic-particle motion reported previously in magnetometric studies of human subjects who are smokers.