The inflammatory response in lungs of rats exposed on the airborne particles collected during different seasons in four European cities.

Epidemiological studies have reported associations of ambient particulate air pollution, especially particulate matter (PM) less than 10 µm with exacerbations of asthma and chronic obstructive pulmonary disease. In an in vivo model, we have tested the toxicity of urban airborne particles collected during spring, summer, and winter seasons in four cities (Amsterdam, Lodz, Oslo, and Rome) spread across Europe. The seasonal differences in inflammatory responses were striking, and almost all the study parameters were affected by PM. Coarse fractions of the urban particle samples were less potent per unit mass than the fine fractions in increasing cytokine [macrophage inflammatory protein (MIP)-2 and tumor necrosis factor (TNF)-α] levels and in reducing Clara-cell secretory protein (CC16) levels. This study shows that PM collected at 4 contrasting sites across Europe and during different seasons have differences in toxic potency. These differences were even more prominent between the fine and coarse fractions of the PM.

Fine ambient particles from various sites in europe exerted a greater IgE adjuvant effect than coarse ambient particles in a mouse model.

In the European Union (EU)-funded project Respiratory Allergy and Inflammation due to Ambient Particles (RAIAP), coarse and fine ambient particulate matter (PM) was collected at traffic dominated locations in Oslo, Rome, Lodz, and Amsterdam, in the spring, summer, and winter 2001/2002. PM was also collected in de Zilk, a rural seaside background location in the Netherlands. The aim of this study was to screen the ambient PM fractions for allergy adjuvant activity measured as the production of allergen- (ovalbumin-) specific immunoglobulin (Ig) E following subcutaneous (sc) injection into the footpad of mice. A second aim was to determine whether the 6-d popliteal lymph node (PLN) assay can be used to detect an allergy adjuvant activity. Allergy screening for IgE adjuvant activity showed that in the presence of ovalbumin (Ova) 12 out of 13 of the fine ambient PM fractions exerted a significant IgE adjuvant activity. In contrast, only 3 out of 13 of the coarse PM fractions had significant adjuvant activity. Overall, fine ambient PM exerted significantly greater IgE adjuvant activity per unit mass than coarse PM. No significant differences were observed between locations or seasons. Substantial higher levels of specific components of PM such as vanadium (V), nickel (Ni), zinc (Zn), ammonium (NH(4)), and sulfate (SO(4)) were present in the fine compared to coarse PM fractions. However, differences in the content of these components among fine PM fractions did not reflect the variation in the levels of IgE anti-Ova. Still, when comparing all seasons overall, positive correlations were observed between V, Ni, and SO(4) and the allergen specific IgE levels. The PLN responses (weight and cell number) to Ova and ambient PM in combination were significantly higher than to Ova or PM alone. Still, the PLN assay appears not to be useful as a quantitative assay for screening of allergy adjuvant activity since no correlation was observed between PLN responses and allergen specific IgE levels. In conclusion, fine ambient PM fractions consistently were found to increase the allergen-specific IgE responses more than the coarse ones. Our finding is in agreement with the notion that traffic-related air pollution contributes to the disease burden in asthma and allergy, and points to fine and ultrafine ambient PM as the most important fractions in relation to allergic diseases.

Occupational exposure to hexabromocyclododecane at an industrial plant.

Occupational exposure to hexabromocyclododecane (HBCD) among workers at an industrial plant producing expandable polystyrene (PS) added HBCD as flame retardant has been assessed in the present study. Airborne dust samples were collected near the breathing zone of 10 male workers during three 8-h work shifts. The HBCD concentrations in the airborne dust varied from 0.2 to 150 microg/m3 (mean 12.2 and median 2.1 microg/m3). Two serum samples were obtained from each of the workers. The mean serum concentration was 190 ng/g lipids; the median was 101 ng/g lipids (range 6 to 856 ng/g lipids). HBCD was not detected above 1 ng/g lipids (LOD) in any samples from persons in a reference group with no occupational exposure to HBCD. The contribution of gamma-HBCD to the total HBCD serum concentration was notably high (39%) compared to what has usually been observed in biological samples. There was no clear correlation of serum levels with average HBCD concentrations in the airborne dust samples collected near the subjects' breathing zone. The elevated exposure levels reported in this study compared to urban air and serum levels in general populations suggest that further and more detailed exposure assessment studies should be initiated in industries where HBCD is applied.

From science to policy--when are scientific results certain enough?

The amount of scientific findings linking air pollution with adverse health effects is continuously growing and indicates a need for action to improve air quality. On 21 September 2005, the European Commission published a new draft directive on air quality, as part of the Thematic Strategy on air pollution. This is a long-term plan on how to reduce air pollution in the European Union in the next 15 years. Immediately after its release, the Commission received criticism for not going far enough from various instances, such as research institutions and nongovernmental organizations concerned with health and environmental effects of air pollution. One policy argument for not taking full measures was the argument of scientific uncertainty. In light of this air quality strategy and the corresponding criticism which ensued, the present article discusses how the ambiguity of scientific uncertainty may contribute to impeding the process of translating scientific findings into concrete policy options. As complete certainty is likely to never be achieved, the question arises whether it is possible to determine and agree on clear and applicable definitions of certain levels of scientific certainty. The case referred to in this paper clearly demonstrates a situation with discordant views on the uncertainty of scientific findings. More discussion on how to define scientific uncertainty and how to deal with it would be beneficial for both the scientific and the political communities. Finally, it is important to recognize that scientific evidence is not the only driver influencing policy decisions.

Relation between sources of particulate air pollution and biological effect parameters in samples from four European cities: an exploratory study.

Given that there are widely different prevalence rates of respiratory allergies and asthma between the countries of Europe and that exposure to ambient particulate matter (PM) is substantial in urban environments throughout Europe, an EU project entitled "Respiratory Allergy and Inflammation Due to Ambient Particles" (RAIAP) was set up. The project focused on the role of physical and chemical composition of PM on release of cytokines of cells in vitro, on respiratory inflammation in vivo, and on adjuvant potency in allergy animal models. Coarse (2.5-10 microm) and fine (0.15-2.5 microm) particles were collected during the spring, summer and winter in Rome (I), Oslo (N), Lodz (PL), and Amsterdam (NL). Markers within the same model were often well correlated. Markers of inflammation in the in vitro and in vivo models also showed a high degree of correlation. In contrast, correlation between parameters in the different allergy models and between allergy and inflammation markers was generally poor. This suggests that various bioassays are needed to assess the potential hazard of PM. The present study also showed that by clustering chemical constituents of PM based on the overall response pattern in the bioassays, five distinct groups could be identified. The clusters of traffic, industrial combustion and/or incinerators (TICI), and combustion of black and brown coal/wood smoke (BBCW) were associated primarily with adjuvant activity for respiratory allergy, whereas clusters of crustal of material (CM) and sea spray (SS) are predominantly associated with measures for inflammation and acute toxicity. The cluster of secondary inorganic aerosol and long-range transport aerosol (SIALT) was exclusive associated with systemic allergy. The present study has shown that biological effect of PM can be linked to one or more PM emission sources and that this linkage requires a wide range of bioassays.

Toxicological comments to the discussion about REACH.

It is the ultimate goal of the intended REACH process (Registration, Evaluation and Authorization of Chemicals) of the European Union to identify substances of hazardous properties and to evaluate the risks of human and environmental exposure. During the last few months there has been a controversial discussion as to what extent in vitro studies and consideration of structure activity relationship provide sufficient information to waive repeated exposure studies. Industry as well as certain regulatory agencies or NGOs support this approach and propose that repeated dose studies may only be required beyond 100 t/a. From a toxicological point of view it has to be stressed that this discussion primarily considers the cost reduction and protection of animals, whereas protection of human health and the environment are secondary. In vitro studies only allow identification of specific hazardous properties which can be detected by the specific test system. Moreover, appropriate information on the dose response of adverse effects, identification of thresholds and NOELs that are essential for risk characterization cannot be obtained from these studies. Consequently, identification of all relevant hazardous properties and endpoints of adverse effects can only be determined in the intact animal by repeated dose studies such as 28-day or 90-day studies. In the absence of such information the hazard identification is incomplete and there is no basis for appropriate risk assessment of human exposure. Thus, any waiving of repeated dose studies in animals bears the probability of unforeseen effects in case of acute or continuous human exposure. From this the undersigning European Toxicologists conclude: 1. The intention of REACH is to identify hazardous properties in order that a reliable risk assessment can be made and measures taken to deal with chemicals posing a significant risk. 2. The recent debate has centered on ways in which the well established in vivo methods for risk assessment can be bypassed. 3. The evidence that the available alternatives would support such replacement is weak. Progress to improve their value for risk assessment purposes is bound to be slow because the issues are very complex. As a group of European Toxicologists we strongly support the need for more research support in these areas, but we believe that over claims for progress is damaging their development. 4. Under the circumstances only two options are available: to reduce very substantially the estimation of hazard and risk with inevitable adverse consequences for human health and environmental protection, or to continue the existing methods until properly validated new methods are available.

Recent outcomes in European multicentre projects on ambient particulate air pollution.

The adverse health effects associated with ambient air pollution have triggered epidemiologists, toxicologists and chemists to combine their experience to investigate the toxicity of ambient PM (particulate matter) from European sites with differing traffic intensity, in order to increase the understanding of the role of fine and coarse PM, the role of chemical characteristics and relate that to health effects. Under the European Union 5th Framework Programme (FP5), the HEPMEAP, RAIAP and PAMCHAR projects have utilised high-volume samplers to collect PM in European locations with contrasting PM sources and performed a range of different laboratory investigations. The PM investigated generally induced significant biological responses, with both coarse (2.5-10 microm) and fine (0.1-2.5 microm) PM being able to induce toxic effects. The chemical composition of the PM (also reflecting the differences in the emission-source contribution) has been suggested to play an important role in these responses. Oxidative and immune effects have been demonstrated in several in vitro and animal models. Investigations have also given support for the assumption that asthmatic and elderly subjects with chronic obstructive pulmonary disease may be more susceptible to PM exposure.

Cytokine release from alveolar macrophages exposed to ambient particulate matter: heterogeneity in relation to size, city and season.

BACKGROUND: Several studies have demonstrated an association between exposure to ambient particulate matter (PM) and respiratory and cardiovascular diseases. Inflammation seems to play an important role in the observed health effects. However, the predominant particle component(s) that drives the inflammation is still not fully clarified. In this study representative coarse (2.5-10 microm) and fine (0.1-2.5 microm) particulate samples from a western, an eastern, a northern and a southern European city (Amsterdam, Lodz, Oslo and Rome) were collected during three seasons (spring, summer and winter). All fractions were investigated with respect to cytokine-inducing potential in primary macrophages isolated from rat lung. The results were related to the physical and chemical parameters of the samples in order to disclose possible connections between inflammatory potential and specific characteristics of the particles. RESULTS: Compared on a gram-by gram basis, both site-specific and seasonal variations in the PM-induced cytokine responses were demonstrated. The samples collected in the eastern (Lodz) and southern (Rome) cities appeared to be the most potent. Seasonal variation was most obvious with the samples from Lodz, with the highest responses induced by the spring and summer samples. The site-specific or seasonal variation in cytokine release could not be attributed to variations in any of the chemical parameters. Coarse fractions from all cities were more potent to induce the inflammatory cytokines interleukin-6 and tumour necrosis factor-alpha than the corresponding fine fractions. Higher levels of specific elements such as iron and copper, some polycyclic aromatic hydrocarbons (PAHs) and endotoxin/lipopolysaccaride seemed to be prevalent in the coarse fractions. However, variations in the content of these components did not reflect the variation in cytokine release induced by the different coarse fractions. Addition of polymyxin B did not affect the particle-induced cytokine release, indicating that the variations in potency among the coarse fractions are not explained by endootoxin. CONCLUSION: The inflammatory potential of ambient PM demonstrated heterogeneity in relation to city and season. The coarse particle fractions were consistently more potent than the respective fine fractions. Though a higher level of some elements, PAH and endotoxin was found in the coarse fractions, the presence of specific components was not sufficient to explain all variations in PM-induced cytokine release.

Past challenges faced: an overview of current educational activities of IUTOX.

Over the past decade, educational programmes have been the main focus of the activities of the International Union of Toxicology (IUTOX). The IUTOX educational programmes are dynamic and have been growing in scope and frequency each year. It is envisaged that this growth will continue with guidance from our member societies and the continuing support of our sponsors. Presently, IUTOX is engaged in the following educational programmes: (1) International congresses that provide the opportunity for direct communication of current toxicological information. Fellowships are sponsored to facilitate attendance at these congresses for toxicologists in need. (2) Workshops that permit interaction on a more localised level of topics of more regional interest. Workshops have served to help stimulate formation of toxicology societies by bringing together sufficient scientists to facilitate these discussions. (3) Continuing educational (CE) programmes at member society meetings. Topics are prioritized based on input received from the local societies. Programmes often are those from CE courses given at meetings, such as conferences of the US Society of Toxicology (US SOT) and EUROTOX from the previous year. (4) Biennial Risk Assessment Summer School (RASS), an intensive week-long interaction between senior toxicologists who serve as faculty with attendees providing individual training. (5) Dissemination of donated printed toxicological books from publishers and syllabi from continuing education courses to regional locations. (6) Web-based interactive training programmes in regions where formal toxicological educational programmes are limited or lacking. (7) Preparation and distribution of monographs on selected topics of very current interest. Monographs on environmental oestrogens and genetically-modified foods have been published. The recent activities in each of these programmes are reviewed in this paper.

Comparison of carcinogenic and in vivo genotoxic potency estimates.

Mutagenic substances classified as carcinogens are primarily regulated on the basis of their carcinogenic effect. Regulation of mutagens that have not been tested for carcinogenicity represents a problem. In cases where a threshold cannot be identified, the substances may be banned or if their uses are deemed to be unavoidable, the exposure may be reduced to as low as technically and economically feasible. In an attempt to develop a procedure that may be helpful in regulation of mutagenic substances when studies on carcinogenicity are lacking, we have compared the lowest effective dose (LED) giving a response in an in vivo genotoxic test after oral or inhalation exposure with the carcinogenic dose descriptor T25 (the chronic daily dose which will give 25% of the animals tumours above background at a specific tissue site). The 34 carcinogens in the present analysis for which genotoxic mechanisms are likely or possible, represent different classes of carcinogens and different genotoxic endpoints, exhibiting carcinogenic and mutagenic potencies both covering a range of 10,000 between the most and least potent substances. A linear correlation was found between the lowest effective dose for in vivo genotoxicity after oral administration or inhalation exposure and the lowest dose descriptor T25 for tumour formation. The finding that the median of the ratio LED/T25 was 1.05 and that the ratio for 90% of the substances studied fell in the range 0.21 to 9.2 shows that the numerical value of LED is similar to the numerical value of T25 within a factor of 5-10. The results suggest that LED may be used as a basis for regulation of mutagens in cases where a threshold cannot be demonstrated or inferred, and where the substance has not been studied in long-term carcinogenicity studies. In such cases LED divided by a specified assessment factor may represent a virtually safe level or a tolerable risk level for a possible carcinogenic effect.

Comparison of carcinogen hazard characterisation based on animal studies and epidemiology.

Recently we have described a simple method for quantitative risk assessment of non-threshold carcinogens based on the dose descriptor T25. In the present report quantitative hazard estimates calculated with the T25 method have been compared with results obtained using quantitative methods based on epidemiological studies. "Known" and "Likely/Probably" human carcinogens were identified from the US EPA database IRIS. In cases were the hazard characterisation was performed on the basis of epidemiological studies, the IARC monographs were used to identify animal studies by oral or inhalation exposure suitable for hazard characterisation by the T25 method. Six agents were identified: benzene, benzidine, 1,3-butadiene, cadmium, nickel subsulfide and vinyl chloride for which US EPA had made their hazard estimation based on epidemiological data. Animal data suitable for hazard characterisation were also available. For comparing hazard characterizations based on epidemiological and animal data, it was pragmatically decided to do this by comparing the chronic doses expressed as those representing a lifetime cancer hazard of 10(-3). In all cases the difference between the chronic doses determined from animal studies by the T25 method differed from those determined from epidemiological studies by a factor of less than three. Although a limited number of carcinogens were studied, the results demonstrate a very good agreement between the hazard characterisation based on epidemiological data and animal experiments over a range of more than 10(4).

Ambient air particles from four European cities increase the primary cellular response to allergen in the draining lymph node.

In the RAIAP (respiratory allergy and inflammation due to ambient particles) project, qualitative properties of ambient air particles from Amsterdam, Oslo, Lodz and Rome were investigated in relation to inflammation and allergy. Most collected particle fractions were found to increase the allergen-specific IgE and IgG2a responses after subcutaneous injection of particles with allergen in mice. However, some fractions appeared to skew the antibody response towards more Th1- or Th2-associated antibody isotypes, and the fine fractions were found to be more potent than the coarse fractions with regard to IgE adjuvant activity. In the present study we investigated the cellular response in the draining lymph node 5 days after a subcutaneous injection of selected RAIAP particle fractions. The particles (100 microg) were injected into both hind footpads of BALB/cA mice, in the presence or absence of the allergen ovalbumin (OVA, 50 microg). We also studied if the coarse and fine RAIAP particle fractions affected the cellular responses to OVA differently. The number of lymph node cells, as well as the relative number of B and T lymphocytes and T helper cells were determined. Expression of cell surface molecules (MHC class II, CD86 and CD23) and ex vivo cytokine production (IL-4, IL-10 and IFN-gamma) by the lymph node cells were measured. Overall, particles in the presence of allergen enhanced the levels of the various cellular parameters compared to allergen alone or particles alone. In the absence of allergen, ambient air particles, in contrast to diesel exhaust particles, marginally affected some cellular parameters. By histological examination of the lymph node, the particles appeared to be scattered between the lymphocytes, often localised within macrophage-like (acid phosphatase positive) cells. The cell parameters measured could, for the individual sample, neither predict the degree of a Th2- or Th1-skewed antibody response, nor the stronger antibody adjuvant capacity of the fine than the coarse particle fractions. In conclusion, we have shown that coarse and fine ambient air particles from different European cities enhance the cellular response in the draining lymph node after injection with an allergen. In the absence of allergen, ambient particles only marginally affected the cellular parameters.

Respiratory allergy adjuvant and inflammatory effects of urban ambient particles.

PM(10) and PM(2.5) fractions were collected by high-volume cascade impactors during 4-week periods in spring, summer and winter seasons in Amsterdam, Lodz, Oslo and Rome and at a Dutch seaside site. The samples were screened for respiratory allergy potential with the mouse popliteal lymph node (PLN) and the ELISA-based IgE antibody assays. For inflammatory screening, release of the cytokine macrophage inflammatory protein-2 (MIP-2) from primary rat type 2 cells was determined. Most fractions gave an increase in the lymph node response with the model allergen ovalbumin indicating adjuvant activity. Some of the coarse fractions gave a lymph node response even in the absence of ovalbumin, caused probably by non-specific inflammation. With the exception of a few of the coarse fractions, all ambient fractions increased the production of specific IgE. Fine particles had stronger adjuvant effect than coarse particles. A significant increase in the allergen specific IgG2a response was observed for the fine and some of the coarse fractions, indicating a non-allergic Th1 response. No consistent differences in adjuvant effects between the locations were observed. Particle samples collected in the different European cities differed in their potency to induce MIP-2 in type 2 cells. Coarse fractions of the urban particles samples, as well as the coarse fraction collected at the seaside, were more potent than the fine fractions to induce MIP-2. With respect to seasonal variations, the coarse fractions collected in summer seemed to be the most potent.

The use of in vitro methods for hazard characterisation of chemicals.

The usual starting points for hazard characterisation are No Observed Adverse Effect Levels (NOAELs)/benchmark doses for threshold effects and risk-specific doses/unit risks for non-threshold effects. In vitro studies are in general of no use in identifying these doses. However, based in part on in vitro investigations toxic equivalency factors have been developed for selected halogenated organic PCDD/PCDF/PCB congeners. Such factors can be used to determine the total toxic equivalent doses of mixtures of these contaminants. Studies with paracetamol illustrate that in vitro systems may help in the identification of the most sensitive species and strain. In vitro methods have been successfully used to studying qualitative and quantitative species differences in the toxicity of agents such as peroxisome proliferators and dichloromethane. Investigations with a number of chemicals show that in vitro systems are excellent models for characterisation of the mode of action of chemicals, but in vitro findings need to be validated in vivo. Experiments with bis(tri-n-butyltin)oxide illustrate that in vitro systems may aid in the extrapolation from high to low dose and from experimental animals to humans. In addition, in vitro approaches can be used to obtain useful information on the disposition of xenobiotics. It is concluded that if sufficient in vivo mechanistic information is available, in vitro studies using sub-cellular fractions/cells/tissue from animals and humans may significantly aid in the hazard characterisation of chemicals.