Jürgen Pauluhn

Multi-walled carbon nanotubes (Baytubes): approach for derivation of occupational exposure limit.

Carbon nanotubes come in a variety of types, but one of the most common forms is multi-walled carbon nanotubes (MWCNT). This paper focuses on the dose-response and time course of pulmonary toxicity of Baytubes, a more flexible MWCNT type with the tendency to form assemblages of nanotubes. This MWCNT has been examined in previous single and repeated exposure 13-week rat inhalation studies. Kinetic endpoints and the potential to translocate to extrapulmonary organs have been examined during postexposure periods of 3 and 6 months, respectively. The focus of both studies was to compare dosimetric endpoints and the time course of pulmonary inflammation characterized by repeated bronchoalveolar lavage and histopathology during the respective follow-up periods. To better understand the etiopathology of pulmonary inflammation and time-related lung remodeling, two metrics of retained lung dose were compared. The first used the mass metric based on the exposure concentration obtained by filter analyses and aerodynamic particle size of airborne MWCNT. The second was based on calculated volumetric lung burdens of retained MWCNT. Kinetic analyses of lung burdens support the conclusion that Baytubes, in principal, act like poorly soluble agglomerated carbonaceous particulates. However, the difference in pulmonary toxic potency (mass-based) appears to be associated with the low-density (approximately 0.1-0.3g/m(3)) of the MWCNT assemblages. Of note is that assemblages of MWCNT were found predominantly both in the exposure atmosphere and in digested alveolar macrophages. Isolated fibers were not observed in exposure atmospheres or biological specimens. All findings support the conclusion that the low specific density of microstructures was conducive to attaining the volumetric lung overload-related inflammatory response conditions earlier than conventional particles. Evidence of extrapulmonary translocation or toxicity was not found in any study. Thus, pulmonary overload is believed to trigger the cascade of events leading to a stasis of clearance and consequently increased MWCNT doses high enough to trigger sustained pulmonary inflammation. This mechanism served as conceptual basis for the calculation of the human equivalent concentration. Accordingly, multiple interspecies adjustments were necessary which included species-specific differences in alveolar deposition, differences in ventilation, and the time-dependent particle accumulation accounting for the known species-specific differences in particle clearance half-times in rats and humans. Based on this rationale and the NOAEL (no-observed adverse effect level) from the 13-week subchronic inhalation study on rats, an occupational exposure limit (OEL) of 0.05 mg Baytubes/m(3) (time weighted average) is considered to be reasonably protective to prevent lung injury to occur in the workplace environment.

Poorly soluble particulates: searching for a unifying denominator of nanoparticles and fine particles for DNEL estimation.

Under the new European chemicals regulation, REACH (Registration, Evaluation, Authorization and Restriction of Chemicals) a Derived No-Effect Level (DNEL), i.e., the level of exposure above which humans should not be exposed, is defined. The focus of this paper is to develop a weight-of-evidence-based DNEL-approach for inhaled poorly soluble particles. Despite the common mode of action of inhaled insoluble, spherical particulate matter (PM), a unifying, most appropriate metric conferring pulmonary biopersistence and toxicity has yet not been demonstrated. Nonetheless, there is compelling evidence from repeated rat inhalation exposure studies suggesting that the particle displacement volume is the most prominent unifying denominator linking the pulmonary retained dose with toxicity. Procedures were developed to analyze and model the pulmonary toxicokinetics from short-term to long-term exposure. Six different types of poorly soluble nano- to submicron PMs were compared: ultrafine and pigmentary TiO₂, synthetic iron oxide (Fe₃O₄, magnetite), two aluminum oxyhydroxides (AlOOH, Boehmite) with primary isometric particles approximately of either 10 or 40 nm, and MWCNT. The specific agglomerate densities of these materials ranged from 0.1 g/cm³ (MWCNT) to 5 g/cm³ (Fe₃O₄). Along with all PM, due to their long retention half-times and associated biopersistence in the lung, even short-term inhalation studies may require postexposure periods of at least 3 months to reveal PM-specific dispositional and toxicological characteristics. This analysis provides strong evidence that pulmonary toxicity (sustained inflammation) is dependent on the volume-based cumulative lung exposure dose. Lung toxicity, evidenced by PMN in BAL occurred at lung doses exceeding 10-times the overload threshold. Furthermore, the conclusion is supported that repeated inhalation studies on rats should utilize an experimental window of cumulative volume loads of respirable PM in the range of 1 μl/lung (no-adverse-effect range); however, not exceeding ≈10 μl/lung that would lead to retention half-times increasing 1 year. This can be targeted best by computational toxicology, i.e., the modeling of particle deposition and lung retention biokinetics during the exposure and recovery periods. Inhalation studies exceeding that threshold volume may lead to meaningless findings difficult to extrapolate to any real-life scenario. In summary, this analysis supports a volume-based generic mass concentration of 0.5 μl PM(respirable)/m³ x agglomerate density, independent on nano- or submicron-sized properties, as a generic no-adverse effect level in both rats and humans.

Pulmonary Toxicity and Fate of Agglomerated 10 and 40 nm Aluminum Oxyhydroxides following 4-Week Inhalation Exposure of Rats: Toxic Effects are Determined by Agglomerated, not Primary Particle Size

Inhaled polydisperse micronsized agglomerated particulates composed of nanosized primary particles may exert their pulmonary toxicity in either form, depending on whether these tightly associated structures are disintegrated within the biological system or not. This hypothesis was tested in a rat bioassay using two calcined aluminum oxyhydroxides (AlOOH) consisting of primary particles in the range of 10-40 nm. Male Wistar rats were nose-only exposed to 0.4, 3, and 28 mg/m(3) in two 4-week (6 h/day, 5 days/week) inhalation studies followed by a 3-month postexposure period. The respective mass median aerodynamic diameter (MMAD) of agglomerated particles in inhalation chambers was 1.7 and 0.6 mum. At serial sacrifices, pulmonary toxicity was characterized by bronchoalveolar lavage (BAL) and histopathology. The retention kinetics of aluminum (Al) was determined in lung tissue, BAL cells, and selected extrapulmonary organs, including lung-associated lymph nodes (LALNs). Significant changes in BAL, lung, and LALN weights occurred at 28 mg/m(3). Histopathology revealed alveolar macrophages with enlarged and foamy appearance, increased epithelial cells, inflammatory cells, and focal septal thickening. The determination of aluminum in lung tissue shows that the cumulative lung dose was higher following exposure to AlOOH-40 nm/MMAD-0.6 mum than to AlOOH-10 nm/MMAD-1.7 mum, despite identical exposure concentrations. The associated pulmonary inflammatory response appears to be principally dependent on the agglomerated rather than primary particle size. Despite high lung burdens, conclusively increased extrapulmonary organ burdens did not occur at any exposure concentration and postexposure time point. Particle-induced pulmonary inflammation was restricted to cumulative doses exceeding approximately 1 mg AlOOH/g lung following 4-week exposure at 28 mg/m(3). It is concluded that the pulmonary toxicity of nanosized, agglomerated AlOOH particles appears to be determined by the size of agglomerated rather than primary particles, whereas the clearance half-time of particles appears to increase with decreased primary particle size. However, in regard to toxicokinetics, this outcome is highly contingent upon the total lung burden and especially whether overloading or non-overloading conditions were attained or not. In order to reliably demonstrate retention-related different characteristics in toxicity and fate of poorly soluble (nano)particles postexposure periods of at least 3 months appear to be indispensible.

Respiratory Allergy: Hazard Identification and Risk Assessment

Various chemicals and proteins of industrial importance are known to cause respiratory allergy, with occupational asthma being the most important manifestation of the disease. This paper describes clinical syndromes, mechanisms associated with occupational respiratory hypersensitivity, and methods available currently for the prospective identification of potential respiratory allergens. Certain classes of chemicals are commonly associated with occupational respiratory allergy. There is insufficient information, however, to predict respiratory sensitization potential from analysis of structure alone, although reactivity with proteins is likely to be relevant. As yet there exist no fully validated or widely applied predictive methods or internationally harmonized guidelines. The most promising predictive animal methods are the mouse IgE test and guinea pig models. Work in mice has focused upon events occurring during the induction phase of sensitization following primary encounter with the test chemical. In contrast, guinea pig models have been used primarily to identify respiratory allergens (chemicals or proteins) as a function of elicitation reactions induced in previously sensitized animals. Given the possible serious health manifestations of respiratory allergy, early identification of respiratory sensitizers is urgently required. The two methods should, as a priority, be developed further and the production of a detailed protocol for these methods be undertaken to facilitate further validation. Together, this information will allow for two types of risk assessment associated with respiratory allergy: the risk that exposure to a material will (1) induce sensitization in an individual and (2) elicit allergic reactions in a previously sensitized individual.

Review Article: Inhalation Studies in Laboratory Animals—Current Concepts and Alternatives

Highly standardized and controlled inhalation studies are required for hazard identification to make test results reproducible and comparable and to fulfill general regulatory requirements for the registration of new drugs, pesticides, or chemicals. Despite significant efforts, the results of inhalation studies have to be analyzed judiciously due to the great number of variables. These variables may be related to technical issues or to the specific features of the animal model. Although inhalation exposure of animals mimics human exposure best, ie, error-prone route-to-route extrapolations are not necessary, not all results obtained under such very rigorous test conditions may necessarily also occur under real-life exposure conditions. Attempts are often made to duplicate as closely as possible these real-life exposure conditions of humans in appropriate bioassays. However, this in turn might affect established baseline data, rendering the interpretation of new findings difficult. In addition, specific use patterns, eg, of inhalation pharmaceuticals or pesticide-containing consumer products, may impose test agent-specific constraints that challenge traditional approaches. Moreover, specific modes of action of the substance under investigation, the evaluation of specific endpoints, or the clarification of equivocal findings in common rodent species may require exposure paradigms or the use of animal species not commonly used in inhalation toxicology. However, particularly in inhalation toxicology, the choice of animal models for inhalation toxicity testing is usually based on guideline requirements and practical considerations, such as exposure technology, expediency, and previous experience rather than validity for use in human beings. Larger animal species, apart from the welfare aspects, may require larger inhalation chambers to accommodate the animals, but for technical reasons and the difficulty of generating homogeneous exposure atmospheres in such inhalation chambers, this may jeopardize the outcome of the study. Some of the many variables and possible artifacts likely to occur in animal inhalation studies are addressed in this paper.

Acute Nose-Only Exposure of Rats to Phosgene. Part I: Concentration × Time Dependence of LC50s, Nonlethal-Threshold Concentrations, and Analysis of Breathing Patterns

Groups of young adult Wistar rats were acutely exposed to phosgene gas using a directed-flow nose-only mode of exposure. The exposure durations used were 10, 30, 60, and 240 min and the corresponding C × t products bracketed a range from 1538 to 2854 mg/m3× min. The postexposure period was 2 wk. Subgroups of rats were subjected to respiratory function measurements. With few exceptions, mortality occurred within 24 h after exposure. The median lethal concentration (LC50) and the estimated nonlethal threshold concentrations (LC01) for 10, 30, 60, and 240 min were 253.3 (105.3), 54.5 (29.2), 31.3 (21.1), and 8.6 (5.3) mg/m3, respectively. With regard to the fixed outcome Cn× t product, the exponent n was found to be ∼0.9 for both the LC50 and the LC01. Due to an apparent rodent-specific transient depression in ventilation, results from 10-min exposures were excluded for the calculation of average C × t products. The average LCt50 (and confidence interval 95%) and LCt01 were 1741 (1547–1929) mg/m3× min and 1075 mg/m3× min, respectively, with a LCt50/LCt01 ratio of 1.6. Respiratory function measurements revealed an increased apnea time (AT), which is typical for lower respiratory tract irritants. This response was associated with transiently decreased respiratory minute volumes. Borderline, although distinct, changes in AT occurred at 1.2 × 30 mg/m3 × min and above, which did not show evidence of recovery during a 30-min postexposure period at 47.6 × 30 mg/m3× min and above. In an ancillary study, one group of rats was exposed to 1008 mg/m3× min (at 4.2 mg/m3 for 240 min; postexposure period 4 wk). Emphasis was on the time course of nonlethal endpoints (bronchoalveolar lavage, BAL) and histopathology of the lungs of rats sacrificed at the end of the 4-wk postexposure period. The climax of BAL protein was on the first postexposure day and exceeded approximately 70 times the control without causing mortality. The changes in BAL protein resolved within 2 wk. Histopathology did not show evidence of lung remodeling or progressive, potentially irreversible changes 4 wk postexposure. In summary, the analysis of the C × t dependent mortality revealed a steep C × t mortality relationship. The C × t product in the range of the nonlethal threshold concentration (1008 mg/m3 × min) caused pulmonary injury as indicated by markedly increased protein in BAL. Changes resolved almost entirely within the 4-wk postexposure period.

Respiratory hypersensitivity in guinea pigs sensitized to 1,6-hexamethylene diisocyanate (HDI): comparison of results obtained with the monomer and homopolymers of HDI.

This study used guinea pigs that were sensitized to the biuret or isocyanurate type homopolymers of 1,6 hexamethylene diisocyanate (HDI). Induction was either by intradermal injection or repeated inhalation exposures. For comparison, groups of guinea pigs were sensitized to monomeric HDI. Naive animals served as negative controls. Two and three weeks following induction, animals were challenged by inhalation with the hapten and homologous protein conjugate of the hapten, respectively. Assessments were based on changes in respiratory rate, serum IgG(1)-antibody titer, and influx of eosinophilic granulocytes in airways. Guinea pigs induced and challenged with the HDI-monomer did not display appreciable changes in respiratory rate, whilst the re-challenge with the HDI-protein conjugate caused unequivocal changes in respiratory patterns, including a marked bronchial influx of eosinophilic granulocytes. In contrast, animals induced and challenged with either the free or conjugated aerosols of HDI-homopolymers failed to elicit specific physiological or morphological pulmonary responses. IgG(1) antibodies were observed in all groups receiving monomeric HDI or HDI-homopolymers. Based on the comparative assessment of antibody titers following intradermal injections, it appeared that monomeric HDI was more potent to induce specific IgG(1) antibodies than the homopolymers of HDI. In summary, with respect to induction of allergy and asthma, the data presented here suggest that the homopolymeric forms of HDI appear to be less potent asthmagens, if any, than monomeric HDI.

Hazard identification and risk assessment of pyrethroids in the indoor environment.

Household insecticide products raise several important considerations concerning safety. These are related to the use of insecticides by untrained individuals, the difficulty of controlling the use of these products once purchased by the consumer and the potential exposure of the very young and very old, possibly with or without pre-existing pulmonary disease. Exposure to pyrethroids contained in mats or vaporizers, being slow release systems, have particular potential for long-term low-level exposure whilst for foggers, spray-cans or sprayed formulations the short-term high-level exposures may be of more concern. According to the volatility of the active ingredient contained in the household insecticide, its persistence in a non-inhalable matrix, i.e. sedimented house dust, may be short or long for highly volatile or low volatile active ingredients, respectively. On the other hand, the potential of exposure is apparently just reciprocal. This demonstrates that the extent and duration of exposure may be highly product-specific. Accordingly, the extent of exposure has to be accounted for and for risk assessment both concentration-dependent (e.g. sensory irritation) as well as concentration x time (= dose) related effects have to be considered and addressed in adequate bioassays. The issue as to whether pyrethroids adhering to house dust is of concern has been addressed in a model study using carpets treated with pyrethroids. This study has demonstrated that the total mass of pyrethroid applied to the carpet and that brushed off within an 18-h period is too small to be of any relevance for risk assessment. Therefore, assessment of health hazards in the indoor environment based simply on methodologies of emptying the household vacuum cleaner and analysing its content, which addresses contamination only, rather than examination of the actual airborne concentration, including other relevant airborne materials, is prone to tremendous errors and misjudgments. Due to the many substances potentially present in house dust and indoor air, e.g. bioaerosols originating from animals, pests and microorganisms, volatile organic substances (VOCs) or metals, prudent expert judgment is needed to assess the relevance of analytical findings. The complex indoor exposure scenario makes it especially difficult to causally relate clinical and epidemiological findings to arbitrarily selected indicator substances contained in a matrix not readily available to inhalation exposure.

Rat model of lung fibrosis: comparison of functional, biochemical, and histopathological changes 4 months after single irradiation of the right hemithorax.

This study investigated changes in lung function, hydroxyproline (OH-pro) content of lung tissue and histopathology in anesthetized, spontaneously breathing rats after a single, selective irradiation of the right hemithorax with a single dose of 20 Gy. The objective of this animal model was to examine as to whether non-invasive lung function measurements (LFM) could be used to analyze the magnitude of the irradiation-related pneumonitis and its long-term sequel occurring in the right lung in the presence of a normal left lung. Four months after irradiation, the OH-pro content in the irradiated right lung was determined and compared with the non-irradiated contralateral left lung, as well as lungs from non-irradiated sham controls. LFM revealed significantly depressed flow-volume curves and reduced quasistatic compliance, suggesting a marked diminution of elastic recoil of the lung. Total lung capacity (TLC) was significantly decreased, while the residual volume (RV) and functional residual capacity (FRC) remained almost unchanged. One of the most predominant dysfunction of the lung was a severe maldistribution of ventilation shown by the single-breath N(2)-wash-out test. Single-breath carbon monoxide diffusing capacity (Dlco) was significantly decreased. The content of OH-pro, a marker of increased collagen, was significantly increased in the irradiated right lung but was indistinguishable from sham controls in the non-irradiated left lung. Histopathological examinations provided evidence of both inflammatory and fibrotic lesions in the irradiated lobes, including bronchiolo-alveolar hyperplasia. No changes were observed in the non-irradiated left lung. In summary, effects observed in the irradiated right lung were largely consistent with effects described in other animal models of human interstitial pulmonary fibrosis. Non-invasive LFM were considered to be particularly sensitive to study the overall extent of changes, however, the interpretation of findings appears to be complicated by the lobar heterogeneity of tissue- and flow-related functional end points.

Predictive testing for respiratory sensitisation

A rat bioassay has been developed to provide an objective approach for the identification and classification of upper and lower respiratory tract irritants, with particular emphasis on the concentration-dependent induction and regression of lesions characteristic of asthma, such as persisting non-specific airway hyperreactivity, inflammation and ensuing mismatch of the ventilation-perfusion relationship. For the identification of respiratory allergy, the established guinea-pig bioassay has been further refined. Refinement focused on procedures making this animal model more robust to changes in study design. Attempts were made to allow differentiation of non-specific and specific bronchial hyperresponsiveness and to minimise the use of hapten-protein conjugates for elicitation of respiratory allergy. It appears that the combined assessment of specific pathologic features such as airway eosinophilia and the evaluation of several breathing parameters during hapten and acetylcholine bronchoprovocation challenge make it easier to distinguish effects caused by irritation and respiratory hypersensitivity. Findings support the conclusion that current guinea-pig models require specific optimisation of sensitisation and challenge procedures for each chemical class tested.