Joachim Bruch

Response to the Reply on behalf of the ‘Permanent Senate Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area’ (MAK Commission) by Andrea Hartwig Karlsruhe Institute of Technology (KIT)

Calculation error in the MAK Commission’s document on GBS [3] when using the rule of three in Pauluhn’s volumetric model (we emphasize that the comment did not dispute the arithmetical error lowering Model B’s GBS limit value erroneously from 2.0 mg/m to 0.5 mg/m). Use of an MPPD2 program version in [3] that is outdated and no longer available to enable to replicate the MAK Commission’s conclusions. Input values in [3] that cannot be reproduced from the references listed in [3] or are not state-of-the-art. Inconsistent use of varying input data by the MAK Commission in [3] although explicitly specified as guideline in the same document [3].

Does lung contusion affect both the traumatized and the noninjured lung parenchyma? A morphological and morphometric study in the pig.

Isolated unilateral lung contusion (LC) was induced in 12 pigs to determine the pathophysiological role of LC in the high mortality after multiple injury. The Horovitz quotient, pulmonary vascular resistance, mean pulmonary artery pressure, mixed venous oxygen consumption, inspiratory pressure and compliance were significantly decreased in the LC group as compared to controls. The number of polymorphonuclear granulocytes, the microvascular permeability of albumine and the Wilhelmy balance as determined by bronchoalveolar lavage were significantly increased after lung contusion. As indicators of a systemic reaction we found elevated plasma levels of the terminal complement complex and decreased levels of the complement factor 3a after LC. The morphological assessment revealed changes such as those encountered during the early phase of adult respiratory distress syndrome, with granulocyte sticking, endothelial cell adhesion and transendothelial diapedesis. Morphometric analysis demonstrated a significant decrease in alveolar diameter in both the injured and the contralateral lung due to impaired surfactant surface activity. A distinct increase in septal diameter, related to edema and caused by increased microvascular permeability, was found in the injured lungs. These findings emphasize that LC leads to a generalized impairment of the entire lung, which may lead to progressive lung failure.

Translational toxicology in setting occupational exposure limits for dusts and hazard classification – a critical evaluation of a recent approach to translate dust overload findings from rats to humans

BackgroundWe analyze the scientific basis and methodology used by the German MAK Commission in their recommendations for exposure limits and carcinogen classification of “granular biopersistent particles without known specific toxicity” (GBS). These recommendations are under review at the European Union level. We examine the scientific assumptions in an attempt to reproduce the results. MAK’s human equivalent concentrations (HECs) are based on a particle mass and on a volumetric model in which results from rat inhalation studies are translated to derive occupational exposure limits (OELs) and a carcinogen classification.MethodsWe followed the methods as proposed by the MAK Commission and Pauluhn 2011. We also examined key assumptions in the metrics, such as surface area of the human lung, deposition fractions of inhaled dusts, human clearance rates; and risk of lung cancer among workers, presumed to have some potential for lung overload, the physiological condition in rats associated with an increase in lung cancer risk.ResultsThe MAK recommendations on exposure limits for GBS have numerous incorrect assumptions that adversely affect the final results. The procedures to derive the respirable occupational exposure limit (OEL) could not be reproduced, a finding raising considerable scientific uncertainty about the reliability of the recommendations. Moreover, the scientific basis of using the rat model is confounded by the fact that rats and humans show different cellular responses to inhaled particles as demonstrated by bronchoalveolar lavage (BAL) studies in both species.ConclusionClassifying all GBS as carcinogenic to humans based on rat inhalation studies in which lung overload leads to chronic inflammation and cancer is inappropriate. Studies of workers, who have been exposed to relevant levels of dust, have not indicated an increase in lung cancer risk. Using the methods proposed by the MAK, we were unable to reproduce the OEL for GBS recommended by the Commission, but identified substantial errors in the models. Considerable shortcomings in the use of lung surface area, clearance rates, deposition fractions; as well as using the mass and volumetric metrics as opposed to the particle surface area metric limit the scientific reliability of the proposed GBS OEL and carcinogen classification.

Deposition behavior of inhaled nanostructured TiO2 in rats: fractions of particle diameter below 100 nm (nanoscale) and the slicing bias of transmission electron microscopy.

Context: In experimental studies with nanomaterials where translocation to secondary organs was observed, the particle sizes were smaller than 20 nm and were mostly produced by spark generators. Engineered nanostructured materials form microsize aggregates/agglomerates. Thus, it is unclear whether primary nanoparticles or their small aggregates/agglomerates occur in non-negligible concentrations after exposure to real-world materials in the lung. Objective: We dedicated an inhalation study with nanostructured TiO2 to the following research question: Does the particle size distribution in the lung contain a relevant subdistribution of nanoparticles? Methods: Six rats were exposed to 88 mg/m3 TiO2 over 5 days with 20% (count fraction) and <0.5% (mass fraction) of nanoscaled objects. Three animals were sacrificed after cessation of exposure (5 days), others after a recovery period of 14 days. Particle sizes were determined morphometrically by transmission electron microscopy (TEM) of ultra-thin lung slices. Since the particles visible are two-dimensional surrogates of three-dimensional structures we developed a model to estimate expected numbers of particle diameters below 100 nm due to the TEM slicing bias. Observed and expected numbers were contrasted in 2 × 2 tables by odds ratios. Results: Comparisons of observed and expected numbers did not present evidence in favor of the presence of nanoparticles in the rat lungs. In simultaneously exposed satellite animals agglomerates of nanostructured TiO2 were observed in the mediastinal lymph nodes but not in secondary organs. Conclusions: For nanostructured TiO2, the deposition of nanoscaled particles in the lung seem to play a negligible role.

Different toxic, fibrogenic and mutagenic effects of four commercial quartz flours in the rat lung

There is still intensive debate on the variability in the biological activities of different quartz species. Therefore we examined in a rat lung model the inflammatory, fibrogenic and genotoxic characteristics of four commercial quartz flours. The samples, two with probably low activity and two with probably high activity were selected from a panel of 16 samples on the basis of in vitro investigations. Rats were exposed by a single intratracheal injection of 0.6, 1.2 and 2.4 mg quartz samples per lung or with 1.2 mg standard quartz DQ12. After 90 days the inflammatory response was measured in the bronchoalveolar lavage fluid, as well as the content of 8-oxoguanine in the DNA of the lung cells. Additionally mutated p53 protein was determined. The four quartz samples revealed specific differences in all parameters investigated. In good agreement with the in vitro results the two samples expected as lowly active showed only weak inflammatory and no genotoxic reactions in the rat lungs. In contrast the two samples suspected as highly reactive induced a pronounced inflammatory response and for one of the samples genotoxic effects could be proven. The results raised here show a broad spectrum of biological activities dependent on the type of quartz from almost inert to genotoxic and highly inflammatory.

Silicotic lymph node reactions in mice: genetic differences, correlation with macrophage markers, and independence from T lymphocytes

Quartz was injected into a hind foot of BALB/c and DBA/2 mice and on days 40, 90, and 180 the progressive response ensuing in the draining popliteal lymph node (PLN) was investigated by histopathology and immunohistopathology. The area of silicotic nodules (ASN) was measured by morphometry, and, by this parameter, strain BALB/c proved to be a high responder to quartz, and strain DBA/2 a low responder, albeit both strains showed a similar degree of reactive lymphoid hyperplasia in the draining PLN. Both strains also showed a similar quartz content in the draining PLN but in BALB/c mice quartz particles were concentrated in the ASN, whereas in DBA/2 mice they were evenly dispersed over the PLN. Because the silicotic response of athymic BALB/c nu/nu mice was even stronger than that of euthymic BALB/c mice, T cells are not required for the development of silicotic nodules. This fits the notion that quartz is not an antigen and that high and low responder strains are MHC‐identical. Because quartz‐treated BALB/c, but not DBA/2 mice, showed a persistent expression of the macrophage differentiation markers MRP8 and MRP14, phenotypically the observed strain difference in silicotic responsiveness seems to be expressed at the level of macrophages.

Toxicological investigations on the respirable fraction of silicon carbide grain products by the in vitro vector model.

Abstract Increased lung cancer incidence with workers at the production site of crude silicon carbide (SiC) using the Acheson process has been reported. Several agents derived from the process were discussed as causative factors. Recently concern had been expressed about the presence of cleavage fragments (CFs) in commercial products fulfilling the WHO criteria for fibers. This study has focused on the toxicological significance of such CFs. The test samples were respirable fractions of five different commercial samples of SiC grains. The CF content (scanning electron microscopy) was in the range 17–493 fibers/µg. Crystalline silica and whiskers could not be detected. Quartz DQ12, cristobalite, SiC whisker, UICC crocidolite and electrocorundum were used as control reference samples. Biological activity was assessed with the in vitro vector model (VM) on ex vivo rat and guinea pig alveolar macrophages (AMs). The dose range of the VM is derived from calculated AM loads from intratracheal instillation experiments and confirmed by measured AM loads from inhalation studies on alumina monohydrate particles with low biological activity: ≤120 pg/AM. The response of the references was clearly different from that of the SiC grains which yielded low toxicity overall. However, the parameter reactive oxygen species secreted by AMs was elevated at the higher SiC doses, but not related to the CF content of these samples. Our data showed that CFs seem to have no biological relevance. This is in agreement with results from recent studies in which no carcinogenic activity had been demonstrated for CFs.

DNA Damage and Inflammation in the Rat Quartz Model: Differences in Inflammatory Response and Formation of Oxidative DNA Adducts to High and Low Dose of DQ12 Quartz

Chronic exposure to poorly soluble particles such as quartz and diesel soot produces dose-dependent inflammatory responses in the rat lung. It has been shown that the inflammation in the rat lung causes persistent oxidative DNA damage and mutations in proliferation-competent cells, which are thought to be critical for tumorigenesis. In measuring various inflammatory parameters to a multidose quartz exposure in parallel with the amount of 8-oxoguanine (8-oxoGua) on the cellular level in rat lung, mechanistic data for understanding the underlying processes could be gained. Rat lungs (female Wistar, 180–220 g/bodyweight) were instilled with quartz DQ12 (doses 0.3, 1.5, and 7.5 mg/animal; controls: corundum at the same doses and physiological NaCl) and analyzed 90 days after intratracheal instillation. The bronchoalveolar lavage (BAL) fluid was determined for inflammation markers (differential cell count, protein, lung surfactant lipids, and tumor necrosis factor alpha); tissue sections of lungs were investigated for the amount of 8-oxoGua on the cellular level using an antibody against 8-oxoGua. The results reflect different responses for quartz versus all controls and show a clear dose-response relationship. Quartz elicited inflammatory reponses determined in the BAL fluid even at the low dose (0.3 mg/animal). In contrast, the level of 8-oxoGua in the lung of animals exposed to 0.3 mg quartz was not statistically increased above controls, whereas doses of 1.5 mg and 7.5 mg caused significant elevations. The data obtained indicate a no-effect level for the persistence of the mutagenic DNA adduct 8-oxoguanine in the epithelial lung cells at a low-dose quartz exposure that is still inflammatoric and fibrogenic.

Biological responses of workplace particles and their association with adverse health effects on miners

Epidemiological research has demonstrated the relationship between exposure to quartz dust and an elevated risk of pneumoconiosis and possible elevated risk of cancer. The current study was designed to evaluate the biological responses of workplace particles containing crystalline silica using an in vitro cell test. Respirable particle samples were sampled from four tin mines, where the standardized mortality ratio (SMR) for pneumoconiosis was 51.6 and SMR for lung cancer was 2.2 in dust-exposed miners. Alveolar macrophages (AM) are considered as the target cells for primary dust effects. The samples were then measured at 15, 30, 60 and 120 microg particle per 10(6) AM for cytoxicity with the release of glucuronidase, lactate dehydrogenase, for reactive oxygen damage with H(2)O(2) release, and for ability to induce fibrosis using the secretion of tumor necrosis factor-alpha (TNF-alpha). Pure quartz (DQ12) and corundum were used as controls. The results showed the samples from tin mines caused a higher cytoxicity when compared to corundum, yet lower when compared to quartz. However, reactive oxygen species release (148-177 nmol/3 x 10(5) AM in high concentration of 120 microg/10(6) AM) induced by the samples were significantly higher than that induced by quartz (57 nmol/3 x 10(5) AM) and corundum (62 nmol/3 x 10(5) AM). Furthermore, particle samples induced higher TNF-alpha secretion than corundum, the samples from Limu tin mine induced much higher TNF-alpha levels than that induced by DQ12 quartz. The results from the in vitro tests help elucidate the degree of hazard of dust particles in tin mines. The in vitro reaction patterns of AM also constitute a powerful tool to monitor biological and pathogenic responses of humans following dust particle exposure.

Particle diameter estimates of Creutzenberg et al. (2012) are distorted due to the slicing bias of transmission electron microscopy

Dear Editor, We read with interest the study by Creutzenberg et al. (2012) and Schaudien et al. (2012) on the presence of nanoparticles in vivo following lung exposure in rats. Particle sizes were determined morphometrically by transmission electron microscopy (TEM) of ultrathin lung slices. A particular methodological challenge is the two dimensional presentation of larger particles in the ultrathin TEM section planes and the analytical approach to extract informative data thereof. The typical thickness of an ultrathin section is about 80 nm or smaller (Hayat, 1986). Therefore, a larger particle (e.g. with a diameter of 300 nm) is cut into a slice at a random section plane. The profiles of this slice visible and measured in the TEM sections predictably underestimate the true particle diameter: this is the TEM slicing bias. This slicing bias is amplified by the systematic oversampling of large particles (Boyce et al., 2010; Cruz-Orive, 1987; Hsia et al., 2010). We developed a model to estimate expected numbers of particle diameters below 100 nm due to the TEM slicing bias and contrasted observed and expected numbers in 2 × 2 tables by odds ratios (Morfeld et al., 2012). Our results showed pronounced overestimates of the prevalence of small particles if the bias is not taken into account. Creutzenberg et al. (2012) and Schaudien et al. (2012) did not take care of the TEM slicing bias in their analyses. The results presented in Table 3, Figure 2 and Figure 3 in Creutzenberg et al. (2012) are distorted suggesting erroneously the presence of too many small particles. This may irritate readers who are interested in the question whether nano-objects are present in the lung after exposure to nanostructured materials. As an example for such an irritation we note that the MAK committee appears to take such an observation for granted: “Ultrafine primary particles are measured according to their mobility-equivalent diameter (DM) <100 nm (corresponds to a diffusionequivalent diameter (Dae) <100 nm). They can occur as single particles in the workplace air or more often as basic units of aggregates and agglomerates. In these forms they can be seen under an electron microscope”. (Deutsche Forschungsgemeinschaft, 2012). We would suggest correcting the particle size distributions given in Creutzenberg et al. (2012) for the immanent TEM slicing bias to avoid confusion.