Systemic inflammatory effects of zinc oxide particles: is a re-evaluation of exposure limits needed?

Exposure to airborne substances such as gases, vapours, and particles remains a relevant health risk in many workplaces. A current topic and cause for discussion is the investigation of the health effects of particles containing zinc oxide (ZnO). Among other data, those collected from our study on human exposure data of ZnO in 2018 prompted the National Research Centre for the Working Environment 2021 to formulate a new, sharply lowered proposed occupational exposure limit (OEL) for zinc in workplaces. Since the publication of the Danish report, further studies have been conducted with ZnO. In the following text, all arguments for deriving this new limit value for zinc from the report are discussed, extended with the more recent data since 2018. It should be noted that especially the application of time extrapolation factors needs further discussion and harmonization between regulatory authorities. From our point of view, the data situation can justify a higher OEL for zinc than that proposed by the Danish National Research Centre for the Working Environment.

Physiological responses, self-reported health effects, and cognitive performance during exposure to carbon dioxide at 20 000 ppm.

In this study, 24 subjects (20-58 years) were exposed to carbon dioxide (CO2 ) at 770 ppm and 20 000 ppm in an exposure laboratory for 4-h, including 2 × 15 min of cycling to investigate the effects on acid-base balance, physiological responses, cognitive performance and acute health. Capillary blood analysis, heart rate, respiratory rate, divided attention, flexibility, and sustained attention from the Test Battery for Attentional Performance (TAP), critical flicker fusion frequency (CFF), and self-reported symptoms were measured before, during, and after the 4-h exposure. Blood pH decreased and partial pressure of carbon dioxide (pCO2 ) increased significantly when exposed to 20 000 ppm CO2 compared to 770 ppm. However, the values remained within the normal range. In addition, respiratory rate increased slightly but significantly at 20 000 ppm CO2 . No significant changes in heart rate, CFF, task performance or acute health were found. In sum, the findings suggest that the observed changes in acid-base balance and ventilation can be classified as physiological adaptation responses. Impairment of cognitive performance is not expected from exposure to 20 000 ppm CO2 , neither as direct effect on central nervous system function nor as a distraction related to perception of health effects.

No inflammatory effects after acute inhalation of barium sulfate particles in human volunteers.

BACKGROUND: Most threshold limit values are based on animal experiments. Often, the question remains whether these data reflect the situation in humans. As part of a series of investigations in our exposure lab, this study investigates whether the results on the inflammatory effects of particles that have been demonstrated in animal models can be confirmed in acute inhalation studies in humans. Such studies have not been conducted so far for barium sulfate particles (BaSO4), a substance with very low solubility and without known substance-specific toxicity. Previous inhalation studies with zinc oxide (ZnO), which has a substance-specific toxicity, have shown local and systemic inflammatory respones. The design of these human ZnO inhalation studies was adopted for BaSO4 to compare the effects of particles with known inflammatory activity and supposedly inert particles. For further comparison, in vitro investigations on inflammatory processes were carried out. METHODS: Sixteen healthy volunteers were exposed to filtered air and BaSO4 particles (4.0 mg/m3) for two hours including one hour of ergometric cycling at moderate workload. Effect parameters were clinical signs, body temperature, and inflammatory markers in blood and induced sputum. In addition, particle-induced in vitro-chemotaxis of BaSO4 was investigated with regard to mode of action and differences between in vivo and in vitro effects. RESULTS: No local or systemic clinical signs were observed after acute BaSO4 inhalation and, in contrast to our previous human exposure studies with ZnO, no elevated values of biomarkers of inflammation were measured after the challenge. The in vitro chemotaxis induced by BaSO4 particles was minimal and 15-fold lower compared to ZnO. CONCLUSION: The results of this study indicate that BaSO4 as a representative of granular biopersistent particles without specific toxicity does not induce inflammatory effects in humans after acute inhalation. Moreover, the in vitro data fit in with these in vivo results. Despite the careful and complex investigations, limitations must be admitted because the number of local effect parameters were limited and chronic toxicity could not be studied.

Health effects after inhalation of micro- and nano-sized zinc oxide particles in human volunteers.

Inhalation of ZnO particles can cause inflammation of the airways and metal fume fever. It is unclear if different sizes of the particles alter these effects. However, various studies report higher biological activity of other nano-sized particles compared to microparticles. No effects at all were observed after inhalation of micro- and nano-sized zinc oxide (ZnO) particle concentrations of 0.5 mg/m3. Studies with different particle sizes of ZnO at higher exposures are not available. Accordingly, we hypothesized that inhalation of nano-sized ZnO particles induces stronger health effects than the inhalation of the same airborne mass concentration of micro-sized ZnO particles. 16 healthy volunteers (eight men, eight women) were exposed to filtered air and ZnO particles (2.0 mg/m3) for 2 h (one session with nano- and one with micro-sized ZnO) including 1 h of cycling at moderate workload. Effect parameters were symptoms, body temperature, inflammatory markers in blood and in induced sputum. Induced sputum was obtained at baseline examination, 22 h after exposure and at the end of the final test. The effects were assessed before, immediately after, about 22 h after, as well as two and three days after each exposure. Neutrophils, monocytes and acute-phase proteins in blood increased 22 h after micro- and nano-sized ZnO exposure. Effects were generally stronger with micro-sized ZnO particles. Parameters in induced sputum showed partial increases on the next day, but the effect strengths were not clearly attributable to particle sizes. The hypothesis that nano-sized ZnO particles induce stronger health effects than micro-sized ZnO particles was not supported by our data. The stronger systemic inflammatory responses after inhalation of micro-sized ZnO particles can be explained by the higher deposition efficiency of micro-sized ZnO particles in the respiratory tract and a substance-specific mode of action, most likely caused by the formation of zinc ions.

Whole Blood Assay as a Tool to Describe the Effects of Zinc Oxide Exposure on Innate Immunity.

Inhalation of high concentrations of zinc oxide (ZnO) particles may cause metal fume fever. A useful tool to characterize the reactivity of innate immune cells of an individual, e.g., after in vivo exposure, is the whole blood assay (WBA). The measurable outcome of WBA is the release of cytokines, especially pro-inflammatory and pyrogenic cytokines induced by stimulation in vitro. The aim of the study was to evaluate whether inhalation of nano-sized zinc oxide particles modifies the results of WBA from healthy blood donors. Sixteen healthy subjects were exposed to filtered air and ZnO particles (0.5, 1.0, and 2.0 mg/m3) for 4 h on four different days. Blood was collected before and 24 h after exposure, and ex vivo stimulation of the whole blood was performed using different endotoxin concentrations. The release of interleukin (IL)-1ß and IL-8 after 22-h incubation was quantified with specific immunoassays. The dose-response relationship of ex vivo stimulation with different endotoxin concentrations was not affected by previous ZnO exposure. However, based on the previously established calculation models, changes due to ZnO exposure could be described. The range of cytokine release in WBA was calculated for the whole group of blood donors, for the subgroups of low and high responders (each n = 8), and on the individual level. Most changes were observed after 0.5 mg/m3 ZnO exposure. Higher ZnO exposure did not yield higher effects. We conclude that the effects of inhalation of nano-sized ZnO particles in blood of healthy donors using the WBA could be determined. However, it should be noted that cytokine release as outcome of WBA is not a marker of disease.

Ethyl acrylate: influence of sex or atopy on perceptual ratings and eye blink frequency.

Occupational exposure limits (OELs) are derived for protection from health hazards, assuming that exposed subjects are healthy adult workers. Whether differences in susceptibility to sensory irritation effects from airborne chemicals have to be taken into account is currently under discussion. Thus, we chose atopics as a healthy but possibly susceptible subpopulation that can be identified with a clinical test. To investigate the influence of sex or atopy on sensitivity to airborne chemicals, 22 subjects were exposed for 4 h to ethyl acrylate at three concentrations: 0.05 ppm (odor threshold; sham), 5 ppm (constant), and varying exposure between 0 and 10 ppm. Odor intensity decreased and eye irritation ratings increased in a dose-dependent manner, reflecting the time course of the exposure scenarios. The reports of moderate-to-strong eye irritation were verified by significant increases in eye blink frequency. Our results show that women reported subjective eye irritation to an increasing degree. However, these sex-related differences in ratings could not be verified by objective assessment of eye blink frequency. Atopic subjects reported higher odor intensity than non-atopic subjects, but only during the sham (odorous but not irritating) exposure condition. Differences in ratings on annoyance, and eye or nose irritation were not found. Furthermore, the study revealed that atopic subjects might belong to a group of subjects with frequent eye blink activity. Although the relative increase in blink rates was more pronounced in non-atopic subjects, atopic subjects had significant higher blink rates at the end of the exposure to varying ethyl acrylate concentrations. Our results do not support that atopy enhances chemosensory effects if only the increase of blink rates and not the absolute height are considered as adverse effect. Nevertheless, the results indicate that individuals with frequent eye blink activity should be distinguished from those with normal eye blink activity while investigating blink rates as objective parameter of eye irritation.

Airway inflammation after inhalation of nano-sized zinc oxide particles in human volunteers.

BACKGROUND: Workers in the zinc production and processing of galvanized sheet steel are exposed to a complex mixture of particles and gases, including zinc oxide (ZnO) that can affect human health. We aimed to study the effects of short-term controlled exposure to nano-sized ZnO on airway inflammatory markers in healthy volunteers. METHODS: Sixteen subjects (8 females, 8 men; age 19-42, non-smokers) were exposed to filtered air and ZnO nanoparticles (0.5, 1.0 and 2.0 mg/m3) for 4 h, including 2 h of cycling with a low workload. Induced sputum samples were collected during a medical baseline and a final examination and also about 24 h after each exposure. A number of inflammatory cellular and soluble markers were analyzed. RESULTS: Frequency and intensity of symptoms of airway irritation (throat irritation and cough) were increased in some subjects 24 h after ZnO exposures when compared to filtered air. The group comparison between filtered air and ZnO exposures showed statistically significant increases of neutrophils and interleukin-8 (IL-8), interleukin-6 (IL-6), matrix metalloproteinase (MMP-9) and tissue inhibitors of metalloproteinases (TIMP-1) in sputum starting at the lowest ZnO concentration of 0.5 mg/m3. However, a concentration-response relationship was absent. Effects were reversible. Strong correlations were found between neutrophil numbers and concentrations of total protein, IL-8, MMP-9, and TIMP-1. CONCLUSIONS: Controlled exposures of healthy subjects to ZnO nanoparticles induce reversible airway inflammation which was observed at a concentration of 0.5 mg/m3 and higher. The lack of a concentration-response relationship warrants further studies.

Effects of Exposure to Carbon Dioxide in Potash Miners.

Potash miners can become exposed to carbon dioxide (CO2) during the blasting of basalt intrusions or loading and transporting the blasted salt. In a cross-shift study, we compared physiological effects of acute exposure to elevated CO2 concentrations in miners after long-term exposure to evaluate the possible health risks. A group of 119 miners was assessed by clinical examination, lung function tests, and blood gas content directly before and after the shift. A cumulative CO2 exposure was measured using personal monitors. The miners were categorized as low (<0.1 vol.%, n = 83), medium (<0.5 vol.%, n = 26), and high (>0.5 vol.%, n = 10) CO2 exposed subjects. We found no significant differences among the three groups. Lung function testing revealed no conspicuous findings, and chronic health effects were not observed in the miners either. In conclusion, no significant adverse effects could be found in potash miners exposed to elevated CO2 concentrations. Therefore, the mining authorities allow potash mining operations for 4 h at ambient CO2 up to 1.0 vol.% and for 2 h at CO2 not exceeding 1.5 vol.% per shift.

Concentration-dependent systemic response after inhalation of nano-sized zinc oxide particles in human volunteers.

BACKGROUND: Inhalation of high concentrations of zinc oxide particles (ZnO) may cause metal fume fever. In an earlier human inhalation study, no effects were observed after exposure to ZnO concentrations of 0.5 mg/m3. Further data from experimental studies with pure ZnO in the concentration range between 0.5 and 2.5 mg/m3 are not available. It was the aim of this experimental study to establish the concentration-response relationship of pure nano-sized ZnO particles. METHODS: Sixteen healthy subjects were exposed to filtered air and ZnO particles (0.5, 1.0 and 2.0 mg/m3) for 4 h on 4 different days, including 2 h of cycling with a low workload. The effects were assessed before, immediately after, and about 24 h after each exposure. Effect parameters were symptoms, body temperature, inflammatory markers and clotting factors in blood, and lung function. RESULTS: Concentration-dependent increases in symptoms, body temperature, acute phase proteins and neutrophils in blood were detected after ZnO inhalation. Significant effects were detected with ZnO concentrations of 1.0 mg/m3 or higher, with the most sensitive parameters being inflammatory markers in blood. CONCLUSION: A concentration-response relationship with nano-sized ZnO particles in a low concentration range was demonstrated. Systemic inflammatory effects of inhaled nano-sized ZnO particles were observed at concentrations well below the occpational exposure limit for ZnO in many countries. It is recommended to reassess the exposure limit for ZnO at workplaces.

Cell Activation and Cytokine Release Ex Vivo: Estimation of Reproducibility of the Whole-Blood Assay with Fresh Human Blood.

The whole-blood assay (WBA) with human fresh blood may provide insight into the features of an individual's innate immunity. To assess this, ex vivo cytokine release is measured after stimulation of whole blood with various stimuli, for instance, endotoxin in vitro. The aim of the present study was to evaluate WBA reproducibility with fresh blood using different calculation models. The blood was collected from 16 healthy volunteers on 6 different days. Ex vivo stimulation was performed in each individual's blood sample for 22 h, using different endotoxin concentrations. Interleukin (IL)-1ß and IL-8 release were quantified using specific immunoassays in the cell-free supernatant. We found that a dose-response relationship between endotoxin and cytokine concentration could be verified for all blood donors in all tests. The median coefficient of variation of the repeated tests was 29% for IL-1ß and 52% for IL-8. Upon stimulation with 40 pg/mL endotoxin, a confidence interval of 60-140% was calculated for IL-1ß and 70-271% for IL-8 regarding test reproducibility. Furthermore, the classification into high or low responder was reproducible. We conclude that repeated blood collection offers an opportunity to evaluate the variability of WBA. Considering a high intragroup variability, an individual range assessment has been suggested to evaluate exposure effects.

Human exposure to airborne aniline and formation of methemoglobin: a contribution to occupational exposure limits.

Aniline is an important starting material in the manufacture of polyurethane-based plastic materials. Aniline-derived methemoglobinemia (Met-Hb) is well described in exposed workers although information on the dose-response association is limited. We used an experimental design to study the association between aniline in air with the formation of Met-Hb in blood and the elimination of aniline in urine. A 6-h exposure of 2 ppm aniline in 19 non-smoking volunteers resulted in a time-dependent increase in Met-Hb in blood and aniline in urine. The maximum Met-Hb level in blood (mean 1.21 ± 0.29 %, range 0.80-2.07 %) and aniline excretion in urine (mean 168.0 ± 51.8 µg/L, range 79.5-418.3 µg/L) were observed at the end of exposure, with both parameters rapidly decreasing after the end of exposure. After 24 h, the mean level of Met-Hb (0.65 ± 0.18 %) returned to the basal level observed prior to the exposure (0.72 ± 0.19 %); whereas, slightly elevated levels of aniline were still present in urine (means 17.0 ± 17.1 vs. 5.7 ± 3.8 µg/L). No differences between males and females as well as between slow and fast acetylators were found. The results obtained after 6-h exposure were also comparable to those observed in four non-smoking volunteers after 8-h exposure. Maximum levels of Met-Hb and aniline in urine were 1.57 % and 305.6 µg/L, respectively. Overall, our results contribute to the risk assessment of aniline and as a result, the protection of workers from aniline-derived adverse health effects at the workplace.

Exposure assessment of potash miners at elevated CO2 levels.

PURPOSE: In a potash mine in the center of Germany, stationary measurements 40 cm above ground level have revealed occasional increases in the carbon dioxide (CO2) levels that exceed the 0.5 vol.% German occupational exposure limit (OEL). This study, therefore, aimed to examine the individual exposures of potash miners to CO2 at their underground workplaces. METHODS: 119 miners were equipped with personal CO2 detectors to log the individual CO2 exposures during underground work. We decided to use electrochemical monitors due to their compactness and minimal mass. Furthermore, generated CO2 measurements with precipitated overshooting and false positive CO2 values were studied using diverse CO2 test gases and different fumigation times. RESULTS: The personal detectors showed short-term CO2 peak exposures at very high concentrations in a limited number of workers. Twenty-two threshold limit value violations were observed according to the present OEL, and the personal CO2 monitoring allowed categorization into three exposure groups, low (n = 83), moderate (n = 26) and high burdens (n = 10) of CO2. CONCLUSIONS: The electrochemical sensors used have numerous properties that can potentially influence the assessment of exposures. The current findings suggest that assessing similar exposure scenarios, with respect to elevated and strongly fluctuating CO2 concentrations, the behavior of electrochemical sensors should be taken into consideration.

Zinc fever in a painter and varnisher: a case report.

BACKGROUND: Zinc fever is well described in medical literature, particularly in workers after handling zinc-containing materials at high temperatures e.g., in the welding of hot-dip galvanized steel sheets. It is not known whether zinc fever also occurs at low temperatures. CASE PRESENTATION: We present the case of a 33-year-old Caucasian atopic painter and varnisher with work-related dyspnea, sweating, as well as multiple occurrences of fever. He was sent to Institute for Prevention and Occupational medicine of the German Social Accident Insurance, Institute of the Ruhr-Universität Bochum (IPA) for the evaluation of isocyanate asthma, but an inhalative challenge with hexamethylene diisocyanate was negative. Since symptoms were closely related to the use of zinc coatings at room temperature without adequate protective measures, the diagnosis of zinc fever was made. After exposure cessation the worker immediately became symptom-free. The work as painter and varnisher may be associated with various exposures to hazardous substances. Besides solvents, epoxy compounds and isocyanates, which can cause obstructive respiratory diseases; additionally, zinc-containing agents should be considered as health hazards. CONCLUSIONS: This case demonstrates that zinc fever may occur also after application of zinc coatings by spray painting at low temperatures.

Relationship of pulmonary function response to ozone exposure and capsaicin cough sensitivity.

CONTEXT: Challenge studies in humans have shown considerable interindividual variability in pulmonary impairment across ozone exposure. OBJECTIVE: Since previous results suggested effect modulation by neural mechanism, we investigated sensory C-fiber reactivity in relationship to ozone-triggered response pattern. METHODS: Cough reflex thresholds reflecting C-fiber sensitivity were evaluated by capsaicin single breath dose-response method. Capsaicin concentrations triggering, respectively, two and five or more coughs (C2, C5) were recorded. Sixteen healthy subjects were randomly exposed in an intermittent exercise protocol to ozone concentrations of 240 and 40 ppb (sham exposure). Ozone responsiveness was defined by a decrease in forced expiratory volume in 1 s (FEV(1)) of more than 5%. RESULTS: Based on a dichotomous classification, subjects with enhanced reactivity to ozone had lower cough thresholds than non-responders (C2, p = 0.035; C5, p = 0.086). Over all, we could demonstrate relationships between capsaicin sensitivity and ozone-triggered changes in FEV(1), peak expiratory flow and maximal expiratory flow at 50% vital capacity but not in specific airway resistance. CONCLUSION: Our results suggest that capsaicin challenge tests might be useful to characterize subjects with enhanced pulmonary function response towards inhalant irritants.

Reproducibility of sensitivity to capsaicin assessed by single breath inhalation methodology.

The hallmark of sensory hyperreactivity is an enhanced capsaicin induced cough reflex. The cough reflex can be modified by activation of nociceptive (capsaicin-sensitive) nerve terminals. The aim of our study was to assess the influence of exposure to CO(2) concentrations up to 2.0 vol% on capsaicin induced cough reflex on four different occasions. Sixteen healthy volunteers were exposed to CO(2) concentrations of 0.5, 1.0, and 2.0 vol% for 4 h and to clean air in a repeated measures cross-over design. After exposure the capsaicin induced cough reflex was assessed by the single breath dose-response method according to ERS 2007 guidelines. After blank solutions, capsaicin doses (n=12, range 0.49 to 1000 µM) were administrated from a nebulizer combined with a provocation system (Masterscope, software APS version 5.02). Doses were doubled every minute and the concentration causing five or more coughs (C5) was fixed as the end point. The inter-individual C5 capsaicin responsiveness reflected a representative range (0.95-1000 µM). On an intra-individual basis, a good reproducibility could be demonstrated for four tests within 3 weeks. There was no influence of CO(2) challenge on the cough reflex. The first capsaicin test demonstrated a lower C5 threshold independent of the CO(2) concentration applied. In conclusion, assessing the capsaicin cough reflex by single breath inhalation is reliable. However, the at cough sensitivity might be overestimated at the first test occasion. Exposure to CO(2) in concentrations of up to 2.0 vol% has no effect on sensory reactivity.

Considerations for the design and technical setup of a human whole-body exposure chamber.

Exposures to air contaminants, such as chemical vapors and particulate matter, pose important health hazards at workplaces. Short-term experimental exposures to chemical vapors and particles in humans are a promising attempt to investigate acute effects of such hazards. However, a significant challenge in this field is the determination of effects of co-exposures to more than one chemical or mixtures of chemical vapors and/or particles. To overcome such a challenge, studies have to be conducted under standardized exposure characterization and real time measurements, if possible. A new exposure laboratory (ExpoLab) was installed at IPA, combining sophisticated engineering designs with new analytical techniques, to fulfill these requirements. Low-dose as well as high-dose exposure scenarios are achieved by means of a calibration-gas-generator. Exposure monitoring can be carried out with a high performance real time mass spectrometer and other suitable analyzers (e.g. gas chromatograph). Numerous automated security facilities guarantee the physical integrity of the volunteers, and the waste atmosphere is removed using either charcoal filtration or catalytic post-combustion. Measurements of sulfur hexafluoride, carbon dioxide, aniline and carbon black are presented to demonstrate the performance of the exposure unit with respect to the temporal and spatial stability of generated atmospheres. The variations of generated contents in the atmospheres at steady state are slightly higher than the measurement precision of the analyzers (the typical standard deviation of generated atmospheres is < 2%). The technical components of ExpoLab and its monitoring systems ensure high quality standards in validity and reliability of generating and measuring exposure atmospheres.

Construction of an In Vitro Air-Liquid Interface Exposure System to Assess the Toxicological Impact of Gas and Particle Phase of Semi-Volatile Organic Compounds.

Anthropogenic activities and industrialization render continuous human exposure to semi-volatile organic compounds (SVOCs) inevitable. Occupational monitoring and safety implementations consider the inhalation exposure of SVOCs as critically relevant. Due to the inherent properties of SVOCs as gas/particle mixtures, risk assessment strategies should consider particle size-segregated SVOC association and the relevance of released gas phase fractions. We constructed an in vitro air-liquid interface (ALI) exposure system to study the distinct toxic effects of the gas and particle phases of the model SVOC dibutyl phthalate (DBP) in A549 human lung epithelial cells. Cytotoxicity was evaluated and genotoxic effects were measured by the alkaline and enzyme versions of the comet assay. Deposited doses were assessed by model calculations and chemical analysis using liquid chromatography tandem mass spectrometry. The novel ALI exposure system was successfully implemented and revealed the distinct genotoxic effects of the gas and particle phases of DBP. The empirical measurements of cellular deposition and the model calculations of the DBP particle phase were concordant.The model SVOC DBP showed that inferred oxidative DNA damage may be attributed to particle-related effects. While pure gas phase exposure may follow a distinct mechanism of genotoxicity, the contribution of the gas phase to total aerosol was comparably low.

In vivo monitoring of epidermal absorption of hazardous substances by confocal Raman micro-spectroscopy.

BACKGROUND: Presently, percutaneous absorption of potentially hazardous chemicals in humans can only be assessed in animal experiments, in vitro, or predicted mathematically. Our aim was to demonstrate the proof-of-principle of a novel quantitative in vivo assay for percutaneous absorption: confocal Raman micro-spectroscopy (CRS). The advantages and limitations of CRS for health risk assessments are discussed. PATIENTS AND METHODS: 2-butoxyethanol, toluene, and pyrene were applied in pure form, diluted in water, or in ethanol on the skin of three healthy volunteers. CRS measurements were done following application for 15 min and 3 hours. The concentrations of the three substances as a function of distance to the skin surface were calculated and further analyzed with regard to mass transport into the stratum corneum (µg/cm(2)) and the flux through the stratum corneum (µg/cm(2)h). The results were compared with the available data from literature. RESULTS: Considering the preliminary nature of these data, good accordance with data from the literature was observed. In addition, we observed that 2-butoxyethanol penetrates markedly faster when dissolved in water as compared to ethanol. This observation is also in agreement with previous results. CONCLUSIONS: CRS has the potential to provide fast, accurate and reliable results for advanced studies of in vivo percutaneous absorption kinetics of hazardous substances in human skin. This will require further research with other substances and under differing conditions.

In vitro genotoxicity of dibutyl phthalate on A549 lung cells at air-liquid interface in exposure concentrations relevant at workplaces.

The ubiquitous use of phthalates in various materials and the knowledge about their potential adverse effects is of great concern for human health. Several studies have uncovered their role in carcinogenic events and suggest various phthalate-associated adverse health effects that include pulmonary diseases. However, only limited information on pulmonary toxicity is available considering inhalation of phthalates as the route of exposure. While in vitro studies are often based on submerged exposures, this study aimed to expose A549 alveolar epithelial cells at the air-liquid interface (ALI) to unravel the genotoxic and oxidative stress-inducing potential of dibutyl phthalate (DBP) with concentrations relevant at occupational settings. Within this scope, a computer modeling approach calculating alveolar deposition of DBP particles in the human lung was used to define in vitro ALI exposure conditions comparable to potential occupational DBP exposures. The deposited mass of DBP ranged from 0.03 to 20 ng/cm2 , which was comparable to results of a human lung particle deposition model using an 8 h workplace threshold limit value of 580 µg/m3 proposed by the Scientific Committee on Occupational Exposure Limits for the European Union. Comet and Micronucleus assay revealed that DBP induced genotoxicity at DNA and chromosome level in sub-cytotoxic conditions. Since genomic instability was accompanied by increased generation of the lipid peroxidation marker malondialdehyde, oxidative stress might play an important role in phthalate-induced genotoxicity. The results highlight the importance of adapting in vitro studies to exposure scenarios relevant at occupational settings and reconsidering occupational exposure limits for DBP.