Assessment of the Acute Inhalation Toxicity of Airborne Particles by Exposing Cultivated Human Lung Cells at the Air-Liquid Interface.

Here, we present a specially designed modular in vitro exposure system that enables the homogenous exposure of cultivated human lung cells at the ALI to gases, particles or complex atmospheres (e.g., cigarette smoke), thus providing realistic physiological exposure of the apical surface of the human alveolar region to air. In contrast to sequential exposure models with linear aerosol guidance, the modular design of the radial flow system meets all requirements for the continuous generation and transport of the test atmosphere to the cells, a homogenous distribution and deposition of the particles and the continuous removal of the atmosphere. This exposure method is primarily designed for the exposure of cells to airborne particles, but can be adapted to the exposure of liquid aerosols and highly toxic and aggressive gases depending on the aerosol generation method and the material of the exposure modules. Within the framework of a recently completed validation study, this exposure system was proven as a transferable, reproducible and predictive screening method for the qualitative assessment of the acute pulmonary cytotoxicity of airborne particles, thereby potentially reducing or replacing animal experiments that would normally provide this toxicological assessment.

Exposure of 19 substances to lung A549 cells at the air liquid interface or under submerged conditions reveals high correlation between cytotoxicity in vitro and CLP classifications for acute lung toxicity.

In vivo experiments are still widely used for the testing of lung toxicity but there is an ethical and legal obligation to replace, reduce and refine animal testing. Lung A549 cells could serve as an in vitro indicator for acute lung toxicity but little data about the correlation of the cytotoxicity in A549 cells and data leading to CLP classifications are available. We exposed A549 cells to 19 CLP-classified substances with doses of 25, 50, and 100 µg/cm2 either under submerged (SME) condition or with aerosols at the air-liquid interface (ALIF) and determined accuracy, precision, sensitivity and the F1 score with the CLP classifications H330, H332, or H335. When data from both exposure methods were combined, we found accuracies of 0.84 ±â€¯0.05, precisions of 0.74 ±â€¯0.1, sensitivities of 0.93 ±â€¯0.08 and F1 scores of 0.82 ±â€¯0.04. Separated from each other, ALIF exposure was more sensitive at any dose but, at higher doses, also less accurate and precise compared to SME. Considering the 19 substances tested, our data suggest that cytotoxicity in A549 cells could be a reliable in vitro indicator for in vivo toxicity. Thus, we discuss how A549 could be integrated into validation test guidelines.

Validation of the CULTEX® Radial Flow System for the assessment of the acute inhalation toxicity of airborne particles.

The CULTEX® Radial Flow System (RFS) is a modular in vitro system for the homogenous exposure of cells to airborne particles at the air-liquid interface (ALI). A former pre-validation study successfully demonstrated the general applicability of the CULTEX® RFS and its transferability, stability and reproducibility. Based on these results, the methodology was optimized, validated and prediction models for acute inhalation hazards were established. Cell viability of A549 cells after ALI exposure to 20 pre-selected test substances was assessed in three independent laboratories. Cytotoxicity of test substances was compared to the respective incubator controls and used as an indicator of toxicity. Substances were considered to exert an acute inhalation hazard when viability decreased below 50% (prediction model (PM) 50%) or 75% (PM 75%) at any of three exposure doses (25, 50 or 100 µg/cm2). Results were then compared to existing in vivo data and revealed an overall concordance of 85%, with a specificity of 83% and a sensitivity of 88%. Depending on the applied PM, the within-laboratory and between-laboratory reproducibility ranged from 90 to 100%. In summary, the CULTEX® RFS was proven as a transferable, reproducible and well predictive screening method for the qualitative assessment of the acute pulmonary cytotoxicity of airborne particles.

Is it the time to study air pollution effects under environmental conditions? A case study to support the shift of in vitro toxicology from the bench to the field.

Air pollution and particulate matter are recognised cause of increased disease incidence in exposed population. The toxicological processes underlying air pollution associated effects have been investigated by in vivo and/or in vitro experimentation. The latter is usually performed by exposing cells cultured under submerged condition to particulate matter concentration quite far from environmental exposure expected in humans. Here we report for the first time the feasibility of a direct exposure of air liquid interface cultured cells to environmental concentration of particulate matter. Inflammatory proteins release was analysed in cell medium while differential expression of selected genes was analysed in cells. Significant association of anti-oxidant genes was observed with secondary and aged aerosol, while cytochrome activation with primary and PAHs enriched ultrafine particles. The results obtained clearly show the opportunity to move from the lab bench to the field for properly understanding the toxicological effects also of ultrafine particles on selected in vitro models.

In vitro exposure systems and dosimetry assessment tools for inhaled tobacco products: Workshop proceedings, conclusions and paths forward for in vitro model use.

In 2009, the passing of the Family Smoking Prevention and Tobacco Control Act facilitated the establishment of the FDA Center for Tobacco Products (CTP), and gave it regulatory authority over the marketing, manufacture and distribution of tobacco products, including those termed 'modified risk'. On 4-6 April 2016, the Institute for In Vitro Sciences, Inc. (IIVS) convened a workshop conference entitled, In Vitro Exposure Systems and Dosimetry Assessment Tools for Inhaled Tobacco Products, to bring together stakeholders representing regulatory agencies, academia and industry to address the research priorities articulated by the FDA CTP. Specific topics were covered to assess the status of current in vitro smoke and aerosol/vapour exposure systems, as well as the various approaches and challenges to quantifying the complex exposures in in vitro pulmonary models developed for evaluating adverse pulmonary events resulting from tobacco product exposures. The four core topics covered were: a) Tobacco Smoke and E-Cigarette Aerosols; b) Air-Liquid Interface-In Vitro Exposure Systems; c) Dosimetry Approaches for Particles and Vapours/In Vitro Dosimetry Determinations; and d) Exposure Microenvironment/Physiology of Cells. The 2.5-day workshop included presentations from 20 expert speakers, poster sessions, networking discussions, and breakout sessions which identified key findings and provided recommendations to advance these technologies. Here, we will report on the proceedings, recommendations, and outcome of the April 2016 technical workshop, including paths forward for developing and validating non-animal test methods for tobacco product smoke and next generation tobacco product aerosol/vapour exposures. With the recent FDA publication of the final deeming rule for the governance of tobacco products, there is an unprecedented necessity to evaluate a very large number of tobacco-based products and ingredients. The questionable relevance, high cost, and ethical considerations for the use of in vivo testing methods highlight the necessity of robust in vitro approaches to elucidate tobacco-based exposures and how they may lead to pulmonary diseases that contribute to lung exposure-induced mortality worldwide.

Phenotypical changes in a differentiating immortalized bronchial epithelial cell line after exposure to mainstream cigarette smoke and e-cigarette vapor.

3D constructs composed of differentiated immortalized primary normal human bronchial epithelial (NHBE) cells (CL-1548) were repeatedly exposed at the air-liquid interface to non-lethal concentrations of mainstream cigarette smoke (4 cigarettes a day, 5days/week, 8 repetitions in total) and e-cigarette vapor (50 puffs a day, 5 days/week, 8 repetitions in total) to build up a permanent burden on the cells. Samples were taken after 4, 6 and 8 times of repeated smoke exposure and the cultures were investigated using histopathological methods Compared to the clean air-exposed cultures (process control) and incubator control, the aerosol-exposed cultures showed a reduction of ciliated, mucus-producing and club cells. At the end of the exposure phase, we even found metaplastic areas positive for CK13 antibody in the cultures exposed to mainstream cigarette smoke and e-liquid vapor, commonly seen in squamous cells as a marker for non-cornified squamous epithelium. The control cultures (incubator cells) showed no comparable phenotypical changes. In conclusion, our in vitro model presents a valuable tool to study the induction of phenotypical changes after exposure to hazardous airborne material.

Improvement of the CULTEX® exposure technology by radial distribution of the test aerosol.

The exposure of cellular based systems cultivated on microporous membranes at the air-liquid interface (ALI) has been accepted as an appropriate approach to simulate the exposure of cells of the respiratory tract to native airborne substances. The efficiency of such an exposure procedure with regard to stability and reproducibility depends on the optimal design at the interface between the cellular test system and the exposure technique. The actual exposure systems favor the dynamic guidance of the airborne substances to the surface of the cells in specially designed exposure devices. Two module types, based on a linear or radial feed of the test atmosphere to the test system, were used for these studies. In our technical history, the development started with the linear designed version, the CULTEX® glass modules, fulfilling basic requirements for running ALI exposure studies (Mohr and Durst, 2005). The instability in the distribution of different atmospheres to the cells caused us to create a new exposure module, characterized by a stable and reproducible radial guidance of the aerosol to the cells. The outcome was the CULTEX® RFS (Mohr et al., 2010). In this study, we describe the differences between the two systems with regard to particle distribution and deposition clarifying the advantages and disadvantages of a radial to a linear aerosol distribution concept.

Metaplastic phenotype in human primary bronchiolar epithelial cells after repeated exposure to native mainstream smoke at the air-liquid interface.

3D constructs composed of primary normal differentiated human bronchiolar epithelial (NHBE) cells as mono- or co-culture in combination with normal human lung fibroblasts were exposed repeatedly at the air-liquid interface with non-lethal concentrations of mainstream cigarette smoke (4 cigarettes a day, 5days/week, 13 times repetition in total) to build up a permanent burden on the cells. Samples were taken after 4, 8 and 13 times of repeated smoke exposure and the cultures were analyzed by histopathological methods In comparison with the clean air exposure (process control) and incubator control cells the cigarette smoke exposed cultures showed a reduction of cilia bearing as well as mucus producing cells. In both mono- as well as co-cultures, hyperplasia was induced showing different histological cell types (undifferentiated secretory and squamous cell types). At the end of the exposure phase, we observed the development of non-hyperplastic areas strongly positive to CK13 antibody, commonly seen in squamous cells as a marker for non-cornified squamous epithelium, thus suggesting a transition of the normal bronchial epithelial cells towards metaplastic cells. The control cultures (clean air exposed and incubator cells) showed no comparable phenotypic changes. In conclusion, our in vitro model presents a valuable tool to study the induction of metaplastic alterations after exposure to airborne material.

A new computer-controlled air-liquid interface cultivation system for the generation of differentiated cell cultures of the airway epithelium.

The increased application of in vitro systems in pharmacology and toxicology requires cell culture systems that facilitate the cultivation process and ensure stable, reproducible and controllable cultivation conditions. Up to now, some devices have been developed for the cultivation of cells under submersed conditions. However, systems meeting the requirements of an air-liquid interface (ALI) cultivation for the special needs of bronchial epithelial cells for example are still lacking. In order to obtain in vivo like organization and differentiation of these cells they need to be cultivated under ALI conditions on microporous membranes in direct contact with the environmental atmosphere. For this purpose, a Long-Term-Cultivation system was developed (CULTEX(®) LTC-C system) for the computer-controlled cultivation of such cells. The transwell inserts are placed in an incubator module (24 inserts), which can be adjusted for the medium level (ultrasonic pulse-echosensor), time and volume-dependent medium exchange, and frequency for mixing the medium with a rotating disc for homogeneous distribution of medium and secretion components. Normal primary freshly isolated bronchial epithelial cells were cultivated for up to 38 days to show the efficiency of such a cultivation procedure for generating 3D cultures exhibiting in vivo-like pseudostratified organization of the cells as well as differentiation characteristics like mucus-producing and cilia-forming cells.

Optimization of the CULTEX(®) radial flow system for in vitro investigation of lung damaging agents.

Exposure of the respiratory tract to airborne particles is gaining more and more importance due to the ubiquitous application of these particles in the field of industry, pharmacy and in daily life. Remarkably, the toxic properties and the underlying pathomechanisms with regard to inhalable substances have been insufficiently investigated so far. Thus, the EU Chemicals Regulation demands toxicological data (including the identification of potential inhalation hazards) for all chemicals placed on the market until 2018 (REACH). This requires extensive, technically complex and expensive inhalation toxicology studies that are usually generated in animal experiments. However, the legislation demands the consideration of the "3Rs" principle. Thus, in vitro-based test systems for the assessment of pulmonary toxicity are required. One promising approach to assess acute pulmonary toxicity of airborne particles is the CULTEX(®) RFS methodology that allows exposure of human lung epithelial cells at the air-liquid interface mimicking the alveolar situation. A prevalidation study showed the general applicability of this method. However, the clean air exposure group, which served as unexposed controls, exhibited some variations with regard to cell viability compared to the incubator control group. The aim of this study was therefore the identification of the possible causes and the improvement of methodological aspects. Several parameters including the general workflow, adjustment of airflow parameters, and cleaning procedures were investigated and adapted. Finally, our results showed the successful optimization of the CULTEX(®) RFS methodology for clean air exposure of A549 cells. However, although viability data in incubator controls and clean air exposures were equal, a distinct difference in cell morphology was observed that required further optimization. Additional experiments identified that open-wall cell culture inserts with a 2-fold pore density were found to be superior compared to the standard inserts and thus the deciding factor for the improvement of cell morphology. The presented findings are an important step in providing the CULTEX(®) RFS methodology as a promising alternative method to current in vivo testing in inhalation toxicology.

Cytotoxic Evaluation of e-Liquid Aerosol using Different Lung-Derived Cell Models.

The in vitro toxicological evaluation of e-liquid aerosol is an important aspect of consumer protection, but the cell model is of great significance. Due to its water solubility, e-liquid aerosol is deposited in the conducting zone of the respiratory tract. Therefore, primary normal human bronchial epithelial (NHBE) cells are more suitable for e-liquid aerosol testing than the widely used alveolar cell line A549. Due to their prolonged lifespan, immortalized cell lines derived from primary NHBE cells, exhibiting a comparable in vitro differentiation, might be an alternative for acute toxicity testing. In our study, A549 cells freshly isolated NHBE cells and the immortalized cell line CL-1548 were exposed at the air-liquid interface to e-liquid aerosol and cigarette mainstream smoke in a CULTEX(®) RFS compact module. The cell viability was analyzed 24 h post-exposure. In comparison with primary NHBE cells, the CL-1548 cell line showed lower sensitivity to e-liquid aerosol but significantly higher sensitivity compared to A549 cells. Therefore, the immortalized cell line CL-1548 is recommended as a tool for the routine testing of e-liquid aerosol and is preferable to A549 cells.

Ciliatoxicity in human primary bronchiolar epithelial cells after repeated exposure at the air-liquid interface with native mainstream smoke of K3R4F cigarettes with and without charcoal filter.

Mucociliary clearance is the primary physical mechanism to protect the human airways against harmful effects of inhaled particles. Environmental factors play a significant role in the impairment of this defense mechanism, whereas cigarette smoke is discussed to be one of the clinically most important causes. Impaired mucociliary clearance in smokers has been connected to changes in ciliated cells such as decreased numbers, altered structure and beat frequency. Clinical studies have shown that cilia length is reduced in healthy smokers and that long-term exposure to cigarette smoke leads to reduced numbers of ciliated cells in mice. We present an in vitro model of primary normal human bronchiolar epithelial (NHBE) cells with in vivo like morphology to study the influence of cigarette mainstream smoke on ciliated cells. We exposed mucociliary differentiated cultures repeatedly to non-toxic concentrations of mainstream cigarette smoke (4 cigarettes, 5 days/week, 8 repetitions in total) at the air-liquid interface. Charcoal filter tipped cigarettes were compared to those being equipped with standard cellulose acetate filters. Histopathological analyses of the exposed cultures showed a reduction of cilia bearing cells, shortening of existing cilia and finally disappearance of all cilia in cigarette smoke exposed cells. In cultures exposed to charcoal filtered cigarette smoke, little changes in cilia length were seen after four exposure repetitions, but those effects were reversed after a two day recovery period. Those differences indicate that volatile organic compounds, being removed by the charcoal filter tip, affect primary bronchiolar epithelial cells concerning their cilia formation and function comparable with the in vivo situation. In conclusion, our in vitro model presents a valuable tool to study air-borne ciliatoxic compounds.

Evaluation of E-cigarette liquid vapor and mainstream cigarette smoke after direct exposure of primary human bronchial epithelial cells.

E-cigarettes are emerging products, often described as "reduced-risk" nicotine products or alternatives to combustible cigarettes. Many smokers switch to e-cigarettes to quit or significantly reduce smoking. However, no regulations for e-cigarettes are currently into force, so that the quality and safety of e-liquids is not necessarily guaranteed. We exposed primary human bronchial epithelial cells of two different donors to vapor of e-cigarette liquid with or without nicotine, vapor of the carrier substances propylene glycol and glycerol as well as to mainstream smoke of K3R4F research cigarettes. The exposure was done in a CULTEX® RFS compact  module, allowing the exposure of the cells at the air-liquid interface. 24 h post-exposure, cell viability and oxidative stress levels in the cells were analyzed. We found toxicological effects of e-cigarette vapor and the pure carrier substances, whereas the nicotine concentration did not have an effect on the cell viability. The viability of mainstream smoke cigarette exposed cells was 4.5-8 times lower and the oxidative stress levels 4.5-5 times higher than those of e-cigarette vapor exposed cells, depending on the donor. Our experimental setup delivered reproducible data and thus provides the opportunity for routine testing of e-cigarette liquids to ensure safety and quality for the user.

Target cell types with stem/progenitor function to isolate for in vitro reconstruction of human bronchiolar epithelia.

Recent advancement in research on stem/progenitor cells of respiratory organs is breathtaking, benefiting from the rapid development of technology to create transgenic mice. There is now a great deal of knowledge capable of direct translation from mice to humans. Nevertheless, one has to be careful, since there may be unexpected pitfalls. First of all, there are differences anatomically, histologically and ultrastructurally in the airway epithelia of the two species. In parallel with these structural differences, regionally specific cell types behave and function, particularly in regenerative instances, differently between the two species, at least to some extent. From the viewpoint of important human respiratory diseases, one of the most susceptible regions of the respiratory tract is the bronchiole. In our approach to develop in vitro systems utilizing human bronchiolar epithelial cells, we are currently leaning on the data obtained from mouse studies in spite of the above-mentioned species differences. With the help of such in vitro systems we should be able to investigate the damaging effects and mechanisms of environmental pollutants in the human respiratory epithelium and consequently achieve results useful for quantitative analyses of the impact on human respiratory health. While pursuing this goal, the mouse data have suggested that it should be worthwhile to pay close attention to the stem/progenitor cells contained in the human bronchiolar epithelia and eventually make use of them. The mouse data have further shown that these stem/progenitor cells possess a very close association with the immature and variant club cells and the neuroendocrine cells, and our own unpublished preliminary data with human cells are, apparently, at least partly consistent with what the mouse data are telling us.

Direct exposure at the air-liquid interface: evaluation of an in vitro approach for simulating inhalation of airborne substances.

In toxicology, the strategies for testing the hazardous potential of substances are changing as a result of the ongoing progress in the development of in vitro methods and the demand of the authorities to reduce animal testing. Even in the complex field of inhalation toxicology with its high requirements on the technical implementation and cell culture models, the preconditions for using such methods are fulfilled. We here introduce a sophisticated technique that enables the stable and reproducible exposure of cultivated cells to airborne substances at the air-liquid interface by means of the CULTEX(®) Radial Flow System (RFS) module. The feasibility and suitability of the experimental setup is demonstrated by dose-response investigations of mainstream cigarette smoke and particulate matter of four substances in different lung epithelial cell lines. A dose-dependent cytotoxcity of the test substances was verified by applying different exposure times. The high reproducibility of the results indicate the reliability of the presented method and recommend the integration of such in vitro approaches in the field of inhalation toxicology by advancing their regulatory validation.

Use of the Cultex® Radial Flow System as an in vitro exposure method to assess acute pulmonary toxicity of fine dusts and nanoparticles with special focus on the intra- and inter-laboratory reproducibility.

Exposure of the respiratory tract to airborne particles (including metal-dusts and nano-particles) is considered as a serious health hazard. For a wide range of substances basic knowledge about the toxic properties and the underlying pathomechanisms is lacking or even completely missing. Legislation demands the toxicological characterization of all chemicals placed on the market until 2018 (REACH). As toxicological in vivo data are rare with regard to acute lung toxicity or exhibit distinct limitations (e.g. inter-species differences) and legislation claims the reduction of animal experiments in general ("3R" principle), profound in vitro models have to be established and characterized to meet these requirements. In this paper we characterize a recently introduced advanced in vitro exposure system (Cultex® RFS) showing a great similarity to the physiological in vivo exposure situation for the assessment of acute pulmonary toxicity of airborne materials. Using the Cultex® RFS, human lung epithelial cells (A549 cells) were exposed to different concentrations of airborne metal dusts (nano- and microscale particles) at the air-liquid-interface (ALI). Cell viability (WST-1 assay) as a parameter of toxicity was assessed 24h after exposure with special focus on the intra- and inter-laboratory (three independent laboratories) reproducibility. Our results show the general applicability of the Cultex® RFS with regard to the requirements of the ECVAM (European Centre for the Validation of Alternative Methods) principles on test validity underlining its robustness and stability. Intra- and inter-laboratory reproducibility can be considered as sufficient if predefined quality criteria are respected. Special attention must be paid to the pure air controls that turned out to be a critical parameter for a rational interpretation of the results. Our results are encouraging and future work is planned to improve the inter-laboratory reproducibility, to consolidate the results so far and to develop a valid prediction model.

Importance of material evaluation prior to the construction of devices for in vitro techniques.

The development and validation of new in vitro methods in the field of toxicology have gained more importance in recent years due to stricter guidelines for animal testing, especially in the European Union. Consequently, advances in the construction of technical devices for the exposure of cell or tissue cultures to test substances are necessary. Here, to obtain reliable results, it is important to exclusively use materials that do not interfere with the cell viability. Thus, similar to the biomaterials testing of medical devices which is regulated in the Directive 93/42/EEC, the biocompatibility of the materials has to be verified prior to the construction of such devices. We present here a novel approach for biomaterials testing which allows the quantitative and qualitative assessment of cytotoxicity of material samples. Stainless steel and silicone are often used for laboratory equipment, due to their high chemical, thermal and mechanical resistance. However, our results highlight that some types of silicone may have adverse effects on cultured cells. Moreover, special methods for the surface treatment of metals may also be a critical factor for in vitro devices. Therefore, the testing of all materials coming in contact with cell cultures is highly recommended.

The CULTEX RFS: a comprehensive technical approach for the in vitro exposure of airway epithelial cells to the particulate matter at the air-liquid interface.

The EU Regulation on Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) demands the implementation of alternative methods for analyzing the hazardous effects of chemicals including particulate formulations. In the field of inhalation toxicology, a variety of in vitro models have been developed for such studies. To simulate the in vivo situation, an adequate exposure device is necessary for the direct exposure of cultivated lung cells at the air-liquid interface (ALI). The CULTEX RFS fulfills these requirements and has been optimized for the exposure of cells to atomized suspensions, gases, and volatile compounds as well as micro- and nanosized particles. This study provides information on the construction and functional aspects of the exposure device. By using the Computational Fluid Dynamics (CFD) analysis, the technical design was optimized to realize a stable, reproducible, and homogeneous deposition of particles. The efficiency of the exposure procedure is demonstrated by exposing A549 cells dose dependently to lactose monohydrate, copper(II) sulfate, copper(II) oxide, and micro- and nanoparticles. All copper compounds induced cytotoxic effects, most pronounced for soluble copper(II) sulfate. Micro- and nanosized copper(II) oxide also showed a dose-dependent decrease in the cell viability, whereby the nanosized particles decreased the metabolic activity of the cells more severely.

Detection of the cytotoxicity of water-insoluble fraction of cigarette smoke by direct exposure to cultured cells at an air-liquid interface.

For the biological evaluation of cigarette smoke in vitro, the particulate phase (PP) and the gas vapor phase (GVP) of mainstream smoke have usually been collected individually and exposed to biological material such as cultured cells. Using this traditional method, the GVP is collected by bubbling in an aqueous solution such as phosphate-buffered saline (PBS). In such a way the water-insoluble GVP fraction is excluded from the GVP, meaning that the toxic potential of the water-insoluble GVP fraction has hardly been investigated so far. In our experiments we used a direct exposure method to expose cells at the air-liquid interface (ALI) to the water-insoluble GVP fraction for demonstrating its toxicological/biological activity. In order to isolate the water-insoluble GVP fraction from mainstream smoke, the GVP was passed through 6 impingers connected in series with PBS. After direct exposure of Chinese hamster ovary cells (CHO-K1) with the water-insoluble GVP fraction in the CULTEX(®) system its cytotoxicity was assayed by using the neutral red uptake assay. The water-insoluble GVP fraction was proven to be less cytotoxic than the water-soluble GVP fraction, but showed a significant effect in a dose-dependent manner. The results of this study showed that the direct exposure of cultivated cells at the air-liquid interface offers the possibility to analyze the biological and toxicological activities of all fractions of cigarette smoke including the water-insoluble GVP fraction.