13 October 2015 FEASIBILITY STUDY SUPPORTING AMENDMENTS TO OECD SUBACUTE AND SUBCHRONIC INHALATION TEST GUIDELINES FOR TESTING OF NANOMATERIALS

1 13 October 2015 2 3 FEASIBILITY STUDY SUPPORTING AMENDMENTS TO OECD SUBACUTE AND SUBCHRONIC INHALATION TEST GUIDELINES FOR TESTING OF NANOMATERIA...
Author: Amy Newton
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13 October 2015

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FEASIBILITY STUDY SUPPORTING AMENDMENTS TO OECD SUBACUTE AND SUBCHRONIC INHALATION TEST GUIDELINES FOR TESTING OF NANOMATERIALS

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Terms of reference

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1. This report provides input for discussions among experts in the OECD working group involved in the ‘feasibility study’ project supporting amendments to OECD subchronic inhalation test guidelines for testing of nanomaterials. This report was subject to revision based upon the discussions among members of this working group. Although the focus is on inhaled nanomaterials, nano- and microsized particles appear to coexist as a continuum because the commercialized forms of engineered nanoparticles commonly consist of aggregated / agglomerated structures rather than as isolated nano-particles (NP). Particular emphasis is directed towards poorly soluble nanoparticles. However, considering the great variety of nanoparticles possible, some nanoparticles are likely to exhibit dissolution rates from poorly to intermediate to highly soluble.

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Purpose

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2. The primary objectives of this feasibility study on lung burden and bronchoalveolar lavage (BAL) are to address the following questions:

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 If lung burden and BAL measurements are added to the OECD repeated exposure toxicity studies, what is the value added to the testing guidelines? How will the lung burden and BAL measurements be used to inform the hazard and risk of nanomaterials?

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 Can lung burden measurements and BAL analyses be assessed in the same animal, or are additional test animals needed?

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 How will lung burden and BAL measurements enhance the robustness and reproducibility of the endpoints currently included in TGs 412 and 413?

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For these aspects, the scientific and technical questions considered include:  What standardized methods for lung burden BAL determinations are available?  Is there a need to compare lung burdens with extrapulmonary organ burdens?  How can lung burden and BAL endpoints be used to bridge short-term studies to predict the outcome of long(er)-term studies?  Is this information amenable to improve the comparability of inhalation studies across different laboratories?  Are the endpoints of relevance for humans?  Are there animal welfare concerns?  What changes in study design to the current TGs 412 and 413 are necessary to achieve this objective?

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This feasibility study includes a brief discussion on the expected regulatory needs/data requirements for the revised OECD Test Guidelines 412 and 413 and animal welfare, including the need to:

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 Generate toxicity information that can be used to understand the potential human health hazards of nanomaterials;  Address data requirements for toxicokinetics studies;  Support approaches for grouping (read-across); and/or  Provide waiving arguments for long-term studies using an optional toxicokinetics add-on to revised TGs 412 and 413.

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3. The results of the project may contribute to an improved sensitivity for identification of NP-induced pulmonary and extra-pulmonary toxicity in the rat. The availability of biokinetic data at an early stage in the regulatory testing schemes for NPs provides invaluable information for designing highly focused, hypothesis-based TGs 412 and 413 compatible studies with fewer animals. The implementation of the endpoints in this TG will enhance the international harmonization of hazard and risk assessment with regard to NP-related inhalation toxicity.

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Lung Burden:

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Background and expected regulatory need/data requirement that will be enhanced by the addition of lung burden measurements

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4. The current TG 413 provides information on the exposure concentration-dependence of adverse effects in the lung and extra-pulmonary organs following repeated inhalation exposure of soluble chemicals (gas, vapours, liquid aerosols). This TG is aimed to also be applicable for any isometric nanosized or microsized particle-like substance, including fibers. The regimen, route, and metric of exposure are similar to human exposure. The study design is based on exposure concentrations for a standardized exposure period of 6 hours/day, on 5 consecutive days/week, for 13 weeks.

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5. As stated in TG 413, subchronic inhalation studies should consist of a control plus three concentration levels of the test chemical: i.e., a high concentration which results in a clear level of toxicity but does not cause lethality; an intermediate concentration; and a low concentration which produces no toxicity (NOAEL). This recommendation is in place to allow risk assessors to use rodent inhalation study data to determine a NOAEL (no observable adverse effect level), a LOAEL (lowest observable effect level), and a level resulting in the MTD (maximum tolerated dose). It is also of great value to use rodent inhalation study data to determine a benchmark dose (a retained pulmonary dose which results in a prescribed risk of developing an adverse response in these exposed rodents). Critical to such analyses is the accurate determination of lung burden to obtain dose-response relationships comparable across different studies (Kuempel and Castranova, 2011).

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6. Lung burden would be expected to decline with time post-exposure due to clearance processes. Clearance of non-soluble particles involves several processes. Particles which deposit in the conducting airways (trachea to the terminal bronchioles) are rapidly cleared (t1/2 of a few days) by the mucociliary escalator. Particles which deposit in the respiratory zone (respiratory bronchioles to the alveoli) are cleared by alveolar macrophages, which slowly migrate to the mucociliary escalator, or by movement into lung-associated lymphatics (Brain, 2

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1986; Ferin and Feldstein, 1978; Henderson, 1989; Henderson and Belinsky, 1993). Both of these processes are relatively slow (t1/2 of 70-90 days in rodents and >1 year in humans). High lung burdens of non-soluble particles have been shown to depress the mobility of particleoverloaded alveolar macrophages, resulting in depression of clearance from the deep lung (Morrow et al., 1988; Yu et al., 1989). At such overload lung burdens, low toxicity, poorly soluble particles have been shown to induce chronic lung pathology, such as fibrosis and cancer (ILSI, 2000). For this reason, risk assessors require information concerning the change in lung burden over time post-exposure to determine if chronic pulmonary responses are the result of specific physiochemical properties of the test particle or a non-specific response to an overload dose of a low toxicity particle. Therefore, one needs to evaluate lung burden over time post-exposure. It is suggested that lung burden be measured over at least three postexposure times. For poorly soluble particles, for example, one could measure endpoints immediately (0-3 days post-exposure), 7-14 days post-exposure, and 90 days post-exposure. This would allow evaluation of the clearance rate from the conducting zone as well as from the respiratory zone. Suggested sampling times for highly soluble particles having no persistent pulmonary effects could be much shorter. Data from range-finding pre-studies would be used to determine the appropriate sampling times for 90 day inhalation studies.

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7. A third objective of risk assessors is to compare the bioactivity of nanoparticles using studies from various laboratories. For inhalation studies from different laboratories on a given nanoparticle, such comparison of acute and chronic dose-response data would strengthen risk assessment. For studies with different nanoparticles, comparison of acute and chronic doseresponse data would allow development of relationships between physicochemical properties and bioactivity, so that informed decisions could be made on control banding (setting ranges of controls for various levels of toxicity where complete data are not available), safety by design, and prioritization of further studies. In these cases, reliable lung burden data are essential for comparison of results from different studies.

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Reason for requiring lung burden measurement as part of an inhalation protocol

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8. Total lung burdens following a single particle exposure may be calculated using this equation (Eq. 1):

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Lung burden = (aerosol concentration) (minute ventilation) (exposure duration) (deposition fraction)

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The concentrations used for estimating lung burden according to Eq.1 are those average total mass concentrations of test substance obtained from the analytical characterization of exposure atmospheres. Minute volume (MV) for the rat, generally taken from the literature, times exposure duration is used to estimate total inhaled dose. The fraction of the inhaled dose deposited in the pulmonary region is preferentially estimated by the MPPD2.11 software (Anjilvel and Asgharian, 1995; RIVM, 2002) based on cascade impactor analyses.

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9. There are several uncertainties when estimating lung burden by using equation 1. First, deposition fraction has been estimated using a model based on cascade impactor analyses rather than actual rodent lung data. This calculation is sensitive to the density of the dispersed nanomaterial and whether the procedures of particle-size analysis were followed as described in OECD GD 39 (2009). Evidence indicates that lung deposition is altered in various 3

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pathological states, such as bronchitis, emphysema, and fibrosis (Sweeney et al., 1987; Sweeney et al., 1995; Newton et al., 2008). All of these conditions are characterized by chronic inflammation. Therefore, induction of an inflammatory state or initiation of pathogenesis during the inhalation exposure period may alter actual particle deposition. Secondly, actual values for minute ventilation in rodents are very variable and differ greatly in the literature due to laboratory-specific experimental variables. In addition, some nanoparticles may evoke reflex bradypnea, causing actual minute ventilation to be lower than literature values. However, most inhalation studies employing Eq. 1 to calculate lung burden use literature values for minute ventilation and do not measure minute ventilation throughout the inhalation exposure period, because this would require specialized instrumentation and is labor intensive. Thirdly, calculated lung burdens estimate pulmonary dose assuming no clearance during the 90 day inhalation exposure. Therefore, if the test nanoparticle were soluble, one could over-estimate lung burden after a 90 day inhalation exposure. Thus, actual measurements of changes in lung burden over time post-exposure are required to distinguish between a highly soluble, semi-soluble, and non-soluble particle. Such information is essential for hazard analysis and risk assessment of pulmonary vs. extrapulmonary responses.

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Methods to determine lung burdens of nanoparticles

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10. It is recommended that the lung burden of nanoparticles be measured at three times postexposure whenever technically feasible quantitative methods exist. Highly specialized methods are available for carbonaceous materials which differ from those used for metals and metal oxides. None of the currently applied analytical methods can unequivocally distinguish between retained particle-like material and dissolved and matrix-bound material. Due to the relatively low rates of systemic translocation of non-soluble nanoparticles (Mercer et al., 2013a; Chen et al., 2006) and the sensitivity limits of chemical detection methods, nanoparticle accumulation in extra-pulmonary tissue may not be quantifiable. In such cases, nanoparticles in extra-pulmonary tissue could be qualitatively identified by imaging techniques, e.g., transmission electron microscopy or enhanced darkfield microscopy (Mercer et al., 2013a). Systemic translocation may be relatively high for soluble particles or in conditions of high pulmonary inflammation (Chen et al., 2006). Therefore, although not required, analysis of particles in extrapulmonary tissue can be optional. The method used for tissue conditioning and/or digestion can affect the physical appearance of the retained nanomaterial. The metric chosen to define lung burdens must be compatible with that of exposure. Reference curves used for specific analytes commonly are established as massbased. Therefore, the most direct measure of the deposited/retained dose is mass. Fiber, filament, and particle number measurements are highly dependent on their definition as to how isometric structures differ from fiber-like structures as well as their state of agglomeration and aggregation, including the method-of-analysis-specific lower limit of quantification. Rigorous digestion methods may affect particle number but not necessarily total mass. As dissolution rates by mass of smaller particles may be faster than that of larger, possibly aggregated particles. Therefore, the lung burden of nanostructures should utilize a mass-based metric as default.

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11. For all methods of quantification of nanoparticles, unexposed lungs should be spiked with various known amounts of the nanoparticle to validate specificity of the detection method, determine recovery, and construct a standard curve for that nanoparticle. Interferences of the test particle with the organic matrix and methods applied for conditioning the sample must be 4

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identified and qualified. The methods of determination regarding the total metal/carbon content of the test particle and that in the lung must be identical to minimize errors when calculating back from the analyte to the actual substance tested.

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Metals and metal oxides:

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12. Metals can be determined using inductively coupled plasma (ICP) atomic emission spectroscopy (ICP-AES), also referred to as inductively coupled plasma optical emission spectrometry (ICP-OES). This is a type of emission spectroscopy that uses the inductively coupled plasma to produce excited atoms and ions that emit electromagnetic radiation at wavelengths characteristic of a particular element. This technology is broadened further using inductively coupled plasma mass spectroscopy (ICP-MS). This type of mass spectrometry is capable of detecting metals and several non-metals at concentrations as low as one part in 1015 (part per quadrillion, ppq) on non-interfered low-background isotopes. This is achieved by ionizing the sample with inductively coupled plasma and then using a mass spectrometer to separate and quantify those ions. Compared to atomic absorption techniques (AAS), ICP-MS has greater speed, precision, and sensitivity. Compared with other types of mass spectrometry, however, ICP-MS introduces interfering species: argon from the plasma, component gasses of air that leak through the cone orifices, and contamination from glassware and the cones. There is minimal potential for artifacts from particle-surface interactions; however, none of these methods can definitively separate nanoparticle-associated metals from dissolved and tissuebound metal.

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13. Organs may also be digested and converted to acid-soluble inorganic salts in a mixture of acids (e.g., nitric acid, hydrochloric acid, sulfuric acid, perchloric acid, and/or hydrogen peroxide). This conversion takes place in sealed reaction vessels in a temperature range from 130 to 300ºC at 200 bar in a microwave-heated autoclave (Ultraclave). After cooling, solubilized organs are diluted with deionized water prior to analysis with a high-performance atomic absorption spectrometer (AAS). The AAS should be equipped with an automated motorized atomizer exchange that allows switching between flame and graphite furnace AAS by a simple software command. Modern graphite furnace systems include True Temperature Control (TTC) and pyro tubes, providing full Stabilized Temperature Platform Furnace (STPF) conditions for almost interference-free trace metal trace analysis.

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Carbonaceous materials

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14. Published evidence suggests that carbon nanotubes (CNT) can be quantified in organic matrices using programed thermo-optical analysis, an analytical methodology that relies on the unique thermal stability of CNT. This technique frequently uses a similar instrumental setup as described for the NIOSH 5040 method for elemental carbon analysis (Doudrick et al 2013; Cassinelli 1998). Optimization of the lung digestion methods by multiple digestion procedures are published (Doudrick et al, 2013; Pauluhn and Rosenbruch, 2014; Saxena et al, 2008; 2009; Tamura et al, 2011) to remove organic, tissue-related carbon (OC) that could potentially be reduced to elemental carbon (EC) with a bias to over-estimate the lung burdens of MWCNT. The lung digestion of carbonaceous material is complicated by the fact that the deposited and retained CNT seemed to precipitate and agglomerate with the progress of digestion of tissue. Visible precipitates must be mechanically removed from microfuge tube surfaces with potential of losses.

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15. Example of CNT analysis as published by Pauluhn and Rosenbruch (2014): The lungs were cryopreserved (@-20°C). Thawed lungs were digested overnight in 4 mL of a 25% 5

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solution of tetramethyl ammonia hydroxide in methanol @ 50 °C and then transferred into stoppered centrifuge tubes containing 5 mL methanol. Following sonication for 10 min, the content was centrifuged at 35615xg at 5 °C for 30 min. Thereafter, the supernatant was discarded; the centrifugate (pellet) was then re-suspended in methanol and centrifuged again. The resultant pellet was re-suspended in 5 mL nitric acid (50% in methanol), sonicated for 10 min, and allowed to react overnight. This cycle of sonication, centrifugation, and resuspension in methanol was repeated twice. At the final step the slurry suspension of the pellet in methanol was metered onto the punch of a quartz filter (1 x 1.5 cm), size-adjusted for elemental carbon/organic carbon (EC/OC) analysis), and heated at 180 °C until complete evaporation of solvent. The filter was then transferred to a microfuge tube and subjected to EC/OC analysis.

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16. The EC/OC analysis is a thermal-optical protocol. At first it evolves OC (organic carbon) in pure helium (He), which is carried into a manganese dioxide oxidizing oven for conversion to carbon dioxide (CO2) which is then catalytically (Ni) reduced to CH4. The CH4 is then quantified by gas chromatography equipped with flame-induced detector (GC-FID). The thermolysis of CNT utilizes a ramped temperature profile. The EC is then desorbed from the quartz filter in an oxygen (O2) blend carrier gas and quantified in the same way as OC. At the end of each run, an internal standard of known volume of methane (CH4) is injected and oxidized to CH4 to ensure accurate quantification of OC and EC. During the initial heating process in the O2-free environment, a fraction of collected OC may be pyrolized into EC. If left unaccounted for, the instrument would consistently report less OC and more EC than actually present in a given sample. To correct for this problem, a tunable diode laser is used to determine the absorbance of the sample throughout the heating ramp cycle. The absorbance increases as OC is pyrolized to EC and decreases as EC and the pyrolized OC are desorbed during the second heating cycle. The point at which the laser absorbance returns to its initial value is considered the split point between OC and EC for quantification. Any carbon measured before the split is assigned as thermal OC, and any carbon measured after the split is assigned as thermal EC. Thermal EC and thermal OC are referred to as EC and OC. The limit of detection was 0.2 µg C/sample. The optimal working range is 5-100 µg CNT/filter. Reference curves with CNT as tested in the inhalation chamber should be based on total carbon (TCS): CCNT = TCS-[OCS/(OCB/TCB]; with S for sample and B for blank. The recovery from spiked and digested lungs is in the range of 89-99%.

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17. A sensitive method to quantify carbon nanotubes in lungs was reported by Ohnishi et al. (2013). This method involves adsorption of a hybrid marker onto nanotubes, followed by desorption and detection of these markers using high performance liquid chromatography (HPLC). Briefly, the first stage of this method involves lung digestion with C99 at room temperature overnight as described by Kohyama and Suzuki (1991). The digested solution is centrifuged at 12,000 rpm for 10 minutes and the supernate removed. A 0.5 ml aliquot of Tween-80 (1%) is then added with stirring, centrifuged, and the supernate removed. Concentrated sulfuric acid (100 µl) is added and the suspension filtered (Whatman 111109 pore size = 0.8 um). The filter containing CNT is treated with 1 ml of Tween-80 (1%) and sonicated to extract the CNT. Benze[ghi]perylene (B[ghi]P, 0.125 µg/ml) and 25 µl of acetonitrile are mixed with the CNT solution with stirring for 15 minutes. The suspension is centrifuged and the supernate removed. The CNT pellet is washed with distilled water and centrifuged. The B[gjhi]P adsorbed onto the CNT is extracted with 0.5 ml acetonitrile and the mixture filtered to remove the CNT. The eluate is analyzed by HPLC using a fluorescence 6

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detector at an excitation of 294 nm and an emission of 410 nm. Details given by Ohnishi et al. (2013) indicate recovery is 92-98% with a limit of quantitation of 0.2 µg MWCNT in a rat lung.

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Human relevance

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18. Measured rodent lung burden can be compared to projected human lung burden by normalizing data to mass burden/alveolar epithelial surface area (Stone et al.,1992; Miller et al., 2011). Such normalization would allow risk assessors to quantitatively compare results from rat and mouse inhalation studies and extrapolate the dose-response data to anticipated human exposures. Therefore, more accurate dosimetry (measurement of lung burden) using doses relevant to those resulting from projected human exposures would make rodent inhalation studies more predictive in identifying human risks associated with inhalation of nanoparticles. This greater predictive value will minimize the number of experimental animals required to develop exposure limits and recommended control measures.

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19. Measurement of rodent lung burden over time post-exposure will provide clearance data to distinguish highly soluble nanoparticles from nanoparticles of low solubility. In addition, clearance information with poorly soluble nanoparticles will distinguish pulmonary responses intrinsically derived from the specific physicochemical properties (inherent toxicity) of a nanoparticle from generic overload-dependent outcomes. Avoiding false positive judgements would improve risk characterization and management.

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Animal welfare

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20. Several possibilities to allow measurement of lung burden, BAL endpoints, and histopathology using a minimal number of animals were discussed during a WNT/WPMN face-to-face meeting from September 21-22, 2015 in Washington, DC. Three proposals are discussed in detail giving the technical feasibility and the number of animals required for each. With each of these three proposals, BAL measurements will be required for all gases, vapours, and aerosols; but lung burden measurements are only required for what are known or likely to be poorly soluble particles.

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21. Proposal 1:

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TG 413 states that inhalation studies should be performed using 10 males and 10 females per group, including a control and 3 exposure dose groups. To evaluate clearance there should be 3 post-exposure intervals. Therefore, in proposal 1 for each nanoparticle tested the total number of animals required would be:

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(n=10) (2 sexes) (4 exposure groups) (3 post-exposure intervals) = 240 rodents

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With 10 lungs/group, experimental analyses could be conducted as follows:

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* Each whole lung will be split by tying off the left or right bronchus

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* All 10 left lungs will be instilled with fixative for histopathological evaluation of

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inflammation, granulomatous lesions, and fibrosis

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* All lobes of 5 right lungs will be dedicated to measurement of lung burden

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* All lobes of the remaining 5 right lungs will be dedicated to BAL as described later

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In this proposal, the total lung would be weighed prior to fixation and the right lung of the 5 rats dedicated to lung burden weighed. Lung burden would be expressed as particle 7

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mass/lung, correcting for the % of the total lung used for the lung burden measurements. Note, it is optional to do histopathology on the right lungs and BAL or lung burden measurements on the left lung if one prefers.

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22. It was the consensus of the workgroup that this proposal was consistent with TG 413 and that the n value = 5 for BAL and lung burden was sufficient to obtain reproducible data (supported by the literature) to allow statistical analysis between exposure and time groups. This procedure was viewed as feasible from a technical and labor allocation point of view. However, it used the most animals, since it required studies on both sexes.

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23. Proposal 2:

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This proposal argues that pulmonary responses of rodents to inhalation exposure to particles generally have not shown great differences between sexes. This should be verified in the range-finding pre-study. Therefore, it was proposed that studies be confined to male rodents since they inhale more air, and thus more particles, than females due to a higher metabolic rate. Since a driver of this proposal was to minimize study costs and animal use, the n value/group was reduced to 5. Therefore, the total number of animals used for each nanoparticle in this proposal would be: (n=5) (1 sex) (4 exposure groups) (3 post-exposure intervals) = 60 rodents

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To use 5 rodents/group, histology, BAL, and lung burden would use separate lung lobes. Oakes et al. (2014) have analyzed airflow delivery to each lobe of the rat lung, i.e., 1 left lung and 4 lobes of the right lung (apical, intermediate, diaphragmatic, and cardiac). In normal rats, the left lung receives 35.8% of the total inhaled volume, the right diaphragmatic lobe 27.9%, and the combination of the right apical, intermediate and cardiac lobes 36.1%. Therefore, the workgroup proposed that the left lung be used for BAL, the right diaphragmatic lobe for lung burden, and the right apical, intermediate, and cardiac lobes for histopathology. It was argued that n values of 5 would be sufficient to obtain reproducible BAL, lung burden and histopathology data. Since lung burden would be measured in the right diaphragmatic lobe receiving 27.9% of the total inhaled volume, measured values of particle mass would be multiplied by 3.58 to determine the total lung burden.

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24. This proposal minimizes the number of rats used. However, it was viewed by several members of the workgroup as being very labor intensive, requiring technical skill to tie off not only the right main bronchus prior to lavage of the left lung but also the lobar bronchus to the right diaphragmatic lobe prior to fixation of the other right lobes. Although technically feasible, the practical feasibility when processing a number of rat lungs at a time has not been evaluated in the current literature. Another concern raised by some members of the workgroup was that using only the diaphragmatic lobe (27.9% of the inhaled volume or deposited particle load) would increase the chance that the particle load would approach the detection limit for quantification at low exposures.

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25. Proposal 3:

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Considering the concerns raised above for proposal 2, i.e., technical feasibility and sensitivity for lung burden, a third proposal was put forward. In this proposal, the left lung of 5 rats would be used for lavage and all the lobes of the right lung would be used for histopathology. Note, it is optional to reverse this if one wishes. Another group of 5 rats would be dedicated to lung burden, maximizing the sensitivity of this vital indicator of dose. Therefore, a total of 10 rats would be needed per group. Since lung burden would use entire lungs of 5 rats, it would 8

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not be necessary to weigh total lungs or individual lobes in the 5 rats used for BAL and histopathology. The total number of rats needed for proposal 3 would be: (n=10) (1 sex) (4 exposure groups) (3 times) = 120 rats

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Data verifying that no gender differences in response to particle inhalation would be obtained in the range-finding pre-study.

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26. Whichever proposal is used, accurate lung burden measurement using validated methods would decrease animal use in the long run by avoiding the need for confirmatory studies where estimated lung burdens are in question. Furthermore, validated lung burden measurements would support comparison of studies from different laboratories on the same or different nanoparticles, decreasing the need for repeat studies. Note that these proposals would only use the lung. It is optional to take blood samples to monitor mediator levels as an indication of systemic inflammation. Additionally, one could analyze lung lymphatics and systemic organs microscopically to evaluate translocation of nanoparticles from the lung. These added endpoints would maximize the data gained from each animal.

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It is the opinion of the writing team for this feasibility study that proposal 3 is the preferred recommendation, since it is technically practical, results in data that have been shown in the literature to be statistically valid, and assures that the critical endpoint of lung burden is measured with the greatest accuracy. Proposal 1 would be optional if one wished to do a larger study on two genders. Proposal 2 would be optional if pre-study information verified that it was feasible for a given laboratory.

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Bronchoalveolar Lavage (BAL) Endpoints

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Background on BAL measurements

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27. The alveolar surface of the lungs is lined with a complex and highly surface-active material: pulmonary surfactant. The lung appears to be one of the most sensitive organs for changes in phospholipid homeostasis, since the turnover of phospholipids is highest in this organ due to the anabolism, catabolism, storage and recycling of pulmonary surfactant. This material consists of 90% lipids (including glycerol) and 10% surfactant-specific proteins. The lipids are mainly phospholipids; among the most important is phosphatidylcholine (approximately 60%), mainly responsible for lowering surface tension. The water-soluble film of the surfactant phospholipids and apoproteins determines both structure and homeostasis of surfactant. The surface film that lines the alveoli, prevents alveolar collapse. The functions of surfactant, including its surfactant apoproteins, in the alveolar lining layer are diverse. By stabilization of the fluid balance and reducing the contractile forces in the curved air-liquid interface, it prevents transudation of fluid into the alveoli (Van Golde et al., 1988). Disturbance of the surfactant system by noxious agents can take place at different stages (Van Golde et al., 1988). A compromised surfactant layer may lead to an increased permeability of the air-blood barrier and subsequent extravasation of plasma proteins (Nieman, 1985, Reasor, 1981). While the physicochemical properties and behavior of nanomaterials can be accurately characterized under idealized conditions, this is no longer the case in complex physiological environments. Site of deposition specific molecules can be adsorbed at the nanomaterial-bio interface to form a corona that critically affects the particles’ (patho)biological entities (Docter et al., 2015).

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28. The deposition of nanomaterials in the pulmonary region of the respiratory tract may result in an intraluminal exudate composed of biosubstances derived from increased transepithelial fluid flux as a reflection of the direct interactions on the nanomaterial surfaces with surfactant, degenerating or dead epithelial cells, and a large variety of cellular and humoral mediators and markers of acute intraluminal inflammation. The precise composition of the exudate depends on the cause and extent of injury and the site at which it occurs. Contemporary lung fixation methods require pressure-controlled instillation of fixative to prevent the collapse of lung resulting in variable septal thickness. However, this may dislocate adhering migratory cells and/or mucus/surfactant from intraluminal surfaces. From that perspective, BAL probes for changes occurring at the intraluminal sites of airways with a much higher resolution than histopathology. However, histopathology is more suitably for discerning structural changes at sites commonly not accessible to BAL-fluid, such as interstitial fibrosis. The strength of BAL is that it integrates the overall intraluminal response to injury in a readily quantifiable manner by determining cellular endpoints (cell viability, cytodifferentiation and activation) and acellular endpoints originating from local inflammatory processes or from plasma as a result of alveolar barrier disruption. While this method is suitable to probe for diffuse injuries, it has limited resolution for focal injuries. Histopathology changes are more complex to integrate area- and intensity-wise. However, it is the most suitable method to identify unequivocally the anatomical locations of structural injury and the response to injury. Protocols that focus on the instant response to particle exposure may benefit from BAL-analysis as the intraseptal changes may occur secondary to alveolitis and acute injury. At that point in time where structural remodeling of lung tissue occurs, histopathology will be the method of choice. From that perspective, it can be concluded that each method has its particular advantages and shortcomings and should be viewed as complementary.

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29. Bronchoalveolar lavage (BAL) has been widely employed by pulmonary toxicologists for more than forty years as a simple and reproducible method to quantify pulmonary injury and inflammation resulting from exposure of the lung to particles and other agents (Brain and Frank, 1973; Henderson, 1989; Henderson and Belinsky, 1993). It has become a common means to evaluate pulmonary inflammation and damage in rats, mice, and guinea pigs (Porter et al., 2001; Mercer et al., 2013b; Castranova et al., 1996). In all these species, BAL endpoints have been shown to be highly reproducible within and among different laboratories (Ma-Hock et al., 2009a; Mercer et al, 2013b; Kasai et al., 2014). Therefore, changes in BAL endpoints have been used effectively to monitor dose and time dependence of lung damage and inflammation following pulmonary exposure to particles, chemicals, and gases. Changes in BAL levels of mediators have identified mechanisms for the initiation and progression of particle-induced lung pathology. Such information is of great value to risk assessment and risk management.

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30. Parameters often reported in the literature include measurement of the following:

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1. Lactate dehydrogenase (LDH) activity in acellular BAL fluid (BALF) as an indicator of lung cell injury and/or membrane damage. Increased endocytosis by phagocytes may also cause increased LDH. 10

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2. Total protein, albumin or soluble collagen levels in acellular BALF as an indicator of a dysfunctional alveolar air/blood barrier. 3. -Glutamyltranspeptidase (GT) in acellular BALF as an indicator of an increased Clara-cell and also Type II pneumocyte activities. 4. -NAG (-N-acetylglucosaminidase) in acellular BALF is most probably the lysosomal enzyme released from resident alveolar macrophages engaged with particle endocytosis. 5. Cytokine, chemokine, or other mediator levels in acellular BALF as indicators of (pro-)inflammatory, proliferative, or (pro-)fibrogenic states. Release of these mediators from BAL cells may also be determined. 6. BAL cell counts and differentials as a measure of an inflammatory or immune response. 7. Although the precise mechanism by which any nanoparticle-induced pulmonary phospholipidosis occurs still needs to be elucidated, determination of phospholipids in the lavage fluid or within BAL cells as surfactant constituents may be useful to assess surfactant dysfunction because these endpoints appear to indicate changes most significantly. Emphasis could also be directed to foamy macrophages.

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Reason for requiring BAL as part of an inhalation protocol

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31. In the past decade, quantification of BAL responses have been reported following exposure to numerous engineered nanomaterials, such as nano-metals, quantum dots, nanometal oxides, carbon black nanoparticles, carbon nanotubes, graphene, etc. (Rushton et al. 2010; Roberts et al. 2013; Zhang et al. 2012; Sager et al. 2008; Sager and Castranova, 2009; Shvedova et al. 2008; Porter et al. 2013; Mercer et al. 2013; Ma-Hock et al., 2009a ;2009b ; 2013 2013; Pauluhn, 2009, 2010). These studies have been used widely for hazard identification and ranking of the toxic potency of various nanoparticles.

27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

32. A major advantage of evaluating BAL parameters is that they are quantifiable and objective measures allowing data to be expressed as means ± standard deviation/error and for changes to be subjected to statistical analyses. In contrast, classical histopathological evaluation of lung response is not continuously quantified since it employs scoring for severity and distribution on a non-continuous 1-5 scale. In addition, since scoring is subjective, variation is seen among readers. In addition, BAL is a simple method to obtain an integrated measure of inflammation and damage responses from the whole lung. For these reasons, significant changes in BAL parameters often occur before substantial pathological changes are noted. For example, in a 6 month inhalation study of rats exposed to crystalline silica, statistically significant elevations in BAL neutrophils, LDH activity, and albumin level were reported at earlier exposure time points and lower lung burdens than for histopathological indicators of inflammation and lung damage (Porter et al. 2001). The ability of BAL to provide reproducible and quantifiable information on dose and time dependence of particle-induced lung injury and inflammation is of great value for quantitative risk assessment and determination of relationships between nanoparticle potency and physicochemical properties. 11

1

Bronchoalveolar lavage method

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

33. There are many variations in the specifics of lavage procedures reported in the literature. BAL can be conducted in many ways, which include volume per lavage, total number of lavages per rodent, medium used for lavage, temperature of the medium, whether lungs are left in the chest cavity or removed prior to lavage, whether or not lungs are massaged during lavage, etc. Similarly, the number of lavages can vary from 2~14 times in different laboratories (for details and references see Song et al., 2010). To estimate the total cell number in the lung, it is important to recognize which BAL fluid fractions most influence the total cell number. The first and the second lavages contain similar number of cells in normal rats, but the first lavage commonly includes more cells than the second lavage in bleomycintreated lungs. Several reports suggested that more cells are present in the second lavage than in the first (Rehn et al., 1992; Kelly et al., 1988). Approximately 70% of total lavaged cells were retrieved in fractions 1~3, from which it was concluded that 3 lavages are sufficient for collecting cells that reflect the lung status in normal and bleomycin-induced inflamed lungs (Song et al., 2010). Hence, studies with a single lavage may not represent the whole lung and may introduce misinterpretation of the results in slightly damaged lungs.

17 18 19 20 21

34. A great deal of valuable information can be obtained from the acellular component of BAL fluid including levels of immunoglobulins, enzymes, inflammatory mediators, and surfactant (Henderson, 1989). However, variable dilutions of BAL fluid can cause both inaccuracy of quantification and difficulties detecting trace amounts of solute. Therefore, acellular components are most concentrated in the first lavage sample.

22 23 24 25 26 27 28 29

35. Song et al. (2010) compared the cell number and protein concentration in the whole lung and right lobes, one lobe for histopathology and the other for BAL for reasons of animal welfare or to simplify the experiments. OECD Guideline No. 39 recommends that left lung be used for histopathology, and the right lung used for BAL. The total cell number of both lungs was approximately twice the cell number of right lobes but the difference in protein levels between the 2 groups was negligible. Prevailing evidence suggests that BAL data from right lobes represent the whole lung. However, this conclusion has only been applied to generalized lung diseases (Song et al., 2010).

30 31 32 33 34

36. There are many BAL-associated details to be further determined, including the issue of erythrocytes. BAL fluid contains some erythrocytes when lung is not exsanguinated fully, and it may contain erythrocytes even when an inflamed lung is exsanguinated fully. Erythrocytes can affect the acellular component of BAL and could lead to faulty cell count results. A possible solution is to separate cells from the fluid as quickly as possible by centrifugation.

35 36 37 38 39 40

37. Brain and Frank (1968a, b) has shown that cell yield increases with increasing number of lavages; however, the yield per lavage rapidly declines after 5 or 6 lavages. Cell yield also appears to increase if Ca 2+ and Mg 2+ free medium is used and if the medium used is kept cool rather than at room temperature, since adherence of phagocytic cells to the lung is decreased (Brain and Frank, 1973). Massaging the lung during lavage is believed to improve the uniform distribution of lavage medium throughout the lung and thus improve cell yield. Some labs 12

1 2

remove the lung for weighing prior to BAL, so that changes in lung weight indicative of edema or severe fibrosis can be recorded (Oberdorster et al. 1994).

3 4 5 6 7 8 9 10 11 12 13 14 15

38. Although methods reported for BAL of rats vary in details, there is a general commonality among laboratories. An NIEHS Nano GO Consortium conducted a round robin evaluation of effects of pulmonary exposure to three different samples of nano TiO2 and three types of multi-walled carbon nanotubes (Bonner et al. 2013). Four labs conducted BAL with mice, while three labs conducted BAL with rats. Using common BAL methods, the labs were consistent in the ranking of the toxic potency of these six test nanoparticles. In addition, a single rodent was used for both BAL and histopathological evaluation; thus, minimizing animal use (5 rats per concentration, time post-exposure, or nanoparticle). The following is a general method, which represents guidance and could be modified to fit the needs of a specific project. The information detailed below is optional and should provide an appreciation of the methodological variability of this method. With regard to the recovered cell counts, as a measure of the quality of the lavage process, those reported by Song et al. (2010) should be achieved.

16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

1. Lavage medium – isotonic saline 2. Temperature – cool to room temperature 3. Number of rats – at least 5 per time, concentration, and nanoparticle 4. Euthanasia – administration of a veterinarian-approved anesthetic 5. Open the chest cavity to remove the lung, to measure lung weight (see the three proposed lung burden protocols). 6. Tie off the right main bronchus to allow for later use for histopathology 7. First lavage – attach a blunt needle to a 10 ml syringe; instill 3 ml of lavage medium via the trachea into the left lung; massage the lung; withdraw the fluid and re-instill with massage; withdraw the first BAL; empty into a centrifuge tube and set aside for for later. Alternatively, acellular BAL may be obtained from the first 2 lavages. 8. Lavage 2-5 – instill 4 ml of lavage medium; massage the lung; withdraw the fluid, empty into a tube and save; repeat for lavage 3, 4, and 5 9. Separate acellular BALF from BAL cells – separate cells from fluid by centrifugation for 5 min. at 500 g; save the supernatant from the first lavage for analysis of LDH (save at room temperature, not frozen, and measured the same day) and total protein or albumin; discard the supernatant from tubes 2-5; suspend the cell pellets from tubes 1-5 and combine for cell counts and differentials.

34 35 36 37 38 39 40

Mandatory BAL endpoints 39. The following endpoints are considered mandatory: LDH activity and total protein or albumin levels in acellular BALF, and cell counts and differentials for alveolar macrophages, lymphocytes and neutrophils. 40. Measurement of LDH Activity and Protein or Albumin Level in the First Acellular BALF: BALF samples for LDH should be kept at room temperature, not frozen, and measured the 13

1 2 3

same day as lavage. LDH activity can be quantified by detection of the oxidation of lactate coupled to the reduction of NAD+ by spectrometry at 340 nm. Protein or albumin level can be quantified by the method of Lowry et al. (1951) using an autoanalyzer.

4 5 6 7 8 9

41. Measurement of Cell Counts and Differentials: Add a drop of the BAL cell suspension onto a hemocytometer and count under low power light microscopy. Add 105 cells and spin onto a slide using a cytocentrifuge. Stain with Diff-Quick or equivalent. Conduct cell differentials by identifying a minimum of 400 cells using low power light microscopy. The number of alveolar macrophages, lymphocytes, and neutrophils are quantified by multiplying the % per cell type by the total number of BAL cells.

10

Optional measurements from BAL

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42. Measurement of eosinophil count from BAL has been suggested to evaluate allergic response. This could be done simply by identifying the % of eosinophils on the cytospin slides discussed above. Measurement of cytokines, chemokines, and mediators has also been suggested. This could be done using a sample of the first acellular BALF discussed above. Mediator levels can be quantified using ELISA kits. These mediators could be evaluated at a later time on frozen BALF samples without increasing the number of rats used in the study.

17

Human relevance

18 19 20 21 22 23 24 25 26 27 28

43. Partial lavage by bronchoscopy has been used as diagnostic tool in humans as it provides cells as well as non-cellular constituents from the lower respiratory tract. It opens a window to the lung. Alterations in lavage fluid and cells reflect pathological changes in the lung parenchyma. Its usefulness, also for clinical applications, has been appreciated worldwide in diagnostic work-up of infectious and non-infectious interstitial lung diseases. Moreover, lavage has several advantages over biopsy procedures. It is a safe, easily performed, minimally invasive, and well tolerated procedure. In this respect, when the clinician decides that a lavage might be helpful to provide diagnostic material, it is mandatory to consider the provided information be obtained from lavage fluid analysis carefully and to have reliable diagnostic criteria. This then would allow a better comparison of research-based animal data with human data.

29 30 31 32 33 34 35 36 37 38 39 40 41

44. A sizable body of information exists relating changes in BAL cells and mediator levels in miners to the disease severity, determined pathologically, of Coal Workers’ Pneumoconiosis (CWP). BAL data have substantially added to the understanding of mechanisms involved in the initiation and progression of lung disease (Castranova, 2000). For example, alveolar macrophages harvested by BAL from coal miners exhibit enhanced surface ruffling, indicating cell activation (Takemura et al. 1989; Lapp et al. 1991). A similar response was reported in rats after inhalation of coal dust (Castranova et al. 1985). Release of reactive species from alveolar macrophages obtained by BAL of coal miners appears to track disease progression with no effect in asymptomatic miners to a progressive increase in oxidant production in macrophages from mines with CWP vs. progressive massive fibrosis (PMF) (Lapp et al. 1991; Rom et al. 1987; 1990; Wallaert et al. 1990). Again, a similar activation of macrophage oxidant production was reported in rats exposed to coal dust (Castranova et al. 1985). Increased BALF levels of arachidonic acid metabolites and inflammatory cytokines 14

1 2 3 4 5 6 7 8

have been reported in coal miners with mediator levels being associated with disease progression (Kuhn et al. 1992; Lassalle et al. 1989; VanHee et al. 1993; Lassalle et al. 1990; VanHee et al. 1995). An increase in levels of inflammatory markers has also been seen in coal dust-exposed rats (Kuhn et al. 1990). Furthermore, a similar association with BAL cells and mediators has been reported in rats vs. humans after silica exposure (Castranova and Vallyathan, 2000). Therefore, the relevance of rat BAL data to humans is well documented. Lastly, human BAL data have been used to support the decision making process for regulation of ambient ozone levels (US EPA, 2013).

9

Animal welfare

10 11 12 13 14

45. Bronchoalveolar lavage would have no impact on animal welfare because it would not affect the normal course taken for euthanasia and necropsy procedures. In addition, the number of animals used in repeated exposure studies would not be affected since the lung, which would have been used for histopathology, would now be shared for both procedures, i.e., the right lung for histopathology and the left lung for BAL.

15

Summary and Conclusions

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46. The deposition of nanomaterials in the pulmonary region of the respiratory tract commonly results in a dose dependent intraluminal exudate composed of bio-substances derived from increased transepithelial fluid flux as a reflection of the direct interactions on the nanomaterial surfaces with surfactant, degenerating or dead epithelial cells, and a large variety of cellular and humoral mediators and markers of acute intraluminal inflammation. Contemporary fixation method of lungs requires pressure-controlled instillation of liquid fixative to prevent the collapse of lung resulting in variable septal thickness. However, this may dislocate adhering migratory cells and/or mucus/surfactant from intraluminal surfaces. From that perspective, BAL probes for changes occurring at the intraluminal sites of airways with a much higher resolution than histopathology. However, histopathology is more suitable for discerning focal and/or structural changes at sites commonly not accessible to BAL-fluid, such as interstitial fibrosis.

28 29 30 31 32 33 34 35 36 37 38 39 40 41

47. Hence, BAL is complementary to histopathology, and each method has its particular advantages and shortcomings. The particular strength of BAL is that it integrates the overall intraluminal response to injury in a compartment of the lung where poorly soluble NPs are deposited and retained. The pool of alveolar macrophages is better quantified and increases in pool size can be taken as evidence for kinetic changes and pulmonary NP accumulation. The reason for requiring BAL as part of an inhalation protocol is that BAL parameters are quantifiable and objective measures allowing data to be expressed as means and standard errors and for changes to be subjected to statistical and benchmark dose analyses. The benchmark dose method has been proposed as an alternative to the NOAEL approach for assessing risks associated with hazardous compounds. This method is a more powerful statistical tool than the traditional NOAEL approach resulting in a more accurate risk assessment. The benchmark dose method involves fitting a mathematical model to all the dose-response data obtained by BAL, and thus more biological information is incorporated in the resulting estimates of guidance values (e.g., as suggested by the U.S. Environmental 15

1 2 3 4 5 6 7

Protection Agency). In contrast, conventional histopathological evaluation of lung response is categorically quantified and employs scoring for severity and distribution on a non-continuous 1-5 scale. In addition, since scoring is subjective, variation is seen among pathologists. For these reasons, significant changes in BAL parameters often occur before substantial pathological sequelae of the intraluminal inflammatory changes are seen. BAL parameters are also amenable to more meaningful statistical analyses. Collectively, BAL is the technique of choice for the interrelating time-course changes of effects with changes in NP lung burdens.

8 9 10 11 12 13 14 15

48. The capacity of the lung to accommodate and clear poorly soluble particles is limited. Generally, this capacity is overwhelmed when the deposition rate exceeds the clearance rate. In case deposited NPs occur as agglomerated structures, their clearance follows generic kinetic principles, which change with lung burden. The estimation of the deposited and retained dose per inhalation day is based on theoretical calculations. Indeed, minute ventilation, required for the estimation of lung burden, is highly variable in rodents. Therefore, lung burden measurements are necessary to determine the degree of particle of particle accumulation and retention in the lung following repeated exposure.

16

49. Responses to Questions to Be Addressed by This Feasibility Study Document

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 If lung burden and BAL measurements are added to the OECD repeated exposure toxicity studies, what is the value added to the testing guidelines? How will the lung burden and BAL measurements be used to inform the hazard and risk of nanomaterials?

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Quantification rather than estimation of lung burden strengthens the accuracy of initial lung dose values. Unlike lung burden estimates, measured values for lung burden provide information concerning clearance and possible overload. Unlike histopathology, BAL data are quantifiable and can provide mechanistic information. Accurate dose values, clearance information, and quantifiable inflammatory and lung damage responses would be of great value to risk assessment and risk management.

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 Can lung burden measurements and BAL analyses be assessed in the same animal, or are additional test animals needed?

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Three proposals are discussed to allow measurement of BAL, ling burden, and histopathology using as few rodents as possible. More accurate and complete data would decrease the need for repeat studies.

32 33

 How will lung burden and BAL measurements enhance the robustness and reproducibility of the endpoints currently included in TGs 412 and 413?

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Quantifiable lung burden and BAL data would allow calculations of benchmark exposure doses, which would improve the accuracy of risk assessment in humans from rodent studies.

37 38 39 40 41

 What standardized methods for lung burden BAL determinations are available? Sensitive and reproducible methods to measure lung burden (metals, metal oxides, and carbonaceous nanoparticles) and BAL endpoints are found in the literature. These methods are discussed in this document. 16

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 Is there a need to compare lung burdens with extrapulmonary organ burdens? Since translocation rates for nanoparticles are generally low, systemic organ burdens may be difficult to quantify. Microscopic evaluation of nanoparticles in systemic tissue is listed as optional and would not require additional animals.  How can lung burden and BAL endpoints be used to bridge short-term studies to predict the outcome of long(er)-term studies? Quantification of lung burden would allow comparison of short-term and longer-term inhalation studies. The time course of changes in BAL endpoints after short-term inhalation of MWCNT has been shown to be predictive of responses after longerterm inhalation (Umeda et al., 2013; Kasai et al., 2014).  Is this information amenable to improve the comparability of inhalation studies across different laboratories? Quantification of lung burden and BAL endpoints would allow comparison of results on a given nanoparticle between different laboratories as well as ranking of potency among different nanoparticles.  Are the endpoints of relevance for humans? Quantified rodent lung burden can be normalized to predict human lung burdens on a “mass/alveolar epithelial surface area” basis. A large body of literature exists comparing BAL results from human subjects and rodents after exposure to coal dust or crystalline silica.  Are there animal welfare concerns? Three proposals are presented in this document which allow measurement of BAL endpoints, lung burden, and histopathology using a minimal number of rodents. The increased quantifiability and comparability of results would be expected to decrease the need for repeat studies; thus, decreasing total animal use.  What changes in study design of the current TGs 412 and 413 are necessary to achieve this objective? The document outlines three proposals to measure BAL endpoints, lung burden, and histopathology. Currently, TG 413 requires 10 rodents/group in both sexes. No additional animals would be needed to measure all three endpoints. However, 3 time points are recommended to obtain critical clearance and response persistence/ progression information. Time points post-exposure were not defined in TG 413. This is considered an oversight that should be rectified. Furthermore, 2 proposals recommend testing in males only rather than 2 sexes. This would decrease animal use without affecting the robustness of the data. 17

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Oberdoerster G, Ferin J, Morrow PE, 1992. Volumetric loading of alveolar macrophages (AM): a possible basis for diminished AM-mediated particle clearance. Exp Lung Res, 18: 87104

8 9

Oberdoerster G, Oberdoerster E, Oberdoerster J, 2007. Concepts of nanoparticle dose metric and response metric. Environ Health Perspect 2007, 115: A290

10 11

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12 13

Oberdorster G. et al. 1994. Correlation between particle size, in vivo particle persistence, and lung injury. Environ. Health Perspect. 102 (Suppl. 5):173-179.

14 15

Oberdörster, G., 2002. Toxicology of ultrafine particles: In vivo studies. Philos. Trans. R. Soc. Lond. A358: 2719-2740.

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OECD. 2009. Environment Health and Safety Publications Series on Testing and Assessment No. 39: Guidance Document for Acute Inhalation Toxicity Testing [ENV/JM/MONO 28; July 21 2009; 71 pp.]. Available at: http://www.oecd.org/ document/30/0,3343,en_2649_34377_1916638 _1_1_1_1,00.html.

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OECD, 2011.Working Party on Manufactured Nanomaterials: A SG7 Case Study for Hazard Identification of Inhaled Nanomaterials: An Integrated Approach with Short-Term Inhalation Studies. 8th WPMN meeting 16-18 March 2011, at the OECD Headquarter Conference Center.

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Ogawara, K-I., Yoshida, M., Higaki, K., Kimura, T., Shiraishi, K., Nishikawa, M., Takakura, Y., and Hashida, M. 1999. Hepatic uptake of polystyrene microspheres in rats: Effect of particle size on intrahepatic distribution. J. Controlled Release 59: 15-22.

27 28 29

Ohnishi M. et al. Novel method using hybrid markers: development of an approach for pulmonary measurement of multi-walled carbon nanotubes. J. Occup. Med. Toxicol. 8:30, 2013.

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Pauluhn J, 2010. Subchronic 13-week Inhalation Exposure of Rats to Multi-walled Carbon Nanotubes: Toxic Effects are determined by Density of Agglomerate Structures not fibrillar Structures. Toxicol, Sci, 113: 226-242

33 34

Pauluhn J, 2011. Poorly Soluble Particulates: Searching for a Unifying Denominator of Nanoparticles and Fine Particles for DNEL Estimation. Toxicology, 279, 176-188

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Pauluhn J, 2014a. The Metrics of MWCNT-Induced Pulmonary Inflammation are Dependent on the Selected Testing Regimen. Reg. Pharmacol. Toxicol. 2014a, 68: 343–352

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Pauluhn J, 2014c. Repeated Inhalation Exposure of Rats to an anionic High Molecular Weight Polymer Aerosol: Application of Prediction Models to better understand Pulmonary Effects and Modes of Action. Exp Toxicol Pathol, 66: 243-256

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Pauluhn J, Rosenbruch M, 2014. Lung burdens and kinetics of multi-walled carbon nanotubes (Baytubes) are highly dependent on the disaggregation of aerosolized MWCNT. Nanotoxicology. http://informahealthcare.com/nan 1743-5404.

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Pauluhn J, Rosenbruch M, 2003. Inhalation toxicity of propineb Part I: Results of subacute inhalation exposure studies in rats. Inhal Toxicol, 5: 411-434

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Pauluhn J, Emura M, Mohr U, Rosenbruch M, 2003. Inhalation toxicity of propineb Part II: Results of mechanistic studies in rats. Inhal Toxicol, 5: 435-460

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Pauluhn, J., 2009. Pulmonary Toxicity and Fate of Agglomerated 10 nm and 40 nm Aluminum Oxyhydroxides (AlOOH) following 4-week Inhalation Exposure of Rats: Toxic Effects are determined by agglomerated, not primary Particle Size. Toxicological Sciences 109:152167.

17 18

Pauluhn, J., and Mohr, U., 2000. Inhalation studies in laboratory animals - current concepts and alternatives. Review. Toxicol Pathol 28: 734-753.

19 20

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21 22

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26 27

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40 41

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24 25

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25

1

APPENDIX I

2 3

TERMINOLOGY

4 5 6

Absorbed dose (of a substance): Amount (of a substance) taken up by an organism or into organs or tissues of interest (synonym: internal dose).

7 8 9 10 11

Absorption (in biology): Penetration of a substance into an organism by various processes, some specialized, some involving expenditure of energy (active transport), some involving a carrier system, and others involving passive movement down an electrochemical gradient: in mammals, absorption is usually through the respiratory tract, gastrointestinal tract, or skin.

12 13 14 15 16 17 18 19 20 21 22 23 24 25

Actual concentration: The concentration of a test article in the test animal’s breathing zone. The sampled mass of the test article is determined by characterizing one or more constituents using either an analytical method specific for a selected component (e.g., chromatography) or a nonspecific, integrating method which addresses all non-volatile components, such as the total mass obtained by filter analysis (see gravimetric concentration). The terms actual concentration and analytical concentrations are commonly used interchangeably. The analytical or gravimetric concentration (not the nominal concentration) is generally used for hazard assessment. The actual concentration is commonly expressed in mass units per unit volume of air (mg/L, mg/m³). The mass of test article per unit mass of test animal (e.g., mg/kg), or inhaled dose, is difficult to define in inhalation toxicity studies since the fraction of test article deposited/absorbed/retained in the respiratory tract is dependent on a number of variables often not defined or measured in acute inhalation studies. Due to these uncertainties, exposure should be defined in terms of the "actual exposure concentration" and not the “exposure dose”.

26 27 28 29

Adverse effect: Change in biochemistry, morphology, physiology, growth, development, or lifespan of an organism which results in impairment of functional capacity or impairment of capacity to compensate for additional stress or increase in susceptibility to other environmental influences.

30 31 32

Aerodynamic diameter: The diameter of a unit density sphere having the same terminal settling velocity as the particle in question, whatever its size, shape, and density. It is used to predict where in the respiratory tract such particles may be deposited (24).

33 34 35

Aerosol: A relatively time-stable suspension of small solid or liquid particles in a gas. The diameter size range of aerosol particles is about 0.001 to 100 µm (24). See also dust, fog, fume, haze, mist, smog, and smoke.

36 37

Agglomerate: A group of particles held together by van der Waals forces or surface tension (24).

38 39

Aggregate: A heterogeneous particle in which the various components are not easily broken apart (24). 26

1 2

Alveolar: The portion of the respiratory system in which gas exchange occurs; alveoli are small sacs at the end of the bronchioles.

3

Analytical concentration: See actual concentration.

4 5 6

Apoptosis: Active process of programmed cell death requiring metabolic energy, often characterized by fragmentation of DNA, and without associated inflammation. See also necrosis.

7 8 9

Benchmark concentration: Statistical lower confidence limit on the concentration that produces a defined response (called the benchmark response or BMR, usually 5 or 10 %) for an adverse effect compared to background, defined as 0 %.

10 11 12

Bioaccumulation: Progressive increase in the amount of a substance in an organism or part of an organism which occurs because the rate of intake exceeds the organism’s ability to remove the substance from the body.

13 14

Bioavailability (general): Extent of absorption of a substance by a living organism compared to a standard system. Synonyms: biological availability.

15 16

18 19

Bioavailability (in pharmacokinetics): Ratio of the systemic exposure from extravascular (ev) exposure to that following intravenous (iv) exposure as described by the equation: A  Div where F is the bioavailability, A and B are the areas under the (plasma) F  ev Biv  Dev concentration-time curve following extravascular and intravenous administration respectively, and Dev and Div are the administered extravascular and intravenous doses.

20 21 22 23 24 25 26 27 28

Biokinetics (in toxicology): Biokinetics plays a critical role in dose response. Any thorough toxicity evaluation (Hazard Assessment) for a substance, and subsequently Risk Assessment, must consider biokinetics and biotransformation. Studies to examine these processes serve at least two general functions in the Risk Assessment process: First, the data collected can be used to help elucidate the mechanism of toxicity, define the target organs, and estimate critical exposure levels such as the Maximum Tolerated Dose (MTD) and the No-Observable-Effect Level (NOEL). The second is to help select the appropriate experimental animal model and procedure to extrapolate from animal toxicity to man (translational toxicology).

29 30 31

Cascade impactor: A device that uses a series of impaction stages with decreasing particle cut size so that particles can be separated into relatively narrow intervals of aerodynamic diameter; used to measure aerodynamic particle size (24).

32 33 34 35

Chronic: Long-term (in relation to exposure or effect). (1) In experimental toxicology, Chronic refers to mammalian studies lasting considerably more than 90 days or to studies occupying a large part of the lifetime of an organism. (2) In clinical medicine, long established or long lasting.

36 37 38 39

Clearance (in toxicology): (1) Volume of blood or plasma or mass of an organ effectively cleared of a substance by elimination (metabolism and excretion) divided by the time of elimination. Total clearance is the sum of the clearances of each eliminating organ or tissue for a given substance. (2) In pulmonary toxicology, the volume or mass of lung

17

27

1 2

cleared divided by the time of elimination is used qualitatively to describe removal of any inhaled substance which deposits on the lining surface of the lung.

3 4 5 6 7

Compartmental analysis: Mathematical process leading to a model of transport of a substance in terms of compartments and rate constants, usually taking the form C  Ae t  Be  t ...... where each exponential term represents one compartment. C is the substance concentration; A, B,…. are proportionality constants, a, b,… are rate constants; and t is time.

8 9

Concentration: The mass of test article per unit volume of air (e.g., mg/L, mg/m3), or the unit volume of test article per unit volume of air (e.g., ppm, ppb).

10 11 12

Critical organ (in toxicology): Organ that attains the critical concentration of a substance and exhibits the critical effect under specified circumstances of exposure and for a given population.

13 14 15

Critical organ concentration of a substance: Mean concentration of a substance in the critical organ at the time the substance reaches its critical concentration in the most sensitive type of cell in the organ.

16 17 18 19 20 21 22 23 24 25 26 27 28 29

Deposition of particles in the respiratory tract: The size and shape of particles are primordial factors that condition their deposition in the lungs. The size is defined the MMAD of a particle by means of four mechanisms: Impaction: This is the physical phenomenon by which the particles of an aerosol tend to continue on a trajectory when they travel through the airway, instead of conforming to the curves of the respiratory tract. Interception: This is mainly the case of fibers, which, due to their elongated shape, are deposited as soon as they contact the airway wall. Sedimentation: This is the physical phenomenon by which particles with sufficient mass are deposited due to the force of gravity when they remain in the airway for a sufficient length of time. It can generally be considered that particles with an MMAD higher than 10μm are deposited in the oropharynx, those measuring between 5 and 10μm in the central airways and those from 0.5 to 5μm in the small airways and alveoli. Therefore, for topical respiratory treatment it is best to use particles with an MMAD between 0.5 and 5μm. This is what is known as the breathable fraction of an aerosol.

30 31

Disposition: Total of the process of absorption of a chemical into the circulatory system, distribution throughout the body, biotransformation, and excretion.

32 33 34 35 36

Dissolution: Mathematical models for the dissolution of solid particles involve accounting for the complicated changes in the surface area and/or shape which occur during dissolution. Solid particles in liquids can be modeled using Nernst-Brunner type kinetics which is an extension of the Noyes and Whitney dissolution kinetics (Brunner and Tolloczko, 1900; Brunner, 1900; Nernst, 1904; Wong, 2007):

37

dM D  S A  (CS  C ) dt Vm h

38 39 40

where M is the mass of solid material at a given time t, SA is the area available for mass transfer, D is the diffusion coefficient of the dissolving material, Vm is the dissolution medium volume, h is the diffusion boundary layer thickness, C is the concentration, and 28

1 2 3 4 5 6 7 8 9 10 11

Cs is the substances saturation solubility. Diffusion-controlled models were further refined for single spherical particle dissolution under sink conditions and pseudo steadystate of the kinetic release of a particles homogeneously dispersed in a matrix into a medium under perfect sink conditions (Wong, 2007). Polydisperse particle sizes and coated particles retained in an inflammatory milieu of the lung may add another dimension of complexity to any model. Due to the longer life-time of humans, time- and dissolution-related changes in particle properties are biased to underestimate the contribution of clearance by slow dissolution. For more details on the distinction between thermodynamic and kinetic equilibrium solubility, and how one can exceed the equilibrium solubility to yield a supersaturated solution, specialized literature should be consulted (Dokoumetzidis and Macheras, 2006;Britztain, 2014; Wong, 2007).

12 13

Distribution volume: Theoretical volume of a body compartment throughout which a substance is calculated to be distributed.

14 15 16

Dust: Dry solid particles dispersed in a gas as a consequence of mechanical disruption of a bulk solid material or powder formed from a single component or mixture. Dust particles are generally irregular and larger than 0.5 µm (27).

17 18

Elimination (in toxicology): Disappearance of a substance from an organism or a part thereof, by processes of metabolism, secretion, or excretion.

19 20

Elimination rate: Differential with respect to time of the concentration or amount of a substance in the body, or a part thereof, resulting from elimination.

21 22

Endocytosis: Uptake of material into a cell by invagination of the plasma membrane and its internalization in a membrane-bounded vesicle.

23 24 25

Exposure chamber: A closed system used to expose animals to a gas, vapour, or aerosol of a test article. See Dynamic inhalation chamber, Nose-only inhalation chamber, and Whole-body inhalation chamber.

26 27

Extrathoracic: The portion of the respiratory tract before the thorax including the nose, mouth, nasopharynx, oropharynx, laryngopharynx, and larynx.

28 29 30

Geometric standard deviation (σg or GSD): A unitless number used to portray the range of particle sizes. A particle distribution is considered to be monodisperse when the σg is 1.01.2, and polydisperse when the σg is >1.2 (38).

31 32

Inhalable aerosol: Fraction of an aerosol that can enter the human respiratory system through the nose and mouth.

33 34 35

Inhalable diameter: The aerodynamic diameter of particles which can be inhaled through the nose and/or mouth of a given organism and deposited anywhere along the respiratory tract.

36

Inhalation chamber equilibrium: see Equilibrium concentration.

37 38

Inhalation: Exposure to a test article by normal respiration. The entire respiratory tract can be exposed. 29

1 2 3 4 5 6 7

Kinetic lung overload: The increase of the pool of macrophages as a result of increased alveolar macrophage-related endocytosis of poorly soluble particles is the first indicator of the “kinetic lung overload” of low-toxicity, poorly soluble isometric particles. Along with the increased in the “volume of distribution, Vd” of particles deposited and retained in the lung the elimination half-time increases above the normal t1/2 of 60-90 days (in rats). Humans have a much larger Vd than rats which makes humans more resistant (Pauluhn, 2011).

8 9 10 11

Kinetic lung overload-induced pulmonary injury: Prevailing experimental evidence obtained in the most sensitive bioassay (rat) with granular biopersistent particles demonstrated that the prevention of overload-like conditions may also prevent from secondary long-term effects to occur (ILSI, 2000).

12 13 14 15 16

Lowest-observed-adverse-effect level (LOAEL): Lowest concentration or amount of a substance (dose), found by experiment or observation, which causes an adverse effect on morphology, functional capacity, growth, development, or life span of a target organism distinguishable from normal (control) organisms of the same species and strain under defined conditions of exposure.

17 18 19

Macrophage: Migratory and phagocytic cell found in many tissues, especially in areas of inflammation, derived from blood monocytes and playing an important role in host defense mechanisms.

20 21 22 23 24 25 26

Mass median aerodynamic diameter (MMAD): Mass median of the distribution of mass with respect to aerodynamic diameter. The median aerodynamic diameter and the geometric standard deviation are used to describe the particle size distribution of an aerosol, based on the mass and size of the particles. Fifty percent of the particles by mass will be smaller than the median aerodynamic diameter, and 50% of the particles will be larger than the median aerodynamic diameter. MMADs of 1-4 μm are recommended for acute inhalation toxicology studies. See also Equivalence diameter.

27 28 29 30 31 32 33 34 35

Maximum tolerated dose (MTD): High dose used in repeated exposure (chronic) toxicity testing that is expected on the basis of an adequate subchronic study to produce limited toxicity when administered for the duration of the test period. It should not induce overt toxicity, e.g. appreciable death of cells or organ dysfunction, or toxic manifestations that are predicted materially to reduce the life span of the animals except as the result of neoplastic development, or 10% or greater retardation of body weight gain as compared with control animals. For particle-induced pulmonary effects, an elimination half-time exceeding 1 year in rats is commonly considered to cause a dysfunction in clearance fulfilling the MTD criterion.

36

MMAD: See Mass Median Aerodynamic Diameter.

37 38

Necrosis: Sum of morphological changes resulting from cell death by lysis and/or enzymatic degradation, usually affecting groups of cells in a tissue. See also apoptosis.

39 40 41 42

Nominal concentration: The concentration of test article introduced into a chamber system. It is calculated by dividing the mass of test article generated by the volume of air passed through the chamber. The nominal concentration does not necessarily reflect the concentration to which an animal is exposed. The resultant actual concentration cannot 30

1 2

be predicted from the nominal concentration by default because of its dependence on laboratory-specific technical variables. See also Actual concentration.

3 4 5 6 7

No-observed-effect level (NOEL): Greatest concentration or amount of a substance, found by experiment or observation, that causes no alterations of morphology, functional capacity, growth, development, or life span of target organisms distinguishable from those observed in normal (control) organisms of the same species and strain under the same defined conditions of exposure.

8 9 10 11 12

Nose-Only Inhalation Chamber: An inhalation chamber system that minimizes dermal exposure and oral exposure (via licking of contaminated fur). Animals are place in a restraining tube during the course of exposure. The design of this tube should not interfere with the thermoregulation of the animal to any appreciable extent. Head-only and snout-only are synonyms of nose-only.

13 14 15

One-compartment model: Kinetic model, where the whole body is thought of as a single compartment in which the substance distributes rapidly, achieving an equilibrium between blood and tissue immediately.

16

Particle size - see Aerodynamic particle size.

17 18

Particle size distribution: A description of how much of an aerosol is in each of a set (or continuum) of size intervals.

19 20

Pinocytosis: Type of endocytosis in which soluble materials are taken up by the cell and incorporated into vesicles for digestion.

21 22 23 24

Polydisperse aerosol: An aerosol composed of particles with a range of sizes. A particle distribution is considered to be monodisperse when the GSD is 1.0-1.2, and polydisperse when the GSD is >1.2 (32). See also Monodisperse aerosol and Geometric Standard Deviation.

25 26 27

Portal-of-entry effect: A local effect produced at the tissue or organ of first contact between the toxicant and a biological system. For the inhalation route, the portal-of-entry can be any part of the respiratory tract from the nose to the terminal alveoli of the lung.

28 29 30 31

Protein corona: Nanoparticles in a biological fluid (plasma, or otherwise) associate with a range of biopolymers, especially proteins, organized into the “protein corona” that is associated with the nanoparticle and continuously exchanging with the proteins in the environment.

32 33

Pulmonary (PU): Pertaining to the lungs, including the respiratory bronchioles, alveolar ducts, and alveoli.

34 35

Respirable diameter: The aerodynamic diameter of particles which are capable of reaching the gas-exchange region in the lungs (the alveoli) for the organism under study.

36 37 38

Respirable fraction: Fraction of aerosol that can reach the gas exchange region of the respiratory system (i.e., the alveoli). For details see European Standard EN 481:1993 (39).

31

1 2

Respirable particulate mass: The mass of material that is deposited in the gas-exchange region of the lungs for the organism under study.

3 4

Retention (lung): Amount of a substance that is left in the lung after deposition from the absorbed or cleared fraction after a certain time following exposure.

5 6

Retention: The amount of deposited particles that are not cleared from the respiratory tract at a particular time after exposure.

7 8

Sighting study: A preliminary study performed using a minimum of animals for the purpose of selecting concentrations to be used in a main study.

9 10 11 12 13 14 15 16 17 18 19 20 21 22

Solubility of any substance is normally determined during the pre-testing stage, and it is crucial to know whether the determined values represent genuine equilibrium solubilities (i.e., thermodynamic values) or whether they represent the values associated with a metastable condition (i.e., kinetic values). An understanding of the distinction between thermodynamic and kinetic solubility requires one to determine if and when the substance is undergoing a physical change during the measurement period, and how any solubility values are to be assigned as reflecting either equilibrium solubility or metastable solubility. The equilibrium solubility of a compound is defined as the maximum quantity of that substance which can be completely dissolved at a given temperature and pressure in a given amount of solvent, and is thermodynamically valid as long as a solid phase exists which is in equilibrium with the solution phase. It is necessary for an investigator to understand the distinction between thermodynamic and kinetic solubility, and to know when a particular measurement represents an equilibrium solubility value, or if the determined value simply represents some type of metastable condition.

23 24 25 26 27 28 29 30

Solubility equilibrium is a type of dynamic equilibrium. It exists when a chemical compound in the solid state is in chemical equilibrium with a solution of that compound. The equilibrium is an example of dynamic equilibrium in that some individual molecules migrate between the solid and solution phases such that the rates of dissolution and precipitation are equal to one another. When equilibrium is stablished, the solution is said to be saturated. The concentration of the solute in a saturated solution is known as the solubility. Units of solubility may be molar (mol dm−3) or expressed as mass per unit volume, such as μg ml−1.

31

Speciation: Distribution of an element amongst chemical species in a system.

32

t95: see Equilibrium concentration.

33 34

Target concentration: The desired chamber concentration. See also Nominal concentration and Actual concentration.

35 36 37 38 39

Test article: A product, substance, preparation or mixture (a formulation of multiple components) used for inhalation testing. Some test articles may be thermally decomposed for the purpose of testing, as in combustion toxicology tests. Atmospheres that result from thermal decomposition are considered to be mixtures. In all other circumstances where a non-destructive test is used, the term test article should be used.

40

Test substance: see Test article. 32

1 2

Thoracic Fraction: Fraction of aerosol that can reach the lung airways and the gas-exchange region. See also Respirable fraction, Inhalable aerosol.

3

Threshold: Dose or exposure concentration below which an effect will not occur.

4 5 6

Toxicokinetics: Process of the uptake of potentially toxic substances by the body, the biotransformation they undergo, the distribution of the substances and their metabolites in the tissues, and the elimination of the substances and their metabolites from the body.

7 8

Ultrafine or nano-particles: Particles larger than 1 nm and smaller than 100 nm in at least one dimension.

9 10

Uptake: Entry of a substance into the body, into an organ, into a tissue, into a cell, or into the body fluids by passage through a membrane or by other means.

11 12 13

Volume of distribution: Apparent (hypothetical) volume of fluid required to contain the total amount of a substance in the body at the same concentration as that present in the plasma, assuming equilibrium has been attained.

14 15 16

Whole-body chamber: An inhalation chamber that exposes the whole animal. Especially for aerosols, this results not only in inhalation exposure, but also dermal exposure and oral exposure (via licking of the fur).

17

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1

APPENDIX II

2 3

Purpose of the Short-Term Inhalation Study (STIS)

4 5 6 7 8 9 10 11 12 13

Given the vast number of nanotechnological products entering the market and the multitude of different nanomaterials already available, hazard and risk assessments of each and every single variant of nanomaterial are impractical and undesirable for economic reasons and stand in contradiction to the legal requirement to reduce animal testing. To meet this data requirement a standard short-term inhalation test (STIS) has been developed, which may either serve as a screening tool enabling grouping and read-across or as a range-finding study for a subsequent subchronic inhalation study. As such, it should enable the differentiation of poorly soluble particles from soluble particles at an early phase. It should also build a solid data base for further testing strategy and facilitate concentration selection and the setting of appropriate study design for studies with a prolonged exposure period (e.g. subchronic study).

14

Study design

15 16 17

The study design for STIS was first endorsed based on evaluation of an extensive data set on nano-TiO2 (Ma-Hock et al. 2009a). The core elements of the study design have since remained unchanged:

18 19 20 21 22 23 24 25

      

Male Wistar rats Head-nose or whole-body inhalation exposure (3 concentrations and control) Exposure for six hours a day on five consecutive days Histological examination (n = 3 rats/concentration/time point) Bronchoalveolar lavage (n = 5 rats/concentration/time point) If technically applicable, determination of lung burden and translocation to respective lymph nodes (n = 3 rats/concentration/time point) Post-exposure period of three weeks.

26 27 28

This study design covers three key elements of particle toxicity testing: 1) inflammation potency in the respiratory tract, 2) reversibility or progression of the effect and 3) deposition and bio-persistence of the particles in lung.

29

Key element 1: Inflammation potency in the respiratory tract

30 31 32 33 34 35 36 37 38 39

Inflammation potency within the respiratory tract is determined by histopathology of the respiratory tract and broncho-alveolar lavage (BAL). Histopathology and BAL analysis, as described in the main text, are two complementary methods that examine portal of entry toxicity of inhaled particles. BAL parameters are very sensitive, especially at the early phase of exposure as in a STIS. For example, at exposure levels of 5 or 25 mg/m³ nano ceria (NM 212), BALF parameters were dramatically increased in STIS after only 5-days of exposure, whereas granulomatous inflammation became obvious only in the 28-day study (Keller 2014). Though the data acquired from BAL fluid analysis is valuable, as discussed in detail in the main text, histological examination of the entire respiratory tract is essential for two key reasons: 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16





Effects to the upper respiratory tract can only be detected by histological examination. Substances like ZnO irritate the upper respiratory tissues. The corresponding morphological change in the upper respiratory tract can only be revealed by histological examination. BALF parameters, as mentioned before, indicate an acute reaction of the lung, which is not necessarily associated to a specific type of morphological change. Reviewing existing data (Landsiedel et al. 2014, Ma-Hock et al. 2009b), the pattern of BALF changes was similar: the number of polymorphonuclear neutrophils was the most sensitive parameter accompanied by increases of BALF lymphocytes and increased BALF protein concentration and enzyme activities (lactate dehydrogenes, γglutamyltransferase, alkaline phosphatase and N-Acetyl glucosaminidase). Histological examination, however, showed distinct morphological changes of the lung tissue between different materials. Granulomatous inflammation was observed in animals exposed to carbon nanotubes or nano ceria but not in those exposed to nano TiO2 (Ma-Hock et al. 2009a and b, Keller et al. 2014(Keller et al. 2014)).

17 18

Thus, both BAL analysis and histological examination of the entire respiratory tract should be incorporated into the STIS protocol.

19

Optional Parameters:

20 21 22 23 24 25 26 27 28 29 30 31 32

Pro-inflammatory cytokines are widely used as indicators of acute or ongoing inflammation or inflammation-like responses in vitro. On the other hand, pro-fibrotic cytokines such as transforming growth factor (TGF)-β1, macrophage colony stimulating factor (M-CSF), or osteopontin, may also be useful to identify beginning fibrotic or neoplastic changes upon particle exposure at an early stage, and increase the diagnostic power of lavage. Moreover, cytokines build a bridge between in vivo and in vitro test systems. For these reasons, 68 cytokines, chemokines and inflammationrelevant enzymes from BALF and lung-tissue homogenates were screened in the beginning of these studies. Only a limited panel seemed to provide added value to our understanding of particle effects inside the lungs. None of them was more sensitive than the neutrophils in lavage fluid. The relevance of a few selected cytokines/chemokines are discussed below for further consideration as potential optional parameters.

33 34 35 36 37 38 39 40

Monocyte chemoattractant protein (MCP-1) and cytokine-induced neutrophil chemoattractant 1 (CINC/IL-8) are both released by epithelial cells and in part by macrophages, reached considerable concentrations in BALF upon particle treatment. MCP-1 belongs to the C-C cytokines and strongly attracts blood monocytes and lymphocytes to the alveolar compartment (Maus et al. 2001). This chemokine displayed a large dynamic range observed in this study and showed a sustained increase during quartz DQ12 induced inflammation. Both findings predestine MCP-1 as a marker for early inflammation and macrophage recruitment to the lungs.

41 42

CINC/IL-8 is a pro-inflammatory C-X-C chemokine that is mainly produced by epithelial cells and exhibits polymorphonuclear neutrophil chemotactic activity 35

1 2 3 4 5 6

(Donaldson et al. 2008). In general, its alterations upon particle inhalation resembled those of MCP-1 in that they showed either no or significant increases at the same particle concentration. Dose dependent increases of CINC/IL-8 and MCP-1 were found after inhalation exposure to quartz DQ12, CeO2, Al-doped CeO2, coated nanoZnO, and micron-scale ZnO. In most cases, increases in MCP-1 outlined those of CINC/IL-8.

7 8 9 10 11 12 13 14 15

Macrophage colony-stimulating factor (M-CSF) is a cytokine that is produced by macrophages and is involved in the differentiation of monocytes into histiocytes or, in conjunction with other factors, of osteoclasts (Abbas et al. 1997). The over-expression in mice was associated with glycolipid-induced granulomas (Matsunaga et al. 1996) and it appears possible that M-CSF might increase the number of alveolar macrophages. Reviewing existing STIS data M-CSF was not expressed upon exposure to Carbon Black, nano-BaSO4, and polyacrylate coated amorphous silica, while strong effects were observed for coated nano-ZnO and micron-scale ZnO and different nano CeO2.

16 17 18 19 20 21 22 23 24 25 26 27 28 29

Osteopontin (OPN) is an arginine-glycine-aspartic acid (RGD)-containing protein occurring not only in the extracellular matrix of mineralised tissues, but also as a cytokine in body fluids (Denhardt and Guo 1993). OPN transcription is, among others, stimulated by IL-1 and/or TNFα and has both pro- and anti-inflammatory properties. OPN is acutely up-regulated in the lung upon inhalation of ultrafine Carbon Black particles in mice (Andre et al. 2006). It is also believed to play a role in granulomatous inflammation through the regulation of histiocyte migration, integrin-mediated cell adhesion, and cellular functions such as phagocytosis (Calson et al. 1997). More specifically, OPN is described to be involved in pulmonary granuloma formation in rodents (Chiba et al. 2000, O´Regan et al. 2001). In rats exposed subchronically to 50 mg/m3 micron-scale TiO2, a concentration that elicits lung inflammation and fibrosis (Bermudez et al. 2002), the immediate and sustained formation of OPN was observed. This and other results strongly suggest that OPN is a useful biomarker for fibroproliferative lung disease in rodents and human (Mangum et al, 2004).

30 31 32 33 34 35 36

In our investigation OPN was increased up to 7-fold directly after inhalation of nanoCeO2, coated nano-ZnO, and micron-scale ZnO. In all these cases OPN increases were transient and accompanied by elevations of other pro-inflammatory cytokines such as MCP-1, or IL-1α. Although in our studies histological sections revealed no signs of beginning granulomatous changes or fibrosis, granulomas and deposition of fibrotic tissue was observed after 28-day exposure to nano CeO2 (Keller et al. 2014) and ZnO nanoparticles, respectively (Cho et al. 2010).

37 38 39

Based on available data, MCP-1, CINC/IL8, M-CSF and osteopontin may be selected as optional BALF parameters for STIS, in order to specify potential morphological changes after subchronic or chronic exposure.

40

Key element 2: Potential reversibility or progression of the effect 36

1 2 3 4 5 6

Potential reversibility or progression of the effect is presented by comparing incidence and severity of the effects after exposure and after a recovery period. The STIS included a recovery period of 3 weeks. The recovery period was installed to examine any reversibility or progression of the effects on the one hand and to determine deposition and clearance of the test material in the lung on the other hand. The recovery period is 3 times as long as the exposure period.

7 8 9 10 11 12 13 14 15

The existing data showed that this time span is sufficiently long to indicate reversibility or progression of the effects. In animals exposed to naked SiO2 or nano ZnO, the effects were only observed directly after the exposure, but not after the recovery period, indicating a rapid recovery (Landsiedel et al. 2014). In those animals exposed to TiO2, the morphological changes have lower incidence and severity after the recovery period, indicating a slow recovery (Ma-Hock et al. 2009a). In animals exposed to quartz DQ12, carbon nanotubes or nano ceria, the effect progressed as incidence and severity of histological findings (granulomas) increased after the 3-week recovery period (Ma-Hock et al. 2009b, Ma-Hock et al. 2013, Keller et al. 2014).

16

Key element 3: Deposition and bio-persistency of the particles in lung.

17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

Determination of lung burden on two time points gives a first indication of whether a test item is readily cleared from the lung or tends to persist. Lung burden immediately after the exposure gives the total deposition for poorly soluble particles, because their daily clearance during the exposure period is negligible. Relating to the value directly after the exposure, lung burden after the recovery period can be used to assess clearance behaviour. The prevailing data show those substances that were rapidly cleared (e.g. nano ZnO and nano BaSO4) and others that were not (e.g. nano TiO2, nano ceria). It should be emphasized that the data on two time points give an indication of potential clearance rate, but are not appropriate for calculation of clearance half time. For instance, the calculated clearance halftime of deposited TiO2 nanoparticles in STIS followed by a 2-week post-exposure period was remarkably faster than that observed at essentially similar concentrations of TiO2 nanoparticles in a 4-week rat bioassay followed by a 3-months post-exposure period (Creutzenberg, 2013). It appears that the rapid clearance observed at the end of the exposure period combined the clearance of particles deposited in the airways (rapid clearance) with those in alveolar space (t1/2 at least 60-90 days). For soluble particles however, the second examination time point might be already beyond the time of complete clearance. For ZnO, the measurement after 3 weeks failed to capture the speed of clearance. To overcome this shortage, one may consider measuring lung burden at 3 time points as mentioned in the main document. The first time point may be on the day after the exposure followed by two additional examinations 7 days and 21 days after the exposure. Assuming the rapid clearance phase is completed during the first few hours after the exposure, this time regime enables a more precise determination of clearance half time for poorly soluble particles.

39

Summary and Conclusions

40 41

Previously, only a few substances tested in STIS were also tested in subchronic inhalation studies. Comparing the STIS data with those of subchronic studies, the Low Observed Effect 37

1 2 3 4 5 6 7

Concentration of nano-TiO2 in STIS was similar to that of subchronic studies (Bermudez et al. 2004). STIS of multiwall carbon nanotubes (MWCNT) could clearly demonstrate their high inflammation potency and the progression and persistency of the effects. Nano BaSO4 was rapidly cleared and did not cause any inflammation in STIS up to 50 mg/m³ (Landsiedel et al. 2014). In an instillation study and in 4-week and 13 week inhalation studies, rapid clearance was confirmed. Only a slight increase of BAL parameters were observed after 13 weeks inhalation exposure (Konduru et al. 2014).

8 9 10 11 12 13 14 15 16 17 18 19 20

STIS must rely on early effects mostly reflecting inflammation, and/or the beginnings of histological changes; while a meaningful comparison of endpoints such as fibrosis and cancer is hardly possible. Although not sufficient alone, sustained inflammation is a necessity for enhanced reactive oxygen species formation, fibrosis, and even cancer in rat lung (ILSI workshop 2000). Therefore, the description of inflammatory effects at an early stage (directly or two days after the exposure) combined with data on reversibility (three weeks after the exposure) may be used as an indicator for the severity of toxic effects. Moreover, STIS is a first stage to better understanding and revealing the impact of dissolution and bioaccumulation. According to these criteria, STIS may serve as a versatile first step in a tiered approach enabling us to prioritize nanomaterials for further testing and decide on further appropriately targeted investigations including subchronic or chronic tests. Steps are taken by ECETOC to establish a tiered grouping strategy in which STIS is proposed to be used in tier 3 (Arts et al. 2015).

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AK Abbas, AH Lichtman, JS Pober. Cellular and molecular immunology. W.B. Saunders Company, Philadelphia, USA, 1997.

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E. Andre, T. Stoeger, S. Takenaka, M. Bahnweg, B. Ritter, E. Karg, B. Lentner, C. Reinhard, H. Schulz, M. Wjst. Inhalation of ultrafine carbon particles triggers biphasic proinflammatory response in the mouse lung. Eur Respir J. 28 (2006), 275-285.

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J. H.E. Arts, M. Hadi, M.A. Irfan, A. M. Keene, R. Kreiling, D. Lyon, M. Maier, K. Michel, T. Petry, U. G. Sauer, D. Warheit, K. Wiench, W. Wohlleben, R. Landsiedel: A decisionmaking framework for the grouping and testing of nanomaterials (DF4nanoGrouping). Regul. Toxicol. Pharmacol. 71(2 Suppl) (2015), S1-S27.

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Bermudez E, Mangum JB, Asgharian B, Wong BA, Reverdy EE., Janszen DB, Hext PM, Warheit DB, Everitt JI: Long-term pulmonary responses of three laboratory rodent species to subchronic inhalation of micron-scale titanium dioxide particles. Toxicol Sci. 70 (2002), 86-97.

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E. Bermudez, JB. Mangum, BA. Wong, B. Asgharian, PM. Hext., DB. Warheit, JI. Everitt. Pulmonary responses of mice, rats, and hamsters to subchronic inhalation of ultrafine titanium dioxide particles. Toxicol Sci. 77 (2004), 347-357.

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I. Carlson, K. Tognazzi, EJ. Manseau, HF. Dvorak, LF. Brown. Osteopontin is strongly expressed by histiocytes in granulomas of diverse etiology. Lab Invest, 77.(1997), 103108.

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S. Chiba, MM. Rashid, H. Okamoto, H. Shiraiwa, S. Kon, M. Maeda, M. Murakami, M. Inobe, A. Kitabatake, AF Chambers, T. Uede. The role of osteopontin in the development of granulomatous lesions in lung. Microbiol Immunol 44 (2000), 319332.

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WS. Cho, R. Duffin, CA. Poland, SEM. Howie, W. McNee, M. Bradley, IL. Megson, K. Donaldson. Metal oxide nanoparticles induce unique inflammation footprints in the lung : important implication for nanoparticle testing. Environ Health Perspect. 118 (2010), 1699-1708.

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Denhardt, DT, Guo X: Osteopontin: a protein with diverse functions. FASEB J, 7 (1993), 1475-1482.

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K. Donaldson, PJ. Borm, G. Oberdorster, KE. Pinkerton, V. Stone, CL. Tran. Concordance between in vitro and in vivo dosimetry in the proinflammatory effects of lowtoxicity, low-solubility particles: the key role of the proximal alveolar region. Inhal Toxicol 20(2008), 53-62.

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J. Keller, W. Wohlleben, L. Ma-Hock, V. Strauss, S. Groters, K. Kuttler, K. Wiench, C. Herden, G. Oberdorster, B. van, Ravenzwaay, and R. Landsiedel. Time course of lung retention and toxicity of inhaled particles: short-term exposure to nano-Ceria. Arch. Toxicol. 88 (2014), 2033-2059.

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N. Konduru, J. Keller, L. Ma-Hock, S. Groters, R. Landsiedel, TC. Donaghey, J.D. Brain, W. Wohlleben and R.M. Molina, (2014). Biokinetics and effects of barium sulfate nanoparticles. Part Fibre. Toxicol. 11 (2014), 55ff

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R. Landsiedel, L. Ma-Hock, T. Hofmann, M. Wiemann, V. Strauss, S. Treumann, W. Wohlleben, S. Gröters, K. Wiench, B. van Ravenzwaay. Application of short-term inhalation studies to assess the inhalation toxicity of nanomaterials. Part. Fibre Toxicol., 11 (2014), 16

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L. Ma-Hock, S. Burkhardt, V. Strauss, A.O. Gamer, K. Wiench, B. van Ravenzwaay, R. Landsiedel. Development of a short-term inhalation test in the rat using nanotitanium dioxide as a model substance Inhal. Toxicol., 21 (2009), 102–118

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L. Ma-Hock, S. Treumann, V. Strauss, S. Brill, F. Luizi, M. Mertler, K. Wiench, A.O. Gamer, B. van Ravenzwaay, R. Landsiedel. Inhalation toxicity of multiwall carbon nanotubes in rats exposed for 3 months. Toxicol. Sci., 112 (2009), 468–481

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APPENDIX III

2 3 4

Optional information about the association of lung burdens and kinetics of nanomaterials in the respiratory tract of rats

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

The toxic potential and potency of inhaled NP depend on multiple factors. These include, but are not limited to, their distribution of deposition within the entire respiratory tract and the associated site-specific response. The specific properties of the alveolar area of the tract facilitate interactions with the fluids lining the alveoli (surfactant) and phagocytic cells responding to chemotactic stimuli (alveolar macrophages, AM). NP may have differing kinetic properties within each microenvironment with resultant facilitated dissolution up to accumulation. Prior to any extended repeated study, the degree of solubility of NPs in the lung should be known (for details see Appendix II). An example is given for BaSO4 which is poorly soluble in water (~3 mg/L H2O). This substance was examined in 1-week rat inhalation studies as nano-sized particles (41.4 m² g-1; BET; postexposure period 3 weeks) (Landsiedel et al., 2014) and as micron-sized particles (3.1 m² g-1; calculated) in a study of 17 and 29 weeks followed by postexposure periods of 3 months (Cullen et al., 2000; Tran et al., 2000). The MMAD and GSD were ~1.5 µm (2.1) and 4.3 (1.7), respectively. Calculated and actually determined lung burdens can differ remarkably as exemplified for BaSO4 in Fig. 2.

20 21 22 23 24 25 26

Figure 2: Modeling of two C x t-adjusted subchronic barium sulfate inhalation studies with different particle size and surface areas (Cullen et al., 2000; measured data were reproduced from figures given by these authors). The elimination half-time of BaSO4 in the lung of t1/2

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