SEM/EDX findings for talc used in the original study (Beck et al. ) were not presented in detail, but the authors made descriptive statements to which our findings can be compared. Our measurements of the toxicity of talc-containing dust and its effect on inflammatory cells were generally consistent with the original Beck et al.  study. No asbestos fibers were found, but we did find that 22% of the instilled talc-containing sample met morphologic criteria for fibers, with overall 15% of the material instilled meeting the morphologic and compositional criteria for fibrous talc. In a study to determine the effects of fibrous talc on Hamster Tracheal Epithelial (HTE) cell colonies, colony number decreased as a sign of cytotoxicity . This study was conducted using fibrous talc that had no detectable asbestos, thus providing evidence that fibrous talc per se has inherent toxicity. There have been concerns, cited in the literature, that long-term exposure to talc (both fibrous and not) may be carcinogenic. Studies of Yamada et al.  suggested a correlation between fibrous talc and female lung cancer. A recent study suggested a correlation between talc exposure and lung cancer regardless of whether asbestos is present at detectable levels .
The International Agency for Research on Cancer (IARC) lists talc containing asbestiform fibers (defined by IARC as talc forming mineral fibers that are asbestiform in their mineral “habit”, not talc containing asbestos) as a class I carcinogen . The term asbestiform was defined mineralogically and discussed in detail as it relates to silicates by Virta . Animal studies supporting the toxicity of asbestos-free talc indicate that talc is cytotoxic to macrophages, and talc may induce fibrosis and chronic inflammation .
In the Beck et al.  study, it was reported that quartz accounted for < 1% of their talc dust composition. In our re-analysis of their material, we found no particles with spectra consistent with silicon dioxide or alpha quartz. Because quartz is a known cause of pulmonary inflammation, toxicity, and fibrogenesis, its absence here is significant and indicates that the pathogenicity of talc in our study cannot be explained by quartz contamination of the dust instilled.
Particles designated as talc by SEM/EDX in this report were required to meet the criterion of Mg/Si atomic weight % ratio being within ±2σ of the mean value, based on the gaussian distribution of this ratio found in our past EDX studies of various talc samples . Previously published studies from our laboratory have used the criterion of being within ±5% of the theoretical atomic weight Mg/Si % ratio (0.649) for the diagnostic identification of talc in human tissues, which compares favorably to a ± 2σ standard [24,25,26]. The ±5% criterion is conservative, especially when assessing micron-sized unknown particles in human tissues, but it assures the identification of talc and limits the possibility of false positive or false negative errors . Magnesium silicates with traces of other elements commonly found with talc were found in the samples analyzed in our study. Such materials were classified as non-talc magnesium silicates, and is consistent with the fact that such silicates are commonly found within the Earth’s crust. Trace amounts of aluminum, iron, and calcium may be found in talc , and these could result in the Mg/Si atomic weight % ratio falling outside the criterion for talc previously described. Traces of magnesium calcium silicates, magnesium titanium silicates, and magnesium aluminum silicates were found in the talc sample as well. These silicates are considered relatively non-toxic, and some have been shown to only cause pulmonary damage when inhaled in large amounts .
A chromium-containing particle was found in our analysis. Chromium is a known contaminant of talc, but the relevance of a single such particle to the inflammatory parameters in our study is uncertain. Chromium is a toxic element , but more chromium would need to be found to allow a fair assessment of toxic contribution. Iron has the potential to generate reactive oxygen species via Fenton Chemistry and hence may contribute to toxicity . In our study, there were trace amounts of iron with some talc particles. Iron in more substantial amounts has also been shown to contribute to the carcinogenicity of asbestos . There is no convincing evidence that trace amounts of iron in talc significantly contribute to toxicity. In our current study, we were not able to analyze the granite dust from the original Beck et al.  study by SEM/EDX, since unlike the talc dust, it was no longer available.
By using polarized light microscopy in this study, we were able to quantify the number and size of particles associated with BAL cells. In the Beck et al. 1987 study  as shown in Table 1, at one day after talc instillation, macrophage numbers did not increase, but PMNs migrated into the lung with almost twice as many PMNs after granite exposure than talc at the highest dose. This suggests that the presence of talc particles was sufficient to induce an inflammatory response. Our data show (Fig. 3, 4a, b, and a) that one day after exposure, talc in both particulate and fiber forms had been ingested by cells which far exceeded that of granite ingestion. This is a significant finding because granite particles as measured by Beck et al.  were considerably smaller than talc particles. Most studies [30,31,32] show smaller particles are phagocytosed to a greater extent and have more toxicity per unit mass compared to larger particles. Some studies  indicate that larger particles may have greater toxicity with some material compositions. However, studies generally do not examine the differences in particle ingestion by cells over time in vivo, while most in vitro studies that make this assessment show no difference in cellular uptake and considerable uptake of particles within hours, regardless of material or particle size (Reviewed by Brain et al. ). Therefore, it is unlikely that the particle size differences between talc and granite particles in this study can explain the difference in cellular uptake of talc and granite on day 1.
Data from the Beck et al.  paper suggest that talc and granite may have different toxic mechanisms. Granite induced more PMNs into the lung initially, and initially inhibited the lambda assay (a measure of phagocytic ability) 1 day after instillation. However, over time (day 4 after instillation and later), phagocytic ability with granite exposure returned to control levels. Also, PMNs eventually returned to control levels with granite instillation. However, importantly with talc exposure, PMN numbers were not as high as with granite exposure, but never returned to control levels. Similarly, phagocytic ability with talc (as shown by the lambda assay) remained depressed throughout the 14 days of observation. Our studies follow that same pattern with cellular uptake of talc being less than granite throughout the study except for day 1. Taken together, these observations suggest there may be subacute levels of persistent toxicity with talc exposure. The decrease in phagocytosis by macrophages and PMNs and the increase in giant cells for the talc-exposed group may be due to particle toxicity and the mechanisms by which talc particles inhibit phagocytosis.
There was relatively little birefringent fiber ingestion overall in the exposed talc group and none at all in the exposed granite groups (Fig. 3b). There were no fibers found in the samples from the animals exposed to granite, but as previously mentioned, the granite dust from Beck et al.’s  original 1987 study could not be analyzed by SEM/EDX and compared. Thus, we do not know if it had any fibrous particles. Clearly, the presence of at least some phagocytosed fibers in the talc-exposed animal group, combined with the SEM/EDX data of the talc dust, indicates that talc fibers were indeed instilled and then ingested by cells.
The patterns in birefringent particle cellular ingestion between Fig. 3, 4a and a (data involving macrophages and other cells) are consistent with the inflammatory response that would be expected. Macrophages are the main cell involved in the early inflammatory response, and the quicker and larger macrophage initial uptake of talc raises the possibility that talc binds more quickly to the scavenger receptors on macrophages, or that talc particles may bind opsins better and thus have more potential receptors available for particle uptake. Finally, talc may have intrinsic qualities as a stimulant of phagocytosis, which merit further investigation. Recent studies of McDonald et al.  of talc distribution in the resected tissues from ovarian cancer patients show macrophages in various pelvic tissues in association with very large numbers of intracellular talc particles.
Could our observation of overall decreased macrophage phagocytosis of talc, of decreased uptake of radioactive particles in Beck’s lambda assay across the time course of that study, and the persistence of neutrophils in the lung be a manifestation of particle overload rather than inherent toxicity of talc? Particle overload is caused by very high concentrations of inhaled particles (reviewed by Brain et al. ) and is a common phenomenon seen in rodent animal studies when particle doses are high . Studies have shown that macrophages are able to ingest most amounts of particles in the first 24 h, unless the macrophage becomes overwhelmed by the volume of ingested particles [36, 37]. However, in this study, intracellular and extracellular birefringent particles, as can be seen in the pictures in Fig. 2, tend to be smaller micron-sized particles, and do not appear to reach the average overload volume required for this to be a significant mechanism in the study. Thus, we believe that the intrinsic toxicity of the particles and the phagocytic process with resultant mediators that follow from this process are the most likely explanation for the inflammatory response to both talc and granite.
Since hamsters with intratracheal instillation are the model used in this study, it is important to consider the merits of this model in regard to humans and other species as well as intratracheal instillation versus aerosol exposure. Intratracheal instillation tends to produce different lung distribution patterns compared to inhalation . However, intratracheal instillation has numerous laboratory advantages, including more facile setup, considerably less test material required, less risk to laboratory personnel, and focused exposure into the animal’s lungs. Instillation tends to result in a more centralized pulmonary distribution of deposition (the degree of which is influenced by the volume of carrier fluid), and a more basilar deposition of particles compared to aerosols which tend to be more apical . These differences tend to be more relevant in the interpretation of some dose related outcomes of experimental studies of pulmonary exposures. Hamsters, as a model, had been regularly used to test carcinogens in the development of experimental lung cancers . Hamsters, compared to rats, appeared to be more susceptible to fiber-induced mesothelioma, but less susceptible to the induction of lung tumors with instillation of carcinogens (Reviewed by Brain et al. ). Hamsters are noted to have a higher rate of particle clearance compared to humans and to have the greatest phagocytic ability among respiratory animal models (Reviewed by Brain et al. ). Although only male hamsters were used in this study, a recent study suggests that macrophage responses, especially phagocytic capability, may be more robust in female hamsters compared to males . Examination of possible sex differences in hamster lung inflammatory responses to environmental particles would be an interesting future study.
The large amount of talc particle ingestion by neutrophils (Fig. 4b) is consistent with talc’s potential ability as a chemoattractant and a phagocytic stimulant. The birefringent particle ingestion rate of talc by neutrophils was much higher on day 1 compared to the comparable value for granite. In our study, talc uptake exceeded granite uptake by neutrophils on most days of analysis and was statistically significant at two of the four studied time points. Neutrophils tend to respond in the first 1–3 days as part of the generalized inflammatory response, which may be why their phagocytic activity was high at that stage.
The dosages of both talc and granite ranged from 0.5 to 2.5% instillation. In the Beck et al.  1987 study, as the dosages for the treated granite group increased, so did the number of neutrophils. With talc exposures, an increase in neutrophils was seen through moderate dosages, but then neutrophils did not further increase at the maximum dose. Since macrophages signal the influx of neutrophils via cytokines and chemokines , it is likely that this macrophage function is also diminished by talc exposure in unison with other aspects of macrophage function as noted. Beck et al.’s  data regarding the time course of neutrophil influx for talc vs. granite suggest partial recovery of the signaling function over time after talc exposure so that neutrophils, which have a very short tissue life span ,may continue to infiltrate the lung from the blood. We found significantly greater ingestion of birefringent particles by neutrophils with talc exposure compared to granite at days 1 and 14, but not on day 4.
The frequent finding of PMNs with cytoplasmically located, apparently phagocytosed exogenous particles in both the talc- and granite-exposed groups, at multiple time points, was an interesting observation. PMNs are well known to have phagocytic properties in the context of defense against microorganisms . PMNs are capable of forming a complex known as extracellular trap (NET) in response to various categories of infectious stimuli and small exogenous particles, including silica . In this process, extracellular DNA and chromatin is released from the PMN along with a complex of histones and antimicrobial proteins; however, the PMN undergoes necrosis as a consequence and it is not typically an internal phagocytic process [44, 45]. In a study of the physiology of phagocytosis, it was shown that human PMNs could phagocytize polystyrene beads through an Fc-antibody-mediated binding process . In short, our PMN morphological findings are not entirely with precedent in the literature and suggest that these cells’ phagocytic function against exogenous particulates may be more common than believed. The similarity of PMN phagocytosis with that of macrophages, as well as the roughly similar courses of uptake and retention within the two cellular categories (faster uptake with talc, longer retention with granite, Fig. 4a and b), is also notable. Ours was an animal (hamster) model, and the aforementioned PMN studies derived from humans, so potential cross-species differences should be regarded as a caveat.
The data from Fig. 4c showing marked persistence of giant cell inflammation in talc-exposed animals compared to granite-exposed animals is significant: Giant cells have a tendency to form when the particles are too large to be phagocytosed by individual macrophages or when an inflammatory stimulus is unusually durable and/or persistent. This could signify that talc particles were larger, more durable, and/or less easily phagocytosed, making it more likely for giant cells to form . The studies of Beck et al.  did not include data on giant cells.
There were a large number of macrophages with ≤3 talc particles (Fig. 5) and a large number of macrophages with > 3 granite particles (Fig. 6). This could be an indicator of an earlier inflammatory response due to stimulation by the talc particles compared to the granite particles. The talc, which caused an earlier inflammatory response, apparently hindered the macrophages from phagocytizing more talc particles. This mechanism did not appear to be a feature of granite particles, which had a later response. Beck et al.  noted that the talc particles seen had a mass diameter that was larger than the granite particles. Seen in Fig. 5 of our study, the macrophages phagocytosed more talc particles at a low intracellular level (< 3) compared to granite particles. The lack of macrophages containing > 3 cell-associated talc particles could be due to the rapid elimination response for such macrophages . This is consistent with earlier observations and discussion that talc, as a whole, may be more toxic to macrophages than granite.
Our study regarding talc and inflammation has relevance to the major current health concern for talc: the previously mentioned association between perineal talc use and ovarian carcinoma [1,2,3,4,5]. This is true given the in vitro demonstration of talc’s pro-inflammatory effects on epithelial ovarian carcinoma cells, and the concomitant effects on point mutational activity, increased cell proliferation, and decreased apoptosis , and the well-known observation that macrophages play an important response in mediating inflammatory effects and cellular damage . Also relevant to the current study’s findings regarding macrophages are the various inflammatory infiltrates, including macrophages and other cells, that have been seen associated with talc in the surgically resected tissues from human patients exposed to perineal talc .