To the best of our knowledge, this is the first subchronic 90-day inhalation study using gold nanoparticles to be reported in the peer-reviewed literature. An important aspect of this and predecessor studies from our laboratory is that the surface of the nanoparticles used for this exposure were not modified and not oxidized. There is one previous inhalation (intratracheal instillation) study which used 1.4 nm and 18 nm particles whose surface was ligated with Ph2PC6H4SO3Na  and one 15-day whole body inhalation study which used citrated 20 nm particles .
Interestingly, a recent publication  found that colloidal gold nanoparticles diluted in ultrapure water were well-dispersed, while agglomerates were formed when the diluent was phosphate buffered saline. In that study, rats were administered 50 nm and 250 nm gold particles by intratracheal injection. Despite differences in the degree of agglomeration due to the two diluents, no major differences in pulmonary and systemic toxicity markers were observed.
It is possible to calculate a deposited dose in the lung for this study. The deposited dose is calculated as follows:
If the minute volume of male and female Sprague-Dawley rats in a subchronic study is 0.19 and 0.15 L/min, respectively  and the fraction of the dose deposited for particles less than 10 nm in diameter is 0.8 , then the deposited dose to the lung would be 0.14 μg, 1.35 μg and 71.1 μg for the low, middle, and high dose males, respectively. For the low, middle, and high dose females, the deposited dose to the lung would be 0.11 μg, 1.07 μg and 56.2 μg, respectively. For comparison purposes and using the same assumptions, we can calculate deposited dose to the lung from our previously reported subchronic inhalation study of 18-19 nm silver particles . The deposited dose to the lung for the silver study would be 170 μg, 470 μg and 1800 μg for the low, middle, and high dose males, respectively. For the low, middle, and high dose females, the deposited dose to the lung would be 140 μg, 370 μg and 1400 μg, respectively. Thus, the high deposited dose in our subchronic gold study is about 40% by weight of the low deposited dose in our subchronic silver study.
There is considerable uncertainty in the fraction of the dose retained for particles less than 10 nm in diameter as opposed to the fraction of the dose deposited as used in the calculation above. It is likely that the retained dose is significantly different than 0.8 due to such factors as translocation to other organs and species-specific deposition patterns. The reader is referred to more comprehensive discussions of retention of nanoparticles and deposition modeling in human and animal models [28–32].
Whole body inhalation studies involve an additional component of exposure; that of ingestion since particles accumulate on the skin and fur. It is well known that rats clean skin and fur by licking, thus introducing particles to the gastrointestinal tract. For particles of gold larger than the nanometer range, it is generally assumed that the particles are not absorbed. Little is known about gastro-intestinal absorption of gold nanoparticles, but there are reports on the permeability of rat intestine to colloidal gold nanoparticles. Sonavane et al.  studied movements of 15, 102, and 198 nm colloidal gold particles across intestine in vitro. Fifteen nm particles were shown to cross the intestine more readily than 102 or 198 nm particles. The permeation of these particles through rat intestine was higher than rat skin. Hillyer et al.  studied absorption of 4, 10, 28, and 58 colloidal gold nanoparticles fed to mice. In general, smaller particles were absorbed more readily and corresponding tissue levels were higher. The applicability of this data to whole body inhalation is not apparent because of the colloidal nature of the tested particles [33, 34]. Clearly, quantitative in vivo data on the absorption of unmodified gold nanoparticles in the rat would be of great value in determining the relative contribution of gastrointestinal absorption to the accumulation of gold from nanoparticles.
Tissue concentrations of gold from control rats have been reported in several studies [24, 35] and tend to be lower than found in our study (Table 8 and 9). Takenaka et al.  reported gold in control lungs to be 0.07 ± 0.003 ng/g and in control blood to be 0.1 ± 0.07 ng/ml (mean ± SD). Yu et al.  reported gold in control lungs to be 5.50 ± 4.44 ng/g and in control blood to be 5.83 ± 3.02 ng/ml. Yu et al.  also reported gold in control olfactory bulb to be 4.44 ± 4.43 ng/g and in control brain to be from 4.67 ± 3.63 to 20.27 ± 15.79 ng/g (presumed to be mean ± SD) depending on the part of the brain analyzed. Because of the low levels of gold in tissues, the authors cannot rule out the possibility of contamination of internal tissues from skin or fur. Mitigating that possibility is previous experience by the authors in whole-body inhalation studies involving nanoparticles. Nevertheless, the reader should consider the possibility for such contamination.
The results of this study indicated that the lungs were the major target tissue; pulmonary effects included a decrease in tidal and minute volume and the presence of mixed inflammatory cell infiltrates. Dose-related changes in tidal and minute volume tend to be obscured by changes over time which also occurred in control animals. In our experience, pulmonary function changes can reproduce poorly over time because they are dependent on so many variables. The decrease in pulmonary function following 90-days of gold nanoparticle inhalation in the current study was similar, although lower, than that reported after 90-days of silver nanoparticle inhalation .
In the present study inhaled gold nanoparticles accumulated in a dose-dependent manner in lungs and kidneys of both male and female rats (p < 0.01), but not in liver, blood, and olfactory bulb. This is in contrast to the study of Yu et al.  where rats are exposed to 20 nm gold nanoparticles at a concentration of 2 × 106/cm3 (mass concentration not reported) for 15 days by inhalation reported that the particles relocate from lungs to liver. Exposure in that study for 5 days resulted in a significant increase of gold in the lungs and olfactory bulb, as detected by ICP-MS; after 15 days of exposure, a significant accumulation of gold was detected in the lungs, esophagus, tongue, kidneys, aorta, spleen, septum, heart, and blood. Five or 15 days of inhalation exposure to gold nanoparticles resulted in a slight accumulation of gold in liver and a minimal increase the gold content of the olfactory bulb indicating a small but significant translocation from lung to blood after 15 days of exposure. Takenaka et al.  exposed rats to 16 nm gold particles at a concentration of 4 × 105/cm3 (88 μg/m3) for 6 hours and then serially sacrificed animals at 0, 1, 4, and 7 days. These data also indicate a small but significant translocation from lung to blood.
The lack of significant increase in gold concentration in the olfactory bulb in our study is interesting with regard to findings in previously reported studies of shorter duration, particularly those of Yu et al. . They showed a small but significant increase in the concentration of gold in the olfactory bulb and parts of the brain. The reason for the differences between our study and that of Yu et al.  are not apparent, but could be related to the surface composition of the nanoparticle itself, duration of exposure or the higher concentrations of gold in found in the olfactory bulbs of control animals.
The results of our 90-day study are not consistent with data from studies of nanoparticles of different composition and duration and stand in contrast to Elder et al.  using inhaled Mn oxides nanoparticles and Balasubramanian et al.  using intravenously injected gold nanoparticles. Elder et al. found that when rats inhaled manganese oxide particles (agglomerates measured 30 nm in diameter with primary particles of 3-8 nm) for 12 days, the particles accumulated in the olfactory bulb . It is not clear if the differences between our study and that of Elder et al. are due to exposure duration, particle size, or solubility. Balasubramanian et al.  reported that a single intravenous injection of gold nanoparticles yielded a large amount of gold in the olfactory bulb (up to 72.2 ng/g) two months after intravenous gold nanoparticle injection. A similar comparative distribution between intravenous injection and intratracheal instillation was also observed in a study by Semmler-Behnke et.al. . Perhaps this concurrence of distribution is related to the fact that both intravenous injection and intratracheal installation deliver a large bolus of nanoparticles whereas normal inhalation delivers a much lower concentration of nanoparticles per unit time.
Throughout this study, male rats were larger than females (Tables 2 and 3, Figures 5A and 5B). There appeared to be no consistent differences between male and female in the organ content of gold and particularly lung indicating that differences in were not likely to have been due to a gender difference in deposition.
The higher accumulation of gold nanoparticles in the kidneys found in the present study was also previously observed in silver nanoparticle inhalation and oral exposure studies [14, 16], suggesting that the kidneys are the major accumulation site for metal nanoparticles whether the particles are ionized, like silver, or non-ionized, like gold. Gender-related accumulation of gold nanoparticles noted in this study was also observed for silver nanoparticles in the inhalation and oral exposure studies [14, 16]. It therefore appears that at least over the range of average nanoparticle sizes of 5 nm (gold, inhalation), 15-20 nm (silver, inhalation), and up to 60 nm (silver, oral) there is a similar gender-related accumulation, and that there is a difference in the pattern of nanoparticle distribution between male and female kidneys. It is not apparent whether this difference is anatomically or hormonally-based.
In contrast to the 0-hypothesis for silver suggested by Wijnhoven et al. , in which silver toxicity mainly originates from silver ions generated from the surface of silver nanoparticles, the present results using gold nanoparticle inhalation provide a different view of nanoparticle toxicity and distribution. Gold nanoparticles (1.4 nm) injected intravenously or administered by intratracheal instillation are excreted into the urine and not likely ionized in the body, they are translocated to tissues as particles rather than in an ionic form . This implies that part or even a majority of the tissue distribution pattern of silver nanoparticles could be due to translocation in particulate form rather than ionic form and that most of the silver captured in kidneys is not in ionic form.
Currently, there are no occupational exposure standards for gold dust, fumes, or nanoparticles other than general particulate standards such as the "particles not otherwise regulated" standard of the US OSHA permissible exposure limit (PEL) of 5 mg/m3 for respirable particles. Changes observed in lung histopathology and function in high-dose animals appear in and of themselves to be minor. In the previously reported study on silver nanoparticles, similar changes were noted  and the authors interpreted them to be transient and not sufficient to establish an effects level. It appears that our original interpretation was not correct since when rats from a recent 12 week exposure study similar to Sung et al.  were allowed to "recover," decreases in tidal and minute volumes from the middle and high dose groups have persisted up to 12 weeks post-exposure (unpublished data). In light of similar changes in this study, it appears that the highest concentration (20 μg/m3) is a LOAEL and the middle concentration (0.38 μg/m3) as a NOAEL.