The present study describes the kinetics of titanium dioxide nanoparticles in rats. Animals were exposed via either the oral or the IV route. As exposure via food-products might contribute significantly to the total exposure of titanium dioxide nanoparticles, evaluation of the absorption from the oral route is relevant for human risk assessment. The IV route was included to obtain insight in the blood and tissue distribution profile under conditions of full systemic bioavailability, and to gain insight in the elimination over a prolonged period of time (90 days).
Oral absorption (oral study)
The dose levels used in our oral study (i.e. 2.3 mg TiO2/animal/day for five days; 6.8-8.5 mg TiO2/kg bw/day for male animals and 10.9-12.0 mg TiO2/kg bw/day for female animals) are much lower and more realistic than doses used by others in toxicity or kinetic studies, as these typically applied doses greater than 100 mg/kg bw/day and even as high as several thousands of milligrams TiO2/kg bw/day [15, 30]. The consumption of titanium dioxide for the UK population was reported to be on average 2–3 mg/kg bw/day for children under the age of 10 years and approximately 1 mg/kg bw/day for other consumer age groups . For USA, these numbers were 1–2 mg/kg bw/day and 0.2-0.7 mg/kg bw/day respectively . The dose levels used in our oral study can thus be considered within a realistic and relevant frame for the human situation.
Our data show that after repeated oral exposure (overall dose of 11.5 mg TiO2) titanium levels were near or below the detection limit in liver and spleen, indicating a very low absorption. Because of the realistic dose levels used in the present study, a quantitative value for oral absorption is difficult to determine due to the many measurement points below the LOD. Only in two out of 30 liver/spleen samples of exposed animals (NM-102 and NM-103 liver) was the Ti level at or above the LOD. In contrast all MLN samples (controls and exposed) contained Ti amounts above LOD (Figure 3). Only a small increase in Ti content was observed, because the background levels in MLN were 2–3 times the LOD. MLN from control rats contained 0.14 μg Ti whereas the highest Ti average was 0.36 μg and was located in MLN from NM-104 exposed rats. This gives an increase of 0.226 μg Ti in MLN or 0.003% of the 6895 μg Ti exposure in the dose.
The total recovery of dosed Ti in all tested organs (expressed as % of the total dose) was estimated to be approximately 0.02%. This was based on calculations using different scenarios (i.e. using LOD or half the LOD for the non-detects; correcting the tissue levels for background levels; using only the positive liver titanium levels). Not all tissues were included as the oral study focused on liver, spleen and mesenteric lymph nodes as target tissues, which seems appropriate based on the results of the IV study. The data of our oral study indicate that some minor absorption may occur in the GI tract after oral exposure, be it to a very limited extent. However, due to the very limited elimination of the nanoparticles even a low absorption should be considered in risk assessment.
Recently, Tassinari et al. (2014)  evaluated, in addition to the reproductive and endocrine effects of nano-sized titanium dioxide particles (commercially available, anatase, primary size < 25 nm, BET surface area 45–55 m2/g), Ti levels in tissues as thyroid, ovaries and spleen after short-term exposure . Rats were orally exposed for 5 days to 0, 1 or 2 mg/kg bw/day titanium dioxide nanoparticles (doses which are even lower than the doses as applied in our study). A significant increase in titanium levels in spleen (0.046 ± 0.008 μg/g) was observed at the high dose group, though the difference with the control animals was small. These data are in the same range as incidentally observed in our study. Single particle-ICP-MS analysis of the target tissue spleen revealed the presence of both titanium dioxide nanoparticles and their aggregates . The kinetics of nano-sized titanium dioxide in rats after repeated 13-weeks oral exposure were studied by Cho et al. . Titanium dioxide particles (commercially available, primary particle size of 26.4 ± 6.1 nm as measured with SEM) were administered to rats in doses of approximately 250, 500 and 1000 mg/kg bw/day, 7 days/week for 13 weeks. Titanium-levels were analyzed using ICP-MS with a reported limit of detection of 0.1-1 ng/L. No clear dose-related increases in titanium levels in liver, spleen, kidney and brain could be observed, indicating very low systemic bioavailability . At high dose levels such as several hundreds to one thousand milligram/kg bw/day, absorption of titanium dioxide might be reduced due to agglomeration/aggregation of the particles in the gastrointestinal tract. For silica particles, it was recently hypothesized that gel-formation occurs, especially at higher particle concentrations, under conditions of a relatively high pH and salt concentration, like in the intestine . This gelation is hypothesized to lead to a decrease in oral absorption with increasing dose. In contrast, Wang et al. showed absorption of titanium dioxide nanoparticles after a single high dose oral exposure in mice . They used a very high and quite unrealistic single dose of 5000 mg/kg bw (approximately 100 times higher than the cumulative dose used in our study). Primary particle size of the titanium dioxide particles included in this study was 25, 80 and 155 nm. After a single oral dose, titanium could be detected in liver, spleen, kidney, lung, brain and red blood cells. They identified liver as the main target tissue, where highest uptake (by far) was shown for the 80 nm particle (approximately 4 μg/g; a concentration about 100 times higher than our LOD) .
Tissue distribution (IV study)
Based on the results of our IV study, liver, spleen and lungs were identified as primary target tissues for titanium dioxide nanomaterials. Highest levels were observed in the liver, but redistribution of the liver to the spleen was observed over the 90 day post-exposure period. A similar kinetic profile after 24 hours for titanium dioxide nanoparticles in rats was observed recently [17, 22]. In these studies, a single IV administration of titanium dioxide nanoparticles (5 mg/kg bw) resulted in highest levels (in descending order) in liver, spleen, lung and kidney as well [17, 22]. After IV administration, titanium levels in blood rapidly declined (both after single as well as repeated administration). These results indicate a fast distribution of titanium dioxide to the various tissues.
The tissue distribution profiles of the four IV tested nanomaterials with different sizes and crystalline forms (NM-100, NM-102, NM-103, NM-104) are in general rather similar, although some differences were observed.
Titanium was detected in all investigated tissues in the present study, i.e. blood, liver, spleen, kidney, lung, heart, brain, thymus and reproductive organs. The total recovery was mainly below 100%. An explanation might be that not all tissues were investigated. Skin and muscle were not included as they were inconsequently measured or an initial experiment showed that the levels were close to the detection limit (data not shown). Bone marrow Ti levels could not be measured due to the small sample size. Based on the relatively constant tissue and low blood levels (after the initial rapid decline), it seems rather unlikely that the incomplete recovery can be explained by elimination processes. This is in agreement with the negligible amount of Ti found in excreta (data not shown).
No large differences in distribution between male and female animals were observed. The major difference between male and female animals was the disappearance of Ti in the reproductive organs. In male animals Ti was not detectable in the testes 30 days after administration, whereas at day 90 Ti was still detectable in the female ovaries. This difference can be explained by the differences in blood supply to the testes and ovaries. On day 6 a similar very low percentage of the dose was distributed to both testes and ovaries.
Elimination (IV study)
Both after single and repeated IV exposure, blood titanium levels in blood decreased rapidly during the first minutes after which the titanium levels slowly decreased and approached the limit of detection at 24 hours post-dose, similar to what was found earlier for silver nanoparticles in blood by Lankveld et al. . In the nanosilver study the sampling period ranged to 17 days instead of 90 days as was the case in our study. During the period of 12 days after exposure, Lankveld et al. found a gradual decrease in organ silver levels but silver was still present in several organs including liver, lungs, spleen and kidney . In our study, the total titanium levels slowly, but definitely, reduced up to Day 90, although such a decrease is not obvious from the data at time point day 14 (i.e. 9 days after exposure). Based on these data it can be concluded that elimination of total TiO2 has a long half-life, which was also shown by the results of the kinetic analysis presenting half-life in days for the various organs (for example for liver as the main target organ: 28–248 days). In contrast to the titanium dioxide particles, as described here, for NM-105 (an anatase rutile mixture) a major decline in various organ levels was noted . In the present study, some reduction in recovery was observed up to Day 90 for the pigment-sized (NM-100) and one of the nano-sized (NM-102) titanium dioxide particles, with a maximum relative decrease (day 90 vs. day 2 or 6) of 26% (NM-100, males).
Redistribution of titanium from the liver to the spleen was observed between Day 2/Day 6 and Day 90, whereas redistribution to remaining tissues was not identifiable. Release of particles from liver and possibly other organs may be responsible for the increase in spleen levels. The data show that at the long run TiO2 particles will accumulate in spleen. The spleen Ti concentration rises during the entire exposure, including the 90 day post exposure period.
In addition, titanium levels as measured in the faeces of IV-treated (single and repeated) animals revealed no clear differences between titanium dioxide-exposed animals and vehicle-treated controls (data not shown). Further, no increase in titanium levels in urine was observed. This further confirms the lack of elimination of the titanium dioxide nanoparticles.
The expected accumulation with daily exposure as a consequence of the negligible elimination might indicate a potential concern for human health risk. Even with very low uptake from the gastrointestinal tract human daily oral exposure can be expected to give rise to a very low but steady increase in titanium levels in tissues in time. Weir et al. estimated the exposure to pigment grade TiO2 via food in the order of 1 mg/kg bw per day . The nanofraction in these pigmentary TiO2 (E171) is estimated to be 10-36% of the number of particles [2, 4]. So, the aspects of oral uptake, limited elimination and anticipated accumulation in man should be further investigated in the risk assessment of nanosized TiO2.
Differences between titanium dioxide particles tested
The present study is the first to evaluate the kinetics of different titanium dioxide nanoparticles after both single as well as repeated oral and intravenous administration in rats. The titanium dioxide nanoparticles as used in this study differed with respect to particle size, with NM-100 being the largest particle (nominally 200–220 nm vs. 7–10 nm for NM-101, 15–25 nm for NM-102 and, 20 nm for NM-103 and NM-104). However it should be realized that in the nanomaterial dispersions used, the TiO2 nanoparticles are present as either agglomerates (loosely bound particles) or aggregates (more or less fixed particles). Only minor differences in kinetic profile, both after single and repeated exposure, were observed in the present study, which may be linked to differences in size, hydrophobicity, crystalline form or just animal or random variation. For gold nanoparticles we previously observed a difference in tissue distribution depending on size, the smaller nanoparticles showing a more wide spread tissue distribution .
In the present study, all particles showed a rapid distribution to the organs from the systemic circulation. The total recovery of NM-103 and NM-104 was higher than NM-100 and NM-102, which might indicate a small difference in tissue distribution. Some indications were noted for a difference in recovery between anatase (NM-102) and rutile nano-TiO2 (NM-103 and NM-104). However, these crystalline forms were not compared in one single experiment, making interpretation of these differences difficult.
Further, elimination from the studied organs was very slow, with no difference between the titanium dioxide particles observed. It should be noted that in the present study we did not systematically change a specific physicochemical characteristic of the selected titanium dioxide particles. Such an approach could facilitate the detection of differences in kinetic behavior in relation to such a characteristic. In the present study commercially available titanium materials were used, which is relevant from a risk assessment point of view. At the moment it is not possible to combine these aspects.