Being a mild endogenous oxidant, NCT proved to be an antiseptic suitable for application to both the skin and mucous membranes of different body cavities such as paranasal sinuses [13, 14, 36, 37]. The substance seems to be particularly applicable to tissues for which other antiseptics are avoided because of toxic side effects. Such a field is inhalative application. For special indications aerosolized antibiotics and antifungals have been used successfully, e.g. for treatment or prophylaxis of Pneumocystis jirovecii, Pseudomonas aeruginosa, and fungal infections [17, 18, 38]. Because of some uncertainties regarding efficacy and toxicity of the presently available therapeutics, the development of improved antimicrobial agents for inhalation seems to be suggestive. Antiseptics may be of advantage because of their broad-spectrum activity and largely the absence of antimicrobial resistance. Toxicity is a major point of concern with these compounds when administered to body cavities, but NCT seems to be well tolerated in the bronchopulmonary system according to our results.
Lung epithelial cells (A549) tolerated about 0.5 mM NCT and were similarly susceptible as other body cells [13, 39], e.g. macrophages and keratinocytes. Moreover, our findings are in accordance with a previous report where taurine protected lung epithelial cells from HOCl-induced injury by conversion of HOCl to the low toxic NCT . It is very important to mention that the clinically applied NCT concentration tolerated by different tissues is about 100-fold higher (55 mM) than that of monolayers in the cell culture [13, 20, 39]. Therefore, cell culture systems are useful for comparison of antiseptics (e.g. chloramine T and NCT, ) and to predict to a certain extent in vivo toxicity, as it was the case with NCT plus ammonium in our study. However, it is finally decisive that a clinically effective dose is tolerated in both animal models and in man. As expected and similar to keratinocytes , the stronger oxidant chloramine T damaged lung epithelial cells much more than NCT.
We chose the pig model since it has high similarity to human, ventilation and circulation parameters can be monitored, inhalations can be performed in a similar way, and sufficient amounts of serum and tissue can be removed for evaluation. A limitation in our unit was that the experimentation in each animal had to be performed within one day, so that dosing and monitoring for longer periods was not possible. This disadvantage may have been compensated for to a certain degree by the performed intensive treatment schedule.
In accordance with our previous clinical studies in both human beings and in animals (e.g. [12–14, 36, 37, 40–42], the concentration of 1% NCT (55 mM) seems to be very well tolerated also in the bronchopulmonary system. Also the inhalative way has to be regarded as a topical one since the amino acid concentrations in the blood, particularly of taurine, did not change in all groups. This was the case despite the high concentration gradient of taurine applied locally (5% NCT equals 275 mM and 400 mM taurine after reduction of the active chlorine) and in the serum of the pigs (85–186 μM). The only hint for a small, negligible resorption of high inhaled concentrations could have been the increase in cystine in the 5% NCT group. A theoretic explanation for that is oxidation of cysteine to cystine, which takes place in the presence of NCT. It may be that a minimal increase of taurine after application of 5% NCT could not be detected within the standard deviation. On the other hand, the blood level of methionine remained constant, although it is oxidized by NCT, too. Presently it can be stated that 1% NCT ± 1% NH4Cl does not reach the circulation in detectable amounts, while minimal amounts are possible with 5% NCT.
Since the reaction with thio groups leads to immediate inactivation of NCT, the oxidative activity decreased quickly and could not be measured in situ approximately 30 min after the last dosing. The short decomposition time in vivo is in accordance with previous studies in the eye (5–15 min, [36, 43]). Longer times of a few hours are only possible if a fluid level containing NCT is kept in body cavities .
No tested clinical and laboratory parameter in the 1% NCT group worsened more than in the saline group during the experimental period. With inhalation of chloramine T, a markedly stronger active chlorine compound than NCT, bronchopulmonary irritation and allergic sensitization with asthmatic symptoms are known . The lower reactivity of NCT and the fact that it deals with an endogenous amino acid derivative are obviously decisive for its improved tolerability. If the concentration was increased to 5% in our pig model, only one parameter (pulmonary artery pressure) worsened significantly compared to placebo. A similarly high safety of NCT could be found for instance in the eye , in the ear  and in the cow udder , where concentrations of 3–10% caused local irritation, too, but no severe or long-term side effects. Allergic reactions against NCT seem improbable since it is produced upon every inflammation by leukocytes and it is a small molecule (amino acid derivative), and such reactions have not yet been observed in clinical studies.
As a more potent chloramine formulation and as a positive control, we chose a 1% combination of NCT and ammonium chloride (NH4Cl). This leads to formation of about 0.025% (4.9 mM) monochloramine (NH2Cl) which is only little more reactive but significantly more lipophilic than the hydrophilic NCT . The consequence is increased penetration of pathogens but also body cells and tissue, which may be wanted in some indications, e.g. in the eye for treatment of viral conjunctivitis [11, 43]. The concentration of 1% of both constituents is very high and cannot be applied to the eye, but a tenfold dilution is very well tolerated . As expected, in the pig model oxygenation and perfusion parameters changed after inhalation with 1% NCT + 1% NH4Cl compared to saline and NCT. Since systemic absorption of NCT ± NH4Cl was not detected and low amounts in the blood would be inactivated immediately, these changes must have been caused by direct impact on lung epithelial and endothelial cells. We expected a thickening of basal membranes, but there were no changes at all found by electron microscopy. Therefore, functional abnormalities of the lung cells must be assumed which did not lead to structural changes. In vitro, lung epithelial cells demonstrated about tenfold higher sensitivity to NCT + NH4Cl than to NCT, which is in accordance to the in vivo results, at which the threshold tolerated concentration of the combination remains to be tested in vivo.
Histologic evaluation did not reveal an explanation for the effects of 1% NCT + 1% NH4Cl and 5% NCT, too. The abnormalities found, mainly infiltration with leukocytes, and local atelectasis and emphysema, are well known to occur during the anaesthesia of pigs . It cannot be excluded that these changes might have overlapped and masked minimal ones caused by the test medication. In any case the absence of a significant impact clearly visible in histology indicates the relatively high tolerance of NCT with and without NH4Cl by tissue.
Furthermore, the function of surfactant seems not to be significantly influenced by the medication. It is true that the test procedure using the captive bubble surfactometer is very complicated, laborious, and affected with high deviations, and it was possible to perform only a few tests in the present study. Therefore, small differences between the tested groups cannot be excluded, but a massive destruction of surfactant or its producing cells did not take place. This would have caused much more intensive clinical problems. In accordance with that, NCT as a mild active chlorine compound mainly reacts with thio and amino groups  and has no targets with the phospholipid constituents of surfactant which are important for its function .
The fact that one oxygenation parameter (AaDO2) was even significantly better at the end of the applications in the pigs treated with 1% NCT compared to saline may be regarded as pure chance. On the other hand, anti-inflammatory effects might be caused by NCT since in its presence proinflammatory cytokines and interleukins have been shown to be produced in lower amounts by stimulated leukocytes in vitro [4, 5, 16]. Some effects in part contrasting these results have also been found, i.e. significant induction of IL-1beta, IL-8, TNF-alpha and HO-1 gene expression in human PBMC (Giese T, Heidelberg, unpublished results). When NCT was applied to treat infections in our clinical studies, specific investigations to quantify a possible anti-inflammatory effect and to differentiate it from the antimicrobial one have not been performed. Some signs for clinically relevant anti-inflammatory effects may be derived from phase 2a studies on chronic rhinosinusitis and, above all, postoperative ear care after tympanoplasty, where deswelling and drying effects were found in the absence of infection [37, 41]. In the present study we tried to address this question specifically for the first time in vivo by measurements of IL-1beta, IL-8, TNF-alpha and HO-1 gene expression. However, no conclusive results could be gained from lung tissue in vivo. As observed during histo-pathological examination, the experimental procedure triggered a variable degree of injury-associated inflammation in the lung tissue. This reaction might have overridden the more subtle changes caused by NCT and NCT+NH4Cl. However, this hypothesis could not be proven in the experiment due the limited number of investigated samples.
Summing up, inhaled NCT at a concentration of 1% was well tolerated in the pig model. Further investigations of its tolerability in human beings and on its possible efficacy in the infected bronchopulmonary system are justified.