Table 8.

CARBON-study; analysis of final product

We claimed that the final product had to meet the following criteria: sterile, pure (no contamination with dust, LPS or metals), and the main fraction of nanoparticle aggregates had to stay nanosized when resuspended in saline (Table 6). Carbon particles tend to form firmly linked aggregates, which in turn join together to form agglomerates. In order to mimic diesel soot, it was critical for our research model that the final product contained mostly loose nanoparticles and nanoparticle aggregates smaller than 100 nm.

The final product was an isotonic suspension of carbon (Printex-U) and sodium chloride (saline) in sterile water, which was manufactured under sterile conditions (laminar airflow cabinet) in three different concentrations: 20, 100, and 200 μg carbon in 10 ml saline. The Printex-U powder was accurately weighed, mixed with pulverized sodium chloride and suspended in water for injection. After manufacturing, the product was sterilized in an autoclave (121 °C, 15 min), and sonicated for 5 min directly before administration to the study participant. Manufacturing, packaging, labelling, and batch certification was done by a qualified pharmacist.

The final product and matching placebo consisting of sterilized saline were tested for characteristics, contamination (heavy metals, dust, endotoxins), and stability.

The stability data showed an increase in particle agglomerates in time. More specifically, when analyzed 1 week after manufacturing, the main part of the particles were smaller than 100 nm, but after 1 month the number of clusters with a larger size was increased. Therefore, we decided to make a fresh sample for each study participant at a maximum of 1 week before administration on the study day.