Nanomedicinal products and immunotoxicity: pitfalls in testing and the regulatory framework
Christina Giannakou1,2,*, Wim H de Jong1, Henk van Loveren2, Margriet VDZ Park1, Rob J Vandebriel1, Robert E Geertsma1

1National Institute for Public Health and Environment (RIVM) Bilthoven, The Netherlands
2Maastricht University, Maastricht, The Netherlands
* Corresponding author

Nanomedicinal products (NMPs) are purposely designed tiny structures–less than 1.000nm across-often consisting of multiple components. They embody one of the numerous applications of nanomaterials today as therapeutic, diagnostic or theranostics pharmaceuticals.

All pharmaceuticals, including NMPs, should undergo safety evaluation during the market authorization procedure. The toxicokinetic profile of NMPs indicates a preference to accumulate in and interact with the immune system. The regulatory guideline used currently for immunotoxicity assessment is the ICHS8-immunotoxicity studies for human pharmaceuticals.

Previously, we identified gaps in ICHS8 for addressing potential immunotoxicity of NMPs, including Complement Activation Related-PseudoAllergy, hypersensitivity and immunosuppression. Also, a reflection paper on liposomal products refers to the importance of studying immunotoxicity of NMPs, including determination of complement activation.

Based on that, a set of in vitro, in vivo and ex vivo assays was developed and their applicability for different types of NMPs was evaluated. Specific attention was paid to known pitfalls related to NMP testing, e.g. potential interference with the read-outs chosen.

Here, an improved and integrated strategy is proposed for assessing the risk of potential immunotoxicity of NMPs. The strategy starts with factors that need to be considered to identify the NMP and determine the extent of immunotoxicity testing needed. Next, a cascade of in vitro assays is recommended. Information gathered in these two parts is used to determine the need and design of further in vivo testing. Finally, the conclusions drawn from the previous steps provide input on the risk assessment and design of clinical studies of NMPs.

Keywords: Nanomedicine, strategy, risk assessment, immunomodulation

In Vitro Cytotoxicity of Ionic Surfactants: The Dose Makes the Poison, but what is the Dose?
Floris A. Groothuis1*, Iris Schaap1, Beate Nicol2, Bas J. Blaauboer1, Nynke I. Kramer1.

1Institute for Risk Assessment Sciences, Utrecht University, P.O.Box 80177, 3508 TD Utrecht, The Netherlands
2Unilever U.K., Safety & Environmental Assurance Centre, Colworth Science Park, Sharnbrook, Beds MK44 1LQ, United Kingdom
*Corresponding author: f.a.groothuis@uu.nl

ABSTRACT
The octanol water partition coefficient (KOW) and Henry's law constant (H) correlate with the bioavailable fraction of neutral organic chemicals in vitro. Because surfactants have a hydrophobic tail and hydrophillic headgroup, the relationship between KOW and H and the distribution in vitro is not applicable. The aim of this study was to measure the fraction over time, associated with exposure medium, medium constituents, well plate plastic and cells for cationic and anionic surfactants with six to eighteen carbons in the alkyl chain using an RTgill-W1 basal cytotoxicity assay. Partitioning behaviour was related to chemical properties including head group and charge, alkyl chain length, pKa and membrane-water partition coefficient (KMW). Results indicate that there was variation in cytotoxic potency between surfactants with differing head groups and alkyl chain length where cationic surfactants, especially quaternary amines, were found to be the most cytotoxic. This phenomenon is likely due, in part, to the mechanism of membrane insertion of these quaternary amines. Association to plastic and serum constituents reduced the bioavailable concentration by up to 97% and 99%, respectively for the tested surfactants with long alkyl chain lengths. Cytotoxic potency rankings were strongly dependent on the dose metric (measured, free and cell associated concentrations) and exposure time. Given the variation in partitioning behaviour, the ranking of toxic potencies based on bioavailable or cell-associated effect concentrations will better reflect potency ranking across in vitro assays and facilitate mechanistically based quantitative in vitro to in vivo extrapolation than when nominal concentrations are used.

Keywords: in vitro assay, dose, ionic surfactants, free concentration, quantitative in vitro-in vivo extrapolation.

Defining exposure of food-producing animals to antimicrobial substances
F.J. Taverne1, N.I. Kramer1, I.M. van Geijlswijk2, J.W. Mouton3, D.J.J. Heederik1

1 Institute for Risk Assessment Sciences, Utrecht University, the Netherlands
2 Veterinary Pharmacy, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands
3 Department of Medical Microbiology and Infectious Diseases, Erasmus MC, Rotterdam, the Netherlands

Knowledge regarding exposure of food-producing animals to antimicrobials is essential for risk-driven benchmarking of antimicrobial use in food-producing animals. The current monitoring system for antimicrobial use in food-producing animals estimates and describes exposure based on antimicrobial sales to certain food-producing animal sectors and/or with animal defined daily dosages established by the Netherlands Veterinary Medicines Institute (SDa). It is questionable how well the exposure of animals is captured by these methods. Modelling techniques may be useful for the investigation of exposure of food-producing animals to antimicrobials. Computer models are adaptable to different scenarios regarding treatment and animal/herd characteristics and the need for animal experiments is restricted. The aim of this project was to assess, through different modelling techniques, the exposure of food-producing animals to antimicrobials.

Allometric scaling and physiologically-based pharmacokinetic (PBPK) modelling were chosen as the modelling techniques to be investigated in this project. Allometric scaling was performed on pharmacokinetic data collected for cephalosporin antimicrobials in both food-producing and companion animals to assess the applicability and accuracy of this method for extrapolating kinetics across animal species. A full PBPK model was constructed to assess the distribution of slow-release oxytetracycline in calves. The model was applied to immediate-release oxytetracycline in adult cattle to assess model robustness.

Overall, it may be concluded that modelling approaches are appropriate to investigate exposure of animals to antimicrobials. Whether a simple model or a full PBPK model must be applied depends primarily on whether the research question is efficacy, toxicology or antimicrobial resistance related.