Determination of the LD₅₀ with the chick embryo chorioallantoic membrane (CAM) assay as a promising alternative in nanotoxicological evaluation

Abstract

Toxicity tests in rodents are still considered a controversial topic concerning their ethical justifiability. The chick embryo chorioallantoic membrane (CAM) assay may offer a simple and inexpensive alternative. The CAM assay is easy to perform and has low bureaucratic hurdles. At the same time, the CAM assay allows the application of a broad variety of analytical methods in the field of nanotoxicological research. We evaluated the CAM assay as a methodology for the determination of nanotoxicity. Therefore we calculated the median lethal dose (LD₅₀), performed in vivo microscopy and immunohistochemistry to identify organ-specific accumulation profiles, potential organ damage, and the kinetics of the in vivo circulation of the nanoparticles. Zinc oxide nanoparticles were intravascularly injected on day 10 of the egg development and showed an LD₅₀ of 17.5 µM (1.4 µg/mL_eggcontent). In comparison, the LD₅₀ of equivalent amounts of Zn²⁺ was 4.6 µM (0.6 µg/mL_eggcontent). Silica encapsulated ZnO@SiO₂ nanoparticles conjugated with fluorescein circulated in the bloodstream for at least 24 h. Particles accumulated mostly in the liver and kidney. In immunohistochemical staining, organ damage was detected only in liver tissue after intravascular injection of zinc oxide nanoparticles in very high concentrations. Zinc oxide nanoparticles showed a different pharmacokinetic profile compared to Zn²⁺ ions. In conclusion, the CAM assay has proven to be a promising methodology for evaluating nanotoxicity and for the assessment of the in vivo accumulation profiles of nanoparticles. These findings may qualify the methodology for risk assessment of innovative nanotherapeutics in the future.

Keywords:

• Zinc oxide nanoparticles
• nanotoxicity
• LD₅₀
• chick embryo chorioallantoic membrane assay
• silica nanoparticles

Acknowledgments

We thank Simone Mendler, Karin Benz, and Rita Gieringer for their excellent technical assistance.

Disclosure statement

The authors report no potential conflict of interest. The data of the NP accumulation experiments were presented at the 90. Jahresversammlung – Deutsche Gesellschaft für Hals-Nasen-Ohren-Heilkunde, Kopf- und Hals-Chirurgie e.V in Berlin (01/06/2019) (DOI 10.1055/s-0039-1685971).

Additional information

Funding

This work was financially supported by the Max Planck Graduate Center with the Johannes Gutenberg-Universität Mainz (MPGC), the focus program BiomaTiCS of the University Medical Center, Mainz, and the Stiftung Tumorforschung Kopf-Hals.