Toxicological assessment of ZnO nanoforms to substantiate grouping


zinc oxide
comet assay

How to Cite

Göpfert, A., Honarvar, N., Ma-Hock, L., Strauss, V., Treumann, S., Wohlleben, W., & Landsiedel, R. (2023). Toxicological assessment of ZnO nanoforms to substantiate grouping. Spanish Journal of Environmental Mutagenesis and Genomics, 27(1), 168. Retrieved from


Various zinc oxide nanoforms were grouped to fulfil information requirements according to the European Chemicals Regulation, REACh. The grouping was to be substantiated by new experimental data on selected nanoforms within this group [1]. The toxicological endpoint was a comet assay using the inhalation route of exposure.

Materials & method
28 Nanoforms of ZnO were characterized: (i) size distribution and aspect ratio (by TEM, according to the NanoDefine Method), (ii) dustiness and aerodynamic diameter (by the small rotating drum method, EN 17199:4), (iii) dissolution rate in lysosomal simulant (by continuous flow system; according to the 2, 3]). Two nanoforms with identical size and shape, but different surface coating (uncoated and hydrophobic) were selected for inhalation testing. Soluble zinc sulfate monohydrate and micron-sized ZnO particles were tested as reference substances at equimolar Zn concentrations. Rats were exposed to up to 8 mg/m³ ZnO for 14 days according to OECD test guideline (TG) no. 489. The target tissues analyzed in the comet assay were the nasal epithelium and the lung (site of contact) as well as the liver and the bone marrow.

Zinc oxide nanoparticles caused local toxicity at the respiratory tract. In animals exposed to 8 mg/m³ coated nano ZnO increased neutrophil counts in blood was observed. Other systemic effects were not observed in any of the tested substances. The COMET assay did not show genotoxic effects in any of the examined tissues. Nor were there any changes after exposure of high concentration of ZnO or ZnSO4 monohydrate.

Both ZnO nanoforms, micron-sized ZnO particles and Zn-ions in solution did not cause genotoxicity. The nanoforms of ZnO were comparable and maybe grouped together. No pronounced difference was found between nanoforms and pigment grade ZnO. This grouping approach helps to minimize the number of animal studies to be performed.


  1. Janer, G. et al. „Rationale and decision rules behind the ECETOC NanoApp to support registration of sets of similar nanoforms within REACH." Nanotoxicology 15.2 (2021): 145-166.
  2. Keller, J. G., et al. (2020). Predicting dissolution and transformation of inhaled nanoparticles in the lung using abiotic flow cells: The case of barium sulfate. Scientific reports, 10(1), 1-15.
  3. Keller, J. G., et al. (2021). Variation in dissolution behavior among different nanoforms and its implication for grouping approaches in inhalation toxicity. NanoImpact, 23, 100341.
Creative Commons License

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

Copyright (c) 2023 Spanish Journal of Environmental Mutagenesis and Genomics


Download data is not yet available.