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. 2024 May 17;21(1):24.
doi: 10.1186/s12989-024-00572-y.

Toxicological inhalation studies in rats to substantiate grouping of zinc oxide nanoforms

Tizia Thoma #  1 , Lan Ma-Hock #  2 , Steffen Schneider  2 , Naveed Honarvar  2 , Silke Treumann  2 , Sibylle Groeters  2 , Volker Strauss  2 , Heike Marxfeld  2 , Dorothee Funk-Weyer  2 , Svenja Seiffert  3 , Wendel Wohlleben  3 , Martina Dammann  2 , Karin Wiench  4 , Noömi Lombaert  5 , Christine Spirlet  5 , Marie Vasquez  6 , Nicole Dewhurst  6 , Robert Landsiedel  7   8
Affiliations

Toxicological inhalation studies in rats to substantiate grouping of zinc oxide nanoforms

Tizia Thoma et al. Part Fibre Toxicol. .

Abstract

Background: Significant variations exist in the forms of ZnO, making it impossible to test all forms in in vivo inhalation studies. Hence, grouping and read-across is a common approach under REACH to evaluate the toxicological profile of familiar substances. The objective of this paper is to investigate the potential role of dissolution, size, or coating in grouping ZnO (nano)forms for the purpose of hazard assessment. We performed a 90-day inhalation study (OECD test guideline no. (TG) 413) in rats combined with a reproduction/developmental (neuro)toxicity screening test (TG 421/424/426) with coated and uncoated ZnO nanoforms in comparison with microscale ZnO particles and soluble zinc sulfate. In addition, genotoxicity in the nasal cavity, lungs, liver, and bone marrow was examined via comet assay (TG 489) after 14-day inhalation exposure.

Results: ZnO nanoparticles caused local toxicity in the respiratory tract. Systemic effects that were not related to the local irritation were not observed. There was no indication of impaired fertility, developmental toxicity, or developmental neurotoxicity. No indication for genotoxicity of any of the test substances was observed. Local effects were similar across the different ZnO test substances and were reversible after the end of the exposure.

Conclusion: With exception of local toxicity, this study could not confirm the occasional findings in some of the previous studies regarding the above-mentioned toxicological endpoints. The two representative ZnO nanoforms and the microscale particles showed similar local effects. The ZnO nanoforms most likely exhibit their effects by zinc ions as no particles could be detected after the end of the exposure, and exposure to rapidly soluble zinc sulfate had similar effects. Obviously, material differences between the ZnO particles do not substantially alter their toxicokinetics and toxicodynamics. The grouping of ZnO nanoforms into a set of similar nanoforms is justified by these observations.

Keywords: Developmental toxicity; Genotoxicity; Inhalation toxicity; Metal oxide; Nanoparticles; Reproductive toxicity; Zinc oxide.

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Conflict of interest statement

DFW, HM, KW, LMH, MD, NH, RL, SG, StS, SvS, ST, VS, WW are employees of BASF SE, a chemical company producing and marketing nanomaterials. TT is an employee of the BASF group company BASF Services Europe GmbH supporting BASF SE. NL and CS are employees of the International Zinc Association, MV and ND are employees of Helix3.

Figures

Fig. 1
Fig. 1
Test item characterization. A Transmission electron microscopic (TEM) images, B Size distribution of the constituent particles by TEM, C Dissolution kinetics in the abiotic CFS method of the test substances, uZnO (top) and cZnO (bottom). Scale bar 200 nm
Fig. 2
Fig. 2
Schematic view of the dust generation and exposure system
Fig. 3
Fig. 3
Timeline of 90-day inhalation exposure followed by 8-week post-exposure observation period
Fig. 4
Fig. 4
Changes (fold of control) of BALF parameters after 90-day inhalation exposure of adult animals. High concentration groups of A male (n = 10) and B female (n = 10) animals. Axis in logarithmic scale. Abbreviations: LDH: lactate dehydrogenase, ALP: alkaline phosphatase, NAG: N-acetyl-β-D-glucosaminidase, GGT: γ-glutamyl transferase, MPH: macrophages, LY: lymphocytes, PMN: polymorphonuclear neutrophils
Fig. 5
Fig. 5
Histologic images of the lungs of male control as well as high dose group animals after 90-day inhalation exposure. A control animal, B uZnO 10 mg/m3 intra-alveolar infiltration of foamy macrophages (black arrow), cellular debris within alveolus (black arrowhead), infiltration of neutrophils (red arrow) and hyperplasia of type II pneumocytes (red arrowhead). C cZnO 10 mg/m3, D µZnO 10 mg/m3, E ZnSO4 22 mg/m3 showed comparable findings as in B, hyperplasia of type II pneumocytes not always present. H&E, scale bar 50 µm
Fig. 6
Fig. 6
Histologic images of olfactory epithelium (nasal cavity) of male control as well as high dose group animals after 90-day inhalation exposure at the tip of a conchae or at the region of the dorsal meatus. A Control animal, B uZnO 10 mg/m3 (multi)focal degeneration/regeneration of the olfactory epithelium (arrow), C cZnO 10 mg/m3, and E ZnSO4 22 mg/m3 showed same findings as in B indicated by arrows. D Animals treated with µZnO did not show any findings in the nasal cavity. H&E, scale bar 50 µm
Fig. 7
Fig. 7
LA-ICP-MS analysis of lungs of control and high concentration group animals after 90-day inhalation exposure. Scale bar A–K 1 mm, AA–KK 500 µm. The panels of the first column (A, F, AA and FF) were from one lung lobe of a control animal, analyzed for Fe (panel A, overview, AA high resolution analysis of the indicated region in A); analyzed for Zn (panel F, overview, FF represented the high resolution analysis), The panels of the second column (B, G, BB and GG) were from one lung lobe of an animal exposed to 10 mg/m3 uZnO, those of the third column from one animal exposed to 10 mg/m3 cZnO, those of the fourth column from an animal exposed to 10 mg/m3 µZnO, and those of the fifth column from an animal exposed to 22 mg/m3 ZnSO4
Fig. 8
Fig. 8
Calculated amount of deposited uZnO, cZnO, µZnO, and ZnSO4 in different regions of the respiratory tract. Deposition after 90-day inhalation exposure at high concentration (10 mg/m3 ZnO materials and 22 mg/m3 ZnSO4). URT: upper respiratory tract (nasal cavity, larynx, and trachea), TB: tracheobronchial region, P: pulmonary region
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