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EC number: 215-222-5
CAS number: 1314-13-2
No reliable study available. Supporting information does not show any neurotoxic effects of nano zinc oxide or zinc sulfate.
Neurotoxicity - Oral
This study represents a non- guideline RDT study (non-GLP) including an endpoint relevant for neurotoxicity with oral administration of zinc in combination with other metals or one dose of ZnSO4 7H2O (results described below) to male SD rats and the evaluation of the effects on general toxicological parameters and the performance in the Morris water maze (MWM) test (Su et al. 2017).
Healthy male SD rats (seven weeks old) were randomly divided into a total of 10 groups (six rats per group) including one control and one ZnSO4 group (only relevant in this context). The rats were orally treated with a once daily dose (33.84 mg/kg body weight) every three days for a total of 10 times in 34 days. The exposure and observation period was about 34 days. Animals were weighed every three days. Morris water maze (MWM) test was carried out four days before the end of the experiment. On the first three days, a hidden platform training was done, and the fourth day was for the hidden platform test. At the end of the experiment each rat was sacrificed, and blood was collected for haematological and biochemical analysis. Organs including brain, heart, liver, spleen, lung, kidneys, and testes were taken out and weighed immediately, and relative organ weights (to bw) were calculated. Organs were processed for histopathological examination (but not analysed). Western blot analysis for expression of p38, JNK, p-JNK, NFB, Nrf2, Akt, and GAPDH was conducted with brain tissue samples.
All rats survived the duration (34 days) of the study, and there was no effect on body weight gain in the ZnSO4 group compared to control. The ZnSO4 group showed a longer latent period compared to other treatment groups although no statistical significance was found when compared to the control group. The central activity time of this group was statistically significantly (p<0.05) lower than the control group. Western Blot analysis revealed a significant decrease in the expression of NF-κB, p-JNK and Akt protein in the brain tissue when compared to the control group. A significant increase of Nrf2 protein expression was observed in the brain tissue of the ZnSO4 group compared to the control group.
In this non-guideline short-term RDT study, no signs of general toxicity were observed in male SD rats treated with ZnSO4 administered 10 times over a 34-day period with a dose level of 33.84 mg/kg bw/d. The MWM results showed that the incubation period of the Zn group was longer than that of the control group though there was no significant difference and the central activity time of the ZnSO4 group was statistically significantly lower than the control group, suggesting that Zn exposure may lead to reduced spatial memory. Western-blot analysis revealed a significant decrease in the expression of p-JNK, NF-κB and Akt in brain tissue and an increased Nrf2 protein expression in brain tissue compared to the control group, but the relevance of this finding is not clear.
The results of this non-regulatory RDT study may indicate an effect of treatment of male rats with ZnSO4 on spatial memory. Although the study was conducted by a relevant route of exposure (oral) it was not designed to evaluate a dose-response relationship or a no effect level because only one dose level was chosen. In addition, the dosing regimen with treatment of animals every three days is not according to guideline recommendation and no pathological or histopathological examinations were conducted in the group treated with ZnSO4. Therefore, the study is only of supportive nature and regarded as not reliable [RL=3] in a regulatory context.
The study by Liang et al. 2018 represents a mechanistic non- guideline sub-acute RD neuro-toxicity study (non-GLP) with administration of ZnO NPs (50 mg/kg bw/d) by tongue instillation or gavage for 30 days and evaluation of the effects on biodistribution, brain histopathology and inflammatory responses.
Test material: ZnO NPs (SigmaAldrich (CAS number: 1314-13-2, USA), diameter: 42.31 ± 17.94 nm (prismatic-shaped), hydrodynamic size in distilled water: 232.8 ± 53.55 nm, zeta potential: 14.6 mV at pH 7.0, specific surface area was 27.342 (m2/g) Male Wistar rats (4 weeks, 130–150 g, Animal Center of Southern Medical University, Guangzhou, China) were randomly allocated into three groups with comparable weights: control group, the ZnO NPs tongue instillation group and the ZnO NPs gavage group. A 50 mg/mL suspension of ZnO NPs (50 mg/kg bw) was instilled onto the surface of the tongue with a microsyringe, and the control group was instilled with an equal amount of distilled water (DW). The instillation procedure lasted approximately one hour, and the tongue was then rinsed with DW to clean the remainder of the NPs. For the ZnO gavage group, the rats were treated with a 50 mg/mL suspension of ZnO NPs (50 mg/kg bw) by oral gavage. These protocols were performed every other day for 30 days. After 30 days of treatment, animals from each group (n=5) were sacrificed and fixed tissue samples were analysed by histopathological examination and immunohistochemistry (IHC). The remaining animals were sacrificed to collect tissues, including the brain (cerebellum, brainstem, cerebral cortex and hippocampus), nerves (CT and glossopharyngeal nerve) and blood, which were analysed for Zn content (n=6), cytokine release (n=6) and quantitative real-time PCR (qRTPCR, n=6). Zn in the blood or sub-brain regions were quantified by inductively coupled plasma mass spectrometry (ICP-MS). The gene expression levels of TNF-α, IL-1β, IL-6, IL-10, IFNG and NOS2 were determined by qRTPCR. The fixed tongue and brain tissues were examined histopathologically and IHC analysis was carried out to identify specific neurotoxic effects.
The Zn brain and nerve levels in the tongue instillation group were significantly higher than those in the gavage and control groups. In the tongue instillation and gavage groups, there were no significant increases in the blood Zn concentrations compared with that in the control group. Furthermore, the TNF-α and IL-1β levels were measured by ELISA, revealing significantly high TNF-α and IL-1β levels induced by ZnO NPs tongue instillation for 30 days. Tongue instillation exposure to ZnO NPs induced no apparent changes in brain histology when compared with the gavage and control groups. However, sparser hippocampal tissues were observed in both ZnO-treated groups compared to that in the control. Fungiform papilla hyperaemia was observed in ZnO tongue instillation group, which may give a hint that the sensory and taste function of tongue was affected. Decreased numbers of Nissl bodies and shrunken nuclei were found in the cortex and hippocampus after ZnO NPs tongue instillation, which showed that neurons were damaged in this group. Immunostaining of the cortex and hippocampus with anti-GFAP and anti-CD11b indicated that GFAP and CD11b expression was increased in the tongue instillation group compared to that in the other groups, suggesting that ZnO NPs stimulate astrocyte and microglial activation or proliferation after translocation to the brain. P2X7 receptor expression was also increased in the ZnO NPs tongue instillation group.
In this subacute non-guideline RD neuro-toxicity study with tongue instillation or gavage application of 50 mg/kg bw/d ZnO NPs for 30 days, the results showed that most of ZnO NPs enter the rat brain via the taste nerve pathway but not via blood and the digestive tract when administered via the tongue. TNF-α, IL-1β and NOS2 were significantly upregulated in the tongue instillation group compared to that in the other groups. ZnO NPs in the brain did not affect the cellular integrity or tissue morphology but still caused some minor damage.
This mechanistic non-guideline RD neurotoxicity study support the evidence that ZnO NPs enter the brain via the taste nerve pathway after tongue instillation and not via blood after oral gavage. After oral gavage only very minimal findings were observed while tongue instillation caused glial cell activation and inflammatory response. Although a relevant route of exposure (oral gavage) was included in the study and the material, methods and results are adequately reported, the study is only of supportive nature, because it was not designed to evaluate a dose-response relationship or a no effect level and only a limited number of endpoints was addressed to fit the purpose of the study and not to fulfil regulatory requirements. Therefore, the study is regarded as not reliable [RL=3].
Neurotoxicity - non-physiological routes of exposureFour non-guideline neuro-toxicity studies in rats or mice with intraperitoneal injection of ZnO NPs for 10 days (Amara et al. 2012) or 4 weeks (Tian et al. 2015) or 8 weeks (Han et al. 2011) or ZnSO4 for 14 days (Bernotienne et al. 2016) are available. All studies are only of supportive nature because a non-relevant route of exposure was chosen and only one dose level was used and are therefore regarded as not reliable [RL=3]. In the first non-guideline sub-acute neuro-toxicity study (Amara et al. 2012), male Wistar rats were treated by intraperitoneal injection with ZnO NPs (20-30 nm) at a dose of 25 mg/kg body weight for 10 days and the anxiety behaviours was evaluated using the elevated plus-maze test 24 h after the last injection. Subacute ZnO NPs treatment caused no significant increase in Zn content in the brain homogenate but significantly decreased the Fe level and the Ca2+ content (Na+ and H+ not affected). ZnO NPs treatment modulates the exploratory behaviours of rats only slightly. However, no significant differences were observed in the anxious index between ZnO NP-treated rats and the control group.In the second mechanistic sub-acute non-regulatory RD neuro-toxicity study (Tian et al. 2015), the neurotoxicity induced by ZnO NP (20-80 nm) in different-aged mice and the interaction between age and ZnO NP exposure was investigated following intraperitoneal injection of 5.6 mg/kg bw/d for 4 weeks. The results of the study showed that that ZnO NPs could induce a systemic inflammatory reaction in both adult and old mice, and there existed a synergistic reaction between age and ZnO NP exposure during induction of systemic proinflammation mediators. In the brain, oxidative stress and inflammation reactions were found after ZnO NP exposure and more remarkable changes were identified in aged individual. In addition, ZnO NP exposure resulted in abnormal cognitive function and neuronal pathological changes in the hippocampus. Old mice exhibited greater susceptibility to ZnO NP-induced damage.In another mechanistic sub-acute non-regulatory RD neuro-toxicity study (Han et al. 2011), intraperitoneal administration of one dose of ZnO NPs (20-80 nm) at 4 mg/kg bw (4 mg/mL) biweekly to Wistar rats for 8 weeks revealed that the escape latency was prolonged in the MWM test in the ZnO NP group. The long-term potentiation (LTP) measured after stereotaxic implantation of an electrode was significantly enhanced, while long-term depotentiation was barely influenced in the DG region of the ZnO NP group. It was concluded that spatial learning and memory ability were attenuated by the alteration of synaptic plasticity in ZnO NP treated rats.The last study represents a mechanistic non-regulatory RD neuro-toxicity study (Bernotienne et al. 2016) with sub-acute intraperitoneal administration of zinc (24 mmol Zn/kg body weight) alone or in combination with cadmium to mice daily for 14 days with determination of metallothionein (MT), gluthatione (GSH) and malondialdehyde (MDA) in brain samples. The results indicated that Zn ions could mitigate Cd-induced peroxidation of lipids via induction of metallothionein synthesis and preservation of reduced glutathione.
Neurotoxicity - Review articlesIn total 10 review article were identified, e.g. in the context of the role of zinc in the pathogenesis of neurodegenerative diseases, which did not give relevant information for this endpoint in a regulatory context:Galasso and Dyk et al. 2007 -review article on either neuroprotective or neurotoxic effects of Zn during global and focal cerebral ischemia – no relevant information for hazard or risk assessment purposes
Kawahara et al. 2018 – review article on the role of Zn in the pathogenesis of neurodegenerative diseases, such as AD, VD, and prion diseases with a focus on the neuroprotective effects of carnosine – no relevant information for hazard or risk assessment purposes
Levenson et al. 2005 -mini review on the role of zinc in traumatic and ischemic brain injury – no relevant information for hazard or risk assessment purposes
Morris and Levenson 2012 – review article on the role of the excitotoxic influx and accumulation of zinc, the mechanisms responsible for its cytotoxicity, and a number of disorders of the central nervous system – no relevant information for hazard or risk assessment purposes
Nuttall and Oteiza 2012 – review article on the evidence of the hypothesis that impaired activation of the extracellular signal-regulated kinases (ERK1/2) contributes to the disruptions in neurodevelopment associated with zinc deficiency – no relevant information for hazard or risk assessment purposes
Sensi and Jeng 2004 – review article on the current state of knowledge regarding cellular Zn2+ physiology and discussion of the established processes implicating this cation in ischemic injury, with particular attention to the most recent evidence, controversies, and pathogenic hypotheses – no relevant information for hazard or risk assessment purposes
Szewczyk 2013 - review article to provide an overview of both clinical and experimental evidence that implicates a dysfunction in zinc homeostasis in the pathophysiology of depression, Alzheimer’s disease and aging – no relevant information for hazard or risk assessment purposes
Szutowicz et al. 2017 – review article on findings gained from cellular and animal models of Alzheimer’s disease and discussion of putative energy/acetyl-CoA dependent mechanism including zinc neurotoxicity in early and late stages of neurodegeneration – no relevant information for hazard or risk assessment purposes
Watt et al. 2010 – review article on neuronal zinc metabolism and the way in which zinc can modulate normal brain activity as well as the contribution of zinc to the formation, aggregation, and degradation of the amyloid-β (Aβ) peptide and to the pathogenesis of AD – no relevant information for hazard or risk assessment purposes
Zhang et al. 2007 – review article on how the activation of 12-lipoxygenase and mitogen-activated protein kinase (MAPK) contribute to the toxicity of liberated zinc to neurons and oligodendrocytes – no relevant information for hazard or risk assessment purposes
Neurotoxicity - In vitro and in vivo studies not considered relevantGroeneveld et al. 2003 - effect of chronic oral administration of zinc sulfate (0.075 and 0.375 g/kg) on disease onset and survival of mSOD1 transgenic mice, a mouse model for amyotrophic lateral sclerosis (ALS)
Hsieh et al. 2017 – evaluation of olfactory function and olfactory epithelial recovery in wild-type vs. mice lacking MT1 and MT2 isoforms (MT KO) by intranasal instillation of zinc gluconate (170 mM) to induce robust damage to the olfactory epithelium, with subsequent recovery
Huang et al. 2014 – evaluation of the contribution of zinc to the development of Tau toxicity in a Drosophila tauopathy model expressing a human Tau mutant (hTauR406W, or Tau)
Ekstein et al. 2012 – acute infusion of ZnCl2 (45 μg (22mM)) over 90 min into the brain of rats by intracerebroventricular (i.c.v.) infusion
Sikora et al. 2020 - vesicular zinc transporter-3 (ZnT3) knockout mice, a model for Parkinson’s disease that lack vesicular Zn2+ were used o investigate the contribution of synaptically released Zn2+ to motor and cognitive deficits caused by nigrostriatal dopamine pathway deafferentation. The extracellular Zn 2+ chelator CaEDTA, and knock-in mice lacking high affinity Zn2+ inhibition of NMDARs (GluN2A-H128S KI mice; Nozaki et al., 2011) were also
Suh et al. 2006 – evaluation of temperature-dependent Zn2+ translocation from presynaptic boutons to postsynaptic neurons after traumatic brain injury (TBI) by autometallographic (AMG) and the N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide (TSQ) fluorescence method in Wistar rats
Hane et al. 2016 - single molecule force spectroscopy (SMFS) was used to probe the kinetic and thermodynamic parameters (dissociation constant, Kd, kinetic dissociation rate, koff, and free energy, ΔG) of the dissociation of an Aβ dimer, the amyloid species which initiates the amyloid cascade.
Chen & Liao 2003. Zinc toxicity on neonatal cortical neurons. Involvement of glutathione chelation. Journal of Neurochemistry, 2003, 85, 443–453
Hu et al. 2017. Pathological concentration of zinc dramatically accelerates abnormal aggregation of full-length human Tau and thereby significantly increases Tau toxicity in neuronal cells. Biochimica et Biophysica Acta 1863 (2017) 414–427
Ji and Weiss 2018. Zn2+-induced disruption of neuronal mitochondrial function: synergism with Ca2+, critical dependence upon cytosolic Zn2+ buffering, and contributions to neuronal injury. Exp Neurol. 2018 April; 302: 181–195.
Li et al. 2019. Zn2+ Aggravates Tau Aggregation and Neurotoxicity. Int. J. Mol. Sci. 2019, 20, 487
Sharma et al. 2013. The effect of Cu2+ and Zn2+ on the Aβ42 peptide aggregation and cellular toxicity. Metallomics. 2013 November; 5(11): 1529–1536
Tanaka and Kawahara 2017. Copper Enhances Zinc-Induced Neurotoxicity and the Endoplasmic Reticulum Stress Response in a Neuronal Model of Vascular Dementia. Frontiers in Neuroscience. February 2017; 11(58):
Zhu et al. 2012. Chronic Zinc Exposure Decreases the Surface Expression of NR2A-Containing NMDA Receptors in Cultured Hippocampal Neurons. PLOS ONE. September 2012; 7 (9): e46012
Neurotoxicity – Conclusion No short- or long-term neuro-toxicity studies allowing to derive a robust NOAEL for zinc compounds were identified. The results of non-guideline studies conducted by the oral route indicate that ZnO NPs (50 mg/kg bw/d) given orally for 30 days (every other day) did not alter cellular integrity or tissue morphology in the brain but still caused some minor damage. In addition, ZnSO4 7H2O at a dose of 33.84 mg/kg bw (every three days for a total of 10 times in 34 days) may lead to reduced spatial memory in the MWM test.
Studies with systemic administration of ZnO NPs via the intraperitoneal route revealed evidence that Zn ions when entering the brain may affect the exploratory behaviour of rats or induce systemic inflammatory reactions and abnormal cognitive function and neuronal pathological changes in the hippocampus of mice or resulted in a prolonged escape latency of rats in the MWM as well as enhanced LTP (but not on LTD). On the other hand, Zn ions could mitigate Cd-induced peroxidation of lipids via induction of metallothionein synthesis and preservation of reduced glutathione.
Several in vivo studies were considered as not relevant in a regulatory context, because they were conducted either in transgenic or knock out mice models of diseases (Groeneveld et al. 2003, Hsieh et al. 2017, Sikora et al. 2020) or in a Drosophila tauopathy model (Huang et al. 2014) or by intracerebroventricular infusion (Ekstein et al. 2012) or in a model of traumatic brain injury (Suh et al. 2006). In addition, several mechanistic in vitro studies, e.g. in different neuronal cells, for evaluation of specific neurotoxicological end points were considered not relevant in a regulatory context and were therefore not evaluated.
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