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Nanoparticles are being used extensively and found in applications to various fields ranging from agriculture to electronic devices, diagnosis to drug delivery, and cosmetics to food packaging. Increasing usage of engineered nanomaterials (ENM) raises potential concern for human health as well as to the environment. The present study aims to explore the effects of intermittent intraperitoneal exposure of ZNP on the spleen of male Wistar rat. Animals were divided into three groups, control and ZNP-treated groups (50 mg/kg and 250 mg/kg body weight), six in each group. Experimental animals were treated with different doses of ZNP once a week for 4 weeks, whereas control groups received water. After 28 days of exposure, animals were sacrificed, spleen tissue was excised, and various parameters such as hematological, genotoxicity, antioxidants, and histopathological were studied for changes in spleen if any. Results showed that ZNP exposure manages to induce alteration in various studied hematological parameters like neutrophils, platelets, and eosinophils which are found to increase significantly after the last treatment compared with the first treatment of ZNP. However, hemoglobin content, PCV, and MCV decrease with increasing dose of ZNP significantly in last treatment, when compared with the first treatment. DNA damage was observed in rats treated with a high dose of ZNPs compared with that in the control when analyzed through comet assay. Flow cytometric study was performed for better understanding of the underlying mechanism of the ZNP-mediated toxicity. From the present investigation, an increase in ROS production, a decrease in MMP, and increased apoptosis were exhibited. Further, altered antioxidant level (SOD, CAT, LDH, CYT P450, and CYT b5 r) has been observed in the studied splenic tissue, also histopathological changes observed in the rats exposed with high doses of ZNP. Therefore, ZNP may have the potential to induce a toxic effect even when exposed intermittently.