Studies suggest that choline has potential to be used as a dietary supplement and a drug for immune inflammatory diseases like asthma and rhinitis. But there are apprehensions regarding adverse effects of choline when given orally in high doses. To address this knowledge gap, toxicity assessment of choline chloride was carried out by intranasal (i.n.), oral and intraperitoneal (i.p.) routes in Balb/c mice for 28days. Body weight, food and water consumption of mice were recorded daily. Hematology and clinical chemistry were assessed to check hepatocellular functions and morphological alterations of the cells. Splenocyte counts were analysed for evaluating cellular immunity. Liver function test was performed by assaying different enzyme systems in serum such as, urea, blood urea nitrogen (BUN), creatinine, alanine aminotransferase (ALT), and aspartate aminotransferase (AST). Body weight, food and water consumption did not differ between mice treated with choline and the saline control group. Hematologic and biochemical variables were not affected with any increase in serum toxicity marker enzymes indicating normal liver functioning. Choline administration did not affect total cholesterol and high density lipoprotein levels as compared to their respective controls. Urea and blood urea nitrogen levels in choline treated mice were not different than controls. Creatinine level was, however, higher than control in i.p. treatment group, but other parameters were normal. In conclusion, the repeated consumption of choline chloride via i.n. and oral or i.p. routes did not cause toxicity in mice in the toxicological endpoints examined.