Acute toxicity values such as LD50 and symptoms of treatment effect are often used as the basis for classifying chemicals into toxicity categories, and their subsequent regulation [19, 20]. In this study, two nanoparticle doses were used to evaluate the acute toxicity of Til-HCO-SLN. The high dose was 5 g/kg b.w, which is the highest dose in an acute toxicity study according to the guidelines for toxicity testing [20, 21]. The low nanoparticle dose of 766 mg/kg.bw, equivalent to 75 mg/kg.bw tilmicosin, was chosen based on the clinic dosage and the median lethal dose (LD50). If the overdose did not induce treatment effect, the clinic dosage should be safe. At the same time, the main focus of the low dose was on observing the treatment symptoms rather than on determining the mortality, and thus the dose was below the LD50°The LD50 of tilmicosin for subcutaneous administration is 97 mg/kg in male ICR mice and 109 mg/kg.bw in female ICR mice .
The results showed that Til-HCO-SLN and blank HCO-SLN was not lethal to mice with the high dose, suggesting that the Til-HCO-SLN and blank HCO-SLN are classified as low-toxic substances according to the toxicity categories of chemicals . This is mainly attributed to the good biocompatibility and biodegradability of the lipid matrix and the emulsifier. HCO can be hydrolyzed in vivo resulting in the formation of fatty acid and glycerin as metabolites . PVA can be degraded into acetic acid by oxidase and hydrolase enzyme . The oral LD50 is in the range of 15-20 g/kg , while the residual PVA of Til-HCO-SLN and blank HCO-SLN was very low.
The high dose of Til-HCO-SLN and blank HCO-SLN induced some slight intoxication signs of transient reversible abnormal clinic symptoms. The liver and spleen were affected as indicated by the organ coefficients and pathological changes, which is in agreement with other report that high doses of SLN could lead to accumulation of the lipid in liver and spleen and subsequently to pathological alterations . This might be due to that liver and spleen are the two dominant organs for distribution and metabolism of SLN [26, 27]. The damage of liver function is also confirmed by some biochemical parameter changes. Til-HCO-SLN caused slight decrease of the albumin (ALB)/globulin (GLB), while blank HCO-SLN leads to minor reduction of ALB and GLB. The hepatocellular damage is often indicated by changes of albumin, globulins and total protein . However, other liver function related parameters such as alanine aminotransferas (ALT), aspartate aminotransferase (AST), ALT/AST, bilirubin and urea were normal, suggesting that the damage of liver was slight and reversible . In addition, Til-HCO-SLN induced heart coefficient and histopathological changes, while blank HCO-SLN resulted in no coefficient change and much less histopathological alteration. This could be due to the acute cardiac toxicity of tilmicosin .
With low dose, the mice in the Til-HCO-SLN and blank HCO-SLN groups did not display any intoxication syndrome as referenced with the high dose groups. All mice survived and showed no clinic symptoms of treatment. There were no changes of organ appearance, organ coefficient, histopathology, biochemical and haematological parameters. The transient decrease of daily food and water consumption after the injections on the 1st and 8th days could be due to the irritation of the drug . These results indicate that the nanoparticle carrier and the formulation were safe at 7.5 times of the clinic dosage.
After administration with native tilmicosin, all mice in the high dose group died within 2 h. The surviving mice in the low dose group showed action retardation, incoordination, accelerated breath and gloomy spirit within 2 h after each administration. Tilmicosin primarily affects the heart, leading to acute heart failure with clinical signs of depression, increased heart rate, pulmonary edema, and death [22, 30]. Tilmicosin did not influence the organ appearance and coefficients, biochemical and hematological parameters at the low dose. The intoxication symptoms could be too low to observe. Another reason might be that the acute toxicity of tilmicosin was temporary , and all the functions were recovered at the end of experiments. In contrast, all mice survived and showed no treatment adverse effect in Til-HCO-SLN group with the low dose. These results demonstrated that this nanoparticle carrier remarkably reduced the acute toxicity of tilmicosin. The lower transient drug concentration of Til-HCO-SLN could account for the reduction of acute toxicity of tilmicosin [7, 8].