5-FU could be beneficial in attaining high drug levels in the tumor immediately after administration of the formulation. Gradual release that follows should serve to maintain the drug level at the site of action for a prolonged period. Studies have shown that C-GP systems can release the entrapped compound over a period of several hours to days depending upon the molecular weight of chitosan used and also that the phenomena of burst release of the entrapped compound is observed initially, followed by gradual release, irrespective of molecular mass of the entrapped drug. Pilocarpine entrapped in C-GP containing systems for ocular delivery showed fast release of the entrapped drug initially, after which the drug was released gradually over a period of 24 h. A similar trend was observed for compounds like chlorpheniramine, calcein, etc. \cite{9}.

\subsection{Acute toxicity studies: Determination of LD$_{50}$}

During the studies used for LD$_{50}$ determination, masses of animals dosed at all levels were found to decrease as the time proceeded (Table~\ref{tab:table_1}). All three animals dosed at the highest dose of 550 mg kg$^{-1}$ died whereas those dosed at the lower level of 175 mg kg$^{-1}$ survived. Based on these results, the LD$_{50}$ of the test formulation was calculated to be 285 mg kg$^{-1}$ as per the AOT425StatPgm. The LD$_{50}$ of 5-FU on intraperitoneal administration to mice is reported to be 100 mg kg$^{-1}$ \cite{16}. Thus, a 2.8-fold decrease in toxicity of the test compound was observed.

The test drug in the present studies has a narrow therapeutic index \cite{17}. Following intraperitoneal administration of 5-FU solution to human subjects, the drug concentration was four times greater in portal venous blood compared to that sampled from a peripheral artery or vein or from the hepatic vein \cite{18}. Furthermore, up to 70\% of the drug was extracted by the liver during the first pass. Seven to 20\% of the parent drug was excreted unchanged in the urine in 6 hours; over 90\% of this was excreted during the first hour. The remaining percentage of the administered dose was metabolized, primarily in the liver. The catabolic metabolism of fluorouracil resulted in degradation products (e.g., CO$_2$, urea and $\alpha$-fluoro-$\beta$-alanine), which were inactive \cite{19}.

In the present study, a decrease in toxicity in terms of reduced mortality and absence of any significant change in the mass of dosed animals was observed compared to conventional injection. This decrease in toxicity can be attributed to a gradual and localized presentation of the cytotoxic agent from the formulation at the site of administration, which reduced systemic exposure and hence side effects. This is supported by earlier findings during phase I clinical studies on intraperitoneal formulations of 5-FU, which revealed that the concentration of the drug in the peritoneal cavity was 300 times greater than that in the plasma \cite{20}.

\subsection{In vitro cell line cytotoxicity studies}

Cytotoxicity of the drug free C-GP systems was found to be concentration dependent when evaluated in vitro in L-929 mouse fibroblast cells by an MTT assay. There was no significant difference between the percent proliferation of controls and those of test samples containing 10 to 50 $\mu$L of C-GP gels ($p < 0.05$), indicating the absence of cytotoxicity. But the test wells containing 100 $\mu$L of the C-GP system showed a significant difference in percent proliferation of fibroblast cells in comparison with the control ($p < 0.05$) and exhibited toxic effects indicated by lower percent proliferation (60.57\%) (Fig.~\ref{fig:figure_4}).