Biotic Safety Evaluation Of Polymers Chitosan

Objectives To evaluate the biotic safety of polymers chitosan by pyrogen test, acute toxicity test, hemolysis test, intradermally stimulus test, cell toxicity test in vitro, potential skin sensitization test, reverse mutation test, chromosome aberration test, teratospermia test, and subchronic toxicity test.Methods In pyrogen test, samples were supplied to rabbits by mainline. The fervescence of rabbits was observed. In acute toxicity test, samples were supplied to mice by mainline or intraperitoneal injection respectively. After 4h, 24h, 48h, and 72h, mice were observed respectively with generic states, toxicity appearance, mortality, and the weight in 72h. In hemolysis test, the fresh anticoagulant blood of rabbit was added in samples. To observed hemolytic crisis in all tubes, and to detect their absorbance. In intradermally stimulus test, samples were supplied to rabbit by endermic injection. The erythema and edema were observed, and the index of response was computed. In cell toxicity test in vitro, samples were added in L cell to evaluate cell toxicity. MTT assay was applied to determine cell relative growth rate. In potential skin sensitization test, guinea pigs were two-steps induced and aroused. In AMES, spot plate evaluation assay was used to select sample with no inhibition to tester strain line, and standard plate incorporate assay was used to analyze growth state of colony. In chromosome aberration test, samples were supplied to mice by intraperitoneal injection. Bone marrow was obtained from both femurs. Smears were observed under immersion objective. PCEs ratio in RBC was used to show marrow toxicity. In teratospermia test, samples were supplied to mice by intraperitoneal injection. Smears of epididymis were observed, and number of teratosperm was counted in 1000 sperm. Rank sum test was used to analyze the difference between chitosan groups and negative control group. In subchronic toxicity test, samples were imbedded hypo sac of rats. During embedding period, rats were weighed in and clinical observed. After this period, laboratory observation and pathologic dissection were applied to analyze generic behavior, gain weight, taking food, routine urine examination, hematology, blood biochemistry, and weight of organs. And statistic assay was used to analyze the results.Results In pyrogen test, the fervescence of rabbits was under 0.6℃, and the sum of fervescence of 3 rabbits was under 1.4℃. It is consider that chitosan can't bring fever. Results of acute toxicity test showed that symptoms and weight losing were acceptable. It is consider that chitosan can't bring acute general toxicity. In hemolysis test, hemolytic crisis was observed in positive control group, and in chitosan group and negative control group, hemolytic crisis wasn't observed. The hemolysis rate of chitosan groups is 0.7%, under 5%. Results showed that chitosan can't bring hemolysis. In intradermally stimulus test, the erythema and edema were observed, and the index of response was computed. And the index 0.89 showed that chitosan can stimulate skin and mucosa slightly. In cell toxicity test in vitro, under microscope, visible cell toxicity was observed in positive control group, and cell morphous was natural in both blank control group and negative control group, and slight cell toxicity was observed in chitosan group. Quantitative analysis results showed that toxicity of chitosan group was class 1, toxicity of blank control group was class 0, and toxicity of positive control group was class 4. All results showed that chitosan possessed very slight cell toxicity. In potential skin sensitization test, the skin in position of provocation was observed, and results showed that the sensitization appearances were same between SC group and CSO group. Erythema and edema of chitosan groups and the negative control groups were class 0, and erythema and edema of positive control groups were equal to or larger than class 1. So in this study, chitosan can't bring skin sensitization. In AMES, happened according to anticipation and that of chitosan group was similar with that of negative control group. T/C value of each strain was between 0.5 and 2, which showed the growth of stains didn't be refrained, and chitosan couldn't induce reversible mutation. So in this study, chitosan haven't inducibility. Results of chromosome aberration test showed that there was no statistical significance between each two groups (P>0.05). The difference of the two MPCEs ratio in PCEs between positive control group and chitosan group (SC or CSO) was significant (P<0.05). There was no statistical significance between chitosan group and negative control group (P>0.05). Results showed that chitosan couldn't induce genetoxic to mice. In teratospermia test, Statistics assay showed that there was no statistical significance between the malformation rates of them (P>0.05). The difference of the two rates between CP control group and negative control group was significant (P<0.05). Results showed that chitosan cannot induce genetoxic. In subchronic toxicity test, Statistics assay showed that there was no statistical significance between all targets of chitosan group and negative control group (P>0.05). In this study, it is can be considered that chitosan cannot induce subchronic toxicity after subcutaneously imbedding to rat.Conclusions The results above prove that in this study chitosan can't bring fever, acute general toxicity, and hemolysis. It has slight stimulation for skin and mucosa, and very slight cell toxicity. And it has no potential skin sensitization, no mutagenesis, and no genetoxic. So it is can be considered that chitosan possess upper biologic security.