|
Objective: pyrogen, acute toxicity, hemolysis, intradermal stimulation in vitro cytotoxic potential skin sensitization, Salmonella reverse mutation, chromosomal aberrations (in vivo mammalian bone marrow micronucleus test), sperm teratogenic and sub-chronic toxicity a more comprehensive evaluation of one aspect of the biological safety of chitosan. : Pyrogen test, a certain dose for the test intravenous injection in rabbits, observed that the three test rabbits elevated body temperature; acute systemic toxicity test, the test by intravenous and intraperitoneal given Kunming mice, 4,24,48,72 h after injection observe the general state of the test and control groups Kunming mice toxicity performance and the number of dead animals, and 72h after the animal body weight; hemolysis test, the freshly prepared anticoagulant rabbit blood to join in for the test, observe each group whether hemolysis and measured absorbance; intradermal stimulation test for the test injection in rabbits back to observe erythema and edema of the inoculation point, and calculate the intradermal stimulation index; specific cell lines in vitro cytotoxicity test for the test, to evaluate its toxic effect on the cells and the degree of relative cell proliferation rate, measured by MTT assay; potential skin sensitization test subjects animals by the test, two-step induction and inspire, observed to stimulate parts of the skin; AMES test the drip board evaluation technology select testcross strains inhibited extracts, using standard plate incorporation test, groups were observed colony growth, and calculate the sample measured colony counting colony count ratio of the average of the mean negative control; micronucleus test, the test article intraperitoneal administration of the test animals, whichever bilateral femur bone marrow prepare smears, oil microscope each group were PCEs account the ratio of the total number of red blood cells, is used as the indication of bone marrow toxicity and statistical comparison; sperm abnormality test, the test substance intraperitoneal administration of the test animals isolated epididymal subchronic toxicity test production smear, observe the structural integrity of the 1000 sperm count which abnormal sperm, to calculate abnormal sperm incidence, rank sum test analysis test group differences between the sperm abnormality rate of the negative control group; , the test sample of the pocket for sterile implants the test animal inguinal surface subcutaneous, throughout the implantation period, weekly, weighed again, and individually detailed clinical examination. After the end of the implantation period, laboratory observations and pathological anatomy, and observe the results of the analysis. Results: pyrogen test, elevated body temperature of three rabbits were lower than 0.6 ° C, and three rabbits sum less than the 0.6 ° C warming that chitosan did not pyrogenic. In the acute toxicity test, observed immediately after injection Kunming mice, no adverse reactions, continue to observe the general state of the test and control groups Kunming mice 4,24,48,72 h after injection, toxic manifestations and death number of animals, as well as 72h after the animal weight, the results show that the symptoms of the test group animals and changes in body weight are within the acceptable range, it is considered that chitosan was no acute systemic toxicity. Hemolysis observed in the positive control tube hemolysis test, test products and the negative control tube no hemolysis. Moved into the closed beta absorbance cuvette, hemolysis rate of the test group was 0.7%, <5%, results showed that the chitosan - the biological hemostatic healing sponge did not cause hemolytic reaction. Intradermal stimulation test, the the intradermal irritation index was 0.89, indicating that the test product has a slight stimulation. In vitro cytotoxicity tests, seen under the microscope of the positive control showed obvious cytotoxic blank and negative control cells with normal morphology, the test group was observed to be extremely light toxicity. Quantitative test results showed that the toxicity of the test samples as a blank control to 0, a positive control for four. The quantitative and qualitative test results showed that the chitosan - with extremely light cytotoxicity the biological hemostatic healing sponge. Sensitization potential skin sensitization test in the SC group and CSO group the same, both erythema and edema of the test group and negative control group scoring level are 0, erythema and edema of the positive control group scoring level ≥ 1. So, under the test conditions, the chitosan - the biological hemostatic healing sponge no potential for skin sensitization. AMES test, various strains of yin and yang of spontaneous revertant colonies in accordance with expected growth within the respective ranges, negative control, and positive control back to change the number of colonies are at least as negative than 2 times the number of revertant colonies, the test sample and negative control is basically no difference. Calculating test sample colony count average and negative control colony counts are the average ratio range of between 0.5 and 2, strain growth is not inhibited, and the test article for each test strain without inducing action. Extract of the test samples in this study did not induce the test strains, does not have a mutagenic effect. Micronucleus test, each group PCEs account the erythrocyte total number of the ratio of the difference was not statistically significant, calculate the share of the number of each group MPCEs in PCEs, the positive control of the male and female two groups with male and female of the sample group (SC), the two groups The difference was statistically significant, the sample group (CSO), the two groups of male and female differences were statistically significant, negative control (SC and CSO) sample groups (SC and CSO), the difference was not statistically significant. The test results show that the test group no signs of toxicity of the cells, extracts of the test sample can be considered not produce genetic toxicity in mice. Sperm abnormality test, test group mice sperm abnormality incidence and negative control group to compare the difference was not significant (P> 0.05), CP positive control group and negative control group were significantly different (P <0.05). The results show that under the conditions of this study, the test sample extracts did not exhibit signs of Genetic Toxicity on mice. Subchronic toxicity test throughout the trial period, the test group and the control group contrast, the general behavior of the rats, weight gain, eating, urine routine, hematology, blood biochemistry, organ weight and pathological examination showed no significant difference (P> 0.05). Under the conditions of this study, we believe that the chitosan implanted in the subcutaneous tissue of 92 days, did not lead to the toxic effects of specific changes, does not cause the subchronic toxicity. Conclusion: chitosan as a biological hemostatic healing sponge under the test conditions of this study, non-pyrogenic, does not cause acute systemic toxicity and hemolytic reaction, mild irritation of the skin, mucous membranes, but within an acceptable range, with slight cytotoxic potential allergenicity of the skin, no mutagenic effect, genetic toxicity, therefore, that the chitosan as a the biological hemostatic healing sponge with a high bio-security. Still at an initial stage of the development and application of chitosan in the field of medical devices, not been similar for chitosan Biosafety comprehensive evaluation reports, and therefore in this study for the development and application of chitosan in the medical field to provide a reliable Security data.
|