| Diaminopyrimidines which are antibacterial can also work as antibacterial potentiator, such as trimethoprim, diaveridine, baquiloprim, ormetoprim. As a new drug of this sort, aditoprim was systematically researched by our lab in domestic firstly. This study was performed to evaluate its safety based on the former research in our lab. Research content involved in this study contains acute toxicity test, genotoxicity test, subchronic toxicity test, reproductive toxicity and teratogenicity test. Then, the acceptable daily intake (ADI) and safe concentration (SC) in different tissues of aditoprim were calculated to provide toxicological safety evaluation for clinical applications.1 Acute toxicity testThe acute toxicity test was performed with up and down procedure on specific pathogen free (SPF) adult Wistar rats and kunming mice. Aditoprim was suspended in 0.5% sodium carboxymethyl cellulose (CMC), and administrated by gavage. The experimental sequence was determined by short-term result and the survival animals were observed for 14 days. Finally, the LD50 value and 95% confidence interval was calculated using AOT 425 Stat Pgm. The LD50 value of aditoprim on Wistar rats was 1400 mg/kg b.w.,95% confidence interval was 954.9 to1750 mg/kg b.w.; that on mice was 1130 mg/kg b.w. and 859.2 to 1900 mg/kg b.w., respectively. Aditoprim turn out to be low toxic.2 Genotoxicity testBacterial reverse mutation assay Five S. typhimurium (TA97a, TA98, TA100, TA102 and TA1535) were selected to test the mutagenicity of aditoprim with and without S9 metabolic activation system. The dose levels of aditoprim were 1.0,0.5,0.25,0.125, 0.0625 μg/plate respectively. The results showed that aditoprim did not increase the number of revertant colonies on all strains. Aditoprim was not mutagenic in this test system.In vivo mouse micronucleus test 7-8 week-old SPF Kunming mice were divided into 5 groups in this test. Negative control, positive control and aditoprim treatment group (141.5,282.5,565 mg/kg b.w.) were set. The test substance was administered twice in 30 h by gavage, while cyclophosphamide was administered once by intraperitoneal injection. Bone marrow cells which obtained from the femurs were observed after slides preparation. The micronuclei ratio of polychromatic erythrocytes (PCE) and the ratio of polychromatic erythrocytes to normochromatic erythrocytes (NCE) (PCE/NCE) were calculated after counting. Results of micronuclei ratios indicated that no changes happened in treatment group compared to control group. It can conclude that aditoprim is not mutagenic in vivo.In vitro mammalian chromosome aberration test To evaluate the mutagenicity of aditoprim on V79 cells in vitro,4 dose levels (300,150,75,37.5μg/mL) were set. The negative control (DMSO) and positive control (CP) were set as well.4 h before harvesting, cell cultures were dealt with colchicine. The results showed that compared to the negative control aditoprim did not cause any increase in aberrations of V79 cells.In vitro mammalian cell gene mutation test The mutagenic effect of aditoprim on Chinese hamster ovary cells (CHO-K1) was determined in this study with and without S9 metabolic activation system. The treated concentrations were 300,150,75,37.5μg/mL respectively, while negative control and positive control were set. After 6 h treatment, the cytotoxicity was evaluated and the mutation frequency on hgprt locus was observed for further research. As a result, aditoprim didn’t show mutagenicity at the concentrations between 37.5-300μg/mL with and without S9 metabolic activation system.Mice testicle cells chromosome aberration test 30 g SPF Kunming mice were divided into 5 groups in this test. Negative control, positive control and aditoprim treatment group (141.5,282.5,565 mg/kg b.w.) were set. The test substance was administered daily by gavage for 5 consecutive days, while cyclophosphamide was administered by intraperitoneal injection. Mice were sacrificed and smear slides were made with testicle cells 12 d after the first dosing. The slides were scored for chromosome aberration in primary spermatocytes.The result shows that aditoprim was not mutagenic at all dose levels. It can conclude that aditoprim is not mutagenic for testicle cells in vivo.3 90-day feeding testAditoprim was administrated to 5-6 week-old specific pathogen free Wistar rats by mixed feeding at a dose level of 0,20,100 and 1000 mg/kg diet. The exposure time is 13 weeks and after that high dose group was kept for another 2 weeks without treatment to recovery from toxic effects. There are 20 rats each sex in control and high dose group and 15 rats each sex in low and medium dose group respectively. General clinical observations should be made at least once a day and all animals and the remaining feed should be weighed at least once a week throughout the treatment period. Food consumption and efficiency were calculated weekly.5 or 10 rats were killed at the interim (45 days) and terminal (90 days) period respectively. The blood samples were taken for haematological examinations and clinical biochemistry determinations. All animals were subjected to a gross necropsy and full histopathology was carried out on the preserved organs and tissues of all animals in the control and high dose groups. After 2 weeks recovery, same parameters were checked in the satellite groups. The results show that there is a significant decrease in body weight of the high dose group (p<0.05), while a significant increase was observed in liver or kidney/body weight ratios of the female high dose group (p<0.05) and that in testis (epididymis contained)/body weight ratios of the male high dose group (p<0.05). Some haematology and clinical biochemistry changes were observed in medium and high dose group, the changes included increased concentrations of total protein (TP), albumin (ALB), alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Histopathological changes were observed in liver of the high dose group, included bile duct hyperplasia, inflammatory cell infiltration and liver cells degenerate and necrosis. No adverse effect was observed in low dose group and the actual dose is 1.44 mg/kg b.w./day. In conclusion, the target organ of ADP is the liver and the No Observed Adverse Effect Level (NOAEL) of ADP is 1.44 mg/kg b.w. in subchronic toxicity study.4 Two-generation reproduction toxicity studyTo perform this study, specific pathogen free Wistar rats were subdivided into 4 groups. Aditoprim was administrated by mixed feeding at a dose level of 0,20,100 and 1000 mg/kg diet. Each group has 25 female rats and 18 male rats in the first generation and 25 female and 20 male rats in the second generation.After 13 weeks of exposure, female shall be placed with a single male from the same dose level (1:1/2:1 mating). Some observations and evaluations of general toxicity were same as that determined in subchronic toxicity study and reproductive parameters were determined in Fo and F1. The results show that there is a significant decrease in body weight of the high dose group (p<0.05). Some toxic effects of reproductive performance observed in high dose group include decreased average number of live fetus in F1 and F2a. decreased body weight on day 4 in F1 and that on day 4 and 21 in F2a. Histopathological examination in main organs showed similar changes with that in the subchronic toxicity study and no histopathological changes were found in reproductive organs of each group. Taking all the results into account, a slight maternal toxicity and developmental toxicity were observed in high dose group. Therefore, the No Observed Adverse Effect Level (NOAEL) of ADP is 7.89 mg/kg b.w. in the two-generation reproduction toxicity study.5 Feeding teratogenicity testThe feeding teratogenicity test combined with the 2nd generation of reproduction toxicity study. F1 female rats mated with male rats after F2a weaned and fetuses should be delivered by hysterotomy. Compared to the control group, a significant decrease in the number of implantations, live fetuses, mean male and female body weights and lengths, tail lengths were observed in high dose group. No treatment related skeletal or visceral anomalies were found. All the results indicate a slightly maternal toxicity and embryotoxicity in high dose group, but teratogenicity was not found. Therefore, the NOAEL of ADP is 8.75 mg/kg b.w. in feeding teratogenicity test.6 Calculating ADI and SCTaking all the results into consideration, the NOAEL of aditoprim was 1.44 mg/kg b.w.,and the calculated value of ADI was 0.00144 mg/kg b.w. according to the FDA given formula. The safe concentration (SC) calculated in muscle, liver, kidney and fat was 0.29,0.86,1.73,1.73 mg/kg respectively.The general toxicity, mutagenicity, reproductive toxicity and teratogenicity of ADP were evaluated in this study. The NOAELs and target organ of ADP were achieved and ADP was not mutagenic. All tests were carried out according to the international and domestic guidelines, the characters of the substance itself were taken into account as well. The achievements can provide a scientific basis for further research of aditoprim in the future. |
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