Evaluation of developmental toxicity of ammonium dinitramide by micromass culture and embryonic stem cells models

Abstract

Objective: To evaluated the potential developmental toxicity and teratogenicity of ammonium dinitroamide (ADN) by micromass test (MM Test) and embryonic stem cell test models. Methods: In September 2018, rat embryos were isolated and limb bud cells were collected. The limb bud cells were treated with different concentrations of ADN (0, 312.50, 625.00, 1250.00, 2500.00, 5000.00, 10000.00 μg/ml) . Half proliferation inhibitory concentration and half differentiation inhibitory concentration were calculated and the teratogenic effects were evaluated according to the criteria. For the embryonic stem cell test, the effects of different concentrations of ADN (0, 39.06, 78.13, 156.25, 312.50, 625.00, 1250.00, 2500.00 μg/ml) on the differentiation of mouse embryonic stem cells (mESCs) into myocardial cells and the cytotoxicity of mESCs and 3T3 cells were detected. The embryonic toxicity was evaluated according to the criteria. In this study, both 5-fluorouracil (5-FU) , a known strong embryonic toxic drug, and penicillin-G (P-G) , a non-embryonic toxic drug, were used to verify the effectiveness of the model, and the validated test model was applied to evaluate the embryonic toxicity of ADN. Results: In the MM Test, the inhibition rates of proliferation and differentiation of limb bud cells in ADN groups were higher than that in control group (P<0.05) . And the half proliferation inhibitory concentration and half differentiation inhibitory concentration of ADN on limb bud cells were 7480.32 and 4526.09 μg/ml, respectively. ADN was determined to be non-teratogenic by standard. In the embryonic stem cell test, the inhibition rates of mESCs proliferation in ADN groups were higher than that in control group, and the inhibition rates of 3T3 cells in 156.25, 312.50, 625.00, 1250.00, 2500.00 μg/ml ADN groups were higher than that in control group (P<0.05) . The half proliferation inhibitory concentration and half differentiation inhibitory concentration of ADN on mESCs were 1851.73 and 1796.39 μg/ml, respectively, and the half proliferation inhibitory concentration on 3T3 cells was 3334.35 μg/ml. ADN was determined to be non-embryotoxic by standard. Conclusion: After evaluation by MM Test and embryonic stem cell models, ADN has no embryo toxicity and is a non-teratogenic substance.