Toxicity evaluation of ultra-diluted Methotrexate (12c&30c): An acute and subacute Oral toxicity study in animals

Abstract Purpose Autoimmune diseases exhibit active and remission phases. Tissues may retain an "inflammatory memory," increasing relapse risk after stopping treatment, necessitating ongoing therapy following remission induction. Understanding inflammatory memory and remission-to-recurrence transitions could guide strategies for achieving lasting remission while minimizing long-term medication use and costs. Integrative approaches may address autoimmune conditions' multifaceted pathophysiology. Literature suggests utilizing modern medicine in ultra-diluted form, with chemotherapy drugs like methotrexate undergoing ultra-dilution. However, extreme dilutions may increase toxicity due to nanoparticles' larger surface area. This study evaluated methotrexate's toxicity at 12c and 30c dilutions. Method An acute toxicity study evaluated single-dose effects of Methotrexate 12c and 30c (2,000 µL/kg) in female mice over 14 days, per OECD 423 guidelines. A repeated dose study investigated impacts of Methotrexate 12c and 30c (200 µL/kg) on male and female mice, following OECD 407 protocol. Mortality, clinical signs, body weight, hematology, biochemistry, and histopathology were comprehensively assessed. Result The acute toxicity study showed no animal fatalities with methotrexate, indicating the median lethal dose exceeded 2000 µL/kg. In the subacute study, methotrexate at 12c and 30c caused no mortality, adverse effects, or abnormal weight changes. Evaluations of hematological parameters, biochemical markers, and histopathological analyses of vital organs revealed no abnormalities. Conclusion The research study indicates that the oral administration of Methotrexate at 12c and 30c to mice over an extended duration, with a dosage of 200 µL/kg, did not exhibit any signs of toxicity or adverse reactions, thus suggesting a safe profile for these potencies.