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

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Gujarathi, Rashmi Korat, Piyush Gujarathi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7098879/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version 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. Methotrexate ultra-dilution secondary effect rebound effect Acute toxicity study Subacute toxicity study Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Highlights Ultra-dilution of methotrexate was prepared according to the secondary action principle. The study revealed that the LD50 of Methotrexate at 12c and 30c potencies surpassed 2000µl/kg in Swiss albino mice, indicating low acute toxicity. Methotrexate, administered repeatedly in 12c and 30c potencies at 200µl/kg for 28 days to Swiss albino mice of both sexes, did not alter body weight or hematological and biochemical profiles. Body weight exhibited a gradual increase. No pathological alterations were detected in the tissue samples obtained from the animal subjects, suggesting that methotrexate dosages up to 200µl/kg of body weight are well-tolerated and demonstrate an acceptable safety profile when administered repeatedly. Introduction Most of the autoimmune diseases possess a relapsing and remission or chronic progressive nature 1 – 3 .Accumulating evidence indicates that tissues can retain an "inflammatory memory" even after overt symptoms have resolved. This lingering inflammatory condition could potentially trigger a clinical recurrence at the original site upon cessation of treatment 4 – 7 . At present, patients remain on immunomodulatory therapies indefinitely, even after achieving clinical remission. Enhancing our understanding of inflammatory memory and the dynamic processes underlying the transition from clinical remission to recurrence could inform strategies to maintain remission while mitigating the long-term burden of medication and healthcare costs. The integrative medicinal approach holds the promising potential to address immunological diseases. The use of ultra-dilutions in treating chronic diseases is ubiquitous and continuously growing, being a globally available form of complementary and alternative medicine, with nearly 70–80% of the world population benefiting from it 8 – 10 . They give an unparalleled benefit, including a potent action, least or non-existent adverse effects at an affordable price 8 , 11 . The homoeopathic system of medicine works on the phenomenon of treatment of diseases depending on the keynotes that vary from those utilised in conventional medicines. The principle of similitude, trials in the healthy subjects, and infinitesimal dynamized doses 12 , 13 . The physiological demonstration ‘natural law of cure’, was given by of Founder of homoeopathy, Dr. Samuel Hahnemann. Homeopathic therapeutics uses the secondary response of the individual in an ameliorative manner by administering the medicine that provokes identical indications in healthful person to those afflicted with illness. This is done with the intent of stimulating a curative reaction from the body in oppose to its own maladies. By focusing on this secondary bodily response (vital reaction) occurs " every single occurrence without exception," whether with perceptible or minuscule doses and in the healthy as well as unwell, Hahnemann elevated the doctrine of treating like with like to the status of a "natural law of healing." He documented the development of biphasic action while utilizing the subjective experimental approach in 12 , 14 qualitative research. Hahnemann observed an initial "direct primary action of the drug," followed by a subsequent "indirect secondary action or vital reaction or counter action of the body" that manifested in an opposite manner, schematizing a ‘universal action of medicines’ 15 , 16 . The utilisation of homoeopathic therapeutics includes stimulation of homeostatic and curative reaction of body. Teixeira reconciled homeopathic pharmacology with conventional pharmacology 12 , 14 , 17 . The researcher asserted that the primary action of a drug on an individual resembles to the curative, adverse, and side effects of modern drugs, while the secondary response by the body is analogous to the rebound effect or paradoxical reaction in modern pharmacology. The analogy between the rebound effect of modern pharmacology and the secondary action of homeopathic pharmacology was evident in various literature sources. Teixeira also suggested the homeopathic utilisation of modern medicines, employing the rebound action for curative purposes, and proposed prescribing a modern drug in dynamized doses that elicit similar symptoms in their primary action to the manifestations of the sick individual 14 , 18 – 22 . In consideration of the aforementioned survey, we have prepared the ultra-dilution of methotrexate. Methotrexate is an antimetabolite which is generally used as a chemotherapeutic agent and as an immunosuppressant in autoimmune conditions. It is also used for clinical indications such as rheumatoid arthritis, refractory psoriasis, leukemia, relapsing non-Hodgkin lymphoma, breast cancer, squamous cell carcinoma, and osteosarcoma 23 – 25 . It shows its versatile action by acting through various mechanisms. For eg, Methotrexate acts as an antifolate antimetabolite by inhibiting enzyme dihydrofolate reductase and ultimately impeding the replication of DNA and RNA to produce the cytotoxic effect in cancer. Analogously, Methotrexate (MTX) also inhibits the enzyme 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transferase, which plays a vital part in the de novo purine synthesis pathway 25 – 27 . This enzyme is essential for the synthesis of the purine bases adenine and guanine, which are fundamental components of DNA and RNA. By impairing purine synthesis, MTX subsequently suppresses the synthesis of nucleic acids, thereby disrupting the rapid proliferation of cells such as immune cells. This disruption primarily targets the S phase of the cell cycle, which is responsible for DNA replication, ultimately hindering the growth and division of these rapidly dividing cell populations 26 – 28 . Methotrexate is administered through oral or parenteral route, such as intramuscular, intravenous, intrathecal, and subcutaneous. Orally, methotrexate is administered as a weekly single dose or divided doses every 8 hours over 24 hours. Generally, the peak plasma concentration is reached within 2 hours. The methotrexate was associated with various adverse effects such as nausea, vomiting, mucosal ulcers, anorexia, hepatotoxicity, and liver cirrhosis. Along with these, alopecia, fatigue, pancreatitis, bone marrow suppression, and renal failure are other side effects 25 , 29 – 32 . To our knowledge, this study represents the first demonstration of utilisation of modern drugs in its ultra-diluted form. The objective behind the preparation of the ultra-dilution of methotrexate is to give a new, usable and convenient homeopathic remedy that can be utilised to cure and/or alleviate the symptoms of aliments after individualisation, after undergoing preclinical and homeopathic pathogenic trials, potentially with minimal side effects. Another aim of this formulation is to provide a formulation that can be administered by oral route in psoriasis and other immune-related diseases. This study will give a cost-effective, easily accessible remedy with ease of use, great patient adherence, devoid/ negligible of side effects or rebound characteristics, and a complete cure for psoriasis. Also, will offer a new remedy to conduct Homeopathic Pathogenic Trials for researchers in the field of homeopathy. The conversion of synthetic drugs in its ultra-dilution will seed a new area of research about the homeopathy system of medicine and utilization of the rebound effect of synthetic drugs as pharmacological action, as per the homeopathic similitude principle for the field of pharmacology. The findings from this investigation hold particular significance, as the medications under examination do not exhibit toxic properties or elicit adverse reactions. Ultra-dilutions are generally considered safe and do not necessitate safety studies. However, empirical evidence is lacking to conclusively determine the safety of herbs, their components, or other medicinal sources, despite their long-standing use and inherent safety. Although methotrexate is not a novel molecule, the developed homeopathic formulation requires validation through preclinical toxicity studies to ensure its safety before being employed to treat specific disease conditions. This ethical obligation arises from the lack of comprehensive safety profile data for these homeopathic preparations to date, necessitating their evaluation in experimental rat models before considering further studies. When weighing the potential risks against the prospective advantages, it is imperative to conduct pre-clinical acute and sub-acute toxicity assessments of ultra-dilutions of methotrexate, particularly at the 12c and 30c potencies, because, as mentioned in literature, the nanoparticles remain at the extreme dilution in homeopathic medicines. Therefore, with consideration of the reduction in particle size and increase in surface area the toxicity might be increased. To date no homeopathic medicine formulated from a synthetic active pharmaceutical ingredient was prepared and assessed for its toxicity and efficacy in any disease by preclinical means. This study was conducted to evaluate the acute and subacute toxicity of the ultra-dilution of methotrexate 12c & 30c potencies prepared by homeopathic means as per the Organisation for Economic Cooperation and development (OECD) test guideline (with subtle alterations) in rats. The particular potencies are prepared and assessed since they were mentioned in the National List of Essential Ayush Medicine, 2022. In view of fact that most homeopathic remedies are administered as alcoholic liquid dilutions, the dosage in this study was quantified in microliters per kilogram (µL/kg) of b.w., rather than the conventional milligrams per kilogram (mg/kg) used in the OECD guidelines. Materials and Methods Chemicals and Drugs All the chemicals utilized for the study are of analytical standards. The methotrexate was procured from Globela Health Care Ltd., Surat, Gujarat. The 12c and 30c potencies of methotrexate were prepared as per the Homoeopathic Pharmacopoeia of India and were used for further experimentation. Experimental animals The Swiss albino mice of both sexes utilized in this procedure were authorized by the animal ethics committee of Trans-Genica Services Pvt. Ltd., Jalgaon, Maharashtra, India (Protocol Number:-TRS/PT/024/055A. The standard guidelines of Committee for Control and Supervision of Experiments on Animals (CCSEA), Government of India, were adapted for animal house like habitation, feeding, ventilation, lighting, sanitation, and management procedures. Through the experiment, the principle of 3Rs (replacement, reduction, and refinement) was also taken into consideration. The study protocol was conducted as per the Organization for Economic Cooperation and Development (OECD) guidelines; therefore, the substitution of animals with another substitute was not practicable. Healthy, male and non-pregnant female mice weighing 20–25 grams obtained from the Central Animal House facility of Trans-Genica Services Pvt. Ltd. During the acclimation period, the mice were maintained under standard laboratory conditions with a temperature of 25 ± 2°C, relative humidity of 60 ± 15%, and a natural light-dark cycle of 12 hours each. They were provided with a commercial pellet diet and water ad libitum. The animals were grouped and housed in polypropylene cages, with a maximum of four mice per cage. Randomization was achieved using a random number generator in Microsoft Excel, and all personnel involved in the experiment were appropriately trained and qualified. Experimental Design Preparation of methotrexate A standard procedure in Homeopathic Pharmacopoeia of India, Volume I was adapted for the preparation of methotrexate (MTX) 12c and 30c. Whole process of MTX 12c and 30c preparation was conducted in in-house pharmacy of Vidhyadeep Homoeopathic Medical College and Research Centre, Suart, India as per the homoeopathic pharmacopoeia of India 33 . Concisely, the homeopathic formulation of methotrexate underwent a meticulous preparation process in compliance with the legal requirements for homeopathic remedies. The centesimal (C) preparation involved a 100-fold dilution of methotrexate. The trituration (potentization of solid components) was conducted manually for 60 minutes with 50–55 rpm, adhering to the guidelines outlined in the Homeopathic Pharmacopeia of India Volume I. The procedure entailed maintaining a c-scale by combining 1 part of the crude drug with 99 parts of milk sugar. Specifically, 3 grams of methotrexate were triturated with 297 grams of milk sugar using a mortar and pestle. For the preparation of the 1c potency and subsequent trituration, 3 grams of methotrexate were mixed with 33 grams of milk sugar (11 parts by weight) in a mortar and pestle, and the blend was thoroughly rubbed for six minutes, followed by scraping. The mixture was then stirred using a pestle for six minutes and stirred again for four minutes. To this mixture, 99 grams (33 parts by weight) of milk sugar were added, and the above procedure was repeated twice. Finally, 165 grams (55 parts by weight) of milk sugar were added to the mixture, and the rubbing and stirring were repeated twice. At the end of this process, the 1c potency of methotrexate was obtained. The entire trituration procedure was repeated for the production of the 2c and 3c triturations of methotrexate. For the conversion of trituration into liquid potencies, the 3c trituration proceeded for the preparation of the liquid potency of methotrexate, following the procedure mentioned in the Homeopathic Pharmacopeia of India Volume I. For methotrexate, the liquid potentization was initiated by dissolving 10 grams (1 part) of the methotrexate 3c trituration in 500 ml of a solvent containing 495 ml (50 parts by volume) of distilled water and 500 ml of dispensing alcohol in 1000 ml Erlenmeyer flask. This liquid was subjected to high-intensity manual 10 succussion. The resulting potency was 4c, which served as the jumping potency for methotrexate and was used for further potentization. To prepare the 5c potency, 1 ml of the 4c potency was combined with 49.5 ml of distilled water and dilute alcohol, each, followed by 10 succussions. All subsequent liquid potencies up to 12c and 30c were prepared by repeating the procedure used for the 5c preparation and the volume of subsequent potency addition was selected according the final volume required. All the procedure were conducted in laminar air flow and all the apparatus used to prepare the medicine were used strictly after sterilization. Dose volume calculation For the acute toxicity study, all mice received doses of 2000 µl/kg of MTX 12c and 30c. Toxic manifestations were critically observed during the initial 30 minutes, followed by periodic observations every 2 hours for 24 hours, with heightened vigilance during the first 4 hours. Subsequently, the animals were monitored for a total duration of 14 days. The sub-acute toxicity study adhered to OECD 407 guidelines with minor modifications. A daily oral dose of 200 µl/kg body weight, diluted with distilled water in a 1:9 ratio, was administered 8 – 10 , 34 Acute toxicity study The acute toxicity study adhered to the OECD 423 guidelines with minor modification and healthy non pregnant, nulliparous female mice were used. Prior to dose administration, all animals were devoid to use of food but for water for 4 hours. The drug and vehicle were orally administered at a single dose of 2000 µl/kg body weight on day one 10 , 34 , 35 . Following administration, water was discontinued for next two hours. The treatment groups were as follows: Group name Treatment Normal control (NC) Distilled water Vehicle control (VC) Dispensing alcohol (90%) Test group 1 (T1) Methotrexate 12c Test group 2 (T2) Methotrexate 30 c Animals were critically observed for any sign’s mortality, moribund status and intoxication signs at 0 minutes (before dosing), 30 (± 5) minutes, 1 hour (± 10 minutes), followed by up to 24 hours post-dose administration. Body weight, food, and water intake were monitored. Body weights were recorded on days 1, 7, and 14 of the study. Observations included skin surface and fur condition, salivation, lacrimation, piloerection, eye/light reflex, lethargy, dehydration, nasal discharge, central nervous system effects like tremors and convulsions, and changes in gait, posture, and walking behavior. On day 14, all animals were sacrificed via excessive CO2 inhalation, and gross pathological alterations in skin folds, natural external orifices, outer organ and tissue surfaces of the thoracic, neck, abdominal, pelvic cavities, and remaining carcass were examined for abnormalities 9 , 10 , 34 , 35 . Sub-acute oral toxicity study This study was conducted as per the OECD 407 guidelines from 2008 with slight modification. Mice were randomly assigned to five groups, each consisting of 20 animals (10 males and 10 females). The grouping of animals was consistent with the acute study 8 – 10 , 34 , 36 . All treatments were administered orally as a single dose for a duration of 28 days. The mice were monitored daily for signs of intoxication, mortality, and comorbid conditions throughout the 28 days. On the 29th day, half of the animals in every group were left untreated and observed until the recovery period of 42 days after the 28-day treatment. This group served as the satellite group. All animals under study were inspected daily within their cages for general signs of toxicity and/or mortality, and the findings were recorded. The body weight of the animals was once a week, and the mean body weight was calculated. In-life observation Urine analysis Before the conclusion of the study, all subjects were individually housed in metabolic cages for 24 hours to facilitate the collection of urine samples. These samples underwent comprehensive physical, chemical, and microscopic analyses. The physical examination evaluated urine color, transparency, specific gravity, and pH levels. Chemical testing determined the presence of albumin, sugar, acetone, bile salts, and bile pigments (Acone, India). Microscopic examination involved the identification of pus cells, red blood cells, epithelial cells, crystals, amorphous phosphates, casts, bacteria, mucus, yeast, trichomonads, and spermatozoa. Hematology and Clinical Biochemistry Before blood sample collection, the animals were fasted for 4 hours while maintaining free access to water. On the 29th day, blood aliquots were obtained via the retroorbital plexus. For hematological analysis, blood samples were collected in K2-EDTA tubes and utilized for the examination of hemoglobin (Hb) and red blood cell (RBC) indices using an automatic cell counter (ABX MICROS60, Horiba). Parameters assessed included: hemoglobin, total red blood cells, packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and total white blood cells (WBC). For clinical chemistry analysis, blood samples were collected in 5 ml clot activator tubes, incubated for 15–20 minutes at room temperature, and centrifuged at 2400 rpm for 10 minutes using an Electra Lab centrifuge (Euro diagnostic system Pvt. Ltd, Chennai, India) to obtain serum. The serum was utilized to evaluate the following parameters: reticulocyte count, total cholesterol, serum triglycerides, high-density lipoprotein (HDL), low-density lipoprotein (LDL), blood urea nitrogen (BUN), urea, serum creatinine, serum calcium, serum glutamic oxaloacetic transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), serum bilirubin (total and direct), serum alkaline phosphatase (ALP), serum proteins (total proteins, albumin, A:G ratio), and plasma blood glucose (GLU). These analyses were performed using a Turbochem-100 analyzer (Awareness Technology, USA). Pathology Necropsy The mice were fasted overnight before necropsy and euthanized by inhalation of carbon dioxide followed by exsanguination. A complete autopsy of the carcass was performed, and observations were recorded. Organ weights The organs in which biodistribution of MTX is occurring were considered for histopathological examination. The Brain, Heart, Spleen, Kidney, Stomach, Liver, Lungs, Ovaries, and Testis were collected, quickly blotted, and trimmed for extraneous fat, and the absolute weight of organs was recorded immediately using an electronic balance. Histopathological evaluation All the isolated organs were fixed in 10% neutral buffered formalin for 24 hours, embedded in paraffin. Embedded tissue samples were cut into 2–4 µm sections and stained (Tissue embedding station, Model No.: -MTC-TE-P-5Ltr) with hematoxylin-eosin (H & E), and sections were observed under a light microscope (Nikon ECLIPSE E200) and microanatomical changes captured (MIchrome 6, 6MP Color Microscope Camera). Statistical analysis The data is a representation of mean ± standard error mean (SEM). Whole data is analyzed by one analysis of variance followed by Dunner’s post hoc analysis using SPSS 25 software. Statistical significance was defined as a value of p < 0.05. Results In vivo toxicity study Acute oral toxicity study (OECD 423) All the animals were observed carefully and critically for any signs and symptoms of toxicity at regular time lapses of 0,30 min, 1,2,4,6,8,12 hours, and then daily for up to 14 days. Mild lethargy and shivering were observed in vehicle control, and MTX (12c & 30c) was administered to animals. The abdominal breathing, vocalization, and dyspnoea were observed in all the animals, irrespective of treatment. There were no treatment-related alterations detected in food consumption, mean body weight, and weight gain in comparison to normal control animals during the study period of 14 days. No moribund state or mortality was detected in any treatment group. Additionally, no abnormalities were found in the gross necropsy among the groups Table 1. These results show that the Median lethal dose (LD50) of MTX 12c & 30c on single administration is under category 5 or unclassified as per Globally Harmonized Systems and higher than 2000µl/kg Body weight 35 . Table 1 Cage side observation in single dose acute toxicity study of MTX 23c and 30c in mice Intoxication signs/parameters Groups Normal control Vehicle control (sac. Lac.) MTX 12c MTX 30c Salivation NAD NAD NAD NAD Lacrimation NAD NAD NAD NAD Piloerection NAD NAD NAD NAD Respiration NAD NAD NAD NAD Eyes/ Light reflexes NAD NAD NAD NAD Fur NAD NAD NAD NAD Lethargy NAD 09 min/12 hrs 10 min/12 hrs 09 min/12 hrs Shivering NAD 2 min/30 min 2 min/30 min 2 min/30 min Dyspnoea Nil Nil Nil Nil Tremors NAD NAD NAD NAD Convulsions NAD NAD NAD NAD Nasal discharge NAD NAD NAD NAD Motor activities NAD NAD NAD NAD Gait NAD NAD NAD NAD Posture NAD NAD NAD NAD Walking pattern NAD NAD NAD NAD Food consumption Normal Normal Normal Normal Water consumption Normal Normal Normal Normal Mortality 0 0 0 0 Subacute oral toxicity study (OECD 407) A 43-day subacute oral toxicity study was conducted in two study phases, including 28 days of the main study and 43 days of the recovery study with MTX 12c and 30c at a dose of 200µl/kg/day. No signs of intoxication, moribund animal, or mortality were observed among the animals in each group at both study phases. Additionally, no alterations in food and water consumption, behavior patterns, or urinary and fecal excretion were observed in any animal throughout the study period 36 . Clinical changes Treatment-associated mortality was not found in any group of animals after regular administration of MTX 12c & 30c. Mice of both sexes did not show any noticeable detrimental signs such as alterations in skin, fur, eyes/ light reflexes, motor and nervous abnormalities, mucus membranes, and natural orifices. Moreover, no behavioral changes were observed inclusive of salivation, lacrimation, piloerection, coma, lethargy, sleep, and diarrhoea throughout the study period in both sexes compared to normal control. Body weight All treatment groups' mean body weight changes were recorded weekly for up to 43 days. The gradual rise in mean body weight in all mice of both sexes in control and treatment groups was observed. There were no significant changes found in the mean body weight of animals in the normal control group and treatment group from the onset to the end of the study period. Figures 1 and 2 represent the absolute body weight of females and males respectively. Urine analysis No statistically significant differences (p ≤ 0.05) were found in pH and the specific gravity of urine Table 2. The urine analysis results did not show any treatment-related changes in other urine parameters. There were no shreds of evidence of glycosuria, proteinuria, bile salt, or acetone observed in the chemical examination of urine in treated groups compared to controls. The microscopic examination of urine did not show the presence of pus cells, R.B.C., epithelial cells, crystals, amorphous phosphate, bacteria, mucus, yeast, trichomonas, and spermatozoa in all experimental animals at the end of the study. Table 2 Urine analysis of females and males for toxicity study of MTX 12c and 30c Sex Parameters Treatment Groups Normal control Vehicle control MTX 12c MTX 30c Female pH 1.004 ± 0.00 1.003 ± 0.00 1.004 ± 0.00 1.004 ± 0.00 Specific gravity 5.6 ± 0.1 5.4 ± 0.2 5.6 ± 0.1 5.4 ± 0.2 Male pH 1.005 ± 0.01 1.004 ± 0.00 1.005 ± 0.01 1.005 ± 0.01 Specific gravity 5.8 ± 0.2 5.6 ± 0.1 5.7 ± 0.2 5.8 ± 0.1 The data are presented as mean ± SEM. One-way ANNOVA was used with Dunnett’s post-hock test with significance p < 0.05. No significant difference is detected between the treatment and control group. Haematological indices The hematological indices measured in female and male mice are mentioned in Table 3. There was no significant (p ≤ 0.05) change found in the red blood cell indices, hemoglobin content, as well as total and differential leucocyte counts, of drug-treated mice in comparison to vehicle control animals. Biochemical investigations The biochemical parameters were analysed at the end of the study period. Statistically, no significant alterations were found in all biochemical parameters of animals treated with MTX in comparison to the control group. All the biochemical parameters are mentioned in Table 4. Table 3 Erythrogram, leukogram and platelet count of female and male mice for toxicity study of MTX 12c and 30c Haematological indices Female Male Normal control Vehicle control MTX 12c MTX 30c Normal control Vehicle control MTX 12c MTX 30c Haemoglobin(Gm/dl) 15.05 ± 0.54 14. 10 ± 0.71 15.13 ± 0.63 15.09 ± 0.55 17.09 ± 0.21 17.21 ± 0.50 17.34 ± 0.18 17.18 ± 0.45 Total RBC (×10 6 /µl) 8.24 ± 1.61 7.31 ± 1.57 8.25 ± 1.69 7.28 ± 1.54 5.61 ± 1.65 4.65 ± 1.42 5.56 ± 1.53 4.68 ± 1.39 M.C.V. (Fl) 54.71 ± 4.88 54.62 ± 5.76 53.73 ± 4.80 53.67 ± 4.83 56.22 ± 6.45 57.57 ± 6.39 58.36 ± 7.43 57.48 ± 6.31 M.C.H.(Pg) 17.23 ± 0.25 17.21 ± 0.22 17.28 ± 0.28 17.02 ± 0.25 17.12 ± 1.18 15.19 ± 2.12 17.16 ± 2.27 16.18 ± 1.22 M.C.H.C. (gm/dL) 32.16 ± 1.52 32.25 ± 1.66 32.04 ± 1.59 32.45 ± 2.61 31.76 ± 0.72 32.79 ± 0.76 31.72 ± 0.73 31.75 ± 0.75 TOTAL WBC (×10 3 /µL) 5.22 ± 0.41 5.25 ± 1.38 5.19 ± 1.34 5.21 ± 0.31 8.32 ± 0.34 8.40 ± 0.38 8.28 ± 0.29 8.35 ± 0.32 Eosinophils (×10 2 /µL) 1.2 ± 0.97 1.27 ± 0.98 1.25 ± 0.90 1.19 ± 0.91 1.32 ± 0.94 1.31 ± 0.95 1.33 ± 0.91 1.29 ± 0.93 Neutrophils (×10 2 /µL) 1.31 ± 0.41 1.27 ± 0.39 1.14 ± 0.42 1.21 ± 0.47 1.33 ± 0.34 1.28 ± 0.42 1.30 ± 0.38 1.34 ± 0.21 Lymphocytes (×10 2 /µL) 6.82 ± 0.53 6.85 ± 0.57 6.88 ± 0.51 6.84 ± 0.58 7.06 ± 0.40 8.83 ± 0.19 7.65 ± 0.22 8.88 ± 0.18 Monocytes (×10 2 /µL) 2.12 ± 0.51 2.01 ± 0.25 2.04 ± 0.26 2.29 ± 0.24 2.12 ± 0.79 2.24 ± 0.75 2.19 ± 0.71 2.15 ± 0.74 Platelet (×10 3 /µl) 0.635 ± 0.07 0.713 ± 0.078 0.065 ± 0.073 0.069 ± 0.067 0.413 ± 0.042 0.422 ± 0.052 0.412 ± 0.049 0.428 ± 0.056 The data are presented as mean ± SEM. One-way ANNOVA was used with Dunnet’s post-hock test with significance p < 0.05. No significant difference is detected between the treatment and control group. Abbreviations: Hb- haemoglobin, RBC- red blood cells, M.C.V.- Mean corpuscular volume, M.C.H.- mean corpuscular haemoglobin, M.C.H.C.- Mean corpuscular haemoglobin concentration. Table 5 Organ weight of female and male mice for toxicity study of MTX 12c and 30c Organ Treatment group (Female) Treatment group (Male) Normal control Vehicle control MTX 12c MTX 30c Normal control Vehicle control MTX 12c MTX 30c Brain 0.4 ± 0.1 0.4 ± 0.3 0.4 ± 0.1 0.4 ± 0.2 0.5 ± 0.2 0.6 ± 0.2 0.5 ± 0.2 0.5 ± 0.1 Heart 0.22 ± 0.01 0.23 ± 0.02 0.22 ± 0.04 0.23 ± 0.05 0.26 ± 0.03 0.24 ± 0.01 0.26 ± 0.02 0.24 ± 0.03 Spleen 0.14 ± 0.20 0.13 ± 0.16 0.14 ± 0.15 0.14 ± 0.19 0.42 ± 0.10 0.39 ± 0.18 0.40 ± 0.12 0.41 ± 0.09 Kidney 1.23 ± 0.30 1.29 ± 0.27 1.32 ± 0.18 1.26 ± 0.32 1.53 ± 0.02 1.55 ± 0.01 1.52 ± 0.03 1.53 ± 0.03 Stomach 0.11 ± 0.07 0.10 ± 0.09 0.11 ± 0.06 0.13 ± 0.01 0.19 ± 0.1 0.15 ± 0.21 0.15 ± 0.24 0.18 ± 0.12 Liver 2.21 ± 1.12 2.42 ± 1.10 1.98 ± 1.18 2.53 ± 2.16 3.12 ± 0.14 3.24 ± 0.16 3.16 ± 0.19 3.15 ± 0.17 Lungs 0.21 ± 0.22 0.22 ± 0.14 0.23 ± 0.28 0.20 ± 0.26 0.34 ± 0.31 0.31 ± 0.12 0.37 ± 0.16 0.32 ± 0.14 Ovaries(F)/Testes(M) 4.98 ± 2.68 5.01 ± 2.21 4.83 ± 1.38 5.74 ± 2.52 0.22 ± 0.04 0.19 ± 0.03 0.21 ± 0.01 0.20 ± 0.02 The data are presented as mean ± SEM. One-way ANNOVA was used with Dunnet’s post-hock test with significance p < 0.05. Necropsy No gross pathological changes were observed in any group of animals. MTX (12c & 30c) treated animals did not show any pathological abnormalities in all the organs in comparison to control animals Organ weight The Brain, Heart, Spleen, Kidney, Stomach, Liver, Lungs, Ovaries, and Testis were subjected to organ weight calculation as these are the sites of distribution of methotrexate. The absolute organ weight was determined at the time of necropsy and mentioned in the table. There was no significant change in absolute organ weight in the drug-treated group of all potencies in comparison to control group mice. Organ weights of females and males are shown in Table 5. Histopathology A microscopic examination of the brain, lungs, liver, kidneys, stomach, spleen, heart, testes, epididymis, ovaries, and uterus was performed on all treatment groups in both sexes. There were no microanatomical alterations found in photomicrographs of all the organs of females and males. Figures 3, 4, and 5 illustrate the histological images of organs from female and male animals. Discussion The ultra-high dilution of methotrexate was prepared as per the homeopathic pharmacopeia of India, Volume I, having potency 12 centesimal (12c) and 30 centesimal (30c). The acute toxicity study of MTX 12c and 30c was conducted in mice as per the OECD 423. 2002 guideline with slight modification. The mice are used to perform the toxicity study as they will be the intended species for further studies. OECD 423 is utilized to evaluate the LD50 range of MTX ultra-high diluted potencies. The MTX 12c and 30c were administered as a single dose of 2000 µl/kg on the 1st day and were critical signs of toxicity for 14 days. During the study period of up to 24 hours, mild lethargy and shivering were noticed in treatment and vehicle-treated animals. The abdominal breathing, vocalization, and dyspnoea were noticed in animals for the first few minutes. There was no treatment-associated mortality noticed in all animals following the treatment with MTX 12c and 30c. As per the literature survey, a 20% decrease in body weight will be the end point for the study, which will further give an idea about the food intake of animals. The body weight was found to be gradually increased similarly compared to the normal control animals throughout the study 34 , 37 – 39 . Concerning in-life, or cage-side observations, no alterations were observed that can relate to the treatment. Moreover, no atypical changes were identified in either the outer body surface or internal organs. In the acute toxicity study, the animals were administered nearly 2.5 times a weekly maximum dose of MTX (25 mg/week MTX for a maximum 24 weeks) used clinically, i.e., 2000 µl/kg body weight, for investigation of detrimental effects due to the ultra-dilution of methotrexate. These results also support the rebuff of the claim that the toxicity of methotrexate might be elevated due to an increase in surface area in the preparation method. The repeated dose 28-day toxicity study was conducted in mice of both sexes as per the OECD 407, with minor alterations, to evaluate the repeated treatment consequences of MTX dilutions on possible toxicity. In this study, the effect of MTX (12c & 30c) was assessed following repeated administration. The body weight, food consumption, hematology, blood biochemistry, urine analysis, organ weight, and histopathology were used as confirmation markers. There are no treatment-associated alterations noticed in behavior as well, and clinical signs viz, gait, fur color, piloerection, salivation, lacrimation, eating, sleep, urine-–feces excretions, as well as autonomic activities, were found throughout the study period. As per the OECD guideline 20% reduction in body weight is contemplated as one of the endpoints in human study 37 , 38 . Consumption of food, food palatability, food nutritional content, digestion, and absorption are the important factors affecting body weight. 34 , 40 . The body weight and food consumption were moving steadily throughout the study period in treatment and vehicle-treated groups. Blood indices are the most essential diagnostic parameters in clinical practice to assess the toxic effects of drugs. Furthermore, the hematological parameters alterations in rodents were found to be prognostic of toxicity in humans, as supported by the data obtained from the animal trials 40 – 43 . As the bone marrow is highly sensitive to toxic drugs, the hematological indices are crucial in monitoring the functioning of bone marrow and the intravascular network 41 – 43 . The treatment-related alterations in blood indices were not found in this study compared to the control. The non-significant alterations in blood indices demonstrate the non-toxic nature of MTX to blood indices. Serum biochemistry is another essential consideration in evaluating the toxic nature of the test substance. Serum biochemistry, such as liver function tests and kidney function tests, is essential in evaluating the functioning of the liver and kidneys as they are important for survival, metabolizing, and detoxifying the majority of chemicals and drugs. (olo, ekanay). The liver is the prime organ associated with drug metabolism, cholesterol biosynthesis, and degradation; even MTX is metabolized by the liver. Additionally, it also assists in glucose synthesis and the production of glucose from glycogen storage 44 . The outputs of this study revealed that MTX ultra-dilution did not have an effect on the metabolism of lipids and carbohydrates. The liver function test was used to assess the toxicity of MTX potencies on liver functionality. The increased serum concentration of SGOT, SGPT, BUN, and ALP is indicative of liver injury, cirrhosis, and myocardial infarction 45 . No alterations in the aforementioned parameters were observed in the present study when the treatment group was compared to control group animals, which is an indication of the nontoxic effect of MTX 12c and 30c on the liver. Total bilirubin is a byproduct of hem catabolism and an indicator of liver function and cholestasis. Also, it reduces aplastic anemia 45 . There were no significant changes observed in total bilirubin and BUN in MTX 12c and 30c-treated animals compared to control animals 45 , 46 . Furthermore, various studies have revealed the interconnection between the amount of urea and creatinine with the glomerular filtration rate, which is the prime indicator of kidney function 45 . MTX 12c and 30c treatment did not change the urea and creatinine concentration compared to control. These results imply that the MTX 12c and 30c did not show any effect on liver and kidney functions. Toxicity leads to alteration in color and architecture, hypertrophy, and edema of the organs when the drug is distributed 45 . The macroscopic assessment of all vital organs during necropsy of MTX 12c and 230c treated animals in both acute and repeated dose toxicity studies did not show any lesion, hypertrophy, color, or texture alteration compared with control animals. Moreover, absolute organ weight is the basic indicator for assessing any treatment-related injury in internal organs 45 , 46 . There were no significant changes in body weight of MTX 12c and 30c-treated animals compared to the control. from the obtained study results, the MTX 12c and 30c with vehicle neither impeded physiology nor produced pathological alterations in animals. The toxic substances directly impact the vital organs of the body and lead to alterations in appearance, morphology, an increase in organ size, and edema. The microscopical scrutiny of brain, lungs, liver, kidneys, stomach, spleen, heart, testes, epididymis, ovaries, and uterus did not show any histopathological alterations following the 28 days of repeated dose of MTX at both dilutions. This data is indicative of repeated administration of MTX at dilutions 12c and 30c, nontoxic. Conclusion The outcome of acute and repeated dose toxicity studies indicates that no mortality was observed at a dose of 2000µl/kg of MTX 12c and 30c, which stipulates that the oral LD50 of MTX 12c and 3c is higher than the administered dose. Repeated administration of MTX 12 c and 30c at a dose of 200 µl/kg p.o. was found safe in mice. No observed Adverse Effect Level (NOAEL) of MTX 12c and 30c after 20 consecutive days or oral administration in mice was found to be not less than 200 µl/kg/day. Additionally, upon the repeated administration of MTX 12c and 30c, no organ-specific toxicity was observed. Moreover, no treatment-associated microanatomical alterations were observed in either sex at 200 µl/kg/day of MTX 12c and 30c. therefore, it may be concluded that there are no detrimental properties of ultra-high diluted preparation of MTX at potency 12c and 30c, and it was found safe at a daily dose of 200 µl/kg/day p.m. Declarations Conflict of interest The authors declare that they have no conflict of interest Ethical approval: The study protocol was reviewed and approved by the animal ethics committee of Trans Services Genica Pvt. Ltd, Maharashtra with approval number -TRS/PT/024/055A Consent to participate: Not applicable Consent to publication: Not applicable Clinical trial number: Not applicable Funding: No funding received for this work Author contribution declaration: PG: Formal writing, data collection, statistics, RK: Supervision & manuscript finalising, PG: Drug sample preparation, formal writing, data acquisition References Lebel Y, Milo T, Bar A, Mayo A, Alon U. Excitable dynamics of flares and relapses in autoimmune diseases. iScience. 2023 Nov;26(11):108084. Wildner G, Kaufmann U. What causes relapses of autoimmune diseases? The etiological role of autoreactive T cells. Autoimmun Rev. 2013 Sep;12(11):1070–5. Elkoshi Z. New insights into the phenomenon of remissions and relapses in autoimmune diseases and the puzzle of benign autoantibodies in healthy individuals. Front Immunol. 2025 May 9;16. Chang MH, Bocharnikov A V., Case SM, Todd M, Laird‐Gion J, Alvarez‐Baumgartner M, et al. 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Supplementary Files Table4.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 08 Sep, 2025 Reviews received at journal 24 Aug, 2025 Reviewers agreed at journal 03 Aug, 2025 Reviewers invited by journal 29 Jul, 2025 Editor assigned by journal 24 Jul, 2025 Submission checks completed at journal 24 Jul, 2025 First submitted to journal 11 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7098879","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":492402505,"identity":"99c8ef86-2b1f-4abc-8178-2bdf53768aaa","order_by":0,"name":"Pranjal P. Gujarathi","email":"data:image/png;base64,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","orcid":"","institution":"Bhagwan Mahavir College of Pharmacy, Bhagwan Mahavir University","correspondingAuthor":true,"prefix":"","firstName":"Pranjal","middleName":"P.","lastName":"Gujarathi","suffix":""},{"id":492402506,"identity":"c8863914-10fd-4838-b0f1-5eaf2d18ec78","order_by":1,"name":"Rashmi Korat","email":"","orcid":"","institution":"Bhagwan Mahavir College of Pharmacy, Bhagwan Mahavir University","correspondingAuthor":false,"prefix":"","firstName":"Rashmi","middleName":"","lastName":"Korat","suffix":""},{"id":492402508,"identity":"7bcc1757-d1a3-4c88-aa5b-fc90f824d7d8","order_by":2,"name":"Piyush Gujarathi","email":"","orcid":"","institution":"Vidhyadeep University","correspondingAuthor":false,"prefix":"","firstName":"Piyush","middleName":"","lastName":"Gujarathi","suffix":""}],"badges":[],"createdAt":"2025-07-11 07:23:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7098879/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7098879/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88038702,"identity":"37f3de24-210a-47e2-ba79-f24071248248","added_by":"auto","created_at":"2025-07-31 16:40:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":16241,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBar graphs represent body weight variation of female mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data are presented as mean ± SEM. One-way ANNOVA was used with Dunnett’s post-hoc test with significance p\u0026lt;0.05, and the weights of all groups were compared with their relevant weight on day 1.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7098879/v1/a6cd1549a32d92e219900212.png"},{"id":88038703,"identity":"9b5d651b-9718-4bb9-a37d-b81663976d1e","added_by":"auto","created_at":"2025-07-31 16:40:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":17332,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBar graphs represents of body weight variation of male mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data are presented as mean ± SEM. One-way ANNOVA was used with Dunnett’s post-hoc test with significance p\u0026lt;0.05, and the weights of all groups were compared with their relevant weight on day 1.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7098879/v1/10a6bf3445e70e6c1b61a48a.png"},{"id":88038709,"identity":"2beca0d9-0ffb-44c9-a912-b08078e05cee","added_by":"auto","created_at":"2025-07-31 16:40:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":8017849,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhotomicrographs of Brain, Heart, Lungs of female and male mice\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7098879/v1/82e3b5b6d4144a8f1f4b6376.png"},{"id":88038708,"identity":"fd218d42-ffb6-44d1-af51-2a38bbec6783","added_by":"auto","created_at":"2025-07-31 16:40:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6055311,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhotomicrographs of Liver, Kidney and Spleen of female and male mice\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7098879/v1/dbe172eef4ee33dddd1173cd.png"},{"id":88038714,"identity":"02f7cfb9-8baf-47a0-b1ff-7e23ca101df5","added_by":"auto","created_at":"2025-07-31 16:40:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":6119367,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhotomicrographs of Stomach, Epididymides, Prostate, uterus, \u0026amp; ovary\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7098879/v1/ec232ad304e6d1e4e3dc2033.png"},{"id":88041623,"identity":"3a5a670d-44b7-41c1-9b9b-b3e64846a760","added_by":"auto","created_at":"2025-07-31 17:12:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":28447283,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7098879/v1/e1e11011-95d4-4605-b839-4bcf09c07294.pdf"},{"id":88038701,"identity":"6e84cdf6-db6a-49ab-93ed-5dac6a267ba6","added_by":"auto","created_at":"2025-07-31 16:40:23","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19198,"visible":true,"origin":"","legend":"","description":"","filename":"Table4.docx","url":"https://assets-eu.researchsquare.com/files/rs-7098879/v1/b2fcbeec5e18ee34eacd3a40.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eToxicity evaluation of ultra-diluted Methotrexate (12c\u0026amp;30c): An acute and subacute Oral toxicity study in animals\u003c/p\u003e","fulltext":[{"header":"Highlights","content":"\u003cul\u003e\n \u003cli\u003eUltra-dilution of methotrexate was prepared according to the secondary action principle.\u003c/li\u003e\n \u003cli\u003eThe study revealed that the LD50 of Methotrexate at 12c and 30c potencies surpassed 2000\u0026micro;l/kg in Swiss albino mice, indicating low acute toxicity.\u003c/li\u003e\n \u003cli\u003eMethotrexate, administered repeatedly in 12c and 30c potencies at 200\u0026micro;l/kg for 28 days to Swiss albino mice of both sexes, did not alter body weight or hematological and biochemical profiles. Body weight exhibited a gradual increase.\u003c/li\u003e\n \u003cli\u003eNo pathological alterations were detected in the tissue samples obtained from the animal subjects, suggesting that methotrexate dosages up to 200\u0026micro;l/kg of body weight are well-tolerated and demonstrate an acceptable safety profile when administered repeatedly.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eMost of the autoimmune diseases possess a relapsing and remission or chronic progressive nature\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.Accumulating evidence indicates that tissues can retain an \"inflammatory memory\" even after overt symptoms have resolved. This lingering inflammatory condition could potentially trigger a clinical recurrence at the original site upon cessation of treatment\u003csup\u003e\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. At present, patients remain on immunomodulatory therapies indefinitely, even after achieving clinical remission. Enhancing our understanding of inflammatory memory and the dynamic processes underlying the transition from clinical remission to recurrence could inform strategies to maintain remission while mitigating the long-term burden of medication and healthcare costs. The integrative medicinal approach holds the promising potential to address immunological diseases.\u003c/p\u003e\u003cp\u003eThe use of ultra-dilutions in treating chronic diseases is ubiquitous and continuously growing, being a globally available form of complementary and alternative medicine, with nearly 70\u0026ndash;80% of the world population benefiting from it\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. They give an unparalleled benefit, including a potent action, least or non-existent adverse effects at an affordable price\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The homoeopathic system of medicine works on the phenomenon of treatment of diseases depending on the keynotes that vary from those utilised in conventional medicines. The principle of similitude, trials in the healthy subjects, and infinitesimal dynamized doses\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. The physiological demonstration \u0026lsquo;natural law of cure\u0026rsquo;, was given by of Founder of homoeopathy, Dr. Samuel Hahnemann. Homeopathic therapeutics uses the secondary response of the individual in an ameliorative manner by administering the medicine that provokes identical indications in healthful person to those afflicted with illness. This is done with the intent of stimulating a curative reaction from the body in oppose to its own maladies. By focusing on this secondary bodily response (vital reaction) occurs \" every single occurrence without exception,\" whether with perceptible or minuscule doses and in the healthy as well as unwell, Hahnemann elevated the doctrine of treating like with like to the status of a \"natural law of healing.\" He documented the development of biphasic action while utilizing the subjective experimental approach in \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003equalitative research. Hahnemann observed an initial \"direct primary action of the drug,\" followed by a subsequent \"indirect secondary action or vital reaction or counter action of the body\" that manifested in an opposite manner, schematizing a \u0026lsquo;universal action of medicines\u0026rsquo;\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. The utilisation of homoeopathic therapeutics includes stimulation of homeostatic and curative reaction of body. Teixeira reconciled homeopathic pharmacology with conventional pharmacology\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The researcher asserted that the primary action of a drug on an individual resembles to the curative, adverse, and side effects of modern drugs, while the secondary response by the body is analogous to the rebound effect or paradoxical reaction in modern pharmacology. The analogy between the rebound effect of modern pharmacology and the secondary action of homeopathic pharmacology was evident in various literature sources. Teixeira also suggested the homeopathic utilisation of modern medicines, employing the rebound action for curative purposes, and proposed prescribing a modern drug in dynamized doses that elicit similar symptoms in their primary action to the manifestations of the sick individual \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan additionalcitationids=\"CR19 CR20 CR21\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn consideration of the aforementioned survey, we have prepared the ultra-dilution of methotrexate. Methotrexate is an antimetabolite which is generally used as a chemotherapeutic agent and as an immunosuppressant in autoimmune conditions. It is also used for clinical indications such as rheumatoid arthritis, refractory psoriasis, leukemia, relapsing non-Hodgkin lymphoma, breast cancer, squamous cell carcinoma, and osteosarcoma\u003csup\u003e\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. It shows its versatile action by acting through various mechanisms. For eg, Methotrexate acts as an antifolate antimetabolite by inhibiting enzyme dihydrofolate reductase and ultimately impeding the replication of DNA and RNA to produce the cytotoxic effect in cancer. Analogously, Methotrexate (MTX) also inhibits the enzyme 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transferase, which plays a vital part in the de novo purine synthesis pathway\u003csup\u003e\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. This enzyme is essential for the synthesis of the purine bases adenine and guanine, which are fundamental components of DNA and RNA. By impairing purine synthesis, MTX subsequently suppresses the synthesis of nucleic acids, thereby disrupting the rapid proliferation of cells such as immune cells. This disruption primarily targets the S phase of the cell cycle, which is responsible for DNA replication, ultimately hindering the growth and division of these rapidly dividing cell populations \u003csup\u003e\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eMethotrexate is administered through oral or parenteral route, such as intramuscular, intravenous, intrathecal, and subcutaneous. Orally, methotrexate is administered as a weekly single dose or divided doses every 8 hours over 24 hours. Generally, the peak plasma concentration is reached within 2 hours. The methotrexate was associated with various adverse effects such as nausea, vomiting, mucosal ulcers, anorexia, hepatotoxicity, and liver cirrhosis. Along with these, alopecia, fatigue, pancreatitis, bone marrow suppression, and renal failure are other side effects\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan additionalcitationids=\"CR30 CR31\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eTo our knowledge, this study represents the first demonstration of utilisation of modern drugs in its ultra-diluted form. The objective behind the preparation of the ultra-dilution of methotrexate is to give a new, usable and convenient homeopathic remedy that can be utilised to cure and/or alleviate the symptoms of aliments after individualisation, after undergoing preclinical and homeopathic pathogenic trials, potentially with minimal side effects. Another aim of this formulation is to provide a formulation that can be administered by oral route in psoriasis and other immune-related diseases. This study will give a cost-effective, easily accessible remedy with ease of use, great patient adherence, devoid/ negligible of side effects or rebound characteristics, and a complete cure for psoriasis. Also, will offer a new remedy to conduct Homeopathic Pathogenic Trials for researchers in the field of homeopathy. The conversion of synthetic drugs in its ultra-dilution will seed a new area of research about the homeopathy system of medicine and utilization of the rebound effect of synthetic drugs as pharmacological action, as per the homeopathic similitude principle for the field of pharmacology. The findings from this investigation hold particular significance, as the medications under examination do not exhibit toxic properties or elicit adverse reactions.\u003c/p\u003e\u003cp\u003eUltra-dilutions are generally considered safe and do not necessitate safety studies. However, empirical evidence is lacking to conclusively determine the safety of herbs, their components, or other medicinal sources, despite their long-standing use and inherent safety. Although methotrexate is not a novel molecule, the developed homeopathic formulation requires validation through preclinical toxicity studies to ensure its safety before being employed to treat specific disease conditions.\u003c/p\u003e\u003cp\u003eThis ethical obligation arises from the lack of comprehensive safety profile data for these homeopathic preparations to date, necessitating their evaluation in experimental rat models before considering further studies. When weighing the potential risks against the prospective advantages, it is imperative to conduct pre-clinical acute and sub-acute toxicity assessments of ultra-dilutions of methotrexate, particularly at the 12c and 30c potencies, because, as mentioned in literature, the nanoparticles remain at the extreme dilution in homeopathic medicines. Therefore, with consideration of the reduction in particle size and increase in surface area the toxicity might be increased. To date no homeopathic medicine formulated from a synthetic active pharmaceutical ingredient was prepared and assessed for its toxicity and efficacy in any disease by preclinical means. This study was conducted to evaluate the acute and subacute toxicity of the ultra-dilution of methotrexate 12c \u0026amp; 30c potencies prepared by homeopathic means as per the Organisation for Economic Cooperation and development (OECD) test guideline (with subtle alterations) in rats. The particular potencies are prepared and assessed since they were mentioned in the National List of Essential Ayush Medicine, 2022. In view of fact that most homeopathic remedies are administered as alcoholic liquid dilutions, the dosage in this study was quantified in microliters per kilogram (\u0026micro;L/kg) of b.w., rather than the conventional milligrams per kilogram (mg/kg) used in the OECD guidelines.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eChemicals and Drugs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAll the chemicals utilized for the study are of analytical standards. The methotrexate was procured from Globela Health Care Ltd., Surat, Gujarat. The 12c and 30c potencies of methotrexate were prepared as per the Homoeopathic Pharmacopoeia of India and were used for further experimentation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExperimental animals\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe Swiss albino mice of both sexes utilized in this procedure were authorized by the animal ethics committee of Trans-Genica Services Pvt. Ltd., Jalgaon, Maharashtra, India (Protocol Number:-TRS/PT/024/055A. The standard guidelines of Committee for Control and Supervision of Experiments on Animals (CCSEA), Government of India, were adapted for animal house like habitation, feeding, ventilation, lighting, sanitation, and management procedures. Through the experiment, the principle of 3Rs (replacement, reduction, and refinement) was also taken into consideration. The study protocol was conducted as per the Organization for Economic Cooperation and Development (OECD) guidelines; therefore, the substitution of animals with another substitute was not practicable.\u003c/p\u003e\u003cp\u003eHealthy, male and non-pregnant female mice weighing 20\u0026ndash;25 grams obtained from the Central Animal House facility of Trans-Genica Services Pvt. Ltd. During the acclimation period, the mice were maintained under standard laboratory conditions with a temperature of 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, relative humidity of 60\u0026thinsp;\u0026plusmn;\u0026thinsp;15%, and a natural light-dark cycle of 12 hours each. They were provided with a commercial pellet diet and water ad libitum. The animals were grouped and housed in polypropylene cages, with a maximum of four mice per cage. Randomization was achieved using a random number generator in Microsoft Excel, and all personnel involved in the experiment were appropriately trained and qualified.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExperimental Design\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ePreparation of methotrexate\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA standard procedure in Homeopathic Pharmacopoeia of India, Volume I was adapted for the preparation of methotrexate (MTX) 12c and 30c. Whole process of MTX 12c and 30c preparation was conducted in in-house pharmacy of Vidhyadeep Homoeopathic Medical College and Research Centre, Suart, India as per the homoeopathic pharmacopoeia of India\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Concisely, the homeopathic formulation of methotrexate underwent a meticulous preparation process in compliance with the legal requirements for homeopathic remedies. The centesimal (C) preparation involved a 100-fold dilution of methotrexate. The trituration (potentization of solid components) was conducted manually for 60 minutes with 50\u0026ndash;55 rpm, adhering to the guidelines outlined in the Homeopathic Pharmacopeia of India Volume I. The procedure entailed maintaining a c-scale by combining 1 part of the crude drug with 99 parts of milk sugar. Specifically, 3 grams of methotrexate were triturated with 297 grams of milk sugar using a mortar and pestle. For the preparation of the 1c potency and subsequent trituration, 3 grams of methotrexate were mixed with 33 grams of milk sugar (11 parts by weight) in a mortar and pestle, and the blend was thoroughly rubbed for six minutes, followed by scraping. The mixture was then stirred using a pestle for six minutes and stirred again for four minutes. To this mixture, 99 grams (33 parts by weight) of milk sugar were added, and the above procedure was repeated twice. Finally, 165 grams (55 parts by weight) of milk sugar were added to the mixture, and the rubbing and stirring were repeated twice. At the end of this process, the 1c potency of methotrexate was obtained. The entire trituration procedure was repeated for the production of the 2c and 3c triturations of methotrexate. For the conversion of trituration into liquid potencies, the 3c trituration proceeded for the preparation of the liquid potency of methotrexate, following the procedure mentioned in the Homeopathic Pharmacopeia of India Volume I. For methotrexate, the liquid potentization was initiated by dissolving 10 grams (1 part) of the methotrexate 3c trituration in 500 ml of a solvent containing 495 ml (50 parts by volume) of distilled water and 500 ml of dispensing alcohol in 1000 ml Erlenmeyer flask. This liquid was subjected to high-intensity manual 10 succussion. The resulting potency was 4c, which served as the jumping potency for methotrexate and was used for further potentization. To prepare the 5c potency, 1 ml of the 4c potency was combined with 49.5 ml of distilled water and dilute alcohol, each, followed by 10 succussions. All subsequent liquid potencies up to 12c and 30c were prepared by repeating the procedure used for the 5c preparation and the volume of subsequent potency addition was selected according the final volume required. All the procedure were conducted in laminar air flow and all the apparatus used to prepare the medicine were used strictly after sterilization.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDose volume calculation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor the acute toxicity study, all mice received doses of 2000 \u0026micro;l/kg of MTX 12c and 30c. Toxic manifestations were critically observed during the initial 30 minutes, followed by periodic observations every 2 hours for 24 hours, with heightened vigilance during the first 4 hours. Subsequently, the animals were monitored for a total duration of 14 days. The sub-acute toxicity study adhered to OECD 407 guidelines with minor modifications. A daily oral dose of 200 \u0026micro;l/kg body weight, diluted with distilled water in a 1:9 ratio, was administered\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eAcute toxicity study\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe acute toxicity study adhered to the OECD 423 guidelines with minor modification and healthy non pregnant, nulliparous female mice were used. Prior to dose administration, all animals were devoid to use of food but for water for 4 hours. The drug and vehicle were orally administered at a single dose of 2000 \u0026micro;l/kg body weight on day one\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Following administration, water was discontinued for next two hours. The treatment groups were as follows:\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTreatment\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNormal control (NC)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDistilled water\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVehicle control (VC)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDispensing alcohol (90%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTest group 1 (T1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMethotrexate 12c\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTest group 2 (T2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMethotrexate 30 c\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eAnimals were critically observed for any sign\u0026rsquo;s mortality, moribund status and intoxication signs at 0 minutes (before dosing), 30 (\u0026plusmn;\u0026thinsp;5) minutes, 1 hour (\u0026plusmn;\u0026thinsp;10 minutes), followed by up to 24 hours post-dose administration. Body weight, food, and water intake were monitored. Body weights were recorded on days 1, 7, and 14 of the study. Observations included skin surface and fur condition, salivation, lacrimation, piloerection, eye/light reflex, lethargy, dehydration, nasal discharge, central nervous system effects like tremors and convulsions, and changes in gait, posture, and walking behavior. On day 14, all animals were sacrificed via excessive CO2 inhalation, and gross pathological alterations in skin folds, natural external orifices, outer organ and tissue surfaces of the thoracic, neck, abdominal, pelvic cavities, and remaining carcass were examined for abnormalities\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSub-acute oral toxicity study\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThis study was conducted as per the OECD 407 guidelines from 2008 with slight modification. Mice were randomly assigned to five groups, each consisting of 20 animals (10 males and 10 females). The grouping of animals was consistent with the acute study\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAll treatments were administered orally as a single dose for a duration of 28 days. The mice were monitored daily for signs of intoxication, mortality, and comorbid conditions throughout the 28 days. On the 29th day, half of the animals in every group were left untreated and observed until the recovery period of 42 days after the 28-day treatment. This group served as the satellite group. All animals under study were inspected daily within their cages for general signs of toxicity and/or mortality, and the findings were recorded. The body weight of the animals was once a week, and the mean body weight was calculated.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn-life observation\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eUrine analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBefore the conclusion of the study, all subjects were individually housed in metabolic cages for 24 hours to facilitate the collection of urine samples. These samples underwent comprehensive physical, chemical, and microscopic analyses. The physical examination evaluated urine color, transparency, specific gravity, and pH levels. Chemical testing determined the presence of albumin, sugar, acetone, bile salts, and bile pigments (Acone, India). Microscopic examination involved the identification of pus cells, red blood cells, epithelial cells, crystals, amorphous phosphates, casts, bacteria, mucus, yeast, trichomonads, and spermatozoa.\u003c/p\u003e\u003cp\u003e\u003cb\u003eHematology and Clinical Biochemistry\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBefore blood sample collection, the animals were fasted for 4 hours while maintaining free access to water. On the 29th day, blood aliquots were obtained via the retroorbital plexus. For hematological analysis, blood samples were collected in K2-EDTA tubes and utilized for the examination of hemoglobin (Hb) and red blood cell (RBC) indices using an automatic cell counter (ABX MICROS60, Horiba). Parameters assessed included: hemoglobin, total red blood cells, packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and total white blood cells (WBC). For clinical chemistry analysis, blood samples were collected in 5 ml clot activator tubes, incubated for 15\u0026ndash;20 minutes at room temperature, and centrifuged at 2400 rpm for 10 minutes using an Electra Lab centrifuge (Euro diagnostic system Pvt. Ltd, Chennai, India) to obtain serum. The serum was utilized to evaluate the following parameters: reticulocyte count, total cholesterol, serum triglycerides, high-density lipoprotein (HDL), low-density lipoprotein (LDL), blood urea nitrogen (BUN), urea, serum creatinine, serum calcium, serum glutamic oxaloacetic transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), serum bilirubin (total and direct), serum alkaline phosphatase (ALP), serum proteins (total proteins, albumin, A:G ratio), and plasma blood glucose (GLU). These analyses were performed using a Turbochem-100 analyzer (Awareness Technology, USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003ePathology\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eNecropsy\u003c/b\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe mice were fasted overnight before necropsy and euthanized by inhalation of carbon dioxide followed by exsanguination. A complete autopsy of the carcass was performed, and observations were recorded.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eOrgan weights\u003c/b\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe organs in which biodistribution of MTX is occurring were considered for histopathological examination. The Brain, Heart, Spleen, Kidney, Stomach, Liver, Lungs, Ovaries, and Testis were collected, quickly blotted, and trimmed for extraneous fat, and the absolute weight of organs was recorded immediately using an electronic balance.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eHistopathological evaluation\u003c/b\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAll the isolated organs were fixed in 10% neutral buffered formalin for 24 hours, embedded in paraffin. Embedded tissue samples were cut into 2\u0026ndash;4 \u0026micro;m sections and stained (Tissue embedding station, Model No.: -MTC-TE-P-5Ltr) with hematoxylin-eosin (H \u0026amp; E), and sections were observed under a light microscope (Nikon ECLIPSE E200) and microanatomical changes captured (MIchrome 6, 6MP Color Microscope Camera).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eThe data is a representation of mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error mean (SEM). Whole data is analyzed by one analysis of variance followed by Dunner\u0026rsquo;s post hoc analysis using SPSS 25 software. Statistical significance was defined as a value of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eIn vivo\u003c/strong\u003e \u003cstrong\u003etoxicity study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcute oral toxicity study (OECD 423)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the animals were observed carefully and critically for any signs and symptoms of toxicity at regular time lapses of 0,30 min, 1,2,4,6,8,12 hours, and then daily for up to 14 days. Mild lethargy and shivering were observed in vehicle control, and MTX (12c \u0026amp; 30c) was administered to animals. The abdominal breathing, vocalization, and dyspnoea were observed in all the animals, irrespective of treatment. There were no treatment-related alterations detected in food consumption, mean body weight, and weight gain in comparison to normal control animals during the study period of 14 days. No moribund state or mortality was detected in any treatment group. Additionally, no abnormalities were found in the gross necropsy among the groups Table 1. These results show that the Median lethal dose (LD50) of MTX 12c \u0026amp; 30c on single administration is under category 5 or unclassified as per Globally Harmonized Systems and higher than 2000\u0026micro;l/kg Body weight\u003csup\u003e35\u003c/sup\u003e.\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 1\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eCage side observation in single dose acute toxicity study of MTX 23c and 30c in mice\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eIntoxication signs/parameters\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eGroups\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNormal control\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eVehicle control (sac. Lac.)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMTX 12c\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMTX 30c\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSalivation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLacrimation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePiloerection\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRespiration\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEyes/ Light reflexes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFur\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLethargy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e09 min/12 hrs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10 min/12 hrs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e09 min/12 hrs\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eShivering\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 min/30 min\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 min/30 min\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 min/30 min\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDyspnoea\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNil\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNil\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNil\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNil\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTremors\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eConvulsions\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNasal discharge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMotor activities\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGait\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePosture\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWalking pattern\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAD\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFood consumption\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWater consumption\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMortality\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eSubacute oral toxicity study (OECD 407)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA 43-day subacute oral toxicity study was conducted in two study phases, including 28 days of the main study and 43 days of the recovery study with MTX 12c and 30c at a dose of 200\u0026micro;l/kg/day. No signs of intoxication, moribund animal, or mortality were observed among the animals in each group at both study phases. Additionally, no alterations in food and water consumption, behavior patterns, or urinary and fecal excretion were observed in any animal throughout the study period\u003csup\u003e36\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical changes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTreatment-associated mortality was not found in any group of animals after regular administration of MTX 12c \u0026amp; 30c. Mice of both sexes did not show any noticeable detrimental signs such as alterations in skin, fur, eyes/ light reflexes, motor and nervous abnormalities, mucus membranes, and natural orifices. Moreover, no behavioral changes were observed inclusive of salivation, lacrimation, piloerection, coma, lethargy, sleep, and diarrhoea throughout the study period in both sexes compared to normal control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBody weight\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll treatment groups\u0026apos; mean body weight changes were recorded weekly for up to 43 days. The gradual rise in mean body weight in all mice of both sexes in control and treatment groups was observed. There were no significant changes found in the mean body weight of animals in the normal control group and treatment group from the onset to the end of the study period. Figures 1 and 2 represent the absolute body weight of females and males respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUrine analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv\u003e\n \u003cp\u003eNo statistically significant differences (p\u0026thinsp;\u0026le;\u0026thinsp;0.05) were found in pH and the specific gravity of urine Table 2. The urine analysis results did not show any treatment-related changes in other urine parameters. There were no shreds of evidence of glycosuria, proteinuria, bile salt, or acetone observed in the chemical examination of urine in treated groups compared to controls. The microscopic examination of urine did not show the presence of pus cells, R.B.C., epithelial cells, crystals, amorphous phosphate, bacteria, mucus, yeast, trichomonas, and spermatozoa in all experimental animals at the end of the study.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 2\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eUrine analysis of females and males for toxicity study of MTX 12c and 30c\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eSex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eParameters\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eTreatment Groups\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNormal control\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVehicle control\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMTX 12c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMTX 30c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eFemale\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003epH\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.004\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.003\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.004\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.004\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSpecific gravity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eMale\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003epH\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.005\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.004\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.005\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.005\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpecific gravity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cem\u003eThe data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. One-way ANNOVA was used with Dunnett\u0026rsquo;s post-hock test with significance p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. No significant difference is detected between the treatment and control group.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHaematological indices\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv\u003e\n \u003cp\u003eThe hematological indices measured in female and male mice are mentioned in Table 3. There was no significant (p\u0026thinsp;\u0026le;\u0026thinsp;0.05) change found in the red blood cell indices, hemoglobin content, as well as total and differential leucocyte counts, of drug-treated mice in comparison to vehicle control animals.\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eBiochemical investigations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe biochemical parameters were analysed at the end of the study period. Statistically, no significant alterations were found in all biochemical parameters of animals treated with MTX in comparison to the control group. All the biochemical parameters are mentioned in Table 4.\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 3\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eErythrogram, leukogram and platelet count of female and male mice for toxicity study of MTX 12c and 30c\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eHaematological indices\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNormal control\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eVehicle control\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMTX 12c\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMTX 30c\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNormal control\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eVehicle control\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMTX 12c\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMTX 30c\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHaemoglobin(Gm/dl)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e15.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e14. 10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e15.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e15.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTotal RBC (\u0026times;10\u003csup\u003e6\u003c/sup\u003e/\u0026micro;l)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.31\u0026thinsp;\u0026plusmn;\u0026thinsp;1.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.28\u0026thinsp;\u0026plusmn;\u0026thinsp;1.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.61\u0026thinsp;\u0026plusmn;\u0026thinsp;1.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.65\u0026thinsp;\u0026plusmn;\u0026thinsp;1.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.56\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.68\u0026thinsp;\u0026plusmn;\u0026thinsp;1.39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM.C.V. (Fl)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e54.71\u0026thinsp;\u0026plusmn;\u0026thinsp;4.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e54.62\u0026thinsp;\u0026plusmn;\u0026thinsp;5.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e53.73\u0026thinsp;\u0026plusmn;\u0026thinsp;4.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e53.67\u0026thinsp;\u0026plusmn;\u0026thinsp;4.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e56.22\u0026thinsp;\u0026plusmn;\u0026thinsp;6.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e57.57\u0026thinsp;\u0026plusmn;\u0026thinsp;6.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e58.36\u0026thinsp;\u0026plusmn;\u0026thinsp;7.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e57.48\u0026thinsp;\u0026plusmn;\u0026thinsp;6.31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM.C.H.(Pg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17.12\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e15.19\u0026thinsp;\u0026plusmn;\u0026thinsp;2.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17.16\u0026thinsp;\u0026plusmn;\u0026thinsp;2.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16.18\u0026thinsp;\u0026plusmn;\u0026thinsp;1.22\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM.C.H.C. (gm/dL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32.16\u0026thinsp;\u0026plusmn;\u0026thinsp;1.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32.04\u0026thinsp;\u0026plusmn;\u0026thinsp;1.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32.45\u0026thinsp;\u0026plusmn;\u0026thinsp;2.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTOTAL WBC (\u0026times;10\u003csup\u003e3\u003c/sup\u003e/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.19\u0026thinsp;\u0026plusmn;\u0026thinsp;1.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEosinophils (\u0026times;10\u003csup\u003e2\u003c/sup\u003e/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.93\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNeutrophils (\u0026times;10\u003csup\u003e2\u003c/sup\u003e/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLymphocytes (\u0026times;10\u003csup\u003e2\u003c/sup\u003e/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMonocytes (\u0026times;10\u003csup\u003e2\u003c/sup\u003e/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePlatelet (\u0026times;10\u003csup\u003e3\u003c/sup\u003e/\u0026micro;l)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.635\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.713\u0026thinsp;\u0026plusmn;\u0026thinsp;0.078\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.065\u0026thinsp;\u0026plusmn;\u0026thinsp;0.073\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.069\u0026thinsp;\u0026plusmn;\u0026thinsp;0.067\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.413\u0026thinsp;\u0026plusmn;\u0026thinsp;0.042\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.422\u0026thinsp;\u0026plusmn;\u0026thinsp;0.052\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.412\u0026thinsp;\u0026plusmn;\u0026thinsp;0.049\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.428\u0026thinsp;\u0026plusmn;\u0026thinsp;0.056\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eThe data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. One-way ANNOVA was used with Dunnet\u0026rsquo;s post-hock test with significance p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. No significant difference is detected between the treatment and control group. Abbreviations: Hb- haemoglobin, RBC- red blood cells, M.C.V.- Mean corpuscular volume, M.C.H.- mean corpuscular haemoglobin, M.C.H.C.- Mean corpuscular haemoglobin concentration.\u003c/p\u003e\n\u003cdiv\u003e\u003cbr\u003e\u003c/div\u003e\n\u003cdiv\u003e\n \u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 5\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eOrgan weight of female and male mice for toxicity study of MTX 12c and 30c\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eOrgan\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eTreatment group (Female)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eTreatment group (Male)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNormal control\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eVehicle control\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMTX 12c\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMTX 30c\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNormal control\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eVehicle control\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMTX 12c\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMTX 30c\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBrain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHeart\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpleen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKidney\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStomach\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLiver\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.21\u0026thinsp;\u0026plusmn;\u0026thinsp;1.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.42\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.98\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.53\u0026thinsp;\u0026plusmn;\u0026thinsp;2.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLungs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOvaries(F)/Testes(M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.98\u0026thinsp;\u0026plusmn;\u0026thinsp;2.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.01\u0026thinsp;\u0026plusmn;\u0026thinsp;2.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.74\u0026thinsp;\u0026plusmn;\u0026thinsp;2.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cem\u003eThe data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. One-way ANNOVA was used with Dunnet\u0026rsquo;s post-hock test with significance p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNecropsy\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv\u003e\n \u003cp\u003eNo gross pathological changes were observed in any group of animals. MTX (12c \u0026amp; 30c) treated animals did not show any pathological abnormalities in all the organs in comparison to control animals\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eOrgan weight\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv\u003e\n \u003cp\u003eThe Brain, Heart, Spleen, Kidney, Stomach, Liver, Lungs, Ovaries, and Testis were subjected to organ weight calculation as these are the sites of distribution of methotrexate. The absolute organ weight was determined at the time of necropsy and mentioned in the table. There was no significant change in absolute organ weight in the drug-treated group of all potencies in comparison to control group mice. Organ weights of females and males are shown in Table 5.\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eHistopathology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA microscopic examination of the brain, lungs, liver, kidneys, stomach, spleen, heart, testes, epididymis, ovaries, and uterus was performed on all treatment groups in both sexes. There were no microanatomical alterations found in photomicrographs of all the organs of females and males. Figures 3, 4, and 5 illustrate the histological images of organs from female and male animals.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe ultra-high dilution of methotrexate was prepared as per the homeopathic pharmacopeia of India, Volume I, having potency 12 centesimal (12c) and 30 centesimal (30c). The acute toxicity study of MTX 12c and 30c was conducted in mice as per the OECD 423. 2002 guideline with slight modification. The mice are used to perform the toxicity study as they will be the intended species for further studies. OECD 423 is utilized to evaluate the LD50 range of MTX ultra-high diluted potencies. The MTX 12c and 30c were administered as a single dose of 2000 \u0026micro;l/kg on the 1st day and were critical signs of toxicity for 14 days. During the study period of up to 24 hours, mild lethargy and shivering were noticed in treatment and vehicle-treated animals. The abdominal breathing, vocalization, and dyspnoea were noticed in animals for the first few minutes. There was no treatment-associated mortality noticed in all animals following the treatment with MTX 12c and 30c. As per the literature survey, a 20% decrease in body weight will be the end point for the study, which will further give an idea about the food intake of animals. The body weight was found to be gradually increased similarly compared to the normal control animals throughout the study \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Concerning in-life, or cage-side observations, no alterations were observed that can relate to the treatment. Moreover, no atypical changes were identified in either the outer body surface or internal organs. In the acute toxicity study, the animals were administered nearly 2.5 times a weekly maximum dose of MTX (25 mg/week MTX for a maximum 24 weeks) used clinically, i.e., 2000 \u0026micro;l/kg body weight, for investigation of detrimental effects due to the ultra-dilution of methotrexate. These results also support the rebuff of the claim that the toxicity of methotrexate might be elevated due to an increase in surface area in the preparation method.\u003c/p\u003e\u003cp\u003eThe repeated dose 28-day toxicity study was conducted in mice of both sexes as per the OECD 407, with minor alterations, to evaluate the repeated treatment consequences of MTX dilutions on possible toxicity. In this study, the effect of MTX (12c \u0026amp; 30c) was assessed following repeated administration. The body weight, food consumption, hematology, blood biochemistry, urine analysis, organ weight, and histopathology were used as confirmation markers. There are no treatment-associated alterations noticed in behavior as well, and clinical signs viz, gait, fur color, piloerection, salivation, lacrimation, eating, sleep, urine-\u0026ndash;feces excretions, as well as autonomic activities, were found throughout the study period. As per the OECD guideline 20% reduction in body weight is contemplated as one of the endpoints in human study\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Consumption of food, food palatability, food nutritional content, digestion, and absorption are the important factors affecting body weight.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. The body weight and food consumption were moving steadily throughout the study period in treatment and vehicle-treated groups.\u003c/p\u003e\u003cp\u003eBlood indices are the most essential diagnostic parameters in clinical practice to assess the toxic effects of drugs. Furthermore, the hematological parameters alterations in rodents were found to be prognostic of toxicity in humans, as supported by the data obtained from the animal trials\u003csup\u003e\u003cspan additionalcitationids=\"CR41 CR42\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. As the bone marrow is highly sensitive to toxic drugs, the hematological indices are crucial in monitoring the functioning of bone marrow and the intravascular network\u003csup\u003e\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. The treatment-related alterations in blood indices were not found in this study compared to the control. The non-significant alterations in blood indices demonstrate the non-toxic nature of MTX to blood indices.\u003c/p\u003e\u003cp\u003eSerum biochemistry is another essential consideration in evaluating the toxic nature of the test substance. Serum biochemistry, such as liver function tests and kidney function tests, is essential in evaluating the functioning of the liver and kidneys as they are important for survival, metabolizing, and detoxifying the majority of chemicals and drugs. (olo, ekanay). The liver is the prime organ associated with drug metabolism, cholesterol biosynthesis, and degradation; even MTX is metabolized by the liver. Additionally, it also assists in glucose synthesis and the production of glucose from glycogen storage\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. The outputs of this study revealed that MTX ultra-dilution did not have an effect on the metabolism of lipids and carbohydrates. The liver function test was used to assess the toxicity of MTX potencies on liver functionality. The increased serum concentration of SGOT, SGPT, BUN, and ALP is indicative of liver injury, cirrhosis, and myocardial infarction\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. No alterations in the aforementioned parameters were observed in the present study when the treatment group was compared to control group animals, which is an indication of the nontoxic effect of MTX 12c and 30c on the liver. Total bilirubin is a byproduct of hem catabolism and an indicator of liver function and cholestasis. Also, it reduces aplastic anemia\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. There were no significant changes observed in total bilirubin and BUN in MTX 12c and 30c-treated animals compared to control animals\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Furthermore, various studies have revealed the interconnection between the amount of urea and creatinine with the glomerular filtration rate, which is the prime indicator of kidney function\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. MTX 12c and 30c treatment did not change the urea and creatinine concentration compared to control. These results imply that the MTX 12c and 30c did not show any effect on liver and kidney functions.\u003c/p\u003e\u003cp\u003eToxicity leads to alteration in color and architecture, hypertrophy, and edema of the organs when the drug is distributed\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. The macroscopic assessment of all vital organs during necropsy of MTX 12c and 230c treated animals in both acute and repeated dose toxicity studies did not show any lesion, hypertrophy, color, or texture alteration compared with control animals. Moreover, absolute organ weight is the basic indicator for assessing any treatment-related injury in internal organs\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. There were no significant changes in body weight of MTX 12c and 30c-treated animals compared to the control. from the obtained study results, the MTX 12c and 30c with vehicle neither impeded physiology nor produced pathological alterations in animals.\u003c/p\u003e\u003cp\u003eThe toxic substances directly impact the vital organs of the body and lead to alterations in appearance, morphology, an increase in organ size, and edema. The microscopical scrutiny of brain, lungs, liver, kidneys, stomach, spleen, heart, testes, epididymis, ovaries, and uterus did not show any histopathological alterations following the 28 days of repeated dose of MTX at both dilutions. This data is indicative of repeated administration of MTX at dilutions 12c and 30c, nontoxic.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe outcome of acute and repeated dose toxicity studies indicates that no mortality was observed at a dose of 2000\u0026micro;l/kg of MTX 12c and 30c, which stipulates that the oral LD50 of MTX 12c and 3c is higher than the administered dose. Repeated administration of MTX 12 c and 30c at a dose of 200 \u0026micro;l/kg p.o. was found safe in mice. No observed Adverse Effect Level (NOAEL) of MTX 12c and 30c after 20 consecutive days or oral administration in mice was found to be not less than 200 \u0026micro;l/kg/day. Additionally, upon the repeated administration of MTX 12c and 30c, no organ-specific toxicity was observed. Moreover, no treatment-associated microanatomical alterations were observed in either sex at 200 \u0026micro;l/kg/day of MTX 12c and 30c. therefore, it may be concluded that there are no detrimental properties of ultra-high diluted preparation of MTX at potency 12c and 30c, and it was found safe at a daily dose of 200 \u0026micro;l/kg/day p.m.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval:\u003c/strong\u003e The study protocol was reviewed and approved by the animal ethics committee of Trans Services Genica Pvt. Ltd, Maharashtra with approval number -TRS/PT/024/055A\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eConsent to participate:\u003c/strong\u003e Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publication:\u003c/strong\u003e Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number:\u003c/strong\u003e Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e No funding received for this work\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthor contribution declaration:\u003c/strong\u003e PG: Formal writing, data collection, statistics, RK: Supervision \u0026amp; manuscript finalising, PG: Drug sample preparation, formal writing, data acquisition\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLebel Y, Milo T, Bar A, Mayo A, Alon U. 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Indian Journal of Research in Homoeopathy. 2017;11(1):48. \u003c/li\u003e\n\u003cli\u003eGoswami A, GV NK, Saka VP, Gupta P, Biswas B, Verma D. Safety of potentised dilutions of Rhus toxicodendron in Wistar albino rats. Indian Journal of Research in Homoeopathy. 2024 Mar 28;18(1):3\u0026ndash;14. \u003c/li\u003e\n\u003cli\u003eLal R, Sharma M, Behera S, Regar RK, Tripathi D, Kumar GVN, et al. Safety Evaluation of Arsenicum album in Acute and Sub-Acute Toxicity Studies in Rats. Toxicol Int. 2023 May 23;233\u0026ndash;47. \u003c/li\u003e\n\u003cli\u003ePrakash P, Gupta N. Therapeutic uses of Ocimum sanctum Linn (Tulsi) with a note on eugenol and its pharmacological actions: a short review. Indian J Physiol Pharmacol. 2005 Apr;49(2):125\u0026ndash;31. \u003c/li\u003e\n\u003cli\u003eTeixeira MZ. \u0026ldquo;Similia Similibus Curentur\u0026rdquo;: The scientific grounding of the homeopathic therapeutic principle through the systematic study of the rebound effect of modern drugs. 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International Journal of High Dilution Research - ISSN 1982-6206. 2021 Dec 21;10(37):338\u0026ndash;52. \u003c/li\u003e\n\u003cli\u003eTeixeira MZ. \u0026lsquo;Paradoxical strategy for treating chronic diseases\u0026rsquo;: a therapeutic model used in homeopathy for more than two centuries. Homeopathy. 2005 Oct;94(4):265\u0026ndash;6. \u003c/li\u003e\n\u003cli\u003eTeixeira MZ. Homeopathic use of modern medicines: utilisation of the curative rebound effect. Med Hypotheses. 2003 Feb;60(2):276\u0026ndash;83. \u003c/li\u003e\n\u003cli\u003eWeinstein GD. Methotrexate. Arch Dermatol. 1971 Sep 1;104(3):236. \u003c/li\u003e\n\u003cli\u003eGubner RS. Introduction of antifolics in psoriasis. A twenty-five year retrospect of antineoplastic agents in nonmalignant disease. Cutis. 1979 Apr;23(4):425\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003eChl\u0026aacute;dek J, Simkov\u0026aacute; M, Vaneckov\u0026aacute; J, Hroch M, Chl\u0026aacute;dkova J, Mart\u0026iacute;nkov\u0026aacute; J, et al. The effect of folic acid supplementation on the pharmacokinetics and pharmacodynamics of oral methotrexate during the remission-induction period of treatment for moderate-to-severe plaque psoriasis. Eur J Clin Pharmacol. 2008 Apr 29;64(4):347\u0026ndash;55. \u003c/li\u003e\n\u003cli\u003eUdawant S, Zilate S. Therapeutic Role of Methotrexate in Effective Treatment of Psoriasis: A Review. J Res Med Dent Sci [Internet]. 2022;10(8):25. Available from: www.jrmds.in\u003c/li\u003e\n\u003cli\u003eLaw JH, Koo B, Koo JYM. Methotrexate Update: Mechanism of Action in Psoriasis Therapy. Psoriasis Forum. 2008 Jun 30;14a(1):17\u0026ndash;28. \u003c/li\u003e\n\u003cli\u003eCipriani P, Ruscitti P, Carubbi F, Liakouli V, Giacomelli R. Methotrexate: an old new drug in autoimmune disease. Expert Rev Clin Immunol. 2014 Nov 22;10(11):1519\u0026ndash;30. \u003c/li\u003e\n\u003cli\u003eY\u0026eacute;lamos O, Puig L. Systemic methotrexate for the treatment of psoriasis. Expert Rev Clin Immunol. 2015 May 4;11(5):553\u0026ndash;63. \u003c/li\u003e\n\u003cli\u003eBaggott JE, Morgan SL. Methotrexate catabolism to 7‐hydroxymethotrexate in rheumatoid arthritis alters drug efficacy and retention and is reduced by folic acid supplementation. Arthritis Rheum. 2009 Aug 30;60(8):2257\u0026ndash;61. \u003c/li\u003e\n\u003cli\u003eShen S, O\u0026rsquo;Brien T, Yap LM, Prince HM, McCormack CJ. The use of methotrexate in dermatology: a review. Australasian Journal of Dermatology. 2012 Feb 29;53(1):1\u0026ndash;18. \u003c/li\u003e\n\u003cli\u003eDogra S, Mahajan R. Systemic methotrexate therapy for psoriasis: past, present and future. Clin Exp Dermatol. 2013 Aug;38(6):573\u0026ndash;88. \u003c/li\u003e\n\u003cli\u003eHOMOEOPATHIC PHARMACOPOEIA OF INDIA Preface to E-book Combined Volume-I st to IX th (Revised \u0026amp; Augmented). \u003c/li\u003e\n\u003cli\u003eSharma M, Behera S, Regar RK, Tripathi D, Singh S, Kumar NGV, et al. Acute and Sub-acute Oral Toxicity Assessment of Ferrum phosphoricum in Rats. Homeopathy. 2024 May 21;113(02):086\u0026ndash;97. \u003c/li\u003e\n\u003cli\u003eOecd. OECD/OCDE 423 OECD GUIDELINE FOR TESTING OF CHEMICALS Acute Oral Toxicity-Acute Toxic Class Method INTRODUCTION. 2001. \u003c/li\u003e\n\u003cli\u003eOecd. OECD/OCDE 407 OECD GUIDELINES FOR THE TESTING OF CHEMICALS Repeated Dose 28-Day Oral Toxicity Study in Rodents INTRODUCTION. \u003c/li\u003e\n\u003cli\u003eWren AF, Cleary M, Frantz C, Melton S, Norris L. 90-Day Oral Toxicity Study of a Grape Seed Extract (IH636) in Rats. J Agric Food Chem. 2002 Mar 1;50(7):2180\u0026ndash;92. \u003c/li\u003e\n\u003cli\u003eChitra B, Ramaswamy RS, Suba V. Toxicity Evaluation of Pũrṇa Cantirotaya Centũram, a Siddha Medicine in Wistar Rats. Int Sch Res Notices. 2015 Jan 26;2015:1\u0026ndash;10. \u003c/li\u003e\n\u003cli\u003eLatt R, Macallum E, Canada Inc WL. CCAC guidelines on: choosing an appropriate endpoint in experiments using animals for research, teaching and testing. 1998. \u003c/li\u003e\n\u003cli\u003eSahota T, Danhof M, Della Pasqua O. Pharmacology-based toxicity assessment: towards quantitative risk prediction in humans. Mutagenesis. 2016 May;31(3):359\u0026ndash;74. \u003c/li\u003e\n\u003cli\u003eOlson H, Betton G, Robinson D, Thomas K, Monro A, Kolaja G, et al. Concordance of the Toxicity of Pharmaceuticals in Humans and in Animals. Regulatory Toxicology and Pharmacology. 2000 Aug;32(1):56\u0026ndash;67. \u003c/li\u003e\n\u003cli\u003eMukinda JT, Syce JA. Acute and chronic toxicity of the aqueous extract of Artemisia afra in rodents. J Ethnopharmacol. 2007 May;112(1):138\u0026ndash;44. \u003c/li\u003e\n\u003cli\u003eRao JV. Toxic effects of novel organophosphorus insecticide (RPR-V) on certain biochemical parameters of euryhaline fish, Oreochromis mossambicus. Pestic Biochem Physiol. 2006 Oct;86(2):78\u0026ndash;84. \u003c/li\u003e\n\u003cli\u003eAnderson N, Borlak J. Molecular Mechanisms and Therapeutic Targets in Steatosis and Steatohepatitis. Pharmacol Rev. 2008 Sep;60(3):311\u0026ndash;57. \u003c/li\u003e\n\u003cli\u003eGuleria M, Pallavi KJ, Gujarathi PP, Das T. Evaluation of acute intravenous toxicity of HEPES: Is Good\u0026rsquo;s buffer good and safe enough for clinical utilization in nuclear medicine? Nucl Med Biol. 2024 May;132\u0026ndash;133:108895. \u003c/li\u003e\n\u003cli\u003eRamaiah SK. Preclinical Safety Assessment: Current Gaps, Challenges, and Approaches in Identifying Translatable Biomarkers of Drug-Induced Liver Injury. Clin Lab Med. 2011 Mar;31(1):161\u0026ndash;72. \u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 4","content":"\u003cp\u003eTable 4 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"naunyn-schmiedebergs-archives-of-pharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nsap","sideBox":"Learn more about [Naunyn-Schmiedeberg's Archives of Pharmacology](https://www.springer.com/journal/210)","snPcode":"210","submissionUrl":"https://submission.nature.com/new-submission/210/3","title":"Naunyn-Schmiedeberg's Archives of Pharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Methotrexate ultra-dilution, secondary effect, rebound effect, Acute toxicity study, Subacute toxicity study","lastPublishedDoi":"10.21203/rs.3.rs-7098879/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7098879/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e\u003cp\u003eAutoimmune 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.\u003c/p\u003e\u003ch2\u003eMethod\u003c/h2\u003e\u003cp\u003eAn acute toxicity study evaluated single-dose effects of Methotrexate 12c and 30c (2,000 \u0026micro;L/kg) in female mice over 14 days, per OECD 423 guidelines. A repeated dose study investigated impacts of Methotrexate 12c and 30c (200 \u0026micro;L/kg) on male and female mice, following OECD 407 protocol. Mortality, clinical signs, body weight, hematology, biochemistry, and histopathology were comprehensively assessed.\u003c/p\u003e\u003ch2\u003eResult\u003c/h2\u003e\u003cp\u003eThe acute toxicity study showed no animal fatalities with methotrexate, indicating the median lethal dose exceeded 2000 \u0026micro;L/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.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThe research study indicates that the oral administration of Methotrexate at 12c and 30c to mice over an extended duration, with a dosage of 200 \u0026micro;L/kg, did not exhibit any signs of toxicity or adverse reactions, thus suggesting a safe profile for these potencies.\u003c/p\u003e","manuscriptTitle":"Toxicity evaluation of ultra-diluted Methotrexate (12c\u0026amp;30c): An acute and subacute Oral toxicity study in animals","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-31 16:40:19","doi":"10.21203/rs.3.rs-7098879/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-08T12:17:22+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-24T07:00:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"257910104301062025617198891282870263474","date":"2025-08-03T08:43:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-29T07:10:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-25T01:06:08+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-25T01:05:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Naunyn-Schmiedeberg's Archives of Pharmacology","date":"2025-07-11T07:17:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"naunyn-schmiedebergs-archives-of-pharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nsap","sideBox":"Learn more about [Naunyn-Schmiedeberg's Archives of Pharmacology](https://www.springer.com/journal/210)","snPcode":"210","submissionUrl":"https://submission.nature.com/new-submission/210/3","title":"Naunyn-Schmiedeberg's Archives of Pharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8813b6ef-54dd-46a2-befd-184aed447efd","owner":[],"postedDate":"July 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-09T06:55:24+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-31 16:40:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7098879","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7098879","identity":"rs-7098879","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}