In vivo Acute Oral Toxicity Assessment of Novel Histone Deacetylase 2 Inhibitor

In vivo Acute Oral Toxicity Assessment of Novel Histone Deacetylase 2 Inhibitor | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article In vivo Acute Oral Toxicity Assessment of Novel Histone Deacetylase 2 Inhibitor Padmini Pai, Rachel Savio D’Mello, Shruthi Nayak, Pallavi Rao, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6988823/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Nov, 2025 Read the published version in BMC Pharmacology and Toxicology → Version 1 posted 21 You are reading this latest preprint version Abstract Histone deacetylases (HDACs) are enzymes responsible for removing acetyl groups from histone proteins, resulting in chromatin condensation and repression of genes. They regulate expression of genes, cell cycle, and multiple cellular processes. Hydroxamic acid is a well recognized moiety characterized by its potent zinc-binding capability, making it an effective inhibitor of HDACs. A novel hydroxamic acid based molecule, N 1 -(2,2'-bipyridin-6-yl)-N 8 -hydroxyoctanediamide (compound 3B) was previously synthesized and the anticancer properties of the compound were examined in vitro in our laboratory. No prior toxicological study has been done on this compound. Therefore, the current investigation concentrated on the acute oral toxicity of compound 3B on female BALB/c mice, adhering to OECD 423 guidelines. In this study, compound 3B was given orally at 300 mg/kg body weight (b.w.) and 2000 mg/kg b.w. The food consumption and body weight of the mice did not differ significantly among the control and treated groups. Variations were observed in the levels of a few of the biochemical markers. Histopathological examination showed inflammatory infiltrate, and lesions in a few vital organs. The comprehensive investigation showed that compound 3B exhibited moderate toxic effects at a higher dosage of 2000 mg/kg in few organs and caused alterations in biochemical markers; however, it did not result in any mortality, indicating an LD 50 value exceeding 2000 mg/kg. The dosage of compound 3B can be administered at levels below 2000 mg/kg for subsequent studies. Acute oral toxicity BALB/c mice Histone deacetylase inhibitor Hydroxamic acid Lethal dose OECD 423 Figures Figure 1 Figure 2 Figure 3 Figure 4 Key highlights The toxicity profile of compound 3B was studied in female BALB/c mice as per the OECD 423 guidelines Compound 3B was given through oral gavage at a dosage of 300 mg/kg and 2000 mg/kg Biochemical marker levels and histopathological examination indicated moderate toxic effects of compound 3B at 2000 mg/kg Compound 3B was classified under category 5 (lowest toxicity) as per the OECD 423 guidelines 1. Introduction Cancer is a global health problem that affects millions of the people worldwide. Around 10 million deaths were reported worldwide in 2020 (Debela et al., 2021). Surgery, radiation therapy, and chemotherapy are some of the approaches used to treat cancer (Debela et al., 2021). In recent decades, numerous anticancer agents have been developed, yet each of them had their own challenges and did not offer a completely effective solution (Hesham et al., 2018). Drugs targeting epigenetic writers and erasers for cancer therapy first started with the concept of inhibiting individual targets with drugs such as HDAC inhibitors (HDACi), histone methyl transferase inhibitors, and DNA methyl transferase inhibitors. The primary obstacle was combatting the issue of drug resistance (Fu et al., 2017; Raghavendra et al., 2018). Hence, there is a constant necessity for the development of more precise, specific, effective and safer drugs. Histone deacetylases (HDACs) are frequently overexpressed in various cancers, contributing to the transcriptional repression of tumor suppressor genes. Consequently, HDACis are commonly employed in the treatment of hematological malignancies and a few solid tumors (Jenke et al., 2021). Hydroxamic acid, known for its strong Zn² + chelating ability, exhibits high binding affinity and potent inhibitory effects, making it a widely adopted zinc-binding moiety in drug design (Bian et al., 2015; Zhang et al., 2018). For a medication to enter clinical practice, preclinical toxicity evaluations are crucial. These investigations employ verified methods and suggested animal models. Correlating animal reactions to those in humans is the ultimate objective of toxicity research (Anwar et al., 2022). The hazardous characteristics of the substance can be evaluated, ranked, and categorized based on the Globally Harmonised System (GHS) for classifying substances that show acute toxicity, based on information provided in Organization for Economic Cooperation and Development (OECD) guidelines 420, 423, and 425. According to OECD Guideline 423 (OECD-423, 2001), the method categorizes the test chemical into classes determined by defined LD 50 cutoff values (Gothe et al., 2023). Previously in our laboratory, we have identified a novel HDAC2 inhibitor N 1 -(2,2'-bipyridin-6-yl)-N 8 -hydroxyoctanediamide ( compound 3B) (Fig. 1 ). To date, pharmacological data on compound 3B has been derived from in vitro studies with no toxicological evaluation conducted in rodent models. This study aims to assess the acute oral toxicity of compound 3B in BALB/c mice, in accordance with OECD 423 guideline. The results are anticipated to yield essential understandings of the safety profile of compound 3B when administered orally, thereby aiding its possible advancement as a new HDAC inhibitor for cancer therapy. 2. Materials and Methods 2.1 Chemical structure Novel hydroxamic acid derivative, compound 3B, had been synthesized and characterized in our laboratory. This compound has been patented by the Manipal Academy of Higher Education, Manipal (Patent ID No. 202441019540), preliminary in vitro studies support the potency of compound 3B. The structure of compound 3B is given in Fig. 1 2.2 Animals Female BALB/c mice with an average weight of 28.55 ± 3.38 g were bred and maintained in the institutional animal house facility (Central Animal House Facility, Manipal Academy of Higher Education, Manipal) under controlled standard laboratory conditions. Ethical approval for animal use in this study was obtained from the Institutional Animal Ethics Committee (IAEC/KMC/46/2025), Kasturba Medical College, Manipal, India. The animals were kept in a controlled environment of 23 ± 2 ℃, humidity levels at 60 ± 5%, and a light/dark cycle of 10 hours and 14 hours respectively. The mice had free accessibility to the food and water. Animal care and handling were carried out in accordance with OECD 423 guidelines. 2.3 Experimental design Toxicological evaluation of compound 3B was conducted in vivo following the OECD 423 (OECD-423, 2001) guidelines for acute oral toxicity. Following these guidelines, three female BALB/c mice were utilized for each dose step. The acute toxicity range of compound 3B was determined based on the mortality status of the mice, which aids in classifying the test compound. The OECD guidelines suggest starting with 300 mg/kg b.w. dosage when no prior toxicological data for the compound exists to comply with ethical considerations. Figure 2 and Table 1 provide a detailed account of the experimental procedure. The mice were fasted for 1–2 hours before dosing. The compound 3B treated group received oral administration of compound 3B via gavage at doses of 300 mg/kg and 2000 mg/kg. The suspension consisted of 10% DMSO (Merck, Sigma-Aldrich) and 10% Cremophor (Merck, Sigma-Aldrich), and it was dissolved with the help of a probe sonicator (Sonics, Vibra cell). The control group was administered a combination of 10% DMSO and 10% Cremophor, excluding the test substance. 1 ml/100 g of b.w., volume was administered and altered based on the mice's weight. Food was given 1 hour after drug treatment. The mice were closely observed for the first 24 hours and subsequently for the next two weeks. The mice were weighed, and food consumption was monitored daily. The fur condition, behavior, respiration, ocular and mucous membranes, urine, and excretion were observed daily. Biochemical parameters, organ-to-body weight ratios, and histological assessments were performed on day 14 of the study. Three mice were taken per step, with a 3-day interval between each step, to observe the toxicity before administration of the next dose. Animals were anesthetized with ketamine–xylazine administered intraperitoneally (ketamine 80 mg/kg; xylazine 10 mg/kg). Upon confirmation of anesthesia, animals were euthanized by transcardial perfusion with phosphate-buffered saline followed by 4% paraformaldehyde (Soueid et al., 2015; Buitrago et al., 2008; Stokes et al., 2002). Histopathological analysis of different organs and biochemical assessment of various parameters were carried out after euthanization. Table 1 Experimental treatment groups for OECD 423 acute toxicity studies of compound 3B Group No Dosage No. of mice Group 1 10% DMSO and 10% Cremophor 3 + 3 Group 2 Compound 3B, 300 mg/kg 3 + 3 Group 3 Compound 3B, 2000 mg/kg 3 + 3 2.4 Biochemical Analysis Animals were anesthetized with ketamine–xylazine administered intraperitoneally (ketamine 80 mg/kg; xylazine 10 mg/kg). Upon confirmation of anesthesia, animals were euthanized by transcardial perfusion with phosphate-buffered saline followed by 4% paraformaldehyde (Soueid et al., 2015; Buitrago et al., 2008; Stokes et al., 2002). On day 14 of the experiment, after anesthetizing the mice, blood samples were taken. A capillary was used to gather the blood from the retro-orbital region. Clot activator (BD vacutainers) vacutainers were used for the collection of blood samples. After the blood had clotted, the tubes were spun at 2000 g for 10 mins, and the serum was extracted. Alanine aminotransferase (ALT), albumin (ALB), alkaline phosphatase (ALP), creatinine (CREA), aspartate aminotransferase (AST), and urea (UR), were measured. 2.5 Histopathological study Vital organs (brain, heart, lungs, stomach, kidney, spleen, liver and intestine) were extracted from the mice that had been sacrificed and stored in a 4% formalin solution. The organs were dehydrated using different concentrations of isopropanol, immersed in xylene, and inserted in paraffin wax. 4 µm sections using a microtome (Leica RM2125 RTS) were made and stained using hematoxylin and eosin (H&E) to observe the pathological changes in the organs using a LX-500 LED trinocular research microscope (Labomed) and images were taken with MiaCam CMOS AR 6pro microscope. 2.6 Statistical Analysis Body weight, dietary intake, organ-to-body weight ratio, and biochemical evaluation were assessed using Graph Pad Prism 8.0 software. The observed results underwent statistical analysis and were depicted as Mean ± SD. One-way and two-way ANOVA were used to assess the significance between the datasets, followed by Dunnett’s multiple comparison test. A p-value of ≤ 0.05 was deemed statistically notable. 3. Results 3.1 Observation of Behavior pattern After administering compound 3B at doses of 300 mg/kg and 2000 mg/kg, all mice were individually monitored for 14 days. No noteworthy alterations were noticed in skin condition, respiration, ocular and mucous membranes, behavior, urine and excretion. Throughout the experiment, no abnormalities or deaths were noted in the mice. Body weight and Food intake All treatment groups maintained relatively stable body weights throughout the 14-day toxicity study, as shown in Fig. 3 A. Furthermore, daily food consumption showed slight variations; however, as shown in Fig. 3 B, no statistically significant differences were noted between control and treatment groups. 3.2 Organ to Body Weight Index Organs such as the brain, heart, lungs, intestine, stomach, spleen, kidneys, and liver, were weighed. For every mouse, the organ-to-body weight index was determined. As shown in Fig. 3 C, the ratio of the heart, lungs, brain, kidneys, and stomach among different treatment groups does not exhibit any statistically significant differences. The groups of mice that received compound 3B at a dosage of 2000 mg/kg, however, exhibited a significant difference in the intestine, spleen, and liver, organ-to-body weight index. Figure 3 D presents representative images of organs from three groups: the control and the two test groups. 3.3 Biochemical Analysis Biochemical assays have been performed to check the toxicity in liver and kidneys. Table 2 demonstrates, no statistical difference in the CREA and UR levels across all the experimental groups. Regarding liver function, ALT, and ALB levels show no statistically significant differences among the three different groups. AST levels showed an increase at 2000 mg/kg, but no statistical significance was observed. ALP levels significantly decreased at higher concentrations Table 2 Effect of administration of compound 3B on renal and liver function tests. Organs Control 300 mg/kg 2000 mg/kg UR (mg/dl) 42.00 ± 24.33 22.00 ± 2.65 36.33 ± 1.53 CREA (mg/dl) 0.27 ± 0.29 0.63 ± 0.12 0.23 ± 0.06 ALT (U/L) 68.00 ± 33.65 40.00 ± 36.72 32.67 ± 6.43 AST (U/L) 69.67 ± 18.61 41.00 ± 32.23 105.00 ± 6.93 ALP (IU/L) 209.00 ± 60.36 146.33 ± 20.11** 157.00 ± 8.66 * ALB (g/dl) 2.10 ± 0.10 2.17 ± 0.06 2.27 ± 0.12 UR: urea; CREA: creatinine; ALT: alanine aminotransferase; AST: aspartate amino transferase; ALP: alkaline phosphatase; ALB: albumin. All data are reported as the mean ± SD for n = 3 per group. The differences between the control and treated groups were analyzed by Two−way ANOVA, followed by Dunnett’s test. The significance levels observed are * p < 0.05, ** p < 0.01, and in comparison to control group values . 3.4 Histopathological Analysis Histopathological examination of major organs revealed that the morphology and color of the liver, lungs, kidneys, heart, and spleen were generally maintained across all test groups. In the colon (Fig. 4 A), histological analysis demonstrated intact architecture without any discernible abnormalities. Similarly, the small intestine (Fig. 4 B) maintained normal morphology. However, notable inflammatory infiltrates were observed in the group receiving a 2000 mg/kg dose. Renal histology (Fig. 4 C, D) presented well-preserved glomeruli, Bowman’s capsules, and renal corpuscles across most groups, while the 2000 mg/kg group displayed signs of tubular degeneration, necrosis, and inflammatory infiltrate. Hepatic sections (Fig. 4 E) exhibited clearly defined portal triads and central veins without evidence of necrosis or hemorrhage. Gastric mucosa (Fig. 4 F) appeared structurally intact, though inflammatory infiltrate was again noted in the 2000 mg/kg group. The myocardial architecture of cardiac tissue (Fig. 4 G) retained organized myocardial fibers across all groups. Splenic histology (Fig. 4 H) showed a reduction in the cellularity of white pulp in both test groups, alongside observations of extramedullary hematopoiesis and acute inflammatory cells. Cerebral sections maintained normal morphology overall, with the exception of a few degenerated neurons observed in the 2000 mg/kg group. Pulmonary histological analysis (Fig. 4 I) confirmed preserved alveolar and bronchiolar structures, though the 300 mg/kg group exhibited hemorrhage, while the 2000 mg/kg group demonstrated inflammatory infiltrate. Figure 4 J compared with Control, there is acute inflammatory infiltration in the test group. 4. Discussion The Acute Toxic Class method, which is an approach aimed at classifying substances according to their acute oral toxicity, is introduced in OECD guideline 423. Using small groups of animals (usually three per step), this method employs known dose levels of 5, 50, 300, and 2000 mg/kg body weight to ascertain the toxicity class of a substance. Based on the mortality observed at each dose, additional testing may involve dosing more animals at either the same or different dose levels (Gothe et al., 2023). In the present study Compound 3B, a novel hydroxamic acid derivative, was evaluated for its acute oral toxicity at doses of 300 mg/kg and 2000 mg/kg. The anticancer potential and HDAC1/2 inhibitory activity of Compound 3B have been published (Pai et al., 2025) and patented (Patent ID No. 202441019540). In the study with respect to all doses no mortality was observed at both the dosages. Similar study by Liu and group showed that almond hull powders exhibited no significant variations in the organ-to-body weight index of mice across all treatment groups, encompassing the vital organs: liver, kidney, heart, lungs, and spleen (Liu et al., 2023). Similarly, in our study compound 3B does not show any alteration in behavior, body weight and, dietary consumption at both the dosage (300 mg/kg and 2000 mg/kg). From a statistical perspective, the 300 mg/kg dosage did not show any notable differences in the ratio of the heart, lungs, brain, kidneys, and stomach. However, for the 2000 mg/kg grouped mice, there was a significant difference in the intestine, spleen, and liver organ-to-body weight index. In another study by Bedi and group showed that evaluating liver and kidney function through markers such as ALT, AST, ALP, ALB, CREA and UR can help determine the toxicity of a compound (Bedi et al., 2020). Our investigation revealed that the kidney and liver function tests did not show any statistically significant differences among the different treatment groups. It is known that AST and ALT levels increase significantly, which may be attributed to the destruction of liver cells within a toxic environment. In another study by Yang and group reiterated that due to its high concentration in liver cells, it is important that ALT and AST are often used as a more precise indicator for measuring suspected liver cell damage. Elevated ALT and AST levels can indicate liver damage (Yang et al., 2014). Our research demonstrated that ALT and AST levels did not differ significantly among the treatment groups. Lala and group demonstrated that elevated ALP levels often indicate biliary tract obstruction linked to cholesterol liver disease (Lala et al., 2023). The slight variations in ALP levels were considered incidental and unrelated to the test substance. This indicates that our compound, 3B will not create any impact on metabolic activity or renal functions. It was proposed that histopathological studies offer supportive evidence for biochemical and hematological observations (Karin et al., 2014). Histopathological analysis of major organs revealed dose-dependent alterations, with inflammatory infiltrates, tubular degeneration, necrosis, and hemorrhage being prominent in the higher dose groups (2000 mg/kg). These findings indicate a potential for dose-dependent toxicological effects, highlighting the importance of dosage optimization for safe therapeutic application. This is the first study to investigate OECD toxicity guidelines for evaluating LD 50 , mortality, and other key toxicological parameters of compound 3B. Acute in vivo toxicity studies provide preliminary safety data, however it’s associated with limitations. Studies are conducted in a single animal species, and due to inter-species metabolic differences, the findings may not reliably predict human responses (Chapman, 2007; Robinson et al., 2008). The observation period is short (up to 14 days), thereby neglecting long term chronic toxic effects. In addition, only a single dose level is tested, which restricts the ability to establish a full dose–response relationship or LD₅₀ with precision (Karmaus et al., 2022). Further research is required to comprehensively assess the long-term effects of the compound. Conclusion In accordance with OECD guideline 423, a 14-day acute oral toxicity study was conducted. The study showed that compound 3B falls under Category 5, with an estimated LD 50 above 2000 mg/kg body weight. Although slight signs of toxicity were observed at the 2000 mg/kg dose, mortality was not seen. Thus, the LD 50 of compound 3B is considered to be greater than 2000 mg/kg. Abbreviations Abbreviation Full form ALB Albumin ALP Alkaline phosphatase ALT Alanine aminotransferase AST Aspartate aminotransferase BW Body weight Compound 3B N 1 -(2,2'-bipyridin-6-yl)-N 8 -hydroxyoctanediamide CREA Creatinine H&E Hematoxylin and Eosin HDAC i Histone deacetylase inhibitor IAEC Institutional Animal Ethics Committee LD 50 Lethal dose 50 OECD Organization for Economic Cooperation and Development SD Standard deviation UR Urea Declarations Acknowledgments The authors thank Manipal Academy of Higher Education (MAHE), Manipal, India for infrastructure. The authors thank Department of Biotechnology (DBT) BioCare grant, GOI and Manipal Academy of Higher Education (MAHE), Manipal, India for infrastructure. We would like to thank the Central Animal Research Facility, Manipal Academy of Higher Education (MAHE), Manipal, India, for infrastructure. Funding - Open access funding provided by Manipal Academy of Higher Education, Manipal. This work was supported by the Department of Biotechnology (DBT) BioCare grant [Grant ID: BT/PR20046/ BIC/101/683/2016], Government of India (GOI), India, to Dr. Babitha KS. Authors thank Intramural funding (Grant ID: MAHE/CDS/PHD/IMF/2023), MAHE, Manipal for financial support. Authors also thank MRB Seed grant (Grant ID: DOR/MRB/2023/SG-03), MAHE, Manipal for funding. Statements and Declaration Conflict of interest The authors declare that a provisional patent application (Application Number: 202441019540.) has been filed related to the chemical compound discussed in this research. Manasa Gangadhar Shetty, Padmini Pai, Babitha Kampa Sundara, Kapaettu Satyamoorthy and Srinivas Oruganti are listed as an inventors on the patent. Ethics approval This study has been approved by the Institutional Animal Ethics Committee of Kasturba Medical College, Manipal Academy of Higher Education, Manipal (IAEC/KMC/46/2025). Author contributions Padmini Pai- Execution of experiments, reviewing and editing. Rachel Savio D’Mello- Execution of experiments, writing and reviewing. Shruthi Nayak- Writing and reviewing, Pallavi Rao- Help with chemistry part of work and reviewing. Srinivas Oruganti- Help with chemistry part of work and reviewing. Kapaettu Satyamoorthy- Editing and reviewing. Babitha Kampa Sundara- Conceptualization, editing, reviewing, data analysis and supervision. References Anwar F, Saleem U, Rehman AU, Ahmad B, Ismail T, Mirza MU, Ahmad S. Acute oral, subacute, and developmental toxicity profiling of naphthalene 2-yl, 2-chloro, 5-nitrobenzoate: Assessment based on stress response, toxicity, and adverse outcome pathways. Front Pharmacol. 2022;12:810704. Bedi O, Krishan P. Investigations on acute oral toxicity studies of purpurin by application of OECD guideline 423 in rodents. N-S Arch Pharmacol. 2020;393:565–71. Bian J, Zhang L, Han Y, Wang C, Zhang L. 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Sundara","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABB0lEQVRIiWNgGAWjYBACAwYehgOMDQwMEgzMBxhAFAM7kOIhpOUgWAtbAkQLMxFaGCBaeAwgQoS0mLOfPXj44w47Bsn+M183/NxhkcffzMD44G0bbi2WPXkJBw6eSWaQlsjddrP3jESxxGEGZsO5eLQYHMgxOHCwjZlBToJ32w3eNonEhsMMbNK8+LScfwPSUs8gx3/m2c2/QC3zDzOw/8ar5QbYlsMM0gw5bLdBtmwA2sKMT4vljHcJB862HeeRnJFmdlu2TaLY8DBjs+Scc7i1mPPnHv5Q2VYtJ3H+8LObb9vq8uSONx/88KYMtxYYgEdEAgMDKDGQAhJIUz4KRsEoGAUjAQAAxBdXJRs+YjQAAAAASUVORK5CYII=","orcid":"","institution":"Manipal Academy of Higher Education","correspondingAuthor":true,"prefix":"","firstName":"Babitha","middleName":"Kampa","lastName":"Sundara","suffix":""}],"badges":[],"createdAt":"2025-06-27 07:08:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6988823/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6988823/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40360-025-01040-9","type":"published","date":"2025-11-28T15:58:51+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":91621058,"identity":"460618d8-b34b-4737-adde-7b64c8f1593f","added_by":"auto","created_at":"2025-09-18 11:28:24","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":13429,"visible":true,"origin":"","legend":"\u003cp\u003eStructure of N\u003csup\u003e1\u003c/sup\u003e-(2,2'-bipyridin-6-yl)-N\u003csup\u003e8\u003c/sup\u003e-hydroxyoctanediamide\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e(compound 3B)\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6988823/v1/88f0b137b5cfbd40c74cd033.jpg"},{"id":91621062,"identity":"8ab64b6e-97d2-4d0c-b4e5-408297b0c494","added_by":"auto","created_at":"2025-09-18 11:28:25","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":108243,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of experimental design for LD\u003csub\u003e50 \u003c/sub\u003ecut-off value determination for compound 3B (OECD-423, 2001)\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6988823/v1/9348a822e299a29ce1e06872.jpg"},{"id":91621057,"identity":"7b5f371c-29ac-4d55-8d94-47ff56ec8ff6","added_by":"auto","created_at":"2025-09-18 11:28:24","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":109528,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of administration of compound 3B on (A) body weight (B) food intake (C) organ/body weight index and (D) Images of different organs of control, treatment (300 mg/kg), treatment (2000 mg/kg); (a) brain, (b) lungs, (c) heart, (d) stomach, (e) intestine, (f) spleen, (g) kidney, (h) liver. All data are reported as the mean ± SD for n = 6 per group. Two-way ANOVA, followed by Dunett’s test. The significance levels observed are * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ***\u003cem\u003ep\u003c/em\u003e \u0026lt;0.001. Organ to body weight index = (organ weight /body weight ×100)\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6988823/v1/c21b1bfa24fffa54d6a2ae83.jpg"},{"id":91621396,"identity":"5da5cdfc-cb39-4839-9dea-582093d68574","added_by":"auto","created_at":"2025-09-18 11:36:24","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":363315,"visible":true,"origin":"","legend":"\u003cp\u003eH\u0026amp;E stained tumor tissue sections from control and treated mice, showing histological evaluation of major organs including control, treatment (300 mg/kg), treatment (2000 mg/kg); (A) colon, (B) small intestine, (C) cortex, (D) medulla (kidney), (E) liver (100x), (F) stomach, (G) heart, (H) spleen, (I) cerebrum, and (J) lungs. Mucosa - M, Submucosa - SM, White pulp - WP, Red pulp - RP, Tubules - T, Glomerulus - G, Inflammatory infiltrate - red arrow, Eroded mucosa- blue arrow, Degenerated tubules - green arrow, Necrosed tubules- blue arrow; Data represented as n = 3, mice were treated with compound 3B at two different doses: 300 mg/kg and 2000 mg/kg. Scale: 50 µm; Spleen-40x, cerebrum-400x, all other organs-100x\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6988823/v1/9f799ba26c29ae9188faa20a.jpg"},{"id":97179501,"identity":"75064ce1-593e-40d6-bae4-c08fc183084d","added_by":"auto","created_at":"2025-12-01 16:15:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1230814,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6988823/v1/6c679c64-306e-4793-b3ca-59b7ed98d480.pdf"},{"id":91621060,"identity":"be0b0f2c-2a69-4d78-a192-a31fe207bb61","added_by":"auto","created_at":"2025-09-18 11:28:24","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":366496,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6988823/v1/c90cc06425f17f426bb67c1a.png"}],"financialInterests":"Competing interest reported. The authors declare that a provisional patent application (Application Number: 202441019540.) has been filed related to the chemical compound discussed in this research. Manasa Gangadhar Shetty, Padmini Pai, Babitha Kampa Sundara, Kapaettu Satyamoorthy and Srinivas Oruganti are listed as an inventors on the patent.","formattedTitle":"In vivo Acute Oral Toxicity Assessment of Novel Histone Deacetylase 2 Inhibitor","fulltext":[{"header":"Key highlights","content":"\u003cul\u003e\n \u003cli\u003eThe toxicity profile of compound 3B was studied in female BALB/c mice as per the OECD 423 guidelines\u003c/li\u003e\n \u003cli\u003eCompound 3B was given through oral gavage at a dosage of 300 mg/kg and 2000 mg/kg\u003c/li\u003e\n \u003cli\u003eBiochemical marker levels and histopathological examination indicated moderate toxic effects of compound 3B at 2000 mg/kg\u003c/li\u003e\n \u003cli\u003eCompound 3B was classified under category 5 (lowest toxicity) as per the OECD 423 guidelines\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eCancer is a global health problem that affects millions of the people worldwide. Around 10\u0026nbsp;million deaths were reported worldwide in 2020 (Debela et al., 2021). Surgery, radiation therapy, and chemotherapy are some of the approaches used to treat cancer (Debela et al., 2021). In recent decades, numerous anticancer agents have been developed, yet each of them had their own challenges and did not offer a completely effective solution (Hesham et al., 2018). Drugs targeting epigenetic writers and erasers for cancer therapy first started with the concept of inhibiting individual targets with drugs such as HDAC inhibitors (HDACi), histone methyl transferase inhibitors, and DNA methyl transferase inhibitors. The primary obstacle was combatting the issue of drug resistance (Fu et al., 2017; Raghavendra et al., 2018). Hence, there is a constant necessity for the development of more precise, specific, effective and safer drugs. Histone deacetylases (HDACs) are frequently overexpressed in various cancers, contributing to the transcriptional repression of tumor suppressor genes. Consequently, HDACis are commonly employed in the treatment of hematological malignancies and a few solid tumors (Jenke et al., 2021). Hydroxamic acid, known for its strong Zn\u0026sup2;\u003csup\u003e+\u003c/sup\u003echelating ability, exhibits high binding affinity and potent inhibitory effects, making it a widely adopted zinc-binding moiety in drug design (Bian et al., 2015; Zhang et al., 2018).\u003c/p\u003e\u003cp\u003eFor a medication to enter clinical practice, preclinical toxicity evaluations are crucial. These investigations employ verified methods and suggested animal models. Correlating animal reactions to those in humans is the ultimate objective of toxicity research (Anwar et al., 2022). The hazardous characteristics of the substance can be evaluated, ranked, and categorized based on the Globally Harmonised System (GHS) for classifying substances that show acute toxicity, based on information provided in Organization for Economic Cooperation and Development (OECD) guidelines 420, 423, and 425. According to OECD Guideline 423 (OECD-423, 2001), the method categorizes the test chemical into classes determined by defined LD\u003csub\u003e50\u003c/sub\u003e cutoff values (Gothe et al., 2023).\u003c/p\u003e\u003cp\u003ePreviously in our laboratory, we have identified a novel HDAC2 inhibitor N\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e-(2,2'-bipyridin-6-yl)-N\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e-hydroxyoctanediamide \u003cb\u003e(\u003c/b\u003ecompound 3B) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). To date, pharmacological data on compound 3B has been derived from \u003cem\u003ein vitro\u003c/em\u003e studies with no toxicological evaluation conducted in rodent models. This study aims to assess the acute oral toxicity of compound 3B in BALB/c mice, in accordance with OECD 423 guideline. The results are anticipated to yield essential understandings of the safety profile of compound 3B when administered orally, thereby aiding its possible advancement as a new HDAC inhibitor for cancer therapy.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Chemical structure\u003c/h2\u003e\u003cp\u003eNovel hydroxamic acid derivative, compound 3B, had been synthesized and characterized in our laboratory. This compound has been patented by the Manipal Academy of Higher Education, Manipal (Patent ID No. 202441019540), preliminary \u003cem\u003ein vitro\u003c/em\u003e studies support the potency of compound 3B. The structure of compound 3B is given in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Animals\u003c/h2\u003e\u003cp\u003eFemale BALB/c mice with an average weight of 28.55\u0026thinsp;\u0026plusmn;\u0026thinsp;3.38 g were bred and maintained in the institutional animal house facility (Central Animal House Facility, Manipal Academy of Higher Education, Manipal) under controlled standard laboratory conditions. Ethical approval for animal use in this study was obtained from the Institutional Animal Ethics Committee (IAEC/KMC/46/2025), Kasturba Medical College, Manipal, India. The animals were kept in a controlled environment of 23\u0026thinsp;\u0026plusmn;\u0026thinsp;2 ℃, humidity levels at 60\u0026thinsp;\u0026plusmn;\u0026thinsp;5%, and a light/dark cycle of 10 hours and 14 hours respectively. The mice had free accessibility to the food and water. Animal care and handling were carried out in accordance with OECD 423 guidelines.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Experimental design\u003c/h2\u003e\u003cp\u003eToxicological evaluation of compound 3B was conducted \u003cem\u003ein vivo\u003c/em\u003e following the OECD 423 (OECD-423, 2001) guidelines for acute oral toxicity. Following these guidelines, three female BALB/c mice were utilized for each dose step. The acute toxicity range of compound 3B was determined based on the mortality status of the mice, which aids in classifying the test compound. The OECD guidelines suggest starting with 300 mg/kg b.w. dosage when no prior toxicological data for the compound exists to comply with ethical considerations. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e provide a detailed account of the experimental procedure. The mice were fasted for 1\u0026ndash;2 hours before dosing. The compound 3B treated group received oral administration of compound 3B via gavage at doses of 300 mg/kg and 2000 mg/kg. The suspension consisted of 10% DMSO (Merck, Sigma-Aldrich) and 10% Cremophor (Merck, Sigma-Aldrich), and it was dissolved with the help of a probe sonicator (Sonics, Vibra cell). The control group was administered a combination of 10% DMSO and 10% Cremophor, excluding the test substance. 1 ml/100 g of b.w., volume was administered and altered based on the mice's weight. Food was given 1 hour after drug treatment. The mice were closely observed for the first 24 hours and subsequently for the next two weeks. The mice were weighed, and food consumption was monitored daily. The fur condition, behavior, respiration, ocular and mucous membranes, urine, and excretion were observed daily. Biochemical parameters, organ-to-body weight ratios, and histological assessments were performed on day 14 of the study. Three mice were taken per step, with a 3-day interval between each step, to observe the toxicity before administration of the next dose. Animals were anesthetized with ketamine\u0026ndash;xylazine administered intraperitoneally (ketamine 80 mg/kg; xylazine 10 mg/kg). Upon confirmation of anesthesia, animals were euthanized by transcardial perfusion with phosphate-buffered saline followed by 4% paraformaldehyde (Soueid et al., 2015; Buitrago et al., 2008; Stokes et al., 2002). Histopathological analysis of different organs and biochemical assessment of various parameters were carried out after euthanization.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eExperimental treatment groups for OECD 423 acute toxicity studies of compound 3B\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\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\u003cdiv align=\"char\" char=\"+\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup No\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDosage\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNo. of mice\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10% DMSO and 10% Cremophor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"+\" colname=\"c3\"\u003e\u003cp\u003e3\u0026thinsp;+\u0026thinsp;3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCompound 3B, 300 mg/kg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"+\" colname=\"c3\"\u003e\u003cp\u003e3\u0026thinsp;+\u0026thinsp;3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup 3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCompound 3B, 2000 mg/kg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"+\" colname=\"c3\"\u003e\u003cp\u003e3\u0026thinsp;+\u0026thinsp;3\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\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Biochemical Analysis\u003c/h2\u003e\u003cp\u003eAnimals were anesthetized with ketamine\u0026ndash;xylazine administered intraperitoneally (ketamine 80 mg/kg; xylazine 10 mg/kg). Upon confirmation of anesthesia, animals were euthanized by transcardial perfusion with phosphate-buffered saline followed by 4% paraformaldehyde (Soueid et al., 2015; Buitrago et al., 2008; Stokes et al., 2002). On day 14 of the experiment, after anesthetizing the mice, blood samples were taken. A capillary was used to gather the blood from the retro-orbital region. Clot activator (BD vacutainers) vacutainers were used for the collection of blood samples. After the blood had clotted, the tubes were spun at 2000 g for 10 mins, and the serum was extracted. Alanine aminotransferase (ALT), albumin (ALB), alkaline phosphatase (ALP), creatinine (CREA), aspartate aminotransferase (AST), and urea (UR), were measured.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Histopathological study\u003c/h2\u003e\u003cp\u003eVital organs (brain, heart, lungs, stomach, kidney, spleen, liver and intestine) were extracted from the mice that had been sacrificed and stored in a 4% formalin solution. The organs were dehydrated using different concentrations of isopropanol, immersed in xylene, and inserted in paraffin wax. 4 \u0026micro;m sections using a microtome (Leica RM2125 RTS) were made and stained using hematoxylin and eosin (H\u0026amp;E) to observe the pathological changes in the organs using a LX-500 LED trinocular research microscope (Labomed) and images were taken with MiaCam CMOS AR 6pro microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Statistical Analysis\u003c/h2\u003e\u003cp\u003eBody weight, dietary intake, organ-to-body weight ratio, and biochemical evaluation were assessed using Graph Pad Prism 8.0 software. The observed results underwent statistical analysis and were depicted as Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. One-way and two-way ANOVA were used to assess the significance between the datasets, followed by Dunnett\u0026rsquo;s multiple comparison test. A p-value of \u0026le;\u0026thinsp;0.05 was deemed statistically notable.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Observation of Behavior pattern\u003c/h2\u003e\u003cp\u003eAfter administering compound 3B at doses of 300 mg/kg and 2000 mg/kg, all mice were individually monitored for 14 days. No noteworthy alterations were noticed in skin condition, respiration, ocular and mucous membranes, behavior, urine and excretion. Throughout the experiment, no abnormalities or deaths were noted in the mice.\u003c/p\u003e\u003cp\u003e\u003cb\u003eBody weight and Food intake\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAll treatment groups maintained relatively stable body weights throughout the 14-day toxicity study, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA. Furthermore, daily food consumption showed slight variations; however, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, no statistically significant differences were noted between control and treatment groups.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Organ to Body Weight Index\u003c/h2\u003e\u003cp\u003eOrgans such as the brain, heart, lungs, intestine, stomach, spleen, kidneys, and liver, were weighed. For every mouse, the organ-to-body weight index was determined. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, the ratio of the heart, lungs, brain, kidneys, and stomach among different treatment groups does not exhibit any statistically significant differences. The groups of mice that received compound 3B at a dosage of 2000 mg/kg, however, exhibited a significant difference in the intestine, spleen, and liver, organ-to-body weight index. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD presents representative images of organs from three groups: the control and the two test groups.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Biochemical Analysis\u003c/h2\u003e\u003cp\u003eBiochemical assays have been performed to check the toxicity in liver and kidneys. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e demonstrates, no statistical difference in the CREA and UR levels across all the experimental groups. Regarding liver function, ALT, and ALB levels show no statistically significant differences among the three different groups. AST levels showed an increase at 2000 mg/kg, but no statistical significance was observed. ALP levels significantly decreased at higher concentrations\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEffect of administration of compound 3B on renal and liver function tests.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOrgans\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e300 mg/kg\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2000 mg/kg\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUR (mg/dl)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e42.00\u0026thinsp;\u0026plusmn;\u0026thinsp;24.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e22.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e36.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCREA (mg/dl)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eALT (U/L)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e68.00\u0026thinsp;\u0026plusmn;\u0026thinsp;33.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e40.00\u0026thinsp;\u0026plusmn;\u0026thinsp;36.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e32.67\u0026thinsp;\u0026plusmn;\u0026thinsp;6.43\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAST (U/L)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e69.67\u0026thinsp;\u0026plusmn;\u0026thinsp;18.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e41.00\u0026thinsp;\u0026plusmn;\u0026thinsp;32.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e105.00\u0026thinsp;\u0026plusmn;\u0026thinsp;6.93\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eALP\u0026nbsp;(IU/L)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e209.00\u0026thinsp;\u0026plusmn;\u0026thinsp;60.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e146.33\u0026thinsp;\u0026plusmn;\u0026thinsp;20.11**\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e157.00\u0026thinsp;\u0026plusmn;\u0026thinsp;8.66\u0026nbsp;*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eALB\u0026nbsp;(g/dl)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e2.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e2.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e2.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\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\u003eUR: urea; CREA: creatinine; ALT: alanine aminotransferase; AST: aspartate amino transferase; ALP: alkaline phosphatase; ALB: albumin. All data are reported as the mean \u0026plusmn; SD for n = 3 per group. The differences between the control and treated groups were analyzed by Two\u0026minus;way ANOVA, followed by Dunnett\u0026rsquo;s test. The significance levels observed are *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, and in comparison to control group values\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Histopathological Analysis\u003c/h2\u003e\u003cp\u003eHistopathological examination of major organs revealed that the morphology and color of the liver, lungs, kidneys, heart, and spleen were generally maintained across all test groups. In the colon (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), histological analysis demonstrated intact architecture without any discernible abnormalities. Similarly, the small intestine (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) maintained normal morphology. However, notable inflammatory infiltrates were observed in the group receiving a 2000 mg/kg dose. Renal histology (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D) presented well-preserved glomeruli, Bowman\u0026rsquo;s capsules, and renal corpuscles across most groups, while the 2000 mg/kg group displayed signs of tubular degeneration, necrosis, and inflammatory infiltrate. Hepatic sections (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE) exhibited clearly defined portal triads and central veins without evidence of necrosis or hemorrhage. Gastric mucosa (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF) appeared structurally intact, though inflammatory infiltrate was again noted in the 2000 mg/kg group. The myocardial architecture of cardiac tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG) retained organized myocardial fibers across all groups. Splenic histology (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH) showed a reduction in the cellularity of white pulp in both test groups, alongside observations of extramedullary hematopoiesis and acute inflammatory cells. Cerebral sections maintained normal morphology overall, with the exception of a few degenerated neurons observed in the 2000 mg/kg group. Pulmonary histological analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI) confirmed preserved alveolar and bronchiolar structures, though the 300 mg/kg group exhibited hemorrhage, while the 2000 mg/kg group demonstrated inflammatory infiltrate. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ compared with Control, there is acute inflammatory infiltration in the test group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe Acute Toxic Class method, which is an approach aimed at classifying substances according to their acute oral toxicity, is introduced in OECD guideline 423. Using small groups of animals (usually three per step), this method employs known dose levels of 5, 50, 300, and 2000 mg/kg body weight to ascertain the toxicity class of a substance. Based on the mortality observed at each dose, additional testing may involve dosing more animals at either the same or different dose levels (Gothe et al., 2023).\u003c/p\u003e\u003cp\u003eIn the present study Compound 3B, a novel hydroxamic acid derivative, was evaluated for its acute oral toxicity at doses of 300 mg/kg and 2000 mg/kg. The anticancer potential and HDAC1/2 inhibitory activity of Compound 3B have been published (Pai et al., 2025) and patented (Patent ID No. 202441019540). In the study with respect to all doses no mortality was observed at both the dosages. Similar study by Liu and group showed that almond hull powders exhibited no significant variations in the organ-to-body weight index of mice across all treatment groups, encompassing the vital organs: liver, kidney, heart, lungs, and spleen (Liu et al., 2023). Similarly, in our study compound 3B does not show any alteration in behavior, body weight and, dietary consumption at both the dosage (300 mg/kg and 2000 mg/kg). From a statistical perspective, the 300 mg/kg dosage did not show any notable differences in the ratio of the heart, lungs, brain, kidneys, and stomach. However, for the 2000 mg/kg grouped mice, there was a significant difference in the intestine, spleen, and liver organ-to-body weight index. In another study by Bedi and group showed that evaluating liver and kidney function through markers such as ALT, AST, ALP, ALB, CREA and UR can help determine the toxicity of a compound (Bedi et al., 2020). Our investigation revealed that the kidney and liver function tests did not show any statistically significant differences among the different treatment groups. It is known that AST and ALT levels increase significantly, which may be attributed to the destruction of liver cells within a toxic environment. In another study by Yang and group reiterated that due to its high concentration in liver cells, it is important that ALT and AST are often used as a more precise indicator for measuring suspected liver cell damage. Elevated ALT and AST levels can indicate liver damage (Yang et al., 2014). Our research demonstrated that ALT and AST levels did not differ significantly among the treatment groups. Lala and group demonstrated that elevated ALP levels often indicate biliary tract obstruction linked to cholesterol liver disease (Lala et al., 2023). The slight variations in ALP levels were considered incidental and unrelated to the test substance. This indicates that our compound, 3B will not create any impact on metabolic activity or renal functions.\u003c/p\u003e\u003cp\u003eIt was proposed that histopathological studies offer supportive evidence for biochemical and hematological observations (Karin et al., 2014). Histopathological analysis of major organs revealed dose-dependent alterations, with inflammatory infiltrates, tubular degeneration, necrosis, and hemorrhage being prominent in the higher dose groups (2000 mg/kg). These findings indicate a potential for dose-dependent toxicological effects, highlighting the importance of dosage optimization for safe therapeutic application. This is the first study to investigate OECD toxicity guidelines for evaluating LD\u003csub\u003e50\u003c/sub\u003e, mortality, and other key toxicological parameters of compound 3B. Acute \u003cem\u003ein vivo\u003c/em\u003e toxicity studies provide preliminary safety data, however it\u0026rsquo;s associated with limitations. Studies are conducted in a single animal species, and due to inter-species metabolic differences, the findings may not reliably predict human responses (Chapman, 2007; Robinson et al., 2008). The observation period is short (up to 14 days), thereby neglecting long term chronic toxic effects. In addition, only a single dose level is tested, which restricts the ability to establish a full dose\u0026ndash;response relationship or LD₅₀ with precision (Karmaus et al., 2022). Further research is required to comprehensively assess the long-term effects of the compound.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn accordance with OECD guideline 423, a 14-day acute oral toxicity study was conducted. The study showed that compound 3B falls under Category 5, with an estimated LD\u003csub\u003e50\u003c/sub\u003e above 2000 mg/kg body weight. Although slight signs of toxicity were observed at the 2000 mg/kg dose, mortality was not seen. Thus, the LD\u003csub\u003e50\u003c/sub\u003e of compound 3B is considered to be greater than 2000 mg/kg.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAbbreviation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eFull form\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eALB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAlbumin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eALP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAlkaline phosphatase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eALT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAlanine aminotransferase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAST\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAspartate aminotransferase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eBW\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eBody weight\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCompound 3B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eN\u003csup\u003e1\u003c/sup\u003e-(2,2'-bipyridin-6-yl)-N\u003csup\u003e8\u003c/sup\u003e-hydroxyoctanediamide\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCREA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCreatinine\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eH\u0026amp;E\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eHematoxylin and Eosin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eHDAC\u003csub\u003ei\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eHistone deacetylase inhibitor\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eIAEC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eInstitutional Animal Ethics Committee\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eLD\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eLethal dose 50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eOECD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eOrganization for Economic Cooperation and Development\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eStandard deviation\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eUR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eUrea\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Manipal Academy of Higher Education (MAHE), Manipal, India for infrastructure. The authors thank\u0026nbsp;Department of Biotechnology (DBT) BioCare grant, GOI and Manipal Academy of Higher Education (MAHE), Manipal, India for infrastructure. We would like to thank the Central Animal Research Facility, Manipal Academy of Higher Education (MAHE), Manipal, India, for infrastructure.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding -\u003c/strong\u003e Open access funding provided by Manipal Academy of Higher Education, Manipal. This work was supported by the Department of Biotechnology (DBT) BioCare grant [Grant ID: BT/PR20046/ BIC/101/683/2016], Government of India (GOI), India, to Dr. Babitha KS.\u0026nbsp;Authors thank Intramural funding (Grant ID: MAHE/CDS/PHD/IMF/2023), MAHE, Manipal for financial support. Authors also thank MRB Seed grant (Grant ID: DOR/MRB/2023/SG-03), MAHE, Manipal for funding.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatements and Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that a provisional patent application (Application Number: 202441019540.) has been filed related to the chemical compound discussed in this research. Manasa Gangadhar Shetty, Padmini Pai, Babitha Kampa Sundara, Kapaettu Satyamoorthy and Srinivas Oruganti are listed as an inventors on the patent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study has been approved by the Institutional Animal Ethics Committee of Kasturba Medical College, Manipal Academy of Higher Education, Manipal (IAEC/KMC/46/2025).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePadmini Pai- Execution of experiments, reviewing and editing. Rachel Savio D’Mello- Execution of experiments, writing and reviewing. Shruthi Nayak- Writing and reviewing, Pallavi Rao- Help with chemistry part of work and reviewing. Srinivas Oruganti- Help with chemistry part of work and reviewing. Kapaettu Satyamoorthy- Editing and reviewing. Babitha Kampa Sundara- Conceptualization, editing, reviewing, data analysis and supervision.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAnwar F, Saleem U, Rehman AU, Ahmad B, Ismail T, Mirza MU, Ahmad S. Acute oral, subacute, and developmental toxicity profiling of naphthalene 2-yl, 2-chloro, 5-nitrobenzoate: Assessment based on stress response, toxicity, and adverse outcome pathways. Front Pharmacol. 2022;12:810704.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBedi O, Krishan P. Investigations on acute oral toxicity studies of purpurin by application of OECD guideline 423 in rodents. N-S Arch Pharmacol. 2020;393:565\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBian J, Zhang L, Han Y, Wang C, Zhang L. Histone deacetylase inhibitors: Potent anti-leukemic agents. Curr Med Chem. 2015;22:22\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBuitrago S, Martin TE, Tetens-Woodring J, Belicha-Villanueva A, Wilding GE. Safety and efficacy of various combinations of injectable anesthetics in BALB/c mice. J Am Assoc Lab Anim Sci. 2008;47:11\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChapman K. (2007). Challenging the regulatory requirement for acute toxicity studies in the development of new medicines. In National Centre for the Replacement, Refinement and Reduction of Animals in Research workshop report (Vol. 7, pp. 1998\u0026ndash;2003).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDebela DT, Muzazu SG, Heraro KD, Ndalama MT, Mesele BW, Haile DC, Kitui SK, Manyazewal T. New approaches and procedures for cancer treatment: Current perspectives. SAGE Open Med. 2021;9:20503121211034366.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFu RG, Sun Y, Sheng WB, Liao DF. Designing multi-targeted agents: An emerging anticancer drug discovery paradigm. Eur J Med Chem. 2017;136:533\u0026ndash;78.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGothe S, Pawade U, Nikam A, Anjankar M. OECD guidelines for acute oral toxicity studies: An overview. Int J Res Ayurveda Pharm. 2023;14:137\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHesham HM, Lasheen DS, Abouzid KAM. Chimeric HDAC inhibitors: Comprehensive review on the HDAC-based strategies developed to combat cancer. Med Res Rev. 2018;38:45\u0026ndash;66.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJenke R, Re\u0026szlig;ing N, Hansen FK, Aigner A, B\u0026uuml;ch T. Anticancer therapy with HDAC inhibitors: Mechanism-based combination strategies and future perspectives. Cancers. 2021;13:634.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKarin M, Bevanda M, Babić E, Mimica M, Bevanda-Glibo D, Volarić M, Pravdić D. Correlation between biochemical and histopathological parameters in patients with chronic hepatitis C treated with pegylated interferon and ribavirin. Psychiatr Danub. 2014;26:8\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKarmaus AL, Mansouri K, To KT, Blake B, Fitzpatrick J, Strickland J, Kleinstreuer N. Evaluation of variability across rat acute oral systemic toxicity studies. Toxicol Sci. 2022;188:34\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLala V, Zubair M, Minter D. (2023). Liver function tests. StatPearls.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu J, Yao Y, Cheng Y, Hua W, Zhu X, Miao Q, Huang G, Mi S, Ruan R. Acute oral toxicity evaluation of almond hull powders in BALB/c mice. Foods. 2023;12:4111.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOECD. (2001). Guideline for Testing of Chemicals, Guideline 423: acute Oral Toxicity-Acute Toxic Class Method 2001.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePai P, Reddy Y, Das I, Venkidesh BS, Bhandari P, Rao P, Oruganti S, Prasad K, Shetty MG, Satyamoorthy K, Sundara BK. Targeting neuroblastoma with hydroxamic acid based HDAC1 and HDAC2 inhibitors: Insights from in vitro and in vivo studies. Investig New Drugs. 2025;1:1\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRaghavendra NM, Pingili D, Kadasi S, Mettu A, Prasad SVUM. Dual or multi-targeting inhibitors: The next generation anticancer agents. Eur J Med Chem. 2018;143:55\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRobinson S, Delongeas JL, Donald E, Dreher D, Festag M, Kervyn S, Chapman K. A European pharmaceutical company initiative challenging the regulatory requirement for acute toxicity studies in pharmaceutical drug development. Regul Toxicol Pharmacol. 2008;50:345\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSoueid J, Nokkari A, Makoukji J. Techniques and methods of animal brain surgery: perfusion, brain removal, and histological techniques. Taylor Francis. 2015;1:167\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStokes WS. Humane endpoints for laboratory animals used in regulatory testing. ILAR J. 2002;43:S31\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang X, Schnackenberg LK, Shi Q, Salminen WF. (2014). Hepatic toxicity biomarkers. In Biomarkers in Toxicology, pp 241\u0026ndash;259.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang L, Zhang J, Jiang Q, Zhang L, Song W. Zinc binding groups for histone deacetylase inhibitors. J Enzyme Inhib Med Chem. 2018;33:714\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e\u003c/ol\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":"bmc-pharmacology-and-toxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"phat","sideBox":"Learn more about [BMC Pharmacology and Toxicology](http://bmcpharmacoltoxicol.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/phat/Default.aspx","title":"BMC Pharmacology and Toxicology","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Acute oral toxicity, BALB/c mice, Histone deacetylase inhibitor, Hydroxamic acid, Lethal dose, OECD 423","lastPublishedDoi":"10.21203/rs.3.rs-6988823/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6988823/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHistone deacetylases (HDACs) are enzymes responsible for removing acetyl groups from histone proteins, resulting in chromatin condensation and repression of genes. They regulate expression of genes, cell cycle, and multiple cellular processes. Hydroxamic acid is a well recognized moiety characterized by its potent zinc-binding capability, making it an effective inhibitor of HDACs. A novel hydroxamic acid based molecule, N\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e-(2,2'-bipyridin-6-yl)-N\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e-hydroxyoctanediamide (compound 3B) was previously synthesized and the anticancer properties of the compound were examined \u003cem\u003ein vitro\u003c/em\u003e in our laboratory. No prior toxicological study has been done on this compound. Therefore, the current investigation concentrated on the acute oral toxicity of compound 3B on female BALB/c mice, adhering to OECD 423 guidelines. In this study, compound 3B was given orally at 300 mg/kg body weight (b.w.) and 2000 mg/kg b.w. The food consumption and body weight of the mice did not differ significantly among the control and treated groups. Variations were observed in the levels of a few of the biochemical markers. Histopathological examination showed inflammatory infiltrate, and lesions in a few vital organs. The comprehensive investigation showed that compound 3B exhibited moderate toxic effects at a higher dosage of 2000 mg/kg in few organs and caused alterations in biochemical markers; however, it did not result in any mortality, indicating an LD\u003csub\u003e50\u003c/sub\u003e value exceeding 2000 mg/kg. The dosage of compound 3B can be administered at levels below 2000 mg/kg for subsequent studies.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e","manuscriptTitle":"In vivo Acute Oral Toxicity Assessment of Novel Histone Deacetylase 2 Inhibitor","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-18 11:28:20","doi":"10.21203/rs.3.rs-6988823/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-30T17:28:58+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-30T06:26:48+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-23T03:07:00+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-20T08:23:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"34308375918654146730050554673669604275","date":"2025-09-14T17:04:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"89673684982880664495506349662895054882","date":"2025-09-13T11:45:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"8926933673238072774373509613383418598","date":"2025-09-12T12:50:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"76723432737548456417274711613619557674","date":"2025-09-12T06:41:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-11T18:35:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"230778514359045027551893574079040321216","date":"2025-09-11T05:47:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"118835149395204551990743021070721516782","date":"2025-09-11T03:32:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"329045622188533974288051829794880352732","date":"2025-09-10T23:51:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"35286765829160153849602318883615301068","date":"2025-09-10T18:30:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"159147866843600991792109995440582549027","date":"2025-09-10T13:34:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"225161128835610082831911747530334309272","date":"2025-09-10T13:33:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"326298317419269564675970130514777736585","date":"2025-09-10T13:31:52+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-10T12:38:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-10T12:01:41+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-10T10:46:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-07T14:36:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Pharmacology and Toxicology","date":"2025-09-07T14:33:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-pharmacology-and-toxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"phat","sideBox":"Learn more about [BMC Pharmacology and Toxicology](http://bmcpharmacoltoxicol.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/phat/Default.aspx","title":"BMC Pharmacology and Toxicology","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1f7a34c7-b3a6-4280-ab9a-179c9893d52e","owner":[],"postedDate":"September 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-01T16:12:03+00:00","versionOfRecord":{"articleIdentity":"rs-6988823","link":"https://doi.org/10.1186/s40360-025-01040-9","journal":{"identity":"bmc-pharmacology-and-toxicology","isVorOnly":false,"title":"BMC Pharmacology and Toxicology"},"publishedOn":"2025-11-28 15:58:51","publishedOnDateReadable":"November 28th, 2025"},"versionCreatedAt":"2025-09-18 11:28:20","video":"","vorDoi":"10.1186/s40360-025-01040-9","vorDoiUrl":"https://doi.org/10.1186/s40360-025-01040-9","workflowStages":[]},"version":"v1","identity":"rs-6988823","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6988823","identity":"rs-6988823","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}