Acute and sub-acute oral toxicity assessment of 5-hydroxy-1,4-naphthoquinone in mice

Abstract 5-hydroxy-1,4-naphthoquinone (5NQ) or juglone is a bioactive molecule found in walnuts and has shown therapeutic effects in various disease models. Limited information is available regarding the toxicity of 5NQ, thereby limiting the clinical development of this drug. In the present study, oral acute (50, 300 and 2000 mg/kg) and sub-acute toxicity (5, 15 and 50 mg/kg) was assessed in mice to evaluate the safety of 5NQ. The acute toxicity study identified 118 mg/kg as the point-of-departure dose (POD) for single oral administration of 5NQ using benchmark dose modeling (BMD). Repeated administration of 5NQ at doses of 15 and 50 mg/kg/day caused reduction in food consumption and body weight of mice along with alterations in liver and renal function. Histopathological assessment revealed significant damage to hepatic and renal tissues at all doses in the acute toxicity study, and at higher doses of 15 and 50 mg/kg in the sub-acute toxicity study. We observed dose dependent mortality in sub-acute toxicity study and the no observed adverse effect level (NOAEL) was established as < 5 mg/kg/day. Modeling the survival response in sub-acute toxicity study identified 1.74 mg/kg/day as the POD for repeated administration of 5NQ. Serum levels of aspartate aminotransferase (AST) were most sensitive to 5NQ administration with a lower limit of BMD interval (BMDL) of 1.1 × 10−3  mg/kg/day. The benchmark doses reported in the study can be further used to determine a reference dose of 5NQ for human risk assessment.

Introduction 5-hydroxy-1,4-naphthoquinone (5NQ) or Juglone is a phenolic compound found abundantly in fresh leaves and green husk of several species of walnut trees belonging to the Juglandaceae family (Juglans nigra, Juglans regia, Juglans cinera and Carya illinoinensis). It is also found in smaller quantities in the roots, bark and nuts of the walnut tree (Bello et al. 2018, McCoy et al. 2018, Jahanban-Esfahlan et al. 2019. As the name suggests, 5NQ belongs to the naphthoquinone class of drugs and contains an aromatic ring hydroxylated at the 5-position and a quinone ring with oxygen at positions 1 and 4. Table 1 describes the physico-chemical characteristics of 5NQ (NCI 2002, Juglone j C10H6O3 -PubChem 2021. The therapeutic potential of 5NQ has been tested in a range of clinical conditions. It exhibits anti-bacterial (Wang, Cheng, et al. 2016, Wang, Liu, et al. 2016, anti-fungal (Gumus et al. 2020) and anti-parasitic (Jha et al. 2015, Moghadaszadeh et al. 2021 activity at concentrations ranging between 0.1 and 0.5 mM. It shows anti-viral (Cui and Jia 2021), anti-inflammatory (Seshadri et al. 2011, Gaikwad et al. 2019, Kim et al. 2020) and anti-cancer activity (Aithal et al. 2012, Catanzaro et al. 2018, Wang et al. 2018) at concentrations ranging between 3 À 25 mM. Notably, 5NQ is a redox active molecule and has a strong ability to generate reactive oxygen species (ROS) (Klotz et al. 2014, Ahmad and Suzuki 2019, Majiene et al. 2019, Mesalam et al. 2020. This redox activity of 5NQ is responsible for its pharmacological actions and affects various cellular pathways (Wang et al. 2018, Kao et al. 2021, causing loss of mitochondrial and plasma membrane potential (Ji et al. 2010, Anaissi-Afonso et al. 2018, Majiene et al. 2019, Mesalam et al. 2020, DNA damage (Kiran Aithal et al. 2009, Ourique et al. 2015, cellular degeneration and apoptosis (Seshadri et al. 2011). Despite being a potent therapeutic agent, clinical application of 5NQ faces many challenges as few reports are available for its toxicity in normal tissues, lethal dose (LD 50 ), favorable solvent and route of administration. Westfall and team (1961), reported a LD 50 of 2.5 mg/kg in mice, however, no information about the route of administration or duration of dosing was specified (Westfall et al. 1961). To our knowledge, the study by Aithal and group (2012), is the only one to have systematically determined single-dose, intravenous (iv) acute toxicity of 5NQ, wherein they reported an iv LD 50 of 4.18 mg/kg in healthy mice (Aithal et al. 2012). Further, using tritium labeled 5NQ, they studied the pharmacokinetics and biodistribution of the drug at an iv dose of 0.02 mg/kg and reported the iv-plasma half-life of 5NQ to be approximately 2 h. They found the highest distribution of 5NQ to be in the kidneys followed by the liver and also reported significant nephrotoxicity in 5NQ (1 mg/kg for 7 days) treated mice (Aithal et al. 2011). Furthermore, the therapeutic effect of 5NQ has also been studied using the intraperitoneal (ip) route of administration. Intraperitoneal administration of 5NQ at doses in the range of 1-4 mg/kg for 10 days was reported to be safe and did not cause mortality due to toxicity (Ourique et al. 2015, Wang et al. 2018. Only one study has reported that oral administration of 5NQ at 0.05 mg/kg in carboxymethylcellulose (CMC) is safe and effective against colon carcinoma when administered for 60 days (Seetha et al. 2020(Seetha et al. , 2021, but no information regarding its toxic effects on other organs was available. Although substantial information regarding the safe dose for intravenous administration of 5NQ is available, one cannot overlook the challenges associated with the iv and ip routes of administration for clinical use (Westfall et al. 1961, Boelkins et al. 1968, Bolton et al. 2000, Aithal et al. 2011. Furthermore, the low solubility and hydrophobic nature of 5NQ, demands the use of solvents like DMSO and methanol for iv and ip administration, making it unfavorable for clinical use (Wong et al. 2008, Patel et al. 2020. Considering the low solubility of 5NQ, the oral route of administration may be the most favorable for the clinical translation of this molecule. However, there is no literature available on the oral LD 50 and oral toxicity of 5NQ on major organs in healthy mice. To bridge this gap and to facilitate the clinical application of 5NQ, our study aimed to evaluate oral acute and 28 days repeat dose toxicity of 5NQ in mice using standard guidelines of Organization for Economic Co-operation and Development (OECD) and to determine the oral LD 50 and a no observed adverse effect level (NOAEL) of 5NQ. Further, we evaluated the dose response relationship of effects of prolonged administration of 5NQ in healthy mice using benchmark dose (BMD) modeling (Crump 1995(Crump , 2008. BMD modeling is a regulatory recommended, scientifically and statistically advanced approach, to estimate a point of departure (POD) or a guidance dose level for hazard risk assessment (Hardy et al. 2017, Haber et al. 2018, Slob 2018, Barali c et al. 2020). The BMDL (lower limit of the benchmark dose) and NOAEL can be used to determine an appropriate safe dosage of 5NQ and to evaluate its therapeutic efficacy in preclinical murine models for its clinical application.

Animals
All animal studies were conducted after approval from the Institutional Animal Ethics Committee, ACTREC, as per the Committee for the Purpose of Control and Supervision of Experiments on Animals, Government of India guidelines (IAEC/10/2021). All mice were housed in polypropylene cages, maintained under standard conditions of 22 ± 2 C, 55 ± 5% relative humidity and 12 h light-dark cycles and were provided with a standard pelleted diet and plain drinking water ad libitum.

Acute oral toxicity study
Acute oral toxicity study was conducted in accordance with annex 2c of the OECD guideline 423 (OECD 2002). Female BALB/c mice were divided into four groups and were treated with single dose of either vehicle (DMSO, n ¼ 3), 50 (n ¼ 6), 300 (n ¼ 6), and 2000 (n ¼ 3) mg/kg body weight of 5NQ dissolved in 0.1 ml of DMSO by oral gavage. Mice were kept fasting for 2 h before administration of 5NQ. After treatment, animals were observed for the first 30 min to 4 h and intermittently every 24 h for 14 days. Behavioral changes were observed consciously for the first 4 h for signs of tremor, convulsions, salivation, diarrhea, dyspnea, piloerection and lethargy. Body weight was recorded at the start of the protocol, on the 7th day and at the end of the protocol. At the end of the study, animals were euthanized via CO 2 asphyxiation, and vital organs (liver, lung, heart, kidney, spleen, intestine, brain and femur) were collected and fixed in 10% formalin solution for histopathological analysis.

Sub-acute toxicity study
Sub-acute toxicity study of 5NQ was conducted in accordance with OECD guideline 407 (OECD 2008). BALB/c mice (5 males and 5 females per group, n ¼ 10) were randomly divided into four groups: Group I (vehicle control) received vehicle [5% DMSO in 0.5% carboxymethylcellulose (CMC)], while groups II, III, and IV received 5, 15, and 50 mg/kg body weight of 5NQ, respectively. The decision for the selection of oral doses of 5NQ for the sub-acute toxicity study was made following the recommendations provided under the dosage section of the OECD guideline 407. Accordingly, the highest dose was the one that induced toxic effects but not severe suffering or mortality post administration of a single dose of the drug. Therefore, 50 mg/kg was selected as the highest dose and 5 mg/kg as the lowest dose (tenfold lower than the highest dose), whereas, 15 mg/kg (geometric mean of the lowest and the highest dose) was selected as the intermittent dose. Mice were administered daily with respective doses of 5NQ using oral gavage for 28 days. After treatment, animals were observed for the first 30 min and every 24 h for mortality. Body weight was recorded at the start of the protocol and once every week. All animals were monitored for clinical signs of toxicity every week, and a clinical score was assigned to each animal using a general score sheet adapted from a report published by JM Vlissingen et al. (Fentener van Vlissingen et al. 2015). All animals were scored based on five general clinical signs (body weight reduction, posture, vocalization, hypokinesia and piloerection), which may indicate the existence of a severe condition in these animals. Food consumption was also monitored weekly. The total food consumed per cage was recorded and the weekly mean intake per mice per day was calculated. All animals were subjected to overnight fasting post the last dose on the 28th day. The next day, the mice were anesthetized and blood was collected through orbital plexuses for haematological and biochemical analysis. Mice were euthanized via CO 2 inhalation and vital organs (liver, lung, heart, kidney, spleen, intestine, brain and femur) were collected and fixed in 10% formalin solution for histopathological analysis.

Analysis of biochemical parameters
Blood was collected in dry microcentrifuge tubes, and allowed to clot at room temperature for 1 h. Serum was separated via centrifugation at 3000 rpm for 10 min. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (TB), albumin (ALB), total protein (TP), blood urea nitrogen (BUN), creatinine (CRE) and uric acid (UA) were quantified by standard clinical chemistry assays using Siemens Autoanalyzer (Dimension EXL 200, Germany).

Histopathologic examination
After sacrifice, tissue samples were fixed in 10% formalin and dehydrated overnight using upgraded ethanol series and embedded in paraffin blocks. Ultrathin sections (5 mm) were de-waxed using xylene, hydrated through a degraded ethanol series, and stained with hematoxylin and eosin (H&E). A pathologist, blinded to the treatments, performed the histopathologic examination with an optical microscope (Zeiss Axio Imager.Z1).

Statistical analysis
Body weight, organ weight, serum parameters, and haematological parameters were expressed as mean ± standard error of mean (SEM). Two-way analysis of variance (ANOVA) with Bonferroni correction was applied to evaluate time and dose dependent differences between groups, one-way ANOVA was applied with Bonferroni correction to evaluate significant differences between groups using Prism 8.2.0 software. Values of p < 0.05 were considered significant.

Benchmark dose modeling
Benchmark dose-response modeling was performed using the EFSA web tool for BMD analysis (https://efsa.openanalytics.eu/), which uses the R-package PROAST, version 70.0, for the related calculations. All the parameters determined in the study, i.e., absolute organ weights, haematological and serum parameters (continuous individual data) and survival (quantal data) were modeled according to the software guidelines (PROAST j RIVM 2022) and the European Food Safety Authority (EFSA) guideline (Hardy et al. 2017). The Benchmark response (BMR) or the critical effect size (CES), is defined as the minimal physiological adverse response that can be distinguished from control responses (Slob 2017). For quantal data, a BMR of 10% at the two-sided 90% confidence level was used as recommended by Scientific Committee of the EFSA (Hardy et al. 2017). For continuous data, CES was defined for each parameter based on the reference values proposed by Buist et al. (2009) and Dekkers et al (2006) (Dekkers et al. 2006, Buist et al. 2009). The software calculated the Benchmark dose interval (BMDI) that consists of lower (BMDL) and upper (BMDU) BMD confidence limits using different mathematical models (Slob 2018, Varewyck andVerbeke 2019). The determined BMDL value was analogous to the POD or NOAEL (Hardy et al. 2017). Akaike information criteria (AIC) were applied to obtain the best model of dose-response. Finally, the model averaging method with 200 iterations was used to fit all available models in one, as per EFSA recommendations (Hardy et al. 2017, Haber et al. 2018, Slob 2018.

In silico ADMET analysis
The pharmacological activity of a molecule can be described by its ADME properties. ADMETlab2.0 uses the complex molecular structure of a molecule to predict its drug-likeness, pharmacokinetics and physicochemical properties of a compound. Table 2 summarizes all the ADMET properties of 5NQ. Briefly, the physicochemical properties of 5NQ showed optimal TPSA (Topological Polar Surface Area), logS (log of aqueous solubility), logP (log of water partition coefficient) and logD (logP at physiological pH 7.4) values. Medicinal chemistry predictions show that 5NQ obeys Lipinski, Pfizer, GSK rule and has 1 alert for thiol reactive compound, indicating it has optimal drug-likeness properties. Prediction of absorption properties of 5NQ showed optimal Caco-2, MDCK permeability and Human Intestinal Absorption (HIA) probability. The probability of Plasma Protein Binding (PPB) of 5NQ was found to be 72.26% and its Volume Distribution (VD) was determined to be 0.405 L/Kg. Distribution profile also depicted 5NQ might penetrate Blood Brain Barrier (BBB). Furthermore, the bioavailability of 5NQ was estimated to be higher than or equal to 30% thus, estimating that the drug may have optimal oral bioavailability. Predictions of the metabolism profile showed 5NQ was neither CYP3A4 substrate nor CYP3A4 inhibitor but the probability of being a CYP1A2 inhibitor was 0.933 indicating it may have low metabolism rate. Additionally, 5NQ was shown to have moderate clearance (CL) of 6.788 mL/min/Kg and a shorter half-life (T 1/2 ) of less than 3 h. Notably, predictions for probable toxicity of 5NQ showed that 5NQ may cause drug induced liver injury (DILI) and mutagenicity. However, the probability of causing human-hepatotoxicity (H-HT) and rat oral acute toxicity were found to be low.

Survival
All animals administered with 2000 mg/kg (n ¼ 3) of 5NQ died within 48 h of administration. In the 300 mg/kg group, 1 animal out of 3 died after 72 h of drug administration. Therefore, according to OECD guideline 423, 3 more animals were administered with 300 mg/kg of 5NQ. In the second cohort, all the animals died within 72 h of drug administration. All animals in the 50 mg/kg (n ¼ 6) group survived the 14 days observation period (Figure 1(A)).

Body weight
No significant changes were observed in the body weight of mice in any of the groups (Figure 1(B)).

Clinical signs
Animals were observed once every 24 h for clinical signs of toxicity. All animals in the 2000 mg/kg group showed behavioral changes like huddling, decreased movement, dyspnea, piloerection and mild tremor however, diarrhea was absent. Similar signs were observed in the mice treated with 300 mg/ kg of 5NQ. However, 2 out of 6 mice recovered 48 h after drug administration. Mice in the 50 mg/kg group showed no such behavioral changes during the observation period.

Histopathological analysis: liver
Histopathological analysis of liver sections from mice treated with vehicle control showed normal architecture and morphology. Liver sections of animals administered with a single oral dose of 50 mg/kg and 300 mg/kg showed hepatocellular degenerative changes in form of ballooning and focal mononuclear infiltration. In addition to these changes, liver sections from animals dosed with 2000 mg/kg showed disruption of normal lobular architecture of the liver and dilated and congested vessels along with previously mentioned degenerative changes (Figure 1(C)).

Kidney
Animals in the vehicle control and 50 mg/kg group showed few dilated and congested vessels in the kidney. Those in the 300 mg/kg and 2000 mg/kg groups showed focal tubular degeneration, tubular edema, mild mononuclear infiltrate and focal glomerular proliferation along with dilated and congested vessels, indicating toxicity related changes in kidney tissue (Figure 1(C)).
No major histopathological changes were observed in other tissues like lung, heart, brain, intestine and femur post single dose of 5NQ ( Figure 2).

Sub-acute toxicity
Body weight, clinical score and food intake The animals treated with 15 mg/kg and 50 mg/kg showed a significant reduction in body weight post 5NQ treatment (Figure 3(A)). This reduction in body weight in the 15 mg/kg and 50 mg/kg groups corroborated with the decrease in food intake observed in these groups (Figure 3(B)). Organ to body weight ratios did not show any significant changes when compared to vehicle control, however, absolute organ weights of liver, spleen and kidney showed significant decrease in animals treated with 5, 15 and 50 mg/kg when compared to vehicle control mice (Table 3). Moreover, animals dosed with 15 mg/kg and 50 mg/kg 5NQ showed clinical signs of toxicity in the first week itself resulting in significant increase in clinical scores, which gradually increased during the period of the experiment. Mice administered with 5 mg/kg 5NQ did not show any clinical signs of toxicity, and thus scored low on the clinical score chart (Figure 3(D)).

Survival
Animals in the vehicle control and 5 mg/kg group did not show any toxicity related mortality. Mice administered with 15 mg/kg and 50 mg/kg of 5NQ for 28 days showed significantly reduced survival with median survival of 27 and 12 days, respectively (Figure 3(C)).

Haematological parameters
Repeated oral administration of 50 mg/kg of 5NQ resulted in significantly high platelet counts as compared to vehicle control mice. Other haematological parameters remained unaffected in all the groups (Table 4).

Serum biochemistry
Assessment of liver function revealed significantly increased levels of AST and ALT in mice treated with 15 mg/kg and 50 mg/kg of 5NQ as compared to vehicle control mice, indicative of hepatic dysfunction. In animals administered with 5 mg/kg, only AST levels were mildly elevated. Additionally, levels of ALP, total bilirubin and total protein significantly decreased in mice treated with 50 mg/kg of 5NQ as compared to vehicle control mice, indicating loss of liver function. Significant increase was observed in uric acid and blood urea nitrogen content in animals administered with 15 mg/kg and 50 mg/kg 5NQ as compared to vehicle control mice, indicating mild loss of kidney function in these animals. Administration of 5NQ did not affect serum creatinine levels (Table 5).

Histopathological analysis: liver
Liver tissues of all animals administered with 50 mg/kg 5NQ, showed severe hepatocellular degeneration in the form of diffuse ballooning of hepatocytes when compared to liver tissues of animals administered with the vehicle. Liver sections of animals administered with 5 mg/kg and 15 mg/kg 5NQ showed mild to moderate hepatocellular degeneration in form of focal to diffuse ballooning of hepatocytes when compared to vehicle control liver tissues. These findings relate to the abnormally high serum levels of AST and ALT observed in animals administered with 15 mg/kg and 50 mg/kg 5NQ (Figure 4).

Kidney
Renal tissues of animals administered with 15 mg/kg and 50 mg/kg 5NQ showed mild to moderate tubular edematous change and scarce mononuclear infiltration compared to vehicle control, indicating renal damage. No histological changes were observed in renal tissues of animals administered with 5 mg/kg 5NQ (Figure 4).

Spleen
Histological examination of spleen tissues of animals administered with 15 mg/kg and 50 mg/kg 5NQ showed an increase in number of megakaryocytes. This finding correlates with the increase in platelet counts observed in peripheral blood of animals administered with 50 mg/kg 5NQ. Animals administered with 5 mg/kg 5NQ did not show increase in megakaryocytes ( Figure 5).
No major histological changes were observed in other tissues like lung, heart, brain, intestine and femur post repeated administration of 5NQ for 28 days ( Figure 5).
Benchmark dose analysis: In the acute toxicity study, dose dependent mortality was observed and BMD modeling using the survival data established a BMDL of 118 mg/kg, which can be considered as the point of departure dose for single oral administration of 5NQ (Figure 6(A), Table 6). Similar dose dependent mortality was observed in the sub-acute toxicity study and BMD modeling using the survival data established a BMDL of 1.74 mg/kg/day (Figure 6(B), Table 7) which could be considered as the point of departure dose for repeated administration of 5NQ and can be deemed an equivalent measure to the NOAEL dose.
In sub-acute toxicity, levels of AST, ALT, ALP, total bilirubin, total protein, blood urea nitrogen, and platelets showed dose response relationship. BMD modeling was carried out and a BMDL was established for each of these parameters. AST levels were most sensitive to 5NQ exposure and showed the lowest BMDL of 1.1 Â 10 À3 mg/kg/day, followed by total bilirubin with BMDL of 0.03 mg/kg/day. BMDL for total protein and uric acid was established as 6.35 and 1.82 mg/kg/ day respectively. Although ALP and ALT levels showed statistically significant differences compared to control levels, no  in all groups at the start of the experiment i.e., day 0 and at day 7, 14, 21 and 28 days. Data is expressed as mean ± SEM (N ¼ 10/group), two-way ANOVA with Bonferroni correction was applied to evaluate time and dose dependent differences between groups ( Ã p < 0.05, ÃÃ p < 0.01, ÃÃÃ p < 0.001, ÃÃÃÃ p < 0.0001) (B) Average food consumption of mice per day in each group. The total food consumed per cage was recorded and the weekly mean intake per mice was calculated. Each data point represents average food consumption per mice/day/week. A single data point in the 50 mg/kg group represents the average food consumption/day in week 1 of 5NQ oral dosing. Data is expressed as mean ± SEM, one-way ANOVA with Bonferroni correction was applied to evaluate dose dependent differences between groups ( ÃÃÃ p < 0.001, ÃÃÃÃ p < 0.0001). (C) Survival of mice in each group was analyzed using Kaplan-Meier plot and log rank test (N ¼ 10) was used to compare the difference between control and treated groups. Mice administered 15 and 50 mg/kg of 5NQ showed significant reduction in survival with a median survival of 27 ( Ã p 0.012) and 12 days ( ÃÃ p 0.001) respectively. No mortality was observed in animals administered with 5 mg/kg 5NQ (p > 0.9). (D) Weekly clinical score of mice in each group expressed as mean ± SEM (N ¼ 10), one-way ANOVA with Bonferroni correction was applied to evaluate dose dependent differences between groups per week ( ÃÃÃ p < 0.001, ÃÃÃÃ p < 0.0001). trend was observed in the BMD modeling and hence a BMDL value for these parameters could not be established. Among the haematological parameters platelet count increased in a dose dependent manner with a BMDL of 0.5 mg/kg/day. The BMD modeling results are summarized in Table 8  Discussion 5-hydroxy-1,4-naphthoquinone is a promising drug candidate with immense therapeutic potential. Its therapeutic effect can be attributed to its ability to produce free radicals which affect various cellular pathways (Ahmad and Suzuki 2019). Our in-silico data indicates that 5NQ has favorable ADME properties and therefore is a good drug candidate for further development, but the toxicity estimates of it being a mutagenic agent, and an eye and skin irritant are high. Additionally, like most quinone compounds, the metabolism of 5NQ is known to produce toxic metabolites (Bolton et al. 2000), and hence its therapeutic effects have to be evaluated in the context of potentially toxic effects. Thus, the use of 5NQ as a therapeutic agent comes with considerable risks making it important to have complete information regarding its toxicity profile. The present study aimed at evaluating the oral acute and sub-acute toxicity of 5NQ and determining a safe dose for conducting extensive preclinical studies in murine models to evaluate the therapeutic potential of 5NQ. Along with using the traditional approach of establishing the NOAEL (lowest dose of the compound at which no adverse effects were observed) we employed benchmark dose modeling for deriving a point of departure dose for the daily administration of 5NQ and to identify the most sensitive marker of toxicity to 5NQ. Our acute toxicity data showed that administration of a single oral dose of 50 mg/kg 5NQ was well tolerated and did not cause mortality in mice. However, histopathological evaluation of these animals revealed significant damage to the liver and to a certain extent to the kidneys. Based on our acute toxicity data, we can classify 5NQ as a category 3 substance using the OECD guideline 423 and the Global Harmonized System of classification of substances and mixtures, which indicates that the compound has an acute LD 50 cutoff dose of > 50 to 300 mg/kg body weight (Organization for Economic Co-Operation and Development (OECD)) 2002). Additionally, the observed BMDL of 118 mg/kg may be considered as the point of departure dose for single dose administration of 5NQ, beyond which mortality may be expected.
Sub-acute toxicity study revealed that daily administration of 5 mg/kg 5NQ for 28 days was safe and did not show toxicity related mortality in mice. A notable fact in the sub-acute toxicity study was that mice administered with 5 mg/kg 5NQ  Values are mean ± SEM for (N ¼ 10/group). The differences between control and treated groups were analyzed by ANOVA with Bonferroni correction. The significance levels observed is ÃÃ p < 0.0021 in comparison to control group values; ns: non significant; ns: non significant. for 28 days showed significant weight loss over the 4 weeks period. Lou and his team reported that mitochondrial uncoupling causes loss of mitochondrial membrane potential, thwarting the energy transduction through the electron transport chain. This results in ATP depletion and leads to more fatty acid oxidation for producing ATP through oxidative phosphorylation (Lou et al. 2007). 5NQ is reported to be a narrow-range mitochondrial uncoupler, and higher doses can cause complete mitochondrial uncoupling, leading to ATP depletion, hyperthermia and even death (Saling et al. 2011). Therefore, the observed decrease in body weight can be possibly the result of its mitochondrial uncoupling action. Mortality of mice observed at higher doses of 15 and 50 mg/ kg in the sub-acute toxicity study may be possibly related to the cellular damage caused by mitochondrial uncoupling action of 5NQ. Even though mortality was not observed in animals administered 5NQ at 5 mg/kg/day, the decrease in body weight indicates mild toxicity. Therefore, we estimate the NOAEL for the daily administration of 5NQ to be < 5 mg/ kg/day. The traditional NOAEL approach for determining the POD is limited by the selection of doses in the study and often results in overestimation of POD especially when the sample size is small (Hardy et al. 2017, Barali c et al. 2020. Modeling the survival response in the sub-acute toxicity study established 1.74 mg/kg/day as the POD, which was 65% lower than the lowest dose of 5 mg/kg/day used in the study. Thus, the mathematical approach of BMD converges with the NOAEL approach for identifying the safe dose, while being more accurate in estimating the POD. This dose can be further used for determining the human equivalent reference dose for risk assessment (Conducting a Human Health Risk Assessment j US EPA 2022). Metabolism of 5NQ using isolated rat liver has been well documented and is known to affect various metabolic pathways via its uncoupling effect on mitochondria of hepatocytes. As with most quinones, 5NQ undergoes one or two electron reductions to give rise to semiquinone radicals or hydroquinone (Bolton et al. 2000, Klotz et al. 2014). This reduction is catalyzed by various flavoprotein enzymes like NADPH-cytochrome P450 reductase and NAD(P)H: quinone oxidoreductase. The hydroquinone thus formed is further catalyzed and forms conjugates with glucuronide or sulfate, leading to detoxification of quinones while the formation of free radicals especially superoxide (Bolton et al. 2000). At the same time, 1,4-naphthoquinones like 5NQ are known to interact and form hydroquinone conjugates with glutathione (GSH) and N-acetyl cysteine (NAC). These GSH conjugates undergo auto-oxidation thus maintaining the redox activity of these compounds which in turn leads to the formation of more free radicals (Chen et al. 2005). Thus, it is evident that biotransformation of 5NQ in the liver and generation of free radicals as by products may have a toxic effect on hepatocytes. Despite this knowledge, no toxicity study has reported hepatic toxicity of 5NQ. Our study is the first to report dose dependent hepatic toxicity of oral 5NQ administration. The abnormal serum levels of liver function markers ALT, AST, ALP and total bilirubin along with marked hepatic degenerative changes observed by histopathology after repeated oral administration of 5NQ, provide proof of the cytotoxic effect of 5NQ on hepatocytes. In addition, our results confirm our findings of in silico study, which predicted a high probability of drug induced liver injury (DILI). Conversely, predicted probability of human-hepatoxicity (HT) and rat oral acute toxicity was very low, and therefore, there is a chance that this toxic effect may not manifest during clinical development of this drug.
The available pharmacokinetic and biodistribution studies with 5NQ have shown the highest accumulation of 5NQ in kidney tissues irrespective of the route of administration (Chen et al. 2005, Aithal et al. 2011, 2012. In a study by Chen et al. (2005), it was reported that the glucuronide and sulfate conjugates formed due to 5NQ metabolism are mainly Figure 5. Representative micrographs of spleen, intestine, lung, heart brain and femur sections stained with H&E, corresponding to mice treated with vehicle control (VC) and with different doses of 5NQ, daily for 28 days (20Â magnification).
excreted via urine and have a potent nephrotoxic effect. This nephrotoxic effect of 5NQ was consistent irrespective of the route of administration and can be attributed to the covalent binding of 5NQ or its metabolites (mono-glucuronide of 4,8dihydroxy-1-tetralone) to cytosolic protein a2u-globulin (lipocalin class of proteins) causing hydrocarbon related nephropathy (Bolton et al. 2000, Chen et al. 2005, Saling et al. 2011, Klotz et al. 2014. Moreover, quinone thiol-ethers are known to cause nephrotoxicity when administered intravenously (Sanders et al. 1998, Monks andLau 1992). Our findings of abnormal levels of renal function markers uric acid and BUN along with marked renal tissue damage observed in   animals administered with 15 mg/kg and 50 mg/kg 5NQ for 28 days are consistent with available reports of nephrotoxic effect of 5NQ.
Our study focused on oral toxicity and provides proof of potential hepatic and renal toxicity of 5NQ; however, these toxic effects were observed at doses much higher than those reported in earlier studies with parenteral administration (Westfall et al. 1961, Aithal et al. 2011, 2012. Additionally, 5NQ may be a good candidate for oral administration because it is likely to have favorable oral bioavailability based on in silico predictions. Also, systemic toxicities observed at relatively low doses in our study point toward good oral bioavailability which needs to be confirmed through pharmacokinetic studies. It is pertinent to note that although administration of 5 mg/kg 5NQ did not cause toxicity related mortality in mice, we observed mild elevation of AST, a marker of liver injury, upon repeated administration. Moreover, dose -response modeling of the serum AST levels, showed that it was the most sensitive marker of toxicity to 5NQ with the lowest BMDL of 1.1 Â 10 À3 mg/kg/day. Having a recovery group would have enabled us to understand the reversibility of this process and should be addressed in future studies. Earlier reports have shown that the liposomal formulation of 5NQ for iv administration improved its anti-cancer efficacy while reducing its systemic toxicity in mice (Aithal et al. 2011). Therefore, it follows that the safety of oral 5NQ administration can be further enhanced using appropriate drug delivery systems to harness its therapeutic potential. Nonetheless, the results of our study provide a reasonable estimate of the safe dose and duration for repeated oral administration of 5NQ which can be used for determining the dosage for pre-clinical evaluation of 5NQ's efficacy in various disease models.

Conclusion
In conclusion, we established 118 mg/kg as the POD for single oral administration of 5NQ. Furthermore, from the subacute toxicity, we established 1.74 mg/kg/day as POD for repeated administration of 5NQ. Serum AST was identified as the most sensitive marker of toxicity to 5NQ. The established POD values can be used as a reference for pre-clinical efficacy assessments and for deriving the human equivalent reference dose of 5NQ to ascertain its safety for clinical use. AST: Aspartate amino transferase; CES: critical effect size; BMR: benchmark response; BMDL: Lower confidence limit of the Benchmark dose; BMDU: upper confidence limit of the Benchmark dose.