Assessment of Acute and Chronic Toxicity in Wistar Rats (Rattus norvegicus) and New Zealand Rabbits (Oryctolagus cuniculus) of an Enriched Polyphenol Extract Obtained from Caesalpinia spinosa

Although herbal drugs are often considered safe for consumption, there is increasing evidence that some can generate undesirable health effects. However, polyphenols found in certain plants have been shown to provide a range of benefits for human health. In previous work, a standardized and quantified extract (P2Et) obtained from Caesalpinia spinosa (Dividivi) plant showed promising antioxidant, immunomodulatory, and anti-inflammatory properties in animal models of cancer and COVID-19 patients. The extract has also been subjected to genotoxicity, mutagenicity, and 28-day oral chronic toxicity evaluations, demonstrating a good safety profile. To advance preclinical and clinical development, further acute and chronic toxicity evaluations of the P2Et extract were performed. Acute toxicity tests were performed orally in Wistar rats at a dose of 2000 mg/kg, indicating that the lethal dose 50% (LD50) value exceeded 2000 mg/kg and classifying the P2Et extract as category 5 according to the Globally Harmonized System of Classification (GHS). In this work, chronic toxicity tests were conducted for 180 days on Wistar rats and New Zealand rabbits at a dose of 1000 mg/kg under Good Laboratory Practice (GLP) conditions. No weight loss or alterations in biochemical and hematological parameters associated with treatment were observed in the animals, suggesting the absence of toxicity in the assessed parameters. These results indicate that the P2Et extract is safe for oral administration at doses up to 1000 mg/kg body weight over a six-month period.


Introduction
Polyphenols are a group of naturally occurring compounds that have drawn signifcant interest in recent years due to their potential health benefts.Extensive research has established their diverse properties, including antitumor, antidiabetic, anti-infammatory, antioxidant, and immunomodulatory efects, which make them an attractive target for therapeutic applications [1][2][3][4].As a result, the development of polyphenol-enriched foods, dietary supplements, and phytomedicines has become an area of active research and development.Te aim of ofering individuals' safe and efective options for improving their health and well-being.Te growing body of evidence on polyphenols supports the notion that these compounds could provide signifcant health benefts to individuals in the coming years [5].
However, one of the major challenges associated with herbal products is ensuring their quality and safety for human consumption.Chemical and biological variability of herbal drugs can afect their efcacy and safety, and toxicological support is needed to assess their safety profles in chronic consumption [6].It has been reported that certain polyphenols can have toxic efects when ingested in large bolus doses in a dose-dependent manner [7].Tis highlights the importance of conducting comprehensive toxicological evaluations for polyphenol-enriched products.Te FDA's (Food and Drug Administration) herbal drug guidance recommends that a complete toxicological profle should be established for herbal products based on the intended duration and frequency of use, ranging from acute toxicity, genotoxicity, mutagenicity to chronic toxicity [8].
Te safety of P2Et extract has been investigated through preclinical studies, including genotoxicity and mutagenicity assays [18].In addition, subchronic oral toxicity studies were conducted in rats and rabbits according to international guidelines.Te results of the 28-day toxicity study showed no signifcant toxicity, as evidenced by the absence of clinical, hematological, and biochemical changes, as well as macroscopic fndings at necropsy [19].Tese fndings are consistent with other reports on polyphenols, either as isolated compounds or as part of complex extracts, which have shown low oral toxicity and no mutagenic or genotoxic efects [20][21][22][23][24].
In a four-week administration of P2Et to healthy volunteers, the maximum tolerated dose was determined to be 600 mg/daily [19].Tese safety fndings in humans are also supported by a multicenter randomized phase II clinical trial in hospitalized patients with COVID-19, where the administration of 500 mg daily of P2Et for 14 days showed no serious adverse events [25].In Phase I clinical studies, polyphenols derived from gallic acid have been shown to be safe even when administered in increasing doses.Tis evidence further underscores the safety of extracts containing gallic acid polyphenols [26].
To complete the safety profle of P2Et, this study evaluated the acute and chronic toxicity of the extract for 14 and 180 days, respectively, in preclinical models.Te LD 50 value was determined using the guidelines set by Te Organization for Economic Cooperation and Development (OECD) and International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) [28].Tis study also contributes to the broader understanding of the safety profle of polyphenols and herbal drugs, which may have important implications for the development of natural products for therapeutic purposes.

P2Et
Extract.Te P2Et extract was previously produced and standardized [12,18], ensuring the consistency of both chemical and biological properties between batches, in compliance with FDA regulations [8].Te drug extraction ratio (DER) was 20-50:1, where the P2Et contains a high concentration of hydrolyzable tannins ranging from 70 to 95%.Tese tannins are present in the form of mono-, di-, and tri-O-galloyl quinic derivatives, with a calculated amount of 5-30% gallic acid and 2-7% methyl gallate and ethyl gallate.Te P2Et extract was produced under Good Manufacturing Practices (GMP) conditions.Furthermore, prior to any experiments, the extract was subjected to physicochemical and microbiological certifcation to ensure consistency of quality.

Toxicity Assessment.
Te toxicity evaluation was conducted in MedLab under Good Laboratory Practice (GLP) conditions.Animals were maintained in the test facilities according to local and international requirements, based on the Guide for the Care and Use of Laboratory Animals [27].Animals that exhibited persistent signs of severe distress or pain were euthanized.Tese signs included abnormal vocalization, heightened aggressiveness, unusual posture, atypical reactions to handling, irregular movements, selfinficted injuries, open wounds or skin ulcerations, respiratory difculties, and corneal ulcerations.Other indicators for euthanasia included bone fractures, reluctance to move, unusual external appearance, rapid weight loss, emaciation, severe dehydration, signifcant bleeding, or any other symptoms, suggesting the animal might be in pain or distress during any test stage.Procedures for animal care and criteria for making the decision to euthanize an animal were based on the Guidance Document on the Recognition, Assessment, and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation [28].Te IACUC approval of the study is under the code FM-CIE-004-19.

Acute Oral Toxicity.
Te study was conducted following the guidelines outlined in the OECD no 423, 2001 [29].Six females, Rattus norvegicus (rats), aged 8-12 weeks were used (Figure 1), average weight (225.10 ± 20.13), and we chose females because they generally exhibit a slightly higher sensitivity to toxic efects.Te sample was prepared by mixing 5 g in 20 mL of 0.9% sodium chloride solution to achieve a concentration of 250 mg/mL.Te dose of the sample administered was 2000 mg/kg body weight (BW).Te rats were housed in a specifc room with a temperature range of 20.1 to 22.2 °C, relative humidity of 70.2 to 81.8%, and photoperiod 12/12 hours and underwent an acclimation period of one week.Feeding of the animals was composed of conventional feed for the species and fltered drinking water.Individual identifcation of the animals was established by random selection and numbering using a hydrographic pen.Te animals were fasted overnight prior to sample administration and for 3-4 hours after administration.
Teir individual body weights were recorded on the day of administration (Day 0) and on Days 7 and 14.In a previous study [18], we identifed the maximum tolerated dose to be 2000 mg/kg BW, leading us to choose this dose for our test.Te sample was given orally as a one-time dose through an appropriate gastric cannula.We began by treating three females with the 2000 mg/kg BW dose.Observing no 2 Journal of Toxicology signifcant clinical signs or mortality, we proceeded to treat an additional three females under the same parameters.Te animals were individually observed for the frst 30 minutes after administration and periodically during the frst 24 hours, with particular attention paid during the frst 4 hours and 14 days.Observations included monitoring changes in skin, eyes, and mucous membranes and in the nervous, respiratory, circulatory, somatomotor, and behavioral systems.Special attention was given to observing tremors, convulsions, salivation, diarrhea, lethargy, and coma.Necropsy was performed, and the macroscopic fndings are described in the Results section.

Chronic Toxicity Evaluation.
Te study was conducted in compliance with ICH M3 (R2) guidelines [30] at a GLPcertifed center.A total of 20 male and female (10 male and 10 female) rats (Rattus norvegicus) at seven weeks, and 20 (10 male and 10 female) rabbits (Oryctolagus cuniculus) at 12 weeks were used for the evaluation, with ten animals distributed evenly between control and experimental groups (Figure 1).Animals in the test group were labeled from 1 to 10, while those in the control group were labeled from 11 to 20.Additionally, a "M" was used to denote male animals, and "F" indicated female animals.In rats, the average weight for the female test group was 195.33 ± 7.16, compared to 191.69 ± 5.49 for the control group (p value: 0.39).In male rats, the average weight of the test group was 316.52 ± 18.54, compared to 317.28 ± 18.85 in the control group (p value: 0.95).For rabbits, given that both genders exhibit comparable weights, the test group had an average weight of 2950.8 ± 227.0, while the control group's average stood at 3097.8 ± 300.3 (p value: 0.23).
Te rats were housed in a specifc room with a temperature range of 17.0-20.6°C and relative humidity between 54.9 and 70.2%, while rabbits were housed in a room with a temperature range of 17.4-20.8°C and relative humidity between 54.9 and 70.2%.Both species were subjected to a photoperiod consisting of 12 hours of light followed by 12 hours of darkness.Rats underwent an acclimation period of at least one week, while rabbits acclimated for at least two weeks.
Te animals were fed conventional food for their respective species and provided with fltered drinking water.
Water and food intake were assessed through 24-hour measurements taken weekly throughout the experimental period for both rats and rabbits.Clinical toxicity signs and death were monitored, and a registry was maintained weekly.At the end of the 180 days, three distinct blood samples were collected for analysis: one for whole blood analysis, one for serum separation, and another for plasma obtaining.Hemograms were conducted on the whole blood sample, collected in EDTA tubes, using both automated and manual techniques.Coagulograms were performed on the plasma, which is obtained from blood collected in sodium citrate tubes, utilizing the thromboplastin (prothrombin time, PT) and ellagic acid (partial thromboplastin time, PTT) methods.Standard hematological internal techniques, practiced in the laboratory, were employed for the measurement of the parameters.
Te biochemical were performed in serum, the blood samples were centrifuged at 5000 rpm for 15 minutes, and the serum was kept at −20 °C until analysis for clinical biochemistry measurements.Standardized diagnostic kits by Analisa ® and a Biotron ® spectrophotometer were used in determination of the biochemical.Euthanasia was carried out on both rats and rabbits using an intraperitoneal injection of xylazine and ketamine at doses of 300 mg/kg BW and 30 mg/kg BW [31], respectively.Subsequently, the organs were weighed.Te liver, left kidney, left adrenal, and spleen's absolute weight (AW) were documented (Semianalytical and Analytical Scale Ohaus Adventurer).Te relative weight (RW) calculation was performed according to the formula: (Organ weight/Final body weight) × 100, in accordance with established internal laboratory procedures.Urine samples were also collected for analysis, and these samples were promptly refrigerated at 2-8 °C, though we aim to analyze them immediately.Te control group received the dilution vehicle in fltered water, with the same conditions applied to the P2Et group.Animal weights were recorded on Day 0 and weekly for six months.P2Et was administered orally once a day for six months at a dose of 1000 mg/kg using a syringe and gastric tube, with the administration volumes adjusted according to the animal's weight.For rats, urine analysis was performed using pooled samples from animals within the same group, as it was not possible to collect sufcient volumes of urine per individual animal.

Acute Toxicity.
During the observation period, two animals showed a decrease in body weight (Figure 2).However, the weight loss observed in these animals was not associated with any clinical signs of toxicity, and no macroscopic organ alterations were identifed during necropsy.Te remaining animals did not show any signifcant changes in their body weight or clinical symptoms throughout the study period.Additionally, no relevant alterations were detected in the diferent physiological systems assessed during the necropsy phase, including the cardiovascular, respiratory, digestive, and nervous systems.According to the Globally Harmonized System of Classifcation and Labelling of Chemicals (GHS), the P2Et was classifed as category 5, indicating a low level of toxicity, with an LD 50 value greater than 2000 mg/kg.

Chronic Toxicity.
Te experimental period was conducted without any observed clinical signs of toxicity, indicating that the experimental conditions were welltolerated by the animals.Te comparison of initial weight, fnal weight, and body weight gain between males and females of both the experimental and control groups in rats and rabbits showed no statistical diferences (Table 1).Moreover, water consumption did not difer signifcantly between the experimental and control groups during the investigation period, supporting the absence of any experimental interference (Table 1).Although there was a notable diference in feed consumption between the experimental and control groups among female rats in the second and sixth months, it did not signifcantly afect the overall study outcomes.Male rats demonstrated a notable variance in food consumption exclusively during the fourth month, while female rats showed this diference at the second and sixth months.However, these fuctuations were deemed to be of no consequence to the overall objectives of the study.Regarding the rabbits, a discernible diference in feed intake was apparent only in the fourth and ffth months.In the fourth month, the control group exhibited a higher consumption rate, whereas in the following month, the experimental group's intake surpassed that of the control group (Tables S5-S8).
In the male rat cohort, analysis revealed no statistically signifcant diferences in both the absolute and relative weights of the liver, spleen, and kidneys when comparing the experimental group with the control group.Conversely, in the female rats, a notable diference was observed in both the absolute and relative weights of the adrenal glands, as detailed in Table 2. Urea levels showed a statistically signifcant difference between the experimental and control groups, with the control group exhibiting higher levels, and potassium levels showing the opposite pattern (Table 3).Te evaluation of hemogram results (erythrogram and leukogram), coagulogram, liver function, and kidney function showed no signifcant systemic alterations in any of the animals (Table 4).
An animal (8M) from the test group exhibited diarrhea on the study's initial day but showed recovery in the following days.Four animals from the test group, specifcally two females (2F and 3F) and two males (7M and 8M), succumbed during the experimental phase due to complications from incorrect substance administration (Table S1).However, no clinical signs of toxicity or fatalities attributable to the test substance's efects were noted.Notably, an animal (4F) from the test group had duodenal congestion.Meanwhile, two control group animals (17M and 19M) exhibited a thickened pancreas appearance, and another control group animal (20M) had nodules and vesicles present in the lungs (Table S2).
In the rabbits group, study commenced with a total of 20 animals, evenly split between the test and control groups, and balanced in terms of gender.However, early in the study, four animals succumbed due to complications from incorrect substance administration.To compensate, four additional animals were introduced.As the study progressed, fve more animals either died or were euthanized due to similar administration issues (Table S3).Additionally, two animals were humanely euthanized because of a facial abscess and a spinal injury sustained while attempting to fee the cage.Consequently, the study concluded with three females and four males in the test group, and two females and three males in the control group (Table S3).Table S4 shows the macroscopic evaluation at necropsy examination after the end of the experimental period.In the test group, animal 6M showed dilation of urinary bladder and strangulation in one border of spleen, animal 7M showed hemorrhagic areas and abscesses in the lung, and animal 11F presented edema and hemorrhagic areas in the lung.In the control group, animal 17M showed congestion in cecum and duodenum and pancreas with dark color; animal 20M showed congestion in cecum and duodenum; animal 12F presented lymph node and pancreas with dark color; and animal 15F presented pancreas with dark color and presence of small vesicle in the left adrenal.In general, all observations matched typical background abnormalities in healthy rabbits of the same age and type used in this research.Tese were seen as natural or random occurrences and were not connected to the P2Et treatment.Te only signifcant diference between the experimental and control groups was the partial thromboplastin time (PTT) test, which was higher in the experimental group (Table 5).However, all values were signifcantly below the reference values for the species in both groups, indicating that this alteration was not clinically relevant.Although atypical values were observed in some animals (high monocyte count in two animals in the experimental group, high aspartate aminotransferase (AST) value of one animal from the control group, low bilirubin of one animal from the experimental group, and two animals from the control group), these changes were not considered relevant to the study since they were isolated cases in the groups and were not associated with clinical signs (Table 6).Additionally, some parameters were found outside the reference value ranges for the species, such as PTT and triglycerides, which may indicate natural variability between the evaluated animals and the literature values.Overall, the absence of signifcant systemic alterations in the animals indicated that the experimental conditions were well-controlled and that the results obtained were reliable.

Discussion
Plant extracts have become a promising source of natural compounds for the development of dietary supplements or herbal drugs, and their preclinical regulatory phases are being fulflled [32,33].Caesalpinia spinosa is a plant that has attracted attention due to its rich content of polyphenolic compounds, which have been linked to various health benefts.P2Et is a standardized and quantifed extract obtained from Caesalpinia spinosa, which has shown antioxidant, immunomodulatory, and antitumor activities in previous studies [3,12,13,[15][16][17].In a previous study, we reported that P2Et is not mutagenic or teratogenic [18]; moreover, we found no signifcant changes in toxicity after administering repeated doses of P2Et for 28 days [19], suggesting that it is relatively safe for chronic use.However, before P2Et can be introduced into the market as a dietary supplement or herbal drug, its safety needs to be evaluated rigorously.To this end, the current study conducted both acute and chronic toxicity evaluations to determine the safety profle of P2Et.
After conducting acute toxicity tests, our fndings indicate that P2Et falls under category 5 in the GHS, with a LD 50 greater than 2000 mg/kg.Although we administered a single dose of 2000 mg/kg to assess acute toxicity, two rats experienced weight loss without exhibiting any other clinical signs or macroscopic abnormalities during autopsy.Interestingly, gallic acid, one of the primary constituents of P2Et, has also been found to have an LD 50 exceeding 2000 mg/kg [34], a trait shared by other polyphenolic compound-rich extracts [32,35].
During our chronic toxicity study, it was observed no signifcant deviations in clinical signs of toxicity or any symptoms pertinent to the objectives of the study between the experimental and control groups, with the exception of elevated potassium levels in rats and increased RBC counts in rabbits.Te heightened potassium levels did not correspond with fndings from previous studies involving other polyphenolic compounds.Tis unusual elevation prompts considerations of renal efciency, as the kidneys are  Regarding the augmented RBC counts, it is recognized that polyphenols can stimulate erythropoiesis via several pathways.Tese pathways include boosting hemoglobin levels [36] and elevating α-tocopherol, a variant of vitamin E that plays a role in the genesis and function of red blood cells [37].Additionally, polyphenols may augment the antioxidant defense of red blood cells by binding to their surfaces, thereby improving their oxidant-scavenging efcacy.Consequently, these mechanisms may collectively contribute to a secondary rise in RBC count by bolstering the blood's overall antioxidant capacity [38].It is important to note that despite the extensive use of Caesalpinia spinosa in various traditional preparations [39], no chronic toxicity studies have been conducted to evaluate the safety of these preparations or standardized extracts.Interestingly, there are no reports of toxicity, either acute or chronic, associated with the use of any other species of the genus Caesalpinia [40,41].

Journal of Toxicology
Te results we obtained play a fundamental role in determining the NOAEL (No Observed Adverse Efect Level) for P2Et, set at 1000 mg/kg BW following 180 days of consistent administration.Tis NOAEL designation derives from the lack of adverse efects observed throughout our  Journal of Toxicology study.While we recognize that a dose as high as 1500 mg/kg might not have manifested adverse efects either, our previous human clinical trials, with daily doses of 600 mg and 500 mg, did not exceed a 10-fold exposure margin to clinical exposure, and the clinical dose remained below 1 g per day as recommended in the guideline to use 1000 mg/kg BW as selected dose.It is also noteworthy that the NOAEL values for other polyphenolic compounds, sourced from green tea, have been documented to be marginally lower than P2Et in earlier research [42].Tese results suggest that P2Et is comparatively less toxic and may have a higher safety margin than other polyphenolic compounds found in green tea.Te results of our toxicity evaluations support the continued development of P2Et as a potential herbal therapeutic agent.Nevertheless, the safety data obtained in this study provide a solid foundation for the future development of P2Et as a natural product-based therapy in chronic use.

Conclusions
Tese fndings provide a basis for the safe use of P2Et extract in potential clinical trials involving human subjects in chronic use.However, it is important to note that further research is needed to fully evaluate the safety of P2Et extract in human subjects.Tese results pave the way for future studies to explore the potential clinical applications of P2Et extract with confdence in its safety profle.

Figure 1 :
Figure 1: Schematic representation of the experimental design used to assess the acute and chronic oral toxicity of the P2Et extract.

Table 1 :
Mean intake of food/water as well as initial and fnal body weight in rats and rabbits following 180 days of administration.: number of animals in the group, data expressed as mean ± standard deviation.* Body weight gain � initial weight-fnal weight.Number of animals per group at the end of the experiment: N* * N � 2, * * * N � 3, * * * * N � 4.

Table 2 :
Absolute and relative weight of liver, left kidney, left adrenal, and spleen in rats and rabbits.

Table 3 :
Efects of P2Et extract on biochemical parameters in rats following 180 days of administration.: number of animals in the group, data expressed as mean ± standard deviation.

Table 4 :
Efects of P2Et extract on hematological parameters in rats following 180 days of administration.
N: number of animals in the group, data expressed as mean ± standard deviation.MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration (MCHC); PT: prothrombin time; PPT: partial thromboplastin time.

Table 6 :
Efects of P2Et extract on biochemical parameters in rabbits following 180 days of administration.