Acute and 28-days repeated dose sub-acute toxicity study of gallic acid in albino mice

Accepted Manuscript Acute and 28-days repeated dose sub-acute toxicity study of gallic acid in albino mice Bhavesh C. Variya, Anita K. Bakrania, Prem Madan, Snehal S. Patel PII:

S0273-2300(18)30302-7

DOI:

https://doi.org/10.1016/j.yrtph.2018.11.010

Reference:

YRTPH 4259

To appear in:

Regulatory Toxicology and Pharmacology

Received Date: 12 April 2018 Revised Date:

13 October 2018

Accepted Date: 15 November 2018

Please cite this article as: Variya, B.C., Bakrania, A.K., Madan, P., Patel, S.S., Acute and 28-days repeated dose sub-acute toxicity study of gallic acid in albino mice, Regulatory Toxicology and Pharmacology (2018), doi: https://doi.org/10.1016/j.yrtph.2018.11.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT Acute and 28-days repeated dose sub-acute toxicity study of gallic acid in albino mice

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1 Bhavesh C. Variya1, Anita K. Bakrania1, Prem Madan2, Snehal S. Patel *

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Gujarat, 382 481, India.

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Department of Pharmacology, Institute of Pharmacy, Nirma University, Ahmedabad,

MD-Pathologist, Sukoon pathology laboratory, Ahmedabad, Gujarat 380052, India.

Running title: Oral toxicity study of gallic acid in mice

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*Corresponding author:

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Dr. Snehal S. Patel,

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Department of Pharmacology, Institute of Pharmacy, Nirma University, Ahmedabad-382481, Gujarat, India

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Phone: 02717-241900-04

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E-mail address: [email protected]

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ACCEPTED MANUSCRIPT Abstract:

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Gallic acid is a phenolic acid ubiquitously present in numerous medicinal plants and food

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beverages. Gallic acid is also a potent anti-oxidant phytochemical possessing numerous

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medicinal potentials against various ailments such as diabetes, hypercholesterolemia and

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other life-threatening diseases including malignancy. Present study was aimed to evaluate

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acute and sub-acute toxicity of gallic acid in albino mice. The primary aim of the study was

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to investigate gallic acid prompted PPAR-α/γ activation associated adverse events. Acute

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toxicity of gallic acid was determined in albino mice and 28-days sub-acute toxicity study

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was carried out in male and female albino mice at three dose levels, 100, 300 and 900

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mg/kg/day, p.o. LD50 of gallic acid was found to be greater than 2000 mg/kg in mice.

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Hematological investigation did not show any alteration in transaminases and other blood

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homeostasis parameters. Gross necropsy showed non-significant alteration upon gallic acid

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administration. Histopathological finding suggested no significant alteration in tissue

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histology with slight fatty cells in bone marrow indicating non-significant bone marrow

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suppression, also no obvious effect was observed on hematological parameters. High dose of

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gallic acid (900 mg/kg/day) for 28 days did not produce any significant alteration in

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morphological and behavioral parameters. Histopathological finding also supports safety of

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gallic acid in mice.

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Keywords: Gallic acid; Gross histopathology; Gross necropsy; OECD 407; Oral toxicity

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ACCEPTED MANUSCRIPT 1. Introduction

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Gallic acid, 3,4,5-trihydroxy benzoic acid, is abundantly present in various plants in form of

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either free form or as an ester-conjugate. Polyphenols are believed to be the most important

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group of secondary metabolites involved in plant defence system. Gallic acid is found in

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different food sources such as; grapes, pomegranate, amla, nuts, berries and many more.

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Apart from plant sources, gallic acid is also present in some beverages like wine and tea

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(Badhani et al., 2015; Variya et al., 2016). Amount of gallic acid in plant tissues is greatly

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affected by external stimuli like storage, exposure of radiation and microbial contamination,

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and because of these external factors, phenolic content in grape juice as well as wine is

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largely variable (Amerine and Ough, 1980).

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Globally, as a means of advancement in various ailment therapies, researchers are focussing

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upon bioactive compounds extracted from various plants and semi-synthetic derivatives of

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natural compounds. Recently, flavonoids have received a great deal of attention due to their

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various pharmacological properties such as antitumor, antiulcer, antiallergic, antioxidant and

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antiviral actions (Ali Khan et al., 2017; Bakrania and Patel, 2015; Burkard et al., 2017; Qian

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et al., 2017). Amongst them, gallic acid, which is a natural plant triphenol has been

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extensively evaluated for its various pharmacological actions in a variety of in vivo and in

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vitro models (Locatelli et al., 2013). Gallic acid has numerous benefits as a medicative drug

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which include; anti-melanogenesis, anti-angiogenesis, anti-inflammatory, anti-mutation, anti-

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oxidant, anti-hyperlipidemic, anti-hepatosteatosis, anti-fungal, anti-chloasma, anti-cancer,

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cardio-protective, allergen deactivator, treatment of fatty liver and diabetes (Badhani et al.,

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2015; Choubey et al., 2015). Additionally, currently available dual therapy i.e. PPARα/γ dual

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agonists like -glitazars, have side effects related to either PPAR-α or PPAR-γ activation.

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Even though they possess efficacious dual agonistic potential, many drug molecules failed in

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therapeutic approach owing to the potential side effects viz. weight gain, fluid retention,

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ACCEPTED MANUSCRIPT increased cardiovascular events including myocardial infarction, elevated serum creatinine

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level and bladder tumour (Wright et al., 2014). To overcome PPARα/γ-associated side

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effects, there is an urgent need to identify a molecule having equilibration between PPAR-α

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or PPAR-γ. In line with previous reports claiming PPARs-agonistic potential of gallic acid

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(Bak et al., 2013; Huang et al., 2005), we also identified PPARα/γ-dual agonistic potential of

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gallic acid in various experimental animals. Previous reports demonstrated in vivo as well as

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in vitro safety profile of gallic acid in mice and rats. (Niho et al., 2001; Rajalakshmi et al.,

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2001; Suganthy et al., 2018). Rajalakshmi et al. (2001) and Suganthy et al. (2018) have

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conducted 28-days repeated dose toxicity of 7-methyl gallic acid and gallic acid at 1000

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mg/kg dose level in mice and observed no adverse events on haematological parameters, as

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well as no histological alteration in heart, liver, kidney, lung and brain. However, these

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studies were focused on vital organs and haematological parameters hence there is still need

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to assess detrimental effects of gallic acid on various other organs which are regulated

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through PPARα/γ activation viz. adipose tissue, cardiovascular system, skeletal muscles and

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bone-marrow. Due to the numerous pharmacological actions of gallic acid along with a vast

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biodistribution for its activity on several organs with dual therapy for diabetes and

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hyperlipidemia, we would aim at determining its PPARα/γ-associated toxicity before

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advancing it as a dual management therapy for diabetes and hyperlipidaemia.

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ACCEPTED MANUSCRIPT 2. Materials and methods

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2.1. Materials

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Gallic acid was procured from Sigma Aldrich, Co. St. Louis, MO, USA. Paraffin wax,

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hematoxylin-eosin stain and glycerine used were of analytical grade. Various diagnostic kits

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were procured from Lab-care Diagnostics Pvt. Ltd. India. Other materials used in the study

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were of commercial grade and procured from local market. All other reagents were of

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analytical grade, unless otherwise stated.

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2.2. Experimental Animals

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Three months old male and female swiss albino mice, weighing 25-30 g, were used and all

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the protocols described in the current study were approved, vide protocol number

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IP/PCOL/PHD/15-1/008 by the Institutional Animal Ethics Committee (IAEC) of Institute of

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Pharmacy, Nirma University, Ahmedabad. All the experimental procedures were in

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accordance with the guidelines of Committee for the Purpose of Control and Supervision of

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Experiments on Animals (CPCSEA), Government of India, New Delhi. The animals were

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procured and housed in the animal house of the Institute under controlled conditions of

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temperature 20°-25°C with 55±5% relative humidity and 12 h light and dark cycle. Acute and

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sub-acute toxicity studies were performed according to the OECD-423 and OECD-407,

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respectively; as described by regulation of the Organization for Economic Co-operation and

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Development (OECD).

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2.3. Acute oral toxicity study

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Female mice were randomly allocated into 2 groups i.e. vehicle control and gallic acid

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control with 3 animals in each group. Gallic acid was dissolved in water and administered at

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2000mg/kg body weight by oral gavage and animals were observed for 14 days for any sign 5

ACCEPTED MANUSCRIPT of toxicity or mortality. Behavioural changes were observed consciously for first 4 hr and

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body weight was recorded at the starting of protocol, on 7th day and at the end of protocol.

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2.4. Sub-acute oral toxicity study

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All the animals were randomized into 4 groups of 10 animals (5 male + 5 female) each

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namely; normal control, gallic acid treated (100mg/kg), gallic acid treated (300mg/kg) and

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gallic acid treated (900mg/kg). Treatment was initiated with 900mg/kg gallic acid, which was

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the highest dose of gallic acid as per OECD 407 guidelines. Gallic acid was dissolved in

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water and administered by oral gavage (p.o.) daily for 28 days. Simultaneously, same volume

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of drinking water (approximately 100 µL) was administered orally to control animals.

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Various evaluation parameters and changes in behaviour were recorded till the end of

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protocol. Morphological changes in skin, fur, eyes, mucous membrane, lacrimation,

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piloerection, pupil size, unusual respiration pattern, gait, posture and response to handling;

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presence of tonic-clonic movements, stereotypies (excessive grooming, repetitive circling)

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and bizarre behaviour (self-mutilation, walking backwards), before treatment and once every

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week till 28 days, were recorded as describe earlier (Quesne, 1987). Continuous assessment

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of body weight as well as food and water consumption were carried out once a week. During

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the 4th week, sensory reactivity to stimuli (auditory, visual and proprioceptive stimuli), grip

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strength and motor activity were assessed by former established methods (Bakrania et al.,

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2017; Gad, 1982). The animals were subjected to fasting preceding the last dose on day 28

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followed by blood and urine collection for various investigations.

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2.4.1. Haematological, biochemical and urine investigation

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At the end of protocol, animals were anaesthetized under light ether anaesthesia and blood

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samples were collected from retro-orbital plexus into polypropylene eppendorf tubes

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containing 20µL ethylene-diamine-tetra-acetic acid (EDTA) for various haematological

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blood cell count, haemoglobin concentration, mean cell haemoglobin (MCH), mean cell

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corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV) and

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packed cell volume (PCV).

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Plasma was separated from blood for biochemical investigation. The following biochemical

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parameters were investigated: random plasma glucose, blood urea, serum creatinine, serum

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total cholesterol, serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic

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transaminase (SGPT), sodium level, potassium level, blood urea nitrogen (BUN), total

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protein, albumin and alkaline phosphatase (ALP). All the analyses were performed on ABX

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Micros 60® (Horiba ABX, Montpellier, France). Urine was collected overnight and analysed

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for appearance, volume, osmolarity, pH (urinalysis strips), protein, glucose and presence of

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any blood cells.

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2.4.2. Gross necropsy and histopathological study

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After blood sampling, animals were sacrificed and various organs were collected for gross

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necropsy. Vital organs viz. heart, brain, kidneys, liver, lungs, reproductive organs such as

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testes and ovaries, and other peripheral organs including adipose tissue, adrenal gland,

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thyroid gland, spleen, thymus, stomach, intestine, pancreas, bones and peripheral nerves were

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examined for the macroscopic morphology and organ weight was carried out.

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Three animals from each group were randomly selected for histopathological studies. Heart,

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brain, kidneys, liver, lungs, reproductive organs like testes or ovary and uterus or prostate,

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white adipose tissue (WAT), trachea, salivary gland, thymus, adrenal gland, thyroid gland,

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spleen, thymus, stomach, small and large intestines, pancreas, vagal nerve, peripheral nerve

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i.e. sciatic nerve, femur bone and lymph nodes (1 located near to route of administration and

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1 located distantly from the route of administration) were fixed in neutral buffered 10%

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ACCEPTED MANUSCRIPT formalin. After fixation, samples were dehydrated with alcohol, embedded in paraffin,

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sectioned and stained with hematoxylin and eosin. After formalin fixation, femur bone was

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treated with 5% nitric acid for 24 hours and embedded in paraffin (Bakrania et al., 2017;

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Variya et al., 2015). All the stained sections were examined for optical microscopy using

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Olympus microscope. The pathologist performing the analysis was blinded to study protocol.

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2.5. Statistical analysis

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Statistical results are represented as mean±standard error of mean (S.E.M.). statistical

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significant between the means of various groups were analyzed by unpaired two-tailed

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Student's t-test using Prism (Graphpad, Version 5.00) statistical software. Data were

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considered statistically significant when p<0.05.

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ACCEPTED MANUSCRIPT 3. Results

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3.1. Acute toxicity study

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Mice receiving 2000mg/kg gallic acid showed no alteration in behavioural parameters after

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oral administration of gallic acid. Oral administration of gallic acid did not show any

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significant alteration in body weight throughout the study protocol (supplementary table 1).

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The mice, receiving 2000mg/kg, survived without any symptoms of toxicity indicating single

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dose safety of gallic acid at 2000mg/kg in mice.

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3.2. 28-days sub-acute toxicity study

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3.2.1. Alteration in body weight and food-water intake

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Weekly examined body weights of gallic acid administered mice were comparable to the

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normal control in both sexes i.e. male and female mice. During the whole study protocol,

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body weight fluctuation was recorded but non-significant change in body weight was

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observed when compared with mice receiving either vehicle or other dose of gallic acid. This

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indicates that gallic acid treatment has no major impact on body weights (Figure 1).

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Likewise, food and water intake were found to be similar across all treated groups for the

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entire study period in both, male and female as compared to the normal control mice (data not

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shown); indicating that gallic acid has no major impact on the food and water consumption.

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3.2.2. Morphological alteration

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No changes in general appearance were observed pertaining to the eyes, skin, fur colour,

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mucous membrane, lacrimation, piloerection, respiratory pattern and pupil size. Gallic acid

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did not show alteration in posture, gait and response to handling. Additionally, there was

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absence of any sign of tonic-clonic movements, stereotypies behaviours such as excessive

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grooming and repetitive circling or any bizarre behaviour like backwards walking and self-

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ACCEPTED MANUSCRIPT mutilation. The motor activities were also unaffected by gallic acid administration. Treatment

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did not alter motor activity as well as grip strength significantly in both sexes, which was a

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positive indication of absence of any variation in the skeletal system. No other apparent

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morphological alterations were observed in gallic acid administered animals.

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3.2.3. Haematological and biochemical examination

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Gallic acid administered and control animals were subjected to the haematology analysis for

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various biochemical parameters. As illustrated in Table 1, all the hematological and

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biochemical parameters were found relative with the control mice. Biochemical estimations

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for determination of various body organ functions such as; serum creatinine, blood urea,

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serum cholesterol, random blood glucose, serum BUN, SGOT, SGPT and ALP indicated

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absence of any deviation from the normal range inferring the normal body function regulation

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was maintained in the gallic acid administered animals. Initially, mice receiving high dose of

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gallic acid (900 mg/kg of body weight) and control mice were sacrificed and studies for

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various toxicity. However, no significant toxicity was observed at highest dose of gallic acid

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hence mice receiving lower dose of gallic acid were not sacrificed.

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3.2.4. Urine analysis

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Gallic acid administered animals when subjected to urinalysis parameters such as; change in

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pH, osmolarity or physical appearance of urine, it was evident that no observable changes

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were seen when compared to the normal control animals. The animals portrayed a pale-

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yellow coloration of urine without pus, glucose, haematocrits or blood cells. Also, pH of

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urine was maintained as slightly acidic indicative of the normal urine physiology and similar

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results were observed in the gallic acid administered animals.

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3.2.5. Macroscopic gross necropsy

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ACCEPTED MANUSCRIPT Macroscopic examination exhibited normal architecture without any sign of the

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inflammation. Gallic acid administration did not alter organ weights relative to the terminal

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body weight and were found to be comparable with the normal control indicating absence of

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any inflammation, atrophy, hypertrophy or oedema; rendering it safe for administration in

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various organs viz. kidneys, liver, adrenals, brain, spleen and heart (Figure 2). The gross

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necropsy evidenced devoid of any lesions or abnormalities in the organ macroscopy in both

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male and female mice treated with gallic acid.

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3.2.6. Histopathological studies

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In gallic acid administered mice brain sections, a symmetric pattern was observed in the left

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and right hemispheres of male and female mice. As illustrated in Figure 3A, the cerebrum,

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cerebellum and grey matter regions contained healthy astrocytic cells with prominent nuclei

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and in close proximity to the neurons. Microscopical examination of the heart sections

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demonstrated no abnormalities in the cardiomyocyte architecture in gallic acid administered

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rats (Figure 3B). The normal architecture of the cardiac myocytes was maintained with the

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absence of cardiac myopathy, myofiber degeneration, necrosis, vacuolation and mononuclear

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cell infiltration which justify gallic acid treatment devoid of any toxicity in the heart of both

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male and female mice.

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Renal sections showed thin and delicate glomerular capillary loops with evidently normal

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number of endothelial and mesangial cells indicating a normal glomerular filtration in the

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gallic acid and normal control animals (Figure 3C). As shown in Figure 3D, liver section

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histology showed a network of hepatocytes which were arranged in single-cell thick plates

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separated by vascular sinusoids. In the gallic acid administered mice hepatic sections, no

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incidences of hepato-diaphragmatic nodules, focal inflammation, focal tension, lipid

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accumulation, periportal hepatocellular vacuolation or infiltration of inflammatory cells were

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ACCEPTED MANUSCRIPT observed rendering gallic acid safe for the hepatic-functioning.. In WAT histological study,

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no significant adipogenesis was observed in the gallic acid administered mice (Figure 3E).

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Morphology of the adipocytes was maintained in a continuous polyhedral pattern with no

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significant variations in the adipocyte morphology and adipocyte intensity in gallic acid

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administered mice compared to normal control sections rendering gallic acid devoid of any

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alterations in the WAT.

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Gallic acid administered mice lung sections are devoid of infiltered lymphocytes, plasma

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cells and histocytes in the sub-pleural regions indicating absence of inflammation (Figure

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3F). As shown in Figure 3G, pancreas histology sections comprised of a uni-layered

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pyramidal shaped cells forming the secretory acini. Also, the endocrine regions contained the

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islets of Langerhans with endocrine cells scattered throughout the exocrine part of the tissue.

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As displayed in Figure 3H, the architecture of the small intestine consisting of the intestinal

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mucosa with villi was present along with Paneth cells which were observed beneath the

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crypts in numerous concentrations implying secretion of the antibacterial substances such as

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lysozymes in gallic acid administered animals. (Figure 3I).. HE-stained stomach sections

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showed an intact mucosal surface consisting of simple columnar epithelium along with

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numerous mucus secreting cells and acid secreting cells comprising closely packed glands

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within the gastric mucosa (Figure 3J). Overall, the histopathological study of GI tract was in

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defence with the aspect that there are no abnormalities viz. ulceration, oedema or

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inflammation associated to the administration of gallic acid in the gastrointestinal tract.

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Gallic acid administered mice spleen sections (Figure 3K) evidently showed normal

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haematopoiesis which did not differ from the spleen sections of the normal control animals.

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Accessory axillary and subiliac lymph nodes were examined for any toxicities due to gallic

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acid administration. Immune cells were located in the lymphoid tissue with blood vessels

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present in the connective tissue trabeculae extending from the hilus into the lymph

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ACCEPTED MANUSCRIPT parenchyma with normal microscopy features implying normal lymph functioning hence

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gallic acid exhibits no significant immunological-toxicity in the lymphatic system. As shown

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in Figure 3L, no thymic lymphoid depletion was observed in the gallic acid administered

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mice thymus sections.

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As depicted in Figure 3M, the relative thickness of the cortical zones was found to be similar

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in the gallic acid administered mice pancreas sections as well as control mice pancreas

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sections implying absence of any abnormalities due to treatment. The femur bone was

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examined for bone-marrow related toxicity due to gallic acid administration and its histology

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showed a uniform layer of dense connective tissue (periosteum) which also contained

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osteoprogenitor cells (Figure 3N). However, the overall examination of the bone sections

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exhibited a normal range of bone marrow, but gallic acid administered mice bone sections

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demonstrated diminutive fatty cells (Figure 3N) indicating displacement of these cells with

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fatty cells but the sections were devoid of significant bone-marrow suppression or related

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deformities.

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Uniform microscopy of the sciatic nerve was evident with a healthy epineurium connective

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tissue (Figure 3O)with absence of axon dilation and axonal defragmentation in the nerve

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sections implying a healthy network in the gallic acid administered mice. The gross histology

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of the thyroid comprised of rounded cysts and follicles separated by scant inter-follicular

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connective tissue. Also, the follicles maintained a uniform cuboidal epithelium surrounded

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with the lumen.

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As shown in Figure 4A, testes histology showed a well organised germinal epithelium in both

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gallic acid administered and normal control male mice. No observable difference in the

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seminiferous epithelium of tubules was seen with a single layer of sertoli cells and

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spermatogonia. The prostate gland architecture comprised of the fibromuscular stroma with

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ACCEPTED MANUSCRIPT smooth muscle separated by strands of connective tissue rich in collagenous and elastic fibres

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(Figure 4A). The corpora amylacea in the secretory alveoli were present as circular and

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eosinophilic bodies without any sign of inflammatory secretions implying no toxicity due to

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gallic acid treatment in the male reproductive organs.

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The gross histology of the ovary comprised of a single layer cover of cuboidal epithelium in

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continuation with the peritoneal mesothelium (Figure 4B). The ovarian follicles comprised of

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an oocyte surrounded by various follicular cells viz. griffin follicle and primordial follicle.

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There were no variations in the ovary histology pertaining to gallic acid administration. As

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displayed in Figure 4B, the uterus sections comprised of a healthy and strong mucosal layer,

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endometrium and a fibromuscular layer (myometrium). The endometrium exhibited simple

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columnar epithelium and stromal cells indicating absence of any alterations in the uterine

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functioning due to gallic acid administration.

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ACCEPTED MANUSCRIPT 4. Discussion

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Gallic acid is a major phyto-tannin present in variety of plants. Gallic acid has shown

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excellent results for hyperlipidaemia and diabetes therapy and it was therefore necessary to

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perform the toxicity study of gallic acid in order to evaluate its pharmacological safety in

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animals. Additionally, having PPAR-α/γ dual agonistic potential, it is more important to look

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after PPAR-α/γ activation associated side effects viz. cardiac dysfunction, variation in blood

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homeostasis, bone marrow depression and alteration in metabolism (Ahmed et al., 2007;

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Home, 2011). Initially, the variation in food and water intake as well as body weight was

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analysed and it was keenly observed that there was no variation pertaining to treatment with

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gallic acid.

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During the gross necropsy analysis of the various organs, no significant alteration in the

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organ weights compared to the normal control organ weights was evident. Also, the various

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organs did not portray any lesions, oedema or observable abnormalities; hence, gallic acid did

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not expose any treatment related adverse effect on the various organs.

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Alteration in haematopoietic system is one of the most crucial parameter for assessment of

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drug toxicity (Yuan et al., 2014). In the present study, various biochemical parameters such

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as; total blood counts, haemoglobin, differentiate leucocyte counts, PCV, MCV, MCH and

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MCHC were estimated and these parameters lied within the respective normal ranges in the

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gallic acid (900 mg/kg/day) administered mice of both sexes which was in comparison to the

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normal control estimates. Liver and kidney are site of drug metabolism and elimination and

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most sensitive organs to be affected by chemical toxicity. Any alteration in liver or kidney

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functions result in the elevation of SGOT, SGPT, ALP, BUN, serum creatinine and

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electrolytes in blood (Liju et al., 2013). We observed non-significant alteration in plasma

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cholesterol, liver and kidney functions in gallic acid administered animals. This further states

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ACCEPTED MANUSCRIPT that gallic acid maintains the normal biochemical homeostasis of the body and is devoid of

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any toxicity to the various organs of the body. The urine analysis also portrayed the normal

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appearance and microscopic characteristics along with absence of pus cells (pyuria),

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concluding that gallic acid treatment did not deteriorate the kidney functioning.

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Histopathological study also showed absence of any lesions, microvacuolar degeneration,

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scalloping of the brush border or frank necrosis of tubular epithelium in renal histology

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indicating no alteration in renal function which confirmed that gallic acid did not produce any

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toxicity in the renal system altering its excretion physiology. The liver also devoid of any

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infiltrated inflammatory mediators rendering gallic acid safe for administration at a dose of

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900 mg/kg. Previous reposts have demonstrated that the hepatocytes which did not possess

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any necrosis and portrayed uniform architectural arrangement do not have any alteration in

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liver biomarkers markers i.e. SGOT, SGPT, ALT and albumin (Pari and Amudha, 2011;

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Yayla et al., 2014; Zirak et al., 2014). These data are in accordance with previous study

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demonstrating the safety of gallic acid in mice at a dose of 1000 mg/kg for 28-days

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(Rajalakshmi et al., 2001). However, in contrast to this, Niho et al. demonstrated increased

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liver weight in rats receiving 5% gallic acid in diet for 13 weeks but no alteration was

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observed in rats receiving 1.7% gallic acid in diet for 13 weeks (Niho et al., 2001).

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During the in-depth microscopic study of cardiac tissues through histopathological

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examination, we did not observe any pathological findings as evidence of toxicity. There was

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no evidence of myocyte hypertrophy indicating a physiological adaptive response to

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normalised blood pressure and manifests absence of any changes in the myocardium. Gallic

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acid administrated mice brain tissue histology has its intact architecture with medium and

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large sized neurons with axons fully intact and systematically arranged devoid of any axonal

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swelling or degeneration. Gallic acid did not demonstrate neuronal toxicity as evidence from

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the brain histology.

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ACCEPTED MANUSCRIPT PPAR-γ agonists induce adipogenesis and WAT differentiation leads to increase in adiposity.

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Simultaneously, PPAR-γ activation decreases osteogenesis and finally leads to bone marrow

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suppression (Watt and Schlezinger, 2015). In the present study, there was non-significant

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increase in WAT cellular size without increase in WAT or body weight. Histopathological

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study demonstrated trivial fatty cells in the bone marrow but haematological parameters were

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not affected and found to be comparable with control animals, indicating non-significant or

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absence bone marrow suppression in the gallic acid administered mice.

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Histology of the gastrointestinal system organs were studied and it was observed that the

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stomach was precisely lined by the gastric mucosa and it did not possess any signs of

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ulceration or infiltration of inflammatory markers which evidences that gallic acid

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administration does not possess any hyperacidity which is an adverse effect observed with

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several drug therapies (Cho and Ogle, 1977; Marshall and Warren, 1984). Further, the small

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and large intestines also did not show any significant signs of ulceration or disruption in the

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normal architecture confirming absence of any abnormalities in the gastrointestinal system

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due to gallic acid administration. Similarly, the adrenals also did not expose to hypertrophy or

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any inflammatory secretions which conclude the proper functioning of the adrenals in the

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gallic acid administered animals.

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Gallic acid administrated mice spleen comprised of normal lymphocyte regularity and no

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evidence of lymphocyte hyperplasia indicating no alteration in spleenocytes. Further, the

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thymus maintained its normal size and weight portraying that there was no splenomegaly

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observed and therefore a normal synthesis of the corticosteroids was also maintained in the

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body. No signs of lymphocyte apoptosis, lymphocyte necrosis and epithelial necrosis of

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thymus was evident which concludes that gallic acid does not alter the immune system

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mechanism in mice at a dose of 900 mg/kg.

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could eventually impair the reproductivity of the animals due to gallic acid treatment. We

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observed no observable difference in the testes architecture as compared to the normal

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control male mice. There were evidences showing large quantities of spermatids in the gallic

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acid administered mice which justify normal spermatogenesis and these results complied with

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the normal control data. Also, the healthy occurrence of spermatids documents the orderly

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progression of spermatogonia to spermatocytes (Abdullah and Bondagji, 2011; Aly et al.,

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2012). Similarly, the prostate gland also exposed a normal histology. The female

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reproductive organs- ovary and uterus, both maintained the normal ovulation and uterine

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endometrium characteristics respectively. These indicated that gallic acid neither had any

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adverse effect on the male nor female reproductive systems. Recently, Suganthy et al. also

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did not observe in vitro genotoxicity of gallic acid at dose of 2000 µg/mL (Suganthy et al.,

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2018).

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ACCEPTED MANUSCRIPT 5. Conclusion:

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Acute and sub-acute toxicity study is essential element for translational research as well to

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determine safety. The LD50 of gallic acid is found to be greater than 2000 (category 5,

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LD50>2000 mg/kg). Gallic acid did not produce any significant toxicity related to

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haematological and biochemical parameters. Despite of having adipogenic potential, gallic

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acid did not show significant increase in body weight or bone marrow suppression in sub-

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acute toxicity study in mice. The results of this study suggest that ingestion of gallic acid has

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no adverse action or health-related acute or sub-acute toxicity.

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ACCEPTED MANUSCRIPT Abbreviations

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ALP- Alkaline phosphatase

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BUN- Blood urea nitrogen

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CPCSEA - Committee for the Purpose of Control and Supervision of Experiments on

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Animals

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EDTA - Ethylene-diamine-tetra-acetic acid

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HE - Hematoxylin and eosin

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LD50 – Lethal dose

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MCH - Mean cell haemoglobin

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MCHC - Mean cell corpuscular haemoglobin concentration

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MCV - Mean corpuscular volume

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OECD - Organization for Economic Co-operation and Development

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p.o. - per oral

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PCV - Packed cell volume

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PPAR - Peroxisome proliferator-activated receptor

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SGOT - Serum glutamic oxaloacetic transaminase

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SGPT -Serum glutamic pyruvic transaminase

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WAT - White adipose tissue

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ACCEPTED MANUSCRIPT Acknowledgment:

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The authors would like to acknowledge the financial support provided by Department of

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Science and Technology (DST, SERB, vide file no. SB/FT/LS-185/2012) for providing

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research grant to carry out presented work. BV would like to offer his sincere gratitude to Dr.

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Prem Madan, MD Pathologist for the assistance in the analysis of the histopathology samples.

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Author contributions

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BV and AB designed, performed and interpreted studies data and wrote the manuscript. PM

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analyzed and interpreted histopathological data. SP and PM analyzed and interpreted

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experimental data, contributed to writing the manuscript and contributed to study supervision.

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Conflicts of interest

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The authors state that there are no conflicts of interest pertaining to this manuscript.

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ACCEPTED MANUSCRIPT Figure captions

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Figure 1: Food consumption of control and gallic acid administered male and female mice.

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Values expressed as Mean ± SEM, n=5. Data are considered statistically significant when

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p<0.05

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Figure 2: Gallic acid did not show macroscopic alteration in various organs. Alteration in

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organ weight with respect to body weight.

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Values expressed as Mean ± SEM, n=5. Data are considered statistically significant when

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p<0.05

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Figure 3: Histopathological studies of brain, heart, kidney, liver, white adipose tissue, lung,

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pancreas, small and large intestine, stomach, spleen, thymus, adrenal gland, bone and sciatic

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nerve in control and gallic acid administered mice.

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Figure 4: Histopathological studies of testes, prostate, ovary and uterus in control and gallic

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acid administered mice.

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Table captions

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Table 1: Effect of gallic acid (900 mg/kg) on hematological and biochemical profile in mice

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from 28 days sub-acute toxicity study

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Gallic acid did not show significant alteration in haematological and biochemical

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parameters in mice. Values expressed as Mean ± SEM, n=5. Data are considered statistically

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significant when p<0.05

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Supplementary Table 1: Change in body weight body weight in acute toxicity in mice

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*results are rounded off to nearest whole number

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Supplementary table 2: Grip strength and motor activity in sub-acute toxicity study of gallic

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acid in mice

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Higher dose of gallic acid did not alter grip strength and motor activity in mice. Values

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expressed as Mean ± SEM, n=5. Data are considered statistically significant when p<0.05

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ACCEPTED MANUSCRIPT References:

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Abdullah, L., Bondagji, N., 2011. Histopathological patterns of testicular biopsy in male infertility: A retrospective study from a tertiary care center in the western part of Saudi Arabia. Urol Ann. 3, 19-23. Ahmed, I., et al., 2007. Dual PPAR α/γ Agonists: Promises and Pitfalls in Type 2 Diabetes. American Journal of Therapeutics. 14, 49-62. Ali Khan, M. S., et al., 2017. Flavonoids and Anti-Oxidant Activity Mediated Gastroprotective Action of Leathery Murdah, Terminalia Coriacea (Roxb.) Wight & Arn. Leaf Methanolic Extract in Rats. Arq Gastroenterol. 54, 183-191. Aly, H. A., et al., 2012. Mitochondrial dysfunction induced impairment of spermatogenesis in LPStreated rats: modulatory role of lycopene. Eur J Pharmacol. 677, 31-8. Amerine, M. A., Ough, C. S., Methods for analysis of musts and wines. 1980. Badhani, B., et al., 2015. Gallic acid: a versatile antioxidant with promising therapeutic and industrial applications. RSC Advances. 5, 27540-27557. Bak, E. J., et al., 2013. Gallic acid improves glucose tolerance and triglyceride concentration in dietinduced obesity mice. Scand J Clin Lab Invest. 73, 607-14. Bakrania, A. K., Patel, S. S., 2015. Combination treatment for allergic conjunctivitis – Plant derived histidine decarboxylase inhibitor and H1 antihistaminic drug. Experimental Eye Research. 137, 32-38. Bakrania, A. K., et al., 2017. Repeated dose 28-day oral toxicity study of DEAE-Dextran in mice: An advancement in safety chemotherapeutics. Regul Toxicol Pharmacol. 88, 262-272. Burkard, M., et al., 2017. Dietary flavonoids and modulation of natural killer cells: implications in malignant and viral diseases. The Journal of Nutritional Biochemistry. 46, 1-12. Cho, C. H., Ogle, C. W., 1977. The effects of zinc sulphate on vagal-induced mast cell changes and ulcers in the rat stomach. Eur J Pharmacol. 43, 315-22. Choubey, S., et al., 2015. Medicinal importance of gallic acid and its ester derivatives: a patent review. Pharm Pat Anal. 4, 305-15. Gad, S. C., 1982. A neuromuscular screen for use in industrial toxicology. J Toxicol Environ Health. 9, 691-704. Home, P., 2011. Safety of PPAR agonists. Diabetes Care. 34 Suppl 2, S215-9. Huang, T. H., et al., 2005. Anti-diabetic action of Punica granatum flower extract: activation of PPARgamma and identification of an active component. Toxicol Appl Pharmacol. 207, 160-9. Liju, V. B., et al., 2013. Acute and subchronic toxicity as well as mutagenic evaluation of essential oil from turmeric (Curcuma longa L). Food Chem Toxicol. 53, 52-61. Locatelli, C., et al., 2013. Alkyl esters of gallic acid as anticancer agents: a review. Eur J Med Chem. 60, 233-9. Marshall, B. J., Warren, J. R., 1984. Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet. 1, 1311-5. Niho, N., et al., 2001. Subchronic toxicity study of gallic acid by oral administration in F344 rats. Food and Chemical Toxicology. 39, 1063-1070. Pari, L., Amudha, K., 2011. Hepatoprotective role of naringin on nickel-induced toxicity in male Wistar rats. Eur J Pharmacol. 650, 364-70. Qian, L., et al., 2017. Optimized microwave-assistant extraction combined ultrasonic pretreatment of flavonoids from Periploca forrestii Schltr. and evaluation of its anti-allergic activity. ELECTROPHORESIS. 38, 1113-1121. Quesne, P. M. L., 1987. Principles and Methods for the Assessment of Neurotoxicity Associated with Exposure to Chemicals. (Environmental Health Criteria No 60.). Journal of Neurology, Neurosurgery, and Psychiatry. 50, 510-510. Rajalakshmi, K., et al., 2001. Assessment of the no-observed-adverse-effect level (NOAEL) of gallic acid in mice. Food and Chemical Toxicology. 39, 919-922.

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Suganthy, N., et al., 2018. Safety assessment of methanolic extract of Terminalia chebula fruit, Terminalia arjuna bark and its bioactive constituent 7-methyl gallic acid: In vitro and in vivo studies. Regulatory Toxicology and Pharmacology. 92, 347-357. Variya, B. C., et al., 2016. Emblica officinalis (Amla): A review for its phytochemistry, ethnomedicinal uses and medicinal potentials with respect to molecular mechanisms. Pharmacol Res. 111, 180-200. Variya, B. C., et al., 2015. Comparative evaluation of HMG CoA reductase inhibitors in experimentally-induced myocardial necrosis: Biochemical, morphological and histological studies. Eur J Pharmacol. 764, 283-91. Watt, J., Schlezinger, J. J., 2015. Structurally-diverse, PPARgamma-activating environmental toxicants induce adipogenesis and suppress osteogenesis in bone marrow mesenchymal stromal cells. Toxicology. 331, 66-77. Wright, M. B., et al., 2014. Minireview: Challenges and opportunities in development of PPAR agonists. Mol Endocrinol. 28, 1756-68. Yayla, M., et al., 2014. Protective effect of Et-1 receptor antagonist bosentan on paracetamol induced acute liver toxicity in rats. Eur J Pharmacol. 726, 87-95. Yuan, G., et al., 2014. Toxicological assessment of combined lead and cadmium: acute and subchronic toxicity study in rats. Food Chem Toxicol. 65, 260-8. Zirak, M. R., et al., 2014. Tropisetron attenuates cisplatin-induced nephrotoxicity in mice. Eur J Pharmacol. 738, 222-9.

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Table 1: Effect of gallic acid (900 mg/kg) on hematological and biochemical profile in mice from 28 days sub-acute toxicity study Female animals

900 mg/kg gallic acid

Control

900 mg/kg gallic acid

Hemoglobin (gm/dl)

14.3 ± 1.16

14.1 ± 2.15

14.2 ± 2.08

13.1 ± 1.34

Total WBC count (per cu.mm)

3200 ± 86.11

2700 ± 311

1200 ± 163

1800 ± 248

Total platelet count (Per cu.mm)

803000 ± 71360

790000 ± 33897

570000 ± 42060

651000 ± 61280

Neutrophils (%)

34 ± 3.20

41 ± 5.34

49 ± 7.22

41 ± 8.10

Lymphocytes (%)

63 ± 5.94

52 ± 5.13

43 ± 6.37

49 ± 6.02

Eosinophils (%)

2 ± 0.07

3 ± 1.00

5 ± 1.04

3 ± 0.57

Monocytes (%)

1 ± 0.00

4 ± 0.67

3 ± 0.39

4 ± 1.67

Total RBC count (mil./cumm)

9.71 ± 1.044

9.57 ± 2.38

9.27 ± 2.79

8.87 ± 3.34

PCV (%)

42.9 ± 4.22

43.3 ± 3.36

40.2 ± 4.37

40.8 ± 5.07

MCV (%)

44.2 ± 5.00

45.1 ± 5.71

43.4 ± 3.99

46.2 ± 6.32

MCH (%)

14.7 ± 2.84

13.8 ± 2.62

15.3 ± 6.18

14.8 ± 4.70

MCHC (%)

33.3 ± 2.97

32.6 ± 6.04

35.3 ± 6.98

32.1 ± 3.34

Random blood glucose (mg/dl)

126.2 ± 35.2

82 ± 11.04

146 ± 15.39

124 ± 8.51

0.47 ± 0.05

0.37 ± 0.07

0.28 ± 0.002

0.35 ± 0.005

38.9 ± 2.33

36.5 ± 5.19

36.4 ± 2.08

47.8 ± 7.83

70 ± 0.99

74 ± 6.34

65 ± 3.5

57 ± 8.32

SGPT (U/L)

32.71 ± 3.16

34.6 ± 6.34

32.3 ± 1.87

39.8 ± 6.40

SGOT (U/L)

77.4 ± 5.04

67.4 ± 7.71

81.8 ± 4.75

79.3 ± 7.67

Blood urea (mg/dl) Serum cholesterol (mg/dl)

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Serum creatinine (mg/dl)

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18.1 ± 3.66

17 ± 3.01

17 ± 2.30

21.3 ± 3.18

Serum sodium (mmol/L)

136 ± 22.08

142 ± 13.15

133 ± 9.46

137 ± 9.91

Serum potassium (mmol/L)

3.8 ± 0.64

4 ± 0.34

4 ± 0.36

4.1 ± 0.83

Serum protein (total) (gm/dl)

5.73 ± 1.97

5.98 ± 0.73

5.53 ± 1.00

5.9 ± 1.25

Serum albumin (gm/dl)

2.61 ± 0.05

2.63 ± 0.62

2.58 ± 0.64

2.7 ± 0.81

Serum alkaline phosphatase (U/L)

69.7 ± 1.55

68 ± 8.41

165 ± 6.58

147.9 ± 21.18

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Serum BUN (mg/dl)

Gallic acid did not show significant alteration in haematological and biochemical parameters in mice. Values expressed as Mean ± SEM, n=5. Data are considered statistically significant when p<0.05 Note: Initially, mice receiving high dose of gallic acid (900 mg/kg of body weight) and control mice were sacrificed and studies for various

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toxicity. We did not observe any significant toxicity hence we did not sacrifice the mice receiving lower dose of gallic acid.

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Highlights… •

Acute toxicity i.e. LD50 of gallic acid, known for various biological potentials, was more than 2000 mg/kg in mice.

use of gallic acid. •

Hematological, biochemical and morphological parameters did not show significant alteration in gallic acid treated mice.

•

28 days repeated dose sub-acute toxicity showed NOAEL (No observed adverse effect level) at a dose of 900 mg/kg in mice.

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potential therapeutic agent.

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No alteration in transaminase and BUN level indicates hepato- and nephro-safety with

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•