Pyrogallol

Repeated dose toxicity: other routes

Administrative data

Endpoint:

repeated dose toxicity: other route

Type of information:

experimental study

Adequacy of study:

weight of evidence

Data source

Reference

Reference Type:

publication

Title:

Pyrogallol-induced hepatotoxicity in rats: a model to evaluate antioxidant hepatoprotective agents

Author:

Gupta Y.K., Sharma M. and Chaudhary G.

Year:

2002

Bibliographic source:

Methods Find Exp Clin Pharmacol. 24(8): 497-500

Materials and methods

GLP compliance:

not specified

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Pyrogallol

EC Number:

201-762-9

EC Name:

Pyrogallol

Cas Number:

87-66-1

Molecular formula:

C6H6O3

IUPAC Name:

benzene-1,2,3-triol

impurity 1

Reference substance name:

unknown impurities

IUPAC Name:

unknown impurities

Test material form:

not specified

Details on test material:

not specified

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Pyrogallol (S.D. Fine Chemicals Ltd, India) and silymarin (Ranbaxy, India)
- Expiration date of the lot/batch: not specified
- Purity test date: not specified

RADIOLABELLING INFORMATION (if applicable) not applicable
- Radiochemical purity: -
- Specific activity: -
- Locations of the label: -
- Expiration date of radiochemical substance: -

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: not specified
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle: not specified
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: not specified
- Preliminary purification step (if any): not specified
- Final dilution of a dissolved solid, stock liquid or gel: pyrogallol (and silymarin) were dissolved in distilled water
- Final preparation of a solid: not specified

FORM AS APPLIED IN THE TEST (if different from that of starting material) : liquid

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) : not applicable

OTHER SPECIFICS: All drugs were freshly prepared

Test animals

Species:

rat

Strain:

Wistar

Remarks:

albino

Details on species / strain selection:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: They were procured from the Central Animal House Facility, A.I.I.M.S, New Delhi.
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation: not specified
- Weight at study initiation: between 200-250 g
- Fasting period before study: not specified
- Housing: The animals were housed in standard laboratory conditions
- Diet (e.g. ad libitum): standard rat chow ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: not specified

DETAILS OF FOOD AND WATER QUALITY: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The animals were housed in standard laboratory conditions
- Humidity (%): The animals were housed in standard laboratory conditions
- Air changes (per hr): The animals were housed in standard laboratory conditions
- Photoperiod (hrs dark / hrs light): natural light/dark cycle

IN-LIFE DATES: From: To: not specified

Note: All procedures described were reviewed and approved by the Institutional Committee for the Ethical Use of Animals

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

water

Remarks:

dissolved in distilled water

Details on exposure:

The volume of drugs administered intraperitoneally using a 25-gauge hypodermic needle did not exceed 0.1 ml/100 g.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

1 time

Frequency of treatment:

1

Doses / concentrations

Dose / conc.:

100 mg/kg bw (total dose)

No. of animals per sex per dose:

Each group consisted of 8 animals

Control animals:

not specified

Details on study design:

- Dose selection rationale: not specified
- Rationale for animal assignment (if not random): an equal number of males and females in each group
- Rationale for selecting satellite groups: not specified
- Post-exposure recovery period in satellite groups: not specified
- Section schedule rationale (if not random): not specified

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes / No / No data
- Time schedule:
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:

BODY WEIGHT: Yes / No / No data
- Time schedule for examinations:

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

WATER CONSUMPTION: Yes / No / No data
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes / No / No data
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes / No / No data
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (identity) / No / No data
- Animals fasted: Yes / No / No data
- How many animals:
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes / No / No data
- Time schedule for collection of blood:
- Animals fasted: Yes / No / No data
- How many animals:
- Parameters checked in table [No.?] were examined.

URINALYSIS: Yes / No / No data
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / No data
- Animals fasted: Yes / No / No data
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes / No / No data
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: No
HISTOPATHOLOGY: rats were sacrificed and the liver removed and processed for MDA and GSH (oxidative stress markers) and tissue histology.
MDA was determined by thiobarbituric acid reaction according to the method of Ohkawa.
GSH was measured according to the method of Ellman.

Other examinations:

blood was withdrawn by cardiac puncture for the estimation of serum markers (AST, ALT, Alkaline phosphatase).
Activity of serum AST and ALT were measured according to the method described by Reitman and Frankel . Alkaline phosphatase was determined according to the method of Kind and King.

Statistics:

The results are expressed as Mean ± SEM. Statistical significance was evaluated by Student’s t-test and p < 0.05 was regarded as significant.

Results and discussion

Results of examinations

Clinical signs:

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The pyrogallol-treated rats showed mild inflammatory changes in the liver. The changes included cellular infiltration of leukocytes and sinusoidal dilation even as early as 1 h after pyrogallol administration.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Effect levels

Dose descriptor:

LOAEL

Effect level:

100 mg/kg bw (total dose)

Based on:

test mat. (dissolved fraction)

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw (total dose)

Organ:

liver

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Any other information on results incl. tables

Free radicals and oxidative stress play an important role in the pathogenesis of various liver diseases.

Pyrogallol administration caused a significant increase in serum AST and ALT compared with control animals, thereby indicating liver damage. This may be due to the fact that damage to the structural integrity of the liver is reflected by an increase in the levels of serum enzymes (AST and ALT) because these are cytoplasmic in location and are released into circulation after hepatocellular damage. However, there was an insignificant change in the levels of alkaline phosphatase in the pyrogallol-treated group compared with the controls.

This may be because alkaline phosphatase is an inducible enzyme, which takes time to be induced, prior to seeing an increased level in the serum.

There was an increased level of MDA and GSH in the liver of rats treated with pyrogallol compared with the control group. The increased MDA levels indicated increased lipid peroxidation by pyrogallol. Raised levels of GSH were also observed 1 h after pyrogallol administration.

The increase in GSH may be due to a compensatory increase by the liver tissue to combat the free radical insult. Pyrogallol is a potent generator of superoxide anion (O2–) and also generates hydroxyl radical (OH•) and hydrogen peroxide by the Haber-Weiss reaction (O2– ±H2O2/OH• ± O2

– ± O2). The increased free radical generation by pyrogallol may lead to its hepatotoxicity.

The histological studies in the liver tissues showed mild inflammatory changes even as early as 1 h after pyrogallol treatment.

Applicant's summary and conclusion

Conclusions:

Intraperitoneal injection of pyrogallol in rats can be used as a model to induce hepatotoxicity.
Pyrogallol (100 mg/kg i.p.) produced significant (i) liver damage, as indicated by a marked increase in serum AST and ALT in comparison with the control group (p < 0.05), (ii) significant oxidative stress in liver tissue as indicated by the marked increase in MDA and glutathione levels compared with the control group and (iii) increasing in the Glutathione levels to 37 ± 2.25 mg/g wet tissue compared with control values of 24.4 ± 3.6 mg/g wet tissue (p < 0.05).
The pyrogallol-treated rats showed mild inflammatory changes in the liver.

Executive summary:

Various hepatic disorders and hepatotoxic agents are associated with increased free radical generation. In the present study, the free radical generator pyrogallol (100 mg/kg i.p.) caused significant hepatic damage. The serum enzymes aspartate aminotransaminase (AST) and alanine aminotransaminase (ALT) increased to 357 ± 30.7 IU/I and 147.8 ± 28.4 IU/I, respectively in the pyrogallol-treated group compared with 208.4 ± 4.1 IU/I and 84.5 ± 19.5 IU/I, respectively in the control rats. Compared with control rats, the liver tissue in the pyrogallol-treated group showed an increased level of malondialdehyde (MDA) as well as glutathione (GSH). The infiltration of white blood cells into the liver tissue, as seen histologically, further substantiated liver damage. Pretreatment with a standard hepatoprotective drug (silymarin, 100 mg/kg i.p.) afforded significant protection against pyrogallol hepatotoxicity, as evidenced by amelioration of the raised serum markers of hepatic function, markers of oxidative stress and normal liver histology. Thus, pyrogallol-induced hepatotoxicity could be used as an appropriate model to evaluate hepatoprotective agents that have an antioxidant property.