Acute oral toxicity defines to those adverse effects occurring following oral administration of a single dose of substances or multiple doses given within 24 hr. The various methods used to evaluate the acute oral toxicity are as follows,
1. Fixed dose procedure (OECD guideline-420)
2 Acute toxic class methods (OECD guideline-423)
3. Ups and down procedure (OECD guideline-425)
OECD Guideline- 42352
IAEC/XXXI/04/CLBMCP/2010-2011: Dated on 22/09/2010
OECD Guidelines for the Testing of Chemicals are periodically reviewed in the light of scientific progress or changing assessment practices. The original Guideline 423 was adopted in March 1996 as the second alternative to the conventional acute toxicity test, described in Test Guideline 401. Based on the recommendations of several expert meetings, revision was considered timely because,
International agreement has been reached on harmonized LD50 cut-off values for the classification of chemical substances, which differ from the cut-offs recommended in the 1996 version of the Guideline,
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Testing in one sex (usually females) is now considered sufficient.
The acute toxic class method set out in this Guideline is a stepwise procedure with the use of 3 animals of a single sex per step. Depending on the mortality and/or the moribund status of the animals, on average 2-4 steps may be necessary to allow judgment on the acute toxicity of the test substance. This procedure is reproducible, uses very few animals and is able to rank substances in a similar manner to the other acute toxicity testing methods (Test Guidelines 420and 425). The acute toxic class method is based on biometric evaluations with fixed doses, adequately separated to enable a substance to be ranked for classification purposes and hazard assessment. The method as adopted in 1996 was extensively validated in vivo against LD50 data obtained from the literature, both nationally and internationally.
Guidance on the selection of the most appropriate test method for a given purpose can be found in the Guidance Document on Acute Oral Toxicity Testing. This Guidance Document also contains additional information on the conduct and interpretation of Test Guideline 423.
5.1.2. Experimental Protocol (Acute toxic class method in rats)
In the present study the acute oral toxicity of the synthesized compounds were performed by acute toxic class method. In this methods the toxicity of the synthesized compounds were tested using a step wise procedure, each step using three rats of a single sex. The rats were fasted prior to dosing (food but not water should be with held) for three to four hrs. Following the period of fasting the animal should be weighed and the synthesized compounds (S1-S10) were administered orally at a dose of 2000 mg/Kg body weight. Animals were observed individually after dosing at least once during the first 30 min; periodically during the first 24 hr with special attention given during the first 4 hr and daily thereafter, for a total of 14 days. As no mortality was observed with the above dose. So, 200 mg/Kg body weight dose were selected for the further pharmacological evaluation.
5.1.3. RESULTS AND DISCUSSION
Acute oral toxicity studies were performed according to the OECD guideline 423 method.
This method has been designed to evaluate the substances at the fixed doses and provides information both for hazard assessment and substances to be ranked for hazard classification purposes.
The synthesized compounds were administered initially at a starting dose of 2000 mg/kg b.w. in 1% CMC (p.o.) and observed 14 days mortality due to acute toxicity.
Careful observation were made atleast twice a day for the effect on CNS, ANS, Motor activity, Salivation, Skin coloration and other general signs of toxicity were also observed and recorded.
Since, no sign of toxicity observed at 2000 mg/kg b.w. to the group of animals, the LD50 value of the title compounds (S1-S10) expected to exceed 2000 mg/kg b.w. and represented as class 5 (2000 mg/kg< LD5< 2500 mg/kg).
From the toxicity studies, the data revealed that all the synthesized compounds proved to be non-toxic at tested dose levels and well tolerated by the experimental animals as their LD5 cut of values > 2000 mg/kg b.w.
EVALUATION OF ANTI-INFLAMMATORY ACTIVITY
It comprises systemic response (involving nervous and hormonal adjustments and proliferation of the lymphoreticulo system; and local response (pain. redness, warmth and swelling). Anti-inflammatory agents are substance which modifies the inflammatory reaction. The main anti-inflammatory agents are the glucocorticoids and the non steroidal anti-inflammatory drugs (NSAIDs).
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Mechanism of action
NSAIDs act by inhibiting arachidonic acid-metabolizing activity of COX which leads to inhibition of production of PG and thromboxanes.
The cyclo oxygenase enzymes are bi functional, having two distinct activities that are:
A peroxidase action which converts PGG2 to PGH2
There are two types of COX (COX-1 and COX-2). Recently COX-3 has been described by Chandrasekaran. Both COX-1 and COX-2 inhibitors inhibit only main cyclo oxygenation reaction. Both COX-1 and COX-2 are associated with the membrane and each consists of long channel with a bend at the end. The channel being wider in COX-2. The opening of channel is largely hydrophobic. Arachidonic acid enters, is twisted round the bend and has two oxygens inserted and a free radical extracted, resulting in the 5-carbon ring characteristic of the PGs.
COX-1 inhibition, in general, is instantaneous and competitively reversible. COX-2 inhibition is time dependent, i.e. it is increasing with time. Some NSAIDs produced other actions besides inhibition of COX may contribute to the anti-inflammatory effects such as,
Reactive oxygen radicals produced by neutrophils and macrophages are implicated in tissue damage in some conditions, and NSAIDs such as sulindac that have particularly strong oxygen-radical scavenging effects as well as COX inhibitory activity may decrease tissue damage.
Aspirin has been shown to inhibit expression of the transcription factor NF-kB, which has a key role in the transcription of the genes for inflammatory mediators.
The three important aspects of inflammation that render themselves readily to measurement are erythema, edema and formation of granulation tissue. Compounds claimed to posses anti-inflammatory activity can be evaluated either by their ability to reduce one or more of these phenomena in experimentally induced inflammation or by testing their anti-inflammatory activity in experimental arthritis produced in animals. The commonly employed methods are:
In this method, irradiation of shaven back skin of guinea pig with UV light causes erythema which can be reduced by anti-inflammatory agents.
The edema can be produced in experimental animals by the local injection of substance like, formaldehyde, carrageenan, histamine, dextron and ovalbumin.
There are two types of granuloma assays such as cotton wool pellet and granuloma pouch method.
Experimental arthritis assays
Poly arthritis induced in rats by injection of dead tubercle bacilli suspended in liquid paraffin is frequently used method. Kaolin, talc and even mercury have also been injected directly into joints of rats and pigeons to induce arthritis.
Localised inflammatory reaction can be produced in rats by,
Intrapleural injection of turpentine
Intraperitonial injection of formaldehyde
Experimental Protocol (Carrageenan induced paw oedema method in rats)
Anti-inflammatory activity was performed by carrageenan- induced paw oedema method in rats. Diclofenac sodium (20 mg/kg, i.p) was administered as standard drug for comparison. The synthesised compounds were administered at dose levels of (200 and 400 mg/kg) orally 30 min. prior to the administration of 0.1ml/kg body weight of carrageenan in saline (1% W/V) into the lateral malleolus of the sub-planter region of the left hind paw. The paw volumes were measured using the mercury displacement technique with the help of a plethysmograph immediately before and 30 min, 1, 2 and 3 hr. after carrageenan injection. The percentage inhibition of paw odema was calculated by using the following formula and the results are depicted in Table.
Percentage protection = [(Control-Test)/Control] X 100