Acute Toxic Class Method Biology

Table of Content

To analyze the acute toxicity of the synthesized compounds by Acute Toxic Class method [ Organization of Economic Co-Operation and Development ( OECD ) guidelines-425 ]

To execute antiulcer rating of the synthesized compounds.

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To execute anticonvulsant rating of the synthesized compounds.

Materials and methods

The assorted animate beings and stuffs used for the present survey and their beginnings are as follow

Wister rats

1 % Carboxy methyl cellulose




Water for injection

Synthesized compounds

Wister rats weighing 150-200 gram were kept in a settlement cages at 25+2oC, comparative humidness of 45-55 % under 12 hour visible radiation and dark rhythm. All the animate beings were fed with standard animate being provender and H2O ad libitum. The trial compounds were administered orally in the signifier of suspension utilizing 1 % CMC as suspending agent. The experimental dosage was selected between the minimal effectual dosage and maximum non lethal dosage. All the carnal experimentation was performed harmonizing to the protocols and recommendation of the institutional animate being ‘s moralss commission.

ANOVA followed by dunnet ‘s trial was performed to determine the significance of the exhibited antiulcer, anticonvulsant activities of the synthesized compounds.

Following activities were carried out to the synthesized compounds by below mentioned method.

acute unwritten toxicity ( acute toxic category method in Rats )

antiulcer activity ( acetylsalicylic acid induced + pylorus ligation method in Rats )

anticonvulsant activity ( maximum electric daze method )

Evaluation of acute unwritten toxicity


Acute unwritten toxicity defines to those inauspicious effects happening following unwritten disposal of a individual dosage of substance or multiple doses given within 24 h. the assorted methods used to measure the acute unwritten toxicity are as follows:

Fixed dose process ( OECD guideline-420 )

Acute toxic category method ( OECD guideline-423 )

Ups and down process ( OECD guideline-425 )

OECD Guidelines- 42351

IAEC/XXXI/03/CLBMCP/2010-2011: Dated on 22/09/2010

OECD Guidelines for the Testing of Chemicals are sporadically reviewed in the visible radiation of scientific advancement or altering assessment patterns. The original Guideline 423 was adopted in March 1996 as the 2nd option to the conventional acute toxicity trial, described in Test Guideline 401. Based on the recommendations of several adept meetings, alteration was considered seasonably because:

International understanding has been reached on consonant LD50 cut-off values for the categorization of chemical substances, which differ from the cut-offs recommended in the 1996 version of the Guideline,

Testing in one sex ( normally females ) is now considered sufficient.

The ague toxic category method set out in this Guideline is a bit-by-bit process with the usage of 3 animate beings of a individual sex per measure. Depending on the mortality and/or the moribund position of the animate beings, on mean 2-4 stairss may be necessary to let judgement on the acute toxicity of the trial substance. This process is consistent, uses really few animate beings and is able to rank substances in a similar mode to the other acute toxicity proving methods ( Test Guidelines 420 and 425 ) . The acute toxic category method is based on biometric ratings with fixed doses, adequately separated to enable a substance to be ranked for categorization intents and hazard appraisal. The method as adopted in 1996 was extensively validated in vivo against LD50 informations obtained from the literature, both nationally and internationally.

Guidance on the choice of the most appropriate trial method for a given intent can be found in the Guidance Document on Acute Oral Toxicity Testing. This Guidance Document besides contains extra information on the behavior and reading of Test Guideline 423.

5.1.2. Experimental Protocol ( Acute toxic category method in rats ) –

In the present survey the acute unwritten toxicity of the synthesized compounds were performed by acute toxic category method. In this methods the toxicity of the synthesized compounds were tested utilizing a measure wise process, each measure utilizing three rats of a individual sex. The rats were fasted prior to dosing ( nutrient but non H2O should be with held ) for three to four hours. Following the period of fasting the animate being should be weighed and the synthesized compounds were administered orally at a dosage of 2000 mg/Kg organic structure weight.Animals were observed separately after dosing at least one time during the first 30 min ; sporadically during the first 24 H with particular attending given during the first 4 H and day-to-day thereafter, for a sum of 14 yearss. As no mortality was observed with the above dosage.

So, 200 mg/Kg organic structure weight dose were selected for the farther pharmacological rating.

Flow Chart For Acute Toxic Class Method ( OECD Guidelines 423 ) Get downing Dose of 2000 mg/kg Body Weight/po



Peptic Ulcer Disease ( PUD ) is the most prevailing GI upset, embracing stomachic and duodenal ulcer. The pathophysiology of PUD involves an instability between violative factors like acid, pepsin and defensive factors like hydrogen carbonate, prostaglandins.

Ulcer has been classified as

Gastric ulcer

Duodenal ulcer

Oesophageal ulcer

Meckel ‘s Diverticulum ulcer

Mechanism of action:

  1. The chief mechanism of action of anti-ulcer drugs is found to be
  2. Inhibition of H+ , K+ -ATPase ( proton pump )
  3. Histamine H2- Receptor Antagonist
  4. Prostaglandin parallels
  5. Anti helicobacter pylori agents.

Experimental protocol ( Aspirin Induced + Pylorus Ligation Method ) :

The animate beings were divided into eight groups of 6 animate beings each and placed in coops with grating to avoid corophagy in environmentally controlled suites with free entree to H2O and nutrient. The animate beings were fasted for 24hrs. Synthesized compounds and standard anti-ulcer drug Lansoprazole were prepared in 1 % w/v of carboxy methylcellulose suspension as vehicle and administered orally one time in a twenty-four hours at a dosage of 10 mg/kg organic structure weight severally. Control group of animate beings were treated with 1 % w/v of carboxy methylcellulose suspension and the negative control group is treated with acetylsalicylic acid at a dosage of 200mg/kg organic structure weight with vehicle likewise to experimental animate beings.

  1. Group I: Control
  2. Group II: Negative control ( aspirin treated )
  3. Group III: Standard ( Lansoprazole treated )
  4. Group IV-VIII: Trial ( synthesized compounds )

Aspirin induced + pylorus ligation method:

The animate beings were fasted for 24hr. Aspirin at a dosage of 200mg/kg organic structure weight was used for the initiation of ulcers. Aspirin was administered orally to the rats after 30 proceedingss of pyloric ligation. The synthesized compounds and standard drug were administered orally 5 proceedingss prior to pyloric ligation.

Rats were anaesthesized with anesthetic quintessence. The rats were secured on the operating tabular array. An scratch of 1cm long in venters merely below breastbone was made. The tummy was exposed and yarn was passed around the pyloric sphincter. While ligating due attention took that no blood vas was tied along the knot. The venters wall was closed by seting the suturas. The tegument from any blood musca volitanss and hemorrhage was cleaned. The rat in separate coop was placed and allowed it to retrieve.

After 4 hours of the pyloric ligation sacrificed by cervical disruption, the venters was opened and the oesophageal terminal of the tummy was tied. The full tummy from the organic structure of the animate beings was separated.

A little cut to the pyloric part merely above the knot was made and the contents of the tummy in a calibrated extractor tubing were collected.

The tummy along the greater curvature was opened and washed easy under the running tap H2O and exposure were taken.

Appraisal of biochemical parametric quantities:

After the aggregation of stomachic content in the extractor tubing it was centrifuged at 1000rpm for 10 proceedingss, the supernant liquids were transferred to the mensurating cylinder and the stomachic volume was measured.

Determination of pH:

1ml of the stomachic juice was diluted to 10ml of distilled H2O and pH was measured by the pH metre.

Determination of sourness:

1 milliliter of stomachic juice was pipetted into a 100ml conelike flask and 2 or 3 beads of topfer ‘s reagent was added as an index and titrated against 0.01N Na hydrated oxide, titrated until the solution turns to orange colour. This volume of Na hydrated oxide responds to the free sourness. Then phenolphthalein 2 beads as an index was added, so titration was farther carried out until the solution regains pink colour. This gives the entire volume of Na hydrated oxide, which corresponds to the entire sourness.

The sourness is calculated by the undermentioned equation:

Vol. Of NaOH X normalcy X 100

Acidity = — — — — — — — — — — — — — — — — — — — — — — – mEq/1/100g


Determination of ulcer mark

The curvature of the tummy were taken and washed with running tap H2O. Put it on the glass slide and observed under 10X magnification for ulcers.

0 = Normal coloured tummy

0.5 = Red color

1 = Spot ulcers

1.5 = haemorrhagic runs

2 = ulcers & A ; gt ; 3 but & A ; lt ; 5

3 = ulcers & A ; gt ; 5

Average ulcer mark for each animate being is expressed as ulcer

Evaluation of Anticonvulsant activity

Epilepsy is the corporate term used for a group of chronic ictus upsets which is holding in common, sudden and transeunt episodes ( ictus of loss or perturbation of consciousness ) , normally but ever with a characteristic organic structure motion ( paroxysm ) and sometimes with autonomic hyper activity. The ictus about ever correlates with an unnatural electrical discharge.

Epileptic ictus can be classified into,

1. Chiefly generalised ictus:

a. Grant -mal epilepsy or Tonic-Clonic ictus

B. Tonic Seizure

c. Clonic ictus

d. Petit-mal epilepsy or absence ictus

e. Atonic ictus or bead onslaught.

f.Myoclonic ictus

2. Partial ictus:

a. Simple Partial ictus

B. Complex Partial ictus

3. Secondarily generalised ictuss or cortical focal epilepsy or Jacksonian epilepsy.

Mechanism of action

The chief mechanism of action is as follows

1. Enhancement of GABA action by

a. Activating GABAA- receptor

b.Inhibition of GABA aminotransferase enzyme

2. Inhibition of Na channel map

3. Inhibition of Ca channel map

a. Ethosuxemide specially barricade T type Ca channel

b. GABA pentan may move on L type Ca channel

The other mechanism is suppression of glutamate release and block of Glutamate receptors.

Evaluation Method

The assorted rating methods are as follows

MES ( Maximal Electric daze ) Method

Chemical ( Letazol ) Method

Kindling Method

Rota rod Trial

Drugs which antagonised paroxysm induced by MES, Leptazol and Kindling Method are normally utile in Grand-Mal.

The experimental protocol ( MES Method in Rats )

In the present survey anti-convulsant activity were screened by MES method. Albino rats of either sex were selected by random trying technique was used for survey. The animate beings were divided in three groups incorporating 4-5 animate beings in each group. One group was used as control, 2nd as standard drug ( diphenylhydantoin ) and 3rd was trial group ( synthesized compounds )

Different phases of the paroxysms were noted as ( a ) tonic flexure, ( B ) quinine water extensor stage, ( degree Celsius ) clonic paroxysms, ( vitamin D ) daze, and ( vitamin E ) recovery or decease. The animate being was held decently and corneal electrodes were placed on the cornea and the prescribed current ( 150 ma for 0.2 seconds ) was applied after half an hr disposal of the trial compounds. Phenytoin at the dosage of 25 mg/kg i.p. was administered as standard drug for comparing. The trial compounds at two dose degrees were administered orally. The clip spent by animate being in each stage of paroxysm was recorded. The decrease in clip and abolishment of tonic extensor stage was recorded.


Evaluation of in vitro anti-bacterial activity


The microbic universe comprises of assorted microorganisms which are microscopic in size. Bacteria, Fungi ( barm and molds ) and microscopic algae are some of micro-organisms. These are distinguished into two wide groups such as procaryotes and eucaryotes. Eukaryotes contain nucleus and cell organs ( such as chloroplast, lysosome, endoplasmic Reticulum, chondriosome and golgi organic structures ) whereas, prokaryotes lacks the above characteristics.

Bacterias are most abundant procaryotic being that is critical to life of life things. In nature bacteriums can accommodate to any sort of life conditions than any other group of beings.

The undermentioned conditions must be accomplished for the finding of proper antimicrobic activity:

There should be proper control between the trial being and the substance to be evaluated.

The needed conditions for the growing of the micro-organism should be provided. Measurement of activity should be done right.

Aseptic should be maintained.

Study conditions should be maintained unchanged throughout the experiment.

Materials and methods

Assorted methods have been used to measure the antimicrobic activity of the drugs. The activity can be evaluated by the following techniques.

Agar streak dilution method.

Consecutive dilution method.

Agar diffusion method.

Cup home base method

Cylinder method

Paper phonograph record method.

Turbidimetric method.


The standard strains were procured from the American type civilization aggregation ( ATCC ) , Rockville, USA, and the pathological strains were procured from the section of microbiology, CEEAL analytical lab, Chennai, India. The anti-microbial activity of the synthesized compounds was screened against the undermentioned bacterium.

Gram-positive being:

  1. Staphylococcus aureus ( ATCC 6538P )
  2. Bacillus substillis ( ATCC 6633 )

Gram negative being:

  1. Escherichia coli ( ATCC 25922 )
  2. Pseudomonas aeruginosa ( ATCC 25619 )


Nutrient agar medium ( hi-media research labs, India ) is used as the media for the survey of anti-bacterial activity. The composing of the medium is as follows.



Peptic digest of carnal tissue


Beef infusion


Sodium chloride




Yiest infusion




Standard drug:


Ampicillin is a beta-lactam antibiotic used extensively to handle bacterial infections. The chemical name of Principen is 6- ( 2-amino-2-phenylacetamido ) -3,3-dimethyl-7-oxo-thia-1-aza-bicyclo heptanes-2-carboxylic acid. It is effectual against both gram positive and gram negative bacterial infection.

The anti-bacterial activity of the compounds Ia-Va and Ib-Vb were studied by the paper phonograph record diffusion method. The trial compounds were used in the concentration of 25µg/ml, 50µg/ml, 100µg/ml. Ampicillin 50µg/ml was used as criterion.

Disc diffusion method

A suspension of Staphylococcus aureus was added to the unfertile food agar at 45 & A ; deg ; C in sterile environment, the mixture was transferred to sterile petridishes and allowed to solidify. Sterile phonograph record of Whatmann filter paper 6mm in diameter was dipped in solutions of compounds Ia-Va and Ib-Vb, criterion were placed on the surface of the agar home bases.

All the home bases were allowed to stand at room temperature for 1 hr, ( This was as a period of preincubation diffusion to minimise the effects of fluctuation in clip between the application of the different solutions ) so plates were placed for incubation for 18 hours at 37 ± 1 & A ; deg ; C and observed for the antibacterial activity. In which plate zone of the suppression observed diameter of that was measured.

Similar processs was carried out for analyzing the antibacterial activity of the compounds Ia-Va and Ib-Vb against. The mean country of the zone of suppression was calculated and compared with the criterion.

In vitro Evaluation of Anti-fungal activity of the synthesized compounds


  1. A fungus is a colorless works which is deficiency of chlorophyll. Fungi that cause infections may be like barm or hyphi ( mould ) and these are called fungous infections.
  2. These are of two types
  3. Superfacial mycotic infection
  4. Systemic mycotic infection
  5. Because of the big widespread prevalence and airborne and soilborne transmittal of the fungous pathogens, healthful methods are non sufficient to eliminate the disease caused. So the hunt for the new and improved agents continues.

Material and Method

By Disc Diffusion Method the fungicidal activity of the synthesized compounds Ia-Va and Ib-Vb were studied. For this undermentioned beings were used.

Aspergillus fumigates ( ATCC 46645 )

Candida albicans ( ATCC 10231 )

Compounds Ia-Va and Ib-Vb were used in the concentration of 25µg/ml, 50µg/ml, 100µg/ml utilizing DMF as dissolver. Fluconazole ( 50µg/ml ) was used as criterion.

The Disc Diffusion Method was employed for the showing of fungicidal activity by utilizing Sabouraud Dextrose Agar medium ( 30gms of sabouraud dextroglucose agar and 1000ml of H2O warming ) .

Disc Diffusion Method

A suspension of microorganism ( Aspergillus nigar ) was added to sterile Sabouraud dextrose agar medium at 45 & A ; deg ; C and the mixture was transferred to sterile petridishes and allowed to solidify. Sterile phonograph record of Whatmann filter paper of 6mm in diameter dipped in solutions of synthesized compound Ia-Va, Ib-Vb and criterion were placed on the surface of agar home bases.

All the home bases were allowed to stand at room temperature for 1hour. Then the home bases were incubated at 37 ± 1 & A ; deg ; C for 18 hours and observed for fungicidal activity. The diameter of zone of suppression was measured. Similar process was carried out for analyzing fungicidal activity of compounds against Aspergillus fumigates. The mean country of zone of suppression was calculated and compared with that criterion.

Determination of Minimum Inhibitory Concentration

The MIC ( minimal inhibitory concentration ) of the synthesized compounds Ia-Va, Ib-Vb were determined by the agar run dilution method. The MIC was determined against the bacterium and Fungi.


Gram +ve

Staphylococcus aureus ( ATCC 6538P )

Bacillus Substilis ( ATCC 6633 )

Gram -ve

Escherichia coli ( ATCC 25922 )

Pseudomonas aurogenousa ( ATCC 25619 )

Fungus kingdoms

Aspergillus fumigates ( ATCC 46645 )

Candida albicans ( ATCC 10231 )

Media used

Media used for bacterium was alimentary agar and for fungi saboraud dextroglucose agar. All the civilization media were sterilized by autoclaving at 15lbs for 20 min.

Agar Streak Dilution:

The stock solutions ( 1mg/ml ) of the synthesized compounds were made by utilizing DMF as dissolver. From this stock solution, needed measures of drug solution were assorted with the known measures of the molten unfertile agar media aseptically to supply the undermentioned concentration 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50 and 100µg/ml. About 20ml of the media incorporating the drug was dispensed into unfertile petridishes. Then the media were acquiring allowed to solidify.

Over the surface of the agar home bases, 1µl of standardized microorganism ( 1-105 CFU/ml ) were poured aseptically. After vaccination, all the home bases were incubated at 37 ± 10C for 24 hours. Then all the home bases were observed for the growing of the being. The lowest concentration of the synthesized compounds suppressing the growing of the bacteria/ Fungi were taken as the minimal repressive concentration of the trial compounds against that bacteria/ Fungi.

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Acute Toxic Class Method Biology. (2017, Jul 06). Retrieved from

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