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    LAB MANUAL OF PHARMACOLGY-3 B.PHARM VI SEMESTER

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    LAB MANUAL OF PHARMACOLGY-3 B.PHARM VI SEMESTER

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    Pharmacology-3 Lab Manual

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    LAB MANUAL OF PHARMACOLGY-3 B.PHARM VI SEMESTER

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    u » GEGKAGEEEEEECEOEEVYTVES SEES LEHSSSES Meerut Institute of Engineering & Technology . MEERUT ment of Phar! al Technolo; Programme: B.Pharmacy Semester-VI : BP6OSP. Course: Pharmacology III- Practical Cod List of Experiments (Session: 2020-21) AZ Dose calculation in pharmacological experiments. ~ BX -4 2. To screen anti allergic activity by mast cell stabilization assay demonstrated by simulated experiments/ videos 36. To study anti-ulcer activity of a drug using pylorus ligation rat model and NSAIDs induced lees, model demonstrated by simulated experimentsivideos. « €x-3 4, To study: the effects of drugs on gastrointestinal motility demonstrated by simulated experiments/videos. 5. To demonstrate effect of agonist and antagonists on guinea pig ileum by simulated experiments/videos - 6. Toestimate serum biochemical parameters by using semi-autoanalyser. * 7. To demonstrate effect of saline purgative on frog intestine by simulated experiments/videos. 8. To demonstrate insulin hypoglycemic effect in rabbit by simulated experiments/videos. 9. To demonstrate test for pyrogens (rabbit method) by simulated experiments/ videos. “3dTo determine acute oral toxicity of a drug from a given data demonstrated by simulated experimenis, Cx-4 : \Bkc Determination of acute skin! corosion of atest substance 13ateaaton of pharmacokinetics pstameters from a given data x Determination of acus eye irtation/eorosion of a test substance:—€x-S — “ 7 Experiment No. 1 Aim: Dose calculation in pharmacological experiments. References: Erhirhie Earnest Oghenesuvwe, Ekene, Nwoke E. and Ajaghaku Daniel Lotanna Guidelines on dosage calculation and stock solution preparation in experimental animals’ studies Journal of Natural Sciences Research Vol.4, No.18, 2014. Theory: Dosage calculation and stock solution preparation in preclinical studies, involving the use of experimental animals is important in screening and development of new drugs. Dosage calculation and stock solution preparation based on dosage rationale formula are prerequisites to drug administration in experimental animals. Dosages are usually calculated from stock solution of the test drugs dissolved in appropriate volume of solvent (vehicle). According to the OECD’s (organization of economic corporation and development’s) guidelines, dosage of drug (mg) should be constituted in an appropriate volume not usually exceeding 10 ml/kg (1 ml/100g) body weight of experimental animals (mice and rats) for non-aqueous solvent in oral route of administration. However in the case of aqueous solvents, 20 ml/kg (2 ml/100g) body weight can be considered (OECD, 2000). 1. Vehicle of choice, drugs dissolution and volume selection rational A vehicle is any substance that acts as a medium in which a drug is administered. Example of suitable vehicles for animal research include; water, normal saline (0.9% sodium chloride), 50% polyethylene glycol, 5 to 10% Tween 80, 0.25% methylcellulose or carboxymethylcellulose, Lower volume (5 ml/kg) can be considered to dissolve highly soluble solute drugs. Such low volume would éase the administration of drug in solution. However, highly viscous drug solution should be diluted, whenever possible, for ease of administration. However, final dilution volume should not exceed 20 ml/kg. Based on 10 ml/kg volume selection, réquiréd dose volume for a 100 g rat can be calculated as follows; 100gm/1000gm X 10mL = 1 mL. 2. Dosage calculation and preparation of stock solution of erude plant extract for experimental animals With reference to table 1 above, stock solutions and doses of a plant extract (with selected doses, 200 mg/kg and 400 mg/kg) for a rat weighing 120 g be calculated as follows; calculation Body weight of animal =120 g Dosage in mg = Body weight of animal/1000 gm X dose (mg) 120 g/1000 g X 200 (ing) = 24 mg ‘Step 2: Dissolution of dose in a suitable vehicle for oral administration From the OECD's guidelines, 120 g rat requires 24 mg of the crude plant extract which should be constituted in not more than 1.2 ml of normal saline according to the OECD guildline. In anut shell, 120 g=24 mg = 1.2 ml of normal saline, Bulk volume of the stock solution required for large number of animals can be calculated by multiplying both sides by a constant value as follows: 24mg=1.2 ml ved in 48 ml of normal saline = 960mg/ 48 mL = 960 mg of crude plant extract will be 20 mg/mL Having successfully prepared a stock solution (960 mg/48 ml = 20 mg/ml) for a selected dose of 200 mg/kg, stock solution of the same plant extract with a higher selected dose (400 mg/kg) can easily be prepared by dissolving 960 mg of plant extract with half the volume (24 ml) used in the previous stock (960 mg/48 ml), thereby yielding a higher concentration (960 mg/24 ml = 40 mg/ml) which is twice the concentration of the formal stock’ as shown in table 2 below. In this case animals with similar body weight from two different selected dose categories (200 mg/kg and 400 mg/kg respectively) will receive the same volume, but different concentrations. Table 2: Showing stock solutions from two selected doses of a crude plant extract. Selected dose Stock solution | Animal body Calculated dose | Equivalent weight (g) (mg) dose in ml Low dose, 200 | 960 mg/48 ml" | 120g 24 mg 1.20 ml mglkg (20 mg/ml) 130g 30mg 1.30 ml 135g 26 mg 1.35 ml 150g 30mg 1.50 ml High dose, 400 | 960 mg/24 ml 120 48 mg 1.20 ml mghkg (40 mg/ml) 130g 60mg 1.30 ml 135g 52mg 1.35 ml 150g 60mg 1.50 ml The above table shows that animals with higher body weight in the same standard dose category will receive higher doses (mg or ml) of plant extract solution. Also, the stock solution for high dose will be thicker and more concentrated than low dose. 3. Dosage calculation and preparation of stock solution of a reference drug (example: Sylimarin) for experimental animals. Sylimarin (figure 1) is a reference drug (with dosage formulations of 70 mg and 140 mg per tablet respectively) used in animal model of screening for agents with hepatoprotective nephroprotective and anti-oxidant properties at standard doses ranging betwé g/kg to 200 mg/kg body weight. a Aor example: The required dose of sylimarin (70 mg per tablet) for a rat weigh standard dose 25 mg/kg can be calculated as follows; ees Step 1: Dosage calculation Acquired dose for 130 g rat = Body weight of animal (g) /1000 (g) X Stand: rd d 130 (g) /1000(g) X 25 mg = 3.25 mg. 7 ae) Step 2: Dissolution of sylimarin in a suitable v ne of vehicle for inis ic ‘a oral adi ic From the above calculation, 130 g rat requires 3.25 mg of sylimarin sana asa G 25 mg) should be constituted in not more than 1.3 ml of normal saline accordi D. guideline (see table | above). ee cence Ina nut shell, 130 g = 3.25 mg = 1.3 ml of normal saline. 1f 3.25 is ee 3.25 mg would be constituted in ‘Then, one tablet of sylimarin (70 mg) would be constituted in 1.3 ml /3.25 mg X 70 mg =28 ml of normal saline That is 70 mg/ 28 mL = 2.5 mg/ml From this stock solution, dosages can be administered to animals of varying body weights based on the OECD's 10 mi/kg standard volume rationale as shown in able 3 below: Table 3: Showing stock solution preparation for sylimarin tablet and required doses for animals of different body weights. STOCK Animal’s body | Calculated dose | Equivalent SOLUTION weight in dose in ml (s) mg 7Omg28ml | 130g 3.25 mg 1.30 mi =(25 mg/ml) [135g 3.38 mg 1.35 ml 140g 3.50 mg 1.40 ml 150g 3.75 mg 1.50 ml From the above table, it shows that animals with higher body weight will receive higher doses (mg or ml) of sylimarin solution. . 4. Dosage calculation and preparation of stock solution for salt compound (example: Alloxan monohydrate). Alloxan monohydrate is a diabetogenic agent at a standard dose of 150 mg/kg for rat via intraperitoneal route. It is in salt form. The required dose of alloxan monohydrate to induce experimental diabetes intraperitoneally in a rat weighing 200 g at a standard dose of 150 mg/kg can be calculated as follows; Required dose for 200 g rat = weight of animal (g) /1000 (g) X Standard dose (mg) = 200 g/ 1000g X 150 mg = 30 mg For intraperitoneal administration, appropriate volume of vehicle ranging between 2 ml/kg to 5 mb/kg in rats is recommended. Based on 2ml/kg volume selection in this example, 30 mg (0.03 g) of alloxan monohydrate would be constituted in = 200 g/ 1000g X 2 mL = 0.4 ml of a vehicle (normal saline) ‘corresponding with the volume required for 200 g rat.. Analytical weighing balance with higher accuracy and sensitivity should be used for weighing alloxan monohydrate. However, dissolution volume of 5 ml/kg can be considered for intraperitoneal administration in mice (due to their low body weight compared to rats). 5. Dosage calculation and preparation of stock solution for Ampoules (example: gentamicin injection). Gentamicin is an aminoglycoside antibiotic used to induce nephrotoxicity in experimental animals (rats and mice) at a standard dose of 80 mg/kg (via intraperitoneal route). The required dose of gentamicin to induce experimental nephrotoxicity intraperitoneally in rat Weighing 100 g at a standard dose of 80 mg/kg can be calculated as follows: Step 1: Dosage calculation Required dose for 100 g rat= weight of animal (g) /1000 (g) X Standard dose (mg) = 100 g/ 1000g X 80 mg =8 mg - Step 2: Required volume calculation for intra-peritoneal injection From the above calculation, 100g rat requires 8 mg of gentamicin and this dosage (8 mg) can be withdrawn directly from the stock of gentamicin which is present as 80 mg/2 mi (40 mg/ml) ampoule. Thus, 40 m, wwewvus weweve Worse eee 100 g rat requires 8 mg which is equivalent to =2mL/ 40 mg X 8 mg = 0.2m, ‘Twice serial dilution can be done as follows = 40mg / 1 mL of Gentamycin + 1 mL of normal saline =40 mg/ 2 mL diluted gentamycin 20mg/mL, ‘Therefore, double volume (for example 0.1 ml for 25 g mouse) is required form the diluted stock of gentamicin for experimental animals. 6. Direct calculation of animals’ dose from human dose Some experimental situation may arise, when animal doses of compounds of different formulations (syrup, tonic, solution, ampoule injection, suspension and tablet) are calculated from average adult (70 kg) human dose. For example, ifthe required dose of chemiron blood tonic for 70 kg human = 10 ml (x3 daily) =30 mV/70 kg/day. Dosage for a rat weighing 150 g can be calculated as follows: 70 kg (70,000 g) adult man requires = 30 ml of chemiron blood tonie per day, Therefore, 150 g rat requires = 30 mL/70000g X 150 g = 0.06mL Since the volume is very small to be measured with one milliliter syringe, it is recommended to cary out ten times (x10) serial dilution for the tonic by; Diluting 1 ml of chemiron blood tonic with 9 ml of normal saline or diluting 10 ml of chemiron blood tonic should be diluted with 90 ml of normal saline Required new volume of chemiron tonic for 150 g rat from the diluted chemiron will be = 0.06 ml x10= 0.6 mi. Subsequent volume of chemiron canbe calulted for rats whose body weighs are diferent m 150 g. ‘However, further serial dilutions (x15 to x 20) are recommended for mice (which weigh far Jess than rats) in this scenario in order to obtain a measurable volume. Result: Proper dosage calculation, stock solution preparation and substance delivery to experimental animals via an appropriate route were studied, CCCCe 2% Co CCUTUUTUTUCUCUUUUUCTU ECCT TY Experiment No. 2 ‘Aim: To screen anti-allergic activity of the drugs by mast cell stabilization assay demonstrated by simulated experiments/videos. References: 1, Prussin C, Metcalfe DD. IgE, Mast cells, Basophils and Eosinophils. J Allergy Clin Immunol. 2003; 111 2) 486-94, 2. Hajare R, Darvhekar VM, Shewale A, Patil V. Evaluation of antihistaminic activity of Piper betel leaf in guinea pig, African J Pharmacy Pharmacol. 2011; 5 (2):113-7. Requirement: Simulated experiments/videos Animals: Guinea pigs of 400-600 g of either sex, Albino rats of 175-200 gofeither sex. Drugs: Histamine dihydrochloride aerosol (0.2% w/v; Chlorpheniramine maleate (2 mg/kg, s..); Disodium chromoglycate (SOmg/kg.i.p.) Reagents: Saline solution (0.9%); RPMI 1640 bu"er medium (PH 7.2-7.4); egg albumin (100 g/mL); toludine blue solution (1%); Instruments: Microscope with 10X magnification lens Theory: In allergic diseases mast cells play an imp. antibodies formed in response to antigen antibody compl and rises calcium influx leading to degranulation of m ‘mediators (also known as local hormones) such as ortant role by defending the antigens. IgE lex attaches to the surface receptors of mast cells ast cells which releases some pro-inflammatory histamine and eicosanoids. Procedure: - Evaluation of bronchoconstriction in guinea pigs by using histamine ‘aerosol Selected guinea pigs are divided into two have to be fasted overnight. Group-1 Chlorpheniramine maleate (2 mg/kg, “exposed to histamine aerosol (0.2% Sroups consisting of 3 animals in each. Animals receives normal saline. Group-2 animals receive S.C), Before the drug treatment animals should be ‘Then determine the end point i.e. of histamine aerosol to onset of ) in histamine chamber, the time of exposure Percentage (%) protection= (1-Ty/T,) x 100 Where 7, drug administration, mean value of PCD before drug administration, Ty= mean value of PCD after Observation Table P VUVUVTTG TSCCVVVUV UV UU UUUUG Group Percentage protection (Group-I (Saline) roup-2 (Chlorpheniramine maleate] 2 mglkg, S.C.) 2. Mast cell stabilization activity: Albino rats of either sex are divided into two groups consisting of 3 animals in each. Group-1 receives normal saline, and Group-2 receives Disodium chromoglycate (50 mg/kg. iip.) for 3 days. Inject 10 ml/kg of 0.9% saline into peritoneal cavity on 4th day to each animal. Massage the peritoneal region of the animal gently for 5 min, then collect the peritoneal fluid and transfer to the test tube which is carrying 7-10 mi of RPMI buffer. Centrifuge the fluid for 400-500 RPM. Discard the supernatant and wash the pellets of mast cells twice with same buffer by centrifugation. Add egg albumin to the above cell suspension and incubate at 37°C for 10 min. Later the suspension has to stain with 1% toluidine blue solution and observe the slide under microscope for calculating number of granulated and degranulated mast cells in each group (otal 100 cells has to be counted from different visual areas). Observation Table Group Total number of cells (n=100) Granulated Degranulated \Group-T Galine) fiow? Disodium Jhromoglyeate (50 mg/kg. LP.) Result: Pretreatment of animals with standard drugs stabilizes mast cell membrane and generates nitric oxide as defensive mechanism that inhibits the release of chemokines, which are responsible for vasoconstriction. GIG x I< bt. WCIISS a USS sh ‘ VVVVNV VS Experiment No. 3 ‘Aim: To study anti-ulcer activity of a drug using pylorus ligand (SHAY) rat model and NSAIDs induced ulcer model demonstrated by simulated experiments/videos. References : 1. Shay H., A simple method for the uniform produ Gastroenterol. 1945; 5:43-61 : a? . 2. Vinothapooshan G, Sundar K. Anti-ulcer activity of Mimosa pudica leaves against gastric ulcer in rats. RJPBCS, 2010; 1(4):606-14. Requirement: Simulated experiments/videos Theory: : : Peptic ulcer is one of the most prevalent gastrointestinal disorders. The aim of the present study is to demonstrate the antiulcer activity of drugs using pylorus ligand (SHAY) rat model. This was first demonstrated by Shay in 1945. Ligation of rat pylorus results gastric acid accumulation in the fore-stomach leads to acute gastric ulcers. This procedure is used to sereen the drugs for their anti-secretary and antiulcer activity. Requirements Animals: Albino Wistar rats of 150-200 g are selected for the study. Drugs: Ether (anaesthetic), Ranitidine 20 mg/kg, p.o, 0.9% normal saline Reagents: 0.1 N NaOH, Phenolphthalein, Topfer’s reagent, Instruments: Dissecting microscope (10X magni_cation lens), Burette, pH meter, Surgical instruments. Procedure Animals are to be divided into two groups consisting of 3 animals in each group. Saline is to be administered to control group and Ranitidine (20 mg/kg, P.O.) to other group. Animals have to be fasted for one day with free access to water. 30 min prior to ligation process, the drug (Ranitidine) should be given. Under light ether anaesthesia a midline abdominal incision is made and pylorus will be ligated with proper care and the wound is closed. Then rats are to be placed individually in separate cages without food and water during this period and allowed them to recover. Sacrifice the animals by decapitation after 4 hours and open the stomach and collect the stomach contents in a centrifuge tube. Determine the PH meter. Open the greater curvature of the stomach and clean the part with saline. Under 10X magnification Jens ulcers are to be observed and ulcer index is calculated by using the formula given below: Ulcer index= (U; + Uz+ Us) x 107 U, = Average of number of ulcers per animal U2 = Average of severity score Us = Percentage (%) of animals with ulcer Intensity of ulcers with scoring was undertaken as following manner: 0 normal coloration; 0.5 —red coloration; of gastric ulceration in the rat. 1 = spot ulcer; 1.5 — hemorrhagic stress; 2 — deep ulcer; 3 — perforations. Ulcer score: 1 mm (exact) = -2mm =2;>2mm=3;>3mm= Non-steroidal anti-inflammatory (NSAIDs) induced ulcer model Aspirin, non-selective COX inhibitor is an example of NSAIDs, Aspirin i my/kg (20 mg/ml) was administered to th the experiment and ces unimals on the day of the experiment and ulcers were scored after 4 h. The animals were sacrificed and the stomach was then excised and cut along the greater curvature, washed carefully with 5.0 ml of 0.9% NaCl and ulcers were scored by a person unaware of the experimental protocol in the glandular portion of the stomach, Ulcer index has been calculated by adding the total number of ulcers per stomach and the total severity of ulcers per stomach. The total severity of the ulcers was determined by recording the severity of each ulcer after histological confirmation as follows: © 0,no ulcer; * +, pin point ulcer and histological changes limited to superficial layers of mucosa and no congestion; ‘* +4, ulcer size less than Imm and half of the mucosal thickness showed necrotic changes; +++, ulcer size 1-2mm with more than two-thirds of the mucosal thickness destroyed with marked necrosis and congestion, muscularis remaining unaffected; © +++, ulcer either more than 2mm in size or perforated with complete destruction of the mucosa with necrosis and hemorrhage, muscularis still remaining unaffected. Analysis of stomach contents Measure total volume of gastric content and centrifuge at 1000 rpm for 10 minutes. Pipette out one ml of supernatant liquid of the centrifuged content and dilute with 10 ml distilled ‘water. Titrate the liquid against 0.01N NaOH using Topfer’s reagent as indicator, till the end point (appearance of orange colour). The volume of NaOH used is to be noted to estimate free acidity. Titration has to be continued till the appearance of pink colour and the volume of NaOH run down is to be noted to calculate total acidity. Calculation of acidity is given below: Observations Group | Volume of pH | Freeacidity | Total acidity | Ulcer index Gastric contents (mEq/l) (mEq/) (nm) Control (saline) Standard (Ranitidine 20 mg/kg P.O) Result: Comparison of ulcer index between study groups estimates the potency of antiulcer activity of test drug. Decrease in volume of gastric contents, free and total acidity determinesy anti-secretory activity of test drug and rise in pH evaluates acid neutralizing action of the drug. ee ee ar er Experiment No. 4 ‘Aim: To study effect of drugs on gastrointestinal motility demonstrated by simulated experiments/videos. References: Peddireddy MKR. Jn-vitro Evaluation Techniques for Gastrointestinal Motility, Indian J Pharmaceutical Education Res. 2011; 45 (2): 184-91 Requirement: Simulated experiments/videos Theory: Intestinal motility is regulated by the enteric nervous system of the gut (AuerbAChs and Meissners plexuses) and the activity of this system can be modified by autonomic nervous system. Hence effect of sympathomimetic and parasympathomimetic drugs on jntestinal motility can be studied by using isolated piece of intestine. Parasympathomimetic drugs stimulate enteric neurons to release acetylcholine at neuromuscular junctions and Gnhance muscle tone and rhythmicity of intestine. Sympathomimetic drugs acts on alpha and beta receptors and releases ‘adrenaline which in turn prevents release of acetylcholine and inhibits muscle tone and rhythmicity. Many animal models can be employed to study intestinal motility of symapathetic and parasympathetic drugs. Guinea pig ileum is advantageous for assay purposes as it produces steady baseline for studying effects of drugs. Rabbit intestine (ileum, deuodenum, jejunum) dular movements (continuous contraction and usually jejunum is used for the effects of pen claxavion- Finkelman method). In the present study rabbit ileum is selected for estimating the effects of selected drugs on intestinal motility. Requirements Animals: Medium sized rabbit Drugs: "Adrenaline/Acetylcholine-L0yg/ml, Atropine _—_sulphate-100yg/ml, ieoproterenolsoprenaline 10ug/ml, Propranolol- Img/ml, Phenylephrine- 10pg/ml, phentolamine- 0.1g/ml ‘Solutions: Tyrode solution ‘Apparatus used: Kymograph, Syringe, Frontal writing lever, aeration tube. Procedure. The procedure adopted for the study is the modified Finkleman method developed by Walker and Scott. Select a medium sized rabbit for the study. Fast the animal od in gut results in messy dissection and flushing of gut for 24 hours prior to experiment as fo contents may damage the intestine, Before sacrificing the rabbit, prepare Tyrodes Ringer Sotiton and place about 250 ml of this solution in an ice cold flask. Sacrifice the animal by se rvical decapitation without use of anesthetic as it may affeet the gut motility. Shave the cedomen of the animal and vacuum the surface to remove adhered fur, Make a midline aicsion through the skin and abdominal muscles. Locate ileum and a part of ileum was taken To em away from ileocaecal valve. An optimal length of tissue (5-6 ems) is cut carefully and tie with the help of thread to antimesenterie border on both sides and place them in’ the Tyrode solution (extra pieces of ileum can be stored in ice cold Tyrode solution so that they ae viable for hours. In ice cold solution the motility will cease but after placing them in swarm solution the tissue gets relaxed and shows motility within 5-10 minutes). Record the rhythmic activity of the ileum by using frontal writing le : a is lever and ‘ Suspend the tissue in organ bath of Tyrode solution (100 ml) at 37 °c with ade kymograph, sup (inixture of 95% O; and 5% of C02). Tie one end of the thread of the tase et point inside the organ bath and the other end to the lever for recording contraction i ie kymograph. Stabilise the tis: he sol e factions on the cymogray ise the tissue in the solution to the conditions for about 30 minutes, F Dissecting board, Dissecting instruments, scissors, petriplates, water bath with temperature controlling unit, organ bath with ee the lever should be placed horizontally and record the normal contractions followed by effects of drugs on muscles. After recording normal contractions inject the drugs one by one and observe for force of contraction and tone (normal, increased or decreased), frequency of contractions (per minute) before and after drug administration. Inject 0.1 ml of drugs in the succession order in the organ bath and the responses are recorded. After noting the effect of every drug, drain the muscle bath and refill with fresh warm Tyrode solution (100 ml). Take the control (without drug) reading before and after each drug response. Maintain wash out period for 15-20 minutes for change of every drug and check the next drug response only when the tone and amplitude returned to original value approximately. The drug and dose name should be mentioned in the recording after taking response of each drug. Order of adding drugs. 1. 0.1 ml of ACh- 1.0 ml of ACh- 0.1 ml of NE- 1.0 ml of NE (these drugs are postganglionic neurotransmitters of parasympathetic and sympathetic system. ACh increases contraction and NE relaxes the tissue) 2. 1.0 ml of phenylephrine- 1.0 ml of isoproterenol (phenylephrine causes contraction by {inhibiting adenylate cyclase- alpha adrenergic agonists; isoproterenol causes relaxation by showing beta agonist action) 3, Add drugs one after one (1 and 2) to check for their activity. After taking response of each drug drain the solution and refill the bath with warm Tyrode solution and proceed with next drug. 4. 1.0 ml of phenatolamine (alpha adding 1.0 ml of phenylephrine (to response is seen), 5. 1.0 ml of propranolol (beta adrenergic blocker). Wait for 2 minutes then proceed for adding 1.0 ml of isoproterenol (to check for beta receptor blockade, if they are blocked no response is seen) 6.1.0 ml of atropine- wait for 3 minutes- add 1.0 ml of ACh- (to check for parasympatholytic activity of atropine) Result: The effect of drugs on intestinal motility can be easily interpreted by the responses taken on kymograph. adrenergic blocker). Wait for 2 minutes then proceed for check for alpha receptor blockade, if they are blocked no . Experiment No. 5 Aim: To demonstrate effect of agonist and antagonists on guinea pig ileum by simulated experiments/videos. References: Ghosh MN. Fundamentals of Experimental Pharmacology. Hilton & Company, Kolkata Requirement: Simulated experiments/videos Theory: Guinea pig ileum is a smooth muscle which receives dual nerve supply from autonomic nervous system (sympathetic and parasympathetic). Parasympathetic tone is dominant in ileum in which ACh causes contraction by stimulating M3-R. The drug induces contraction of ileum. It is very sensitive to histamine unlike the rabbit due to the presence of histaminase enzyme. Cholinergic, Serotonergic or tryptaminergic receptors (5-HT1—5-HT7), Histaminergic and Adrenergic receptors are present in Guinea pig ileum. ‘Types of receptors in guinea-pig ileum: Receptor ‘Agonist ‘Antagonist Cholinergic receptors: jcotinie neuronal receptor | Dil. Nicotine Cone. Nicotine (Nn-R) “Muscarinic receptor(M3-R) | ACh ‘Atropine ‘Serotonergic receptors: ‘S-HT2A (Gqll) serotonin “Methysergide & ketanserin (selective) . -cyproheptadine (non selective block both 5-HT2A_ &5-HT receptors) Histaminergie receptors Hy Histamine Mepyramine cyproheptadine Ha Histamine ‘Cimetidine; Ranitidine ‘Adrenergic receptors [at ‘Adrenaline Phentolamine [oz ‘Adrenaline - Result: Cholinergic (ACh), Histaminergie (Histamine), Serotonergic (Serotonin) and direct acting stimulants (Barium chloride, Caleium chloride) contract the ileum of guinea-p MgClo and Papaverine are direct acting Spasmolytics. Experiment No. 6 Aim: To estimate serum biochemical parameters by using semi- auto analyser. References: Requirement: semi- auto analyser Sample (Serum creatinine): Unhaemolysed serum; Reagent Composition: Surfactant 1.00% HCL 100 mmol/L Sulphanilic acid 5 mmoV/L Reagent 2: direct bilirubin reagent Sulphanilic acid 10 mmol/L HCL 100 mmoV/L Reagent 3: sodium nitrite reagent Sodium nitrite 144 mmol/L Reagent 1: total bilirubin reagent Theory: ‘Semi automated (batch) discrete analysers In the case of these analysers the initial part of the procedure i.e. pipetting of reagent and specimen, mixing and incubation is carried out by the technician, Rest of the procedure i.e. setting of incubation temperature (for kinetic determinations), zero setting, photometric readings, result display, automatic printing and data management and processing is carried ‘out by the analyser. Advantage of semiautoanalysers 1) The semiautoanalysers are cheap and compact, compared to other fully automated analyses. 2) Specimen analysis is cheap, since volume of reagent used is 0.5 to 1.0ml. 3) Enzyme determinations by kinetic methods are performed accurately in 1 to 3 minutes. 4) The enzymatic reagents are not corrosive and involve monostep testing. In blood quantitative estimation of glucose is done with serum and several methods have been in use. Bilirubin (formerly referred to as hematoidin) is the yellow breakdown product of normal heme catabolism. Bilirubin is excreted in bile and urine, and elevated levels (more than 1.5 mg/dL or more than 26 umoV/L 5 may indicate certain liver diseases, obstructive condition of the bile duct, hepatitis, cirthosis, in hemolytic disorders and several inherited enzyme deficiency. It is responsible for the the background straw-yellow colour of urine (via its reduced breakdown product, urobilin — the more obvious but variable bright yellow colour of, urine is due to thiochrome, a breakdown product of thiamine), the brown color of feces (via its conversion to stercobilin), and the yellow discoloration in jaundice. Total bilirubin is now often measured by the 2,5-dichlorophenyldiazonium (DPD) method. Principle Bilirubin reacts with diazotized sulphanilic acid in acidic medium to form pink coloured azobilirubin with absorbance directly proportional to bilirubin concentration. Direct bilirubin, being water soluble directly reacts in acidic medium. However indirect or unconjugated bilirubin is solubilized using a surfactant then it reacts similar to direct bilirubin. Precaution: Avoid hemolysis as it causes falsely low results. Sample should be protected from bright light as bilirubin is photo labile, Samples may be stored refrigerated for 3 days or frozen for 1 month. Glucose: An important carbohydrate in biology, cells use it as a secondary source of energy and a metabolic intermediate. Glucose is a major source of energy for most cells of the body; insulin facilitates glucose entry into the cells. Glucose increases due to diabetes mellitus, in ' ’ ’ » » , > > > > > > 2 > > a > a 2 2 2 ? 2 2 2 2 2 ? ? ? ? ? 2 patients receiving glucose containing fluids intravenously, during severe stress and cerebro vascular accidents, It decteases on insulin administration, as a result of insulinoma, inborn errors of carbohydrate metabolism or on fasting. Creatinine is a waste product formed in muscle by creatine metabolism. Creatine is synthesized in the liver which then passes into circulation where it is taken up by skeletal muscle for conversion to creatine phosphate which serves as storage form of energy in skeletal muscles. Creatine and creatine phosphate are spontaneously converted to creatinine at a rate of about 2% the total per day. This is related to muscle mass and body weight. Creatinine formed is excreted in the urine. On a normal diet almost all creatinine in urine are endogenous. Its excretion is fairly constant from day to day and has been used to check the accuracy of 24 hours urine collection. It is independent of urine flow rate and its level in plasma is quite constant. Estimation of serum creatinine by Jaffe's Alkaline Picrate Method Prineiple: Creatinine in alkaline medium reacts with picric acid to form a red tautomer of creatinine picrate the intensity of which is measured at 520nm. The two chief disadvantages with Jaffe's reaction are - Lack of specificity:- Only 80% of the colour developed is due to creatinine in serum. other ‘non specific chromogens that react with picric acid are proteins, ketone bodies, pyruvate, glucose, ascorbate etc. ~ Sensitivity to certain variables like PH temperature etc. Reagents: 1) Picric acid — 0.04M (9.16g/L) 2) Sodium hydroxide — 0.75N 3) Sodium tungstate — 10% 4) 2/3 N H2 SO4 5) Creatinine standard stock ~ 100mg% 6) Working standard ~ 3mg% Procedure :- In a centrifuge tube, take 2ml of serum with 2 ml of distilled water. Precipitate proteins by adding 2m! sodium tungstate and 2ml of 2/3 N sulphuric acid drop with constant shaking. Stand for 10 minutes and filter. Use protein free filtrate for test. Make the test tubes as follows and add : B SL S2 S3 S4 bd Filtrate Standard 3mg% D. Water NaOH Picric acid Mix well Allow to stand for 15 min. and read absorbance at 520nm, Calculation : O.D. Test Amount of Std, Serum creatinine (mg%) = 7 O.D.Std. Vol, of Serum x 100 Interpretation : Normal serum creatinine levels are ma :0,7-1 4mg/100m 1 Yee VVVbbbbbbbbUVbbULULVUVULUVUUYUYVUUTEwvst -1.2me/100m 1 in males since it is related to body size. rum levels ar urea. Creatinine, howev. Increased ser f seen in renal failure and other renal diseases in a manner similar to er, does not increase with age, dehydration and catabolic states (eg fever, sepsis, haemorrhage) to the same extent as urea, It is also not affected by diet. But the Jafie's reaction for measuring serum creatinine is not as sensitive andreliable as wnt for urea, It is interfered with by Ketone bodies and glucose and hence false high Values may be obtained in diabetes ketoavidesis, Low Serum creatinine is not significant. Tis associated with muscle wasting diseases, Estimation of Serum Albumin and Globulin Object: To estimate serum albumin and globulin, Bromocresol Green Method Principle : The method is based on the protein error of indicators. Biding of a protein to an cofur. Among serum proteins, only albumin binds to BCG this binding produces a change in the is measured colorimetrically. The PH is ‘maintained during the reaction by a buffer, Reagents ( Succinate buffer - 11, is adjusted to 4.0 with 0. The volume is made up This solution should be stored in refrigerator. 8) BCG solution - 419 mg of bromocresol green is dissolved ix 10 ml of water. The solution is stored in refrigerator. Gi) Buffered BCG solution — 250 ml of BCG solutions is mi buffer. The pH is adjuste 'd to 4.2 with 0.1 N sodium hydi solution (30%) is added. (iy) Standard albumin solution — an aqueous solution of Prepared and used as a standard, 8 gm of soccinic acid is 1N sodium hydroxide. to 1 litre with water, dissolved in about 800 ml of water. The pH tuman albumin with a concentration Sodium azide should be included in led human serum (preserved with rum having an albumin concentration of 4 gm/100 mi ean aley ‘Blank’, 0.02 ml of serum into 'Unknown', and 0.02 ml of water into 'Blank’, be used as a standard. Procedure : Level 3 test tubes ‘Unknown’, ‘Standard’ and BCG solution into each. Wash solution into ‘Standard’ 5 minutes, Read ‘Unknown’ and 'Standard’ against ‘Blank! Calculations : Au Serum albumin (gm/100 ml)=x 4 As Interpretation : ‘The normal range of serum albumin is 3, Measure 4 ml of buffered 0.02 ml of standard albumin Mix and allow the tubes to stand for at 630 nm or ig a red filter, 75.3 gm/100 ml. Serum globulin ranges from 1.8 to 2.6 ein/100 ml. the A:G ratio is roughly 2:1 though it may range from 1.2:1 tod se Decrease in serum albumin may occur in protein under nutrition, intestinal malabsorption, Protein-losing enteropathy, liver disease, wasting diseases, nephritie _ syndrome and haemodilution. A severe decrease or near ~ absence may be seen in analbuminaemia which ig 8 genetic disease with autosomal recessive in-hertence. A rise in serum albumin may cere in dehydration due to haemoconcentratior se gies tat decrease due to haemodiution in shock, burns, haemorthage ete serum slobulin increases in multiple myeloma, macroglobulinaemia, chronic liver disease, chrome infections and autoimmune diseases. A:G ratio may be decreased or reversed in theo conditions. Me ke NN EEE NEE i that of globulin, the Since the colorimetric measurement of albumin is much simpler than ally Jobulin is obtained {foncentrations of total proteins and albumin are measured in serum, and gl by difference. Technical contents : Using kit of serum albumin, in by kit and Method of teaching : Demonstration and its measurement of serum albumin y taking readings. Using a colorimeter or semi auto analyser. Evaluation : Giving exercise of serum albumin estimation. CE PSST WIN ITN N00 SY SUN Se UH 0 WW Experiment No. 7 : ‘Aim: To demonstrate effect of saline purgative on frog intestine by simulated experiments! videos. References: Requirement: simulated experiments/videos s comprised of highly charged ions and do not cross cell membrane freely. They remain inside the lumen and retain water through osmotic forces. They increase the volume of the content of the bowel, stretch the colon and produce the normal stimulus for contraction of the muscle that leads to defecation. ‘Theory: Saline purgatives are the salts Requirements: Software Animal: Frog intestine s ; Reagent: 0.9% to 0.45% of saline (hypotonic), 27 % hypertonic, Frog ringer solution (isotonic) Procedure: Pith the frog and place it on dissecting board. Expose the abdominal cavity and carefully trace the small intestine. Make the small intestine in three comportments by tying the threads of different colours in such a way that no fluid can move from one compartment to the other. Inject 0.2 ml of each hypnotic solution into first compartment, 0.2 ml of hypertonic solution into second compartment and 0.2 ml of isotonic solution into third compartment. Wait for 20 min. and the observations are to be recorded. Observations Drug ‘Compartment. Effect Hypotonic solution (0.2ml | first compartment Shrunken of 0.9% of saline) Hypertonic solution (0.2ml | second compartment ‘Swollen | of 27 % MpSOs) Isotonie solution (0.2ml of | third compartment No Change Frog ringer solution) Result: Hypotonic solution causes the fluid to move from lumen into the circulation by process osmosis Uereby shrinks the tisue, Hypertonic solution moves from eel ito lumen and ‘swells the tissue and Isotonic solution did not show any fuk ii 0 uid movement across “ hacer y ‘ment across the intestinal t f t t if t [ t [ 6 Experiment No. 8 Aim: To demonstrate insulin hypoglycemic effect in rabbit by simulated experiments/videos. References: Requirement: simulated experiments/videos Theory: Insulin is a peptide hormone produced by the beta cells of pancreas in response to high glucose levels in the blood. A released insulin acts on the insulin receptors on body cells and activates glucose transporters to absorb more glucose into the cells thereby regulates carbohydrate, protein and fat metabolism in body cells. Reduced blood glucose levels inhibit insulin release and stimulate alpha cells of pancreas to release glucagon to maintain glucose levels in the blood by glycogenolysis and gluconeogenesis. Requirements ‘Animals required: Healthy rabbits weighing 1800-3000 gms Drugs: 20 units of insulin preparation. One unit contains 0.04082 mg of insulin Reagents: Normal saline, HCI, 0.5% phenol, 1.4-1.8% glycerin. Procedure 1. Select healthy rabbits weighing 1800-3000 gms for the study. They should be maintained in uniform diet for 7 days. Fast the animals for 18 hrs with no access to Water before starting the procedure. Select three animals for the study and inject 1 univ/ml of insulin. 2 2. Prepare drug solution freshly. Weigh 20 units of insulin accurately and dissolve it in normal saline. Acidify the solution by using HCl to pH 2.5. Add 0.5% phenol as preservative and 1.4 tol.8% glycerine and make the final volume to 20 units/ml of solution. 3, Withdraw 2 ml blood from marginal ear vein of each rabbit and estimate blood lucose level by using suitable biochemical method and the concentration of glucose cam be noted down as initial blood glucose level. Then inject lunit/mL insulin to the gnimal and check the blood sugar level up to 5 hrs at the interval of Ihr each and determine blood glucose levels as final blood sugar level and compare both the final and initial blood glucose levels. Observation Table Final blood glucose level at different time intervals Animals Initial blood (in mg/ml) in hours Glucose level (in mg/ml) 1 1 2 3 4 S 2 _3 Average Mean Value Result: Mean percentage decrease of blood glucose level at different time intervals 4 “termines the effect of insulin. ro 6 w e « S S S ° e af a Mh S S s ¢ e e 1 q ¢ o & Experiment No. 9 ‘Aim: To demonstrate test for | Pyrogens (rabbit method) by simulated experiments/videos. ferences: Test for pyrogens, i a gens, The International Pharmacopoeia - Ninth Edition, 2019. ‘The pyrogen test is designed to limit the rs ; Miminisration of drugs. Teuee mit the risk of a febrile reation following parenteral the test rabbit in a dose of 10 mL per kg, injected for liquid products that can be tolerated bY tr more than 4 minutes. For products th een generally within a period of eal conditions of administration, additonal ure preliminary preparation of a oe be a ns given in the monograph should be Test animal Use healthy, te rabbits, preferably ofthe same variety. House the animals individually in fa area of uniform temperature (2 °C), possibly with uniform humidity, and free from Saturbances likely to excite them. The animals are given ad fae er and food, vnmonly used for laboratory animals. One to 3 d Se er bal comm to 3 days before using an animal that has not previously been used for a pyrogen test, condition it by conducting a training exercise 95 described under the recommended procedure, omitting the injection. Do not use animals for pyrogen tests more frequently than once every 48 hours. After @ pyrogen test in the course of which a rabbit's temperature has raised by 0.5 °C or more, OF Fore rabbit has been given a test substance that was adjudged ‘pyrogenic, at least 2 weeks a be allowed to elapse before the animal is used again : Tomperature recording: Use an accurate thermometer graduated in 0.1 °C that has been tested to determine the time necessary to reach the maximum reading, or any: other temperaturerecording device of equal sensitivity. Inset the temperature-sensing device into frevecum ofthe test animal toa depth of about 6 em If the temperature-sensing device is to remain inserted throughout the sensing period, restrain the rabbit with lightly-fitting neck vey tht allows it to assume a natural resting posture: When & thermometer is used, allow SMfcent time for it to reach a maximum temperature, a5 previously determined, before toking the reading. Procedure Perkam tke test in the area where the animals are housed oF under similar environmental conditions For 2 hours before the test and during the test, ‘withhold all food from the animals teing used. Access to water may be allowed. The ‘animals should be placed under the conditions of the test at least 1 hour before the injection. Por tthe test, 40 minutes before the injection of the test ‘material, determine the temperature of each animal by taking 2 measurements af 2 interval of 30 minutes. The mean ofthe temperatures serves asthe "control temperature” ofthe animal. The control temperature recorded for each rabbit constitutes the temperature from which any subsequent rise following the injection of the ‘material is calculated. In any one test, use only those animals the control temperatures ‘of which do not deviate by etn |.0°C fom eachother ‘Ga animals for which the 2 temperatures used to determine the control temperature have more by more than +0.2 °C from the mean ‘should not be used in the test, nor should any Rede 4 control temperature below 38.0 °C or above 39.8 °C. 2 aringes, needles, and glassware fee from pyrogens by heating at 250 °C for not i ‘table method. Warm the solution to be tested to P C a “4 Inject into a marginal vein of the ear of each of 3 rabbits 10 mL of the solution per kg of body ‘weight or the amount specified in the monograph. The injection should last not longer than 4 minutes, unless otherwise specified in the monograph. ‘When the injection has been completed, record the temperature of the animal during a period of 3 hours, taking the measurements continuously or every 30 minutes. ‘The maximum temperature recorded for each rabbit is considered to be its response; if the temperature readings taken after the injection are all below the control temperature, the response is tre as a zero temperature rise. If no rabbit shows an individual rise in temperature of 0.6 °C or more above its respective control temperature, and if the sum of the 3 temperature rise does not exceed 1.4 °C, the tested material meets the requirements for the absence of pyrogens. If 1 or 2 rabbits show a temperature rise of 0.6 °C or more, or if the sum of the temperature rise exceeds 1.4 °C, continue the test using 5 other rabbits. If not more than 3 of the 8 rabbits show individual rises in temperature of 0.6 °C or more, and if the sum of the 8 temperature rise does not ‘exceed 3.7 °C, the tested material meets the requirements for the absence of pyrogens. Result: Test for pyrogens (rabbit method) was studied. fr 4 rrneerser 4K Experiment No, 10 Aim: To determine acute oral toxicity (LDso) of a drug from a given data. Reference: 1.OECD guid ines for the Testing of Chemicals, 2. Kulkami S. K."Hand Book of Experimental Pharmacology" Third edition 1999. Vallabh Prakshan New Delhi, Page no. 168-170. Requirement: Acute oral toxicity (LDs0) data of drug Theory: The determination of EDso (the dose effective in producing certain expected response in 50% of animal group) values helps in ascertaining the potency of a drug in terms of a reference standard. The calculation of EDso value is done when a drug is showing graded response. But when the response is quantal or all or none, the EDsp value becomes LDso (the dose lethal to 50% of the animal group).Both these values EDso and LDSO are important for knowing the safety of a drug. The ratio between LDsp and EDso (LDso/EDso) represents therapeutic index. Grater is the therapeutic index, safer is the drug. The therapeutic index of most of the drugs which have low margin of safety is generally close to unity. When no information is available to make a preliminary estimate of the LDSO and the slope of the dose-response curve, results of computer simulations have suggested that starting near 175 mg/kg and using half-log units (corresponding to a dose progression of factor 3.2) between doses will produce the best results, This starting dose should be modified if the substance is likely to be highly toxic. The half-log spacing provides for a more efficient use of animals, and increases accuracy in the prediction of the LDSO value. Ideally one would like to determine a dose that is effective in most of the animals (ED99) and least toxic to most of the animals of the group LDjq). These values can, however, be calculated from the graph. Graphical, arithmetical methods and statistical approach are various methods used to calculate LD50 value. For calculating LDSO by either method find out the least tolerated dose (smallest) dose (100%) mortality and most tolerated (highest) dose (0% mortality) by hit and trial method, Once these two doses are determined, select at least five doses in between the least tolerated ‘nd most tolerated doses, and observe the mortality due to these doses. Acute oral toxicity (LD50 value) is determined, Procedure: 1, Take data of overnight fasted mice in which drug was given orally, , 2, Each dose group consists of 10 mice’s data, 3. The given data give the result of LD50 study done with drug in mice. Note that a correction factor is applied to 0 and 100 percent mortality group. The percent mortality values are converted to probit values by reading the corresponding probit units from the probit table, 4, Plot the probit values against the log doses and read the LDSO value as the dose that corresponds to probit 5, Result: Acute oral toxicity (LDS0) of a drug from a given data was determined. ee a a ae ee ee . — ___ Experiment No. 11 Aim: Determination of acute skin irritation / corrosion of a test substance. Reference: Draize J. H., Woodard G., Clavery, H. O. Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes, J. Pharmacol. Exp. Ther., 82, 377-390 (1944), Requirements: Animals: 1-3 rabbits (sequential testing); Test substance (Thioanisole): solid oF liquid applied on cm? skin surface; Exposure time: 3 minutes, 1 hr or 4 hrs (skin corrosion) Theor Skin irritation and skin corrosion refer to localized toxic effects resulting from a topical exposure of the skin to a substance. Irritation (Reversible damage) is characterized macroscopically by erythema (redness) and oedema. Damage to keratinocytes & dermal cells leads to inflammation. Corrosion (irreversible damage) is visible necrosis through the epidermis and into dermis - macroscopically typified by ulcers, bleeding, etc. Substances that are caustic (alkali) or of extreme pH are typically classified as corrosive without animal testing. Various assays are available depending on the purpose of the testing: Joble 1: Organisation for Economic Co-operation and Development (OECD) Test Guideline (TG) f In vitro assays validated for regulatory purposes OECD Test Guideline | (TG) i Skin corrosion Transcutaneous Electrical Resistance Test (TER) #30 Reconstructed Human Epidermis (RHE) Test Method 431 [Membrane Barrier Test Method (Corrositex® 435 i ‘Skin irritation RHE Test Method SIT) 439 Strategies for the assessment of skin corrosion and irritation potential + Based on the particular in vitro assay(s) used, skin corrosion shall be tested first, in case of positive results, no further testing is necessary; the substance shall be classified accordingly. * If the result of the skin corrosion test is negative, then a skin irritation study according to European method B46 shall be performed. If the result is positive, no further testing is necessary but classification of substance as category 2-H315 causes skin iritation. * A negative result in the B46 test does not need to be confirmed by additional testing. * Category 1 -H314 causes severe skin burns and eye damage, Test. substance under category 1A / 1B / 1C may also be classified by the relevant packing group (Packing group J, I, Il). OCD Test Guideline (TG) 404 Acute Dermal Irritation/Corrosion in-vivo Deseription of the evaluation and testing strategy 1G 404 notes that “in the interest of both sound science and animal welfare, in vivo testing should not be undertaken until all available data relevant to the potential dermal Suosivity / inrtation of the substance have been evaluated in a weight-of-the-evidence tnt” The weightof-evidence approACh includes analysis of all existing human and rabbit sera pH extremes, and in vitro data. TG 404 also describes a stepwise approACh to ing. aa SSNS Step 2: Analysis of structure activity relationships Step 3: Physicochemical properties and chemical reactivity Step 4: Dermal toxicity Step 5 and 6: Results ftom in-vitro or ex-vivo tests Step 7 and 8: Jn-vivo test in rabbits Procedure for Acute Dermal Irritation / Corrosion A test substance is applied to the shaved bare skin (about 6 cm?) of healthy young adult albino rabbits and the area is covered with gauze. The substance is removed after four hours and the rabbit's skin is observed at specific times (4 hr for irritant responses for as many as days. “Value (erythema+ oedema) Dermal Irritation Score = No. of animals X No. of observations The evaluation for development of any oedema or erythema from 0 day onward for 14-28 days was scored as follows: Table 2: Scoring |») Erythema and eschar formation No erythema’ Very slight erythema (barely perceptible) Well defined erythema [1 Moderate to severe erythema : : | Severe erythema (beef redness) to eschar formation preventing grading of | enthema eee No elena = very slight oedema (barely perceptible) oppBitcedema (edges or area well defined By de rise iema (raised approximately | mm) te oedema (raised more than | mm and extending beyond area of exposure Resut ineviy >))=5) raising) =[o)e}—o| EUH066 (repeated exposure may cause skin dryness or cracking) was observed in an» invitation study. Thioanisole is not corrosive, its classification H315 is confirmed, 7 y q I : y Vv : ? 5 3 a > > > > a a ? > Experiment No, 12 ‘Aim: Determination of acute eye irvtation / corrosion ofa test substance, Reference ttonConee ae for Testing af Chemicals. Test Guideline 405. Acute a 5 ‘conomic Cooperation and Development, Requirements: Animals: White New ‘Theory: Eye invitation: Eye irritation is a general term used to describe the feeling when something is bathering eyes or the surrounding area. Most common symptoms of eye irritation include itchy eves duting the day or at night; watery or teary eyes; eye redness; eye pain; blurred vision and light sensitivity, 7 : The guide for the evaluation of chemical subst Cooperation and Development was used, degree of acute eye irritation/corrosion, 405. Ocular corrosive:(a) A substance that causes irreversible tissue damage to the eye; (b) Substances that are classified as GHS eye irritants Category 1, or EPA Category I ocular 'mitants, or EU Category 1 (18)(19) (20). Category 1-H318 causes serious eye damage. Ocular irritant:(a) A substance that produces a reversible change in the eye: (b) Substances that are classified as EPA Category II or III ocular irritants; or GHS eye irritants Category 2, 2A or 2B ; or EU Category 2 ( 18) ( 19) (20). Category 2-H319 causes serious eye irritation, Zealands rabbits tances issued by the Organization for Economic following the methodology for determining the Procedure 24 hr before treatment, the eyes of all animals were examined for potential eye lesions with opthalmoscopy and healthy animals were subsequently earmarked for individual identification. A total of three rabbits per test group were subjected to a rigorous study of the ocular structures: cornea, iris, and conjunctiva. A volume of 0.1 mL of test substance was instilled to the bottom of the right conjunctival sac, keeping eyelids together over the next 20 minutes. Both eyes of each animal were examined at the time and 24, 48, and 72 hours after, always by the same specialist. Corneal damage was determined in a dark room with the use of solution of 2% sodium fluorescein, and physiological saline was used to remove excess Solution from the instilled developer substance. Finally an ultraviolet light was used for observation. Observations were made up to five days to assess reversibility of the effects, and animals were weighed at the end of the study to compare variations in this parameter. The Ocular Irritation Score (OIS) was determined using the formula below: Individual Observations Ocular Iritation Scores (OIS) = No. of animals X No. of observations Table 1; Ocular Irritation Scores ranges established under the Cuban method for classifi ication of eye irritation/corrosion Ocular Irritation Score fication of eye irritability 0

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