Contents

Experiment Name of experiment Page

No. Date No.
1 22.04.2021 Study the requirements of student’s organ bath and
preparation of Physiological salt solution.
2 28.04.2021 To record the DRC of acetyl choline using
chicken/guinea pig/ rat ileum.
3 29.04.2021 To study the concentration response curve (CRC) of
Acetylcholine using Guinea pig / Rat ileum.
4.1 05.05.2021 To study the LD50 value of drug by Miller-Tainter
method
4.2 05.05.2021 To study the LD50 value of drug by Karber
method
5 06.05.2021 To determine the unknown concentration of
acetylcholine using chicken/ guinea pig/ ratileum by
matching method.
6 12.05.2021 To determine the strength of an unknown sample of
Acetylcholine by Interpolation Bioassay employing
isolated chicken/ guinea pig/ rat ileum by bracketing
method
7 13.05.2021 To calculate the pA2 value for atropine using acetyl
choline using chicken / guinea pig /rat ileum.
8 19.05.2021 To determine the strength of an unknown sample of
Acetylcholine by Interpolation Bioassay employing
isolated chicken/ guinea pig/ rat ileum by interpolation
method.
9 20.05.2021 To study the effect of drug on isolated frog heart
preparation.
10 02.06.2021 To study the designing of ADR monitoring protocol.

11 09.06.2021 Study the clinical trial protocol development.

12 10.06.2021 To determine the potency of unknown solution of

Acetylcholine by Three-point bioassay using isolated
chicken ileum preparation.
13 16.06.2021 To determine the potency of unknown solution of
Acetylcholine by Four-point bioassay using isolated
chicken ileum preparation
14 17.06.2021 To study the Oral Acute Toxicity –Acute Toxic Class
Method (OECD TG 423)
15 30.06.2021 To study the Oral Acute Toxicity –Up and Down
Procedure (OECD TG 425)
16 01.07.2021 To Study the Sub Acute Toxicity A Repeated Dose 28‐
day Oral Toxicity
Study in Rodents (OECD TG407)
17 09.07.2021 To study the drug absorbed in the everted ileum with
UV Spectrophotometry
 Experiment 1

Objective:
Study the requirements of student’s organ bath and preparation of Physiological salt solution.

Principle:

Organ Bath: The tissue bath used to put the animal tissue for studying the drug actions is called Student organ
bath. This was first designed by Rudolph Magnus in 1904. An organ chamber, organ bath, or isolated tissue bath is
a chamber in which isolated organs or tissues can be administered with drugs, or stimulated electrically, in order to
measure their function. The tissue in the organ bath is typically oxygenated with carbogen and kept in a solution such
as Tyrode's solution or lactated Ringer's solution. The use of organ bath preparations for the measurement of
physiological tissue responses to drug concentrations allows the generation of dose response curves. This in turn allows
the quantification of a drug's pharmacological profile in the tissue in question, such as the calculation of the drug's EC50,
IC50 and Hill coefficient.

Physiological salt solution is used to maintain tissue viability outside the animal body to mimic the internal environment
of ions and nutrients because this solution is isotonic with body fluids, it may also be used as a solvent or diluent, for
antibiotics and other pharmaceuticals and biologicals where compatible, and for washing mucous membranes and
other tissue surfaces.

The parts of organ bath:

a. Outer jacket
b. Inner organ bath
c. Thermostat
d. Stirrer
e. Oxygen delivery tube
f. Glass coil
g. Recording levers

The recording procedures:

a. Magnification value
b. Application of load
c. Smoking of kymograph drums
d. Fixing of tracings
e. Contact time
f. Time Cycle
Procedure :

1. We pour tap water inside the student organ bath assembly & the temperature of the water should be
37˚c & the temperature remain fixed by using thermostat & heater is used to heat the water and set the
temperature upto 37˚c.
2. Clamp holder is also used to fix the clamp at any position of the organ bath, stirrer is also fitted with
it which propelled mixed and throughout uniformly distributed the temperature of water inside the
organ bath.
3. Now tissue is placed inside the organ tube & one aeration tube is placed into the organ tube for oxygen
supply to the tissue.
4. Now one lever is set on the student organ bath (simple/ frontal) & load is added for maintaing the
balance of the lever.
5. Intact tissue’s one part is attached with the lever by using thread, glass coil used to provide temperature
to the upcoming water or physiological salt solution
6. Physiological salt solution holder which hold pss & enters to the organ tube.
7. Due to the rotation of kymograph drum or Rotating drum drugs effect (contraction or relaxation) can
be recorded & showing kymographic graph.

Reference
S.K Kulkarni, Handbook of Experimental Pharmacology, Vallabh Prakashan, 2-12.

Teacher’s Signature:
 Experiment 2

Objective:

To record the DRC of acetyl choline using chicken/guinea pig/ rat ileum

Principle:
Dose response curves demonstrate graded responses to drugs or agonists where an increase in response is
recorded with a subsequent increase in the dose or the concentration of the drug.
The dose-response curve is sigmoid or S-shaped. The first part (25% of the graph) of the curve has poor
discrimination between the doses whereas the middle portion of the curve shows greater sensitivity to
different concentration, and the responses to increasing concentrations are linearly differentiated. The last
part of the curve (plateau) shows the ceiling effect where no more increase in the response is seen with
further increase in the dose.
Sometimes cumulative dose response curves are employed for the study. The cumulative study is obtained
by increasing the concentration of the drug in the organ bath step by step without washing the preceding
doses.
The study of concentration or dose response curve indicates: (1) relative potency of the drug or agonist,
when the curve is more towards the left, it indicates that the drug is more potent. (2) Slope of the curve
indicates error and reliability (precision) of the bioassay.

Requirements: Tissue: Ileum,

Drug: Acetyl choline (stock 1 mg/ml),
Physiological solution: Tyrode solution

Procedure:
1. Sacrifice the animal and cut open the abdomen exposing the ileocaecal junction.
2. Take a section of the ileum about 2 – 3 cm and wash with Tyrode solution with proper
aeration.
3. Fix the tissue to the oxygen tube and wash it at intervals with Tyrode and stabilize the
tissue.
4. Give proper oxygen in the organ bath maintaining the temperature at 37°C.
5. A tension of 0.5g is applied for magnification of the reading.
 6. Apply the drug at various concentrations and record the response by keeping in contact for
30 sec. washing is given after every concentration.
7. The recording is done till the ceiling effect is observed.

Observation & Result:

Dose of ACh (μg) log (dose) Response (cm) % Response

10 1 2.0 39.21
20 1.301 2.8 54.90
40 1.602 3.3 64.70
80 1.903 3.9 76.47
160 2.204 4.8 94.4
320 2.505 5.1 100

log dose vs % Response

120

100

80
% response

60

40

20

0
0 0.5 1 1.5 2 2.5 3
log dose

Conclusion:
 Shape of graded DRC when plotted as dose vs response is parabola.
 Shape of log dose vs %response curve is sigmoid line or having S like shape.

 The first part of the curve has poor discrimination between the doses where as middle portion of
curve show greater sensitivity to different concentration and the responses to increasing conc.are
linerarly differentiated,the last part of the curve show the celling effect where no more increase in the
response is seen with further increase in dose.

Reference:

1. Kharjul Mangesh D et al., Isolated cock preparations: Economical and effective alternative
in India for undergraduate in-vitro Pharmacology, Int J Med Pharm Sci, Nov 2012,vol-3
issue 03,1-6
2. S.K Kulkarni, Handbook of Experimental Pharmacology, Vallabh Prakashan, 85-88

Teacher’s Signature
 Experiment No. 3

Objective
To find the effect of antagonist (atropine) using acetyl choline using chicken /guinea pig
/ rat ileum.

Principle: Rat ileum is an intestinal smooth muscle.Acetylcholine causes the contractions of the smooth
muscle by acting on muscarinic recptors.Atropine blocks muscarinic recptors in the smooth
muscle.Therefore ,atropine blocks acetylcholine induced contractions in rat ileum.The concentration
response curve of acetylcholine will be shifted to the right in the presence of atropine.the nature of
antagonism is of competitive type .
The spontaneous contractions of the preparations can be reduced by reducing the calcium content in
physiological solution and maintaining the bath at room temperature (23+ 2 0 C).The muscle (ileum)
preparation obtained from an unstarved rat gives more stable.

Requirements: Tissue: Ileum,

Drug: Acetyl choline (stock 1 mg/ml), atropine stock solution 1 mg/ml
Physiological solution: Tyrode

Procedure:
1. Sacrifice the animal and cut open the abdomen exposing the ileocaecal junction.
2. Take a section of the ileum about 2 – 3 cm and wash with Tyrode solution with proper
aeration.
3. Fix the tissue to the oxygen tube and wash it at intervals with Tyrode and stabilize the
tissue.
4. Give proper oxygen in the organ bath maintaining the temperature at 37°C.
5. A tension of 0.5g is applied for magnification of the reading.
6. Apply the drug at various concentrations and record the response by keeping in contact for
30 sec. Washing is given after every concentration.
7. The recording is done till the ceiling effect is observed.
8. The atropine solution is added to the bath and then concentrations of acetylcholine is added
9. The response of acetylcholine is recorded.
10. The graph is plotted and percent inhibition of response is recorded.

Observation & Result:

Dose of ACh (μg) log (dose) Response (cm) % Response

10 1 2.0 39.21
20 1.301 2.8 54.90
40 1.602 3.3 64.70
80 1.903 3.9 76.47
160 2.204 4.8 94.4
320 2.505 5.1 100
Dose of Atropine
(μg) + Dose of ACh
(μg)
10+20 1.477 1.5 60

10+40 1.698 2.1 84

10+80 1.954 2.5 100

Conclusion: From the above experiment we see that presence of acetylcholine on rat ileum increase
the activity but when atropine is used with acetylcholine then decrease the activity of acetylcholine.
Hence atropine is a antagonist of acetylcholione.

References:

1. Kharjul Mangesh D et al., Isolated cock preparations: Economical and effective alternative
in India for undergraduate in-vitro Pharmacology, Int J Med Pharm Sci, Nov 2012,vol-3
issue 03,1-6
2. S.K Kulkarni, Handbook of Experimental Pharmacology, Vallabh Prakashan, 109-111.

Teacher’s Signature
 Experiment No: 4.1

Objective: To study the LD50 value of drug by Miller and Tainter method

Principle: This method is well described by the Miller and Tainter (1944) as well as Litchfield
and Wilcoxon (1949). This is depending on the toxic log dose response curve and the
interpretation of result is done by the curve. The preferred method is, first convert the percentage
response into the probit, then plot the curve log dose on ‘x’-axis and ‘y’-axis contains probit
scale.
The LD50 is the antilog (ld) value which falls on the ‘x’-axis.

Requirements: Unknown drug, Animal-mice

Procedure:
1. Take two groups of animals with 10 mice in each group.
2. Inject the drug by intra-peritoneal route and keep the animal under observation until
death after 2 hours of drug administration.
3. Study the LD50 is done by plotting a graph with log dose in X-axis and probit scale on
Y-axis.

Observation:

Group Dose (mg/kg) Log Dose Dead/Total % dead Corrected Probit

%
1. 20 1.30 0 0 2.5 3.04
2. 40 1.60 20 20 20 4.16
3. 60 1.77 60 60 60 5.25
4. 80 1.90 70 70 70 5.52
5. 100 2 80 80 80 5.84
6. 120 2.07 100 100 97.5 6.96

from log dose vs probit graph we get LD50 = 1.80

antilog LD50= 63.09 mg/kg

standard error of LD50 = (Log LD84 – Log LD16)/ √2𝑁

N= number of animal each group
Probit 16 = 4.01 (4)
Probit 84 = 5.99 (6)
LD 84 = 2
Antilog LD 84 = 100
LD 16 = 1.55
Antilog LD16 = 35.48
Standard error = 14.42
Final LD50 = 63.09 ± 14.42 (77.51-48.67)

log dose vs Probit

8

7
y = 4.6644x - 3.1432
R² = 0.9552
6

5
probit

0
0 0.5 1 1.5 2 2.5
log dose

Conclusion: The LD50 value is 63.09 ± 14.42 (77.51-48.67) by Miller and Tainter Method.

Reference:S.K Kulkarni, Handbook of Experimental Pharmacology, Vallabh Prakashan, 213-215.

Teacher’s Signature
 Experiment No: 4.2

Objective: To study the LD50 value of drug by Karber method

Principle: The LD50 may be calculated by Karber's method that does not involve any plotting
of dose-response curve. It is the simplest and rapid though crude method of deriving LD50 value
particularly when the number of animals is small. The interval mean of the number dead in each
group of animals is used as well as the difference between the doses for the same interval. The
product of the interval mean and the dose difference is obtained. Results from doses larger than
the least dose lethal to all in a group and from doses smaller than the maximal tolerated dose are
excluded. The sum of the product is divided by the number of animals in a group and the
resulting quotient is subtracted from the least lethal dose in order to obtain the LO50 value.
Requirements: Unknown drug, Animal-mice
Procedure:
1. Take two groups of animals with 10 mice in each group.
2. Inject the drug by intra-peritoneal route and keep the animal under observation until
death after 2 hours of drug administration.
3. Study the LD50 by the following calculation

LD50 = Max. Dose – (Sum of the product / 10)

Observation:

Group Dose (mg/kg) Dose No of dead Mean Product

Difference (a) mortality (axb)
(b)
1. 20 - 0 - -
2. 40 20 2 1 20
3. 60 20 4 3 60
4. 80 20 6 5 100
5. 100 20 8 7 140
6. 120 20 10 9 180
Total = 500

LD50 = least dose – (sum of product /10)

= 120 – (500/10)

=70 µg/kg

Conclusion: The LD50 value of drug is 70 µg/kg by Karber method

Reference:
S.K Kulkarni, Handbook of Experimental Pharmacology, Vallabh Prakashan, 213-215.

Teacher’s Signature
 Experiment No. 5

AIM: To determine the unknown concentration of acetylcholine using chicken/ guinea pig/ rat
ileum by matching method.

Principle:
Rat fundus is a very sensitive tissue for the study of the action of several naturally occurring substances
like 5-hydroxytryptamine, histamine, Acetylcholine and bradykinin. Unlike the intestinal smooth muscle
(ileum) this preparation is slow contracting and slow relaxing type. Rat fundus preparation is generally
employed for the bioassay of serotonin. Fundus (the upper part of the stomach) is grey in color and
therefore, easily identified from pyloric part (pink in color). A zig-zag preparation of the fundal strip is
prepared so as to expose maximum portion of the tissue to drug.

Requirements:
a) Apparatus/glassware : Organ bath assembly, kymograph, Sherrington drum,
b) Chemical: Acetylcholine (1mg/ml)
c) Isolated tissue: chicken ileum
d) Physiological Salt Solution(PSS) :Ringer Lock
Procedure:

1. Fresh entire gastrointestinal tract of healthy cock was obtained from a local slaughterhouse
and was transported in Ringer Lock Solution.
2. Aeration was provided immediately in it.
3. The caecum was lifted forward and the ileocaecal junction was identified.
4. A few centimeters of the ileal portion was cut and removed and immediately placed it in
the watch glass containing physiological salt solution.
5. The mesentery and adhering tissues were removed with gentle care. Utmost care was taken
to avoid any damage to the gut muscle.
6. The ileum was cut into small segments of 2-3 cm long.
7. To one piece of ileum the thread was tied to top and bottom ends without closing the ileum,
and mounted the tissue in the organ bath containing PSS maintained at 32-35˚C and
bubbled with air.
8. The magnification value is 3,load/tension is 1.5 gm and bath volume of about 25 ml was
maintained, and the tissue was allowed to equilibrate for 30 min before adding Acetylcholine
to the organ bath.
9. Take 0.2ml of the test Acetylcholine solution and record the DRC using frontal writing lever.
Keep 90 sec Contact time and 5 minutes time period for proper response.
10. Then take different concentration DRC of the standard Acetylcholine
11. Match the DRC of the test Acetylcholine with the DRC of the standard and determined the
respective volume.
12. Find the concentration of the test solution with respect to standard solution.
Observation & Result:

Standard: Acetylcholine 1mg/ml

Dose (µg) Dose volume (ml) Response height (mm) % Response

10 0.1 1.4 28.57

20 0.2 1.9 38.77
40 0.4 2.3 46.93
80 0.8 2.8 57.14
160 1.6 3.5 71.42
320 3.2 4.1 83.67
640 6.4 4.9 100
X(unkmown) 3.7 75.51

Conclusion: The unknown concentration of acetylcholine using chicken/ guinea pig/ rat ileum by
matching method is determined

References:

1. V.R.Undale et al., An isolated chicken ileum: Alternative to laboratory animals for isolated
tissue experimentation, IOSR Journal of Pharmacy, Volume 2 Issue 5 ,Sep-Oct. 2012
,PP.39-45

Teacher’s Signature
 Experiment No. 6

AIM: To determine the strength of an unknown sample of Acetylcholine by Interpolation Bioassay

employing isolated chicken/ guineapig/ rat ileum by bracketing method
Principle: In Bracketing method a constant dose of the test is bracketed by varying doses of standard till the
exact match is obtained between test dose and the standard dose. Initially, two responses of the standard are
taken. The doses are adjusted such that one is giving response of approximately 20% and other 70% of the
maximum. The response of unknown which lies between two responses of standard dose is taken. The panel
is repeated by increasing or decreasing the dose s of standard till all three equal responses are obtained. The
dose of test sample is kept constant. At the end, a response of the double dose of the standard and test which
match each other are taken.

Requirements:
a) Apparatus/glassware : Organ bath assembly, kymograph, Sherrington drum,
b) Chemical: Acetylcholine
c) Isolated tissue: chicken ileum

Procedure:

1. Fresh entire gastrointestinal tract of healthy cock was obtained from a local slaughterhouse
and was transported in Ringer Lock Solution.
2. Aeration was provided immediately in it.
3. The caecum was lifted forward and the ileocaecal junction was identified.
4. A few centimeters of the ileal portion was cut and removed and immediately placed it in
the watch glass containing physiological salt solution.
5. The mesentery and adhering tissues were removed with gentle care. Utmost care was taken
to avoid any damage to the gut muscle.
6. The ileum was cut into small segments of 2-3 cm long.
7. To one piece of ileum the thread was tied to top and bottom ends without closing the ileum,
and mounted the tissue in the organ bath containing PSS maintained at 32-35˚C and bubbled
with air.
8. The magnification value is 3,load/tension is 1.5 gm and bath volume of about 25 ml was
maintained, and the tissue was allowed to equilibrate for 30 min before adding Acetylcholine
to the organ bath.
9. Take 0.2ml of the test Acetylcholine solution and record the DRC using frontal writing lever.
Keep 90 sec Contact time and 5 minutes time period for proper response.
10. Then take different concentration DRC of the standard Acetylcholine
11. Match the DRC of the test Acetylcholine with the DRC of the standard and determined the
respective volume.
12. Find the concentration of the test solution with respect to standard solution.

Observation & Result:

Dose of ACh (μg) log (dose) Response (cm) % Response

Conclusion: The Strength of unknown sample of Acetylcholine by Interpolation bioassay employing
isolated chicken/guineapig/ rat ileum by bracketing method is determined.

References:

3. Kharjul Mangesh D et al., Isolated cock preparations: Economical and effective

alternative in India for undergraduate in-vitro Pharmacology, Int J Med Pharm Sci, Nov
2012,vol-3 issue 03,1-6
4. S.K Kulkarni, Handbook of Experimental Pharmacology, Vallabh Prakashan, 111-113.

Teacher’s Signature
 Experiment No: 7

Objective:
To calculate the pA2 value for atropine using acetyl choline using chicken / guinea pig /rat ileum.

Principle:

pAx value is calculated to compare the potency of antagonists acting on the same receptor.
The pAx value is defined as the negative logarithm of the molar concentration of the antagonists required to
reduce the effect of a multiple dose (x) of the agonist to that of a single dose in the absence of antagonist. Higher
the pAx value, more potent is the antagonist. The determination of pA2 (X=2) and pA10 (X=10) values have wider
application

Requirements: Tissue: Ileum,

Drug: Acetyl choline (stock 1 mg/ml), atropine stock solution 1 mg/ml
Physiological solution: Tyrode

Procedure:
2. Sacrifice the animal and cut open the abdomen exposing the ileocaecal junction.
3. Take a section of the ileum about 2 – 3 cm and wash with Tyrode solution with proper
aeration.
4. Fix the tissue to the oxygen tube and wash it at intervals with Tyrode and stabilize the
tissue.
5. Give proper oxygen in the organ bath maintaining the temperature at 37°C.
6. A tension of 0.5g is applied for magnification of the reading.
7. Apply the drug at various concentrations and record the response by keeping in contact for
30 sec. Washing is given after every concentration.
8. The recording is done till the ceiling effect is observed.
9. The atropine solution is added to the bath and then concentrations of acetylcholine is added
10. The response of acetylcholine is recorded.
11. The graph is plotted with negative logarithm of molar concentration of the atropine.

Observation & Result:

 DRC + 0.04 DRC + 0.08 DRC + 0.16
Dose of Acetylcholine DRC of µg/ml µg/ml µg/ml
(2-20 µg/ml) Acetylcholine ATROPINE ATROPINE ATROPINE
0.2 2 0 0 0
0.4 3 1 0 0
0.6 5 3 2 0
0.8 10 7 14 1
1.6 20 16 19 7
3.2 38 29 30 12
6.4 38 38 18
12.8 27
24.6 38

Dose of DRC + 0.04 DRC + 0.08 DRC + 0.16

Acetylcholine (2-20 µg/ml µg/ml µg/ml
µg/ml) DRC of Acetylcholine ATROPINE ATROPINE ATROPINE
-0.698970004 5.263157895 0 0 0
-0.397940009 7.894736842 2.631578947 0 0
-0.22184875 13.15789474 7.894736842 5.26315789 0
-0.096910013 26.31578947 18.42105263 36.8421053 2.631578947
0.204119983 52.63157895 42.10526316 50 18.42105263
0.505149978 100 76.31578947 78.9473684 31.57894737
0.806179974 100 100 47.36842105
1.10720997 71.05263158
1.390935107 100

Conclusion: The pA2 value for atropine using acetyl choline using chicken / guinea pig /rat ileum
has been calculated.

Reference:
12. Kharjul Mangesh D et al., Isolated cock preparations: Economical and effective alternative
in India for undergraduate in-vitro Pharmacology, Int J Med Pharm Sci, Nov 2012,vol-3
issue 03,1-6
13. S.K Kulkarni, Handbook of Experimental Pharmacology, Vallabh Prakashan, 109-11

Teachers signature
 Experiment No. 8

AIM: To determine the strength of an unknown sample of Acetylcholine by Interpolation Bioassay

employing isolated chicken/ guineapig/ rat ileum by interpolation method

Principle:Interpolation method of bioassay is less time consuming and yet reliable as compared to
matching type of bioassay. One of the main advantage of this assay is trgat sensitivity of the tissue is
first determined by prior plotting of concentration -response curve with the known agonist as is the
case with acetylcholine.If the linearity of the curve is good ,one can do very accurate estimate of the
test substance(unknown sample).

Requirements:
a) Apparatus/glassware : Organ bath assembly, kymograph, Sherrington drum,
b) Chemical: Acetylcholine
c) Isolated tissue: chicken ileum

Procedure:

13. Fresh entire gastrointestinal tract of healthy cock was obtained from a local slaughterhouse
and was transported in Ringer Lock Solution.
14. Aeration was provided immediately in it.
15. The caecum was lifted forward and the ileocaecal junction was identified.
16. A few centimeters of the ileal portion was cut and removed and immediately placed it in
the watch glass containing physiological salt solution.
17. The mesentery and adhering tissues were removed with gentle care. Utmost care was taken
to avoid any damage to the gut muscle.
18. The ileum was cut into small segments of 2-3 cm long.
19. To one piece of ileum the thread was tied to top and bottom ends without closing the ileum,
and mounted the tissue in the organ bath containing PSS maintained at 32-35˚C and bubbled
with air.
20. The magnification value is 3,load/tension is 1.5 gm and bath volume of about 25 ml was
maintained, and the tissue was allowed to equilibrate for 30 min before adding Acetylcholine
to the organ bath.
21. Take 0.2ml of the test Acetylcholine solution and record the DRC using frontal writing lever.
Keep 90 sec Contact time and 5 minutes time period for proper response.
22. Then take different concentration DRC of the standard Acetylcholine
23. Match the DRC of the test Acetylcholine with the DRC of the standard and determined the
respective volume.
24. Find the concentration of the test solution with respect to standard solution.

Observation & Result:

Dose of ACh (μg) log (dose) Response (cm) % Response

10 1 1.2 24
20 1.30 1.5 30
40 1.60 2.0 40
80 1.90 2.6 52
160 2.20 3.1 62
320 2.51 4.5 90
640 2.80 5.0 100
unknown 2.37 3.9 78
Log conc. Is 2.37 ml
Antilog (2.37) is 234.42

Log conc. vs %Response (interpolation Method)

120

100

80
% responsee

60

40 % Response

20

0
0 0.5 1 1.5 2 2.5 3
Log Conc (ml)

Conclusion: The strength of an unknown sample of Acetylcholine is 234.42 µg/ml by Interpolation

Bioassay employing isolated chicken/ guineapig/ rat ileum by interpolation method

References:
1. Kharjul Mangesh D et al., Isolated cock preparations: Economical and effective
alternative in India for undergraduate in-vitro Pharmacology, Int J Med Pharm Sci, Nov
2012,vol-3 issue 03,1-6
2. S.K Kulkarni, Handbook of Experimental Pharmacology, Vallabh Prakashan, 111-113.

Teacher’s Signature
 Experiment No: 9

Objective: To study the effect of drug on isolated frog heart preparation

Principle:
Adrenaline: It is sympathomimetic having mixed agonist action .It produces increase in heart
rate (positive chronotropic effect) and force of contraction (positive ionotropic effect). Thus
adrenaline produces direct excitatory action on myocardial muscles mediated through
receptors present in heart.

Acetylcholine: It is parasympathomimetic drug. Muscarinic receptor found in heart. In the

heart acetylcholine causes activation of potassium ion channel,account for negative
chronotropism and negative ionotropism. Thus the heart is inhibited.In perfused frog heart
preparation stoppage of heartis seen on upper side, while in isolated preparation it stops in
diastolic condition.

Calcium chloride:In lower doses it increase heart rate and force of contraction but in high dose
it inhibit the heart in systole characterized by staright line on upper side in isolated heart and
on lower side in purfused frog heart.

Potassium chloride: It has also inhibitory effect on heart. In perfused frog heart preparation, it
stop in systole while in isolated preparation it stops in diastole.

Requirements: Adrenaline (10µg/ml), Acetylcholine (10µg/ml), Calcium chloride

(10µg/ml), Potassium chloride (10µg/ml), Frog ringer solution

Procedure:
1. The frog is killed and pithed.
2. Through the midline incision and after cutting the pectoral girdle, the heart is exposed.
3. The inferior vena cava is traced and the venous cannula is inserted supplying frog
Ringer.
4. The heart is isolated from the thoracic cavity and kept in hanging position.
5. A thin pin hook is passed through the ventricle which is tied with a thread.
6. Connect the thread with the lever and keep in contact with the kymograph.
7. The drugs are added to the cannula and readings are recorded.

Observation:

Reference:
S.K Kulkarni, Handbook of Experimental Pharmacology, Vallabh Prakashan, 194-195.

Conclusion: The effect of drug on frog heart is observed and seen the result.

Teacher’s Signature
 Experiment No: 10

Objective: To study the designing of ADR monitoring protocol.

Principle: ADR monitoring and reporting helps in detection and prevention of reoccurrence
of ADRs. The objective of this study is to study the designing of ADR monitoring protocol.
The detection of adverse drug reactions (ADRs) has become increasingly significant because
of introduction of a large number of potent toxic chemicals as drugs in the last two or three
decades. This observational, cross-sectional, descriptive study was based on an analysis of
medical records. Several parameters were utilized in the data evaluation, including patient
demographics, drug and reaction characteristics, and reaction outcomes. The reaction
severity and predisposing factors were also assessed.

ADR- “An appreciably harmful or unpleasant reaction, resulting from an intervention related
to the use of a medicinal product, which predicts hazard from future administration and
warrants prevention or specific treatment, or alteration of the dosage regimen, or withdrawal
of the product.”
Objectives of ADR monitoring:
1. To detect the nature and frequency of ADRs
2. To assist the Drug Regulatory Authority, Public Health Programs, Scientists and
Consumer Society to minimize ADRs.
3. Providing updated Drug Safety Information to Health Care Professionals.
4. Dissemination of information by designing proper education program to consumers
5. To identify risk factors that may predispose, induce or influence the development, severity
and incidence of ADRs
Collection of ADR-
Adverse drug reactions reporting tools or monitoring is a process of continuously monitoring
of undesirable effect suspected to be associated with use of medical products. ADR reporting
covers all pharmaceutical products, biological, herbal drugs, cosmetics and medical devices.
Who can Report?
All healthcare professionals(clinicians, dentists, pharmacists, nurses etc) and Non-healthcare
professionals including consumers can report suspected adverse drug reaction

Reporting of ADR-
How to report?
1. Report should be on a standardized ADR reporting form.
2. Dully filled the ADRs in the reporting from when an ADR is encountered.
3. Use a separate from for each patient and filled with the complete information.
4. The completed ADR form is then returned to the nearest adverse drug reaction monitoring
Centre (AMC) or to National Coordinating Centre. (NCC)
5. Any follow-up information for an ADR case that has already been reported can be sent on
another ADR form, or communicated by telephone, fax or e-mail.
6. Follow-up reports should be identifiable and the following should be indicated on the
report
a) Follow-up Information
b) Date of Original Report
c) Patient Identity
Submission of documents-

Attach a protocol format-

Teacher’s Signature
 Experiment No: 11

Objective: To study the protocol design for clinical trial.

Principle:
Protocol design is done to describe the detail of scientific rationale for a research activity involving
human subjects.
The protocols that is prepared-
 To clarify research question
 To compile existing knowledge
 To formulate a hypothesis and objectives
 To decide about a study design
 To clarify ethical consideration
 To apply for funding
 To have guidelines and tools for research team

Clinical trial-
The parts of clinical trial protocol-
1. Title page
2. Signature page
3. Content page
4. List of abbreviations
5. Introduction/Abstract
6. Objectives
7. Background/ Rationale
8. Eligibility Criteria
9. Study Design/ Methods
10. Safety/ Adverse events
11. Regulatory Guidance
12. Statistical section
13. Human subjects protection/ Informed consent

1. Title Page
 Title page introduces the document, its title, precise number, sponsor and author to the
reader.
 Protocol identifying number and date. Any amendment should also bear the amendment
number and date.
 The protocol number must clearly indicate the version number, whether it is final or draft
and date of this version.
Title page should include-
 Summary study design, medicinal products, nature of treatment, comparator placebo,
randomized double bind multiple studies.
 Title page also includes -name and address of sponsor and monitor, Sponsor names
and list of responsibilities.
 Name and address of the person authorized to sign the protocol and protocol
amendment for the sponsor.
 Name, title, address and contact no of sponsor medical expert for the trial.
 Name, title of investigator who is responsible for conducting the trial, the address and
contact no of the trial site also should be mentioned.
 Detail of qualified physician who is responsible for all trial site related medical
 decisions.
 Name and address of clinical laboratory and other medical/technical/institution
involved in the trial.

2. Signature Page
 Signature page of all healthcare professionals in the trial including contact details of
participating site, sponsor and sponsor medical advisor if not already given above.
3. Content Page
 This help navigation through the document by large number of different people that will be
needed throughout the life of the trial.

4. List of Abbreviations
 All abbreviations used should be listed and defined. Accepted international medical
abbreviations should be standardized within each project.

5. Introduction/Abstract
 This summary should be only one to two pages long. It should give the reader sufficient
information to understand the rationale for the trial, its objective and methods that will be
used to achieve this objective.

6. Objectives
 Objectives should be stated clearly as hypothesis should be tested.
 It should have a corresponding discussion in the statistical section.

7. Background/Rationale
 All protocols require a section detailing the scientific rationale for a protocol and
justification in medical and scientific literature for the hypothesis being proposed.
 Describe why it make sense to study this product in this population an why the information
is needed.
 Risk benefit assessment is done to clarify the potential risk and benefit.

8. Eligibility Criteria
 Subjects with a diagnosis of the specific disease intended to take in study, who meet the
inclusion and exclusion criteria will be eligible for participation in study.
 Reasons for imposing eligibility criteria can include scientific rationale, safety concerns,
regulatory issues and practical considerations.
 It should be clearly defined and verifiable by an external auditor.

9. Study designs/ Methods

 The study design section of the protocol should contain a stepwise description of all procedures
required by the study.
Parts of the study design are-
 Initial evaluations
 Screening tests
 Required lab tests
 Details of treatment and supplementary procedures
 Agent information or device specifications
 Dose scheduling and modifications
Types of study design-
 Observational study
 Interventional study
  Descriptive study
 Analytical study
 Cross sectional study
 Cohort study

10. Safety/ Adverse events

 Adverse effect and side effect are terms commonly associated with drugs. They are used by
nurses and doctors, to refer to undesirable effects of a medication on a patient.
 The safety (or adverse events) section should include –
o Detailed information for reporting adverse events, including reporting to FDA and/or the
sponsor
o Unblinding processes
o List of expected adverse events

11. Regulatory Guidance

The study should be conducted according to-
 Declaration of Helsinki
 Protection of human values
 Institutional Review Board
 Institutional Ethics Committee
 Obligation of Clinical Investigator
 Good clinical Practices

12. Statistical Section

 The study objectives and study design elements in the statistical section should be described in
the objectives sections.
 The descriptions and definitions of toxicities in the statistical section match those in the safety/
adverse event section.

13. Human Subject Protection/Informed Consent

 Informed consent should be prepared in accordance to declaration of Helsinki, ICH, GCP, FDA,
health insurance portability and accountability act and local regulations.
 A proper executed, written, informed will be obtained from each subject prior to entering the
subject into the trial.
 Information should be given in both oral and written form in their native language to subjects/ or
their legal representatives about study.
The protocol’s informed consent must provide-
o Statement that the study involves trials
o Purpose of trial and length of the study
o Description of risks and benefits
o Discussions of alternative therapies
o Subject Confidentiality: In order to maintain subject confidentiality, only a site number and
subject initials will identify all study subjects.
To maintain confidentiality- all study records should be kept in a locked file cabinet.
o Compensation for injury
o Contact for further questions/information
o Statement of voluntary participation

Teacher’s Signature
 Experiment No. 12

AIM: To determine the potency of unknown solution of Acetylcholine by Three point

bioassay using isolated chicken ileum preparation.
Principle: Three point Bioassay is a method based on the assumption of dose-response relationship.Log
dose response curve is plotted and the dose of the standard producing the same response as produced by the
test sample is directly read from the graph so to estimate the potency of the test sample.
In three point bioassay,the DRC of standard ,test samples is first obtained from the responses due to graded
doses.From the DRC of standard,two standard doses are selected in such a way that they have produced 20%
and 80 % of he maximal response respectively and are designated as S1 and S2. The responses of these
doses lie on the steepest and straightest part (linear ) of the curve. From The DRC of test sample one test
dose is selected such that it gives a response which lies in between the two standard responses i.e. it gives a
greater response than S1 and a smaller response than S2 and is designated as T.
After selecting the standard and test doses, the bioassay is performed by recording the standard and test
responses in a randomized fashion. The pattern of addition of doses is S1 S2 T; S2 ,T, S1 and T, S1, S2 in 3
successive cycles.The mean values of height of the contraction for all the 3 doses are calculated and are used
in plotting the graph so as to estimate the potency of the test sample.

Requirements:

a. Apparatus/glassware : Organ bath assembly, kymograph, Sherrington drum,

b. Chemical: Acetylcholine (1mg/ml) & Acetylcholine test sample
c. Isolated tissue: chicken ileum
d. Physiological Salt Solution(PSS) :Tyrode solution
Procedure:

1. Fresh entire gastrointestinal tract of healthy cock was obtained from a local
slaughterhouse and was transported in Ringer Lock Solution.
2. Aeration was provided immediately in it.
3. The caecum was lifted forward and the ileocaecal junction was identified.
4. A few centimeters of the ileal portion was cut and removed and immediately placed it
in the watch glass containing physiological salt solution.
5. The mesentery and adhering tissues were removed with gentle care. Utmost care was
taken to avoid any damage to the gut muscle.
6. The ileum was cut into small segments of 2-3 cm long.
7. To one piece of ileum the thread was tied to top and bottom ends without closing the
ileum, and mounted the tissue in the organ bath containing PSS maintained at 32-35˚C
and bubbled with air.
8. The magnification value is 3,load/tension is 1.5 gm and bath volume of about 25 ml was
maintained, and the tissue was allowed to equilibrate for 30 min before adding
Acetylcholine to the organ bath.
9. Record graded responses with the standard solution of acetylcholine until ceiling effect
is obtained.
10. Select two concentration (A,B) of the standard drug, eliciting sub- maximal responses
(S1,S2) and bearing a dose ratio of 1: 2 preferentially.
11. Select two suitable volumes of the test solution by trial and error method in such a way
that the response (T1) due to the lower dose of the test (C) lies preferentially between S1
& S2.
12. Record three sets of responses following Latin square design
13. Label and fix the tracing
14. Take the average of the responses.
15. Calculate The Potency Ratio (M) using the following formula
Log Potency ratio [M] = [(T –S1) / (S2-S1)] X log (dose ratio)
 Observation & Result:

Standard Acetylcholine 1mg/ml

Response:

n1 n2 t
32 50 45

n2 t n1
50 46 31

t n1 n2
46 32 52

Concentration of test solution

=
Conc.(ml) Avg. response (cm)
n1 0.4 31.66
n2 1.6 50.66
t 0.8 45.66
(n1/t) x antilog{(T- S1) / (S2-S1) x log(n2/n1) x std. dose
= (0.4/0.8) x antilog {(45.66 – 31.66) / (50.66 – 31.66) x log( 1.6 /0.4) x 15
= 20.55 µg/ml

References:

1. Gaurav Jain et al., Development of an ex vivo model for pharmacological

experimentation on isolated tissue preparation, J Adv Pharm Technol Res. 2012 Jul-
Sep; 3(3): 176–181.
2. S.K Kulkarni, Handbook of Experimental Pharmacology, Vallabh Prakashan, 99-101

Conclusion: The potency of unknown solution of Acetylcholine is 20.55 µg/ml by Three point
bioassay using isolated chicken ileum preparation

Teacher’s Signature
 Experiment No. 13

AIM: To determine the potency of unknown solution of Acetylcholine by Four point

bioassay using isolated chicken ileum preparation.

Principle: The principle of four point bioassay is to compare the test substances to the
international standard preparation of the same and to find out how much test substance is required
produce same biological effect, as produced by standard. Where two standard and two test doses
are taken and latin square method is used to minimize the chance of having error.
In this bioassay, DRC is recorded by using graded doses of the standard until the ceiling effect is
obtained.

Requirements:

a. Apparatus/glassware : Organ bath assembly, kymograph, Sherrington drum,

b. Chemical: Acetylcholine (1mg/ml) & Acetylcholine test sample
c. Isolated tissue: chicken ileum
d. Physiological Salt Solution(PSS) :Tyrode solution

Procedure:

1. Fresh entire gastrointestinal tract of healthy cock was obtained from a local
slaughterhouse and was transported in Ringer Lock Solution.
2. Aeration was provided immediately in it.
3. The caecum was lifted forward and the ileocaecal junction was identified.
4. A few centimeters of the ileal portion was cut and removed and immediately placed it
in the watch glass containing physiological salt solution.
5. The mesentery and adhering tissues were removed with gentle care. Utmost care was
taken to avoid any damage to the gut muscle.
6. The ileum was cut into small segments of 2-3 cm long.
7. To one piece of ileum the thread was tied to top and bottom ends without closing the
ileum, and mounted the tissue in the organ bath containing PSS maintained at 32-35˚C
and bubbled with air.
8. The magnification value is 3,load/tension is 1.5 gm and bath volume of about 25 ml was
maintained, and the tissue was allowed to equilibrate for 30 min before adding
Acetylcholine to the organ bath.
9. Record graded responses with the standard solution of acetylcholine until ceiling effect
is obtained.
10. Select two concentration (A,B) of the standard drug, eliciting sub- maximal responses
(S1,S2) and bearing a dose ratio of 1: 2 preferentially.
11. Select two suitable volumes of the test solution by trial and error method in such a way
that the response (T1) due to the lower dose of the test (C) lies preferentially between S1
& S2. The higher volume of the test solution selected would be D such that the dose ratio
B/A= D/C. All the four responses (S1,S2,S3,S4) due to the doses thus selected (A,B,C,D)
must lie on the linear part of the standard ( sigmoid ) curve.
12. Standardise the tissue with the concentration A.( Tissue is said to be standardised when
it response identically to the same concentration, when repeated)
13. Record four sets of responses due to A,B,C and D adding them to the organ bath in a
random fashion(Latin square design)
14. Label and fix the tracing
15. Measures various responses to calculate the mean of each response (S1,S2,S3 & S4)
16. Calculate The Potency Ratio (M) using the following formula
 X1 T2-S2 + T1-S1

Potency Ratio = X antilog X log X2 =M

Y1 T2-T1 + S1-S2 Y2

Where X1 = Lower Volume Of Standard Drug (A)

X2 = higher volume of standard drug (B)

Y1= lower volume of test solution (C)

S and T = represent the mean responses

Observation & Result:

Standard Acetylcholine 1mg/ml
Acetylcholine Dose Response height (mm) Average
response(mm)
S1 0.4 4 4.3 4.2 3.9 4.1
S2 3.2 10.5 10 10.4 10.4 10.32
T1 0.6 5.6 6 5.8 5.5 5.72
T2 1.2 8.3 8.5 8 8.5 8.32

Four point bioassay formula:

= 0.4/0.6 antilog [{(10.32 – 8.32) + (4.1 – 5.72)} /{ (10.32 – 4.1) + (8.32 – 5.72)} x log(3.2/0.4)] x 10

= 7.02 µg/ml

References:

1. Gaurav Jain et al., Development of an ex vivo model for pharmacological

experimentation on isolated tissue preparation, J Adv Pharm Technol Res. 2012 Jul-
Sep; 3(3): 176–181.
2. S.K Kulkarni, Handbook of Experimental Pharmacology, Vallabh Prakashan, 113-115.

Conclusion: The potency of unknown solution of Acetylcholine is 7.02 µg/ml by Four point bioassay
using isolated chicken ileum preparation.

Teacher’s Signature
 Experiment No. 14

Aim: To study the Oral Acute Toxicity –Acute Toxic Class Method (OECD TG 423)

Principle:

Oral acute toxicity-Acute Toxic Class Method is based on a stepwise procedure with the use of a minimum
number of animals per step, sufficient information is obtained on the acute toxicity of the test substance
to enable its classification. The substance is administered orally to a group of experimental animals at one
of the defined doses. The substance is tested using a stepwise procedure, each step using three animals of
a single sex (normally females). Absence or presence of compound-related mortality of the animals dosed
at one step will determine the next step, i.e.; − no further testing is needed, − dosing of three additional
animals, with the same dose − dosing of three additional animals at the next higher or the next lower dose
level.

In principle, the method is not intended to allow the calculation of a precise LD50, but does allow for the
determination of defined exposure ranges where lethality is expected since death of a proportion of the
animals is still the major endpoint of this test. The method allows for the determination of an LD50 value
only when at least two doses result in mortality higher than 0% and lower than 100%. The use of a
selection of pre-defined doses, regardless of test substance, with classification explicitly tied to number
of animals observed in different states improves the opportunity for laboratory to laboratory reporting
consistency and repeatability.

Procedure:
1. Normally female rats (five in number) are used or depending upon the sensitivity either sex can
be used with adequate justification. Females should be nulliparous and non-pregnant. At the
commencement of its dosing, each animal should be between 8 and 12 weeks old and its weight
should fall in an interval within ± 20 % of the mean initial weight of any previously dosed animals.
2. Requisite housing and feeding condition should be maintained
3. For acclimatisation to the laboratory conditions they should be kept for 5 days before dosing.
4. In general test substances should be administered in a constant volume over the range of doses to
be tested. In rodents, the volume should not normally exceed 1 ml/100g of body weight; however
in the case of aqueous solutions, 2 ml/100g body weight can be considered.
5. Single dose is to be administered to overnight fasten animal by gavage using a stomach tube or a
suitable intubation canula
6. The limit test is done for 2000mg/kg and 5000 mg/kg.
7. The main test is done with the starting dose of 5, 50, 300 and 2000 mg/kg. After dosing at least
once during the first 30 minutes, Special attention given during the first 4 hours, periodically
during the first 24 hours / 14 days Signs of toxicity appear and disappear are important.
8. OBSERVATIONS: Skin and fur Eyes and mucous membranes Respiratory & circulatory
Autonomic and central nervous systems Somatomotor activity and behaviour pattern Tremors,
convulsions, salivation, diarrhoea , lethargy, sleep and coma Urine analysis
9. Pathology: All test animals (survive / death) – gross necroscopy (Microscopical examinations of
each organs – evidence of gross pathology) Organs - brain, colon, heart, kidneys, liver, lungs,
 oesophagus , rectum, sciatic nerve, spleen, sternum with bone marrow, stomach, thyroid /
parathyroid, trachea, urinary bladder and uterus. They were subjected to histopathological
examination.
10. Body weight: Individual weights of animals – before test substance admn , one week after & end
of the test – survive animals Wt. of the organs - liver, kidneys, adrenals, epididymis , thymus,
spleen, brain, heart, uterus and testes/ovaries

Reference: https://ntp.niehs.nih.gov/iccvam/suppdocs/feddocs/oecd/oecd_gl423.pdf

Teacher’s Signature
 Experiment No. 15

Aim: To study the Oral Acute Toxicity –Up and Down Procedure (OECD TG 425)

Test Guideline 425 was undertaken concurrently with revisions to the Test Guidelines 420 and 423.

Principle:

The test procedure described in this Guideline is of value in minimizing the number of animals required
to estimate the acute oral toxicity of a chemical. In addition to the estimation of LD50 and confidence
intervals, the test allows the observation of signs of toxicity. Revision of Test Guideline 425 was
undertaken concurrently with revisions to the Test Guidelines 420 and 423.

The Limit Test is a sequential test that uses a maximum of 5 animals. A test dose of 2000, or exceptionally
5000 mg/kg, may be used. The procedures for testing at 2000 and 5000 mg/kg are slightly different (see
paragraphs 23 -25 for limit test at 2000 mg/kg and paragraphs 26-30 for limit test at 5000 mg/kg). The
selection of a sequential test plan increases the statistical power and also has been made to intentionally
bias the procedure towards rejection of the limit test for compounds with LD50s near the limit dose; i.e.,
to err on the side of safety. As with any limit test protocol, the probability of correctly classifying a
compound will decrease as the actual LD50 more nearly resembles the limit dose.

The main test consists of a single ordered dose progression in which animals are dosed, one at a time , at
a minimum of 48-hour intervals. The first animal receives a dose a step below the level of the best estimate
of the LD50. If the animal survives, the dose for the next animal is increased by [a factor of] 3.2 times the
original dose; if it dies, the dose for the next animal is decreased by a similar dose progression. (Note: 3.2
is the default factor corresponding to a dose progression of one half log unit). Each animal should be
observed carefully for up to 48 hours before making a decision on whether and how much to dose the
next animal. That decision is based on the 48-hour survival pattern of all the animals up to that time. A
combination of stopping criteria is used to keep the number of animals low while adjusting the dosing
pattern to reduce the effect of a poor starting value or low slope. Dosing is stopped when one of these
criteria is satisfied, at which time an estimate of the LD50 and a confidence interval are calculated for the
test based on the status of all the animals at termination.

Procedure:

1. Normally female rats(five in number) are used or depending upon the sensitivity either sex can be
used with adequate justification. Females should be nulliparous and non-pregnant. At the
commencement of its dosing, each animal should be between 8 and 12 weeks old and its weight
should fall in an interval within ± 20 % of the mean initial weight of any previously dosed animals.
2. Requisite housing and feeding condition should be maintained
3. For acclimatisation to the laboratory conditions they should be kept for 5 days before dosing.
4. In general test substances should be administered in a constant volume over the range of doses to
be tested. In rodents, the volume should not normally exceed 1 ml/100g of body weight; however
in the case of aqueous solutions, 2 ml/100g body weight can be considered.
5. Single dose is to be administered to overnight fasten animal by gavage using a stomach tube or a
suitable intubation canula
6. Unusual circumstance that a single dose is not possible - dose may be given in smaller fractions
over a period not exceeding 24 hours
7. The first animal is dosed a step below the best preliminary estimate of the LD50. If the animal
survives, the second animal receives a higher dose. If the first animal dies or appears moribund,
the second animal receives a lower dose. The dose progression factor should be chosen to be the
antilog of 1/(the estimated slope of the dose-response curve) and should remain constant
throughout testing (a progression of 3.2 corresponds to a slope of 2). When there is no information
on the slope of the substance to be tested, a dose progression factor of 3.2 is used. Using the default
progression factor, doses would be selected from the sequence 1.75, 5.5, 17.5, 55, 175, 550, 2000
(or 1.75, 5.5, 17.5, 55, 175, 550, 1750, 5000 for specific regulatory needs). If no estimate of the
substance’s lethality is available, dosing should be
initiated at 175 mg/kg
8. After dosing at least once during the first 30 minutes, Special attention given during the first 4
hours, periodically during the first 24 hours / 14 days Signs of toxicity appear and disappear are
important.
9. OBSERVATIONS: Skin and fur Eyes and mucous membranes Respiratory & circulatory
Autonomic and central nervous systems Somatomotor activity and behaviour pattern Tremors,
convulsions, salivation, diarrhoea , lethargy, sleep and coma Urine analysis
10. Pathology: All test animals (survive / death) – gross necroscopy (Microscopical examinations of
each organs – evidence of gross pathology) Organs - brain, colon, heart, kidneys, liver, lungs,
oesophagus , rectum, sciatic nerve, spleen, sternum with bone marrow, stomach, thyroid /
parathyroid, trachea, urinary bladder and uterus. They were subjected to histopathological
examination.
11. Body weight: Individual weights of animals – before test substance admn , one week after & end
of the test – survive animals Wt. of the organs - liver, kidneys, adrenals, epididymis , thymus,
spleen, brain, heart, uterus and testes/ovaries

Reference:

https://ntp.niehs.nih.gov/iccvam/suppdocs/feddocs/oecd/oecd_gl425-508.pdf

Teacher’s Signature
 Experiment No. 16

Aim: To Study the Sub Acute Toxicity A Repeated Dose 28‐day Oral Toxicity Study in Rodents
(OECD TG407)

Principle

The test substance is orally administered daily in graduated doses to several groups of experimental
animals, one dose level per group for a period of 28 days. During the period of administration the animals
are observed closely, each day for signs of toxicity. Animals that die or are euthanised during the test are
necropsied and at the conclusion of the test surviving animals are euthanised and necropsied. A 28 day
study provides information on the effects of repeated oral exposure and can indicate the need for further
longer term studies. It can also provide information on the selection of concentrations for longer term
studies. The data derived from using the TG should allow for the characterization of the test substance
toxicity, for an indication of the dose response relationship and the determination of the No-Observed
Adverse Effect Level (NOAEL).

Procedure:

1. Selection of animal species Preferred rodent species is the rat. Other rodent species may be used
but a detailed justification should be given. Weight variation of animals used should not exceed ±
20% of the mean weight of each sex.
2. Housing and feeding Temperature : 22°C (± 3°C). Relative humidity : 30% - 70%. Lighting should
be artificial, the photoperiod being 12 hours light, 12 hours dark. For feeding, conventional
laboratory diets are used with an unlimited supply of drinking water. No more than five animals
should be housed per cage
3. Preparation of animals Healthy young adult animals are randomly assigned to control and
treatment groups. Animals are kept in their cages for at least five days prior to start of study to
allow for acclimatisation to laboratory conditions.
4. Preparation of doses Test compound is administered by gavage or via the diet or drinking water.
Method of oral administration is dependent on the purpose of study, & physical/chemical/ toxico
-kinetic properties of the test material.
5. Number and sex of animals At least 10 animals (five female & five male) should be used at each
dose level. If interim euthanasia are planned, the number should be increased by the number of
animals scheduled to be euthanised before the completion of study.
6. Dosage At least three test groups and a control group should be used. If there are no suitable data
available, a range finding study may be performed to aid the determination of the doses to be used.
If a vehicle is used in administering test substance, control group should receive vehicle in the
highest volume used.
7. The highest dose level should be chosen to induce toxic effects but not death. Thereafter, a
descending sequence of dose levels should be selected with a view to demonstrate any dosage
related response and no-observed-adverse effects at the lowest dose level (NOAEL).
8. Administration of doses The animals are dosed with test substance daily 7 days each week for a
period of 28 days. The volume should not exceed 1 ml/100g body weight except in the case of
aqueous solutions where 2 ml/100 g body weight may be used
9. Observations General clinical observations should be made at least once a day. At least twice
daily, all animals are observed for morbidity and mortality. In the fourth exposure week sensory
reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli), assessment
of grip strength and motor activity should be conducted.
10. Functional observations conducted in fourth exposure week may be omitted when the study is
conducted as a preliminary study to a subsequent subchronic (90-day) study. At necropsy, the
oestrus cycle of all females could be determined (optional) by taking vaginal smears which can
assist in histological evaluation of estrogen sensitive tissues.
11. Body weight and food/water consumption All animals should be weighed at least once a week.
Measurements of food consumption should be made weekly. If the test substance is administered
via the drinking water, water consumption should also be measured weekly.
12. At the end of the experiment (on 29th day), blood samples were collected from overnight fasted
rats (only water allowed) by retro-orbital bleeding into heparinized and non-heparinized tubes for
hematological analysis and biochemical analysis.
13. Haematology: Haematocrit, Haemoglobin concentrations, Erythrocyte count ,Reticulocytes, Total
and differential leucocyte count, Platelet count Measure of blood clotting time.
14. Clinical biochemistry To investigate major toxic effects in tissues such as kidney and liver.
Investigations of plasma or serum include Na, K, glucose, total cholesterol, urea, creatinine , total
protein and albumin, at least two enzymes indicative of hepatocellular effects (such as alanin
aminotransferase , aspartate aminotransferase , alkaline phosphatase , γ- glutamyl trans-peptidase
and glutamate dehydrogenase ), and bile acids.
15. Pathology Gross necropsy :The liver, kidneys, adrenals, testes, prostate + seminal vesicles,
thymus, spleen, brain and heart of all animals should be trimmed of any adherent tissue & their
wet weight taken as soon as possible after dissection to avoid drying. Histopathology Full
histopathology should be carried out on the preserved organs & tissues of all animals in the control
and high dose groups.

Reference:
https://ntp.niehs.nih.gov/iccvam/suppdocs/feddocs/oecd/oecdtg407-2008.pdf

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 Experiment No: 17

Objective: To study the drug absorbed in the everted ileum with UV- spectrophotometry

Principle: The absorption of drug can be assayed using this method of analysis. This method
involves isolating a small segment of the intestine of a laboratory animal such as rat, inverting
the intestine and filling the sac with a small volume of drug free buffer solution. Both the
segments are tied off and the sac is immersed in an Erlenmeyer Flask containing a large volume
of buffer solution that contains the drug. The flask and its contents are then oxygenated and the
whole preparation is maintained at 37°C and shaken mildly. At predetermined time intervals, the
sac is removed and the concentration of drug in the serosal fluid is determined/assayed for drug
content using UV- spectrophotometry.

Requirements: Unknown drug, Standard concentration of drug, everted ileum, Tyrodesalt

solution

Procedure:
1. Sacrifice the animal and cut open the abdomen exposing the ileocaecal junction.
2. Take a section of the ileum about 2 – 3 cm and wash with Tyrode solution with proper aeration.
3. With a thin glass rod evert the ileum with the mucous layer on the outer surface.
4. Fix the tissue to the oxygen tube and wash it at intervals with Tyrode and stabilize the tissue.
5. Give proper oxygen in the organ bath maintaining the temperature at 37°C.
6. Add drug to the bath at the required concentration.
7. At different type intervals 1 ml of Tyrode solution is collected and the absorbance is measured
with UV-spectrophotometer.
8. A standard curve of absorbance of the drug is plotted and the absorbance of the new solution is
measured.

Observation:

Conclusion: The drug absorbed in the everted ileum with UV- spectrophotometry has been
studied.

Reference:
M.A., Alam, F.I., Al-Jenoobi, A.M., Al-Mohizea. Everted gut sac model as a tool in pharmaceutical
research: limitations and applications. 2011, J Phar Pharmacol, 64, 326-336.

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