Toxicity studies are generally performed to determine drug effects that cannot be evaluated through standard pharmacology profiles or that only occur with repeated administration. Most toxicity tests are performed in two species, such as a rodent and non-rodent, to avoid overlooking unexpected adverse effects before introducing new chemical entities into humans. While animal testing has faced controversy, alternatives using biotechnology tools such as transgenic animal models, cell cultures, and in silico methods are being developed wherever possible to reduce animal use. These alternative techniques include cell line techniques, full thickness skin models, and patch clamp methods to study ion channels at a cellular level.

1. General background:
TOXICITY studies are generally performed to determine drug -related effects
that cannot be evaluated in standard pharmacology profile or occur only after
repeated administration of the agent.
Theoretically, toxicity profiling in animals would be most useful if the test
model responded in a fashion that was identical to the human. However, such
is seldom true in practice, even when the route of administration and vehicle
used are identical to the clinical use because deposition (pharmacokinetics) can
vary dramatically among species, and even between strains.
Therefore, most toxicity tests are performed in two species – a rodent and a
non-rodent – to ensure that any unexpected adverse effects are not overlooked
before new chemical entities (NCE) are introduced into man.
The sequence of toxicity testing proceeds from the simple to the complex.
This is because toxicity testing is desirable at an early stage in the development
process but large quantities of drug samples are usually not available at that
time, limiting the advancement of medical science during the last 100 years
which largely depended on research with animals.

2. Use of animals for research and testing:
 Use of animals in scientific research and testing has raised controversy and
criticism for long.
 Use of animals in medical research has been objected to and a number of
legislative initiatives have been proposed from time to time to limit animal
research, or ensure proper treatment of animals. Animal protection movement
began in England during the nineteenth century.
 Antivivisection groups, sometimes referred to as abolitionists, opposed all
forms of animal research. Animal welfare groups, or reformers, opposed
various forms of animal research due to the increased use of animals for
developing drugs and safety tests for pesticides.
 However, use of non-animal alternatives is also being developed wherever
possible to meet the mandatory regulation of animal experimentation

3. BIOTECHNOLOGY: A better option..!
 Biotechnology in its broadest sense is the use of living organisms (cells,
microbes, plants and animals) to make useful products.
 The application of biotechnological knowledge and in particular gene
technology is especially important to pharmaceuticals in the manufacture of
vaccines, development of more diagnostic aids and therapeutic agents, and
ultimately in gene dosing and expression of genes; peptide engineering
generation and use of various antibodies, mammalian cell cultures; and
transgenic animal techniques.
 These tools are already being used to some degree by pharmaceutical firms, but
as their use expands and knowledge accumulates, many disciplines, including
toxicology will undergo substantial changes.
 Transgenic animals for mutagenecity testing are already on the market.
 Transgenic animal models of carcinogenesis, as well as a number of human
diseases are also available, and increasing use of such models will provide a
powerful tool for assessing the potential toxicity of drugs and chemicals.

4. Animal Alternatives:
 It is not possible to replace whole animal models with in vitro systems to
evaluate drug effects on major organ systems.
 However, techniques can greatly reduce the number of animals needed, and
refined protocols can improve the design efficiency and quality of studies,
and lessen stress and discomfort experienced by lab animals.
 In order to monitor physiological functions in conscious animals, survival
surgery may be performed to implant catheters, electrodes, flow probes or
other devices.
 While chronically instrumented animal models can reduce the numbers of
animals used per study and reduce numbers associated with acute
procedures, these models are resource-intensive to prepare and maintain.
 Generally instrumented animal models can be reused in major organ
systems toxicology (MOST) for studies to evaluate more than one drug.

5. Alternative techniques: definition and
overview:
The term ‘alternative’ is used to refer to those techniques or methods
that replace the use of laboratory animals altogether, reduce the
numbers of animals required, or refine an existing procedure or
technique to minimize the level of stress endured by the animal.
The concept of alternative techniques is now widespread throughout
the scientific community.
This is due largely to regulations and standards which require
consideration and support to alternatives.
However, the field of alternatives study particularly in vitro
toxicology has evolved into a respected discipline and is attracting
competent and motivated scientists around the world.
The Johns Hopkins Center for Alternatives to Animal Testing
(CAAT) was founded in 1981 and is structured to support four core
programmes.

6. Different techniques to be used as an alternatives to
animal testing….
1) Full thickness skin model (invitro method)*¹.
2) In sillico methods*².
3) cell line technique.
4) Patch clamp method.
*¹ and *² are used by Henkel and Phenion medical laboratory

7. In-vitro methods:
 Instead of using animals, cell and tissue
cultures can be used to test product
ingredients.
 Cell culture experiments can show, for
example, the lowest concentration at
which an ingredient causes damage to
cells.
 The results enable conclusions to be
drawn about the ingredient’s
compatibility with tissue.
 Cell cultures are now also used routinely
to test substances for mutagenic
properties.
 Tissue cultures are additionally used to
test substances for compatibility with
mucous membranes.
 A familiar example is the Hen’s Egg
Test, which scientists are now further
developing so that it can be used to test
for mutagenic properties as well.

8. Full Thickness Skin Model:
It has been developed as a robust full thickness skin model based on human cells. It can be
produced to a constant and very high level of quality. A test substance is applied to the Full
Thickness Skin Model so that its effect on the skin tissue can be systematically evaluated.
The substance, e.g. a cream
formulation, is applied topically
using a brush. This can be done
several times over a period of at
least nine days.
In this way, the effect of the
substance on the cell layers
in the skin can be studied.
The standardized production of
the model, in combination with
its special properties, make it
suitable for use as an in-vitro
alternative to animal testing.
Hen’s Egg Test

9. In-sillico methods:
 Substances with similar chemical
structures often have similar properties.
 In these cases, therefore, a knowledge of
the properties of a few representative
substances is sufficient to be able to
deduce the properties of a series of similar
substances.
 By analogy, certain properties of these
representative substances can also be
assumed to be properties of the other
substances in the series.
 The required calculations are performed
using specially developed computer
programs.
 It is anticipated that combinations of such
calculations will make it possible to
narrow down the number of substances to
be tested.
 Only these selected substances will then
have to be tested according to the legally
prescribed test methods. In sillico method

10. Cell line technique:
 The term cell line refers to the propagation of culture after the first
subculture.
 Once the primary culture is sub cultured, it becomes a cell line
 A cell line derived by selection or cloning is referred to as cell strain.
 Cell strain do not have infinite life, as they die after some divisions.
 Types of cells used in cell line:
a) Precursor/ stem cells/ master cells.
b) Undifferentiated but committed precursor cells.
c) Mature differentiated cells.

11. Types of cell lines:
 Mainly two types;
1) Finite cell line.
2) Continuous cell line.
(1) FINITE CELL LINE:
 The cell line with limited culture life spans are referred to as
Finite cell line.
 The cell normally divide 20-100 times before extinction.
 The actual number of doublings depends on the species, cell
lineage differences, culture conditions etc.

12. (2) CONTINUOUS CELL LINE:
The continuous cell lines are transformed, immortal and
tumorigenic.
A few cells in culture may acquire different morphology and get
altered. Such cells are capable of growing faster resulting in an
independent culture.
The progeny derived from these altered cells have unlimited life.

13. Comparison of properties of finite and continuous cell lines:
SERIAL NO PROPERTIES FINITE CELL
LINE
CONTINEUOUS
CELL LINE
1 Growth rate Slow Fast
2 Mode of growth Monolayer Suspension
3 Yield Low High
4 Transformation Normal Immortal,
Tumorigenic
5 Cloning efficiency Low High
6 Serum requirement High Low
7 Markers Tissue specific Chromosomal,
antigenic.

14. APPLICATIONS OF CELL LINE:
 Screening of anti cancer drugs
 Cell based bioassays
 To determine the cytotoxicity
 In vitro screening of several drugs
 Production of anti viral vaccines
 Cancer research, which require the study of uncontrolled cell
division in culture
 Cell fusion technique
 Genetic manipulation
 Gene therapy
 Recombinant DNA therapy
 Biotechnology
 Molecular biology……etc.

15. PATCH CLAMP TECHNIQUE:
 The Technique was developed by Erwin neher & bert
Sakman.
 Patch clamp technique is a technique in electrophysiology that
allows the study of individual ion channels in cells.
 The technique is used to study excitable cells such as neurons,
muscle fibers and the beta cells of the pancreas.

16. What is Patch Clamp….?
 Use a pipette to pinch off a small region of membrane.
 Provides access to:
 the inside of the cell
 a small region of membrane
 Used to measure current through ion channels.
 Why to use patch clamp method..??
 Provides access to the inside of the cell
 Can insert an electrode into the cell
 Can change the intracellular fluid
 Creates a seal impermeable to ion flow
 High electrical resistance
 Allows one to measure current through ion channels vs. voltage,
time, temperature.

17. The patch clamp consists of an electrode inside a glass pipette.
The pipette, which contains a salt solution resembling the fluid
normally found within the cell, is lowered to the cell membrane
where a tight seal is formed.
When a little suction is applied to the pipette, the "patch" of
membrane within the pipette ruptures, permitting access to the
whole cell.
The electrode, which is connected to specialized circuitry, can
then be used to measure the currents passing through the ion
channels of the cell.
Varieties of ion channels can be studied by the patch clamp
technique.

18. PATCH CLAMP

19. TYPES OF PATCH CLAMP:
A. On-cell
B. Inside Out
C. Whole Cell
D. Outside-Out

20. Patch clamp technique in kidney cells:
PRINCIPLE:-
 In the different parts of the kidney fluid is reabsorbed and
substances may be transported either from the tubule lumen to
the blood side (reabsorption) or vice versa (secretion).
 Besides active transport and coupled transport systems, ion
channels play an important role in the function of kidney cells.
 The various modes of the patch clamp technique (cell-attached,
cell-excised, whole-cell mode) allow the investigation of ion
channels.

21. PROCEDURE:
 The patch clamp technique can be applied to cultured kidney
cells freshly isolated kidney cells or to cells of isolated perfused
kidney tubules.
 Segments of late superficial proximal tubules of rabbit kidney
are dissected and perfused from one end with a perfusion
system.
 The non-cannulated end of the tubule is freely accessible to a
patch pipette the patch pipette can be moved through the open
end into the tubule lumen and is brought in contact with the
brush border membrane.
 After slight suction of the patch electrode, gigaseals form
instantaneously and single potassium or sodium channels can be
recorded in the cell-attached or inside- out cell-excised modal.

22. In order to obtain exposed lateral cell membranes suitable to the
application of the patch clamp method, pieces of the tubule are
torn off by means of a glass pipette .
As to facilitate the tearing off, the tubules are incubated for
about 5 min in 0.5 g/L collagenase at room temperature.
After tearing off part of the cannulated tubule, clean lateral cell
membranes are exposed at the non-cannulated end.
The patch pipette can be moved to the lateral cell membrane
and gigaseals can be obtained.
It was possible, to investigate potassium channels and
nonselective cations channels in these membranes.

23. EVALUATION:
 In isolated perfused renal tubules, concentration response
curves of drugs which inhibit ion channels can be
obtained with the patch clamp technique.
 In isolated cells of the proximal tubule, the whole-cell
mode of the patch clamp technique enables the
investigation of the sodium-alanine cotransport system.

24. Applications of Patch Clamp Technique:
 For the evaluation of antiarrhythmics agents.
 In kidney cells.
 Used for isolated ventricular myocytes from Guinea pigs to
study a cardio selective inhibition of the ATP sensitive
potassium channel.
 To identify multiple types of calcium channels.
 To measure the effect of potassium channel openers.
 Used in the molecular biology.
 Voltage clamp studies on sodium channels.
 Used to investigate a wide range of electrophysiological cell
properties.
 Measurement of cell membrane conductance.

25. REFERENCES:
1. Vogel H. Gerhard “Drug Discovery & Evaluation” Pharmacology assay.
Second edition, Page No.83, 84, 319, 321, 480.
2. Alternatives to animal testing, Henkel and Phenion medical laboratory,
www.henkel.com and www.phenion.com
3. http://www.sciencedisplay.com/patchclamp.html
4. Cell culture – wikipedia, free encyclopedia. www.wikipedia.com
5. www.google-image.com