TOXICITY AND

IDENTIFICATION TEST
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LEARNING OBJECTIVES

• Describe the methods of identification tests of

different pharmaceuticals (in-vivo)
• Describe the methods of toxicity tests.
• Depends on biological reactions
1.Atropine tablets
2.Insulin injection
3.Isoflurophate solution
• Take sufficient tablets/injection volume to
present 0.6-1mg of Atropine sulphate
dissolved in 10ml water and filter the solution.

• Place one drop of the solution into cat’s eye,

dilation of the pupil of eye results within 2
hours.
• Six rabbits having weight 1.8-2.2kg
• A quantity of insulin injection that may cause
convulsions in at least three of the rabbits is
injected subcutaneously into each rabbit.
• Immediately after convulsions occur in a
rabbit, 5ml of 50% solution of dextrose is
injected IV .
• The convulsions should be relieved and at
least 4 rabbits should remain alive for at least
3 days.
• The identification test is based on meiotic
activity
• Place 100mg ointment in right eye of 3
rabbits.
• For positive result the diameter of the pupil of
treated eye should be at least 2mm smaller
than average diameter of un treated eye.
• Performed on finished dosage form
• Detect unforeseen errors in formulation
• Conducted for specific Toxoids and Plastic
containers
• Select at least 4 healthy guinea pigs.
• Inject subcutaneously 5 times of dose of
Diphtheria toxoids use for human
immunization.
• No general symptoms of diphtheria toxin
poisoning should occur within 30 days.
• Select at least 4 healthy guinea pigs.
• Inject subcutaneously 5 times of dose of
Tetanus toxoids use for human immunization.
• No general symptoms of tetanus toxin
poisoning should occur within 21 days.
• See USP.
• <1031> THE BIOCOMPATIBILITY OF
MATERIALS USED IN DRUG CONTAINERS,
MEDICAL DEVICES, AND IMPLANTS
• <88> BIOLOGICAL REACTIVITY TESTS, IN
VIVO
THE BIO-COMPATIBILITY OF
MATERIALS USED IN DRUG
CONTAINERS, MEDICAL DEVICES,
AND IMPLANTS
• At the end of this lecture students will be
able to
• Define the purpose of biocompatibility
tests
• Explain the tests to determine the toxicity
of the plastic.
 Biocompatibility

“Tendency of the product to remain biologically

inert throughout the duration of their contact
with the body”.
The testing procedure used to
Detect the non specific, biologically reactive,
physical and chemical characteristics of medical
product and material used in the construction.
To identify and detect the toxicity of medical products prior
to use.
Specific in-vitro and in-vivo testing procedures used to
evaluate the biocompatibility of medical products,

1. Biological reactivity test IN-VITRO

2. Biological reactivity test IN-VIVO
 Biological reactivity test
IN VIVO
The USP Class tests are a set of in-vivo, screening“ tests to
characterize the basic biocompatibility of the plastic under
investigation.
• Six classes of plastics are defined, based on responses to a series
of in vivo tests for which extracts, materials and routes of
administration are specified.
• Types
The following three in-vivo tests make up the USP Class test.
o ACUTE SYSTEMIC TOXICITY TEST:
o IRRITATION TEST – INTRACUTANEOUS INJECTION TEST:
o IMPLANTATION TEST:
• Purpose : determine the biological
response of animals to elastomerics,
plastics, and other polymeric material with
direct or indirect patient contact, or by the
injection of specific extracts prepared from
the material under test
• USP reference standard: high density polyethylene
• Extracting media:
1. sodium chloride injection containing 0.9% of NaCl.
2. 1 in 20 solution of alcohol in sodium chloride injection
3. polyethylene glycol 400
4. vegetable oil— use freshly refined sesame oil or
cottonseed oil
5. drug product vehicle (where applicable).
6. Water for injection
• Apparatus: autoclave, oven, extraction container.
 Sample preparation

• Select and subdivide into portions Sample

• Place the Sample into In a clean, glass-stoppered, 100-mL
graduated cylinder of Type I glass,
• add about 70 mL of Water for Injection.
• Agitate for about 30 seconds, and drain off the water.
• Repeat this step,
• dry those pieces prepared for the extraction with Vegetable Oil in an
oven at a temperature not exceeding 50°.
• [NOTE—Do not clean the Sample with a dry or wet cloth or by
rinsing or washing with an organic solvent,
 PREPARATION OF EXTRACTS

• Place a prepared Sample to be tested in an extraction container,

• add 20 mL of the appropriate extracting medium
• prepare 20-mL blank of each medium for parallel injections and
comparisons.
• Extract by heating in an autoclave at 121° for 60 minutes, in an oven at
70° for 24 hours, or at 50° for 72 hours.
• Allow adequate time for the liquid within the container to reach the
extraction temperature.
• Cool to about room temperature but not below 20°, shake vigorously for
several minutes, and decant each extract immediately, using aseptic
precautions, into a dry sterile vessel,
• Store the extracts at a temperature between 20° and 30°, and do not use
for tests after 24 hours.
 1.ACUTE SYSTEMIC TOXICITY TEST:

PURPOSE:

In-vivo systemic tests evaluate the impairment or activation of a system

– rather than the impairment of individual cells or organs.

In “acute” systemic toxicity tests, the test material (extract) is tested for
systemic toxic effects as a result of a single, acute exposure.
Test Animals—

• Use healthy, not previously used albino mice weighing

between 17 and 23 g.
• For each test group period use only mice of the same
source.
• Allow water and food easily.
 Procedure
• Prepare EXTRACT in 4 different extraction media: a Saline solution,
a 1:20 Ethanol/Saline solution, Polyethylene Glycol 400 and
Cottonseed oil/sesame oil.

Mice are injected(5mice)

• intravenously (saline solution and 1:20 ethanol/saline solution)
• intra-peritoneal (Polyethylene Glycol 400 and Cottonseed
oil/sesame oil) with the test and control materials over 72 h.
• consider test as negative if none of the animals injected with the
test material shows a greater biological reaction than animals
treated with the negative control material (blank).
 2.IRRITATION TEST - INTRACUTANEOUS
INJECTION TEST

PURPOSE:

The irritation tests are in-vivo screening tests to evaluate the potential
of test materials – or their extracts – to cause irritation on the
exposed part of the body.
• Designed to evaluate local responses(inflammatory reactions) to
the extracts of materials under test following intracutaneous
injection into rabbits
• Test Animals—
Select healthy, thin-skinned albino rabbits with fur that
can be clipped closely and skin that is free from
mechanical irritation or trauma.
• Extracts of the test material are prepared in 4 different extraction
media:
• a Saline solution,
• a 1:20 Ethanol/Saline solution,
• Polyethylene Glycol 400 and
• Cottonseed oil/sesame oil.
 Procedure

• For each Sample use two animals and inject each

intracutaneously,
• using one side of the animal for the Sample and the
other side for the Blank,
• Rabbits are injected intracutaneously with the test
and control materials and observed over a 72h period.
• A primary irritation index is determined based on the
defined evaluation criteria in USP<88>.
• Examine injection sites for evidence of any tissue reaction such as
erythema, edema, and necrosis. Observe all animals at 24, 48, and
72 hours after injection.
•
• The requirements of the test are met if the difference between the
Sample and the Blank mean score is 1.0 or less.
• If at any observation period the average reaction to the Sample is
questionably greater than the average reaction to the Blank, repeat
the test using three additional rabbits. The requirements of the test
are met if the difference between the Sample and the Blank
• mean score is 1.0 or less.
 3.IMPLANTATION TEST:

PURPOSE:

evaluate the local pathological effects on living tissue, at both the

gross and microscopic level of a test article that is surgically
implanted into an appropriate implant site.

• designed for the evaluation of plastic materials and other polymeric

materials in direct contact with living tissues.
Test Animals—

Select healthy, adult rabbits weighing not less than 2.5 kg, and with
paravertebral muscles that are sufficiently large in size to allow for
implantation of the test strips..
1. Intramuscular Implantation in Rabbits (4 strips /animal)

Prepare for implantation 8 strips of the Sample and 4 strips of USP High
Density Polyethylene RS.
Each strip should measure not less than 10 ´ 1 mm.
Strips of the specified minimum size are implanted
• Implant four strips of the Sample into the paravertebral muscle on one
side of the spine of each of two rabbits,
• Similarly implant two strips of USP High-Density Polyethylene RS in the
opposite muscle of each animal.
• Keep the animals for a period of not less than 120 hours,
• and sacrifice them at the end of the observation.
• Examine macroscopically the area of the tissue surrounding the center
portion of each implant strip.
• the Sample and Control implant sites for hemorrhage, necrosis,
discolorations, and infections, and record the observations.
• Measure encapsulation, if present, by recording the width of the
capsule (from the periphery of the space occupied by the implant
Control or Sample to the periphery of the capsule) rounded to the
nearest 0.1 mm.
2.Subcutaneous Implantation in Rats

• Prepare for implantation 10 sample specimens and 10 control specimens.

The size and shape of the control specimens shall be as similar to that of
the test specimens as practically possible.

• Test Animals—Select healthy rats weighing between 225 - 350 g at the

time of implantation
• Procedure—(5rats)
• Perform the test in a clean area. Anesthetize the animal until
a surgical plane is achieved.
• Clip the fur of the animals on both sides of the spinal column.
• Remove the loose hair by means of vacuum.
• Using aseptic technique, by dissecting underneath the skin
form two pockets
• Insert a sterile sample into each pocket, close the incision
with wound clips or sutures
• Implant two test samples and two control samples in each of
five rats.
• Keep the animals for a period of at least seven days, and
sacrifice them at the end.
• Carefully examine macroscopically the area of the tissue
surrounding the implant.
• Observe the Sample and Control implant sites for
hemorrhage, necrosis, discolorations, and infections,
and record the observations.
• Measure encapsulation, if present, by recording the
width of the capsule (from the periphery of the space
occupied by the implant Control or Sample to the
periphery of the capsule) rounded to the nearest 0.1 mm.
 Biological reactivity test
IN VITRO
• To determine the biological reactivity of
mammalian cell cultures following contact with
the elastomeric plastics and other polymeric
materials with direct or indirect patient contact or
of specific extracts prepared from the materials
under.
 TESTS
Three tests are
• Agar Diffusion Test,
• Direct Contact Test,
• Elution Test.
 Agar Diffusion Test

Designed for elastomeric closures in a variety of shapes.

The agar layer acts as a cushion to protect the cells from
mechanical damage while allowing the diffusion of
leachable chemicals from the polymeric specimens.
Ex-tracts of materials that are to be tested are applied to a
piece of filter paper.
 Reactivity grades for
Direct Contact Test/ Agar Diffusion
Test
Grade Reactivity Description of Reactivity Zone

No detectable zone around or under

0 None specimen

Some malformed or degenerated

1 Slight cells under specimen

Zone limited to area under speci-

men and less than 0.45 cm beyond
2 Mild specimen

Zone extends 0.45–1.0 cm beyond

3 Moderate specimen

Zone extends greater than 1.0 cm

4 Severe beyond specimen
 Direct Contact Test

Designed for materials in a variety of shapes.

The procedure allows for simultaneous extraction and
testing of leachable chemicals from the specimen with a
serum-supplemented medium.
The procedure is not appropriate for very low- or high-
density materials that could cause mechanical damage
to the cells.
 Elution Test

• This test is designed for the evaluation of extracts of

polymeric materials.
• The procedure allows for extraction of the specimens
at physiological or non physiological temperatures for
varying time intervals.
• It is appropriate for high-density materials and for
dose-response evaluations.
•
 Reactivity grades for
Elution Test
Grade Reactivity Conditions of All Cultures
Discrete intracytoplasmic granules;
0 None no cell lysis
Less than or equal to 20% of the
cells are round, loosely attached,
and without intracytoplasmic gran-
ules; occasional lysed cells are pres-
1 Slight ent
Greater than 20% to less than or
equal to 50% of the cells are round
and devoid of intracytoplasmic
granules; no extensive cell lysis and
2 Mild empty areas between cells
Greater than 50% to less than 70%
of the cell layers contain rounded
3 Moderate cells or are lysed
Nearly complete destruction of the
4 Severe cell layers