How Is Intracutaneous

Cytotoxicity Testing
Performed?
What Is In-Vivo Cytotoxicity Testing, And Why Is It Needed For Your Medical
Device Or Product?
Plastic polymers evaluated for toxicity fall under six classes. Plastics are
classified based firstly on their extractants (solutions in which polymer
extracts will be prepared). The extractant(s) choice is based on any solutions
in the preparations the plastics are likely to be in contact with. Secondly,
plastics are classified by their route of administration (the end product the
plastics will be used for). Lastly, plastics are ranked based on their material
properties. All cytotoxicity tests are directly related to the intended end-use
of the plastics under assessment. Table 1 below (a reproduction of Table 1 of
USP 88) summarizes the six plastic classes based on test material, animal
model, dosage, and procedure (route of administration). These classifications
summarize the tests that will be needed for each category of plastic. In Table
1, the tests required for each plastic class are indicated by “x” in the
appropriate columns. Under the procedure column in Table 1, A (IP) stands
for a systemic injection test (intraperitoneal), B (IC) stands for an
intracutaneous test (intracutaneous), and C stands for implantation test
(intramuscular or subcutaneous implantation). Intracutaneous allergy testing
and tests that meet USP 88 and ISO 10993 intracutaneous reactivity tests in
animals will be covered.
Classification Of Plastics
Plastic polymers evaluated for toxicity and intracutaneous reactivity fall
under six classes. Plastics are classified based on their extractants (solutions
in which polymer extracts will be prepared). Plastics are also classified based
on the route of administration (the end product the plastics will be used for).
Lastly, plastics are classified by their material properties. These tests are
directly related to the intended end-use of the plastics. The extractant(s)
choice is based on any solution(s) in the preparations of the plastics which
they are likely to be in contact with. Table 1 below (a reproduction of Table 1
of USP 88) summarizes the six plastic classes based on test material, animal
model, dosage, and procedure (route of administration). These classifications
summarize the tests needed for various plastics.
In Table 1, the tests required for each plastic class are indicated by “x” in the
appropriate columns. Under the procedure column, A (IP) stands for systemic
injection test (intraperitoneal), B (IC) stands for intracutaneous test
(intracutaneous), and C stands for implantation test (intramuscular or
subcutaneous implantation).

Table 1. Classification of plastics

Introduction To Intracutaneous Testing For Cytotoxicity
Intracutaneous allergy and reactivity testing is designed to determine the
local biological responses of animals to plastics (or other polymers) by a
single-dose injection of sample extracts from medical devices or therapeutic
products. An intracutaneous injection is an injection between the layers of the
skin. Systemic injection testing and intracutaneous testing may be performed
using the same extracts. Extracts are prepared depending on the heat
resistance of the material being assessed. Thus, extracts are prepared at either
50°, 70°, or 121°C. Extracts are often classified by the type of plastic (Table
1) and extract temperature. For example, a class IV plastic extracted at 121°
would be referred to as IV-121°. Plastics used for containers for oral or
topical products do not apply for Table 1 classification. Additionally, Table 1
classifications do not apply to natural elastomers, which are tested in sodium
chloride injection and vegetable oils only. Sample sizes for extract
preparation are detailed in Table 3 of USP 88 (reproduced as Table 2 below).

Table 2. USP 88 sample extraction surface area

Extraction conditions should not cause physical changes such as fusion or
melting of the sample pieces. Fusion or melting of sample pieces would result
in a decrease in the available surface area and jeopardize the results.
In general, extraction conditions of most importance are:
1. Contact of the extracting medium with the available surface area of the
plastic
2. Extraction temperature
3. Extraction time
4. Proper extract cooling
5. Proper extract agitation
6. Proper extract decanting
 7. Aseptic handling and storage of the extracts following extraction
Skin Reaction Evaluation For Intracutaneous Cytotoxicity Testing
For intracutaneous reactivity and allergy testing, the edema scoring scale
described in Table 2 of USP 88 (reproduced as Table 3 below) excludes
noninflammatory (mechanical) edema from the blank or extraction fluid. Oil
residue at the injection site should not be misinterpreted as edema.
Edematous tissue blanches when gentle pressure is applied.
The scale in Table 3 below uses the intracutanous reactivityscoring system
described in “Methods for the study of irritation and toxicity of substances
applied topically to the skin and mucous membranes” (Draize JH).

Table 3. Skin reaction evaluation

How Are Intracutaneous Cytotoxicity Tests Performed?
Intracutaneous allergy and reactivity testing determines the local response of
an animal (rabbits or guinea pigs) to intracutaneous injection of material
extracts. For testing, animals are shaved, and any loose hair is vacuumed
away. Care is taken to prevent any mechanical irritation or trauma of the
animal’s skin before injection. Each extract is shaken vigorously before
injection to ensure an even distribution of the extracted matter. More than one
extract from a given material can be used per rabbit or guinea pig (if the test
results will not be affected). For each sample, two animals are injected
intracutaneously. One side of the animal is injected with the sample extract,
and the other side is injected with the negative control (Blank). Table 5 of
USP 88 describes this process and is reproduced in Table 4 below. Note that
blanks are composed of the media used for extract preparation. Media used
for blanks has not come in contact with the sample materials being assessed.

Table 4. Intracutaneous test

After injection, injection sites are assessed for evidence of any tissue reaction
such as erythema, edema, and necrosis (see Table 3 above for scoring
system). All animals are observed at 24, 48, and 72 hours after injection for
tissue reaction. The average erythema and edema scores for the extract and
blank injection sites are determined at every scoring interval (24, 48, and 72
hours) for each rabbit or guinea pig.
After the 72-hour scoring, all erythema scores plus edema scores are totaled
separately for each extract sample and blank. Each total is divided by twelve
(two animals × three scoring periods × two scoring categories) to determine
the overall mean score for each sample extract versus each corresponding
blank. Suppose the average reaction to the sample extract is questionably
greater than the average reaction to the blank (at any observation period). In
that case, the test is repeated using three additional rabbits or guinea pigs.
The test requirements are met if the difference between the mean score of the
extract samples and the mean score of the blanks (negative controls) is 1.0 or
less.