ISO 10993-10

The international standard's four-tier approach to irritation testing protects patients and minimizes
animal studies.
Three types of testingcytotoxicity, sensitization, and irritation are mandated for all medical device
materials by the international biocompatibility standard ISO 10993-1, "Guidance on the Selection of
Tests," and its FDA counterpart, blue book memorandum #G95-1. Specific tests that can be used to
satisfy these requirements are then provided in ISO 10993-5, which covers cytotoxicity, and ISO
10993-10, which addresses both sensitization and irritation. This article focuses on that part of ISO
10993-10 devoted to irritation tests. (Earlier articles in this MD&DI series outlined the other two types
of required testingcytotoxicity and sensitization.)

IRRITATION RESPONSES

The chemicals released from device materials that contact the body may produce skin, mucosal, or
eye irritation. In general terms, such irritation is a local tissue response characterized by the usual
signs of inflammationredness and swellingand sometimes accompanied by heat and pain (Figure
1). Numerous chemicals are capable of causing irritation, either immediate or delayed, and some of
these may be present in materials as additives, processing or manufacturing aids, or inadvertent
contaminants. For example, the organotin stabilizers used in nonmedical vinyl formulations are
corrosive chemicals capable of causing irritation and necrosis when applied to mucosal surfaces;
residual concentrations of ethylene oxide present in gas-sterilized devices can produce an irritant
response if they are not reduced to acceptable levels before the device is used; and residues of such
contaminants as chemical detergents in a particular batch of materials or devices can cause
unexpected irritation responses in users or patients.
Figure 1. Some individuals exhibit a marked response to the
chemicals present in such products as latex gloves. This case
was judged to be an irritation, not a sensitization, response.

With regard to irritation testing, the ISO standard defines irritation as a "localized inflammatory
response to single, repeated, or continuous application of the test substance, without involvement of
an immunological mechanism." ISO 10993-10 also proposes a four-tier approach to assessing the
potential of a material to cause irritation. A device manufacturer should first conduct a review of the
literature to determine whether others have reported that the chemical or material under
consideration, or structurally related chemicals or materials, can cause irritation. It is essential that the
chemical or material of interest already be sufficiently characterized that it can be correlated to those
described in the literature. The second step is to use available, validated in vitro tests (such as
cytotoxicity assays using mammalian cells in culture) to identify, whenever possible, severely irritating
materials without using test animals. When materials have not been ruled out by the first two steps,
they should be evaluated using the in vivo tests described in the standard. The final step is the use of
noninvasive clinical studies in human subjects, but this is not presented in the standard as a routine
part of an irritation testing program. Rather, it is presumably reserved for samples with unusual
characteristics or those with equivocal test results.

IN VIVO TEST METHODS

The intracutaneous, primary skin, and ocular irritation tests are the three in vivo, nonclinical tests
commonly used to evaluate materials for possible contact hazards. The intracutaneous test has been
described in the United States Pharmacopeia (USP) for more than 30 years as a means to assess the
suitability of plastic pharmaceutical containers for their intended use. The test is conducted much like
allergy testing in human patients. Fluid extracts of the test material are prepared under controlled
conditions of temperature, time, and ratio of the material surface area to the volume of extraction fluid.
Using small-bore needles, a volume of fresh extract is then injected intracutaneously (that is, into the
skin) at multiple sites on the shaved backs of two albino rabbits. An equal number of control sites are
injected with an untreated volume of the extraction vehicle. At 24, 48, and 72 hours after injection, the
test and control sites are observed and scores are given for the severity of any redness (erythema) or
swelling (edema) (Figure 2). Extracts that produce a significantly greater response than controls are
considered irritants. The USP method describes the use of up to four extraction fluidssaline, salinealcohol, polyethylene glycol 400, and vegetable oilfor evaluating pharmaceutical containers. For
medical device materials, saline and vegetable oil are used to ensure extraction of both water-soluble
and fat-soluble chemicals.
Figure 2. In intracutaneous irritation tests using albino rabbits, the
raised blebs caused by injecting extracts of test materials are
either resolved without causing any visible changes or produce
inflammation marked by redness and swelling in the 24 to 72
hours following injection.

The intracutaneous reactivity test is aggressive in that it makes use of extracts prepared under
exaggerated conditions and places them directly into the skin of the test animal, thereby maximizing
the chance of finding irritant chemicals if they are present. The primary skin irritation test is less
aggressive in that portions of the test material itself are simply placed on the shaved backs of albino
rabbits. The samples are then covered with an occlusive dressing and left in place for at least 4 hours
but more commonly for 24. The contact sites are observed for an additional period of up to 72 hours
and scored for erythema and edema. After these scores are totaled, they are compared with known
values for primary skin irritation (available in table format), and the response is categorized as
negligible, slight, moderate, or severe. When necessary, the method may be modified to use fluid
extracts rather than the material itself on the contact sites.

Generally reserved for eye-contact products, the ocular irritation test is usually performed with fluid
extracts prepared as described above, although some materials may be tested directly as solids or
powders. A small volume of fresh extract (or solid) is placed directly into the pocket formed by
withdrawing the lower eyelid of an albino rabbit. The rabbit's other eye is left untreated as a control.
Observations are made at regular intervals for up to 72 hours, and scores are compiled for redness
and swelling of the eye's conjunctiva, response of the iris to light, corneal opacity, and presence of
discharge. These scores are then compared with a classification table to determine which test
materials are considered eye irritants.

In addition to the intracutaneous, primary skin, and ocular irritation tests, oral, rectal, penile, and
vaginal irritation tests are described in annex D of the standard as complements to, not replacements

for, the primary tests. They are considered relevant for medical devices intended to be applied to
these respective mucosal areas of the body. Of the four, the vaginal irritation test in the rabbit is of
particular importance for several reasons. Extracts applied to the vaginal mucosa remain in contact
with the tissue for an extended time, exaggerating exposure; the vaginal epithelium of the rabbit is
only one cell thick and thus particularly sensitive to irritants; and a microscopic scoring system is
available, providing a cellular basis for judging the irritant potential of a material.

CONCLUSION

Considerable effort has been expended during the past 20 years to identify suitable in vitro
alternatives to animal tests for irritation. However, none of the methods reported on thus far has
duplicated the complex physiology of the animal model, so the search continues. Meanwhile, the
tiered approach described in ISO 10993-10 offers a means of minimizing both the potential for
exposure of human patients to irritating medical devices and the amount of animal testing that must
be conducted.

ISO 10993-10
Testing for sensitivity to chemical extractables from medical devices is a key element of the
biocompatibility standards. Note: this is the fourth installment in an ongoing series of articles on ISO
10993. If you haven't done so already, you might like to read part one, ISO 10993: An Introduction to
the Standard.
According to International Organization for Standardization (ISO) 10993-1the first in the set of
international standards covering the biological evaluation of medical devices and the basis for FDA's
blue book memorandum on this subject (#G95-1)all device materials must undergo cytotoxicity,
sensitization, and irritation testing. Methods for performing such tests are described in other parts of
ISO 10993, including ISO 10993-10, "Tests for Sensitization and Irritation."

This article focuses on those test methods currently being used to determine whether sensitization
reactions are elicited by any chemicals that may be released from specific biomaterials and medical
devices. Earlier articles addressed materials characterization and cytotoxicity.

SENSITIZATION REACTIONS

Sensitization or hypersensitivity reactions usually occur as a result of repeated or prolonged contact

with a chemical substance that interacts with the body's immune system. Because most such
reactions to biomaterials have been of the dermal cellmediated type, rather than the humoral or
antigen-antibody type, the skin of laboratory animals is used in sensitivity testing. Dermal sensitization
reactions in laboratory animals are marked by redness and swelling.

Biomaterials and devices that cause sensitization reactions do so by means of their extractable
chemicals. In some cases, an individual may develop a reaction only after encountering a material
repeatedly or after continuous, prolonged contact, such as with an implant. Or, after wearing natural
latex gloves daily for several weeks or months, a previously unaffected person may develop a

persistent rash on the hands and wrists (Figure 1). This sensitization may be caused by one of
several chemical components of the gloves acting as an allergen.

Figure 1. After wearing natural latex gloves daily for several

weeks or months, a person may develop a rash.

In other cases, when an individual has already become sensitized to a chemical, such as one present
in the environment, he or she will experience a reaction when first exposed to a device that contains
that chemical. Thus, an individual previously sensitized to nickel will develop a rash along the temples
a few days after beginning to wear eyeglasses with nickel-plated frames.

TEST METHODS

Biomaterials and other device materials are tested for the presence of sensitizing chemicals using
guinea pigs, a species known to be nearly as responsive to dermal sensitizers as human beings are.
Guinea pig sensitization tests require six to eight weeks and thus take the longest time to complete of
all the acute biocompatibility tests described in the 10993 standards.

The repeated-patch, or Buehler, test method involves exposing the shaved backs of guinea pigs
directly to the test material under occlusive dressings for a minimum of six hours. This procedure is
repeated as many as three times a week for three weeks. This part of the test is often referred to as
the induction phase. Following a two-week rest or recovery period to allow for the development of a
delayed response, the animals are challenged by a final exposure to a patch of the biomaterial. The
repeated-patch model is used primarily for topical devices such as dermal electrodes and surgical
gowns and drapes, since in these cases the method of applying test materials to the animals
simulates clinical use.

In the maximization, or Magnuson-Kligman, test method, fluid extracts of the test material are
prepared in saline and vegetable oil, and separate groups of guinea pigs are exposed repeatedly to
the two types of extracts. The guinea pigs are first injected with an extract along with an adjuvant
intended to enhance an immune response, then receive a topical application. Following a two-week
rest or recovery, the animals are covered with a topical patch containing the extract. Generally
considered more sensitive than the repeated- patch model, the maximization test is used for device
materials that will contact areas other than skin. The use of both a saline extract and an oil extract
simulates extraction by bodily fluids and by intravenous liquids and other pharmaceutical products
that first contact the device and then the patient.

Figure 2. Positive response to a positive-control substance in a

guinea pig, seen in the maximization, or Magnuson-Kligman, test.

In both techniques, the area of the challenge patches is examined for reactions (redness and
swelling) that are not present in negative-control animals. In addition, known sensitizing chemicals are
used periodically to validate the model and the technician (Figure 2).

A CAVEAT AND FUTURE POSSIBILITIES

The guinea pig sensitization tests described above are useful methods for eliminating the possibility
that patients will be exposed to strong sensitizing chemicals extracted from medical device materials.
However, these methods are far from perfect in their ability to detect weak sensitizers or chemicals,
and they do not detect chemicals that act as adjuvants, enhancing an immune response to other
chemicals to which a patient might be exposed. Nor are they able to detect responses to antigens
such as the plant proteins found in natural latex, which have been responsible for the severe, even
life-threatening, systemic immune responses that occasionally have been reported.

Additional test methods currently under development also may prove useful for evaluating
biomaterials for sensitization. One test that shows promise is the local lymph node assay in mice. In
this method, the ears of mice are treated and then the surrounding lymph nodes are examined for a
lymphocyte proliferative response as demonstrated by an accumulation of a radio-labeled marker.

Much effort also has been devoted to developing in vitro alternatives to the guinea pig sensitization
models, but thus far no suitable replacements have been identified.