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á87ñ BIOLOGICAL REACTIVITY TESTS, IN VITRO

The following tests are designed 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 test. It is essential that the tests be performed on the specified surface area. When the surface area of the
specimen cannot be determined, use 0.1 g of elastomer or 0.2 g of plastic or other material for every mL of extraction fluid.
Exercise care in the preparation of the materials to prevent contamination with microorganisms and other foreign matter.
Three tests are described (i.e., the Agar Diffusion Test, the Direct Contact Test, and the Elution Test).1 The decision as to which
type of test or the number of tests to be performed to assess the potential biological response of a specific sample or extract
depends upon the material, the final product, and its intended use. Other factors that may also affect the suitability of a
sample for a specific use are the polymeric composition; processing and cleaning procedures; contacting media; inks;
adhesives; absorption, adsorption, and permeability of preservatives; and conditions of storage. Evaluation of such factors
should be made by appropriate additional specific tests before determining that a product made from a specific material is
suitable for its intended use. Materials that fail the in vitro tests are candidates for the in vivo tests described in Biological
Reactivity Tests, In Vivo á88ñ.
PROCEDURES
• TEST CONTROL
Positive control: Polyurethane film containing zinc diethyldithiocarbamate (ZDEC)2 or zinc dibutyldithiocarbamate (ZDBC)

Cell culture preparation: Prepare multiple cultures of L-929 (ATCC cell line CCL 1, NCTC clone 929; alternative cell lines
obtained from a standard repository may be used with suitable validation) mammalian fibroblast cells in

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serum-supplemented minimum essential medium having a seeding density of about 105 cells per mL. Incubate the cultures
at 37 ± 1° in a humidified incubator for NLT 24 h in a 5 ± 1% carbon dioxide atmosphere until a monolayer, with greater
than 80% confluence, is obtained. Examine the prepared cultures under a microscope to ensure uniform, near-confluent
monolayers. [NOTE—The reproducibility of the in vitro biological reactivity tests depends upon obtaining uniform cell
culture density.] ci
Extraction solvents: Sodium Chloride Injection [see monograph—use Sodium Chloride Injection containing 0.9% of sodium
chloride (NaCl)]. Alternatively, serum-free mammalian cell culture media or serum-supplemented mammalian cell culture
media may be used. Serum supplementation is used when extraction is done at 37° for 24 h.
• APPARATUS
Autoclave: Employ an autoclave capable of maintaining a temperature of 121 ± 2°, equipped with a thermometer, a pressure
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gauge, a vent cock, a rack adequate to accommodate the test containers above the water level, and a water cooling system
that will allow for cooling of the test containers to about 20°, but not below 20°, immediately following the heating cycle.
Oven: Use an oven, preferably a mechanical convection model, that will maintain operating temperatures in the range of
50°–70° within ±2°.
Incubator: Use an incubator capable of maintaining a temperature of 37 ± 1° and a humidified atmosphere of 5 ± 1% carbon
dioxide in air.
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Extraction containers: Use only containers, such as ampuls or screw-cap culture test tubes, or their equivalent, of Type I
glass. If used, culture test tubes, or their equivalent, are closed with a screw cap having a suitable elastomeric liner. The
exposed surface of the elastomeric liner is completely protected with an inert solid disk 50–75 µm in thickness. A suitable
disk can be fabricated from polytef.
Preparation of apparatus: Cleanse all glassware thoroughly with chromic acid cleansing mixture and, if necessary, with hot
nitric acid followed by prolonged rinsing with Sterile Water for Injection. Sterilize and dry by a suitable process for containers
and devices used for extraction, transfer, or administration of test material. If ethylene oxide is used as the sterilizing agent,
allow NLT 48 h for complete degassing.
• PROCEDURE
Preparation of sample for extracts: Prepare as directed in the Procedure in á88ñ.
Preparation of extracts: Prepare as directed for Preparation of extracts in á88ñ using either Sodium Chloride Injection [0.9%
sodium chloride (NaCl)] or serum-free mammalian cell culture media as Extraction solvents. [NOTE—If extraction is done at
37° for 24 h in an incubator, use cell culture media supplemented by serum. The extraction conditions should not in any
instance cause physical changes, such as fusion or melting of the material pieces, other than a slight adherence.]
• AGAR DIFFUSION TEST
This test is 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. Extracts of
materials that are to be tested are applied to a piece of filter paper.
Sample preparation: Use extracts prepared as directed, or use portions of the test specimens having flat surfaces NLT
100 mm2 in surface area.
Positive control preparation: Proceed as directed for Sample preparation.
Negative control preparation: Proceed as directed for Sample preparation.
Procedure: Using 7 mL of cell suspension prepared as directed in Cell culture preparation, prepare the monolayers in plates
having a 60-mm diameter. Following incubation, aspirate the culture medium from the monolayers, and replace it with
serum-supplemented culture medium containing NMT 2% of agar. [NOTE—The quality of the agar must be adequate to

1 Further details are given in the following publications of the American Society for Testing and Materials, 1916 Race St., Philadelphia, PA 19103: Standard
test method for agar diffusion cell culture screening for cytotoxicity, ASTM Designation F 895-84; Standard practice for direct contact cell culture
evaluation of materials for medical devices, ASTM Designation F 813-83.
2 ZDEC and ZDBC polyurethanes are available from the Food and Drug Safety Center, Hatano Research Institute, Ochiai 729–5, Hadanoshi, Kanagawa
257, Japan.

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support cell growth. The agar layer must be thin enough to permit diffusion of leached chemicals.] Place the flat surfaces
of Sample preparation, Positive control preparation, and Negative control preparation or their extracts in an appropriate
extracting medium, in duplicate cultures in contact with the solidified agar surface. Use no more than three specimens
per prepared plate. Incubate all cultures for NLT 24 h at 37 ± 1°, preferably in a humidified incubator containing 5 ± 1%
of carbon dioxide. Examine each culture around each sample, negative control, and positive control under a microscope,
using a suitable stain, if desired.
Interpretation of results: The biological reactivity (cellular degeneration and malformation) is described and rated on a
scale of 0–4 (see Table 1). Measure the responses of the cell cultures to the Sample preparation, the Positive control
preparation, and the Negative control preparation. The cell culture test system is suitable if the observed responses to the
Negative control preparation is grade 0 (no reactivity) and to the Positive control preparation is at least grade 3 (moderate).
The sample meets the requirements of the test if the response to the Sample preparation is not greater than grade 2 (mildly
reactive). Repeat the procedure if the suitability of the system is not confirmed.

Table 1. Reactivity Grades for Agar Diffusion Test and Direct Contact Test
Grade Reactivity Description of Reactivity Zone
0 None No detectable zone around or under specimen

1 Slight Some malformed or degenerated cells under specimen

2 Mild Zone limited to area under specimen and less than 0.45 cm beyond specimen

3 Moderate Zone extends 0.45–1.0 cm beyond specimen

4 Severe Zone extends greater than 1.0 cm beyond specimen

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• DIRECT CONTACT TEST
This test is 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.
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Sample preparation: Use portions of the test specimen having flat surfaces NLT 100 mm2 in surface area.
Positive control preparation: Proceed as directed for Sample preparation.
Negative control preparation: Proceed as directed for Sample preparation.
Procedure: Using 2 mL of cell suspension prepared as directed in Cell culture preparation, prepare the monolayers in plates
having a 35-mm diameter. Following incubation, aspirate the culture medium from the cultures, and replace it with 0.8 mL
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of fresh culture medium. Place a single Sample preparation, a Positive control preparation, and a Negative control
preparation in each of the duplicate cultures. Incubate all cultures for NLT 24 h at 37 ± 1° in a humidified incubator
containing 5 ± 1% of carbon dioxide. Examine each culture around each Sample preparation, a Positive control
preparation, and a Negative control preparation, under a microscope, using a suitable stain, if desired.
Interpretation of results: Proceed as directed for Interpretation of results in Agar Diffusion Test. The sample meets the
requirements of the test if the response to the Sample preparation is not greater than grade 2 (mildly reactive). Repeat the
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procedure if the suitability of the system is not confirmed.

• 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 nonphysiological temperatures for varying time intervals. It is appropriate for high-density materials
and for dose-response evaluations.
Sample preparation: Prepare as directed in Preparation of extracts, using either Sodium Chloride Injection [0.9% sodium
chloride (NaCl)] or serum-free mammalian cell culture media as Extraction solvents. If the size of the sample cannot be
readily measured, a mass of NLT 0.1 g of elastomeric material or 0.2 g of plastic or polymeric material per mL of extraction
medium may be used. Alternatively, use serum-supplemented mammalian cell culture media as the extracting medium
to simulate more closely physiological conditions. Prepare the extracts by heating for 24 h in an incubator containing 5
± 1% of carbon dioxide. Maintain the extraction temperature at 37 ± 1°, because higher temperatures may cause
denaturation of serum proteins.
Positive control preparation: Proceed as directed for Sample preparation.
Negative control preparation: Proceed as directed for Sample preparation.
Procedure: Using 2 mL of cell suspension prepared as directed in Cell culture preparation, prepare the monolayers in plates
having a 35-mm diameter. Following incubation, aspirate the culture medium from the monolayers, and replace it with
extracts of the Sample preparation, Positive control preparation, or Negative control preparation. The serum-supplemented
and serum-free cell culture media extracts are tested in duplicate without dilution (100%). The Sodium Chloride Injection
extract is diluted with serum-supplemented cell culture medium and tested in duplicate at 25% extract concentration.
Incubate all cultures for 48 h at 37 ± 1° in a humidified incubator preferably containing 5 ± 1% of carbon dioxide. Examine
each culture at 48 h, under a microscope, using a suitable stain, if desired.
Interpretation of results: Proceed as directed for Interpretation of results in Agar Diffusion Test but use Table 2. The sample
meets the requirements of the test if the response to the Sample preparation is not greater than grade 2 (mildly reactive).
Repeat the procedure if the suitability of the system is not confirmed. For dose-response evaluations, repeat the procedure,
using quantitative dilutions of the sample extract.

Table 2. Reactivity Grades for Elution Test

Grade Reactivity Conditions of All Cultures
0 None Discrete intracytoplasmic granules; no cell lysis

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Table 2. Reactivity Grades for Elution Test (continued)

Grade Reactivity Conditions of All Cultures
Less than or equal to 20% of the cells are round, loosely attached, and without intracytoplasmic
1 Slight granules; occasional lysed cells are present

Greater than 20% to less than or equal to 50% of the cells are round and devoid of intracyto-
2 Mild plasmic granules; no extensive cell lysis and empty areas between cells

3 Moderate Greater than 50% to less than 70% of the cell layers contain rounded cells or are lysed

4 Severe Nearly complete destruction of the cell layers

ADDITIONAL REQUIREMENTS
• USP REFERENCE STANDARDS á11ñ
USP High-Density Polyethylene RS
(Negative Control)

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