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11.2 PLASTIC CONTAINERS

          Plastic containers for pharmaceutical products are made from plastics based on the following polymers: polyethylene, polypropylene, polyvinyl chloride, polystyrene and, to a lesser extent, polymethyl methacrylate, polyethylene terephthalate, polytetrafluoroethylene, the amino formaldehydes and polyamides, to which certain additives are added such as antioxidants, antistatic agents, colours, impact modifiers, lubricants, plasticizers and stabilizers. For pharmaceutical purposes antistatic agents are not normally used, and colours are also not normally used in containers for injections. These containers should not impart any potential hazards to the pharmaceutical products contained therein and should meet the requirements set up for each type of those products.

Plastic Containers for Pharmaceutical Products and Their Requirements

1. Containers for solid dosage forms

Comply with the physico-chemical tests 1.2, 1.3.1 and biological test 2.6.

2. Containers for semisolid dosage forms

Comply with the physico-chemical tests 1.2, 1.3.2 and biological test 2.6.

3. Containers for oral liquid dosage forms

Comply with the physico-chemical tests 1.2, 1.3.3 and biological test 2.6.

4. Containers for ophthalmic preparations

Comply with the physico-chemical tests 1.2 and either 1.3.2 or 1.3.4, and biological test 2.6.

5. Containers for parenteral preparations

Comply with the physico-chemical tests 1.1, 1.2, 1.3.4 and biological tests 2.

6. Containers for irrigations

Comply with the test under parenteral preparations.

7. Containers for human blood and blood components

Comply with the physico-chemical tests 1.1, 1.2, 1.3.5 and biological tests 2 and tests under Appendix 11.3.

1. Physico-chemical Tests

          The following tests, designed to determine physical and chemical properties of plastics and their extracts, are based on the extraction of the plastic material, and it is essential that the designated amount of the plastic be used. Also, the specified surface area must be available for extraction at the designated temperature.

          1.1 Transparency and Appearance The containers have a transparency which does not practically interfere with the test according to “Particulate Matter in Injections” (Appendix 4.27). For the test of observed light transmission for the plastic containers intended for parenteral use, perform the test according to “Light Transmission” under Glass Containers (Appendix 11.1). The containers do not have scratches, cracks, bubbles, or other faults which make them difficult to be used. 

1.2 Tests of Extractive Substances

          Extracting medium Unless otherwise directed in a specific test below, use Purified Water as the extracting medium, maintained at a temperature of 70º during the extraction of the prepared sample.

Apparatus

          – WATER-BATH

          – EXTRACTION CONTAINERS Use only containers, such as ampoules or screw-capped culture test-tubes, of Type I glass. If used, culture test tubes are closed with screw caps having suitable elastomeric liners. The exposed surface of the elastomeric liner is completely protected with an inert solid disc 0.05 mm to 0.075 mm in thickness. A suitable disc may be fabricated from a polytef resin.

          Preparation of apparatus Cleanse all glassware thoroughly with chromic acid cleansing mixture, or if necessary with hot nitric acid, followed by prolonged rinsing with water. Clean cutting utensils by an appropriate method (e.g., successive cleaning with acetone and dichloromethane) prior to use in subdividing a sample. Clean all other equipment by thorough scrubbing with a suitable detergent and prolonged rinsing with water.

          Preparation of sample From a homogeneous sample, use a portion, for each 20.0 ml of extracting medium, equivalent to 120 cm2 in total surface area (both sides combined), and subdivide into strips approximately 3 mm in width and as near to 5 cm in length as is practical. Transfer the subdivided sample to a glass-stoppered, 250-ml graduated cylinder of Type I glass, and add about 150 ml of water. Agitate for about 30 seconds, drain off and discard the liquid, and repeat with a second washing.

          Transfer the prepared sample to a suitable extraction flask, and add the required amount of extracting medium. Extract by heating in a water-bath at the temperature specified for the extracting medium for 24 hours. Cool, but not below 20º, and use this solution as the test solution. Pipette 20 ml of test solution into a suitable container. Use this portion in the test for Buffering Capacity. Immediately decant the remaining extract into a suitably cleansed container, and seal.

          Blank Use Purified Water where a blank is specified in the following tests.

          1.2.1 Nonvolatile residue Transfer, in suitable portions, 50.0 ml of the test solution to a suitable, tared crucible (preferably a fused-silica crucible that has been acid-cleaned), and evaporate the volatile matter on a steam bath. Similarly evaporate 50.0 ml of the blank in a second crucible. (Note If an oily residue is expected, inspect the crucible repeatedly during the evaporation and drying period, and reduce the amount of heat if the oil tends to creep along the walls of the crucible.) Dry at 105º for 1 hour: the difference between the amounts obtained from the sample and the blank does not exceed 15 mg.

          1.2.2 Sulfated ash (Note It is not necessary to perform this test when the nonvolatile residue test result does not exceed 5 mg.) Proceed with the nonvolatile residue obtained from the sample and from the blank, using, if necessary, additional sulfuric acid but adding the same amount of sulfuric acid to each crucible and perform the test according to “Sulfated Ash (Method II, Appendix 5.3): the difference between the amounts of residue on ignition obtained from the sample and the blank does not exceed 5 mg.

          1.2.3 Heavy metals Pipette 20 ml of the test solution, filtered if necessary, into one of two matched 50-ml comparison tubes. Adjust with 1 M acetic acid or 6 M ammonium hydroxide to a pH between 3.0 and 4.0, using short-range pH paper as an external indicator, dilute with water to about 35 ml, and mix. Into the second comparison tube pipette 2 ml of lead standard solution (1 ppm Pb), and add 20 ml of the blank. Adjust with 1 M acetic acid or 6 M ammonium hydroxide to a pH between 3.0 and 4.0, using short-range pH paper as an external indicator, dilute with water to about 35 ml, and mix. To each tube add 1.2 ml of thioacetamide reagent and 2 ml of acetate buffer pH 3.5, dilute with water to 50 ml, and mix: any brown colour produced within 10 minutes in the tube containing the extract of the prepared sample does not exceed that in the tube containing the Lead Standard Solution (1 ppm Pb), both tubes being viewed downward over a white surface (1 ppm in extract).

          1.2.4 Buffering capacity Titrate the previously collected 20-ml portion of the test solution potentiometrically to a pH of 7.0, using either 0.010 M hydrochloric acid or 0.010 M sodium hydroxide, as required. Treat a 20.0-ml portion of the blank similarly: if the same titrant was required for both sample and blank, the difference between the two volumes is not greater than 10.0 ml; and if acid was required for either the sample or the blank and alkali for the other, the total of the two volumes required is not greater than 10.0 ml.

1.3 Water Vapour Permeability

1.3.1 Containers for solid dosage forms

          PROCEDURE Prepare a quantity of granular anhydrous calcium chloride (nominal width of aperture of test sieves between 1.20 mm and 0.71 mm, and free from fine powder) by drying for 1 hour at 110º and cooling subsequently in a desiccator.

          Select a sample of 13 containers at random from the containers to be tested, fill 10 of the containers with the calcium chloride to within 13 mm of the closure, and close the containers tightly.

          Fill the remaining three containers with a sufficient amount of glass beads, or other suitable inert material, so that when the closure has been applied, the weights of the closed containers are approximately equal to the mean weight of the 10 filled test containers; two of these containers serve as controls and the third is used as a counterpoise. Weigh each of the test containers and the control containers separately, using the counterpoise container and appropriate weights, and immediately place the test and control containers in a chamber in which the air is controlled at a temperature of 30º±1º and a relative humidity of 80±3 per cent, keep the counterpoise container under ordinary laboratory conditions.

          After an interval of 7 days, measured to the nearest hour, remove the test and control containers from the chamber, and immediately reweigh each test and control container against the counterpoise container.

          From the gross increase in weight in each test container subtract the mean increase in weight of the two control containers, and from the net increases in weight, calculate the rate of passage of water vapour into each test container, in milligrams per 24 hours (mg/d).

          ASSESSMENT OF RESULTS The containers shall be deemed to pass the test if in no case the rate of passage of water vapour into the containers in the sample exceeds the appropriate value indicated in Table 1.

          If more than one container in the sample gives a value exceeding the appropriate figure, the containers shall be deemed to fail the test. If only one container in the sample gives a figure exceeding this value, then a further 10 containers shall be tested and if the appropriate figure is not exceeded with any of these further containers, the containers shall be deemed to pass the test.

Table 1 Water-Vapour Permeability of Solid Dosage Form Containers

          1.3.2 Containers for semisolid dosage forms

          PROCEDURE Select a sample of 10 containers at random from the containers to be tested and weigh each separately with its closure. Fill each container to approximately 90 per cent of its capacity with cream having the following formula: cetrimide 5 g; cetostearyl alcohol 50 g; Purified Water, freshly boiled and cooled 445 g; liquid paraffin 500 g. Apply the closure to each container and weigh the closed containers separately. Place them in a chamber in which the air is controlled at a temperature of 30º±1º and at a relative humidity of 65±3 per cent. After 14 days, reweigh each closed container.

          ASSESSMENT OF RESULTS The containers shall be deemed to pass the test if none of the contents of the closed containers shows a loss in weight during the test greater than 1.5 per cent of its weight.

          If more than one container in the sample gives a figure exceeding this value, the containers shall be deemed to fail the test. If only one container in the sample gives a figure exceeding the value, then a further 10 containers shall be tested and if this value is not exceeded with any of these further containers, the containers shall be deemed to pass the test.

          1.3.3 Containers for oral liquid dosage forms

          PROCEDURE Select a sample of 10 containers at random from the containers to be tested and weigh each separately with its closure. Fill each container with water equivalent to the labelled volume. Close the containers tightly and weigh each container separately. Immediately place the test containers in a chamber in which the air is controlled at a temperature of 30º±1º and at a relative humidity of 65±3 per cent. Allow to stand for 14 days and reweigh each test container. Calculate the net decrease in weight.

          ASSESSMENT OF THE RESULTS The loss in weight should not exceed 0.2 per cent of the contents in 14 days.

          If more than one container in the sample gives a figure exceeding this value, the containers shall be deemed to fail the test. If only one container in the sample gives a figure exceeding the value, then a further 10 containers shall be tested and if this value is not exceeded with any of these further containers, the containers shall be deemed to pass the test.

          1.3.4 Containers for parenteral preparations

          PROCEDURE Select a sample of 10 containers at random from the containers to be tested and weigh each separately with its closure. Fill each container with water equivalent to the labelled volume. Close the containers tightly and weigh each container separately. Heat the test containers for 30 minutes at 110º and place them in a chamber in which the air is controlled at a temperature of 30º±1º and at relative humidity of 65±3 per cent. Allow to stand for 14 days and reweigh each test container. Calculate the net decrease in weight.

          ASSESSMENT OF THE RESULTS The loss in weight should not exceed 0.2 per cent of the contents in 14 days.

           If more than one container in the sample gives a figure exceeding this value, the containers shall be deemed to fail the test. If only one container in the sample gives a figure exceeding the value, then a further 20 containers shall be tested and if this value is not exceeded with any of these further containers, the containers shall be deemed to pass the test.

          1.3.5 Containers for human blood and blood components

          PROCEDURE Select a sample of 10 containers at random from the containers to be tested and weigh each separately. Fill each container with the labelled amount of anticoagulant and a volume of saline TS equal to the volume of blood to be collected. Seal the containers as for issue, weigh separately, and place them in a chamber at a temperature of 10º in still air conditions at approximately zero humidity, using self-indicating silica gel as a desiccant. Allow to stand for 28 days and reweigh each test container. Calculate the net decrease in weight.

          ASSESSMENT OF THE RESULTS The loss from the specified combined volume should not exceed 5 per cent w/w of the contents in 28 days.

          If more than one container in the sample gives a figure exceeding this value, the containers shall be deemed to fail the test. If only one container in the sample gives a figure exceeding the value, then a further 20 containers shall be tested and if this value is not exceeded with any of these further containers, the containers shall be deemed to pass the test.

          Note

          1. Circulating the air in a closed chamber over a saturated solution of ammonium sulfate will give the required relative humidity of 80±3 per cent at 30º.

          2. Circulating the air in a closed chamber over a saturated solution of ammonium chloride and potassium nitrate, as by dissolving 365 g of each substance in 600 ml of water, will give the required relative humidity of 65±3 per cent at 30º.

2. Biological Tests

          Perform the in vitro biological tests according to the procedures set forth under In vitro biological reactivity tests. Materials that meet the requirements of the in vitro tests are not required to undergo further testing. No plastic class designation is assigned to these materials. Materials that do not meet the requirements of the in vitro tests are not suitable for containers for pharmaceutical products.

          The tests are designed for plastics in condition in which they are received. If, however, a plastic is to be exposed to any sterilization process prior to its use, then the tests are conducted on plastics preconditioned by the appropriate sterilization procedure.

          The in vivo biological reactivity tests are designed to determine the biological response of animals to elastomerics, plastics and other polymeric materials with direct or indirect patient contact, or by the injection of specific extracts prepared from the material under test. The Acute Systemic Toxicity Test and the Intracutaneous Reactivity Test are used for elastomeric materials, especially elastomeric closures for which the appropriate in vitro biological reactivity tests, have indicated significant biological reactivity. These two tests are used for plastics and other polymers in addition to a third test, the Acute Intramuscular Tissue Toxicity Test, to test the suitability of these materials intended for use in fabricating containers and accessories thereto, for use in parenteral preparations, and for use in medical device, implants and other systems.

          The in vivo biological reactivity tests do not apply to plastics that are intended for use as containers for oral or topical products, or that may be used as an integral part of a drug formulation.

          Extracting media

          PYROGEN-FREE WATER (Diluent A, Appendix 8)

PYROGEN-FREE SALINE TS

ETHANOL IN PYROGEN-FREE SALINE TS (1 in 20)

POLYETHYLENE GLYCOL 400

          VEGETABLE OIL Use freshly refined sesame oil or cottonseed oil. The oil meets the following additional requirements. Obtain, if possible, freshly refined oil. Use three properly prepared animals (see under Intracutaneous Reactivity Test) and inject the oil intracutaneously in a dose of 200 μl into each of 10 sites per animal, and observe the animals at 24, 48 and 72 hours following injection. No site shows a tissue reaction, such as edema and erythema, that is larger than 5 mm in overall diameter.

Apparatus The apparatus for the test includes the following.

          AUTOCLAVE Use an autoclave capable of maintaining a temperature of 121º±2º, equipped with a thermometer, a pressure 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, but not below, 20º immediately following the heating cycle.

OVEN Use an oven, preferably of a forced-circulation model, that will maintain operating temperatures of 50º±2º.

          EXTRACTION CONTAINERS Use containers, such as ampoules or screw-capped culture test-tubes, of borosilicate glass. Culture test-tubes used are closed with screw caps having suitable rubber liners. The exposed surface of the rubber liner is completely protected with an inert solid disc 50 to 75 μm in thickness. A suitable disc may be fabricated from a polytetrafluoroethylene resin.

Preparation of apparatus Clean all glassware thoroughly with chromic acid cleansing mixture, or if necessary with hot nitric acid, followed by prolonged rinsing with water. Clean cutting devices by an appropriate method (e.g., successive cleaning with acetone and dichloromethane) prior to use in subdividing a specimen. Clean all other equipment by thorough scrubbing with a suitable detergent and prolonged rinsing with water.

          Render containers and devices used for extraction, and in transfer and administration of test material, sterile and dry by a suitable process. If ethylene oxide is used as the sterilizing agent, allow adequate time for complete degassing.

Preparation of sample From a homogeneous plastic sample use a portion equivalent to 120 cm² when the thickness is 500 μm or less, or 60 cm² when the thickness is greater than 500 μm, and subdivide into strips of approximately 0.3 × 5 cm. Remove particulate matter, such as lint and free particles, by treating each subdivided sample as follows: transfer the subdivided sample to a clean, glass-stoppered 100-ml graduate cylinder of Type I glass and add about 70 ml of pyrogenfree water. 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 50º.

(Note Do not clean the plastic with a dry or wet cloth or by rinsing or washing with an organic solvent or surfactant.)

Extracts Place two properly prepared samples of the plastic to be tested in separate extraction flasks, and add to each flask 20 ml of the appropriate extracting medium (i.e., the fluid nearest in properties and composition to the products intended to be held). Repeat these directions for each extracting medium required for testing. Also prepare one 20-ml blank of each medium for parallel injections and comparisons. Extract by heating in an autoclave at 121º, or in an oven at 50º (Table 2). Allow adequate time for the liquid within the container to reach the extraction temperature.

(Note The extraction conditions should not in any instance cause physical changes such as fusion or melting of the plastic pieces which result in a decrease in the available surface area. A slight adherence of the pieces can be tolerated. Always add the cleaned pieces individually to the extracting medium. If culture tubes are used for autoclave extractions with sesame oil, seal their screw caps adequately with pressure-sensitive tape.)

          Cool all containers at the end of the extraction period to about, but not below, 20º. Agitate vigorously for several minutes, transfer each extract immediately to a dry, sterile container, and test within the following 24 hours. No extract should be stored at any time at temperatures below 20º 

Table 2 Test Conditions

          2.1 Acute Systemic Toxicity Test

          Test animal Use healthy, not previously used, white mice weighing between 17 and 23 g. For each test group use only mice of the same source. Allow water and food ad libitum.

          Procedure Inject each extract of the sample, and the corresponding blank, into groups of five mice each in the amount and by the route set forth in Table 3, except to dilute the extracts of sample prepared with polyethylene glycol 400, and the corresponding blank with 4.6 volumes of a pyrogen-free saline TS to obtain a solution having a concentration of 200 mg of polyethylene glycol per ml. The extract prepared with pyrogen-free water should be rendered isotonic prior to injection by the addition of 900 mg of pyrogen-free sodium chloride for each 100 ml. 

Table 3 Test to Be Conducted

          Evaluation Observe the animals immediately after injection, and at least at 24, 48 and 72 hours. If during the observation period all five of the animals treated with the extract of the sample show no significantly greater reaction than the animals treated with the blank, the sample meets the requirements of this test.

          If any animals treated with the sample show only slight signs of toxicity and not more than one animal shows gross symptoms of toxicity or death, repeat the test using groups of 10 mice each. On the repeated test, the requirements of the test are met if none of the animals treated with the sample shows a significantly greater reaction than that observed in the animals treated with the blank.

          2.2 Intracutaneous Reactivity Test

          Test animal Select healthy, thin-skinned albino rabbits, not previously used for any test, whose fur can be clipped closely and whose skin is free from mechanical irritation or trauma. In handling the animals during observation periods, avoid touching the injection sites.

          Procedure On the day of the test, closely clip the fur on the animal’s back on both sides of the spinal column over a sufficiently large test area. Avoid mechanical irritation and trauma. Remove loose hair by means of vacuum. If necessary, swab the skin lightly with ethanol (70 per cent), and dry the skin prior to injection.

          For each sample use two rabbits. Inject intracutaneously into each of five sites on one side of each animal 200 μl of the sample. Similarly, inject into each of five sites on the other side 200 μl of the corresponding blank (Table 3). The extract prepared with polyethylene glycol 400 and the corresponding blank should be diluted prior to injection with 8.3 volumes of pyrogen-free saline TS to obtain a concentration of 120 mg of polyethylene glycol per ml.

          Evaluation Examine the injection sites for evidence of any tissue reaction such as erythema, edema and necrosis. Swab the skin lightly, if necessary, with ethanol(70 per cent)   to facilitate reading of the injection sites. Observe animals at 24, 48 and 72 hours after the injection. Clip the fur as necessary during the observation period. Rate the observations on a numerical scale for the extract of the sample and for the blank, respectively, using Table 4.

          If each animal at any observation period shows an average reaction to the sample which is not significantly greater than that to the blank, the sample meets the requirements of the test.

          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. On the repeated test, the average reaction to the sample in any of the three animals should not be significantly greater than that to the blank.

Table 4 Evaluation of Skin Reactions

2.3 Acute Intramuscular Tissue Toxicity Test (Implantation Test)

          The acute intramuscular tissue toxicity test is designed for the evaluation of a plastic material in direct contact with living tissue. Of importance are the proper preparation of the implant strips and their proper implantation under aseptic conditions. Prepare for implantation eight strips of the sample and four strips of Negative Control Plastic. (Note A Negative Control Plastic is a plastic sample that gives no reaction under the conditions of the test.) Each strip should be not less than 10 × 1 mm. The edges of the strips should be as smooth as possible to avoid additional mechanical trauma upon implantation. Strips of the specified minimum size are implanted by means of a hypodermic needle such as a 15-gauge needle with intravenous point and of 19-mm cannula length, and a sterile trocar. Use either pre-sterilized needles into which the sterile plastic strips are aseptically inserted, or insert each clean strip into a needle, the cannula and hub of which are protected with an appropriate cover, and then subjected to the appropriate sterilization procedure. Allow for proper degassing if agents such as ethylene oxide are used.

          Test animal Select healthy, adult rabbits weighing not less than 2.5 kg, and whose paravertebral muscles are sufficiently large in size to allow for implantation of the test strips. Do not use any muscular tissue other than the paravertebral site. The animal may be anesthetized with a commonly used anesthetic agent to a degree deep enough to prevent muscular movements, such as twitching.

          Procedure Perform the test in a clean area. On the day of the test or up to 20 hours before testing, clip the fur of the animals on both sides of the spinal column. Remove loose hair by means of vacuum.

          Implant four strips of the sample into the paravertebral muscle on one side of the spine of each of two rabbits, 2.5 to 5 cm from the midline and parallel to the spinal column, and about 2.5 cm apart from each other. In a similar fashion implant two strips of Negative Control Plastic in the opposite muscle of each animal. Insert a sterile stylet into the needle to hold the plastic strip in the tissue while withdrawing the needle. If excessive bleeding is observed after implantation of a strip, place a duplicate strip at another site. Close the incision after implantation is complete.

          Keep the animals for a period of not less than 72 hours, and sacrifice them at the end of the observation period by administering an overdose of an anesthetic agent. Allow sufficient time to elapse for the tissue to be cut without bleeding. Examine macroscopically the area of the tissue surrounding the centre portion of each implant strip. Use a magnifying lens if necessary.

          Evaluation The tissue immediately surrounding the Negative Control Plastic strips appears normal and entirely free from hemorrhage, film or encapsulation. The requirements of the test are met if, in each rabbit, the reaction to not more than one of the four sample strips is significantly greater than that to the strips of Negative Control Plastic.

2.4 Pyrogen Test

Perform the test according to the “Pyrogen Test” (Appendix 8.2) as specified in Table 3.

2.5 Hemolysis Test

Preparation of sample Proceed as described in the general method of Preparation of sample but using homogeneous plastic material 200 cm² in total surface area as a sample.

Extracts Proceed as described in the general procedure for Extracts, using 50 ml of pyrogen-free water as the extracting medium.

Procedure Dilute sodium phosphate-sodium chloride buffer TS as follows:

          Dilute further the intermediate solution a₀ and b₀ as follows:

          To each of three centrifuge tubes add 1.40 ml of the extract of the sample, and to a fourth centrifuge tube add 1.40 ml of the corresponding blank. To the first tube add 1.0 ml of a₀, to the second and the fourth tubes add 0.10 ml of b₀, and to the third tube add 0.01 ml of c₀.

          (Note The osmotic effects of the solutions correspond to those of sodium chloride solutions having a concentration of 0.5 per cent w/v, 0.4 per cent w/v, and 0.1 per cent w/v for the first tube, the second and the fourth tubes, and the third tube, respectively.) To each tube add 0.02 ml of freshly prepared, well mixed, heparinized human blood TS and place the tubes in a waterbath at 30º±1º for exactly 40 minutes. Immediately to the first tube add 1.50 ml of a₁, to the second and the fourth tubes add 1.50 ml of b₁, and to the third tube add 1.50 ml of c₁. Centrifuge the tubes for 5 minutes, preferably in a horizontal centrifuge.

          Measure the absorbance at the maximum at about 540 nm, using sodium phosphate-sodium chloride buffer TS as a blank (Appendix 2.2).

          Calculate the degree of hemolysis (h_n) from the formula:

where A₁₀₀ is the absorbance of the solution in the third tube,

n is the tube number, and

A_n is the absorbance of the solutions in the first, the second and the fourth tubes, respectively.

          Evaluation The degree of hemolysis (h1) in the first tube should not exceed 10, and the difference between the degrees of hemolysis in the second and the fourth tubes should not exceed 10.

          The degree of hemolysis (h₄) for the fourth tube should be between 60 and 75.

          2.6 In Vitro Biological Reactivity Tests

          The following tests are designed to determine the biological reactivity of mammalian cell cultures following contact with the elastomeric plastics and otherpolymeric 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 sample 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 micro-organisms 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 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.

          Reference substances High-Density Polyethylene RS and Positive Bioreaction RS.

          Cell culture preparation Prepare multiple cultures

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¹ Further details are given in the following publications of the American Society of 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. 

of L-929 (ATCC cell line CCL 1, NCTC clone 929) mammalian fibroblast cells in serum-supplemented minimum essential medium having a seeding density of about 10⁵ cells per ml. Incubate the cultures at 37º±1º in a humidified incubator for not less than 24 hours in a 5±1 per cent carbon dioxide atmosphere until a monolayer, with greater than 80 per cent 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.)

          Extraction solvents Sodium Chloride Injection (use Sodium Chloride Injection containing 0.9 per cent 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 37º for 24 hours.

Apparatus

          AUTOCLAVE Use an autoclave capable of maintaining a temperature of 121º±2º, equipped with a thermometer, a pressure 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 20º, but not below 20º, immediately following the heating cycle.

OVEN Use an oven, preferably of a mechanical convection model, that will maintain operation temperatures in the range of 50º to 70º within ±2º.

INCUBATOR Use an incubator capable of maintaining a temperature of 37º±1º and a humidified atmosphere of 5±1 per cent carbon dioxide in air.

          EXTRACTION CONTAINERS Use only containers, such as ampoules or screw-capped 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 disc 50 to 75 μm in thickness. A suitable disc 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 containers and devices used for extraction, transfer, or administration of test material. If ethylene oxide is used as the sterilizing agent, allow not less than 48 hours for complete degassing.

Procedure

PREPARATION OF SAMPLE FOR EXTRACTS Prepare as directed in Preparation of sample.

PREPATATION OF EXTRACTS Prepare as directed in Extracts using either Sodium Chloride Injection (0.9 per cent NaCl) or serum-free mammalian cell culture media as an extracting medium.

          (Note If extraction is done at 37º for 24 hours 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.)

2.6.1 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 samples. 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 not less than 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 suspensio prepared as directed under 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 not more than 2 per cent for agar.

          (Note The quality of the agar must be adequate to support cell growth. The agar layer must be thin enough to permit diffusion of leached chemicals.) Place the flat surfaces of sample preparation, negative control preparation, and positive 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 samples per prepared plate. Incubate all cultures for not less than 24 hours at 37º±1º, preferably in a humidified incubator containing 5±1 per cent carbon dioxide. Examine each culture around each-sample. Negative control, and positive control, under a microscope, using a suitable stain, if desired.

          Evaluation The biological reactivity (cellular degeneration and malformation) is described and rated on a scale of 0 to 4 (Table 5). Measure the responses of the cell cultures to the sample preparation, the negative control preparation, and the positive control preparation. The cell culture test system is suitable if the observed responses to the positive control preparation is grade 0 (no reactivity) and to the positive control preparation is at least grade 3 (moderate). The requirements of the test are met 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 5 Reactivity Grades for Agar Diffusion Test and Direct Contact Test

          2.6.2 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 sample 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.

          Sample preparation Use portions of the test sample having flat surfaces not less than 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 under 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 0.5 ml of fresh culture medium. Place a single sample preparation, negative control preparation, and a positive control preparation in each of duplicate cultures. Incubate all cultures for not less than 24 hours at 37º±1º in a humidified incubator containing 5±1 per cent carbon dioxide. Examine each culture around each sample, negative control, and positive control preparation, either visually or under a microscope, using a suitable stain, if desired.

          Evaluation Proceed as directed for Evaluation under Agar diffusion test. The requirements of the test are met 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.

          2.6.3 Elution test

          This test is designed for the evaluation of extracts of polymeric materials. The procedure allows for extraction of the sample at physiological or non-physiological 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 per cent NaCl) or serum-free mammalian cell culture media as extracting medium. If the size of the sample cannot be readily measured, a mass of not less than 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 hours in an incubator containing 5±1 per cent carbon dioxide. Maintain the extraction temperature at 37º±1º 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 under 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, negative control preparation, or positive control preparation. The serum-supplemented and serum-free cell culture media extracts are tested in duplicate without dilution (100 per cent). The Sodium Chloride Injection extract is diluted with serum-supplemented cell culture medium and tested in duplicate at 25 per cent extract concentration. Incubate all cultures for 48 hours at 37º±1º in a humidified incubator preferably containing 5±1 per cent carbon dioxide. Examine each culture at 48 hours, under a microscope, using a suitable stain, if desired.

           Evaluation Proceed as directed for Evaluation under Agar diffusion test but using Table 6. The requirements of the test are met 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 denaturation of the sample extract.

Table 6 Reactivity Grades for Elution Test