USP87细胞毒性体外试验

87 BIOLOGICAL REACTIVITY TESTS, IN VITROThe following tests are designed to determine the biological reactivity of mammalia n cell cultures followi ng con tact with the elastomeric plastics and other polymeric materials with direct or in direct patie nt con tact 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 specime n cannot be determ in ed, 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 preve nt con tam in ati on with microorga nisms and other foreign matter. Three tests are described (i.e., the Agar Diffusi on Test , the Direct Con tact 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 inten ded use. Other factors that may also affect the suitability of sample for a specific use are the polymeric composition; processing and cleaning procedures; con tact ing media; in ks; adhesives; absorptio n, adsorptio n, and permeability of preservatives; and con diti ons of storage. Evaluatio n of such factors should be made by appropriate additi onal specific tests before determining that a product made from a specific material is suitable for its in ten ded use. Materials that fail the in vitro tests are can didates for the in vivotests described in Biological Reactivity Tests, In Vivo 88 .USP R EFERENCE S TANDARDS 11 —USP High-Density Polyethylene RS.USP Positive Bioreaction RS.Cell Culture Preparation —Prepare multiple cultures of L-929 (ATCC cell lineCCL 1, NCTC clone 929; alternative cell lines obtained from a standard repository may be used with suitable validation) mammalian fibroblast cells inserum-suppleme nted minimum esse ntial medium hav ing a seedi ng den sity of about 10 5 cells per mL. Incubate the cultures at 37 1 一in a h±midifiedin cubator for NLT 24 h in a 5 1% c±b on dioxide atmosphere un til amono layer, with greater tha n 80% con flue nee, is obta in ed. Exam ine the prepared cultures un der a microscope to en sure uniform, n ear-c on flue ntmono layers. [NOTE ——The reproducibility of the In Vitro Biological Reactivity Tests depends upon obtaininguniform cell culture density. ]Extractio n Solvents —Sodium Chloride Injectio n (see mono graph —useSodium Chloride Injection containing 0.9% of NaCl). Alternatively, serum-free mammalia n cell culture media or serum-suppleme nted mammalia n cell culture media may be used. Serum suppleme ntati on is used whe n extracti on 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 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 about20 -, but not below 20 -, immediately following the heating cycle.Oven —Use an ove n, preferably a mecha ni cal con vect ion model, that will maintain operating temperatures in the range of 50 -刁0 within ±.In cubator —Use an in cubator capable of maintaining a temperature of 37 1土and a humidified atmosphere of 5 1% carbon dioxide in air.Extracti on Containers —Use only contain ers, such as ampuls or screw-cap culture test tubes, or their equivale nt, of Type I glass. If used, culture test tubes, or their equivale nt, are closed with a screw cap hav ing a suitable elastomeric liner. The exposed surface of the elastomeric liner is completely protected with an inert solid disk 50 —5 m in thickness. A suitable disk can be fabricated from polytef.Preparati on of Apparatus ——Clea nse all glassware thoroughly with chromic acid clea nsing mixture an d, if n ecessary, with hot n itric acid followed by proIon ged rinsing with Sterile Water for Injectio n. Sterilize and dry by a suitable process for containers and devices used for extracti on, tran sfer, or adm ini strati on of test material. If ethyle ne oxide is used as the steriliz ing age nt, allow NLT 48 h for complete degass ing.Procedure —Preparati on of Sample for Extracts —Prepare as directed in the Procedure under Biological Reactivity Tests, In Vivo 88 .Preparati on of Extracts ——Prepare as directed for Preparati on of Extracts inBiological Reactivity Tests, In Vivo 88 using either Sodium Chloride Injection (0.9% NaCl) or serum-free mammalia n cell culture media asExtraction Solvents. [NOTE —If extraction is done at 37 U 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 Diffusi on TestThis test is desig ned for elastomeric closures in a variety of shapes. The agar layer acts as a cushion to protect the cells from mechanical damage while allow ing the diffusi on of leachable chemicals from the polymeric specime ns. Extracts of materials that are to be tested are applied to a piece of filter paper. Sample Preparati on —Use extracts prepared as directed, or use porti ons of the test specime ns hav ing flat surfaces NLT 100 mm in surface area. Positive Control Preparation —Proceed as directed for Sample Preparation . Negative Con trol Preparatio n ——Proceed as directed for Sample Preparati on Procedure —Using 7 mL of cell suspe nsion prepared as directed un der CellCulture Preparati on , prepare the mono layers in plates hav ing a 60-mm diameter. Following incubation, aspirate the culture medium from the mono layers, and replace it withserum-suppleme nted culture medium con tai ning NMT 2% of 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 ofSample Preparati on, Negative Con trol Preparati on , and Positive Con trolPreparation or their extracts in an appropriate extracting medium, in duplicate cultures in con tact with the solidified agar surface. Use no more tha n threespecimens per prepared plate. Incubate all cultures for NLT 24 h at 37 1- ,±preferably in a humidified in cubator containing 5 1% of carbb n dioxide.Examine each culture around each Sample, Negative Control , and PositiveControl, under a microscope, using a suitable stain, if desired.In terpretati on of Results ——The biological reactivity (cellular dege nerati on and malformati on) is described and rated on a scale of 0 — (see Table 1). Measurethe responses of the cell cultures to the Sample Preparation , the NegativeControl Preparation , and the Positive Control Preparation . The cell culture test system is suitable if the observed resp on ses to the Negative Con trolPreparation is grade 0 (no reactivity) and to the Positive Control Preparation isat least grade 3 (moderate). The Sample meets the requireme nts 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 con firmed.Table 1. Reactivity Grades for Agar Diffusio n Test and Direct Con tact TestGrade Reactivity Descripti on of Reactivity Zone0 None No detectable zone around or un der specime nSome malformed or dege nerated cells un der1 Slight specime nZone limited to area un der specime n and lesstha n2 Mild 0.45 cm bey ond specime n3 Moderate Zone exte nds 0.45 to 1.0 cm bey ond specime n4 Severe Zone exte nds greater tha n 1.0 cm bey ondspecime nDirect Con tact TestThis test is desig ned for materials in a variety of shapes. The procedure allows for simulta neous extract ion and testi ng of leachable chemicals from the specime n with a serum-suppleme nted medium. The procedure is not appropriate for very low- or high-de nsity materials that could cause mecha ni cal damage to the cells.Sample Preparati on —Use porti ons of the test specime n hav ing flat surfaces2NLT 100 mm in surface area.Positive Control Preparation —Proceed as directed for Sample Preparation . Negative Con trol Preparatio n ——Proceed as directed for Sample Preparati on Procedure —Using 2 mL of cell suspe nsion prepared as directed un der Cell Culture Preparati on , prepare the mono layers in plates hav ing a 35-mm diameter. Following incubation, aspirate the culture medium from the cultures, and replace it with 0.8 mL of fresh culture medium. Place a single Sample Preparation , a Negative Control Preparation , and a Positive Control Preparation in each of duplicate cultures. Incubate all cultures for NLT 24 h at 37 ±1〕in a humidified in cubator containing 5 1% of carb on dioxide.Examine each culture around each Sample, Negative Control , and Positive Control Preparation , under a microscope, using a suitable stain, if desired.In terpretati on of Results —Proceed as directed for In terpretati on of Results under 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 procedure if the suitability of the system is not con firmed.Eluti on TestThis test is designed for the evaluation of extracts of polymeric materials. The procedure allows for extract ion of the specime ns at physiological or。