主题:【分享】USP<87>体外生物反应测试中英文对照

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第一章87 BIOLOGICALREACTIVITY TESTS, IN VITRO

The followingtests are designed to determine the biological reactivity of mammalian cellcultures following contact with the elastomeric plastics and other polymericmaterials with direct or indirect patient contact or of specific extractsprepared from the materials under test. It is essential that the tests be performedon the specified surface area. When the surface area of the specimen cannot bedetermined, use 0.1 g of elastomer or 0.2 g of plastic or other material forevery mL of extraction fluid. Exercise care in the preparation of the materialsto prevent contamination with microorganisms and other foreign matter.

<87>生物反应测试,体外



以下试验旨在确定哺乳动物细胞培养物在与弹性体塑料和其他聚合物材料接触(直接或间接与患者接触)或从被测材料制备的特定提取物接触后的生物反应性。测试必须按规定的表面积进行。当试样的表面积不能确定时,每mL萃取液中使用0.1g弹性体、0.2g塑料或其他材料。在材料的准备过程中要小心,防止微生物和其他外来物质的污染。

Three tests aredescribed (i.e., the Agar Diffusion Test, the Direct Contact Test, and theElution Test). The decision as to which type of test or the number of tests tobe performed to assess the potential biological response of a specific sampleor 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 specificuse are the polymeric composition; processing and cleaning procedures;contacting media; inks; adhesives; absorption, adsorption, and permeability ofpreservatives; and conditions of storage. Evaluation of such factors should bemade by appropriate additional specific tests before determining that a productmade from a specific material is suitable for its intended use. Materials thatfail the in vitro tests are candidates for the in vivo tests described in BiologicalReactivity Tests, In Vivo 88.

描述了三种测试(即:琼脂扩散试验,直接接触试验,洗脱试验),可作为评估特定样品或提取物的潜在生物反应,而测试类型、测试数量取决于材料、最终产品及其预期用途。另外可能影响样品对特定用途适应性的其他因素是聚合物组合物,加工和清洗程序,接触介质、油墨、粘合剂、防腐剂的吸收、吸附和渗透性,以及储存条件。在确定由特定材料制成的产品适合其预期用途之前,应通过适当的附加特殊试验对这些因素进行评估。未通过体外试验的材料可选择按Biological Reactivity Tests, In Vivo 88中所述进行体内试验。

PROCEDURES

TEST CONTROL

Positivecontrol: Polyurethane film containing zinc diethyldithiocarbamate (ZDEC) orzinc dibutyldithiocarbamate (ZDBC)

Cell culturepreparation: Prepare multiple cultures of L-929 (ATCC cell line CCL 1, NCTCclone 929; alternative cell lines obtained from a standard repository may beused with suitable validation) mammalian fibroblast cells in serum-supplementedminimum essential medium having a seeding density of about 10 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 toensure uniform, near-confluent monolayers. [NOTE—The reproducibility of the invitro biological reactivity tests depends upon obtaining uniform cell culturedensity.]

Extractionsolvents: Sodium Chloride Injection [see monograph—use Sodium ChlorideInjection containing 0.9% of sodium chloride (NaCl)]. Alternatively,serum-freemammalian cell culture media or serum-supplemented mammalian cell culture mediamay be used. Serum supplementation is used when extraction is done at 37° for24 h.

程序

测试控制

正控


含二乙基二硫代氨基甲酸锌(ZDEC)或二丁基二硫代氨基甲酸锌(ZDBC)的聚氨酯膜。

细胞培养制备

制备L-929 (ATCC细胞系CCL 1, NCTC克隆929;替代细胞系获得从一个标准库可以使用合适的验证)哺乳动物成纤维细胞serum-supplemented最低必要的媒介有播种密度每毫升约10细胞。孵化文化在37±1℃湿润孵化器NLT 24 h 5±1%二氧化碳气氛中直到单层,融合,大于80%。在显微镜下检查准备好的培养物,以确保均匀,接近融合的单层。[:体外生物反应性试验的可重复性取决于获得均匀的细胞培养密度。]

萃取溶剂

氯化钠注射液[见专著-使用含0.9%氯化钠(NaCl)的氯化钠注射液]。或者,也可以使用无血清哺乳动物细胞培养基或添加血清的哺乳动物细胞培养基。在37℃提取24小时时使用补充血清

PROCEDURE

Preparation ofsample for extracts: Prepare as directed in the Procedure in 88.

Preparation ofextracts: Prepare as directed for Preparation of extracts in 88 using either Sodium Chloride Injection[0.9% sodium chloride (NaCl)] or serumfree mammalian cell culture media asExtraction solvents. [NOTE—If extraction is done at 37° for 24 h in anincubator, use cell culture media supplemented by serum. The extractionconditions should not in any instance cause physical changes, such as fusion ormelting of the material pieces, other than a slight adherence.]

过程

提取样品的准备:按照<88>中的步骤进行制备。

提取物的准备: 按照<88>中的提取步骤进行制备。使用氯化钠注射液[0.9%氯化钠(NaCl)]或无血清哺乳动物细胞培养基作为提取溶剂。[-如果在培养箱中37℃提取24小时,使用含血清的细胞培养基。萃取条件在任何情况下都不应引起物理变化,如材料块除轻微粘连的熔化或熔化。

AGAR DIFFUSION TEST

This test isdesigned for elastomeric closures in a variety of shapes. The agar layer actsas a cushion to protect the cells from mechanical damage while allowing the diffusionof leachable chemicals from the polymeric specimens. Extracts of materials thatare to be tested are applied to a piece of filter paper.

Interpretationof results: The biological reactivity (cellular degeneration and malformation)is described and rated on a scale of 0–4 (see Table 1). Measure the responsesof the cell cultures to the Sample preparation, the Positive controlpreparation, and the Negative control preparation. The cell culture test systemis suitable if the observed responses to the Negative control preparation isgrade 0 (no reactivity) and to the Positive control preparation is at least grade3 (moderate). The sample meets the requirements of the test if the response tothe Sample preparation is not greater than grade 2 (mildly reactive).Repeat theprocedure if the suitability of the system is not confirmed.

琼脂扩散试验

该测试是为各种形状的弹性体密封件设计的。琼脂层起到缓冲作用,保护细胞免受机械损伤,同时允许可从聚合标本中分离的化学物质扩散。将待测材料的提取物涂在一张滤纸上。

结果解释

生物反应性(细胞变性和畸形)0-4的范围内进行描述和评分(见表1)。测量细胞培养物对样品制备、阳性对照制备和阴性对照制备的反应。如果阴性对照制剂的反应为0(无反应性),阳性对照制剂的反应至少为3(中度),则该细胞培养试验系统适用。如果对样品制备的反应不大于2(轻度反应),则该样品满足测试的要求。如果系统的适用性没有得到确认,则重复该程序。

DIRECT CONTACT TEST

This test isdesigned for materials in a variety of shapes. The procedure allows forsimultaneous extraction and testing of leachable chemicals from the specimenwith a serum-supplemented medium. The procedure is not appropriate for verylow- or high-density materials that could cause mechanical damage to the cells.

直接接触试验

这个测试是为各种形状的材料设计的。该程序允许用补充血清的培养基同时从标本中提取和测试可浸出的化学物质。这种方法不适用于会对细胞造成机械损伤的非常低或高密度的材料。

ELUTION TEST

This test isdesigned for the evaluation of extracts of polymeric materials. The procedureallows for extraction of the specimens at physiological or nonphysiologicaltemperatures for varying time intervals. It is appropriate for high-densitymaterials and for dose-response evaluations.

Samplepreparation: Prepare as directed in Preparation of extracts, using eitherSodium Chloride Injection [0.9% sodium chloride (NaCl)] or serum-free mammaliancell culture media as Extraction solvents. If the size of the sample cannot bereadily measured, a mass of NLT 0.1 g of elastomeric material or 0.2 g ofplastic or polymeric material per mL of extraction medium may be used.Alternatively, use serum-supplemented mammalian cell culture media as theextracting medium to simulate more closely physiological conditions. Preparethe extracts by heating for 24 h in an incubator containing 5 ± 1% of carbondioxide. Maintain the extraction temperature at 37 ± 1°, because highertemperatures may cause denaturation of serum proteins.

Samplepreparation: Prepare as directed in Preparation of extracts, using eitherSodium Chloride Injection [0.9% sodium chloride (NaCl)] or serum-free mammaliancell culture media as Extraction solvents. If the size of the sample cannot bereadily measured, a mass of NLT 0.1 g of elastomeric material or 0.2 g ofplastic or polymeric material per mL of extraction medium may be used.Alternatively, use serum-supplemented mammalian cell culture media as theextracting medium to simulate more closely physiological conditions. Preparethe extracts by heating for 24 h in an incubator containing 5 ± 1% of carbondioxide. Maintain the extraction temperature at 37 ± 1°, because highertemperatures may cause denaturation of serum proteins.

Positive controlpreparation: Proceed as directed for Sample preparation.

Negative controlpreparation: Proceed as directed for Sample preparation.

Procedure: Using2 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 extractsof the Sample preparation, Positive control preparation,or Negative controlpreparation. The serum-supplemented and serum-free cell culture media extractsare tested in duplicate without dilution (100%). The Sodium Chloride Injectionextract is diluted with serum-supplemented cell culture medium and tested induplicate 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, ifdesired.

Interpretationof results: Proceed as directed for Interpretation of results in Agar DiffusionTest but use Table 2. The sample meets the requirements of the test if theresponse to the Sample preparation is not greater than grade 2 (mildlyreactive). Repeat the procedure if the suitability of the system is not confirmed.For dose-response evaluations, repeat the procedure, using quantitativedilutions of the sample extract.

洗脱测试

本试验旨在评价高分子材料的提取物。该程序允许在生理或非生理温度下、在不同的时间间隔内提取标本。它适用于高密度材料和剂量反应评估。

样品制备

按照提取液制备的指导进行制备,使用0.9%氯化钠注射液或无血清哺乳动物细胞培养基作为提取溶剂。如果样品的面积不易测量,则可以使用每毫升萃取介质中不少于0.1g弹性材料或0.2g塑料或其他高分子材料。或者,使用补充血清的哺乳动物细胞培养基作为提取培养基,以模拟更接近的生理条件。在含有5±1%二氧化碳的培养箱中加热24小时制备提取物。保持萃取温度在37±1℃,因为更高的温度可能导致血清蛋白变性。

阳性对照制备:按照样品制备的指导进行。

阴性对照制备:按照样品制备的指导进行。

操作步骤

用细胞培养制剂中指示配制的2mL细胞悬液,在直径35mm的平板上制备单层膜。孵育后,从单分子膜中吸取培养基,用样品制备液、阳性对照制备液或阴性对照制备液替代。补充血清和无血清细胞培养基抽提物重复,不稀释(100%)。氯化钠注射液用血清补充细胞培养基稀释,以25%的提取物浓度重复试验。将所有培养物在37±1℃的湿化培养箱中孵育48小时,最好含有5±1%的二氧化碳。如果需要的话,在显微镜下用合适的染色剂检查每一个培养物48小时。

结果解释

生物反应性(细胞变性和畸形)0-4的范围内进行描述和评分(见表2)。测量细胞培养物对样品制备、阳性对照制备和阴性对照制备的反应。如果阴性对照制剂的反应为0(无反应性),阳性对照制剂的反应至少为3(中度),则该细胞培养试验系统适用。如果对样品制备的反应不大于2(轻度反应),则该样品满足测试的要求。如果系统的适用性没有得到确认,则重复该程序。对于剂量-反应评价,重复上述步骤,使用样品提取物的定量稀释。

等级

反应

症状描述

0

离散的胞浆内颗粒;没有细胞溶菌作用

1

轻微的

少于或等于20%的细胞呈圆形,松散附着,胞浆内无颗粒;偶有裂解细胞

2

温和的

大于20%至小于或等于50%的细胞为圆形且无胞浆内颗粒;无广泛的细胞裂解,细胞间无空洞

3

中等的

大于50%到小于70%的细胞层包含圆形细胞或裂解

4

严重的

细胞层几乎完全或完全破坏



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