〈88〉 BIOLOGICAL REACTIVITYTESTS, IN VIVO
Thefollowing tests are designed to determine the biological response of animals toelastomerics, plastics, and other polymeric material with direct or indirectpatient contact, or by the injection of specific extracts prepared from thematerial under test. It is essential to make available the specific surfacearea for extraction. 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. Also, it is essential to exercise care in thepreparation of the materials to be injected or instilled to preventcontamination with microorganisms and other foreign matter. Three tests aredescribed. The Systemic Injection Test and the Intracutaneous Test are used forelastomeric materials, especially to elastomeric closures for which theappropriate Biological Reactivity Tests, In Vitro 〈87〉 have indicatedsignificant biological reactivity. These two tests are used for plastics andother polymers, in addition to a third test, the Implantation Test, to test thesuitability of these materials intended for use in fabricating containers andaccessories thereto, for use in parenteral preparations, and for use in medicaldevices, implants, and other systems.
Thesethree tests are applied to materials or medical devices, if there is a need forclassification of plastics and other polymers based on in vivo biologicalreactivity testing.
Forthe purpose of this chapter, these definitions apply: the Sample is thespecimen under test or an extract prepared from such a specimen. A Blankconsists of the same quantity of the same extracting medium that is used forthe extraction of the specimen under test, treated in the same manner as theextracting medium containing the specimen under test. A Negative Control is aspecimen that gives no reaction under the conditions of the test.
以下测试旨在确定动物对与患者直接或间接接触的弹性体、塑料和其他高分子材料的生物反应，或注射被测材料制备的特定提取物。使萃取的比表面积可用是很重要的。当试样的表面积不能确定时，每mL萃取液使用0.1g弹性体或0.2g塑料或其他材料。此外，在准备注射或灌输的材料时要小心谨慎，以防止微生物和其他异物的污染。本文描述了三个测试。系统注入测试和皮内的测试用于弹性材料,尤其是弹性体密封件或经〈87〉体外生物反应测试具有显著的生物反应性的。除了第三项试验(植入试验)，这两项试验用于塑料和其他聚合物，以测试这些材料用于制造容器及其附件、用于肠外制剂以及用于医疗器械、植入物和其他系统的适用性。
如果需要根据体内生物反应性测试对塑料和其他聚合物进行分类，则这三种测试适用于材料或医疗设备。
就本章而言，这些定义适用于:样品是被测样品或从该样品制备的提取物。空白由用于提取待测试样的相同数量的提取介质组成，处理方式与加入被测试样的提取介质相同。阴性对照是指在试验条件下没有反应的样品。
CLASSIFICATIONOF PLASTICS
SixPlastic Classes are defined (see Table 1). This classification is based onresponses to a series of in vivo tests for which extracts, materials, androutes of administration are specified. These tests are directly related to theintended end-use of the plastic articles. The choice of extractants isrepresentative of the vehicles in preparations with which the plastics arelikely to be in contact. The Table 1 classification facilitates communicationamong suppliers, users, and manufacturers of plastics by summarizing the teststo be performed for containers for injections and medical devices if a need forclassification exists.
塑料的分类
塑料被定义为六类(见表1)。这种分类是基于对一系列体内试验的反应，这些试验的提取物、材料和给药途径都被指定。这些测试直接关系到塑料制品的预期最终用途。萃取剂的选择代表了制备过程中塑料可能与之接触的载体。表1的分类总结了注射容器和医疗器械在需要分类时需要进行的测试，从而促进了塑料供应商、用户和制造商之间的交流。
表1
| 塑料分级 | 测试项目 |
| 1 | 2 | 3 | 4 | 5 | 6 | 试验材料 | 动物 | 剂量 | 给药途径 |
| X | X | X | X | X | X | 氯化钠注射液浸提 | 小鼠 | 50ml/kg | A（Iv） |
| X | X | X | X | X | X | 家兔/豚鼠 | 0.2 mL/动物，每10或6个位点 | B（Ic） |
| X | X | X | X | X | 用1/20的乙醇/氯化钠注射液提取 | 小鼠 | 50ml/kg | A（Ip） |
| X | X | X | X | X | 家兔/豚鼠 | 0.2 mL/动物，每10或6个位点 | B（Ic） |
| X | X | X | 聚乙二醇400浸提 | 小鼠 | 10/kg | A（Ip） |
| X | X | 家兔/豚鼠 | 0.2 mL/动物，每10或6个位点 | B（Ic） |
| X | X | X | X | 植物油浸提 | 小鼠 | 10/kg | A（Ip） |
| X | X | X | 家兔/豚鼠 | 0.2 mL/动物，每10或6个位点 | B（Ic） |
| X | X | 样品条 | 家兔 | 4个点/动物 | C |
| X | X | 样品 | 大鼠 | 2个点/动物 | C |
注：每个类所需的测试在相应列中由“x”表示。
图例: A(Iv)-全身注射试验(静脉)；A(Ip)-全身注射试验(腹腔)；B(Ic)-皮内试验(皮内)；C--植入试验(肌肉或皮下植入)。
With the exception of the Implantation Test, theprocedures are based on the use of extracts that, depending on the heatresistance of the material, are prepared at one of three standard temperatures:50°, 70°, and 121°. Therefore, the class designation of a plastic must beaccompanied by an indication of the temperature of extraction (e.g., IV-121°,which represents a class IV plastic extracted at 121°, or I-50°, whichrepresents a class I plastic extracted at 50°).
Plastics may be classified as USP Plastic ClassesI–VI only on the basis of the response criteria prescribed in Table 1.
This classification does not apply to plastics thatare intended for use as containers for oral or topical products, or that may beused as an integral part of a drug formulation. Table 1 does not apply tonatural elastomers, which are to be tested in Sodium Chloride Injection andvegetable oils only.
The Systemic Injection Test and the IntracutaneousTest are designed to determine the systemic and local, respectively, biologicalresponses of animals to plastics and other polymers by the single-doseinjection of specific extracts prepared from a Sample. The Implantation Test isdesigned to evaluate the reaction of living tissue to the plastic and otherpolymers by the implantation of the Sample itself into animal tissue. Theproper preparation and placement of the specimens under aseptic conditions areimportant in the conduct of the Implantation Test.
These tests are designed for application to plasticsand other polymers in the condition in which they are used. If the material isto be exposed to any cleansing or sterilization process prior to its end-use,then the tests are to be conducted on a Sample prepared from a specimenpreconditioned by the same processing.
Factors such as material composition, processing andcleaning procedures, contacting media, inks, adhesives, absorption, adsorptionand permeability of preservatives, and conditions of storage may also affectthe suitability of a material for a specific use. Evaluation of such factorsshould be made by appropriate additional specific tests to determine thesuitability of a material for its intended use.
除植入测试外，该程序基于提取物的使用，根据材料的耐热性，在以下三种标准温度之一制备：50℃、70℃和121℃。因此，塑料的类别指定必须伴有萃取温度的指示(例如，IV-121℃，表示在121℃下提取的IV类塑料，或I-50℃，表示在50℃下提取的I类塑料)。
只有根据表1中规定的响应标准，塑料才可被归类为USP塑料I-VI类。
本分类不适用于作为口服或外用产品容器的塑料，或可能用作药物制剂的组成部分的塑料。上表不适用于只在氯化钠注射液和植物油中进行测试的天然弹性体。
系统注射试验和皮内试验旨在通过单剂量注射从样品中制备的特定提取物，分别确定动物对塑料和其他聚合物的系统和局部生物反应。植入试验是通过将样本本身植入动物组织来评估活体组织对塑料和其他聚合物的反应。在无菌条件下正确的准备和放置标本在植入试验中是很重要的。
这些测试是设计用于塑料和其他聚合物在使用条件下的应用。如果材料在最终使用前要经过清洗或灭菌过程，则需要使用经过同样处理的试样进行测试。
材料组成、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等因素也可能影响材料对特定用途的适宜性。对这些因素的评价应通过适当的附加特殊试验来确定材料对其预期用途的适宜性。
USP Reference Standards〈11〉— USP High-Density Polyethylene RS.
Extracting Media—
SODIUM CHLORIDE INJECTION (see monograph). UseSodium Chloride Injection containing 0.9% of NaCl.
1 IN 20 SOLUTION OF ALCOHOL IN SODIUM CHLORIDEINJECTION.
POLYETHYLENE GLYCOL 400 (see monograph).
VEGETABLE OIL— Use freshly refined Sesame Oil (seemonograph) or Cottonseed Oil (see monograph) or other suitable vegetable oils.
DRUG PRODUCT VEHICLE (where applicable).
WATER FOR INJECTION (see monograph).
[NOTE—The Sesame Oil orCottonseed Oil or other suitable vegetable oil meets the following additionalrequirements. Obtain, if possible, freshly refined oil. Use three properlyprepared animals, and inject the oil intracutaneously in a dose of 0.2 mL intoeach of 10 sites per animal, and observe the animals at 24, 48, and 72 hfollowing injection. Rate the observations at each site on the numerical scaleindicated in Table 2. For the 3 rabbits or guinea pigs (30 or 18 injection sites),at any observation time, the average response for erythema is not greater than0.5 and for edema is not greater than 1.0, and no site shows a tissue reactionlarger than 10 mm in overall diameter. The residue of oil at the injection siteshould not be misinterpreted as edema. Edematous tissue blanches when gentlepressure is applied.]
参考标准：USP〈11〉高密度聚乙烯RS。
提取溶剂
氯化钠注射液(见专著)：使用含0.9% NaCl的氯化钠注射液。
含1/20酒精的氯化钠注射液。
聚乙二醇400(见专著)。
植物油：使用新鲜精炼的芝麻油(见专题)或棉籽油(见专题)或其他合适的植物油。
药品载体(如适用)。
注射用水(见专著)。
[注-芝麻油或棉籽油或其他合适的植物油符合下列附加要求。如果可能的话，获取新鲜精炼的油。使用3只动物，每10个部位用0.2mL的油腔内注射，24、48、72h观察。按表2所示的数值比例尺对每个场址的观测结果进行评分。3只兔或豚鼠(30或18个注射部位)，在任何观察时间，红斑反应均不大于0.5，水肿反应均不大于1.0，所有部位的组织反应均不大于直径10mm。注射部位的油渣不应被误解为水肿。当轻轻按压时，水肿组织变白。]
表2
| 红斑 | 分值 |
| 无 | 0 |
| 非常轻微的红斑(几乎察觉不到) | 1 |
| 轻度红斑 | 2 |
| 中度-重度红斑 | 3 |
| 严重红斑(甜菜红)到轻微的焦痂形成(深度损伤) | 4 |
| 水肿形成 | 分值 |
| 无 | 0 |
| 非常轻微的水肿(几乎觉察不到) | 1 |
| 轻微水肿(边缘区域明显升高) | 2 |
| 中度水肿(约1mm) | 3 |
| 严重水肿(肿胀超过1毫米，并超过曝光) | 4 |
Apparatus—Theapparatus for the tests includes the following.
AUTOCLAVE—Usean autoclave capable of maintaining a temperature of 121 ± 2.0°, equipped witha thermometer, a pressure gauge, a vent cock, a rack adequate to accommodatethe test containers above the water level, and a water cooling system that willallow for cooling of the test containers to about, but not below, 20° immediatelyfollowing the heating cycle.
OVEN—Use anoven, preferably a forced-circulation model, that will maintain operatingtemperatures of 50° or 70° within ±2°.
EXTRACTIONCONTAINERS—Use only containers, such as ampuls or screw-cap culture test tubes,of Type I glass. If used, culture test tubes are closed with screw caps havingsuitable elastomeric liners. The exposed surface of the elastomeric liner iscompletely protected with an inert solid disk 0.05–0.075 mm in thickness.Asuitable disk may be fabricated from a polytef resin.
Preparationof Apparatus—Cleanse all glassware thoroughly with chromic acid cleansingmixture, or if necessary, with hot nitric acid, followed by prolonged rinsing withwater. Clean cutting utensils by an appropriate method (e.g., successivecleaning with acetone and methylene chloride) prior to use in subdividing a specimen.Clean all other equipment by thorough scrubbing with a suitable detergent andprolonged rinsing with water.
Rendercontainers and equipment used for extraction, and in transfer andadministration of test material, sterile and dry by a suitable process.[NOTE—If ethylene oxide is used as the sterilizing agent, allow adequate timefor complete degassing.]
设备
用于测试的设备包括以下设备。
高压灭菌器：使用一台能够保持温度121±2.0℃高压蒸汽灭菌锅，配备一个温度计，压力表，气阀，一架足以容纳测试容器于水位以上的架子；并配备水冷却系统，用于测试容器在加热后冷却至不低于20℃。
烘箱：使用烘箱，最好是强制循环的，将工作温度保持在50℃或70℃，温度波动在±2℃内。
提取容器：仅使用I型玻璃的容器，如安瓿或螺旋盖培养试管。如果使用，培养试管用具有适当弹性衬垫的螺旋帽封闭。弹性体衬垫的暴露表面完全由厚度0.05-0.075毫米的惰性隔垫保护。可以用聚四氟乙烯制作合适的隔垫。
设备准备：用铬酸洗液彻底清洗所有玻璃器皿，必要时用热硝酸清洗，然后长时间用大量水冲洗。用适当的方法清洗切割器具(例如，连续用丙酮和二氯甲烷清洗)，然后再用它来分割标本。用适当的洗涤剂彻底擦洗其他设备，并长时间用大量水冲洗。
将用于提取、转移和其他用于测试的容器和设备用适当的方法进行消毒和干燥。[注:如果使用环氧乙烷作为灭菌剂，要有足够的时间进行完全脱气。]
Procedure—
PREPARATIONOF SAMPLE—Both the Systemic Injection Test and the Intracutaneous Test may beperformed using the same extract, if desired, or separate extracts may be madefor each test. Select and subdivide into portions a Sample of the sizeindicated in Table 3. Remove particulate matter, such as lint and freeparticles,by treating each subdivided Sample or Negative Control as follows.Place the Sample into a clean, glass-stoppered, 100-mL graduated cylinder ofType I glass, and add about 70 mL of Water for Injection. Agitate for about 30s, and drain off the water. Repeat this step, and dry those pieces prepared forthe extraction with Vegetable Oil in an oven at a temperature not exceeding50°. [NOTE—Do not clean the Sample with a dry or wet cloth or by rinsing orwashing with an organic solvent, surfactant, etc.]
样品制备
如果需要，可使用相同的提取物进行全身注射试验和皮内试验，或为每次试验采用单独的提取物。根据表3所示的大小，选择样本并将其分成若干部分。通过以下方法处理每个细分样本或阴性对照，去除颗粒物，如棉絮和自由颗粒。将样品放入一个干净的装有玻璃塞子的100mlI型玻璃量筒中，加入大约70ml的水进行注射。搅拌大约30秒，然后沥干水分。重复以上步骤后，在50℃以下将准备用植物油提取的碎片放入烤箱中烘干。[注意:不要用干布、湿布或用有机溶剂、表面活性剂等漂洗或洗涤样品。]
| 材料的形状 | 厚度mm | 每20 mL萃取介质的样品量 | 细分 |
| 膜、薄片或管壁 | ＜0.5 | 120cm2（两面共计） | 约5×0.3 cm的条带 |
| 0.5-1 | 60cm2（两面共计） |
| 管子、管道 | ＜0.5（管壁） | 长度(厘米)= 120cm2 /(内径和外径周长之和) | 切片约5×0.3 cm |
| 0.5-1（管壁） | 长度(厘米)= 60cm2 /(内径和外径周长之和) |
| 板材、管材和模压制品 | ＞1mm | 相当于60cm2的总表面积(所有暴露表面的总和) | 约为5×0.3厘米 |
| 弹性体密封件 | ＞1mm | 相当于25cm2的总表面积(所有暴露表面的总和) | 不细分 |
注：当由于样品的结构不能确定表面积时，每1ml萃取液加入0.1g弹性体、0.2g塑料或其他聚合物；模制弹性体密封件测试完好无损。
PREPARATIONOF EXTRACTS—Place a properly prepared Sample to be tested in an extractioncontainer, and add 20 mL of the appropriate extracting medium. Repeat thesedirections for each extracting medium required for testing. Also, prepare one20-mL blank of each medium for parallel injections and comparisons. Extract byheating in an autoclave at 121° for 60 min, in an oven at 70° for 24 h, or at50° for 72 h. Allow adequate time for the liquid within the container to reachthe extraction temperature. [NOTE—The extraction conditions should not in anyinstance cause physical changes such as fusion or melting of the Sample pieces,which result in a decrease in the available surface area. A slight adherence ofthe pieces can be tolerated. Always add the cleaned pieces individually to theextracting medium. If culture tubes are used for autoclave extractions withVegetable Oil, seal screw caps adequately with pressure-sensitive tape.]
Cool to aboutroom temperature but not below 20°, shake vigorously for several minutes, anddecant each extract immediately, using aseptic precautions, into a dry, sterilevessel. Store the extracts at a temperature of 20°–30°, and do not use fortests after 24 h. Of importance are the contact of the extracting medium with theavailable surface area of the plastic and the time and temperature duringextraction, the proper cooling, agitation, and decanting process, and theaseptic handling and storage of the extracts following extraction.
提取液的制备
将准备好的待测样品放入提取容器中，加入20mL合适的提取介质。对测试所需的每一种提取介质重复上述操作。同时，每种培养基制备一个20ml空白，用于平行注射和对比。在121℃的高压蒸汽灭菌锅中加热60分钟，在烘箱中70℃加热24小时或50℃加热72小时。留出足够的时间使容器内的液体达到提取温度。[注:萃取条件不应在任何情况下引起物理变化，如试样的熔化或熔化，这将导致可用表面积的减少。碎片的轻微粘连是可以容忍的。一定要将清洗后的切片分别加入提取液中。如果使用培养管进行植物油的高压釜提取，应使用压敏胶带适当密封螺旋帽。
冷却到室温，但不要低于20℃，用力摇晃几分钟，然后使用无菌预防措施，立即将每种提取物倒入干燥、无菌的容器中。存储提取的温度20-30℃，24小时内测试。以下操作需要注意：提取介质与可用塑料的表面接触、提取过程中时间和温度适当的冷却和搅拌、减压过程、提取后的无菌处理和存储提取。
SYSTEMICINJECTION TEST
Thistest is designed to evaluate systemic responses to the extracts of materialsunder test following injection into mice. Alternate routes of injection may be usedwith justification.
TestAnimals—Use healthy, not previously used albino mice weighing 17–23 g. For eachtest group use only mice of the same source. Allow water and food, commonlyused for laboratory animals and of known composition, ad libitum.
Procedure—[NOTE—Agitateeach extract vigorously prior to withdrawal of injection doses to ensure evendistribution of the extracted matter.] Inject each of the five mice in a testgroup with the Sample or the Blank as outlined in Table 4, except to diluteeach g of the extract of the Sample prepared with Polyethylene Glycol 400, andthe corresponding Blank, with 4.1 volumes of Sodium Chloride Injection toobtain a solution having a concentration of about 200 mg of polyethylene glycolper mL.
Table4. Injection Procedure—Systemic Injection Test
系统注入测试
该试验旨在评估注射到小鼠体内后对所测材料提取物的系统反应。可以合理地使用不同的注射路径。
实验动物：使用健康的、未使用过的体重17-23克的白鼠。每个试验组只使用同一来源的小鼠。允许随意使用通常用于实验室动物已知成分的水和食物。
程序：[注:混匀模拟提取溶液，以确保提取物的均匀分布。]注入测试组中的每一个的五个老鼠与样品或空白如表4中列出,除了稀释每克样品的提取准备聚乙二醇400和相应的空白,4.1卷的氯化钠注射液获得解决方案有一个集中的每毫升200毫克的聚乙二醇。
表4：注射程序-系统注射试验
| 提取液/空白 | 使用量/kg | 测试方式 |
| 氯化钠注射液 | 50 mL | IV |
| 含1/20酒精的氯化钠注射液 | 50 mL | IV |
| 聚乙二醇400 | 10g | IP |
| 制剂溶液 | 50 mL | IV |
| 50 mL | IP |
| 植物油 | 50 mL | IP |
Observe theanimals immediately after injection, again 4 h after injection, and then atleast at 24, 48, and 72 h. If during the observation period none of the animalstreated with the extract of the Sample shows a significantly greater biologicalreactivity than the animals treated with the Blank, the Sample meets the requirementsof this test. If two or more mice die, or if abnormal behavior such asconvulsions or prostration occurs in two or more mice, or if a body weight lossgreater than 2 g occurs in three or more mice, the Sample does not meet therequirements of the test. If any animals treated with the Sample show only slightsigns of biological reactivity, and not more than one animal shows grosssymptoms of biological reactivity or dies, repeat the test using groups of 10 mice.On the repeat test, all 10 animals treated with the Sample show no significantbiological reactivity above the Blank animals during the observation period.
注射后立即观察，4小时再次观察，之后至少在24、48、72小时再观察。如果在观察期内，样品提取液处理的所有动物的生物反应性都没有显著高于空白处理的动物，则样品满足本试验的要求。如果两只或更多的小鼠死亡，或两只或更多的小鼠出现异常行为，如惊厥或虚脱，或三只或更多的小鼠体重减轻超过2克，则样品不符合试验要求。如果用该样品处理的任何动物只显示出轻微的生物反应迹象，并且不超过一只动物表现出明显的生物反应症状或死亡，则用10只小鼠组重复试验。在重复试验中，10只动物在观察期间均不得表现出明显的高于空白动物的生物反应性。
INTRACUTANEOUS TEST
This test isdesigned to evaluate local responses to the extracts of materials under testfollowing intracutaneous injection into rabbits or guinea pigs.
TestAnimals—Select healthy, rabbits or guinea pigs with fur that can be clippedclosely and skin that is free from mechanical irritation or trauma. In handlingthe animals, avoid touching the injection sites during observation periods,except to discriminate between edema and an oil residue.
Procedure—[NOTE—Agitateeach extract vigorously prior to withdrawal of injection doses to ensure evendistribution of the extracted matter.] On the day of the test,closely clip thefur on the animal's back on both sides of the spinal column over a sufficientlylarge test area. Avoid mechanical irritation and trauma. Remove loose hair bymeans of vacuum. If necessary, swab the skin lightly with diluted alcohol, anddry the skin prior to injection. More than one extract from a given materialcan be used per rabbit or guinea pig, if it is determined that the test resultswill not be affected. For each Sample use two animals and inject each intracutaneously,using one side of the animal for the Sample and the other side for the Blank,as outlined in Table 5. [NOTE—Dilute each g of the extract of the Sampleprepared with Polyethylene Glycol 400, and the corresponding Blank, with 7.4volumes of Sodium Chloride Injection to obtain a solution having a concentrationof about 120 mg of polyethylene glycol per mL.]
皮内的测试
本试验旨在评估皮肤内注射兔或豚鼠后对待测材料提取物的局部反应。
实验动物
选择健康的兔子或豚鼠，它们的皮毛可以剪得很短，皮肤没有机械刺激或外伤。在处理动物时，在观察期间避免接触注射部位，除非要区分水肿和油渣。
程序
[注:混匀模拟提取溶液，以确保提取物的均匀分布。]在测试当天，在动物背部脊柱两侧的皮毛上剪一个足够大的测试区域。避免机械刺激和外伤。用真空吸尘器除去松散的毛发。如有必要，用稀释的酒精轻拭皮肤，并在注射前擦干皮肤。如果确定测试结果不会受到影响，那么每只兔子或豚鼠都可以从一种给定的材料中提取一种以上的提取物。每个样品用两只动物进行腔内注射，用动物一侧注射样品，另一侧注射空白，如表5所示。[注-用聚乙二醇400配制的样品的每g提取物和相应的空白稀释7.4体积的氯化钠注射液，得到浓度约为120mg聚乙二醇/mL的溶液]。
表5皮内的测试
| 提取液/空白 | 每只动物测试点 | 注射体积 |
| 样品 | 5 | 200μL |
| 空白 | 5 | 200μL |
Examineinjection sites for evidence of any tissue reaction such as erythema, edema,and necrosis. Swab the skin lightly, if necessary, with diluted alcohol to facilitatereading of injection sites. Observe all animals at 24, 48, and 72 h afterinjection. Rate the observations on a numerical scale for the extract of the Sampleand for the Blank, using Table 2. Reclip the fur as necessary during theobservation period. The average erythema and edema scores for Sample and Blanksites are determined at every scoring interval (24, 48, and 72 h) for eachrabbit or guinea pig. After the 72-hour scoring, all erythema scores plus edemascores are totalled separately for each Sample and Blank. Divide each of thetotals by 12 (2 animals × 3 scoring periods × 2 scoring categories) todetermine the overall mean score for each Sample versus each correspondingBlank. The requirements of the test are met if the difference between theSample and the Blank mean score is 1.0 or less. If at any observation periodthe average reaction to the Sample is questionably greater than the averagereaction to the Blank, repeat the test using three additional rabbits or guineapigs. The requirements of the test are met if the difference between the Sampleand the Blank mean score is 1.0 or less.
检查注射部位是否有组织反应，如红斑、水肿和坏死。如有必要，用稀释的酒精轻拭皮肤，以便观察注射部位。分别于注射后24、48、72h观察。使用表2在样本提取和空白的数值尺度上对观察值进行评分。在观察期间，如有需要，须将皮毛重新拢起。在每一个评分间隔(24、48、72h)，测定每只兔或豚鼠样本和空白部位的平均红斑和水肿评分。在72小时评分后，分别对每个样本和空白样本的红斑评分和水肿评分进行汇总。将每个总数除以12(2只动物、3个评分时段、2个评分类别)，以确定每个样本相对于每个相应空白的总体平均得分。样本与空白平均分之差小于1.0即满足测试要求。如果在任何观察期间，确认样本的平均反应大于空白样本的平均反应，再用三只兔子或豚鼠重复这个测试。样本与空白平均分之差小于1.0即满足测试要求。
IMPLANTATION TEST
Theimplantation test is designed for the evaluation of plastic materials and otherpolymeric materials in direct contact with living tissue. Of importance are theproper preparation of the implant strips and their proper implantation underaseptic conditions. The intramuscular implantation test requires healthy adult NewZealand rabbits. The test specimens are placed into needles as the deliverysystem for implantation. Although most materials lend themselves readily to thismethod, there are a number of materials that are unsuitable for intramuscularimplantation. For materials with physical characteristics unsuitable for routineintramuscular implantation, the subcutaneous rat implantation model is a viablealternative.
植入试验
植入试验是为了评价塑料材料和其他与活组织直接接触的高分子材料。需要注意的是种植条的适当准备和在无菌条件下的适当种植。肌肉内植入试验需要健康的成年新西兰兔子。将试样放入针中作为植入的传递系统。虽然大多数材料很容易用于这种方法，但仍有一些材料不适合肌内植入。对于物理特性不适合常规肌内植入的材料，可选择大鼠皮下植入模式。
IntramuscularImplantation in Rabbits
Prepare forimplantation 8 strips of the Sample and 4 strips of USP High-DensityPolyethylene RS. Each strip should measure not less than 10 × 1 mm. The edgesof the strips should be as smooth as possible to avoid additional mechanicaltrauma upon implantation. Strips of the specified minimum size are implanted bymeans of a hypodermic needle (15–19 gauge) with intravenous point and a steriletrocar. Use either presterilized needles into which the sterile plastic stripsare aseptically inserted, or insert each clean strip into a needle, the cannulaand hub of which are protected with an appropriate cover, and then subjected tothe appropriate sterilization procedure. [NOTE—Allow for proper degassing ifagents such as ethylene oxide are used.]
兔肌内植入
准备植入样品8条，4条USP高密度聚乙烯，每条测量不小于10×1mm。贴片的边缘应尽可能光滑，以避免植入时额外的机械损伤。通过带静脉点的皮下注射针(15-19号针)和无菌套管针植入指定最小尺寸的带。使用预先消毒的针，无菌地将无菌塑料条插入针内，或将每条清洁的针插入针内，用适当的盖子保护套管和针毂，然后进行适当的消毒程序。[注:如果使用环氧乙烷等试剂，可以进行适当的脱气。]
TestAnimals—Select healthy, adult rabbits weighing not less than 2.5 kg, and withparavertebral muscles that are sufficiently large in size to allow forimplantation of the test strips. Do not use any muscular tissue other than theparavertebral site. The animals must be anesthetized with a commonly usedanesthetic agent to a degree deep enough to prevent muscular movements, such astwitching. See the Association for Assessment and Accreditation of LaboratoryAnimal Care (AAALAC) guidelines.
Procedure—Performthe test in a clean area. On the day of the test or up to 20 h before testing,clip the fur of the animals on both sides of the spinal column.Remove loosehair by means of vacuum. Swab the skin lightly with diluted alcohol, and drythe skin prior to injection.
Implant fourstrips of the Sample into the paravertebral muscle on one side of the spine ofeach of two rabbits, 2.5–5 cm from the midline and parallel to the spinalcolumn, and about 2.5 cm apart from each other. In a similar fashion implanttwo strips of USP High-Density Polyethylene RS in the opposite muscle ofeach animal. Insert a sterilestylet into the needle to hold the implant strip in the tissue whilewithdrawing the needle. If excessive bleeding is observed after implantation ofa strip, place a duplicate strip at another site.
Keep theanimals for a period of not less than 120 h, and sacrifice them at the end ofthe observation period by administering an overdose of an anesthetic agent orother suitable agents. Allow sufficient time to elapse for the tissue to be cutwithout bleeding. Examine macroscopically the area of the tissue surroundingthe center portion of each implant strip. Use a magnifying lens and auxiliarylight source. Observe the Sample and Control implant sites for hemorrhage,necrosis, discolorations, and infections, and record the observations. Measureencapsulation, if present, by recording the width of the capsule (from theperiphery of the space occupied by the implant Control or Sample to theperiphery of the capsule) rounded to the nearest 0.1 mm. Score encapsulation accordingto Table 6.
试验动物
选择体重不少于2.5公斤的健康成年兔，并有足够大的椎旁肌肉来植入试验条。不要使用除椎旁部位以外的任何肌肉组织。必须用常用麻醉剂麻醉动物，麻醉深度要足够深，以防止肌肉运动，如抽搐。参见实验室动物护理评估和认证协会(AAALAC)指南。
程序
在干净的地方进行测试。在试验当天或试验前20小时内，将动物脊柱两侧的皮毛剪除。用真空吸尘器除去松散的毛发。用稀释的酒精轻拭皮肤，并在注射前擦干皮肤。
将4条标本置入两只兔各一侧脊柱的椎旁肌，距中线2.5-5cm，与脊柱平行，彼此相距约2.5cm。以类似的方式在每只动物相对的肌肉中植入两条USP高密度聚乙烯条。将无菌针头插入针中，在拔出针时将植入条固定在组织中。如果在植入条后观察到出血过多，则在另一个位置重新植入一条。
饲养动物的时间不少于120小时，在观察结束时，用过量的麻醉剂或其他合适的药物将它们宰杀。留出足够的时间，使组织能在不出血的情况下被切开。从整体上检查每个种植条中心部分周围的组织区域。使用放大镜和辅助光源。观察样本和对照植入部位是否有出血、坏死、变色和感染，并记录观察结果。如果存在囊肿，通过记录囊肿的宽度(从种植体或样品植入部位的外围到囊肿的外围)，修约至0.1mm来测量囊肿程度。根据表6统计囊肿分数。
计算样本和对照点的平均得分的差异。如果差异不超过1.0，或者任何植入动物四个植入位点中样本和对照均值得分的差异均不超过1，则满足测试的要求。
表6植入试验中封装性的评估
| 囊肿宽度 | 得分 |
| 没有 | 0 |
| ≤0.5mm | 1 |
| 0.6-1.0mm | 2 |
| 1.1-2.0mm | 3 |
| ＞2.0mm | 4 |
Calculate thedifferences between average scores for the Sample and Control sites. Therequirements of the test are met if the difference does not exceed 1.0, or ifthe difference between the Sample and Control mean scores for more than one ofthe four implant sites does not exceed 1 for any implanted animal
SubcutaneousImplantation in Rats
Prepare forimplantation 10 sample specimens and 10 control specimens. The size and shapeof the control specimens shall be as similar to that of the test specimens aspractically possible. For example, specimens made of sheeting material shall be10–12 mm in diameter and from 0.3–1 mm in thickness. The edges of the specimensshould be as smooth as possible to avoid additional mechanical trauma uponimplantation.
Test Animals：Selecthealthy albino rats weighing 225–350 g at the time of implantation.
Procedure：Performthe test in a clean area. Anesthetize (see AAALAC guidelines) the animal untila surgical plane is achieved. Clip the fur of the animals on both sides of thespinal column. Remove loose hair by means of vacuum. Clean the clipped areawith povidone–iodine solution. Using aseptic technique, make two midlineincisions (approximately 1.0 cm long) through the skin at the cranial andcaudal regions on the dorsal surface. Using blunt dissection, separate thefascia connecting skin to muscle to form a pocket underneath the skin lateralto each side of the incision (base of pocket approximately 20 mm from the lineof implant). Insert a sterile sample into each pocket, and close the incisionwith wound clips or sutures. Implant two test samples and two control samplesin each of five rats. Keep the animals for a period of at least seven days, andsacrifice them at the end of the observation period by CO induced hypoxia oradministering an overdose of an anesthetic agent. Allow sufficient time toelapse for the tissue to be cut without bleeding. Cut the skin (dorsal surface)longitudinally and lay back. Carefully examine macroscopically the area of thetissue surrounding the implant. Cut the sample in half and remove for closeexamination of the tissue in direct contact with the sample. Use a magnifyinglens and auxiliary light source, if appropriate. Observe the Sample and Controlimplant sites for hemorrhage, necrosis, discolorations, and infections, andrecord the observations. Measure encapsulation, if present, by recording thewidth of the capsule (from the periphery of the space occupied by the implantControl or Sample to the periphery of the capsule) rounded to the nearest 0.1mm. Score encapsulation according to Table 6. Calculate the differences betweenaverage scores for the Sample and Control sites. The requirements of the testare met if the difference does not exceed 1.0.
大鼠皮下植入
准备植入10个样本标本和10个对照标本。控制试样的尺寸和形状应尽可能与试验试样的尺寸和形状相似。例如，用薄板材料制成的试样直径应在10-12mm，厚度应在0.3-1mm之间。标本的边缘应尽可能光滑，以避免植入时额外的机械损伤。
实验动物
选择植入时体重225-350克的健康白化大鼠。
程序
在洁净区进行测试。麻醉动物(参见AAALAC指南)，直到达到手术要求。将动物的皮毛夹在脊柱两侧。用真空吸尘器除去松散的毛发。用聚维酮碘溶液清洁剪发区域。使用无菌技术，在皮肤的背侧头部和尾部做两个中线切口(大约1.0厘米长)。采用钝性剥离，将连接皮肤和肌肉的筋膜分离，在切口两侧外侧的皮肤下方形成一个袋(袋的底部距植入线约20毫米)。将无菌样本放入每个袋中，用伤口夹或缝合线封闭切口。在5只大鼠中各植入2个测试样本和2个对照样本。将这些动物饲养至少7天，在观察期结束时，通过CO致其缺氧或使用过量麻醉剂将它们宰杀。留出足够的时间，使组织能在不出血的情况下被切开。纵向切开皮肤(背部表面)，放平。仔细观察种植体周围组织的全部区域。将样本切成两半，取出与样本直接接触的组织进行密切检查。如果合适，使用放大镜和辅助光源。观察样本和对照植入部位是否有出血、坏死、变色和感染，并记录观察结果。如果存在囊肿，通过记录囊肿的宽度(从种植体或样品植入部位的外围到囊肿的外围)，修约至0.1mm来测量囊肿程度。根据表6统计囊肿分数。计算样本和对照点的平均得分的差异。如果差值不超过1.0，则满足测试的要求。
SAFETYTESTS—BIOLOGICALS
The safetytest set forth here is intended to detect in an article any unexpected,unacceptable biological reactivity. This in vivo test is provided for the safetyassessmentof biotechnology-derived products.
SafetyTest
Select fivehealthy mice not previously used for testing, weighing 17–23 g, unlessotherwise directed in the individual monograph or elsewhere in this chapter, andmaintained on an adequate balanced diet. Prepare a test solution as directed inthe individual monograph. Unless otherwise directed in the individual monographor elsewhere in this chapter, inject a dose of 0.5 mL of the test solution intoeach of the mice, using a 26-gauge needle of suitable length, or of the lengthspecified below as applicable. Observe the animals over the 48 h following theinjection. If, at the end of 48 h, all of the animals survive and not more thanone of the animals shows outward symptoms of a reaction not normally expectedof the level of toxicity related to the article, the requirements of this testare met. If one or more animals die or if more than one of the animals showssigns of abnormal or untoward toxicity of the article under test, repeat thetest using at least another 10 mice similar to those used in the initial test,but weighing 20 ± 1 g. In either case, if all of the animals survive for 48 hand show no symptoms of a reaction indicative of an abnormal or undue level oftoxicity of the article, the requirements of the test are met. Body weights ofmice before and at the end of the test should be obtained to detect anyuntoward effects. Animals that show signs of toxicity should be grosslynecropsied and subjected to histopathology if necessary.
For biologics,perform the test according to the procedures prescribed in the Code of FederalRegulations, Section 610.11.
生物制剂安全测试
本文提出的安全测试是为了检测一份样品中任何意想不到的、不可接受的生物反应。此体内试验是为生物技术衍生产品的安全性评估而提供的。
安全测试
除另有规定外，选择5只先前未用于测试的健康小鼠，体重17-23克，并保持适当的均衡饮食。按照各专著中的指示准备测试溶液。
除另有规定外，使用适当长度的26号针或以下规定长度的针，向每只小鼠注射0.5mL试验溶液。在注射后的48小时内观察动物。如果在48小时后，所有的动物都存活了下来，并且只有不超过一只的动物表现出与该物品有关的正常毒性水平不同的外部反应症状，则符合该试验的要求。如果一只或多只动物死亡，或有多只动物表现出被测物品的异常或有害毒性，至少再用10只体重为20±1g的小鼠重复试验。在任何一种情况下，如果所有的动物存活了48小时，并且没有表现出表明该物品的异常或过度毒性水平的反应症状，那么测试的要求就满足了。试验前和试验结束时的小鼠体重应记录，以检测任何不良影响。有毒性迹象的动物应进行肉眼解剖，必要时进行组织病理学检查。
对于生物制剂，按照联邦法规第610.11节规定的程序进行试验。
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