Q1：What is the difference between ‘dried’ and ‘anhydrous’ substances?
问1：“干燥”和“无水”物质有何不同？
A1：The term ‘dried substance’ takes into account the loss on drying test (including class 3 solvents), whereas ‘anhydrous substance’ refers to the result obtained by water determination. It is important to note that, when a quantitative determination of a residual solvent is carried out and a test for loss on drying is not carried out, the content of residual solvent is taken into account for calculation of the assay content of the substance, the specific optical rotation and the specific absorbance, even if not explicitly stated in the definition.
答1：术语“干燥物质”考虑了干燥试验（包括第 3 类溶剂）的损失，而“无水物质”是指通过水分测定获得的结果。需要注意的是，当对残留溶剂进行定量测定并且不进行干燥损失测试时，即使定义中没有明确说明，在计算物质的测定含量、比旋光度和比吸光度时，也会考虑残留溶剂的含量。
Q2: Could you explain what pyrogens are and why testing for them is crucial in pharmaceutical products?问2：你能解释一下什么是热原以及为什么在药品中检测热原至关重要吗？
A2: Pyrogens are contaminant substances in injectable drugs or implants that can provoke harmful reactions in patients, ranging from mild fever to severe, potentially life-endangering symptoms. Among the various pyrogens, endotoxins—lipopolysaccharides of gram-negative bacteria—are the most commonly found in pharmaceutical products. All other pyrogens are classed as non-endotoxin pyrogens (NEPs). This heterogeneous group includes compounds such as peptidoglycans, lipoteichoic acids, and lipoproteins from gram-positive bacteria, polysaccharides from fungi, components of myxoviruses, but also microscopic rubber or plastic particles and metal compounds in elastomers. Some NEPs remain unidentified.
答2：热原是注射剂或植入物中的污染物质，可在患者体内引发有害反应，从轻微发热到严重的、可能危及生命的症状不等。在各类热原中，内毒素 —— 革兰氏阴性菌的脂多糖 —— 是药品中最常见的一种。其他所有热原都被归类为非内毒素热原（NEPs）。这一异质性群体包括多种化合物，如革兰氏阳性菌的肽聚糖、脂磷壁酸和脂蛋白，真菌的多糖，粘液病毒的成分，还有弹性体中的微小橡胶或塑料颗粒以及金属化合物。一些非内毒素热原尚未被识别出来。
Q3: What exactly was the Rabbit Pyrogen Test (RPT), and why did the European Pharmacopoeia Commission decide to eliminate it from its monographs by July 2025?问3：兔热原试验（RPT）究竟是什么，为什么欧洲药典委员会决定在 2025 年 7 月前将其从各论中删除？
A3: The RPT has been used for decades to detect pyrogens in pharmaceutical products by measuring changes in rabbits’ body temperature following injection. Its removal from the EP was largely motivated by the 3Rs principle—aimed at replacing, reducing, or refining animal use in pharmaceutical testing wherever feasible. Beyond ethical concerns, the RPT has notable limitations, including low sensitivity, significant result variability, and sometimes non-human specificity, which can result in false positives or negatives.
答3：几十年来，兔热原试验一直被用于检测药品中的热原，其方法是测量兔子注射后体温的变化。它从欧洲药典中被移除，很大程度上是受 3Rs 原则的推动 —— 该原则旨在在可行的情况下，替代、减少或优化药品测试中动物的使用。除了伦理方面的担忧，兔热原试验还有明显的局限性，包括灵敏度低、结果变异性大，有时还缺乏人类特异性，这可能导致假阳性或假阴性结果。
Q4: What are the key alternative methods to the Rabbit Pyrogen Test, and how do they differ in their ability to detect various types of pyrogens?问4：兔热原试验的主要替代方法有哪些，它们在检测各类热原的能力上有何不同？
A4: The EP now recommends the monocyte activation test (MAT) as the replacement pyrogen test for the RPT. Introduced in chapter 2.6.30, the MAT was first included in the EP in 2010 as an alternative method and is now the compendial test method in the EU for the full range of pyrogens. Other in vitro assays, such as the bacterial endotoxin test (BET), also known as the limulus amebocyte lysate (LAL) test, the recombinant factor C (rFC) test, and the recombinant cascade reagent (r-CR) test, have recently been introduced in USP <86> and have also been adopted by pharmacopoeias; however, these only detect endotoxins. According to EP chapter 5.1.13, an endotoxin-specific test may be used for pyrogen testing only if a thorough risk assessment rules out the presence of NEPs.
答4：欧洲药典现在推荐单核细胞激活试验（MAT）作为兔热原试验的替代热原检测方法。单核细胞激活试验在 2.6.30 章中介绍，于 2010 年首次作为替代方法纳入欧洲药典，如今已成为欧盟范围内用于检测所有热原的法定检验方法。其他体外检测方法，如细菌内毒素试验（BET，也称为鲎阿米巴细胞溶解物试验）、重组因子 C（rFC）试验和重组级联试剂（r-CR）试验，最近已被纳入美国药典 < 86>，并被其他药典采用；然而，这些方法仅能检测内毒素。根据欧洲药典5.1.13 章，只有在通过全面的风险评估排除非内毒素热原存在的情况下，才能使用内毒素特异性试验进行热原检测。
In addition to avoiding the ethical concerns associated with animal-based tests, the MAT addresses many of the shortcomings associated with the RPT and the BET. Since it is based on the immune response of humans, its results more accurately reflect human fever reactions. The test is also highly sensitive, reproducible, and compatible with a broader range of product types for testing than the RPT, BET, and rFC.除了能避免与动物试验相关的伦理担忧外，单核细胞激活试验还解决了兔热原试验和细菌内毒素试验的许多缺点。由于它基于人类的免疫反应，其结果能更准确地反映人类的发热反应。与兔热原试验、细菌内毒素试验和重组因子 C 试验相比，该试验灵敏度高、可重复性好，且适用于更广泛的产品类型检测。
Q5: How will the pharmaceutical industry need to adapt to comply with the new European Pharmacopoeia requirements after July 2025?问5：2025 年 7 月后，制药行业需要如何调整才能符合欧洲药典的新要求？
A5: Any company that sells or manufactures parenteral drugs, biologics, cs, or medical devices in the European Union now has to switch to the MAT for full-spectrum pyrogen testing. The MAT is a quantitative in vitro method that mimics the human immune response to both endotoxins and NEPs. It measures the release of inflammatory cytokines—such as TNF-α, IL-1β, or IL-6—produced by activated human monocytes. These cytokines are subsequently identified and quantified through an immunological assay (ELISA), which uses specific antibodies and an enzyme-driven colorimetric reaction. Only monocytes from validated sources may be used for the MAT, including whole human blood (fresh or cryopreserved), peripheral blood mononuclear cells (PBMCs), or established monocytic cell lines like Mono-Mac-6 (MM6).
答5：现在，任何在欧盟销售或生产注射剂、生物制品、化妆品或医疗器械的公司都必须改用单核细胞激活试验进行全谱热原检测。单核细胞激活试验是一种定量体外方法，可模拟人类对内毒素和非内毒素热原的免疫反应。它测量被激活的人类单核细胞产生的炎性细胞因子（如 TNF-α、IL-1β 或 IL-6）的释放量。随后，通过免疫测定法（ELISA）对这些细胞因子进行鉴定和定量，该方法使用特异性抗体和酶驱动的比色反应。单核细胞激活试验只能使用来自经过验证的来源的单核细胞，包括全人类血液（新鲜的或冷冻保存的）、外周血单个核细胞（PBMCs）或已建立的单核细胞系，如 Mono-Mac-6（MM6）。
Q6: What challenges might companies face in transitioning from the Rabbit Pyrogen Test to in vitro alternatives like the Monocyte Activation Test?问6：企业从兔热原试验过渡到像单核细胞激活试验这样的体外替代方法时可能会面临哪些挑战？
A6: The MAT is compendial in the EU, so manufacturers do not need to perform full validation. The supplier should be able to provide comprehensive validation data.
答6：单核细胞激活试验在欧盟是法定方法，因此制造商无需进行全面验证。供应商应能提供全面的验证数据。
Q7：How might other global regulatory bodies respond to the European Pharmacopoeia’s decision to ban the Rabbit Pyrogen Test?问7：其他全球监管机构可能会对欧洲药典禁止兔热原试验的决定做出怎样的回应？
A7: The Ph. Eur. Commission moved first to effectively abolish the RPT, and there seems to be a general willingness among the larger nations to follow suit. Whether this will materialize in the coming years remains uncertain at this time. In the US, the MAT has been accepted as an alternative method for pyrogen detection since the release of an FDA industry guidance in 2012, with similar recommendations made in USP <151> in 2017. The MAT is also accepted as a compendial method in the Pharmacopoeia of Russia and Eurasia, as an alternative method in the Pharmacopoeia of India, China, Brazil, and as a supplementary method in Japan.
答7：欧洲药典委员会率先采取行动，实际上废除了兔热原试验，而且各大国家似乎普遍愿意效仿。未来几年这是否会成为现实，目前尚不确定。在美国，自 2012 年美国食品药品监督管理局发布行业指南以来，单核细胞激活试验已被接受为热原检测的替代方法，2017 年美国药典 < 151 > 也提出了类似建议。单核细胞激活试验在俄罗斯和欧亚药典中被列为法定方法，在印度、中国、巴西的药典中被列为替代方法，在日本被列为补充方法。
Q8: Microbiology texts (e.g. chapters 2.6.1, 2.6.12, 2.6.13, 2.7.2, monograph 0008): can microbial strains other than those that are cited in the Ph. Eur. be used?问8：微生物章节（例如第 2.6.1、2.6.12、2.6.13、2.7.2 章，专论 0008）：可以使用欧洲药典中引用的微生物菌株以外的微生物菌株吗？
A8: The micro-organisms that are prescribed in these texts must be used. Strains from other culture collections may be used if they have been shown to be equivalent to those prescribed in the Ph. Eur.答8：必须使用这些章节中规定的微生物。如来自其他菌种保藏单位的菌株已被证明与欧洲药典中规定的菌株相同，则可以使用它们。
Q9: The name of a micro-organism in the Ph. Eur. does not match the ones used by culture collections (e.g. ATCC 6633 is named Bacillus subtilis in the Ph. Eur. and Bacillus spizizenii on the ATCC website). Which strain should I use?问9：欧洲药典中微生物的名称与菌种保藏中心使用的微生物名称不相同（例如，ATCC 6633 在欧洲药典中被命名为枯草芽孢杆菌，ATCC 网站上被命名为斯皮泽尼芽孢杆菌）。我应该使用哪种菌株？
A9: It is important when performing microbial tests to check that the strains used are the same as the ones listed in the relevant Ph. Eur. text. We acknowledge that micro-organism taxonomy may evolve over time, and that this can result in potential differences between the strain names stated in the Ph. Eur. texts and those used by culture collections; however, the culture collection references (e.g. ATCC 6633) cited in the Ph. Eur. take precedence over the strain names. Therefore, when purchasing micro-organisms from culture collections, please refer to the culture collection references listed in the Ph. Eur.
答9：在进行微生物测试时，重要的是要检查所使用的菌株是否与相关欧洲药典中列出的菌株相同。我们承认微生物可能会随着时间的推移而演变，这可能导致欧洲药典中陈述的菌株名称与菌种保藏中心使用的菌株名称之间存在潜在差异;然而，欧洲药典中引用的菌种索引号（例如 ATCC 6633）优先于菌株名称。因此，在从菌种保藏中心购买微生物时，请参考欧洲药典中列出的菌种索引号。
Q10：How should I report the results of my test?问10：我应该如何报告我的测试结果？
A10: The Ph. Eur. is not prescriptive on the reporting of the results of the TYMC and TAMC. The results must be reported based on what you observe in the test.答10：欧洲药典对 TYMC（霉菌和酵母总数） 和 TAMC（需氧菌总数） 结果的报告没有规定。应根据在测试中观察到的内容报告结果。
Standard reporting criteria would dictate that you should report, for example, <1 CFU/g in cases where no visible CFU were observed, because of the inherent variability of any microbiological examination method. However, the actual figure reported is affected by the dilution of the sample for testing purposes, and this must be taken into account when reporting the result, e.g. if 1 mL of a 1/10 dilution is tested, then the result would be <10 CFU/g.标准报告标准规定，例如，在未观察到可见 CFU 的情况下，应该报告 <1 CFU/g，因为任何微生物检查方法都存在固有的可变性。然而，报告的实际数字会受到用于测试目的的样品稀释度的影响，在报告结果时必须考虑到这一点，例如，如该结果是测试1 mL 的 1/10 稀释液，则结果将为 <10 CFU/g。
Q11：What is the meaning of “102 CFU: maximum acceptable count = 200”?问11：“102 CFU：最大可接受计数 = 200”是什么意思？
A11: This is the way in which experts have decided to express results/counts. Microbiological methods have a degree of variability and expressing the results in this way takes account of the variability of the method.
答11：这是专家决定表达结果/计数的方式。微生物方法具有一定程度的变异性，以这种方式表达结果考虑了方法的变异性。
If, for example, the specification in a product monograph is 102 CFU/g or mL, the product could be released if up to 200 CFU/g or mL are counted.例如，如果产品专论中的标准是 102 CFU/g 或mL，则如果计数高达 200 CFU/g 或 mL，该产品可能会被放行。
Q12：Should a reduction from 2.9 × 105 CFU to 4.6 × 103 CFU be interpreted as meeting the A criteria, since this is the same as a reduction from 104.5 to 103.7 and 4.5 - 3.7 = 1.8, which can be rounded up to 2? Or should it be interpreted as failing to meet the A criteria because the result has not decreased to 2.9 × 103 CFU?
问12：（消毒剂微生物杀灭试验）从 2.9 × 105 CFU 减少到 4.6 × 103 CFU 是否符合 A 标准，因为这与从 104.5 减少到103.7，即 4.5 - 3.7 = 1.8，可以四舍五入为2？还是不符合 A 标准，因为结果没有下降到 2.9 × 103 CFU？
A12: From a mathematical point of view, logarithmic values should not be rounded. To illustrate the effect, the value of 1.8 represents a factor 63 loss, whereas a value of 2.0 represents a factor 100 loss.
答12：从数学的角度来看，对数值不应四舍五入。为了说明效果，值 1.8 表示下降63倍，而值 2.0 表示下降 100倍。
We recommend that borderline results are approached on a case-by-case basis. A specific borderline result may be considered acceptable when taking into account the preservative efficacy test as a whole and the precision of the method. The outcome of the test should not be based on a single test result and you may decide to collect additional data.我们建议根据具体情况处理临界结果。当考虑到整个消毒剂功效测试和方法的精确度时，特定的临界结果可以被认为是可以接受的。测试结果不应基于单个测试结果，可以决定收集其他数据。
Q13: When is the negative control performed? When testing the suitability of the method, when testing the product, or in both situations?问13：何时进行阴性对照？在测试方法的适用性时？在测试产品时？还是在两种情况下？
A13: It is performed in both situations. Chapter 2.6.13, section 3-2 states: “To verify testing conditions, a negative control is performed using the chosen diluent in place of the test preparation. [...] A negative control is also performed when testing the products as described in section 4.”
答13：它在两种情况都应进行阴性对照。第 2.6.13 章第 3-2 节规定：“为了确认测试条件，使用所选稀释剂代替测试制剂进行阴性对照。[...]在测试第4 节所述的产品时，也进行了阴性对照。
Q14: All incubations times are given as periods. How should these periods be interpreted? For example, 18-72 hours: do I need to incubate for 72 hours unless growth occurs sooner? Or is it always sufficient to incubate for 18 h?问14：所有培养时间都以时间范围的形式给出。这些时间范围应如何解释？例如，18-72 小时：除非生长更快发生，否则我是否需要培养72 小时？还是孵育18小时是足够的？
A14: The periods are given to be more flexible for the testing laboratories. If the suitability test shows compliant results at 18 h, the minimal incubation time for routine use is 18 h.
答14：给出时间范围是为了让测试实验室更加灵活。如果适用性测试在18小时时显示结果，则日常使用的最小培养时间为18小时。
