Q1：What is the difference between ‘dried’ and ‘anhydrous’ substances?
問1：“干燥”和“無水”物質(zhì)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：術(shù)語“干燥物質(zhì)”考慮了干燥試驗(yàn)（包括第 3 類溶劑）的損失，而“無水物質(zhì)”是指通過水分測定獲得的結(jié)果。需要注意的是，當(dāng)對(duì)殘留溶劑進(jìn)行定量測定并且不進(jìn)行干燥損失測試時(shí)，即使定義中沒有明確說明，在計(jì)算物質(zhì)的測定含量、比旋光度和比吸光度時(shí)，也會(huì)考慮殘留溶劑的含量。
Q2: Could you explain what pyrogens are and why testing for them is crucial in pharmaceutical products?問2：你能解釋一下什么是熱原以及為什么在藥品中檢測熱原至關(guān)重要嗎？
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：熱原是注射劑或植入物中的污染物質(zhì)，可在患者體內(nèi)引發(fā)有害反應(yīng)，從輕微發(fā)熱到嚴(yán)重的、可能危及生命的癥狀不等。在各類熱原中，內(nèi)毒素 —— 革蘭氏陰性菌的脂多糖 —— 是藥品中最常見的一種。其他所有熱原都被歸類為非內(nèi)毒素?zé)嵩∟EPs）。這一異質(zhì)性群體包括多種化合物，如革蘭氏陽性菌的肽聚糖、脂磷壁酸和脂蛋白，真菌的多糖，粘液病毒的成分，還有彈性體中的微小橡膠或塑料顆粒以及金屬化合物。一些非內(nèi)毒素?zé)嵩形幢蛔R(shí)別出來。
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?問3：兔熱原試驗(yàn)（RPT）究竟是什么，為什么歐洲藥典委員會(huì)決定在 2025 年 7 月前將其從各論中刪除？
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：幾十年來，兔熱原試驗(yàn)一直被用于檢測藥品中的熱原，其方法是測量兔子注射后體溫的變化。它從歐洲藥典中被移除，很大程度上是受 3Rs 原則的推動(dòng) —— 該原則旨在在可行的情況下，替代、減少或優(yōu)化藥品測試中動(dòng)物的使用。除了倫理方面的擔(dān)憂，兔熱原試驗(yàn)還有明顯的局限性，包括靈敏度低、結(jié)果變異性大，有時(shí)還缺乏人類特異性，這可能導(dǎo)致假陽性或假陰性結(jié)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?問4：兔熱原試驗(yàn)的主要替代方法有哪些，它們在檢測各類熱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：歐洲藥典現(xiàn)在推薦單核細(xì)胞激活試驗(yàn)（MAT）作為兔熱原試驗(yàn)的替代熱原檢測方法。單核細(xì)胞激活試驗(yàn)在 2.6.30 章中介紹，于 2010 年首次作為替代方法納入歐洲藥典，如今已成為歐盟范圍內(nèi)用于檢測所有熱原的法定檢驗(yàn)方法。其他體外檢測方法，如細(xì)菌內(nèi)毒素試驗(yàn)（BET，也稱為鱟阿米巴細(xì)胞溶解物試驗(yàn)）、重組因子 C（rFC）試驗(yàn)和重組級(jí)聯(lián)試劑（r-CR）試驗(yàn)，最近已被納入美國藥典 < 86>，并被其他藥典采用；然而，這些方法僅能檢測內(nèi)毒素。根據(jù)歐洲藥典5.1.13 章，只有在通過全面的風(fēng)險(xiǎn)評(píng)估排除非內(nèi)毒素?zé)嵩嬖诘那闆r下，才能使用內(nèi)毒素特異性試驗(yàn)進(jìn)行熱原檢測。
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.除了能避免與動(dòng)物試驗(yàn)相關(guān)的倫理擔(dān)憂外，單核細(xì)胞激活試驗(yàn)還解決了兔熱原試驗(yàn)和細(xì)菌內(nèi)毒素試驗(yàn)的許多缺點(diǎn)。由于它基于人類的免疫反應(yīng)，其結(jié)果能更準(zhǔn)確地反映人類的發(fā)熱反應(yīng)。與兔熱原試驗(yàn)、細(xì)菌內(nèi)毒素試驗(yàn)和重組因子 C 試驗(yàn)相比，該試驗(yàn)靈敏度高、可重復(fù)性好，且適用于更廣泛的產(chǎn)品類型檢測。
Q5: How will the pharmaceutical industry need to adapt to comply with the new European Pharmacopoeia requirements after July 2025?問5：2025 年 7 月后，制藥行業(yè)需要如何調(diào)整才能符合歐洲藥典的新要求？
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：現(xiàn)在，任何在歐盟銷售或生產(chǎn)注射劑、生物制品、化妝品或醫(yī)療器械的公司都必須改用單核細(xì)胞激活試驗(yàn)進(jìn)行全譜熱原檢測。單核細(xì)胞激活試驗(yàn)是一種定量體外方法，可模擬人類對(duì)內(nèi)毒素和非內(nèi)毒素?zé)嵩拿庖叻磻?yīng)。它測量被激活的人類單核細(xì)胞產(chǎn)生的炎性細(xì)胞因子（如 TNF-α、IL-1β 或 IL-6）的釋放量。隨后，通過免疫測定法（ELISA）對(duì)這些細(xì)胞因子進(jìn)行鑒定和定量，該方法使用特異性抗體和酶驅(qū)動(dòng)的比色反應(yīng)。單核細(xì)胞激活試驗(yàn)只能使用來自經(jīng)過驗(yàn)證的來源的單核細(xì)胞，包括全人類血液（新鮮的或冷凍保存的）、外周血單個(gè)核細(xì)胞（PBMCs）或已建立的單核細(xì)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?問6：企業(yè)從兔熱原試驗(yàn)過渡到像單核細(xì)胞激活試驗(yàn)這樣的體外替代方法時(shí)可能會(huì)面臨哪些挑戰(zhàn)？
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：單核細(xì)胞激活試驗(yàn)在歐盟是法定方法，因此制造商無需進(jìn)行全面驗(yàn)證。供應(yīng)商應(yīng)能提供全面的驗(yàn)證數(shù)據(jù)。
Q7：How might other global regulatory bodies respond to the European Pharmacopoeia’s decision to ban the Rabbit Pyrogen Test?問7：其他全球監(jiān)管機(jī)構(gòu)可能會(huì)對(duì)歐洲藥典禁止兔熱原試驗(yàn)的決定做出怎樣的回應(yīng)？
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：歐洲藥典委員會(huì)率先采取行動(dòng)，實(shí)際上廢除了兔熱原試驗(yàn)，而且各大國家似乎普遍愿意效仿。未來幾年這是否會(huì)成為現(xiàn)實(shí)，目前尚不確定。在美國，自 2012 年美國食品藥品監(jiān)督管理局發(fā)布行業(yè)指南以來，單核細(xì)胞激活試驗(yàn)已被接受為熱原檢測的替代方法，2017 年美國藥典 < 151 > 也提出了類似建議。單核細(xì)胞激活試驗(yàn)在俄羅斯和歐亞藥典中被列為法定方法，在印度、中國、巴西的藥典中被列為替代方法，在日本被列為補(bǔ)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?問8：微生物章節(jié)（例如第 2.6.1、2.6.12、2.6.13、2.7.2 章，專論 0008）：可以使用歐洲藥典中引用的微生物菌株以外的微生物菌株嗎？
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：必須使用這些章節(jié)中規(guī)定的微生物。如來自其他菌種保藏單位的菌株已被證明與歐洲藥典中規(guī)定的菌株相同，則可以使用它們。
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?問9：歐洲藥典中微生物的名稱與菌種保藏中心使用的微生物名稱不相同（例如，ATCC 6633 在歐洲藥典中被命名為枯草芽孢桿菌，ATCC 網(wǎng)站上被命名為斯皮澤尼芽孢桿菌）。我應(yīng)該使用哪種菌株？
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：在進(jìn)行微生物測試時(shí)，重要的是要檢查所使用的菌株是否與相關(guān)歐洲藥典中列出的菌株相同。我們承認(rèn)微生物可能會(huì)隨著時(shí)間的推移而演變，這可能導(dǎo)致歐洲藥典中陳述的菌株名稱與菌種保藏中心使用的菌株名稱之間存在潛在差異;然而，歐洲藥典中引用的菌種索引號(hào)（例如 ATCC 6633）優(yōu)先于菌株名稱。因此，在從菌種保藏中心購買微生物時(shí)，請(qǐng)參考?xì)W洲藥典中列出的菌種索引號(hào)。
Q10：How should I report the results of my test?問10：我應(yīng)該如何報(bào)告我的測試結(jié)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：歐洲藥典對(duì) TYMC（霉菌和酵母總數(shù)） 和 TAMC（需氧菌總數(shù)） 結(jié)果的報(bào)告沒有規(guī)定。應(yīng)根據(jù)在測試中觀察到的內(nèi)容報(bào)告結(jié)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.標(biāo)準(zhǔn)報(bào)告標(biāo)準(zhǔn)規(guī)定，例如，在未觀察到可見 CFU 的情況下，應(yīng)該報(bào)告 <1 CFU/g，因?yàn)槿魏挝⑸餀z查方法都存在固有的可變性。然而，報(bào)告的實(shí)際數(shù)字會(huì)受到用于測試目的的樣品稀釋度的影響，在報(bào)告結(jié)果時(shí)必須考慮到這一點(diǎn)，例如，如該結(jié)果是測試1 mL 的 1/10 稀釋液，則結(jié)果將為 <10 CFU/g。
Q11：What is the meaning of “102 CFU: maximum acceptable count = 200”?問11：“102 CFU：最大可接受計(jì)數(shù) = 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：這是專家決定表達(dá)結(jié)果/計(jì)數(shù)的方式。微生物方法具有一定程度的變異性，以這種方式表達(dá)結(jié)果考慮了方法的變異性。
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，如果產(chǎn)品專論中的標(biāo)準(zhǔn)是 102 CFU/g 或mL，則如果計(jì)數(shù)高達(dá) 200 CFU/g 或 mL，該產(chǎn)品可能會(huì)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?
問12：（消毒劑微生物殺滅試驗(yàn)）從 2.9 × 105 CFU 減少到 4.6 × 103 CFU 是否符合 A 標(biāo)準(zhǔn)，因?yàn)檫@與從 104.5 減少到103.7，即 4.5 - 3.7 = 1.8，可以四舍五入為2？還是不符合 A 標(biāo)準(zhǔn)，因?yàn)榻Y(jié)果沒有下降到 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：從數(shù)學(xué)的角度來看，對(duì)數(shù)值不應(yīng)四舍五入。為了說明效果，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.我們建議根據(jù)具體情況處理臨界結(jié)果。當(dāng)考慮到整個(gè)消毒劑功效測試和方法的精確度時(shí)，特定的臨界結(jié)果可以被認(rèn)為是可以接受的。測試結(jié)果不應(yīng)基于單個(gè)測試結(jié)果，可以決定收集其他數(shù)據(jù)。
Q13: When is the negative control performed? When testing the suitability of the method, when testing the product, or in both situations?問13：何時(shí)進(jìn)行陰性對(duì)照？在測試方法的適用性時(shí)？在測試產(chǎn)品時(shí)？還是在兩種情況下？
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：它在兩種情況都應(yīng)進(jìn)行陰性對(duì)照。第 2.6.13 章第 3-2 節(jié)規(guī)定：“為了確認(rèn)測試條件，使用所選稀釋劑代替測試制劑進(jìn)行陰性對(duì)照。[...]在測試第4 節(jié)所述的產(chǎn)品時(shí)，也進(jìn)行了陰性對(duì)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?問14：所有培養(yǎng)時(shí)間都以時(shí)間范圍的形式給出。這些時(shí)間范圍應(yīng)如何解釋？例如，18-72 小時(shí)：除非生長更快發(fā)生，否則我是否需要培養(yǎng)72 小時(shí)？還是孵育18小時(shí)是足夠的？
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：給出時(shí)間范圍是為了讓測試實(shí)驗(yàn)室更加靈活。如果適用性測試在18小時(shí)時(shí)顯示結(jié)果，則日常使用的最小培養(yǎng)時(shí)間為18小時(shí)。
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