1. Introduction

For thousands of years, people have been increasingly exposed to numerous diseases. Millions of people died from these illnesses, which frequently escalated into epidemics. During this period, individuals began to contemplate infectious diseases and their causes. However, efforts to prevent, treat, or control the spread of such diseases were initially unsuccessful.¹One of the most effective medical breakthroughs against infectious diseases is the discovery of antibiotics. The number of lives they have saved and their profound impact on combating infectious diseases—which throughout most of human history were the main causes of morbidity and mortality—cannot be overstated.²

    Salvarsan, the first antibiotic, was introduced in 1910. Antibiotics have significantly transformed modern medicine and increased average human life expectancy by approximately 23 years within just over a century.³ Salvarsan has remained an enigmatic compound due to its extensive use. By 1920, two million doses of Salvarsan were produced annually in the United States (US) alone, and it was also demonstrated to be effective against certain parasitic diseases. Salvarsan remained in high demand until 1943, when penicillin, a more tolerable and equally effective drug, became available.⁴ Following Fleming’s discovery of penicillin in 1928, numerous other antibiotics were subsequently developed and approved for human use. However, both Gram-positive and Gram-negative bacteria have developed resistance to many of these agents.⁵

    Given the slow pace in the discovery of novel antibiotic classes over recent decades,⁶ there is a growing concern about their effectiveness and the emergence of resistant pathogens. Recent in vitro studies have demonstrated that certain antibiotics, particularly those targeting penicillin-binding protein 3, can promote endotoxin release.⁷⁸ It is well established that cell wall–active antibiotics facilitate the release of endotoxins.The study of endotoxins began in the late 19th century when Richard Pfeiffer, a student of Robert Koch, discovered that lysates of heat-inactivated Vibrio cholerae contained a toxic substance capable of inducing death in experimental animals.⁹ Additionally, two other scientists, Eugenio Centanni and Hans Buchner, independently isolated the same toxin.¹⁰ Centanni made two important contributions: first, he observed that the toxin could be isolated from lysates of many Gram-negative bacteria,⁷ but not from Gram-positive species.¹¹ Second, he emphasized the remarkable pyrogenic properties of endotoxin.

    Since it is well recognized that pyrogenic substances elevate body temperature, bacterial endotoxins exert multiple physiological effects.¹²¹³ Endotoxins are lipopolysaccharide (LPS) components derived from the outer membrane of Gram-negative bacteria.¹⁴¹⁵ They are released at a low, steady rate from living bacteria; however, substantially larger amounts of endotoxins are released following bacterial cell lysis.¹⁶

    The rabbit pyrogen test (RPT) and bacterial endotoxin test (BET) are compendial methods that share the same origin—“injection fever”¹⁷—a quality defect that was once prevalent in parenteral therapy. Injection fever results from contamination with Gram-negative bacterial endotoxins, as evidenced by patient febrile reactions caused by contaminated medicinal products, biologicals, and medical devices over the past century.¹⁸ All parenteral drug products and devices administered via intravenous, intraventricular, intra-arterial, intra-articular, subcutaneous, intramuscular, intrathecal, intracisternal, and intraocular routes must comply with an appropriate endotoxin limit specification to obtain marketing authorization from health authorities.¹⁹

    There are variations in the pharmacopeial guidelines and standard limits for bacterial endotoxin testing across different pharmacopeias—including the Indian Pharmacopeia (IP), British Pharmacopeia (BP), Japanese Pharmacopeia (JP), European Pharmacopeia (Ph. Eur.), US Pharmacopeia (USP), and International Pharmacopeia (Ph. Int.)—which directly and indirectly affect the cost of testing, increase the complexity of compliance, and hinder the global transport and trade of pharmaceutical products.

    Therefore, the harmonization of pharmacopeial standards is urgently needed, involving the exchange and alignment of methods and requirements to yield comparable results and ensure patient safety by upholding quality standards for pharmaceuticals. Specifically, the harmonization of BET guidelines and limits among different pharmacopeias is crucial to achieve consistency and uniformity in the manufacturing and regulatory processes of pharmaceutical products.Based on the recent updates to the core Q4B guideline and its annexes by the Pharmacopeial Discussion Group, the BET procedures described in the USP, JP, and Ph. Eur. can now be used interchangeably, leading to the recognition of pharmacopeial BET methods by regulatory authorities within the International Council for Harmonization (ICH) regions.²⁰

2. Pyrogen and Endotoxin

Pyrogens are substances, such as endotoxins (lipopolysaccharides, LPS), released from Gram-negative bacteria that can cause fever. They pose a serious risk when present in pharmaceutical products.²¹ Pyrogens can be either endotoxins or non-endotoxin pyrogens (NEPs); however, bacterial endotoxins are the most prevalent and pose the greatest threat to patient safety.²²²³Endotoxins are intrinsic components of the outer membrane of Gram-negative bacteria.²⁴²⁵ They are considered a heterogeneous group of biomolecules that are released upon bacterial cell lysis, resulting in toxic effects such as fever, septic shock, multiple organ failure, and death.²⁶ Due to the growing number of resistant bacterial strains, endotoxin-induced septic shock syndrome continues to pose a significant global health threat and is associated with an unacceptably high mortality rate.²⁷

    The primary component of the outer membrane of Gram-negative bacteria, LPS, becomes hazardous when it enters the bloodstream, or when released into the environment, or when used in industrial applications such as pharmaceuticals.²⁸ Examples of LPS-producing bacteria include species of Escherichia, Salmonella, Yersinia, Shigella as well as Enterobacter, Proteus, and pathogens such as Vibrio cholerae, Yersinia pestis, and Brucella abortus.²⁹

    Endotoxin units (EU) are used to measure endotoxin levels. The Second International Endotoxin Standard—which establishes that 1 EU is equivalent to 1 International Unit (IU)—was approved by the World Health Organization’s Expert Committee on Biological Standardization. The expression K/M is used to determine the endotoxin limit for a particular test preparation, where M is the maximum dose administered to an adult (estimated to be 70 kg for this purpose) per kilogram of body weight per hour, and K is the threshold pyrogenic dose of endotoxin per kilogram of body weight. K has a value of 5.0 EU/kg for parenteral drugs (except those administered intrathecally) and 0.2 EU/kg for preparations intended for intrathecal administration.²⁹–³¹ It has been reported that approximately 2 × 10⁶ LPS molecules, equivalent to approximately 20 femtograms, can be found in a single E. coli cell.²⁹ The physiologically active LPS is associated with or embedded in phospholipids, lipoproteins, and other proteins that constitute the Gram-negative bacterial outer membrane.³²³³ LPS is a structural component of the cell wall of Gram-negative bacteria and plays a major role in the pathogenesis of septic shock in humans. The hydrophobic lipid region of the membrane contains a portion of the LPS molecule embedded within it, while the hydrophilic polysaccharide portion is exposed to the external environment of the cell.³⁴³⁵

    The most widely used test for detecting endotoxins is the Limulus amebocyte lysate (LAL) assay.³⁶³⁷ In this test, horseshoe crab-derived LAL reacts with bacterial endotoxin (LPS) to form a measurable gel clot.³⁷ The unit of measurement for endotoxins is EU/mL, with 1 EU equivalent to approximately 0.1–0.2 ng of endotoxin/mL of solution.³⁸³⁹ The LAL test is currently available in three formats, each with distinct sensitivity levels. While the kinetic turbidimetric and chromogenic LAL assays can detect concentrations as low as 0.01 EU/mL, the gel-clot method detects concentrations as low as 0.03 EU/mL.²⁴

3. Overview of Endotoxin Testing

Endotoxin testing of parenteral medications and implantable devices is essential for detecting contamination and ensuring patient safety. It is also a mandatory release test for the batch production of therapeutic products.⁴⁰

3.1. Rabbit Pyrogen Test (RPT)

The US Food and Drug Administration approved the RPT as the first method for detecting LPS. It was developed in the 1920s, based on the observation that rabbits exhibit an increase in body temperature following the intravenous injection of a test solution.⁴¹ In practice, the RPT has been gradually replaced by the LAL assay due to its greater efficiency in terms of cost and testing time.²⁶⁴² However, the RPT has been phased out in many regions. According to the recent updates to the Ph. Eur., the RPT has been officially removed to promote animal welfare and scientific advancement.⁴³–⁴⁵

3.2. Limulus amebocyte lysate assay or BET

In 1964, Levin and Bang made the groundbreaking discovery that the lysate of the Atlantic horseshoe crab (Limulus polyphemus) coagulates upon exposure to bacterial endotoxins. Their findings were instrumental in the development of the LAL test.⁴⁶ Despite being classified as “vulnerable,” Atlantic horseshoe crabs are crucial for maintaining global public health. Currently, humans use these blue-blooded marine invertebrates to ensure that endotoxins do not contaminate vaccines, injectable medications, and medical devices used in both human and veterinary care.⁴⁷ Over the past decade, LAL has been used to quantify endotoxin levels in pharmaceutical products and biological fluids.⁴⁸ The LAL assay can detect not only Gram-negative bacterial endotoxins but also fungal components containing β-D-1,3-glucan.⁴⁹

    All major pharmacopeias worldwide recommend the LAL test for the detection of bacterial endotoxins.⁵⁰ Endotoxins are also contaminants found in certain organic dusts and environmental media that support the growth of Gram-negative bacteria.⁵¹ Among the pharmacopeias—IP, BP, JP, Ph. Eur, Ph. Int, and USP—three assay methods are described: (i) the turbidimetric method, which is based on the development of turbidity following the cleavage of an endogenous substrate; (ii) the chromogenic method, which is based on color development that occurs after a synthetic peptide–chromogen complex is cleaved; and (iii) the gel-clot method, which is based on gel formation (Figure 1).⁵²

    Several ethical concerns are associated with the harvesting of LAL from horseshoe crabs. Overharvesting LAL from crabs may cause injury and mortality, leading to population decline and adverse ecological impacts. Although the crabs are returned to the ocean after blood collection, a significant number of crabs die because of the procedure or suffer from adverse impacts. These ethical issues emphasize the need for alternative methods for the BET.⁴⁷

3.2.1. Legislative classification of Limulus amebocyte lysate assay

The LAL test is categorized at an equivalent level across national pharmacopeias, including the 2024 BP⁵³, 2025 USP⁵⁴, JP⁵⁵, 2022 Ph. Eur.⁵⁶, 2022 Ph. Int.⁵⁷, and 2022 IP⁵⁸ and its Addendum.

3.3. Qualitative analysis

3.3.1. Gel-clot limit test method

Gel‐clot testing is a manual process. The BET performed using the gel-clot method is a 60-min test conducted at an incubation temperature of 37°C.⁵⁹ LAL reagent water, LAL, and control standard endotoxin (CSE) are combined to create an endotoxin standard series for the experiment. The sensitivity unit of the LAL employed for the test is lambda (λ), which is printed on the LAL vial and the certificate of analysis. The standard curve dilutions tested are 2λ, 1λ, ½λ, and ¼λ. The standard curve must be positive within a two-fold dilution of λ to be valid.⁵³⁻⁵⁸

3.3.2. Specific reagents

(a) Endotoxin reference standard (ERS) and control standard endotoxin
The ERS and commercially produced CSE are isolated from Gram-negative bacterial cell membranes using the Westphal hot phenol method and processed to eliminate membrane components.⁶⁰ Furthermore, the purified preparations are typically stabilized with suitable agents. A CSE that is appropriately standardized against the ERS may be used for routine bacterial endotoxin testing.⁶¹

(b) Lysate
Lysate is an aqueous extract of blood cells (amoebocytes) from one of the following horseshoe crab species⁶²—L. polyphemus, Tachypleus gigas, Tachypleus tridentatus, or Carcinoscorpius rotundicauda—and is reconstituted according to the instructions on the label.⁶⁴

(c) BET-grade water
The BET, which is used to identify and measure endotoxins produced by Gram-negative bacteria, depends heavily on water (comprising 80–95% of the test system).⁶⁵ If endotoxins contaminate certain products, particularly those used in medications and medical equipment, they may pose a risk to human health.⁶⁶ It is crucial to use water that is free from endotoxins and other impurities that could skew the test results to ensure reliability. If endotoxins are present in this water, they should be below 0.005 EU/mL to prevent false-positive results.⁶⁷

3.3.3. Endotoxin limit calculation for the gel-clot method

(a) Endotoxin limit calculation
The product’s dosage and route of administration are considered when calculating the endotoxin limit. The standard equation is given below (Equation [I]):

where:
(i) EL is the endotoxin limit (EU/mL or EU/unit).
(ii) K is the threshold pyrogenic dose of endotoxin per kilogram of body weight. Standard K values are 0.2 EU/kg for intrathecal medications and 5 EU/kg for intravenous injections, depending on the administration route.⁶⁸
(iii) M is the maximum dose of the product per kilogram of body weight per hour.

(b) Dilution preparation
A series of dilutions must be prepared to determine whether the product exhibits any interfering effects. The dilution factor should be selected so that the endotoxin concentration falls within the LAL reagent’s detection range.

The maximum valid dilution can be calculated using Equation (II):

Where:
(i) MVD is the maximum dilution that still permits the detection of endotoxins.
(ii) EL is the endotoxin limit.
(iii) λ is the sensitivity of the LAL reagent.

3.4. Photometric quantitative techniques

3.4.1. Kinetic turbidimetric method

The kinetic turbidimetric method—a photometric assay—measures the increase in turbidity resulting from the reaction between endotoxin and lysate. It also determines either the rate of turbidity development or the time required to reach a predefined absorbance or transmission value of the reaction mixture. The test is conducted at the incubation temperature recommended by the lysate manufacturer (37 ± 1°C).⁵³⁻⁵⁵,⁵⁸

3.4.2. Kinetic chromogenic method

The kinetic chromogenic method is a photometric assay that measures the rate of color development or the time (onset time) required to reach a predetermined absorbance, which results from the reaction between endotoxin and lysate. This reaction releases chromophores from a chromogenic substrate.⁵³⁻⁵⁵,⁵⁸

3.4.3. End-point chromogenic assay

The end-point chromogenic assay measures the color intensity at the end of the incubation period, after the reaction has been stopped by the addition of a suitable reagent.⁵³⁻⁵⁵,⁵⁸

4.Evidence acquisition

In this study, data were collected from multiple databases. The official websites of national and regional pharmacopeial authorities were examined using the World Health Organization index,⁵² and library resources were utilized to obtain additional information. Furthermore, databases such as Google Scholar and PubMed were also assessed to identify relevant materials.

5.Findings and harmonization recommendations

Based on the comparative data obtained from this study, recommendations for harmonizing pharmacopeial updates related to BET are presented in Table 1. In addition, acceptance criteria, specifications, and endotoxin limits for various antibiotics are summarized in Table 2.

6.Endotoxin test methods in different pharmacopeias

In terms of microbiological requirements for bacterial endotoxin testing, the 2024 BP, 2022 IP, 2021 JP, 2022 EP, 2022 Ph. Int., and 2025 USP were reviewed and compared with respect to bacterial endotoxin limit acceptance criteria and microbial enumeration specifications for antibiotics and antibiotic drug preparations (Table 1). Since all comparisons in this study were based on data from previously published pharmacopeias, ethical approval was not required.

(i) BP: The BP published similar monographs and contained the same microbiological standards as those described in General Text 5.8.⁵³
(ii) IP: The IP Chapter 2.2.3, “Biological Methods,” provides BET values for all types of antibiotics listed in Table 1.⁵⁸
(iii) USP: The USP Biological Text <85> and General Chapter 2025 describe the microbiological characteristics of both nonsterile and sterile products. Each product type should have distinct bacterial endotoxin limits.⁵⁴
(iv) JP: The JP General Test 4.01 (page V-A764) provides BET methods for all types of antibiotics listed in Table 1.⁵⁵
(v) Ph. Eur.: The 2022 Ph. Eur. Chapter 2.6.14 (page 226) provides bacterial endotoxin testing guidelines for all types of antibiotics listed in Table 1.⁵⁶
(vi) Ph. Int.: The 2022 Ph. Int. Text 3.4 provides bacterial endotoxin testing procedures for all types of antibiotics listed in Table 1.⁵⁷

7.BET limits for antibiotic preparations in different pharmacopeias

This comparative study focused on pharmacopeial quality standards concerning the BET for antibiotics. It aims to evaluate the differences and similarities among the IP, BP, JP, Ph. Eur., Ph. Int., and USP regarding the endotoxin limit specifications for injectable antibiotics (Table 2).

The methods used for endotoxin detection, such as the RPT and LAL assay, are discussed in detail. These methods ensure patient safety by preventing pyrogenic reactions caused by endotoxins, which are LPS released from Gram-negative bacteria. This study compares the endotoxin limits across different antibiotics and identifies both commonalities and gaps among the pharmacopeial standards.

Comparison of different pharmacopeias revealed significant variations in the guidelines and standard limits for BET methods, indicating regional disparities in the BET specifications across different pharmacopeias (Table 2). Differences in regulatory guidelines, policies, historical developments, and national health priorities have resulted in discrepancies between the pharmacopeias. Additionally, epidemiological factors such as disease outbreaks and pandemics may also drive pharmacopeial content, contributing to these disparities.Therefore, updating and aligning pharmacopeial guidelines to ensure better regulation of bacterial endotoxin limits across various pharmacopeias from different regions is crucial. This harmonization of bacterial endotoxin standards across different pharmacopeias is essential, as it provides consistent and comparable results for the same product at different locations, ensures patient safety, and reduces trade barriers by establishing universally recognized standards for pyrogen detection.

8.Recombinant factor C (rFC) assay

The rFC assay is an animal-free alternative method used for bacterial endotoxin testing. The rFC enzyme, cloned from the horseshoe crab, is used in this assay. The solution emits fluorescence when the activated rFC enzyme cleaves a synthetic fluorogenic substrate in response to endotoxin binding. Without the use of horseshoe crabs, the rFC test provides the same level of reliability as the LAL approach, using a single enzymatic step. Compared with conventional LAL test methods, rFC exhibits higher affinity and sensitivity to LPS, which enhances assay specificity.⁶⁹ Compared with the LAL test, the rFC assay provides reliable results and is more readily accepted than other bacterial endotoxin testing methods.⁷⁰

9.Discussion

The methods used for endotoxin detection have evolved over time, with the initial Rabbit Pyrogen Test (RPT) being replaced by more efficient and reliable methods, such as the LAL test and the BET.⁷¹ These methods—including the qualitative gel-clot limit test and photometric quantitative techniques such as kinetic turbidimetric, kinetic chromogenic, and end-point chromogenic methods—have become essential tools for ensuring the safety and effectiveness of medical and pharmaceutical products through the detection of bacterial endotoxins.⁷²

The comparative study of BET standards for antibiotics across the 2022 IP, 2025 USP, 2021 JP, 2022 Ph. Eur., 2022 Ph. Int., and 2024 BP reveals both similarities and differences in the allowable endotoxin limits for different antibiotics. Overall, while there are slight differences between the pharmacopeias, the USP generally provides the most detailed and comprehensive specifications for endotoxin limits across a wide range of antibiotics,⁵³ whereas the IP, JP, and BP occasionally lack specific details for certain drugs.This comparison underscores the importance of harmonizing pharmacopeial standards to ensure consistent quality control and patient safety in antibiotic production worldwide.

10.Harmonization of microbiological bacterial endotoxin acceptance

10.1. Criteria for antibiotics

From the above review, it can be seen that all pharmacopeias demonstrate both similarities and differences in bacterial endotoxin acceptance criteria, including those for antibiotics and their active pharmaceutical ingredients (APIs). The similarities and differences in endotoxin limits and their acceptance criteria, as specified in the various pharmacopeias, need to be harmonized and streamlined to ensure that if a test is within the specified limit according to the harmonized monographs, it satisfies the requirements of all pharmacopeias, as well as the regulatory stipulations of the relevant countries.The International Council for Harmonisation (ICH) has already harmonized pharmacopeial texts for use in the ICH regions through the BET General Chapter Q4B Annex 14. These ICH quality guidelines can be used interchangeably within ICH regions.²⁰

10.2. Practical barriers to harmonization

Each pharmacopeia has evolved independently, which has led to discrepancies in pharmacopeial standards. In addition, variations in legal frameworks, rigid regulatory requirements, and national priorities act as major hindrances to achieving consistency in drug quality and testing standards. This regulatory inertia serves as a significant practical barrier to pharmacopeial harmonization.Furthermore, harmonization of pharmacopeias also affects manufacturing processes, testing methods, regulatory compliance, and international trade, ultimately influencing production costs.

11.LPS variability complicating standardization

LPSs released by bacteria are complexed with LPS-binding protein, a plasma protein primarily produced by hepatocytes and one of the most extensively studied soluble proteins with LPS-binding capacity.²⁸ LPS is composed of an O-specific polysaccharide chain, a core oligosaccharide region, and lipid A, which contributes to its toxicity. Lipid A, an amphiphilic glycolipid, exhibits structural variations under different temperature and pH conditions.²⁸ Thus, LPS variability in chemical structure and molecular interactions leads to complications in the quantification of endotoxin measurements.⁷³

12.Clinical impacts of divergent endotoxin limits

When administered to patients, pharmaceutical drugs containing high concentrations of bacterial endotoxins—a type of pyrogen—can cause severe and sometimes fatal immune reactions. Excess endotoxins in injectable drugs, contaminated medical equipment, or implanted materials can result in septic shock and systemic inflammation.⁷⁴

When Gram-negative bacteria die, their cell walls release endotoxins, which are LPS. Lipid A, the toxic component of LPS, binds to host immune cells such as macrophages through Toll-like receptor 4. This interaction triggers a cascade of hyperinflammatory responses that may lead to the following detrimental clinical effects:⁷⁴

(i) Flu-like symptoms and fever (septic fever)
(ii) Shock accompanied by hypotension
(iii) Disseminated intravascular coagulation
(iv) Multiple organ dysfunction syndrome
(v) Inflammatory diseases.

13.Cost impact of harmonization on manufacturers

The main benefits of harmonizing bacterial endotoxin standards for manufacturers include reduced production costs due to simplified procedures, streamlined regulatory compliance, and enhanced market accessibility. However, several drawbacks persist, such as the initial expenses associated with implementing harmonized procedures and the potential challenges in validating complex samples that require specialized testing. The currently employed qualitative BET method (gel-clot limit test) is labor-intensive; however, the universal acceptance of quantitative methods by all pharmacopeias may alleviate costly manufacturing challenges through prior standardization.⁴⁰

14.Recommendations for key stakeholders

While regulatory authorities should implement Good Pharmacopeial Practices and the Common Technical Document format for data submission, pharmacopeial commissions should adopt Good Pharmacopeial Practices and align with ICH guidelines. Collaboration should be fostered through forums such as the Pharmacopeial Discussion Group. In cooperation with their stakeholders, pharmacopeias and regulatory bodies worldwide have been actively and effectively pursuing compendial harmonization for decades. These continuing efforts to harmonize compendial standards provide perspectives that may be useful when considering pharmacopeias’ future trajectory.

15.Conclusion

It can be concluded that the test methods and specifications used to ensure the bacterial endotoxin limits of antibiotic preparations in various pharmacopeias need to be harmonized. A harmonized pharmacopeia would provide a globally standardized procedure for establishing bacterial endotoxin acceptance criteria for antibiotics and antibiotic preparations. This approach would offer a more convenient option for global drug manufacturers involved in the import and export of antibiotic raw materials or injections and would also eliminate the burden of performing multiple analytical methods to comply with different endotoxin acceptance criteria set by various pharmacopeias.