Establishment of In Vitro Alternative Methods for Cysteine Pyrogen Testing and Comparison with the Rabbit Method
10.12300/j.issn.1674-5817.2026.029
- VernacularTitle:半胱氨酸热原检查体外替代方法的建立及其与家兔法的比较研究
- Author:
Yating GAI
1
;
Lihua LIN
1
Author Information
1. Department of Pharmacology, Xiamen Institute for Food and Drug Quality Control, Xiamen 361012, China
- Publication Type:Journal Article
- Keywords:
Cysteine;
Bacterial endotoxin test;
Micro kinetic chromogenic assay;
Pyrogen test;
Monocyte activation test;
Rabbits
- From:
Laboratory Animal and Comparative Medicine
2026;46(5):740-749
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveThis study attempts to establish a cysteine bacterial endotoxin test as an in vitro alternative method for cysteine pyrogen detection, so as to reduce the use of rabbits in pyrogen detection and ensure the welfare of experimental animals. MethodsAccording to the Chinese Pharmacopoeia (2025 edition) Volume Ⅳ, General Chapters 1142 and 1143, and Guidelines 9251 and 9301, methodological studies on pyrogen and bacterial endotoxin testing were conducted on three batches of cysteine raw material. We used the pyrogen test (rabbit method, RPT) and the in vitro pyrogen test (monocyte activation test, MAT) to detect pyrogens in cysteine and assessed cell viability during the MAT for one batch. Bacterial endotoxin testing was performed using the gel-clot method (limit test) and the bacterial endotoxin photometric assay (microkinetic chromogenic assay, kinetic chromogenic assay, kinetic turbidimetric assay). ResultsPyrogen testing of cysteine using the two pyrogen test methods yielded false-negative results. In the RPT, all the test articles spiked with endotoxin did not show a positive pyrogen response. The MAT results showed that cysteine inhibited interleukin-6 (IL-6) secretion by human peripheral blood mononuclear cells (hPBMCs) after incubation, with recovery rates below 50%. Interference was observed in bacterial endotoxin testing using the gel-clot method: all three batches showed interference at dilutions not exceeding the maximum valid dilution (MVD). In the interference test using the microkinetic chromogenic method, when cysteine was prepared at a concentration of 1.05 mg/mL, the recovery rates for all three batches were between 50% and 200%, indicating that the interference was eliminated and the method was feasible. The recovery rates measured by the microkinetic chromogenic assay, kinetic chromogenic assay and kinetic turbidimetric assay fell within the acceptable range of 50%–200% when the cysteine concentration was 1.05 mg/mL. The bacterial endotoxin levels in the three batches met the specified limits. ConclusionThis study establishes a bacterial endotoxin assay for cysteine and preliminarily demonstrates that the photometric techniques can replace RPT for the bacterial endotoxin testing of cysteine.
