Comparison of the different protocols of the Hen's Egg Test-Chorioallantoic Membrane (HET-CAM) by evaluating the eye irritation potential of surfactants.

The Hen's Egg Test - Chorioallantoic Membrane (HET-CAM) is a valid alternative method used to assess the potential for eye irritation from chemicals. This method is the only one that mimics the conjunctivae of the eye and aims to semi-quantitatively evaluate the irritant potential of a chemical on the chorioallantoic membrane surrounding the chicken embryo in egg by observing the irritation effects on the membrane immediately after the pure or diluted chemical is applied. The purpose of this study was to compare the different protocols of the HET-CAM, the French and German protocols, by evaluating the eye irritation potential of surfactants. The comparison led to the optimization of the French protocol, generating an adapted one, to reduce subjectivity in the test evaluation, ensuring more accurate results and greater quality control. The comparison showed that there are no significant differences between the results obtained in the French and German protocols. HET-CAM is known to overestimate the results and to be able to accurately identify non-irritant products and it is a great candidate to be part of a Bottom-up test strategy. It also can be used in a battery of tests to completely replace rabbits.

Applicability of the Monocyte Activation Test (MAT) in the quality control of the 17DD yellow fever vaccine.

The need for alternatives to animal use in pyrogen testing has been driven by the Three Rs concept. This has resulted in the inclusion of the monocyte activation test (MAT) in the European Pharmacopoeia, 2010. However, some technical and regulatory obstacles must be overcome to ensure the effective implementation of the MAT by the industry, especially for the testing of biological products. The yellow fever (YF) vaccine (17DD-YFV) was chosen for evaluation in this study, in view of: a) the 2016-2018 outbreak of YF in Brazil; b) the increase in demand for 17DD-YFV doses; c) the complex production process with live attenuated virus; d) the presence of possible test interference factors, such as residual process components (e.g. ovalbumin); and e) the need for the investigation of other pyrogens that are not detectable by the methods prescribed in the YF vaccine monograph. The product-specific testing was carried out by using cryopreserved and fresh whole blood, and IL-6 and IL-1ß levels were used as the marker readouts. After assessing the applicability of the MAT on a 1:10 dilution of 17DD-YFV, endotoxin and non-endotoxin pyrogens were quantified in spiked batches, by using the lipopolysaccharide and lipoteichoic acid standards, respectively. The quantitative analysis demonstrated the correlation between the MAT and the Limulus amoebocyte lysate (LAL) assays, with respect to the limits of endotoxin recovery in spiked batches and the detection of no pyrogenic contamination in commercial batches of 17DD-YFV. The data demonstrated the applicability of the MAT for 17DD-YFV pyrogen testing, and as an alternative method that can contribute to biological quality control studies.

Applicability of the Monocyte Activation Test (MAT) for hyperimmune sera in the routine of the quality control laboratory: Comparison with the Rabbit Pyrogen Test (RPT).

Pyrogen tests are safety assays performed during the routine quality control of injectable products required by regulatory agencies. Currently, there are three available testing possibilities: 1) the Rabbit Pyrogen Test (RPT); 2) the Bacterial Endotoxin Test (BET); and 3) test systems using human whole-blood or monocytes, termed Monocyte Activation Test (MAT). Although BET is often considered as a replacement for the animal test, it is unable to detect pyrogens other than endotoxin. MAT is based on the human fever reaction and thus, most closely reflects the human response. The aim of this study was to conduct a parallel comparison of the RPT and MAT for hyperimmune sera (HS) batches analyzed during the routine of a quality control laboratory. MAT was performed in the same 43 batches of HS previously tested using RPT. The results showed that MAT presented 100% sensitivity and approximately 85% specificity as compared to RPT, i.e., no false-negative results were obtained. Few suspicious samples, which were negative in the RPT after retesting, provided divergent positive results suggesting a lower limit of detection of MAT. MAT is thus able to detect contaminants in biological products such as HS batches.