Relationship between Endotoxin Content in Vaccine Preclinical Formulations and Animal Welfare: An Extensive Study on Historical Data to Set an Informed Threshold

Abstract

The most widely known pyrogen impurity in vaccines is the Gram-negative bacterial endotoxin lipopolysaccharide (LPS). When administered at toxic doses, endotoxin triggers inflammatory responses, which lead to endotoxic shock. The literature on endotoxic content (EC) for preclinical vaccines' formulations used in animal studies is very poor, and the recommended thresholds are solely based on commercial vaccine limits set for humans and are, therefore, not connected to the actual impact of EC on animal welfare for species used in preclinical research studies. An extensive study to evaluate the presence of a potential relationship between endotoxin content in formulations administered to mice (the most common species used in preclinical research studies) and their welfare was conducted to calculate an EC threshold for formulations of candidate vaccines. Three years of historical data, from more than 500 formulations of different antigen types (i.e., proteins, glycoconjugates, OMV/GMMA) injected into more than 5000 mice, was evaluated with two alternative statistical methodologies, both demonstrating that there is no significant relationship between actual endotoxin levels and mouse welfare. The calculation of thresholds was, therefore, performed by consistency versus formulations that demonstrated no impact on animal welfare.

Keywords: animal welfare; endotoxins; in vivo; preclinical studies; threshold.

Figure 1

Percentage of animals with at least one VetCare report potentially correlated to the presence of endotoxins vs. median EC of the formulation (DP). Stars indicate not potentially intrinsically pyrogenic formulations, and dots indicate potentially intrinsically pyrogenic formulations.

Figure 2

Percentage of animals with at least one VetCare report potentially correlated to the presence of endotoxins vs. different EC level groups (Group 1 EC ≤ 10 EU/mL, Group 2 10 EU/mL < EC ≤ 25.5 EU/mL and Group 3 EC > 25.5 EU/mL) with the 95% confidence intervals.

Figure 3

EC of the formulation (DP) injected for each animal. Stars indicate not potentially intrinsically pyrogenic formulations and dots indicate potentially intrinsically pyrogenic formulations. Dashed line corresponds to the proposed threshold for not potentially intrinsically pyrogenic formulations, while dotted line corresponds to the proposed threshold for potentially intrinsically pyrogenic formulations.

Figure 4

Percentage of animals with at least one VetCare report potentially correlated to the presence of endotoxins vs different EC level (classes: EC ≤ 20 EU/mL, EC lower than the limit reported in [1] and 20 EU/mL < EC ≤ 200 EU/mL, EC higher than the limit reported on [1] and lower than the proposed threshold) with the 95% confidence intervals.

Figure 5

Percentage of animals with at least one VetCare report potentially correlated to the presence of endotoxins vs different EC levels EC ≤ 200 EU/mL, proposed threshold for not potentially intrinsic pyrogenic formulations and 200 EU/mL < EC ≤ 89,110 EU/mL, proposed threshold for intrinsic pyrogenic) with the 95% confidence intervals.