D2.186 - Toxicity Assessment of Repeated Doses of Apis mellifera Venom for Immunotherapy

Preclinical studies are essential in vaccine development to ensure safety before human application. They allow us to identify and characterize potential adverse effects related to vaccine components (allergens, adjuvants, excipients), as well as establishing the maximum doses that can be used in human treatment without toxic effects. Hymenoptera venom is an extract that, in addition to causing allergic reactions in sensitized patients, may contain compounds that are toxic depending on the administered dose. Therefore, such studies are crucial for Hymenoptera immunotherapy vaccines to minimize the risk of severe adverse effects while inducing venom tolerance.

In this work we have studied the local and systemic effect of repeated doses administration of the immunotherapy with Apis mellifera venom obtained by electrostimulation of Spanish bees.

The study was performed in Sprague Dawley rats following the 3R principle, receiving 5 repeated subcutaneous administrations (days 1, 15, 29, 43, and 57) with placebo,100 and 300 µg of venom/animal/dose in 1 mL. Evolution of any adverse events is studied after 2 weeks of recovery following the last administration.

Changes in body weight and organs, haematological and biochemical parameters, as well as organ histopathology or mortality are evaluated.

No treatment-related mortality was recorded during the study. Additionally, no biologically significant differences were observed in body weight, food intake, clinical pathology, biochemical parameters or organ weights at necropsy between the placebo and test item-treated animals. Histopathologic examination of organs from all animals revealed no toxicologically meaningful test item-related effects. Although no irritation symptoms were noted at the injection site, histological findings indicated minor physiological responses to tissue damage. Except for two moderate instances in Group C, these events were classified as low severity and resolved within the two-week recovery period.

Both doses, 100 and 300 µg/animal, administered via repeated intermittent subcutaneous injections, were well tolerated and did not cause any signs indicative of local or systemic toxicity. In the case of the higher dose, transient local reactions were observed, which resolved by the end of the recovery period. Considering the absence of systemic effects at both dose levels, a systemic No-Observed-Adverse-Effect Level (NOAEL) can be established at 300 µg/animal/administration.