Histological analysis of the toxic effects of commercial formulations of topical dental anesthetics in a chicken embryo chorioallantoic membrane (CAM) model

• Grant number: 23/13866-8
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: March 01, 2024
• End date: December 31, 2024
• Field of knowledge: Health Sciences - Dentistry
• Principal Investigator: Michelle Franz Montan Braga Leite
• Grantee: Isabella França Quadrado
• Host Institution: Faculdade de Odontologia de Piracicaba (FOP). Universidade Estadual de Campinas (UNICAMP). Piracicaba , SP, Brazil

Abstract

Pain has always been present in Dentistry and is one of the primary hindering factors in performing various clinical treatments. Local anesthesia is a technique that allows these procedures to be carried out painlessly; however, paradoxically, the pain of needle insertion and injection of the solution for its administration are major sources of stress and anxiety for dental patients. Therefore, improving techniques that contribute to the development of new topical anesthetics is essential to provide greater comfort to the patient, considering that commercially available formulations are not 100% effective in eliminating the discomfort of this technique. The use of animals in research has been increasingly questioned. As a result, the development of alternative research methods has emerged, such as the HET-CAM method, which uses the chorioallantoic membrane of chicken eggs for toxicological tests. In the master's thesis defended by the student Arthur Antunes Bezerra Costa (defense in August 2023), it was demonstrated that the HET-CAM model is feasible for analyzing the toxicity of commercially available topical dental anesthetics. However, the model still needs to be validated, which will be carried out during the same student's doctoral research. Among the assays to be performed in the methodological validation process, the histological analysis of the membranes exposed to commercial formulations was proposed. Thus, the aim of this study is to assess whether this microscopic analysis is necessary as a complement to the in vivo HET-CAM method to evaluate the toxicity of commercial formulations of topical dental anesthetics. The toxicity assay (experiments completed during the mentioned master's degree) was conducted according to protocol number 96 of the European Centre for the Validation of Alternative Methods (ECVAM), named Hen's Egg Test on the Chorioallantoic Membrane (HET-CAM). The membranes exposed to treatments (controls and commercial topical anesthetics) were removed from the eggs and immersed in 10% buffered formalin for the preparation of histological slides proposed in this research project. The integrity of the layers of the chorioallantoic membrane and its blood vessels, as well as the presence of intravascular and extravascular thrombi, will be examined. The histological findings will be compared to those obtained with the results of the HET-CAM method. This study is part of an ongoing research project aimed at validating the use of this alternative method to replace the irritation test of the oral mucosa in hamsters, which is the gold standard recommended by the International Organization for Standardization - ISO and described in the ISO 10993-10:2010 protocol, which is advocated to evaluate the toxicity of topical formulations for use in the oral cavity, including topical dental anesthetics.