Usage of the method HET-CAM as an alternative model for the oral mucosa irritation test in hamsters in evaluate the irritation potential of toothpaste

Certos ensaios toxicológicos utilizados no controle de qualidade de produtos têm sido questionados por grupos defensores do bem estar dos animais por levarem ao sacrifício e sofrimento injustificado de um número extraordinário de animais. Além de questões éticas, abusca por métodos alternativos é uma questão de grande relevância para os laboratórios oficiais de controle da qualidade. O método HET CAM (Hens Egg Test-Chorion Allantoic Membrane) vem sendo estudado como método alternativo ao teste de irritação ocular em coelhos. No entanto, estudos que busquem avaliar a relação entre os efeitos induzidos in vivo em outras mucosas (e.g. mucosa oral) e o HET CAM são ainda escassos. Neste sentido, o objetivo do presente estudo foi avaliar o potencial de irritação de dentifrícios de uso adulto, apreendidos por Secretarias Estaduais e/ou Municipais de Saúde, e de uso infantil, adquiridos comercialmente no Município do Rio de Janeiro; comparando os resultados obtidos em ensaio in vivo com aqueles obtidos no método HET CAM, abordando as vantagens e desvantagens de cada método. O teste in vivo de irritação da mucosa oral foi realizado em hamsters, conforme preconizado pela ISO 10.993 Parte 10. Dependendo do seu potencial irritante, um produto é classificado como nãoirritante(NI), minimamente irritante (IMin), irritante leve (IL), irritante máximo (IM) e irritante severo (IS). Para o teste in vitro, seguiu-se o método oficial de avaliação do potencial irritanteno ovo de galinha, tendo como princípio a observação dos efeitos irritantes quando em contato com a membrana córion-alantóide do ovo no décimo dia de incubaçãoAnimal welfare groups criticize some toxicological assays, applied in the quality control of products, because of the unjustified suffering of an extraordinary number of animals. Apart from the ethical reasons, the search for alternative methods is a matter of great relevance to the official quality control laboratories. The method HET-CAM (Hens Egg Test-Chorion Allantonic Membrane) is under study as an alternative for the eye irritation test, which is normally performed in rabbits. Studies that intend to evaluate the correlation between the effects induced in vivo in other mucosa (for example oral mucosa) and the HET-CAM, however, are scarce. In this logic, the main goal of the present study was to evaluate the irritation potential of adult’s toothpaste, confiscated by the Secretary of Health from Rio de Janeiro State and/or City, and of commercially available infant’s toothpaste, comparing the results obtained in vivo with the ones obtained with the HET-CAM method. The advantages and disadvantages of both methods were also considered. The oral mucosa irritation test in vivo was performed in hamsters, as established by ISO 10.993-Part 10. The products were classified, according to the irritability potential, as follows: not irritant (NI), minimally irritant (IMin), slightly irritant (IL), maximally irritant (IM) and severely irritant (IS). Concerning the in vitro test, the official method for the evaluation of the irritant potential for application of products on the chorion allantonic membrane of hen eggs was performed and the principle followed was that only eggs at the tenth incubation day were used for the application of substances and observation of effects. In the HET-CAM method, two autonomous analysts tested the products pure (100%) and 10% (triplets) diluted, in independent assays. This test allows the following classification: NI, IL, IM and IS. Twenty products were analyzed, 10 products for adults and the other 10 for infants. In the in vivo method, all products for adults were considered irritants. Within the infant’s products, 60% were considered not irritant or minimally irritant, whereas all the rest were classified as slightly irritant. Except for one adult product, all the other toothpastes studied in this monograph presented a pH level within the security range recommended by the international norm ISO11.609. Concerning HET-CAM, there was no disagreement between the two analysts for the final classification of the products 10% diluted. For the analyses of the pure products, however, divergence on the classification established by the two analysts was observed. The study of the correlation between the values of the scores obtained in vivo and in vitro showed that the irritation index obtained in hamsters associated better with the HET-CAM method performed with the diluted products when compared to the results obtained for the pure products. With the purpose of evaluating the grade of predictability of the in vitro test in comparison to the animal model, a cut-off was established for differentiation between products with low irritancy potential (Ii ≤4,99) and the ones with irritant potential (Ii ≥5,00). Parameters like precision (85%), sensibility (100%) and specificity (50%) were determined. Our study allow us to conclude that, although a satisfying correlation between the results obtained in vitro with the oral mucosa irritation test in hamsters was observed, there is a tendency of overestimation of the results with the HET-CAM method. It is, therefore, recommended that products classified as positive with the HET-CAM might be submitted to further investigation using other in vitro assays (such as citotoxicity, RBC, isolated eye etc) in order to confirm the label of irritan