RESUMO
Snake antivenoms are parenterally administered; therefore, endotoxin content must be strictly controlled. Following international indications to calculate endotoxin limits, it was determined that antivenom doses between 20 mL and 120 mL should not exceed 17.5 Endotoxin Units per milliliter (EU/mL) and 2.9 EU/mL, respectively. The rabbit pyrogen test (RPT) has been used to evaluate endotoxin contamination in antivenoms, but some laboratories have recently implemented the LAL assay. We compared the capability of both tests to evaluate endotoxin contamination in antivenoms, and we found that both methods can detect all endotoxin concentrations in the range of the antivenom specifications. The acceptance criteria of RPT and LAL must be harmonized by calculating the endotoxin limit as the quotient of the threshold pyrogenic dose and the therapeutic dose and the dose administered to rabbits as the quotient of the threshold pyrogenic dose and the endotoxin limit. Since endotoxins from Gram-negative bacteria exert different pyrogenicity, if contamination occurred, antivenom batches that induce pyrogenic reactions may be found in spite of passing LAL specifications. Although LAL assay can be used to assess endotoxin content throughout the antivenom manufacturing process, we recommend that the release of final products be based on the results of both methods.
Assuntos
Antivenenos/administração & dosagem , Endotoxinas/toxicidade , Caranguejos Ferradura/metabolismo , Proteínas de Membrana , Pirogênios/toxicidade , Animais , Cavalos , CoelhosRESUMO
The rabbit pyrogen test was used to evaluate veterinary products, suggesting the temperature rise of 0.6°C as the ending point for the positive results. The test doses were calculated based on the therapeutic dose increased from three to seven times. The semi-quantitative Limulus amoebocyte lysate (LAL) gel clot test was performed and compared to the LAL spectrophotometric chromogenic, quantitative assay. The comparative evaluation of the samples showed correlation and reproducibility of the results. The interference test was carried out, the procedure validated and the maximum valid dilution established for the analysis of the products without Pharmacopoeial specifications. Two of the twenty-eight products of different pharmacological groups evaluated didn't meet the requirements and were reproved. The specifications investigated are suggested to be used for the purity evaluation of the veterinary parenteral products, as a contribution to assure the quality and safety of the products.
Realizou-se a avaliação de pirogênios em produtos veterinários de uso parenteral, pelo método da hipertermia em coelhos, calculando-se, para o teste das amostras, doses com concentrações de três a sete vezes superiores à terapêutica. Preconizou-se como resposta positiva o aumento de temperatura de 0,6°C. Utilizou-se também o ensaio do lisado de amebócitos do Limulus (LAL) por geleificação, semiquantitativo, executando o teste de interferentes, validando o procedimento e estabelecendo a máxima diluição válida para a análise de cada produto. Paralelamente, efetuou-se avaliação comparativa de amostras com o método do LAL cromogênico, quantitativo, demonstrando correlação e reprodutibilidade dos resultados. Avaliaram-se vinte e oito produtos de diferentes classes farmacológicas, observando-se que dois não cumpriram as especificações, sendo reprovados. Sugere-se que as especificações estudadas sejam adotadas, contribuindo para aprimorar o controle de contaminantes, garantindo a qualidade e a segurança dos produtos veterinários.
RESUMO
The rabbit pyrogen test was used to evaluate veterinary products, suggesting the temperature rise of 0.6°C as the ending point for the positive results. The test doses were calculated based on the therapeutic dose increased from three to seven times. The semi-quantitative Limulus amoebocyte lysate (LAL) gel clot test was performed and compared to the LAL spectrophotometric chromogenic, quantitative assay. The comparative evaluation of the samples showed correlation and reproducibility of the results. The interference test was carried out, the procedure validated and the maximum valid dilution established for the analysis of the products without Pharmacopoeial specifications. Two of the twenty-eight products of different pharmacological groups evaluated didn't meet the requirements and were reproved. The specifications investigated are suggested to be used for the purity evaluation of the veterinary parenteral products, as a contribution to assure the quality and safety of the products.
Realizou-se a avaliação de pirogênios em produtos veterinários de uso parenteral, pelo método da hipertermia em coelhos, calculando-se, para o teste das amostras, doses com concentrações de três a sete vezes superiores à terapêutica. Preconizou-se como resposta positiva o aumento de temperatura de 0,6°C. Utilizou-se também o ensaio do lisado de amebócitos do Limulus (LAL) por geleificação, semiquantitativo, executando o teste de interferentes, validando o procedimento e estabelecendo a máxima diluição válida para a análise de cada produto. Paralelamente, efetuou-se avaliação comparativa de amostras com o método do LAL cromogênico, quantitativo, demonstrando correlação e reprodutibilidade dos resultados. Avaliaram-se vinte e oito produtos de diferentes classes farmacológicas, observando-se que dois não cumpriram as especificações, sendo reprovados. Sugere-se que as especificações estudadas sejam adotadas, contribuindo para aprimorar o controle de contaminantes, garantindo a qualidade e a segurança dos produtos veterinários.