2017, Vol.11, No.1

Journal of Alternatives to Animal Experiments 2023 KCI Impact Factor : 0.07

pISSN : 1975-9657
https://journal.kci.go.kr/ksaae

In order to establish a pig-a gene mutation assay as a regulatory test for evaluating in vivo genotoxicity in Korea, the transferability study as a stage II validation was performed using the test substance, N-ethyl-N-nitrosourea (ENU) across three laboratories. The lead laboratory (KIT) standardized the protocol using Rat MutaFlow® Kit and transferred the methodology to two participating laboratories (Hoseo University and Biotoxtech Co. Ltd.). Sprague Dawley rats were treated with vehicle or three dose levels of ENU (10, 20, 40mg/kg) by oral gavages on the 3 consecutive days. On the day 15 after the first treatment, bloods were collected and analyzed to estimate the percentage of reticulocytes (%RETs), mutant type of RETs and erythrocytes (RBC) using flowcytometer. In results, the exposure of ENU induced the dose-dependent increases in the mutant frequencies of RBC CD59- and RET CD59- , while %RETs were not affected. Data comparisons among three participating laboratories indicated the good transferability with the high correlation coefficients (0.97-0.99) between the lead and participating laboratories. In conclusion, the pig-a gene mutation assay may be the effective test for the estimation of in vivo gene mutation and the validation studies may be needed using more test substances in Korea.

This study was carried out in accordance with OECD guidelines on skin absorption test for the comparison of reconstructed human skin models, EFT-306™ and KeraSkinFT™ with human skin and pig skin. Three OECD standard compounds were used for penetration and absorption: Caffeine, benzoic acid and testosterone, all of which differ vastly in their lipophilicity. Reconstructed human skins were more permeable to low lipophilic compounds such as caffeine than human and pig skin. Differences became most obvious with KeraSkinFT™ showing a relatively weak penetration barrier on caffeine. EFT-306™ and KeraSkinFT™ both showed similar penetration patterns in the case of lipophilic compounds, benzoic acid and testosterone compared with human and pig skins. High correlation was observed on the penetration flux between the reconstructed human skin models and human and pig skin, even though different penetration rates existed. The reconstructed human skin models can be regarded as generally useful on lipophilic compounds for in-vitro penetration studies.

Drug-induced torsades de pointes (TdP) have been linked to a sudden cardiac death. Prolongation of the action potential duration (APD) and the QT interval (PQT), in nonclinical assays, have been investigated before drug development. However, TdP can often occur without any appreciable QT prolongation, and drug development companies and researchers may ask better accurate models or methods. We wish to verify whether it is effective to determine the cardiac toxicity as a result of the measurement using the TRIaD assay in the rabbit cardiac monophasic action potential. ‘TRIaD’ means Trianguation, Reverse use dependence, Instability and Dispersion. In this study, to confirm this assay, control, negative control and positive control were classified based on the degree of cardiotoxicity. Control group was perfused by only normal Tyrode’s solution. Diltiazem and Amoxicillin were used as negative controls. Terfenadine, Quinidine, Amiodarone, and Nifedipine were used as positive controls. Based on these, proarrhythmic scores were calculated as follow: instability 20 points, triangulation 10 points, and RUD 5 points. Both in control and negative groups, proarrhythmic score did not exceed the 25 points, but exceeded in all positive groups. These data propose that this is robust and reliable assay that can add value to drug-induced prolongation/TdP risk assessment. Additionally, this assay provides detailed information on the overall profile of drug-induced electrophysiological effects. Therefore, TRIaD assay can be a valuable to establish for the cardiovascular risk assessment of drugs.