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Evaluation of the transferability of in vivo Pig-a gene mutation assay across three laboratories

  • Journal of Alternatives to Animal Experiments
  • Abbr : J Altern Anim Exp
  • 2017, 11(1), pp.1-8
  • DOI : 10.23032/jaae.2017.11.1.004
  • Publisher : Korean Society for Alternatives to Animal Experiments
  • Research Area : Medicine and Pharmacy > Veterinary > Basic Veterinary Medicine > Laboratory Animal Medicine
  • Received : November 28, 2017
  • Accepted : December 21, 2017
  • Published : December 29, 2017

Young-Shin Chung 1 Bum-Soo Park 1 Se-Hee Han 1 Ji-Yeon Lee 1 Ji-Young Kim 2 Seung-Min Back 2 Cho-Rong Park 3 Su-Hwan Kim 3 Jong-Kwon Lee 4

1호서대학교 생명공학과
2안전성평가연구소
3(주)바이오톡스텍
4식품의약품안전평가원 특수독성과

ABSTRACT

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.

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