Development and assessment of new risk assessment methods

Title of Research:
13_PT01-02

Applied research of a novel in vitro method for developmental
toxicity to facilitate the industrial utilization
Principal Investigator:
Kohji Yamakage (Division of Alternative Toxicology Test, Hatano Research Institute, Food andDrug Safety Center)
Ochiai 729-5, Hadano-shi, Kanagawa 257-8523, Japan(tel) +81-(0)463-82-4751, (e-mail) yamakage.k@fdsc.or.jp
Collaborators:
Noriho Tanaka (Hatano Research Institute, Food and Drug Safety Center); Hajime Kojima(National Institute of Health Sciences, Japan); Koichi Saito, Noriyuki Suzuki (Sumitomo Chemical,Co., Ltd); Naohiro Ikeda (Kao Corporation); Kazunori Yanagi (Sumika Chemical Analysis Service,Ltd.); Takashi Omori (Doshisha University)
Summary of Research:
We are developing the Hand1-Luc Embryonic Stem Cell Test (Hand1-Luc EST) which uses ES
cells introduced the reporter gene into as a test method to detect embryonic toxicants. To
familiarize the Hand1-Luc EST in the chemical industry, the Hand1-Luc EST is currently undervalidation process. To obtain objective evaluation and reliability of this test from experts, it isnecessary to accumulate data and validate the test about the robustness, the predictability and theinter- and intra- laboratory reproducibility.
In 2013 as a project supported by the Ministry of Economy, Trade and Industry, the validation
management team, composed of experts on developmental toxicity or alternative test methods
from Japan and other counties, has been organized. The international validation study was started
with the collaboration of Japanese three laboratories to optimize the test protocol, to establish
acceptance criteria, and to verify the technical transfer and inter- and intra-laboratory
reproducibility. In the phase 0 and phase 1 studies, the technical transfer and intra-laboratory
reproducibility has been cleared respectively.
In Phase2a study, which was started as the project supported by LRI, the necessity of multiple
experiments for prediction of the positivity or the negativity of chemicals was investigated because
at the very beginning, we proposed that the prediction can evaluate with only one experiment.
Experiments in each chemical were performed two or three times according to the revised protocol
and the results of four coded chemicals were compared between three laboratories. In eachlaboratory, all of four chemical results were consistent with the in vivo results and the accuracy andthe inter-laboratory reproducibility were 100%. Thus, the improved protocol was confirmed to bevalid. Following those results, to verify the intra- and the inter-laboratory reproducibility, 24 codedchemicals (8 kinds of chemicals, 3 bottles per chemical were prepared) were distributed to the 3laboratories and the phase 2b/2c studies were started (May 2014 – February 2015). By gathering
the results from phase 2a, 2b and 2c studies, the inter-laboratory reproducibility reached 83.3%(10/12 chemicals were consistent with the in vivo data) and the intra-laboratory reproducibility was
higher than 75% in each laboratory (the number of chemicals of which the results were consistent
between three tests in each chemical, Lab A: 7/8 chemicals, Lab B: 7/8 chemicals, Lab C: 6/8chemicals).
Timeline:
November 1, 2013Topics:
Presented at the 2nd Annual Conferences of New LRI (29th August, 2014, Tokyo)
Publications:27th Annual Meeting, the Japanese Society for Alternatives to Animal Experiments, Yokohama,