Development and assessment of new risk assessment methods
Title of Research:
13_PT01-02-2

Applied research of a novel in vitro method for developmental
toxicity to facilitate the industrial utilization
Principal Investigator:
Koji Yamakage, PhD (Director, Hatano Research Institute, Food and Drug Safety Center)
Ochiai 729-5, Hatano-shi, Kanagawa 257-8523, Japan(tel) +81-(0)463-82-4751, (e-mail) yamakage.k@fdsc.or.jp
Collaborators:
Noriho Hanaka (Hatano Research Institute, Food and Drug Safety Center) ; Hajime Kojima(National Institute of Health Sciences, Japan); Koichi Saito, Noriyuki Suzuki (Sumitomo Chemical,Co., Ltd); Yuichi Ito (Kao Corporation); Kazunori Yanagi (Sumika Chemical Analysis Service, Ltd.);
Takashi Omori (KOBE University)
Summary of Research:
The purpose of the present research is to lead the Hand1-Luc EST, test using ES cells transfectedwith a reporter gene to a wide use in the chemical industry. To achieve this goal, the Hand1-Luc
EST is currently under validation process. According to experts, to satisfy objective valuation andreliability of the test, it is necessary to gradually gather data, and verify the robustness, thepredictability and the inter- and intra- laboratory reproducibility of the method. Before adoption of
the test in 2013 by the Ministry of Economy, Trade and Industry, the validation management team,composed of Japanese and international expert on developmental toxicity or alternative test
methods, has been organized. The international Validation was started to verify, with the aid ofJapanese collaborating labs, the improvement of the protocol, the satisfaction of the establishedacceptance criteria, the technical transfer and inter- and intra- laboratory reproducibility. Until now,
the technical transfer and high intra-laboratory reproducibility has been cleared by tests done withphase 0 and phase 1 study. With the support of LRI, by gathering the results from phase 2a, 2band 2c, 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.
This semester, firstly, when phase2 was finished, we improved the way of analyzing the data after
considering the advice from experts. Concretely, the way of fitting curves was changed from a 2
parameter curve fitting to a 3 parameter curve fitting. Thanks to this revision, the relation between
the IC50 and ID50 ratio was improved and thus, it was decided that the 3 parameter curve fitting
shall be used for further experiments. The IC50 and the ID50 values being changed by the newanalysis, the prediction model was revised. The phase 3 consisted in 16 chemicals tested in 3
participant laboratories to test the within laboratory reproducibility of the Hand1-Luc EST. One
dose finding study and two or three definitive studies were carried out according to the results
given by the introduction of the data (IC50, ID50, Maximum dose) in the prediction model. 4
chemicals did not meet the requirements of the within laboratory reproducibility (1 laboratory had adifferent prediction from the 2 other labs). By analyzing more in details the data obtained, some
explanations were found. The first point was the determination of the maximum dose of somechemicals that precipitated at this concentration. The second point was a high dilution ratio chosen
by a laboratory leading to a lesser accuracy for the IC50 and ID50 determination. Facing those facts,
it was acknowledged that the reproducibility would be significantly improved by minor modificationof the protocol concerning those points. The phase 3 satisfied the criteria fixed in the study plan(within laboratory reproducibility of 75%, 12/16 chemicals).The predictivity and the applicability of
Hand1-Luc EST were also discussed. On the whole, it was concluded that the positive predictive
value was very high and thus this test would be useful to detect positive embryotoxicants by aTop-Down approach. With a common agreement about the end of the validation, the validation
report will be prepared so as to, then, submit the Hand1-Luc EST to the OECD to become a test
guideline.
Timeline: March, 2015Topics: Presented at the 3rd Annual Conferences of New LRI (28th August, 2015, Tokyo)

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