A n nu a lRepor t2 0 2 0

2020
Japan Chemical Industry Association

Annual Report 2020

Long-range Research Initiative

Title of Research:
18_S01-01

The validation study of EpiSensA (Epidermal Sensitization Assay);the in vitro skin sensitization assay based on reconstructed humanepidermis
Principal Investigator:
Hideyuki Mizumachi, PhD (Safety Science Research Laboratory, Kao Corporation)
2606, Akabane, Ichikai-Machi, Haga-Gun, Tochigi 321-3497, Japan(tel) +81-70-3297-2574, (e-mail) mizumachi.hideyuki@kao.com
Collaborators:
Sho Suzuki (Safety Science Research Laboratory, Kao Corporation)
Summary of Research:
Lipophilic chemicals are difficult to correctly evaluate by existing in vitro tests because these tests
employ aqueous-phase systems. To overcome the limitation, we focused on a reconstructed
human epidermis (RhE) and developed the Epidermal Sensitization Assay (EpiSensA) based on
the expression of four marker genes related to induction of skin sensitization. Based on thecomparison with the results of animal test, we confirmed that EpiSensA has better predictive
performance for a variety of chemicals including lipophilic chemicals than existing in vitro tests.
Therefore, the validation study of EpiSensA was started from July 2018 at JaCVAM (JapaneseCenter for the Validation of Alternative Methods) to adopt it for OECD test guideline. We havereported that the validation management team (VMT) concluded the technical transfer from thelead laboratory to three participating laboratories (Food and Drug Safety Center, KOSÉ
Corporation and LION Corporation) was successfully completed.
For evaluation of within laboratory reproducibility (WLR), three laboratories test 15 codedchemicals in three independent experiments each. This Phase I was conducted in three parts(Phase I-A, I-B, and I-C), each including 5 chemicals. The Phase I-A and I-B were finished, andsubsequent Phase I-C was performed. As a result, the WLR was not confirmed at one out of thefive test chemicals in two laboratories, so the data analysis of the chemical was performed. In
consequence, it was confirmed that the cross-contamination of volatile strong sensitizer was thecause of unreproducible result. Therefore, protocol modification was proposed to avoid thepotential cross-contamination effect of volatile strong sensitizers, and approved by the VMT.
Finally, the WLR using 15 coded test chemicals satisfied the target criteria or 85% in alllaboratories. The validation management team concluded that the entire Phase I study wascompleted successfully.
Timeline:
March 1, 2020 - February 28, 2021
Topics:Oral presentation at JCIA LRI Annual Workshop 2020 “The validation study of EpiSensA(Epidermal Sensitization Assay); the in vitro skin sensitization assay based on reconstructed
human epidermis” (On-line, August 21st, 2020)
Publications:
The 33rd Annual Meeting of the Japanese Society for Alternatives to Animal Experiments, On-line,
Japan, Nov. 2020.

1

Title of Research:
18_R04-01

Exploring roles and simple estimation methods of species
sensitivity distribution for deriving PNECs
Principal Investigator:
Masashi Kamo. National Institute of Advanced Industrial Science and Technology. ResearchInstitute of Science for Safetymasashi-kamo@aist.go.jp
Collaborators:
Yuichi Iwasaki. National Institute of Advanced Industrial Science and Technology. ResearchInstitute of Science for Safety
Wataru Naito. National Institute of Advanced Industrial Science and Technology. ResearchInstitute of Science for Safety
Summary of Research:
We developed models to predict species sensitivity distribution (SSD) parameters from limited
data (research 1), and quantified the uncertainties associated with SSD-based assessment(research 2). In the research 1, to predict mean and standard deviation (SD) of acute SSDs, wedeveloped multiple linear regression models that included, in addition to readily obtainable
descriptors, the mean and SD of the log10-transformed concentrations that are acutely toxic to one
algal, one crustacean, and one fish species, as predictors. We also found that the means ofchronic SSDs were, one average, 10 times lower than acute SSD means, and the SDs of chronic
and acute SSDs were similar. In the research 2, a condition that we need uncertainty factor todetermine a predicted no effect concentration (PNEC) by SSD approach was argued.
Timeline:
March 1, 2020 - February 28, 2021
Topics:Oral presentation at JCIA LRI Annual Workshop 2020 “Exploring roles and simple estimation
methods of species sensitivity distribution for deriving PNECs” (On-line, August 21st, 2020)
Publications:
1. Yuichi Iwasaki, Kiyan Sorgog (2021). Estimating species sensitivity distributions on the basisof readily obtainable descriptors and toxicity data for three species of algae, crustaceans, andfish. PeerJ 9: e10981 https://doi.org/10.7717/peerj.10981
2. Kyoshiro Hiki, Yuichi Iwasaki (2020). Can we reasonably predict chronic species sensitivity
distributions from acute species sensitivity distributions? Environmental Science &Technology 54(20):13131-13136. doi: 10.1021/acs.est.0c03108.
3. Masashi Kamo, Kiyan Sorgog (2020). Response to: Quantifying the precision of ecologicalrisk: Misunderstandings and errors in the methods for assessment factors versus species
sensitivity distributions by Drs. Scott E. Belanger and Gregory J. Carr. Ecotoxicology andEnvironmental Safety. Vol.207, No. 111542
4. 加茂将史、岩崎雄一、Kiyan Sorgog、内藤航 (2021). 生態リスク評価における種の感受性分布の活用について. 第 55 回日本水環境学会年会. オンライン開催 (in Japanese)
5. 日置恭史郎、岩崎雄一（2021）. 慢性毒性に基づく種の感受性分布を急性毒性に基づく種の感受性分布から予測できるか？日本環境毒性学会第１回オンライン研究発表会、オンライン開催(inJapanese)
6. Kiyan Sorgog, Yuichi Iwasaki (2020). Developing models to estimate parameters of species
sensitivity distribution by three species ecotoxicity data. 41st annual meeting on Society ofEnvironmental Toxicology and Chemistry (SETAC) North America. virtual conference.

2

Title of Research:
18_R05-01

Study on the contribution of microplastics to bioaccumulation andbiological magnification towards fish
Principal Investigator:
Tatarazako Norihisa（Professor, Ehime University, Graduate School of Agriculture）
3-5-7 Tarumi, Matsuyama, Ehime, 790-8566 Japan
Collaborators:
Tatsuhiro Niino (LSI Medience Corporation)
1000 Kamoshidacho, Aoba-ku, Yokohama-shi, Kanagawa 227-0033, Japan
Shin Takahashi（Ehime University, Graduate School of Agriculture）
3-5-7 Tarumi, Matsuyama, Ehime, 790-8566 Japan
Yoshifumi Horie (Akita Prefectural Univesity)
241-438 Kaidobata-Nishi Nakano Shimoshinjo Akita City 010-0195 Japan
Summary of Research:
The microplastics (MPs) are known to adsorb chemical substances and there is a concern about
those chemicals may be taken more efficiently to organisms intermediated by MP and that
biological concentration or biological magnification is accelerated. Our study will try to clarify
whether the chemical substances adhered to MP are eluted, and absorbed / transferred /accumulated in the body of organisms.
In previous report, the concentration analysis of the PAHs which is adsorbed to MP, and uptaken
and bioaccumulated by fish were conducted presuming that comparison of the applied dose andchemical concentration of fish meat calculates the accumulation factor. It was found by analyzing
the 13 kinds of PAHs that the amount of chemical substances adsorbed varies depending on thetype of MP and environmental conditions, and that the more hydrophobic the substance, the moredifficult it is to desorb once it is adsorbed on the MP surface.From the result of examining theuptake of MP, medaka did not affected by MPs of all sizes and Daphnia did not affected by MPs
with a diameter of 1 ㎛ or more. By using the fluorescent plastics, the MP beads were observedto accumulate in the gastrointestinal tract but not transferred to the outside of the gastrointestinal
tract. Also, no translocation of MP from Daphnia to medaka was observed by feeding the Daphnia
which had exposed (and taken) MP, to medaka.
There were difficulties to explain the presence or absence of the vector effect only by analyzing
the chemical concentration accumulated in the body, we employed new method to detect whether
the toxicity changes under the condition of coexistence of the MP and chemical, and to confirm thevector effect indirectly. As a result, in the closed experimental system, the presence of MP withsizes of uningested by living organisms reduced the concentration of chemical substances byadsorption, and the toxicity was mitigated. In the case of medaka, the toxic effect was mitigatedeven with the sizes of ingested MP and any harmful effect due to the vector effect were notconfirmed. Only Daphnia exhibited adverse effects when the intakes were high, but it was due tointestinal obstruction by MP.
In conclusion, the vector effect of MP exists in the theory, though it is estimated that the amount of
MP in the environment has no harmfulness in reality.
Timeline:
March 1, 2020 - February 28, 2021
Topics:Oral presentation at JCIA LRI Annual Workshop 2020 “Study on the contribution of microplastics
to bioaccumulation and biological magnification towards fish” (On-line, August 21st, 2020)
Publications:
1. Norihisa Tatarazako, ”Ecotoxicity of microplastics”, Society of Environmental Hormone,News
Leter 23-2（2020）3

2. Norihisa Tatarazako, ”Ecotoxicity of microplastics”, Microfiber lecture（Japan Chemical Fiber
Association）, on web, 27/11/2020
3. Norihisa Tatarazako, Yukiyo Okazaki, Ecotoxicity of microplastics, The 22nd UK-Japan Annual
Scientific Workshop,Research into Environmental Endocrine Disrupting Chemicals &Chemicals of Emerging Concern, on web, 27/10/2020

4

Title of Research:
19_R01-01

Development of rapid, accurate, and low-cost AI drug hazard
assessment method by human stem cell test
Principal Investigator:
Wataru Fujibuchi, Ph.D.
Professor, Center for iPS Cell Research and Application (CiRA), Kyoto University
53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507
Tel: 075-366-7012
email: fujibuchi-g@cira.kyoto-u.ac.jp
Summary of Research:
We developed a new method “hEST”, which has more than 95% accuracy predicting neurotoxicity,and genotoxic/non-genotoxic carcinogen categories, using machine learning of gene network dataobtained from exposure of human embryonic stem cells to chemicals. This year, we selected 9
hepatotoxins and 10 non-hepatotoxins as negative control for SVM. 1) We determined IC10
concentration for all chemicals and obtained gene expression data from 19 chemical exposure
samples. As a results, 2) we can predict at more than 90 % accuracy.
Timeline:
March 1, 2020 - February 28, 2021
Topics:Oral presentation at JCIA LRI Annual Workshop 2020 “Development of rapid, accurate, andlow-cost AI drug hazard assessment method by human stem cell test” (On-line, August 21st,
2020)
Publications:Panina, Y., Yamane, J., Kobayashi, K., Sone, H., Fujibuchi, W. 2021. Human ES and iPS cells
display less drug resistance than differentiated cells, and naïve-state induction further decreases
drug resistance. J. Toxicol. Sci. 46 (3), pp. 131-142.

5

Title of Research:
19_R03-01-2

Development of a novel alternative method for evaluation ofsensitizing potential and allergenicity by measuring human T cell
activation and differentiation (Key event 4)
Principal Investigator:
Takayuki Yoshimoto, PhD (Professor, Tokyo Medical University, Institute of Medical Science)
6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan(Tel) +81-3-3351-6141 Ext. 431, (E-mail) yoshimot@tokyo-med.ac.jp
Collaborators:
Izuru Mizoguchi, PhD (Research Associate, Tokyo Medical University, Institute of Medical
Science) 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan(Tel) +81-3-3351-6141 Ext. 448, (E-mail) imizo@tokyo-med.ac.jp
Summary of Research:
Several in vitro assays to predict the sensitizing potential of chemicals have been developed sofar. However, these animal testing alternatives cannot distinguish chemical respiratory and skinsensitizers, although the risk management systems for them are quite different. Therefore, wehave been aiming at developing a novel in vitro assay, which can discriminate them by taking
advantage of the fundamental differences between their modes of function; development of helper
T (Th) 2 immune responses, which are critically important for respiratory sensitization. Recently,
we established a new 3-dimentional (3D) dendritic cell (DC) coculture system consisting of human
airway epithelial cell line, immature DCs derived from human peripheral monocytes, and lungfibroblast cell line. This coculture system was shown to successfully discriminate respiratory
sensitizers from skin sensitizers using 6 representative chemical sensitizers by more enhanced
mRNA expression of key costimulatory molecule OX40 ligand (OX40L), which is important for Th2
differentiation, in DCs (Mizoguchi et al. Front Immunol. 2017). In this project, we have been further
trying to establish a new 2-step DC/T coculture system by introducing T cells in the DC coculture
system, in which the Key event 4, that is T cell, in the adverse outcome pathway of sensitization
can be used as a marker. To increase the versatility, we are also trying to generate DC progenitor
cell lines and T cell lines to apply for it.
In the 8th term, as a DC progenitor cell line, we eventually generated monocytic cell lines byintroducing genes related to cell survival and cell cycle into peripheral CD14 + monocytes. Whenthese cell lines were applied for the 3D DC coculture system, the similar more enhanced mRNA
expression of OX40L was observed by respiratory sensitizers than skin sensitizers. When thesecell lines were then applied for the 3D DC/T coculture system using primary CD4+T cells, thesimilar more enhanced mRNA expression of IL-4 was also observed by respiratory sensitizers. As
the possible molecular mechanism to enhance IL-4 mRNA expression, augmented expression of
the transcription factor GATA-3, that is important for Th2 differentiation, was also observed byrespiratory sensitizers. Furthermore, we have generated allogenic Th1 and Th2 cells andsyngeneic cedar pollen antigen (Cryj1)-specific Th2 cells, and are currently cloning them.
Timeline:
March 1, 2020 - February 28, 2021
Topics:Oral presentation at JCIA LRI Annual Workshop 2020 “Development of a novel alternative method
for evaluation of sensitizing potential and allergenicity by measuring human T cell activation anddifferentiation (Key event 4)” (On-line, August 21st, 2020)
Publications:
Yoshimoto T. Expectations for immune on-chip. Symposium on New Technology for Cell-based
Drug Assay (Web meeting, Jan. 26, 2021)

6

Title of Research:
19_R05-01

Establishment of medaka kinetic model for aged microplastic andadsorbed chemical
Principal Investigator:
Yuji Oshima (Kyushu University, Faculty of Agriculture, Laboratory of Marine Environmental
Science Professor)
744 Motooka, Nishi-ku, Fukuoka 8020345, Japan(tel&fax)+81-092-802-4607
Collaborators:
Yohei Shimasaki (Faculty of Agriculture, Kyushu University, Associated Professor)
Kang Ik Joon (School of Interdisciplinary Science and Innovation, Kyushu University, AssociatedProfessor)
Summary of Research:
Plastic has an extensive use in our daily life due to its low cost, lightweight and hard todecompose. Due to their high production and a quite slow degradation, plastic pollution has been
expanded worldwide in aquatic environments. Pollution of plastic from large items to small particle(microscopic plastic MP, < 5mm) were emerged. Effect of MP and its vector effect with toxicants
are of concerns. Many researchers have studied these topics. However, most of study were usedpristine MPs. Thus, risk analysis of aged MP is required.
In this study, three kinds of aged plastics (sphere polyethylene (PE), grind PE, grind
polystyrene (PS)) were prepared by exposure to ultraviolet for 180 and 460 hours. Changes on
chemical characters were detected by aging. Decrease in sorption of anthracene on aged MPs
were observed, especially aged grind PS showed 1/8 of sorption constant (ke) compared withthat of pristine grind PS. Bioaccumulation studies were performed in medaka mix-exposed withanthracene and MPs, respectively. As results, no effect was observed in maximum concentrationof anthracene in medaka body among aging, quality or shape of MPs. However, anthracene was
detected in grind-aged PE and PS even at depuration periods. These results suggested that small
particle of PE or PS in grind MPs may remained inside of body after depuration period. Further
study on another type of aged MP is required.
Timeline:
March 1, 2020 - February 28, 2021
Topics:Oral presentation at JCIA LRI Annual Workshop 2020 “Establishment of medaka kinetic model foraged microplastic and adsorbed chemical” (On-line, August 21st, 2020)
Publications:
1. Qiu, X., Saovany, S., Takai, Y., Akasaka, A., Inoue, Y., Yakata, N., Liu, Y., Waseda, M.,
Shimasaki, Y., Oshima, Y., 2020. Quantifying the vector effects of polyethylene microplastics
on the accumulation of anthracene to Japanese medaka (Oryzias latipes). Aquatic Toxicology
228, 105643.
2. Assas, M., Qiu, X., Chen, K., Ogawa, H., Xu, H., Shimasaki, Y., Oshima, Y., 2020.
Bioaccumulation and reproductive effects of fluorescent microplastics in medaka fish. Mar.
Pollut. Bull. 158, 111446.
3. Liu, Y., Qiu, X., Xu, X., Takai, Y., Ogawa, H., Shimasaki, Y., Oshima, Y., 2021. Uptake anddepuration kinetics of microplastics with different polymer types and particle sizes inJapanese medaka (Oryzias latipes). Ecotoxicol. Environ. Saf. 212, 112007.

7

Title of Research:
19_D08-01

Elucidation of the formation mechanism of microplastics
Principal Investigator:
Kuroda Shinichi (Graduate School of Science and Engineering, Gunma University)
29-1 Honmachi, Ota City, Gunma Prefecture 373-0057(tel) 0276-50-2434 (e-mail) skuroda@gunma-u.ac .jp
Collaborators:
Kouzai Hiroaki (Department of Science and Engineering, Kanto Gakuin University), Kuriyama
Takashi (Graduate School of Organic Materials and Systems Science, Yamagata University)
Ohtani Hajime (Graduate School of Engineering, Nagoya Institute of Technology), Igarashi Toshio(Research Division for Science and Technology, Kanazawa University), Hiejima Yusuke (Research
Division for Science and Technology, Kanazawa University), Tokumitsu Katsuhisa (Materials
Science Division, Faculty of Engineering, University of Shiga Prefecture)
Summary of Research:
The following conclusions were obtained with using various methods to clarify the mechanisms
by which secondary microplastics are formed from the viewpoint of polymer science and polymer
engineering.
In the Py-APGC-MS measurement of plastics, by focusing on fragment ions characteristic ofplastics and recording extracted ion chromatograms, we succeeded in extracting individualcomponent information from plastic mixture samples of PP, PE, PVC, PS, and PET. We also
succeeded in detecting PS taken up by Daphnia magna (adult).
As a result of comparative study of seawater degradation and photo-degradation of various
plastics, it was found that the presence of seawater suppressed the progression of degradation of
HDPE, PP, and PET by inhibiting the increase in sample temperature and oxygen diffusion, while
the elongation at break of PA66 decreased significantly with irradiation time in seawater
degradation. However, PA66 showed a significant decrease in elongation at break with irradiation
time, suggesting that seawater was the main factor in the degradation.
In the case of PP, the surface of the micro-cut powder showed a brittle fracture surface and the"crack pattern" grew inside, suggesting that MP was generated from the consolidated growth ofcracks, similar to the outdoor exposure. Crack patterns were also observed in the river MPs after
pulverization by glass beads, but no brittle fracture surfaces were observed in most of the leaking
marine MPs, suggesting that the MPs have smooth surfaces and are less prone to collapse-type
weathering mechanisms.
On the other hand, the PET bottles recovered from the river retained their shape despite being
left in the environment for nearly 20 years. The outer surface of the PET bottles showed no signsof becoming microplastics, although some degradation such as a decrease in molecular weight
was observed.
Finally, it was confirmed that all types of plastics, including elastomers, could be miniaturized toa spherical shape of less than 1 mm in a short time by treatment with a new type of jet mill device
with multiple blades arranged in an air flow channel and additional treatment with a regular jet mill.
In addition, it was confirmed that the bead-mill treatment efficiently progressed the plastic
refinement in a water atmosphere.
Timeline:
March 1, 2020 - February 28, 2021.
Topics:Oral presentation at JCIA LRI Annual Workshop 2020 “Elucidation of the formation mechanism of
microplastics” (On-line, August 21st, 2020)
Publications: Presentations: (Only the PI's presentation)
1. Shinichi Kuroda, "Elucidation of the formation mechanism of microplastics," 69th Symposiumon Macromolecules, September 16, 2020 (online)8

2.

Shinichi Kuroda, "Elucidation of the formation mechanism of microplastics," MICRO 2020,
November 25, 2020 (online)

9Title of Research:
20-1-11

Proposal of a new AOP for the neurotoxicity and developmental
neurotoxicity assessment of glutamate receptor binding agoniststhat cause learning and memory impairment.
Principal Investigator:
Yuko Sekino, PhD (Project Professor, Endowed Laboratory of Human Cell-Based Drug Discovery,
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan(tel)+81-3-5841-4355, (e-mail) yukos@mol.f.u-tokyo.ac.jp
Collaborators:
Hiroyuki Yamazaki, PhD (Assistant Professor, Department of Pharmacology, Gunma University
Graduate School of Medicine, 3-39-22, Showa-machi, Maebashi, Gunma, 371- 8511, Japan(tel)+81-27-220-8052, (e-mail) spikar@gunma-u.ac.jp
Yonehiro Kanemura, MD, PhD (Director, Department of Biomedical Research and Innovation
Institute for Clinical Research, National Hospital Organization Osaka National Hospital, 2-1-14
Hoenzaka, Chuo-ku, Osaka 540-0006, Japan(tel)+ 81-6-6942-1331, (e-mail) kanemura.yonehiro.hk@mail.hosp.go.jp
Daiju Yamazaki, PhD (Section Chief, Division of Pharmacology, National Institute of Health
Sciences, 3-25-26 Tonomachi, Kawasaki-ku, Kawasaki City, Kanagawa, 210-9501, JAPAN(tel)+81-44-270-6642, (e-mail) daiju-y@nihs.go.jp
Summary of Research:
This study proposes an adverse outcome pathway (AOP) in which the molecular initiating event(MIE) is the binding of compounds to glutamate receptors and the key event (KE) is drebrin loss.
The adverse event (AO) is the learning and memory impairment caused by morphologicalchanges in dendritic spines. Drebrin is an actin-binding protein that governs dendritic spine
formation of CNS neurons and is responsible for the morphological plasticity of dendritic spines
associated with learning and memory. The subcellular localization of drebrin is determined byglutamate receptor activity, and when drebrin is lost, the learning and memory mechanism does
not function properly. In this study, we have established an experimental system using frozen
hippocampal neurons prepared from rat embryo. We will construct an in vitro method to evaluate
compounds that cause learning and memory impairment, and will replace animal experiments forneurotoxicity. We have developed an image processing algorithm for quantitative analysis ofneuron count, dendrite length, and drebrin clusters from high-content image data using a confocal
image cytometer. In particular, the brightness distribution analysis of drebrin clusters is highly
sensitive. We have started to develop a machine learning platform for AI. From the images of
immunocytochemical staining, we will clarify the indices for quantitatively evaluating the structuralchanges of neurons, and provide SOPs for culture techniques and analysis methods. In the future,
we are planning to build an experimental system using neurons derived from human iPS cells.
Timeline:
March 1, 2020 - February 28, 2021
Topics:Oral presentation at JCIA LRI Annual Workshop 2020 “Proposal of a new AOP for the neurotoxicity
and developmental neurotoxicity assessment of glutamate receptor binding agonists that cause
learning and memory impairment.” (On-line, August 21st, 2020)
Publications:
1. Shogo Mase，Yuko Sekino，Tomoaki Shirao, Toshinari Mitsuoka, “Optimization of algorithms
to quantify changes in drebrin distribution induced by glutamate stimulation of cultured
neurons for a confocal quantitative image cytometer.” The 63rd Annual Meeting of theJapanese Society for Neurochemistry, Web, September, 202010

2.

Toshinari Mitsuoka, Shogo Mase，Noriko Koganezawa, Yuichi Kato, Tomoaki Shirao, Yuko
Sekino “Assessment of CB agonist CP55940 in maturity for rat hippocampal neurons using ahigh-throughput immunocytochemical assay and image digital analysis.” The 94th Annual
Meeting of the Japanese Pharmacological Society, Web＋On Site, Sapporo, March 2021.

11

Title of Research:
20-3-02

Development of a short-term in vivo assay for thyroid hormone
disrupting activity in maternal rats and their fetus/pups asprescreening for potential of developmental neurotoxicity.
Principal Investigator:
Tomoya Yamada, DVM, PhD (Fellow, Sumitomo Chemical Company, Ltd., Environmental Health
Science Laboratory)
3-1-98 Kasugadenaka, Konohanaku, Osaka 554-8558, Japan(e-mail) yamadat8@sc.sumitomo-chem.co.jp
Collaborators:
Hiroaki Aoyama, PhD (Executive Director, Toxicology Division, The Institute of Environmental
Toxicology)
4321 Uchimoriya-machi, Joso-shi, Ibaraki 303-0043, Japan(e-mail) aoyama@iet.or.jp
Hidenori Suto, PhD (Research Associate, Sumitomo Chemical Company, Ltd., Environmental
Health Science Laboratory)
3-1-98 Kasugadenaka, Konohanaku, Osaka 554-8558, Japan(e-mail) sutoh@sc.sumitomo-chem.co.jp
Summary of Research:
While thyroid hormones (THs) are essential for brain development, effects of mild suppression ofmaternal blood THs by hepatic enzyme inducers on the infant brain developments is not fully
understood. Conducting guideline study to identify of developmental neurotoxicity induced bychemicals requires significant resources (animals, time and costs). A simple screening test forinvestigating whether maternal chemical exposure reduces brain THs in fetal and neonatal rats
would be valuable. To verify reliability of the Comparative Thyroid Assay with additional
examination of the brain, THs and histology, propylthiouracil (PTU, 10 ppm) and phenobarbital (PB,
1000 ppm) were dosed by feeding. Clearly suppressed brain THs in rat fetuses and pups andbrain abnormality (heterotopia) in pups were noted with PTU but not with PB. Reproducibility andeffects of PB at higher dose level will be examined in further study.
Timeline:
March 1, 2020 – February 28, 2021.
Topics:Oral presentation at JCIA LRI Annual Workshop 2020 “Development of a short-term in vivo assay
for thyroid hormone disrupting activity in maternal rats and their fetuses/pups as prescreening forpotential of developmental neurotoxicity.” (On-line, August 21st, 2020)
Publications:
1. Hidenori Suto1, Akira Sato2, Keiko Ogata1, Kenta Minami1, Tadashi Kosaka2, Hitoshi Hojo2,
Naofumi Takahashi2, Naruto Tomiyama2, Katsumasa Iwashita1, Hiroaki Aoyama2, Tomoya
Yamada1 (1 Sumitomo Chemical Company, Ltd. 2 The Institute of Environmental Toxicology)“Development of a short-term in vivo assay for thyroid hormone disrupting activity in maternalrats and their fetuses/pups as prescreening for potential developmental neurotoxicity:
Propylthiouracil and phenobarbital examples. I. Findings in maternal rats and their fetuses”.
The 48th Annual Meeting of the Japanese Society of Toxicology, Kobe, Japan, July 2021.
2.

Akira Sato1, Hidenori Suto2, Keiko Ogata2, Kenta Minami2, Tadashi Kosaka1, Hitoshi Hojo1,
Naofumi Takahashi1, Naruto Tomiyama1, Katsumasa Iwashita2, Hiroaki Aoyama1, Tomoya
Yamada2 (1 The Institute of Environmental Toxicology 2 Sumitomo Chemical Company, Ltd.)“Development of a short-term in vivo assay for thyroid hormone disrupting activity in maternalrats and their fetuses/pups as prescreening for potential developmental neurotoxicity:
12

Propylthiouracil and phenobarbital examples. II. Findings in maternal rats and their pups”.
The 48th Annual Meeting of the Japanese Society of Toxicology, Kobe, Japan, July 2021.

13

Title of Research:
20-3-06

Evaluation methods for toxicity using indices of developing neurons
Principal Investigator:
Yaichiro Kotake, Ph.D (Professor, Hiroshima University, Graduate School of Biomedical and
Health Sciences)1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan(tel)-+81-82-257-5325 (e-mail)yaichiro@hiroshima-u.ac.jp
Collaborators:
Yasunari Kanda, Ph.D (Division Head, National Institute of Health Sciences, Division of
Pharmacology)
3-25-26 Tonomachi, Kawasaki-ku, Kawasaki 210-9501, Japan(tel)-+81-44-270-6640 (e-mail)kanda@nihs.go.jp
Summary of Research:
In recent years, basic research in the field of neuroscience has revealed many molecules involvedin neurite outgrowth and neuronal reorganization, but there have been few attempts to evaluate
the toxicity of chemical substances using these molecules as indicators. The purpose of this study
was to identify better indicators of developmental neurotoxicity of chemicals from key molecules inneurodevelopment and to clarify their usefulness in assessing developmental neurotoxicity ofchemicals.
First, we established culture conditions for primary cultured neurons of the rat cerebral cortex andexamined changes in mRNA expression of 12 key molecules which play an important role inneurodevelopment until days in vitro 21. The results showed three major patterns: genes whose
expression was low in early culture and increased with culture, genes whose expression was
maximum in mid-culture, and genes whose expression was maximum in early culture anddecreased with culture. Next, we used methylmercury, a known developmental neurotoxin, toinvestigate the effects of these molecules on gene expression. We found that methylmercury
suppressed the expression of three genes, Dlg4, Syp and Bdnf. In the next year, we will
investigate whether these gene expression changes are also observed with other developmental
neurotoxicants and whether these phenomena are reproduced in human iPS neurons, which will
provide clues for the development of evaluation indices for developmental neurotoxicity.
Timeline:
March 1, 2020 - February 28, 2021
Topics:Oral presentation at JCIA LRI Annual Workshop 2020 “Evaluation methods for toxicity using
indices of developing neurons” (On-line, August 21st, 2020)14

Title of Research:
20-3-08

Development of a novel test for the assessment ofneuroinflammation useful to elucidate adverse outcome pathways indevelopmental neurotoxicity
Principal Investigator:
Yuhei Nishimura (Department of Integrative Pharmacology, Mie University Graduate School of
Medicine)
2-174 Edobashi, Tsu, Mie 514-8507, Japan
Tel: 81-59-231-5006 email: yuhei@med.mie-u.ac.jp
Collaborators:
Takashi Shiromizu (Department of Integrative Pharmacology, Mie University Graduate School of
Medicine)
Summary of Research:
The incidence of neurodevelopmental disorders such as autism, attention deficit hyperactivity
disorder, and learning disabilities is increasing year by year and has become a major social
problem. Although chemical exposure during development has been suggested to increase therisk of developing these neurodevelopmental disorders, the detailed mechanisms underlying thedevelopmental neurotoxicity remain largely unclear. Various molecular initiating events (MIEs) are
involved in the developmental neurotoxicity mechanisms of chemical substances, but different
MIEs often exert toxicity through a common key event (KE). Developing a test method that canevaluate such a common KE is an effective strategy that will lead to the elucidation of the adverse
outcome pathway (AOP) of chemical substances. Impaired differentiation of neural stem cells into
neurons and astrocytes, and neuroinflammation mediated by microglia, which are macrophages
resident in the central nervous system, are attracting attention as the KE common to thedevelopmental neurotoxicity of chemical substances. The purpose of this study is to develop anovel test method that can assess the developmental neurotoxicity of chemicals focusing on
neuroinflammation through microglia and the differentiation of neural stem cells into neurons andastrocytes. In 2020, we were able to produce a one-color zebrafish that selectively expressed afluorescent protein mVenus in macrophages/microglia. We also produced two-color zebrafish that
expressed fluorescent proteins Cerulean and mCherry in neurons and astrocytes, respectively. In
addition, we decided the optimal concentrations of valproic acid, chlorpyriphos, ethinylestradiol,and ethanol, which are positive chemical substances that induce neuroinflammation, to assess thedevelopmental neurotoxicity related to the neuroinflammation in zebrafish. In 2021, we will try to
produce a three-color zebrafish that expresses mVenus, Cerulean, and mCherry inmacrophages/microglia, neuron, and astrocytes, respectively.
Timeline:
March 1, 2020 - February 28, 2021
Topics:Oral presentation at JCIA LRI Annual Workshop 2020 “Development of a novel test for theassessment of neuroinflammation useful to elucidate adverse outcome pathways indevelopmental neurotoxicity” (On-line, August 21st, 2020)
Publications:
Wakai E, Suzumura Y, Ikemura K, Mizuno T, Watanabe M, Takeuchi K, Nishimura Y: An Integrated
In Silico and In Vivo Approach to Identify Protective Effects of Palonosetron in Cisplatin-Induced
Nephrotoxicity. Pharmaceuticals 2020, 13(12):480.

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Title of Research:
20-3-10

Establishment of an adverse outcome pathway for the evaluation ofdevelopmental neurotoxicity in chemical-induced hypothyroidism
Principal Investigator:
Tsuyoshi Nakanishi (Laboratory of Hygienic Chemistry and Molecular Toxicology, Gifu
Pharmaceutical University)
Collaborators:
Daisuke Matsumaru (Laboratory of Hygienic Chemistry and Molecular Toxicology, Gifu
Pharmaceutical University)
Summary of Research:
In humans, environmental chemicals that induce hypothyroidism can be a significant risk
during pregnancy, since fetal brain development in early pregnancy depends on thyroid hormones
supplied by the mother. However, the detailed causal relationship between abnormalities in thyroid
function during pregnancy and adverse effects on the development of the baby's brain is unknown.
In order to solve such a problem, we focused on differentiation markers of mature neurons andproduced reporter transgenic mice (Tg mice) carrying luciferase (Luc2) and LacZ genes
downstream of a differentiation marker promoter. In this fiscal year, we verified the usefulness of
Tg mice by characterizing the expression of reporter genes in Tg mice. Among the three lines
obtained, the highest Luc2 activity was observed in the mature males and females of line #1 at thesites responsible for higher brain functions, including the cerebral cortex. Although, in males, Luc2
activity was also observed in the testes as in brain tissue, Luc2 activity was rarely detected inother organs. In the brain, Luc2 expression peaked immediately after birth and dropped sharply as
the age progressed. When the brain slices were X-gal stained to confirm the expression of the
LacZ gene, lacZ also showed the same expression pattern as Luc2. These results indicate that
tracing luc2 and LacZ expression may provide some understanding of neuronal differentiation. In
the future, in vivo imaging immediately after birth of this Tg mouse will be performed to verify theusefulness of this mouse, and the adverse effects induced during hypothyroidism during
pregnancy will be examined by tracing the expression fluctuations of the reporter genes.
Timeline:
April 1, 2020 – March 31, 2021
Topics:Oral presentation at JCIA LRI Annual Workshop 2020 “Establishment of an adverse outcome
pathway for the evaluation of developmental neurotoxicity in chemical-induced hypothyroidism”(On-line, August 21st, 2020)
Publications:
Ishida K, Minamigawa Y, Mori K, Matsumaru D, Nakanishi T, Establishment of neuronaldifferentiation tracer mouse for evaluation of developmental neurotoxicity, The 141st Annual
Meeting of the Pharmaceutical Society of Japan (Hiroshima), 2021.

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Title of Research:
20-6-04

Development of a conceptual model for environmental risk
assessment of microplastics and a trial risk assessment in TokyoBay

Principal Investigator:
Wataru Naito. National Institute of Advanced Industrial Science and Technology (AIST),
Research Institute of Science for Safety and Sustainability (RISS), Group Leader
w-naito@aist.go.jp
Collaborators:
Masashi Gamo, AIST, RISS
Kiyotaka Tsunemi, AIST, RISS
Hideo Kajihara, AIST, RISS
Kyoko Ono, AIST, RISS
Isamu Ogura, AIST, RISS
Bin-Le Lin, AIST, RISS
Xue Mianqiang, AIST, RISS
Hiroyuki Mano, AIST, RISS
Yuichi Iwasaki, AIST, RISS
Naohide Shinohara, AIST, RISS
Yuriko Ishikawa, AIST, RISS
Summary of Research:
The purpose of this study project is to review the existing domestic and international literature forenvironmental risk assessment of microplastics (MPs), to develop a conceptual model that
concretely shows the risk assessment procedure of MPs, and to conduct a trial risk assessment
for Tokyo Bay. In FY2020, we have reviewed a couple of assessment documents published byinternational organizations and the latest literature, and presentation materials found inacademic conferences (SETAC Europe, SETAC North America, Water Environment Society ofJapan). We have exchanged opinions with MPs experts to understand current researchchallenges. After reviews and discussions among co-researchers, current problems andchallenges in conducting environmental risk assessment of MPs were identified under six
categories, i.e., MPs in the ocean, emission estimates, exposure analysis, analysis andmeasurement methods of MPs, hazard assessment, and environmental risk assessment casestudies and critical reviews. The key elements identified include lack of knowledge on the origin
and formation mechanism of MPs in the ocean, determination and validation of emission factors
necessary for estimating environmental emissions of various MPs, characterization of MP types,
sizes, shapes, lack of information on vertical distribution of MPs in water and changes in density ofMPs, lack of standard measurement methods for MPs, and lack of ecological relevance between
toxicity tests and the real environment, and the need to clarify the purpose and target of theassessment. In addition, a draft framework for the environmental risk assessment of MPs wasdeveloped in order to propose a conceptual model that shows specific procedures for risk
assessment considering the characteristics of MPs. In the next fiscal year, while reviewing thelatest findings that will contribute to the environmental risk assessment of MPs, we will examine
the methodology at each stage of the risk assessment, determine the important parameters, andstart the trial risk assessment for Tokyo Bay.
Timeline:
March 1, 2020 - February 28, 2021
Topics:Oral presentation at JCIA LRI Annual Workshop 2020 “Development of a conceptual model forenvironmental risk assessment of microplastics and a trial risk assessment in Tokyo Bay” (On-line,
August 21st, 2020)17

Publications:
Wataru Naito (2020) Challenges to Environmental Risk Assessment of Marine Plastics andMicroplastics. BIOINDUSTRY 37(9): 40-49 (In Japanese)18

A n n u a l R e p o r t 2 020
Japan Chemical Industry Association
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