A n nu a lRepor t2 0 2 1

2021
Japan Chemical Industry Association

Annual Report 2021

Long-range Research InitiativeDevelopment and assessment of new risk assessment methods
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)
Makoto Mizuno, Megumi Sakuma, PhD, Momoko Shibata (KOSÉ Corporation)
Mika Watanabe, Michiyo Yasuda, Michika Kajiwara (Hatano Research Institute, Food and Drug
Safety Center)
Shin-ichi Watanabe, Junko Ueno (LION 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 tested 15 codedchemicals in three independent experiments during Phase I study, and target criteria wasestablished at 85% by the VMT. Several protocol modifications and additional experiments were
performed as appropriate, and the mean WLR resulted in 91% and satisfied the target criteria. The
VMT concluded that the Phase I study was successful. Regarding subsequent Phase II study forevaluation of between laboratory reproducibility (BLR), three laboratories tested 12 codedchemicals once, and target criteria was established at 80%. The BLR was calculated using 27 test
chemicals including Phase I and Phase II chemicals, and the final prediction for the chemicals thatwere tested 3 times in each laboratory was based on the median classification. As a result, theBLR was 89% and satisfied the target criteria. In addition, protocol modification was proposed to
avoid the potential cross-contamination effect of volatile strong sensitizers. Furthermore, hazard
predictive performances at participating laboratories were evaluated using 27 test chemicals, theperformances were similar to that of the lead laboratory. From these results, the VMT concluded
that the Phase II study was successful, and the protocol modification was accepted.
Timeline:
April 1, 2018Topics:
Presentations at 2018,2019, 2020, and 2021 JCIA-LRI Workshop
Publications:
The 34th Annual Meeting of the Japanese Society for Alternatives to Animal
Experiments, Okinawa and On-line, Japan, Nov. 2021.
1Title of Research:
19_R03-01

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:
Although several in vitro assays that predict the sensitizing potential of chemicals have beendeveloped, none can distinguish between chemical respiratory and skin sensitizers. Previously,
we established a new three-dimensional dendritic cell (DC) coculture system consisting of ahuman airway epithelial cell line, immature DCs derived from human peripheral monocytes, and ahuman lung fibroblast cell line. In this coculture system, compared to typical skin sensitizers,
typical respiratory sensitizers showed enhanced mRNA expression in DCs of the key
costimulatory molecule OX40 ligand (OX40L), which is important for T helper 2 (Th2) celldifferentiation. Herein, we established a new two-step DC/T cell coculture system by adding
peripheral allogeneic naive CD4+ T cells to the DCs stimulated in the DC coculture system. In this
DC/T cell coculture system, typical respiratory sensitizers but not skin sensitizers enhanced mRNA
expression of the predominant Th2 marker IL-4 and its transcription factor GATA-3. To improve theversatility, in place of peripheral monocytes, monocyte-derived proliferating cells called CD14-ML
were also used in the DC coculture system. Similar to peripheral monocytes, enhanced mRNA
expression of OX40L was observed by typical respiratory sensitizers compared to skin sensitizers.
In the 9th term, these cell lines were applied to the DC/T cell coculture system with peripheral
allogeneic naive CD4+ T cells, and it was revealed that typical respiratory sensitizers but not skinsensitizers enhance the mRNA expression of IL-4. When allogeneic Th2 cell line was also applied
to it, typical respiratory sensitizer enhanced IL-4 mRNA expression, as well as IL-4 secretion. Thus,
this DC/T cell coculture system might be useful for discriminating between respiratory and skinsensitizers by differential upregulation of IL-4 in T cells.
Timeline:
March 1, 2019-Feberary 28, 2022
Topics:2021 Annual Meeting of The Japan Chemical Industry Association LRI, oral presentation“Development of a novel alternative method for evaluation of sensitizing potential and allergenicity
by measuring human T cell activation and differentiation (Key event 4)” (Web meeting, Aug. 20,
2021)
Publications:
1. Mizoguchi I, et al. A novel coculture system for assessing respiratory sensitizing potential by
IL-4 in T cells. ALTEX in press.
2. Yoshimoto et al. Development of a novel alternative method for evaluation of sensitizing
potential and allergenicity by measuring human T cell activation. The 34th Annual Meeting of
the Japanese Society for Alternatives to Animal Experiments, Web Oral Presentation (Nov.
11-13, 2021, Okinawa)
3. Mizoguchi et al. Development of a novel alternative method for evaluation of sensitizing
potential and allergenicity by measuring human T cell activation and differentiation. The 34th2

Annual Meeting of the Japanese Society for Alternatives to Animal Experiments, Web Oral
Presentation (Nov. 11-13, 2021, Okinawa) President’s Special Award

3

Assessment on the effects on ecosystems and the environment

Title of Research:
19_R05-01
Establishment of medaka kinetic model for aged microplastic and adsorbed 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,
Associated Professor)

Summary of Research:
Plastic has extensive use in our daily life due to its low cost, lightweight, and hard todecompose. Due to their high production and slow degradation, plastic pollution has
expanded worldwide in aquatic environments. Pollution of plastic from large items to small
particles (microscopic plastic MP, < 5mm) emerged. The effect of MP and its vector effectwith pollutants are of concerns. Many researchers have studied these topics. However,most of study were used pristine MPs. Thus, the study on effect of aging on vector effect of
MP is required.
First, commercially available polyethylene pellets were used to produce grind
polyethylene microplastics (ｃgPE-MP, 200 µm), and then this MP was aged by exposure to
UV irradiation equivalent to 5 years in a real environment (aｃgPE-MP).
Next, effect of aging on anthracene (ANT) uptake on medaka fish was examined using the
cgPE-MP and acgPE-MP. No apparent difference was observed in ANT concentrations offish between cgPE-MP and acgPE-MP co-exposure treatments. This result might be
attributed to decrease of ANT concentration in water phase caused by absorption of ANT
to cgPE.
The results of a follow-up test using sphere PE-MP (sPE-MP) and cgPE-MP showedthat the concentration of ANT in the water was reduced to 87% and 73%, respectively.
Simulation results showed a vector effect of sPE-MP and cgPE-MP co-exposure group.
3-year studies have confirmed the vector effect on ANT for PE and PS-MP(approximately 200 µm, 40 mg/L). There seemed to be no significant difference in vector
effect due to shape (sphere or grind) or aging. The cPE-MP also showed that it could
strongly bind to ANT in water and reduce ANT concentration in the fish.
Furthermore, a PE-MP-ANT dual one-compartment model was constructed to predict
vector effect in real environment, and it was estimated that the vector effect would beapparent at high MP concentrations (40 mg/L), but that the vector effect on ANT would be
weak at low MP concentrations (0.1 mg/L, similar to the real environment). In the future
work, it is necessary to construct vector effect prediction model that considers MP quality,4Assessment on the effects on ecosystems and the environment
particle size, vector chemicals.

Timeline:
March 1, 2019 - Feb 1, 2022

Topics: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 and depuration kinetics of microplastics with different polymer types andparticle sizes in Japanese medaka (Oryzias latipes). Ecotoxicol. Environ. Saf. 212,
112007.

5

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) , Kawai Takahiko(Graduate School of Science and Engineering, Gunma University)
Summary of Research:
The purpose of this study is to clarify the mechanisms by which secondary microplastics are
generated from plastic products from the viewpoints of polymer science and polymer engineering.
The study focused on analyzing microplastics (MP) in the environment and elucidating themechanism of the miniaturization of the MP in the environment.
The use of deactivated SiO2 as a diluent was effective in Py-APGC-MS measurements for
pyrolysis analysis of plastics. An algorithm for analyzing MP in the environment was developedand its usefulness for analyzing unknown marine plastic samples was demonstrated.
Next, MP shapes recovered in the estuary were divided into pellets and flake fragments, withpellets recovered in greater numbers than flakes. In addition, crack patterns were observed in 13%
of all PP pellets. Most of the spherical pellets showed an isotropic crackle pattern, indicating thatthese pellets were manufactured by hot cutting. In contrast, about 30% of the flakes developed
parallel crack patterns, indicating the influence of molecular orientation during flow molding. In
order to evaluate the mechanical collapsibility of the pellets, ball milling tests using glass beads
were conducted, and it was found that the collapsibility can be evaluated by the actual MP surface
degradation layer peeling off due to mechanical action.
Finally, a diamond disk grinder was installed and rotated in the flow path of the high-speed
swirling airflow of the conventional jet mill system to increase the efficiency of accelerated MP
formation. The PS elastomer was successfully milled to a fine particle size of approximately 5 μm
in diameter using this improved jet mill system. It is expected that this improved jet mill can beused to grind any type of plastic into fine particles of a few microns in diameter, and is expected to
be used as a standard MP for biotoxicity evaluation.
Timeline:
March 2021-.
Topics:Online presentation "Elucidation of the mechanism of microplastic formation" at the 2021LRI
Research Report Meeting
Publications:
Presentations: (Only the PI's presentation)
1) Shinichi Kuroda, "Accelerated Production of Micro- and Nanoplastics Using Impact Pulverization", Academic
Symposium on Marine Plastic Litter, March 3, 2021 (online)
2) Shinichi Kuroda, "Issues and Research Trends on Microplastics", Suga Weathering Webinar
2021, December 1, 2021 (online)

6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@g.ecc.u-tokyo.ac.jp
Collaborators
Hiroyuki Yamazaki, PhD (Associate Professor, Faculty of Social Welfare, Gunma University of
Health and Welfare) 191-1, Kawamagari-machi, Maebashi, Gunma, 371- 0852, Japan(tel) 027-253-0294 (e-mail) yamazaki@shoken-gakuen.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
Noriko Koganezawa, PhD (Associate Professor, Department of Pharmacology, Gunma
University Graduate School of Medicine) 3-39-22, Showa-machi, Maebashi, Gunma, 371- 8511,
Japan(tel) 027-220-8052, (e-mail) n-koganezawa@gunma-u.ac.jp
Summary of Research:
The purpose of this study is to propose an adverse outcom pathway (AOP) in which the molecular
initiating event (MIE) is the binding of a compound to the glutamate receptors, resulting in theadverse outcome (AO) defined as learning and memory impairment. First, we have established analgorithm for high content imaging to detect dendritic spines with drebrin immunoreactivity, and an
image analysis method by machine learning. Then, morphological changes of dendritic spines
were quantitatively evaluated using frozen hippocampal nerve cells from rat embryos and neurons
derived from human iPS cells. We will validate the possibility as an alternative method fordevelopmental neurotoxicity/neurotoxicity testing, using known compounds with learning andmemory impairment.
Timeline:
March 1, 2020 –Topics:Presentation in LRI Research Report Meeting 2021 “Establishment of an in vitro test method forpredicting the neurotoxicity and developmental neurotoxicity of glutamate receptor-binding compounds
that cause learning and memory impairment.”

Publications:Shogo Mase, Izuo Tsutsui, Toshinari Mitsuoka, Noriko Koganezawa, Hiroyuki Yamazaki, Yuuichi
Kato, Hiroshi Kawabe, Tomoaki Shirao, Yuko Sekino, “Developmental neurotoxicity assessment ofglutamate receptor binding agonists that cause learning and memory impairment：analysis ofdrebrin immunoreactivity in rat hippocampal cultured neuron” The 48th Annual Meeting of theJapanese Society of Toxicology, Kobe (Web), July 2021
Shogo Mase, Toshinari Mitsuoka, Noriko Koganezawa, Hiroyuki Yamazaki, Yuuichi Kato, Izuo
Tsutsui, Tomoaki Shirao, Hiroshi Kawabe, Yuko Sekino “Upregulation of drebrin in dendritic spinesand neuronal death induced by a synthetic cannabinoid, CP55940, in cultured rat hippocampal
neurons” The 44th Annual Meeting of the Japan Neuroscience Society, Kobe (Web), July 2021.
Shogo Mase, Toshinari Mitsuoka, Noriko Koganezawa, Hiroyuki Yamazaki, Yuuichi Kato, Izuo
7

Tsutsui, Tomoaki Shirao, Hiroshi Kawabe, Yuko Sekino “Effects of a synthetic cannabinoid,
CP55940, on synaptogenesis of cultured hippocampal neurons: imaging analysis of drebrin
immunocytochemistry” The 64th Annual Meeting of the Japanese Society for Neurochemistry,
Nara (Web), September 2021

8

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 as prescreening for potential of developmental neurotoxicity.
Principal Investigator:
Tomoya Yamada, DVM, PhD (Fellow, Environmental Health Science Laboratory, Sumitomo
Chemical Company, Ltd.)
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, Environmental Health Science Laboratory, Sumitomo
Chemical Company, Ltd.,)
3-1-98 Kasugadenaka, Konohanaku, Osaka 554-8558, Japan(e-mail) sutoh@sc.sumitomo-chem.co.jp
Summary of Research:
As thyroid hormones (THs) are essential for brain development, concern has been raised that mild
THs disrupting chemicals may have potential to interfere with the developing brain. Since
guideline studies to evaluate developmental neurotoxicity require significant resources, a simple
screening assay would be valuable. The Comparative Thyroid Assay (CTA) is a candidate
screening test, but it requires several animals and advanced techniques of sampling and THs
measurements. We are currently attempting to develop a downsized CTA that reduces the animal
number, while adding extra parameters in offspring (brain THs levels and histology). We had
shown that 6-propylthiouracil (PTU, 10 ppm) induced severe (> 50%) reduction of THs andoffspring brain abnormalities (heterotopia), while phenobarbital (PB, 1000 ppm) induced mild
(approx. 30%) reduction of THs but did not induce brain abnormalities. Recently, the previous
findings at PB 1000 ppm were reproduced, and detection of heterotopia was improved by step
section. These findings suggest that the modified CTA is feasible and reliable and mild
suppression of THs may have little impact on offspring brain development. Further studies should
be investigated.
Timeline:
March 1, 2021 – February 28, 2022.
Topics:Oral presentation at JCIA LRI Annual Workshop 2021 “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 20th, 2021)
Publications:〇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 maternal rats
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 9th, 2021.
〇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 maternal rats
9

and their fetuses/pups as prescreening for potential developmental neurotoxicity: Propylthiouracil
and phenobarbital examples. II. Findings in maternal rats and their pups”. The 48th Annual Meetingof the Japanese Society of Toxicology, Kobe, Japan, July 9th, 2021.
Tomoya Yamada (Sumitomo Chemical Company, Ltd.)“A short-term in vivo assay for thyroid hormone disrupting activity in maternal rats and theirfetuses/pups as prescreening for potential developmental neurotoxicity”. The 61st Annual Meetingof the Japanese Society Teratology, On-line, August 8th, 2021.
Tomoya Yamada (Sumitomo Chemical Company, Ltd.)“Comparative Thyroid Assay: Current situation of a short-term in vivo assay for thyroid hormone
disrupting activity in maternal rats and their offspring as prescreening for potential developmental
neurotoxicity”. The 49th Annual Meeting of the Japanese Society of Toxicology, Sapporo, Japan,
July 2022.
Kenta Minami1, 〇Hidenori Suto1, Akira Sato2, Keiko Ogata1, Kenta Minami1, Tadashi Kosaka2,
Hitoshi Hojo2, Naofumi Takahashi2, Naruto Tomiyama2, Hiroaki Aoyama2, Tomoya Yamada1 (1Sumitomo Chemical Company, Ltd. 2 The Institute of Environmental Toxicology)“Feasibility and reliability of a downsized comparative thyroid assay for evaluating thyroid hormone
disrupting activity in maternal rats and their offspring: reproducibility study with sodium
phenobarbital”. The 49th Annual Meeting of the Japanese Society of Toxicology, Sapporo, Japan,
July 2022.

10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.
In the second year of the project, a validation check of the experiment was conducted due to achange in the experimenter in order to eliminate the variation in gene expression data. By strictly
standardizing the position of fetal cerebral cortex sections and the method of cell counting before
seeding, we were able to obtain data with less variation than before. Next, changes in mRNA
expression encoding 12 molecules that play important roles in neurodevelopment were examined
up to day 21 of culture when exposed to acrylamide, a candidate positive control substance. The
results suggest that the expression of three genes, Dlg4, Syp, and Bdnf, is altered at similar time
points as during methylmercury exposure. Since the experimental conditions for human iPS
neurons are now in place, we plan to increase the number of compounds evaluated in rat corticalneurons in the next year and compare them with human iPS neurons to clarify a better evaluationindex.
Timeline:
March 1, 2021-Februery 28, 2022
Topics:Publications:11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 E-mail: yuhei@med.mie-u.ac.jp
Collaborators:
Takashi Shiromizu (Department of Integrative Pharmacology, Mie University Graduate School of
Medicine)
Munekazu Komada (Department of Life Science, Faculty of Science and Engineering, Kindai
University)
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
developmental neurotoxicity (DNT) remain largely unclear. Various molecular initiating events(MIEs) are involved in the developmental neurotoxicity mechanisms of chemical substances, butdifferent MIEs often exert toxicity through a common key event (KE). Developing a test methodthat can evaluate such a common KE is an effective strategy that will lead to the elucidation of theadverse outcome pathway (AOP) of chemical substances. Impaired differentiation of neural stem
cells into neurons and astrocytes, and neuroinflammation mediated by microglia, which areresident immune cells of the brain parenchyma, 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 DNT of chemicals focusing on neuroinflammation throughmicroglia and the differentiation of neural stem cells into neurons and astrocytes. In 2021, we wereable to generate a transgenic zebrafish line selectively expressing fluorescent proteins mVenus,
Cerulean, and mCherry in microglia, neurons, and astrocytes. In 2021, we will try to establish amethod to assess the DNT of chemicals on the microglial dynamics and the differentiation ofneurons and astrocytes, which can be useful to elucidate the adverse outcome pathways related
to neuroinflammation.
Timeline:
March 2020 ~Topics:JCIA LRI Annual Workshop (2021)
Publications:

1.

2.
3.
4.

[1-4]

Nishimura Y, Kanda Y, Sone H, Aoyama H: Oxidative Stress as a Common Key Event in
Developmental Neurotoxicity. Oxidative Medicine and Cellular Longevity 2021,
2021:6685204.
Higuchi A, Wakai E, Tada T, Koiwa J, Adachi Y, Shiromizu T, Goto H, Tanaka T, Nishimura Y:
Generation of a Transgenic Zebrafish Line for In Vivo Assessment of Hepatic Apoptosis.
Pharmaceuticals 2021, 14(11):1117.
Adachi Y, Higuchi A, Wakai E, Shiromizu T, Koiwa J, Nishimura Y: Involvement of homeobox
transcription factor Mohawk in palatogenesis. Congenital Anomalies 2022, 62(1):27-37.
Nishimura Y, Kurosawa K: Analysis of gene-environment interactions related todevelopmental disorders. Front Pharmacol 2022, in press.

12

Development and assessment of new risk assessment methods
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:
Recently, the concern over environmental chemicals that induce hypothyroidism has been
increased, since hypothyroidism in pregnancy can have a significant impact on the development of
the fetal brain in human. However, detection of abnormalities in thyroid function of pregnant
animals in developmental toxicity tests have not been sufficiently utilized for risk assessment ofchemical substances because the detailed causal relationship between the thyroid function
abnormalities and adverse effects on the development of the fetal brain remains unclear. In orderto solve such a problem, we focused on differentiation markers of mature neurons and produced
reporter transgenic mice (Tg mice) carrying luciferase (Luc2) and LacZ genes downstream of aneuronal differentiation marker promoter. In this fiscal year, we characterized the expression
profile of in vivo imaging in the brain during the developmental stage of Tg mice and analyzed theexpression profile when hypothyroidism was induced in pregnancy. The daily profiles of in vivo
imaging in the brain of Tg pups consisted with those of in vitro reporter assay in the removed brain.
In addition, when antithyroid drugs are administered to pregnant Tg mice under conditions thatinduce hypothyroidism, in vivo imaging was possible to capture their effects on the brain of Tg
pups. These results suggest that in vivo imaging with our Tg mice may be able to noninvasively
detect the effects of chemical exposure during pregnancy on the brain development in pups. In thenext fiscal year, the effects of hypothyroidism during pregnancy on the brain development in pups
will be examined in detail using the expression profile of reporter gene.
Timeline:
April, 2021 – March, 2022
Topics:
None
Conference Presentations:
1) Ishida K, Minamigawa Y, Mori K, Matsumaru D, Nakanishi T, Characterization of neuronaldifferentiation tracer mouse for novel developmental neurotoxicity evaluation system, The
48th Annual Meeting of the Japanese Society of Toxicology (Kobe), 2021.
2) Minamigawa Y, Ishida K, Mori K, Tatsumi K, Matsumaru D, Takuma K, Nakanishi T, Validationof neuronal differentiation tracer mice for a novel developmental neurotoxicity in vivo
evaluation system, Forum 2021 Pharmaceutical Health Sciences/Environmental Toxicology(Chiba), 2021.

13

Development and assessment of new risk assessment methods
Title of Research:
20-6-04

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

Principal Investigator:
Wataru Naito. National Institute of Advanced Industrial Science and Technology (AIST), ResearchInstitute 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
FY2021, a literature review was conducted to highlight the latest trends and parameters for emission
estimation, exposure analysis, hazard assessment and risk assessment of MP. In order to conduct a
trial risk assessment for Tokyo Bay, we attempted to estimate emissions from major sources, evaluate
key parameters in exposure concentration analysis, gather and compile a dataset for thedevelopment of species susceptibility distributions (SSD), and calculate effect concentrations basedon SSD. In addition, an online survey was conducted to investigate public perceptions of marine
plastic litter and MP. In the next fiscal year, while verifying the methods and data that contribute to
the environmental risk assessment of MPs studied in the current fiscal year, we will present a
conceptual model (framework and approach) for environmental risk assessment and managementthat takes into account the characteristics of MPs by illustrating a trial risk assessment for Tokyo Bay.
Furthermore, based on recent trends, we will update research issues and points in MP risk
assessment and management, which are necessary for more realistic risk assessment.
Timeline:
March 2020Topics:
JCIA-LRI Annual Workshop “Development of a conceptual model for environmental risk assessment of
microplastics and a trial risk assessment in Tokyo Bay”, August 2021 (Online)

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Development and assessment of new risk assessment methods

Publications:
1. Iwasaki Y., Mano H., Lin BL, Naito W. “Current status and issues of hazard assessments focusing
on the effects of microplastic particles on aquatic organisms” Jpn. J. Environ. Toxicol. 24: 53-61,
2021.
2. Takeshita KM., Iwasaki Y, Sinclair TM, Hayashi TI, Naito W “Developing a species sensitivity
distribution for nano- and microplastic particles by using Bayesian hierarchical modeling”
SETAC NA 42nd Annual Meeting、Oral Presentation, Nov. 2021 (Virtual Meeting)
3. Takeshita KM., Iwasaki Y, Sinclair TM, Hayashi TI, Naito W“Illustrating a Species Sensitivity
Distribution for Nano- and Microplastic Particles Using Bayesian Hierarchical Modeling” Env. Tox.
Chem. doi:10.1002/etc.5295, 2022
4. Ono K, Xue M, Naito W, Tsunemi K. “Preliminary source analysis of microplastics entering TokyoBay”, 56th Annual Meeting of Japanese Society of Water Environment, Poster Presentation,
March 2022, (Univ. Toyama, On-line)

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Title of Research:
21-2-01
Development of evaluation method of inflammatory particles based on alveolar
macrophage function.
Principal Investigator:
Etsushi Kuroda, PhD (Professor and Chairman, Hyogo College of Medicine, Department of
Immunology)
1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan
tel: +81-798-45-6574, e-mail: kuroetu@hyo-med.ac.jp
Collaborators:
Koubun Yasuda (Hyogo College of Medicine)
Kazufumi Matsushita (Hyogo College of Medicine)
Masakiyo Nakahira (Hyogo College of Medicine)
Takumi Adachi (Hyogo College of Medicine)
Yasuo Morimoto (University of Occupational and Environmental Health)
Hiroto Izumi (University of Occupational and Environmental Health)
Yoshitaka Shirasaki (The University of Tokyo, Faculty of Pharmaceutical Siences)
Summary of Research:
The number of patients with allergic diseases have increased in developed countries. Many
factors are thought to be involved in this issue, and multiple studies have demonstrated thatparticulate pollution such as PM2.5 might be one of factors of exacerbation of allergic
inflammation. These particulates function as adjuvant and induce allergic immune responses.
However, the mode of action of particulate and the mechanisms by which they induce allergic
responses remain to be elucidated. In general, inhaled particles are engulfed by alveolar
macrophages as sentinel cells of the lung mi mune systems, and then excreted. Recently we foundthat inflammatory particulate alum induced cell death in alveolar macrophages after phagocytosis
and then IL-1α was released ad dead cell factor, and also that released IL-1α was involved inallergen-specific IgE production in vivo. These results suggest that responses of alveolar
macrophages against particulates are a useful tool for detecting particulates that cause
inflammation.
In this term, we performed four types of experiments, those are 1) study of in-vitro-differentiatedalveolar macrophages and of establishment of alveolar macrophage cell line, 2) search for new
factor(s) specifically induced in response to inflammatory particulates, 3) establishment of in vivo
experiment systems to evaluate in vitro study, and 4) study of live cell imaging as a new method
for monitoring alveolar macrophages in response to particulates. In these experiments, we found
new factor for evaluating inflammatory particulates by comprehensive analysis of lipid mediatorrelease from alveolar macrophages. This lipid mediator was released both in vitro and in vivo. We
are currently investigating its efficacy for in vivo evaluation method, along with IL-1α. For live cell
imaging analysis, we observed that IL-1αwas released form alveolar macrophage death by alum.
We will perform same experiments using other particulates such as Al2O3, ZnO, silica, TiO2, sanddust and so on to understand detailed mechanisms of alveolar macrophage function in response
to particulates.
Timeline:
March 1, 2021－February 28, 2022
Topics:Research meeting of LRI by JCIA, oral, “Development of evaluation method of inflammatory
particles based on alveolar macrophage function”, online, August 31, 2021.
Publications:
Kuroda Etsushi “Lung immune responses and allergic inflammation induced by fine particles”, The
62nd Annual Meeting of Japan Society for Atmospheric Environment, special meeting, online,
September 16, 2021.
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TitleofResearch:
21-3-01
Establishment of in silico model to predict skin absorption of chemical compounds with twolayered diffusion model
Principal Investigator:
Hiroaki Todo, PhD (Associate Professor, Faculty of Pharmacy and Pharmaceutical Sciences, Josai
University)
1-1 Keyakidai, Sakado, Saitama 350-0295, Japan(tel)+81-049-271-7367
Collaborators:
Kenji Sugibayashi, PhD (Josai University)
Kozo Takayama, PhD (Josai University)
Shoko Itakura, PhD (Josai University)
Kenji Mori, PhD (Josai International University, 1 Gumyo, Tougane-shi, Chiba, 283-8555, Japan)
Takeshi Oshizaka, PhD (Josai International University)
Chihiro Takei (Josai International University)
Tetsuya Watanabe (Ohu University) 31-1 Koriyama-shi, Fukushima, 963-8611, Japan
Masahiro Sugino (Ohu University)
Summary of Research:
The skin is the site of exposure to various chemicals. Therefore, the prediction of the chemical's
permeability to the ski and local concentration in the skin is very important to assure ensure thesafety of the chemicals. Many in silico models for predicting these parameters are based on theprediction of the permeability coefficient (P) of the exposed chemical. On the other hand, theamount of chemicals absorbed in the body after dermal exposure is calculated from the sum of theamount of the chemical permeated through the skin and the amount of chemical in the viableepidermis and dermis (VED). The amount of chemical permeated through the skin can becalculated by the P value. However, the amount of chemical distributed in the VED layer, which isalso related to the local safety of the exposed substance, cannot be predicted.
We have already reported that two-layer diffusion model based on Fick’s second law of diffusion
could predict blood concentration- and skin concentration-time profiles. However, this model has
the drawback that skin permeation parameters (chemical diffusivity in stratum corneum (SC) and
Ved layers, chemical partitioning into SC and VED layers) must be obtained from in vitro skinpermeation experiment.
Therefore, this year, we aimed to develop an in silico model by clarifying the relationship between
skin permeation parameters and characteristic values of chemical substances (molecular weight,
fat solubility, molecular volume, energy difference between the highest occupied molecular orbital(HOMO) and the lowest unoccupied molecular orbital (LUMO) of the molecule, etc.).
The approach with linear regression analysis, it was not possible to obtain a highly correlated
relationship between skin permeation parameters and characteristic values of the chemical
substance. On the other hand, the regression analysis using random forest regression analysis
revealed that good correlations were obtained with all skin permeation parameters whenmolecular weight, lipophilicity, HOMO, and LUMO were selected. The permeability coefficients of
30 compounds were calculated based on the results using random forest regression analysis, andwhile two chemicals showed more than 5-fold difference in permeability coefficients, 22 chemicals
showed a difference within 2-fold. In the future, we would like to evaluate the predictability of skinpermeability of various compounds that listed in the EDETOX database to clarify the applicabilityof this method.
Timeline:
March 1st, 2021Topics:
Publications:
ICCA-LRI Workshop (June 20, 2022)
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A n n u a l R e p o r t 2 021
Japan Chemical Industry Association
1-4-1 Shinkawa, Chuo-ku Tokyo, 104-0033, Japan
TEL. +81-3-3297-2575

FAX. +81-3-3297-2612

URL：https://www.nikkakyo.org/
URL for LRI : https://www.j-lri.org/