A n nu a lRepor t2 0 2 2

2022
Japan Chemical Industry Association

Annual Report 2022

Long-range Research Initiative

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, , The University of Tokyo) 1-1-1 Yayoi, Bunkyo-ku, Tokyo
113-8657, 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:
When the actin-binding protein drebrin which forms dendritic spines of neurons is eliminated by
NMDA-type glutamate activity, the spines become thinner and eventually disappear. Loss ofdendritic spines impairs learning and memory. In this study, we developed an image analysis
algorithm that automatically counts the number of drebrin immunostained clusters in 3-weeks
cultured rat fetal hippocampal-derived neurons. Next, we measured changes in the number ofdrebrin clusters caused by chemical substances. This research resulted in a proposal submitted
an Adverse Expression Pathway (AOP) to OECD. The AOP started from the binding of thecompound to the NMDA-type glutamate receptor as a molecular initiation event (MIE) , and end at
the impairment of learning and memory as the adverse event (AO). The new proposal wad
registered as Wiki475. We will collect evidence to complete AOP-Wiki475 in the future.
Timeline:
March 1, 2020 –Topics:Poster presentation in 2022 ICCA-LRI & NITE Workshop “Proposal of a new AOP for theneurotoxicity and developmental neurotoxicity assessment of glutamate receptor binding agoniststhat cause learning and memory impairment.”
Publications:
Mase S, Mitsuoka T, Koganezawa N, Shirao T, Sekino Y, "Drebrin cluster analysis using cultured
hippocampal neurons"ISRN-2022, Web, March 2022
Sekino Y., "The neurochemistry of addiction and strategies for substance abuse prevention" 69th
Annual meeting of Hokkaido Pharmaceutical Sciences, Web, May 2022
Shogo Mase, Toshinari Mitsuoka, Noriko Koganezawa, Hiroyuki Yamazaki, Yuuichi Kato, Izuo
Tsutsui, Hiroshi Kawabe, Tomoaki Shirao, Yuko Sekino "Quantitative analysis of developmental
neurotoxicity using drebrin immunocytochemical images of cultured rat hippocampal neurons" The49th Annual Meeting of the Japanese Society of Toxicology, Sapporo, June 2022
Shogo Mase, Toshinari Mitsuoka, Noriko Koganezawa, Hiroyuki Yamazaki, Yuuichi Kato, Izuo
Tsutsui, Hiroshi Kawabe, Tomoaki Shirao, Yuko Sekino, "High-content analysis using drebrin
immunocytochemical images of cultured rat hippocampal neurons" Japan Basic and Clinical1Pharmacology Week 2022, Dec 2022
Yuko Sekino, "Collecting Evidence for Enlightenment Activities to Prevent Illegal Drug Use by
Youth" Research Report on Subsidies for the Ministry of Health, Labor and Welfare Administration
Promotion Research Project (FY2022)2

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 developmental neurotoxicity potential.
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:
As thyroid hormones (TH) are essential for fetal brain development, the chemicals which induce
reduction of serum TH levels may potentially interfere with the developing brain. Since
standardized studies to identify developmental neurotoxicity (DNT) require significant resources, a
simple screening test is eagerly awaited for sorting chemicals to be examined in the DNT studies.
Recently, we began verifying the feasibility of a modified Comparative Thyroid Assay (CTA) by
downsizing the number of rats but with the addition of examination of the brain TH levels and brain
histology. The 1st study showed that the modified CTA could detect 6-propylthiouracil (6-PTU, 10ppm)-induced severe (>70%) reduction of serum TH in dams, with >50% suppressed serum/brain
TH levels and brain abnormality (heterotopia) in offspring. The modified CTA also detected sodium
phenobarbital (NaPB, 1000 ppm)-induced mild (<35%) reduction of serum TH levels in dams, withmild (<35%) reduction of serum/brain TH levels in fetuses but not in pups, and without increased
brain heterotopia. The 2nd study showed that the findings by NaPB at 1000 ppm were generally
reproducible in dams and offspring. To investigate what degree of serum/brain TH disruptions will
be adverse to brain morphology, we performed a 3rd study focusing on heterotopia formation byusing treatment with a wider range of doses of 6-PTU (0, 1, 3, and 10 ppm in feed) during thecritical time window for heterotopia formation (GD19-LD2). 6-PTU dose-dependently reduced
serum TH in dams and serum/brain TH in offspring, and induced heterotopia formation in PND21
pups. Although detailed analysis is still in progress, our findings suggest that the modified CTA canbe a potential short-term in vivo assay for sorting offspring TH disruptors.
Timeline:
March 1, 2022 – February 28, 2023.
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 26th, 2022)
Publications:
Kenta Minami1, Hidenori Suto1, Akira Sato2, Keiko Ogata1, Tadashi Kosaka2, Hitoshi Hojo2,
Naofumi Takahashi2, Naruto Tomiyama2, Takako Fukuda 1, Katsumasa Iwashita 1, Hiroaki
Aoyama2 and Tomoya Yamada1 (1 Sumitomo Chemical Company, Ltd. 2 The Institute ofEnvironmental Toxicology), 2023. Feasibility study for a downsized comparative thyroid assay withmeasurement of brain thyroid hormones and histopathology in rats: case study with
6-propylthiouracil and sodium phenobarbital at high dose. Regulatory Toxicology and3

Pharmacology. 137, 105283.
Kenta Minami1, Akira Sato2, Naruto Tomiyama2, Keiko Ogata1, Tadashi Kosaka2, Hitoshi Hojo2,
Naofumi Takahashi2, Hidenori Suto1, Hiroaki Aoyama2 and Tomoya Yamada1 (1 Sumitomo
Chemical Company, Ltd. 2 The Institute of Environmental Toxicology), 2023. Feasibility study for adownsized comparative thyroid assay with measurement of brain thyroid hormones andhistopathology in rats: Part II. Within laboratory reproducibility for effects of sodium phenobarbital(submitted, under review)
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.
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.
〇Keiko Ogata1, Kenta Minami1, Hidenori Suto1, Hiroyuki Asano1, Masahiko Kushida1, Keiko
Maeda1, Akira Sato2, Naofumi Takahashi2, Hiroaki Aoyama2, Tomoya Yamada1 (1 Sumitomo
Chemical Company, Ltd. 2 The Institute of Environmental Toxicology)“A method evaluating brain morphology in a screening study of low thyroid hormone-related
developmental neurotoxicity”. The 39th Annual Meeting of the Japanese Society of Toxicologic
pathology, Tokyo, Japan, January 2023.
〇Tomoya Yamada1 and Hiroaki Aoyama2, (1 Sumitomo Chemical Company, Ltd. 2 The Institute ofEnvironmental Toxicology)“A proposal for the use of a modified comparative thyroid assay with reduced number of animals
and additional parameters”. The 62nd Annual Meeting of the Society of Toxicology, Nashville,
United States, To be presented on March 2023.
〇Kenta Minami1, Hidenori Suto1, Akira Sato2, Keiko Ogata1, Tadashi Kosaka2, Hitoshi Hojo2,
Naofumi Takahashi2, Naruto Tomiyama2, Hiroaki Aoyama2, Tomoya Yamada1 (1 Sumitomo
Chemical Company, Ltd. 2 The Institute of Environmental Toxicology)“Effects of sodium phenobarbital in a downsized comparative thyroid assay with additional
examination of brain thyroid hormone levels and brain histology”. The 62nd Annual Meeting of theSociety of Toxicology, Nashville, United States, To be presented on March 2023.

4

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.
The third year of the study, mRNA expression changes encoding 12 molecules that play importantroles in neurodevelopment, mainly acrylamide and tributyltin, were examined up to day 21 ofculture. The results suggested that the expression of three genes, Dlg4, Syp, and Bdnf, changed
at the same time as in the methylmercury-exposed group, although some genes showed different
expression patterns from those in the methylmercury-exposed group. Considering the possibility
that gene expression may be altered by other suspected developmental neurotoxicants, we
examined the effects of suspected developmental neurotoxicants on Dlg4 gene expression in
DIV10, 14, and 21, and found that Dlg4 gene expression was suppressed in some cases. Dlg4
expression in DIV14 was shown to be sensitive to chemical substances and may be a good
indicator of developmental neurotoxicity. In addition to the 12 gene markers, we performed acomprehensive gene expression analysis using RNA-Seq to investigate the possibility that there
are other genes that may be indicators of developmental neurotoxicity. The results revealed theexistence of genes that were significantly up-regulated in common with the three developmental
neurotoxicants and, conversely, genes that were significantly down-regulated in common with thethree substances.
Timeline:
March 1, 2020-Februery 28, 2023
Topics:Publications:5Title of Research:
20_PT03-08

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. In this study, we have developed a novel test
method that can assess the DNT of chemicals on the morphology of microglia using a transgenic
zebrafish line expressing mVenus, Cerulean, and mCherry in microglia, neurons, and astrocytes,
respectively. Using the assay, we were able to detect the DNT of ethanol on the formation of
microglia. We will apply this method to assess the DNT of other chemicals, such as valproic acid,ethinylestradiol, and chlorpyrifos. We will also analyze the correlation among the morphologicalchange of microglia, neuroinflammation, and neurodifferentiation.
Timeline:
March 2020 ~ February 2023
Topics:JCIA LRI Annual Workshop (2022)
Publications:1.
2.

Komada M, Nishimura Y: Epigenetics and Neuroinflammation Associated With
Neurodevelopmental Disorders: A Microglial Perspective. Front Cell Dev Biol 2022,
10:852752.
Nishimura Y, Kurosawa K: Analysis of Gene-Environment Interactions Related to
Developmental Disorders. Frontiers in pharmacology 2022, 13:863664.

6

Development and assessment of new risk assessment methods
Title of Research:
20_PT03-05

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)(e-mail) nakanishi@gifu-pu.ac.jp
Collaborators:
Daisuke Matsumaru (Laboratory of Hygienic Chemistry and Molecular Toxicology, Gifu
Pharmaceutical University)(e-mail) matsumaru-da@gifu-pu.ac.jp
Summary of Research:
Recently, the concern over the toxicity of environmental chemicals that induce hypothyroidism
has been increased, since hypothyroidism during pregnancy can have a significant impact on thedevelopment of the fetal brain in human. However, the results of thyroid function disruption
detected by toxicity testing have not been sufficiently utilized for risk assessment of chemical
substances because the correlation between the degree of hypothyroidism during pregnancy andadverse effects on the development of the fetal brain remains unclear. In order to solve theproblem, we focused on differentiation markers of mature neurons and produced reportertransgenic mice (Tg mice) carrying luciferase (Luc2) and LacZ genes downstream of a neuronaldifferentiation marker promoter. In the past year, we have confirmed the usefulness of Tg mice inassessing developmental neurotoxicity and found the possibility of evaluating the effects ofhypothyroidism from early pregnancy on the infant brain using the antithyroid drug propylthiouracil(PTU). In this fiscal year, we analyzed the correlation between the expression profile of in vivo
imaging in the infant brain and maternal thyroid-related parameters when hypothyroidism was
induced by exposure to various doses of PTU from early pregnancy.
PTU induced a dose-dependent decrease in triiodothyronine (T3)/thyroxine (T4) and anincrease in thyroid-stimulating hormone (TSH) in maternal blood. In histological analysis of the
thyroid gland, symptoms of hypothyroidism such as thickening of follicular cells were observed
even at low-dose PTU exposure prior to significant changes in serum T3/T4/TSH levels. In
addition, the expression of reporter molecules in the brain of the infants was also increased in adose-dependent manner of PTU. Furthermore, similar to the results of thyroid histological analysis
in the mother, the effects were observed even at low-dose without significant changes in the serum
levels of thyroid-related hormones.
These results suggest that in chemical-induced hypothyroidism, thyroid histology is a moresensitive indicator for detecting their effects than serum thyroid-related hormones, and that someadverse effects on brain development in the child may occur prior to changes in maternal serum
thyroid-related hormones.
Timeline: April, 2022 – March, 2023
Topics:1) 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 26th, 2022)
2) Report in the Yomiuri Shimbun Morning Edition “Environmental Chemicals and the Brain:A
New Approach Methodology Using "Glowing Mice" - Gifu Pharmaceutical University, etc.,
Verify Effects on Children” (February 7th, 2023)
Publications:
7

1)
2)
3)

Development and assessment of new risk assessment methods
Ishida K, Tatsumi K, Minamigawa Y, Mori K, Matsumaru D, Nagase H, Kanda Y, Takuma K,
Nakanishi T, Neuronal differentiation reporter mice as a new methodology for detecting in vivo
developmental neurotoxicity. Biochem. Pharmacol., 206:115332 (2022)
Ishida K, Matsumaru D, Shimizu S, Hiromori Y, Hisamitsu Nagase H, Nakanishi T, Evaluationof the estrogenic action potential of royal jelly by genomic signaling pathway in vitro and invivo. Biol. Pharm. Bull., 45:1510–1517 (2022)
Ishida K, Furukawa M, Kunitani M, Yamagiwa R, Hiromori Y, Matsumaru D, Hu J, Nagase H,
Nakanishi T, Novel, highly sensitive, in vivo screening method detects estrogenic activity at
low doses of bisphenol A. J. Hazard. Mater. 445:130461 (2023).

Conference Presentations:
1) Ishida K, Minamigawa Y, Tatsumi K, Mori K, Matsumaru D, Nagase H, Kanda Y, Takuma K,
Nakanishi T, Validation of brain neuronal differentiation reporter mice for improved
developmental neurotoxicity evaluation，2022 ICCA-LRI & NITE Workshop, Yokohama/Japan,
June 2022
2) Tatsumi K, Ishida K, Minamigawa Y, Mori K, Matsumaru D, Nagase H, Kanda Y, Takuma K,
Nakanishi T, Validation of brain neuronal differentiation tracer mice for improvement
developmental neurotoxicity evaluation, The 49th Annual Meeting of the Japanese Society of
Toxicology (Sapporo), 2022.
3) Mori K, Ishida K, Minamigawa Y, Tatsumi K, Matsumaru D, Murashima A, Nagase H, Kanda Y,
Nakanishi T, Imaging analysis of the offspring brain in a model of maternal hypothyroidism,
The 49th Annual Meeting of the Japanese Society of Toxicology (Sapporo), 2022.
4) Ishida K, Minamigawa Y, Tatsumi K, Mori K, Matsumaru D, Nagase H, Kanda Y, Takuma K,
Nakanishi T, Establishment of in vivo neuronal differentiation tracing method for improved
developmental neurotoxicity evaluation, The 62nd Annual Meeting of the Japanese
Teratology Society (Kanazawa), 2022.
5) Ishida K, Matsumaru D, Nakanishi T, Perinatal hypothyroidism and DOHaD: Aiming for therisk assessment of neurodevelopmental disorders in offspring, Forum 2022 Pharmaceutical
Health Sciences/Environmental Toxicology (Kumamoto), 2022.
6) Ishida K, Tatsumi K, Minamigawa Y, Mori K, Matsumaru D, Murashima A, Nagase H, Kanda Y,
Takuma K, Nakanishi T, Neuronal differentiation reporter mice as a new methodology for
detecting in vivo developmental neurotoxicity, In vivo imaging forum 2022 (Tokyo), 2022.

8

Development and assessment of new risk assessment methods
Title of Research:
XXXX

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
FY2022, we established methods for estimating MP loadings from major sources, which isindispensable for considering priorities for source control measures, etc. We estimated loadings for
product-containing MP (PCP), laundry-derived fiber, and tire-wear particles (TWP) entering Tokyo Bay,and showed the range of possible values for the estimates. We investigated to understand the MP
monitoring levels along the Japanese coast (especially in Tokyo Bay) and a method for estimating the
distribution of MP concentrations in the unmeasured range and estimated the distribution of thenumber of unmeasured MP concentrations from available data. We derived an SSD based onhierarchical Bayes using toxicity values listed in the new MP ecotoxicity database. Furthermore, as atrial ecological risk assessment in Tokyo Bay, we compared the corrected MP particle concentrations
in Tokyo Bay with threshold values using Food Dilution as an effect index. Through the environmentalload estimation, analysis of the monitoring data, consideration of data correction methods, and trial
risk assessment in Tokyo Bay, we were able to clarify items with large uncertainties and future
research issues. In the future, we plan to establish an assessment framework that enables us topredict the future environmental burden and risk of MP by improving the accuracy of the estimation
method and understanding the particle size distribution of environmental MP that matches the actual
situation.
Timeline:
March 2020Topics:
JCIA-LRI Annual Workshop “Development of a conceptual model for environmental risk assessment of9

Development and assessment of new risk assessment methods
microplastics and a trial risk assessment in Tokyo Bay”, August 2022 (Online)

Publications:
1. Naito W “Risk Assessment in Practice : Opportunities, Challenges and Evolving Roles for Complex
Environmental Issues” SETAC AP 2022, Plenary Speech, Sep. 2022 （Virtual Meeting）
2. Naito W “Development of a conceptual model for environmental risk assessment of microplastics
and a trial risk assessment in Tokyo Bay” MARII Workshop on “Advancements and steps towards aholistic, quantitative risk assessment on microplastics” Oral presentation、 Oct. 2022、Barcelona,
Spain
3. Ueda K, Iwasaki Y, Uesaka M, Naito W “Which concentration unit should be used for environmental
risk assessment of microplastics?” 57th Annual Meeting of Japanese Society of Water Environment,
Poster Presentation, March 2023, (Univ. Ehime, Japan)
4. Naito W, Iwasaki Y, Ono K, Ogura I “Framework of environmental risk assessment for microplastics
and an illustrative example” 57th Annual Meeting of Japanese Society of Water Environment, Oral
Presentation, March 2023, (Univ. Ehime, Japan)

10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 Medical University, School 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 Medical University)
Kazufumi Matsushita (Hyogo Medical University)
Masakiyo Nakahira (Hyogo Medical University)
Takumi Adachi (Hyogo Medical University)
Hinata Inoue (Hyogo Medical University)
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, and it issuggested that particulate pollution such as PM2.5 might be one of factors of exacerbation ofallergic inflammation. These particulates function as adjuvant and induce allergic immune
responses. In general, inhaled particles are thought to be engulfed by alveolar macrophages andthen excreted. Therefore, in this study we aim to develop methods of evaluation of inflammatory
particles focusing on alveolar macrophage functions in response to particles. In addition,
animal-free toxicity testing is recommended in these days, so we also aim to develop in vitro
evaluation methods using alveolar macrophage cell lines which is useful tools for detecting
inflammatory particles.
Freshly isolated alveolar macrophages were immortalized by SV40 large T gene transfer using
lentiviral vector system, and then cultured with GM-CSF and PPAR-γ agonist to maintain theircharacteristics of alveolar macrophage. After screening of obtained cells, and we establishedalveolar macrophage cell line, ALV-3. The same as primary alveolar macrophages, ALV-3 cells
responded to inflammatory particles and induced cell death and subsequently IL-1α release.
Furthermore, ALV-3 cells expressed Siglec F and CD11c antigens, which are unique antigens foralveolar macrophages, on their surface. MH-S cells, which are commercially available alveolar
macrophage cell line, did not expressed Siglec F antigen and did not induce IL-1α release in
response to inflammatory particles. These results suggest that ALV-3 cells might be useful cell line
to evaluate inflammatory particles, as an alternative in vitro method to animal testing. Now we arefurther investigating the characteristics of ALV-3 cells, as alternative to primary alveolar
macrophages.
Timeline:
March 1, 2022－February 28, 2023
Topics:Research meeting of LRI by JCIA, oral, “Development of evaluation method of inflammatory
particles based on alveolar macrophage function”, online, August 26, 2022.
Publications:
Kuroda Etsushi “Analysis of alveolar macrophage functions in response to fine particles" The127th Annual meeting of the Japanese Association of Anatomists, Online Symposium, 2022,
March 28.
Kuroda Etsushi “DAMPs release and allergic airway inflammation caused by inhaled fine particles”
SOT 61st Annual Meeting, Online Symposium, 2022, March 29.

11Title of Research:
21-3-01
Establishment of in silico model to predict skin absorption of chemical compounds with
two-layered 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)
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)
Hiroyuki Teramae, PhD (Josai University)
Summary of Research:
Purpose: The skin is exposed to many chemical substances, including pharmaceuticals,
cosmetics, and fragrances, and is inadvertently exposed to many chemical substances in daily life.
Therefore, it is extremely important to evaluate the skin permeability of chemical substances that
may contact the skin. Next-generation risk assessment (NGRA), a risk assessment method thatcombines various methods without using animals, has attracted much attention in recent years. In
the present study, development of in silico model that predict dermal absorption after chemical
exposure under real-world condition, finite dose, was investigated by considering changes inconcentration of the applied substance on the skin surface.
Methods: Prediction of skin permeation parameters: Physicochemical parameters of various
chemical substances were calculated using Scigress software. Diffusion and distribution
coefficients, which are skin permeation parameters of chemicals, were predicted by random forest
regression analysis, an ensemble learning algorithm, using the statistical software JMP Pro16 withphysicochemical parameters as descriptors.
Results and conclusion: The study using random forest regression analysis showed that all of theskin permeation parameters were well predicted when regression analysis was performed using
M.W., ClogP, HOMO, and LUMO as descriptors. Skin permeation profiles, calculated by two-layer
diffusion model after chemical exposure under finite dose condition, showed good perdition results
when water volatilization rate constant was considered. There were, however, some deviations
between the predicted and observed skin permeation profiles, the overall prediction results were
good. These results suggest that the constructed model would be used to predict the dermal
absorption of finite dose systems.
Timeline:
March 1st, 2021Topics:
2022 ICCA-LRI & NITE Workshop（June 20, 2022）
In silico estimation of skin permeation of chemicals with their diffusion and partition parameters2022 年 LRI Annual Workshop（August 26, 2022）
Establishment of in silico model to predict skin absorption of chemical compounds with
two-layered diffusion model

12

Title of Research:
XX-XX-XX

Establishment of Human iPSC Reporter-Based Developmental
Toxicity Assay that Detects FGF Signal Disruption
Principal Investigator:
Junji Fukuda (PhD, Professor, Yokohama National University, Faculty of Engineering)79-5 Tokiwadai, Hodogaya Ward, Yokohama, Kanagawa 240-8501, Japan
Collaborators:
Yusuke Okubo (PhD, Principle Investigator, National Institute of Health Sciences)
3-25-26 Tono-machi, Kawasaki-ku, Kawasaki, Kanagawa 210-9501, Japan
Yoshihiro Nakajima (PhD, Principle Investigator, National Institute of Advanced Industrial Scienceand Technology)2217-14 Hayashi-cho, Takamatsu, Kagawa, 761-0395, Japan
Summary of Research:
In the 10th period, we reported that the compounds listed in ICH (S5), including thalidomide, could
be detected by using RTK-SRF signal reporter cells (S. Kanno, et al., Journal of Bioscience andBioengineering, 133, 3, 291- 299 (2022)). As a first step to spread this approach, we also reported
a detailed experimental protocol in a scientific paper (S. Kanno, et al., Star Protocols, 3, 2, 101439(2022)). As for the experiments, a real-time luminescence measurement system was introduced to
improve the data accuracy of the kinetic assay, which was a feature of this method. In other words,
the amount of luminescence was measured manually every few hours in the past, but the
introduced device made it possible to perform real-time measurement. When measurements were
continued for three days instead of the conventional one day using this device, it was also
confirmed that the luminescence peak occurred not once, but at least twice. This is a fact that had
been overlooked manually. In the 11th period, we would like to add a discussion on the biologicalsignificance of this phenomenon and the measurement mechanism.
Timeline:
March 1, 2022 Topics:
Poster presentation at 2022 ICCA-LRI and NITE Workshop, “Establishment of developmental
toxicity test based on the integration of FGF signal disruption effects for safety evaluation of drugs
and chemicals using human iPS cells”
Publications:Seiya Kanno, Kashu Mizota, Yusuke Okubo, Tatsuto Kageyama, Lei Yan, Junji Fukuda,“Luciferase assay system to monitor fibroblast growth factor (FGF) signal disruption in human
iPSCs”, Star Protocols, 3, 2, 101439, 2022
Seiya Kanno, Yusuke Okubo, Tatsuto Kageyama, Lei Yan, Junji Fukuda, “Integrated FGF signal
disruptions in human iPS cells for prediction of teratogenic toxicity of chemicals”, Journal of
Bioscience and Bioengineering, 133, 3, 291-299, 202213

Title of Research: 22-3-01

Prediction of internal concentrations of chemicals orally administered using
data-driven pharmacokinetic modeling
Principal Investigator: Prof. Hiroshi Yamazaki, PhD (Showa Pharmaceutical University,
Laboratory of Drug Metabolism and Pharmacokinetics), 3-3165, Higashi-tamagawa Gakuen,
Machida,Tokyo
194-8543,
Japan.
(phone)+81-42-721-1406,(e-mail)hyamazak@ac.shoyaku.ac.jp.
Collaborator: Makiko Shimizu, ibid, (e-mail) shimizu@ac.shoyaku.ac.jp
Summary of Research: To evaluate internal exposures in humans without any reference toexperimental data, physiologically based pharmacokinetic (PBPK) modeling could be used.
The input parameters for PBPK models (i.e., fraction absorbed x intestinal availability,
absorption rate constants, volumes of the systemic circulation, and hepatic intrinsic
clearances) were estimated for a panel of 355 chemicals using a light gradient boostingmachine learning algorithms (LightGBM) based on between 11 and 29 in silico-calculated
chemical descriptors. Parameters for human PBPK models for a diverse range of compounds
could be successfully estimated using chemical descriptors. This approach topharmacokinetic modeling has potential for application in computational toxicology and in theclinical setting for assessing the potential risk of general chemicals.
Timeline: From March 1, 2022 to February 28, 2023
Topics: The principal Investigator has been the recipient of Scientific Achievement Award from theInternational Society of Study of Xenobiotics (ISSX) for major contributions to the field of
xenobiotics in the Asia Pacific Region (Bangalore, India, 2023).
Publications:(1) Adachi, K., Shimizu, M., and Yamazaki, H. (2022) Updated in Silico Prediction Methods for
Fractions Absorbed and Key Input Parameters of 355 Disparate Chemicals for Physiologically
Based Pharmacokinetic Models for Time-Dependent Plasma Concentrations after Virtual Oral
Doses in Humans. Biol Pharm Bull 45, 1812-1817.
(2) Kamiya, Y., Handa, K., Miura, T., Ohori, J., Kato, A., Shimizu, M., Kitajima, M., and Yamazaki, H.
(2022) Machine Learning Prediction of the Three Main Input Parameters of a Simplified
Physiologically Based Pharmacokinetic Model Subsequently Used to Generate Time-Dependent
Plasma Concentration Data in Humans after Oral Doses of 212 Disparate Chemicals. Biol Pharm
Bull 45, 124-128.
(3) Shimizu, M., Hayasaka, R., Kamiya, Y., and Yamazaki, H. (2022) Trivariate Linear Regression andMachine Learning Prediction of Possible Roles of Efflux Transporters in Estimated Intestinal
Permeability Values of 301 Disparate Chemicals. Biol Pharm Bull 45, 1142-1157.
(4) Kamiya, Y., Handa, K., Miura, T., Ohori, J., Shimizu, M., Kitajima, M., Shono, F., Funatsu, K., andYamazaki, H. (2022) Correction to "An Updated In Silico Prediction Method for Volumes of
Systemic Circulation of 323 Disparate Chemicals for Use in Physiologically Based
Pharmacokinetic Models to Estimate Plasma and Tissue Concentrations after Oral Doses in Rats".
Chem Res Toxicol 35, 1433.
(5) Adachi, K., Shimizu, M., and Yamazaki, H. (2022) Updated in silico prediction methods for
fractions absorbed and absorption rate constants of 372 disparate chemicals for use inphysiologically based pharmacokinetic models for estimating internal concentrations in rats. J
Toxicol Sci 47, 453-456.
(6) Miura, T., Uehara, S., Shimizu, M., Suemizu, H., and Yamazaki, H. (2022) Forward and reverse
dosimetry for aniline and 2,6-dimethylaniline in humans extrapolated from humanized-liver mouse
data using simplified physiologically based pharmacokinetic models. J Toxicol Sci 47, 531-538.

14

Assessment on the effects on ecosystems and the environment

Title of Research:
22-5-03

Constructing a model for estimating a vector effect of microplastics using an artificial
bioconcentration device and predicting impact in the real environment

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 daily life due to its low cost, lightweight, and rigid
decomposing. Due to their high production and slow degradation, plastic pollution has
expanded worldwide in aquatic environments. Plastic pollution from large items to small
particles (microscopic plastic MP, < 5mm) emerged. The effect of MP and its vector effecton pollutants are of concern. Many studies have examined these topics. However, most of
the study were used pristine MPs. Thus, a study on the effect of aging on the vector effectof MP is required.
This fiscal year, freshwater and marine medaka were co-exposed to microplastics (MP)with chlorobenzenes (CBs) and anthracene (ANT) under various conditions, and vector
effects were investigated. No vector effect could be observed in CBs (logPow 4.195.73). However, a vector effect was confirmed with anthracene (logPow4.45). Therefore, it
was concluded that the vector effect differs greatly depending on the character of thechemicals, even if the LogPow is close. Further studies may be required on PAHs, POPs,etc., concerned about the influence of vector effects. The details are described below.
1) Freshwater medaka-CBs + degraded crushed PE-MP 3-day co-exposure experiment:
CBs group (CBs; TriCB: 10 µg/L, TetCB: 10 µg/L, PentCB: 10 µg/L, HexCB: 8 µg/L), CBs+ aged fragmented PE-MP (1 mg/L) co-exposure group, CBs + unaged fragmented PEMP (1 mg/L) co-exposure group were set, and after equilibrating with MP for one day,medaka ( 3 fish/group) were exposed for 3 days. As a result of the analysis, CBs in theunaged group was 1.6 times higher than in the CBs group. Moreover, there was no effecton aging.
2) 7-day co-exposure experiment with different concentrations of freshwater medakaCBs+PE-MP : CBs group (CBs; TriCB: 10 µg/L, TetCB: 10 µg/L, PenCB: 10 µg/L, HexCB:

15

Assessment on the effects on ecosystems and the environment
8 µg/L ), CBs + low-concentration fragmented PE-MP (0.1 mg/L) Co-exposure group,and CBs+ high-concentration crushed PE-MP (1.0 mg/L) group were set. They were
exposed to Japanese medaka (33 fish/section) for 7 days and excreted for 5 days.
Sampling was performed on the 3rd, 5th, 7th, 9th, and 12th days. The water exchange wassemi-static (once a day, full exchange). There was no significant difference in CBs
concentrations compared with the water-exposed group, indicating no vector effect.
3) Freshwater medaka-CBs + PS-MP co-exposure experiment with different particle
sizes: CBs (CBs; TriCB: 10 µg/L, TetCB: 10 µg/L, PenCB: 10 µg/L, HexCB: 8 µg/L)
exposure group, CBs+10-µm PS-MP co-exposure group,and a CBs+45-µm PS-MP coexposure group were set.. After 7 days of exposure, they were excreted for 5 days andsampled on the 7th, 8th, 9th, 10th, and 12th days. The water exchange was semi-static(once a day, full exchange). As a result, the CBs concentration in the co-exposed groups
did not differ from that in the CBs-exposed group and somewhat decreased, indicating novector effect.
4) Freshwater medaka-ANT+58-µm PE-MP co-exposure experiment with different MP
concentrations: ANT (200 µg/L) group, ANT+ low-concentration PE-MP (0.6 mg/L) coexposure group, ANT+ middle-concentration PE-MP ( 6 mg/L) co-exposure group, and
ANT+ high-concentration PE-MP (60 mg/L) co-exposure group were set. After 10 daysof exposure, fish were excreted for 4 days and sampled on the 10th, 11th, 12th, 13th, and14th days. The water exchange was semi-static (once a day, full exchange). The vector
effect was confirmed from the obtained concentrations of ANT in the medaka. A twincompartment vector model was used to analyze the effect of MP concentration, and as aresult, the vector coefficient decreased as the MP concentration increased.
5) Prediction of ANT-MP vector effect in the real environment: The data set (Takai, PhD.
thesis, Kyushu University, 2023.3) was used obtained from marine Java medakaanthracene (ANT) + different size (2, 10-µm) polystyrene MP (PS-MP) exposure studies.
As a result of evaluating the vector effect from the different concentrations of PS-MP
using a twin-compartment vector model, it was estimated that the vector effect would
hardly occur in the real environment.
6) Effect of PS-MP particle size on ANT adsorption: As a result of examining theadsorption of ANT to PS-MP with particle sizes of 10, 45, and 90 µm. The smaller theparticle size PS-MP show a larger surface area and adsorption potency to ANT,
suggesting a vector effect. However, it was expected that the larger the particle size PSMP has minor adsorption potency and the weaker the vector effect.

Timeline:
March 1, 2022- Feb28, 2023

Topics:Publications:16

Assessment on the effects on ecosystems and the environment
1. Takai Y., Tokusumi, H., Sato, M., Inoue D., Chen K., Takamura T., Enoki S., Ueno Y., Kang
I. J., Shimasaki Y., Qiu X., Oshima Y., Combined effect of diazepam and polystyrene
microplastics on the social behavior of medaka (Oryzias latipes), Chemosphere, 299
134403-134403.
2. Takai Y., Tokunaga M., Honda M., Qiu X, Shimasaki Y., Kang I.J., Oshima Y., Size effect ofpolystyrene microplastics on the accumulation of anthracene for Java medaka (Oryzias

javanicus). Chemosphere (Under reviewing)
3. Takai Y., Tominaga A., Honda M, Qiu X., Shimasaki Y., Kang I.J, Oshima Y., Combined effectof anthracene and polyethylene microplastics on Java medaka (Oryzias javanicus).
Ecotoxicology. (2023.3.31，submit)
4. Takai Y., Honda M., Qiu X., Shimasaki Y., Kang I.J., Oshima Y., Concentration effect ofpolystyrene microplastics on the accumulation of anthracene for Java medaka (Oryzias

javanicus). Mar poll bull (2023.4, submit)

17

Title of Research:
22-6-02

Development of an objective read-across method based on
statistical and mathematical sciences for evaluation of
repeated-dose toxicity
Principal Investigator:
Kouichi Yoshinari (School of Pharmaceutical Sciences, University of Shizuoka)
52-1 Yada, Suruga-ku, Shizuoka 422-8526 Japan. E-mail: yoshinari@u-shizuoka-ken.ac.jp
Collaborators:
Jun-ichi Takeshita (Research Institute of Science for Safety, National Institute of Advanced
Industrial Science and Technology (AIST))
Tomomichi Suzuki (Department of Industrial Administration, Tokyo University of Science)

Summary of Research:
Repeated-dose toxicity (RDT) test is important for chemical safety evaluation. Currently, it is highly
recommended to develop alternatives to rat RDT tests, but it remains very difficult to develop sucha method largely due to the diversity and complexity of the toxicity. The aim of this project is todevelop an objective read-across method, in which the toxicity of an untested substance ispredicted from the toxicity information of similar substances (called “source substances”), usingchemical information and in vitro assays as an alternative to rat RDT tests. In this year, weselected test substances from HESS and REACH databases and set 8 group endpoints (6hepatotoxicity- and 2 hematotoxicity-related) as targets. Studies were conducted on criteria for
judging the presence or absence of toxicity of test substances from source substances, molecular
descriptors to be used for similarity assessment, and methods for selecting source substances.
Results obtained indicate that the criteria for judging the toxicity of test substances should be
adjusted based on the positive rate of the group endpoints, source substances differ depending on
the type of inter-substance distances used, the descriptors should be selected by an appropriate
method, and the accuracy of toxicity prediction is improved by appropriately selecting descriptors.
Timeline:
March 1, 2022 - February 28, 2023
Topics:Publication #1 (Yu Harakawa) received Presentation Award for Students at the 35th annual
meeting of the Japanese Society for Alternatives to Animal Experiments.
Publications:
1. Y Harakawa, N Omura, J Takeshita, R Shizu, T Hosaka, Y Kanno, K Yoshinari. Developmentof a read-across method for evaluating repeated-dose toxicity using molecular descriptors:
Examination of methods of selecting molecular descriptors for better prediction. The 35th
annual meeting of the Japanese Society for Alternatives to Animal Experiments. Shizuoka,
Japan, 2022.11.18-20.
2. K Yoshinari. Evaluation of developmental neurotoxicity of chemical substances by aread-across method using chemical structure information. The 35th annual meeting of theJapanese Society for Alternatives to Animal Experiments. Shizuoka, Japan, 2022.11.18-20.
3. K Yoshinari. Safety assessment of chemical compounds by in vitro and in silico methods:
challenges and future prospects. ILSI Japan symposium: Alternatives to animal experimentsin the food science area -current situation, trends and initiatives for the future. Online,
2023.2.3.
4. Y Harakawa, N Omura, R Shizu, T Hosaka, J Takeshita, K. Yoshinari. Examination of theselection method of molecular descriptors for the evaluation of repeated-dose toxicity using aread-across approach. The Society of Toxicology 62nd Annual Meeting and ToxExpo.
Nashville, TN, USA. 2023.3.19-23.
18

Title of Research:
22-D-01 Elucidation of the mechanism and rate of microplastic formation contributing to risk
assessment and preparation of standard 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:
Kuriyama Takashi (Graduate School of Organic Materials and Systems Science, Yamagata
University), 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) ,Ohtani Hajime (Graduate School of Engineering, Nagoya Institute of Technology), Kouzai Hiroaki(Department of Science and Engineering, Kanto Gakuin University), Kawai Takahiko (Graduate
School of Science and Engineering, Gunma University)
Summary of Research:
MP collected in the lower reaches of the Arakawa River were sorted and observed. The MP
pellets were ball-milled with glass beads. The results showed that as the size of the MPs
decreased, they became more spherical in shape. All MPs that were surface peeled off in the ball
milling test were less than 1 mm in diameter, indicating that the MPs changed from flaky MPs toparticle-like MPs. This is interpreted to indicate that the formation mechanism of MPs is similar tothat of rocks converted to gravel by river transport (i.e., collapse by mechanical action).
The iPP sheets were exposed to high temperatures, and the results showed that the thermallydeteriorated sheets had localized bleaching and yellowing in the peripheral areas, while a healthy
area remained in the central part. In the bleached areas, cracks were formed due to shrinkage
caused by chemical crystallization and propagated in one direction, forming a columnar structure.
In the yellowing area, chemical crystallization progressed further and some of the cracks collapsed
to form fine powder of about 0.2 mm in diameter.
Dumbbell pieces punched from PET bottles, PE sheets, and PP sheets were exposed to axenon weather meter, and the results showed that photo-oxidation degradation progressedsignificantly in PP, with a marked decrease in molecular weight and mechanical properties, andcracks appeared and developed. The molecular weight of PET also decreased, and cracks
appeared on the surface in a mosaic-like pattern, and some of the cracks appeared to be peelingoff and collapsing.
Based on the above findings, it is considered that the collapse and exfoliation of the"weathering layer" generated by the cracks that occur with the degradation of plastics are deeply
related to the generation of MP. The formation mechanism of this "weathering layer" can be
explained by a pattern formation in which shrinkage propagates, as is the case with dry cracks.
The aerosols were classified and collected for analysis by pyrolysis GC/MS, and the pyrolysis
GC/MS allowed the identification and quantification of atmospheric MPs without any special
pretreatment. The fact that no pretreatment is required is also important to prevent contamination.
On the other hand, the use of a blade-type jet mill with many blades in a swirling stream ofcompressed air, which is an improvement over conventional jet mills, has made it possible toreduce various plastics to near-spherical particles of approximately 10 μm in diameter.
Furthermore, UV irradiation of atomized plastics in a mixed solvent of acetone and alcohol
enabled efficient surface oxidation.
Timeline: March 2022-.
Topics: Online presentation at the 2022 LRI Research Report Meeting
Publications: Presentations: (Only the PI's international presentation)
1) Shinichi Kuroda，”Elucidation of the formation mechanism of microplastics”， Microplastics
Advance Research and Innovation Initiative (MARII) Workshop on “Advancements and steps
towards a holistic, quantitative risk assessment on microplastics”, 12 – 13 October 2022 (Online).
19

A n n u a l R e p o r t 2 022
Japan Chemical Industry Association
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TEL. +81-3-3297-2575

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