A n nu a lRepor t2 0 1 92019Japan Chemical Industry Association

Annual Report 2019

Long-range Research InitiativeDevelopment and assessment of new risk assessment methods
Title of Research:
17_PT01-01

Chip device for in vitro systemic toxicology
Principal Investigator:
Junji Fukuda, PhD (Professor, Yokohama National University, Engineering)79-5 Tokiwadai, Hodogaya, Yokohama, Kanagawa, 240-8501, Japan(tel) +81-045-339-4008, (e-mail) fukuda@ynu.jp
Collaborators:
Kazutomo Iijima, PhD (Associate Professor, Yokohama National University, Engineering)79-5 Tokiwadai, Hodogaya, Yokohama, Kanagawa, 240-8501, Japan
Yasunari Kanda, PhD (Head of Cardiac pharmacology, National Institute of Health Sciences,
Division of Pharmacology)
3-25-26 Tonomachi, Kawasaki-ku, Kawasaki, Kanagawa, 210-9501, Japan
Yusuke Okubo, PhD (Senior staff scientist, National Institute of Health Sciences, Division of
Cellular and Molecular Toxicology)
3-25-26 Tonomachi, Kawasaki-ku, Kawasaki, Kanagawa, 210-9501, Japan
Kiyomi Ohmori, PhD (Adjunct Professor, Yokohama National University, Research Initiatives andPromotion Organization)
79-5 Tokiwadai, Hodogaya, Yokohama, Kanagawa, 240-8501, Japan
Summary of Research:
Body-on-a-chip has emerged as a novel approach for Alternatives to Animal Testing. The aim of
this study is to develop a body-on-a-chip for in vitro drug testing to evaluate systemic toxicity ofchemicals. In our chip devices, several tissues including the liver, the cardiac tissue, and the small
intestine were fabricated as either cell layer tissues or spherical tissues to replicate in vivo kinetics
and interactions. One of our results showed that improved oxygen supply and a selection ofenergy metabolisms significantly matured human iPS-derived cardiomyocyte spheroids. Changes
in beating of these spheroids were characterized using cell motion imaging on the chip device,indicating that this can be an important toxicity testing. In addition, human iPS cell-based signaling
reporter system was also developed for prediction of chemical teratogenicity.
Timeline:
March 1, 2019 - February 29, 2020
Topics:Poster presentation at JCIA LRI Annual Workshop 2019 “Chip devices for in vitro systemic
toxicology” (Tokyo, August 30th, 2019)
Publications:
1. Y. Sawada, Y. Miura, E. Kobayashi, K. Iijima, J. Fukuda, H. Itagaki, “Evaluating
immunotoxicity in co-culture system of THP-1 and undifferentiated / keratinized NHEK”, The
46th Annual Meeting of The Japanese Society of Toxicology, Tokushima, Japan, June 2019
2. T. Shinagawa, J. Fukuda, S. Toyama, J. Fujita, K. Fukuda, Y. Kanda, A. Satsuka, “Maturationof human iPS-derived cardiomyocytes on oxygen-permeable spheroid culture device”,
Yokohama Branch Meeting of Society for Chemical Engineers, Yokohama, Japan, August
2019
3. T. Usui, D. Qin, J. Fukuda, “Co-culture of CACO-2 cell sheets and E. coli-encapsulated
microbeads for in vitro intestine model”, APCChE2019, Sapporo, September 2019
4. Y. Yamamura, J. Fukuda, “Drug evaluation chip device using multiple 3D tissue models”, The
32nd Annual Meeting of the Japanese Society for Alternatives to Animal Experiments,
Tsukuba, Japan, November 2019
5. D. Qin, T. Usui, J. Fukuda, “Co-culture of intestinal epithelial cells and bacteria-embedded
beads for in vitro small intestine model”, The 32nd Annual Meeting of the Japanese Society
for Alternatives to Animal Experiments, Tsukuba, Japan, November 20191

Development and assessment of new risk assessment methods
Title of Research:
17_PT01-02

Development of novel screening system to predict the effects ofenvironmental factors in each organ by using cell-free circulating
DNA in serum -- Advanced system for prediction of hazards fromenvironmental factors in fetus using cell free fetal DAN in mother’s
blood
Principal Investigator:
Wataru Miyazaki, PhD (Professor, Department of Bioscience and Laboratory Medicine, Graduate
School of Health Sciences, Hirosaki University)
66-1 Hon-cho, Hirosaki, Aomori 036-8564, Japan(tel) +81-172-39-5955, (e-mail) miya@hirosaki-u.ac.jp
Collaborators:
Noriyuki Koibuchi (Professor, Department of Integrative Physiology, Graduate School of Medicine,Gunma University)
Izuki Amano (Assistant Professor, Department of Integrative Physiology, Graduate School of
Medicine, Gunma University)
Takahiko Katoh (Professor, Department of Public health, Graduate School of Medicine, Kumamoto
University))
Summary of Research:
To prevent the adverse effects of several toxic chemicals at an early stage, it is important todevelop a novel screening system for detecting organ injury. For such purpose, we have focusedon the cell-free circulating DNA (cfDNA), whose origin may be identifiable if they harbortissue-specific epigenetic modification. This system is simple and minimally invasive because weutilized cfDNA in serum to detect the injuries. Furthermore, our system may have the potential todetect fetal organ injury using cfDNA of fetal origin in maternal serum. Previously, we identified
several candidate regions as the adult and fetal organ-specific markers (specific un-methylated
regions). We performed bisulfite amplicon sequence assay with cfDNA derived from organ-specificinjury mouse model, and detect the organ injuries. In this term, we have continued the assessmentof the novel system using cfDNA from several chemical-exposure mouse models (nicotine, ethanol,high fat). The injuries in the liver and cerebellum were detected in ethanol and/or high fat exposedgroups using our system. On the other hand, we could not detect the injuries of fetal organs frommaternal cfDNA. To improve the labor, time, and cost of our system, we have attempted an
improved method with Oligoribonucleotide Interference-PCR (ORNi-PCR). Both the cost and timeof the improved method were smaller than the previous. These results indicate that our system
may be useful to detect specific injured organs just by using serum, although further experiments
are needed to detect the injuries of fetal organs using maternal blood.
Timeline:
March 1, 2019 – February 29, 2020
Topics:Poster presentation at JCIA LRI Annual Workshop 2019 “Development of novel screening system
to predict the effects of environmental factors in each organ by using cell-free circulating DNA inserum” (Tokyo, August 30th, 2019)
Publications:
1. Poster presentation at 9th Federation of Asian and Oceanian Physiological Societies
Congress: FAOPS2019, title: A novel screening system to predict injured organs using
cell-free DNA in serum2

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:
Masaaki Miyazawa, PhD (Safety Science Research Laboratory, Kao Corporation)
2606, Akabane, Ichikai-Machi, Haga-Gun, Tochigi 321-3497, Japan(tel) +81-80-8466-6147, (e-mail) miyazawa.masaaki@kao.com
Collaborators:
Hideyuki Mizumachi, PhD (Safety Science Research Laboratory, Kao Corporation)
Summary of Research:
Lipophilic chemicals are difficult to correctly evaluate by existing in vitro tests because these tests
employ aqueous-phase systems. To overcome the limitation, we focused on a reconstructed
human epidermis (RhE) and developed the Epidermal Sensitization Assay (EpiSensA) based on
the expression of four marker genes related to induction of skin sensitization. Based on thecomparison with the results of animal test, we confirmed that EpiSensA has better predictive
performance for a variety of chemicals including lipophilic chemicals than existing in vitro tests.
Therefore, the validation study of EpiSensA was started from July 2018 at JaCVAM (JapaneseCenter for the Validation of Alternative Methods) to adopt it for OECD test guideline. At the last year,
we reported 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.
Therefore, For the evaluation of with-in laboratory reproducibility (WLR), the Phase I study wasstarted from November 2018. The Phase I study using 15 coded test chemicals is divided into
three parts (Phase I-A, I-B, and I-C) and each part includes three repetition tests of 5 chemicals.
Regarding the Phase I-A, two out of three laboratories didn’t meet the target criteria of WLR(>85%) established by the VMT. However, the following points of the protocol were revised andagreed by the VMT; i) statistically determined acceptance criteria of vehicle control, ii) clarified
criteria of retesting at a low dilution rate and iii) retesting when internal control gene fails to meetthe criteria. After reanalysis and retesting based on the revised protocol, all laboratories met thetarget criteria. In the subsequent Phase I-B, all laboratories showed 100% WLR according to therevised protocol. Finally, the mean WLR using 10 coded test chemicals was 93% and satisfied thetarget criteria. Currently, the Phase I-C study is under-evaluation.
Timeline:
March 1, 2019 – February 29, 2020
Topics:Poster presentation at JCIA LRI Annual Workshop 2019 “The validation study of EpiSensA(Epidermal Sensitization Assay); the in vitro skin sensitization assay based on reconstructed
human epidermis” (Tokyo, August 30th, 2019)3

Development and assessment of new risk assessment methods
Title of Research:
19_R01-01

Development of rapid, accurate, and low-cost AI drug hazard
assessment method by human stem cell test
Principal Investigator:
Wataru Fujibuchi, Ph.D.
Professor, Center for iPS Cell Research and Application (CiRA), Kyoto University
53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507(tel) 075-366-7012, (e-mail) fujibuchi-g@cira.kyoto-u.ac.jp
Summary of Research:
We developed a new method “hEST”, which has more than 95% accuracy predicting
neurotoxicity, and genotoxic/non-genotoxic carcinogen categories, using machine learning ofgene network data obtained from exposure of human embryonic stem cells to chemicals. This firstyear, we 1) selected 4 Japanese male/female ES/iPS cell lines as the test cells for 10 hepatotoxic
substances. Surprisingly, cell death occurred with 8 hepatotoxic substances when ES cells wereused, however, the sensitivity of iPS cells to chemical substances was low. In order to improve iPS
cells’ sensitivity and standardize the test cell lines, we 2) tested 24 existing naïve celltransformation methods, and 3) developed our own culture method. Although iPS cells’ sensitivity
increased, it has decreased again as cells were passaged. We will continue to develop morestable, high sensitivity iPS cells, and will obtain gene expression data from ES cells as well.
Timeline:
March 1, 2019 - February 29, 2020
Topics:Poster presentation at JCIA LRI Annual Workshop 2019 “Development of rapid, accurate, andlow-cost AI drug hazard assessment method by human stem cell test” (Tokyo, August 30th, 2019)
Publications:
1. Wataru Fujibuchi “代替法としてのヒト幹細胞への大きな期待-その驚くべき潜在能力” The 32nd
Annual Meeting of the Japanese Society for Alternatives to Animal Experiments (Tsukuba,
November, 2019)
2. Panina Yulia, Junko Yamane, Kenta Kobayashi, Wataru Fujibuchi “Development of a fast,
reliable and low-cost toxicity prediction system using AI and reset iPS cells.” Kyoto University
Medical Innovation Graduate School Program Medical research exchange salon, Poster
presentation (Kyoto, January, 2020)4

Research on the safety of new chemicals such as “Nano materials” etc.
Title of Research:
17_PT02-01

Establishment of mechanism based assay protocol for hazard andcarcinogenic risk of carbon based nanomaterials
Principal Investigator:
Hiroyuki Tsuda, MD, PhD (Professor, Nagoya City University, Nanotoxicology Project Lab)
3-1, Tanabedohri, Mizuho-ku, Nagoya, 467-8603, Japan(tel)+81-52-836-3496, (e-mail) htsuda@phar.nagoya-cu.ac.jp
Summary of Research:
To evaluate the toxicity and carcinogenicity of carbon nanotubes (CNT), we are establishing an
inexpensive intratracheal intrapulmonary spraying (TIPS) method for administration of CNTs via
the airway. TIPS will greatly expand research into the mechanism of CNT-mediated tissue injury
and carcinogenesis beyond what is currently possible using conventional expensive inhalation
exposure testing. Work in our laboratory has shown that among multi-walled carbon nanotubes(MWCNT), MWCNT-7 (M-H company, 40 wall layers) and MWCNT-N (N company, about 30-50
layers), which form needle-like aggregates, are carcinogenic to the lung and pleura of rats. Morerecently, work in our laboratory and by others using TIPS have shown that MWCNT-A (Company C,
150 layers) and MWCNT-B with fewer layers (Company C, 15 layers) are also lung carcinogens. In
the current study, double-walled carbon nanotubes (DWCNT) (diameter 1-3 nm) (Company A)
were compared with the carcinogenic CNT, MWCNT-7, using the TIPS method: acute / subchronic/ chronic toxicity / carcinogenicity was analyzed.
Methods: 10-week-old male rats were administered a total dose of 0.25 and 0.5 mg DWCNT and
MWCNT-7 per rat for the short-term test, and 10-week-old male rats were administered a total
dose of 0.125, 0.25, and 0.5 mg DWCNT or 0.5 mg for MWCNT-7 per rat for the long-term test.
CNTs were suspended in a solvent (saline + 0.5% dispersant PF68PF copolymer) andadministered every other day over the course of 15 days (total of 8 TIPS administrations). The
control groups were untreated rats and rats administered the solvent. MWCNT-7, which is known
to be carcinogenic in the lung and pleural mesothelium, was used as a positive control.
Short-term test: 3 and 8 weeks after beginning administration (1 and 6 weeks after the final TIPS
administration): The distribution and shape of the administered CNTs was examined.
Histopathological analysis of lung tissue was performed to determine lung tissue pathology.
Tracheal and lung lavage fluid, pleural lavage fluid supernatant, inflammatory markers in the cellpellets, and biochemical material prepared from lung tissue were analyzed to determine thedegree of inflammation. Inflammatory cytokines, DNA damage, and oxidative stress markers
revealed that DWCNT was less toxic than MWCNT-7.
Subchronic test (52W): The number of granules and alveolar Mφ of the encapsulated granulations
in lung specimens was greater in the DWCNT treated rats compared to the MWCNT-7 treated rats.
Notably, the number of PCNA-positive alveolar epithelial cells was less in the DWCNT treated rats
compared to the MWCNT-7 treated rats. Many MWCNT-7 fibers were not encased in granulations
and were often deposited in the alveoli. No neoplastic lesions were observed in either group.
Chronic toxicity. Carcinogenicity test (104w):
Lung tumor incidence (adenoma + adenocarcinoma) was significantly higher in the 0.5mg
DWCNT group, 7/24, (29.2%) compared to vehicle group, 1/25, (P <0.048). The incidence of
pleural malignant mesothelioma was significantly higher in the MWCNT-7 group, 16/25, (64%)
compared to the vehicle group, 0/25, (P <0.0001).
Less acute toxicity and more pronounced granulation encapsulation in the lungs of rats
administered DWCNT compared to rats administered MWCNT-7 suggests that malignant pleural
mesothelioma developed relatively early in the MWCNT group, resulting in death before lungtumors were able to develop, however, this is a topic that requires further study. We are also
studying carbon nanohorns and nanobrushes containing SWCNT structures using this same
method (currently 46w).
Timeline:
March 1, 2019 – February 29, 2020
Topics:5

Research on the safety of new chemicals such as “Nano materials” etc.
Poster presentation at JCIA LRI Annual Workshop 2019 “Establishment of mechanism based
assay protocol for hazard and carcinogenic risk of carbon based nanomaterials” (Tokyo,
August 30th, 2019)
Publications:
1. Abdelgied M., Elgazzar AM., Alexander D., Alexander W., Numano T., Iigo M., Naiki-Ito A.,
Takase H., Abdou KB., Hirose A., Taquahashi Y., Kanno J., Abdelhamid M., Tsuda H.,
Takahashi S. Pulmonary and pleural toxicity of potassium octatitanate fibers, rutile titanium
dioxide nanoparticles, and MWCNT-7 in male Fischer 344 rats, Arch. Toxicol., 93(4): 909-920,
2019
2. Abdelgied M., Elgazzar AM., Alexander TW., Numano T., Iigou M., Naiki-Ito A., Takase H.,
Hirose A., Taquahashi Y., Kanno J., Abdelhamid M., Khaled AA., Takahashi S., Alexander BD,
Tsuda H. Carcinogenic effect of potassium octatitnate (POT) fibers in the lung and pleura ofmale Fischer 344 rats after intrapulmonary administration, Particle and Fibre Toxicology,
https://doi.org/10.1186/s12989-019-0316-2 20196

Title of Research:
19_R03-01

Research on the effects of chemical substances on children, elderly
people, and those with gene disorders

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:
Recently, several in vitro assays to predict the sensitizing potential of chemicals have beendeveloped. However, these animal testing alternatives cannot distinguish chemical respiratory andskin sensitizers, although the risk management systems for them are quite different. Therefore, wehave been aiming at developing a novel in vitro assay, which can discriminate them by taking
advantage of the fundamental differences between their modes of function; development of helper
T (Th) 2 immune responses, which are critically important for respiratory sensitization. Recently,
we established a new 3-dimentional (3D) dendritic cell (DC) coculture system consisting of human
airway epithelial cell line, immature DCs derived from human monocytes, and lung fibroblast cellline. This coculture system was shown to successfully discriminate respiratory sensitizers fromskin sensitizers using 6 representative chemical sensitizers by more enhanced expression of key
costimulatory molecule OX40 ligand (OX40L), which is important for Th2 differentiation, in DCs. In
the present study, we are currently trying to establish a new 2-step DC/T coculture system byintroducing T cells in the DC coculture system, in which the Key event 4, that is T cell, in theadverse outcome pathway of sensitization can be used as a marker. To increase the versatility, we
are also trying to generate iPS-derived DC progenitors and T cell lines to apply for it.
In the 7th term, DCs stimulated by 6 representative sensitizers in the DC coculture system were
further reacted with allogeneic naive CD4+ T cells in the 2-step DC/T coculture system. As a result,selective up-regulation of IL-4, Th2 differentiation marker, by respiratory sensitizers could be seen
after 5-day culture. Furthermore, we generated several iPS-derived DC progenitor lines bytransduction of genes related to cell survival and cell cycle. Some of them showed a tendency toselectively up-regulate OX40L by respiratory sensitizer in the 3D DC coculture system as primarymonocyte-derived immature DCs did. We are currently still continuing to investigate them.
Timeline:
March 1, 2019 - February 29, 2020
Topics:Oral presentation “Establishment of a novel in vitro evaluation system for the prediction of
respiratory sensitizing potential of chemicals” and poster 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)” at JCIA LRI Annual Workshop 2019 (Tokyo,
August 30th, 2019)
Publications:
1. Mizoguchi I, Yoshimoto T, et al. Development of a novel 3D co-culture system to evaluatesensitizing potential of chemicals. The 32th Annual Meeting of the Japanese Society for
Alternatives to Animal Experiments. (Tsukuba, Nov. 20-22, 2019)
2. Mizoguchi I, Yoshimoto T, et al. Development of a novel alternative method for evaluation ofsensitizing potential and allergenicity by measuring human T cell activation and differentiation
7

Research on the effects of chemical substances on children, elderly
people, and those with gene disorders
in vitro. The 48th Annual Meeting of the Japanese Society for Immunology. (Hamamatsu, Dec.
12-13, 2019)8

Assessment of the effects on ecosystems, ecotoxicity and environment.
Title of Research:
18_S04-01

Exploring roles and simple estimation methods of species
sensitivity distribution for deriving PNECs
Principal Investigator:
Masashi Kamo (National Institute of Advanced Industrial Science and Technology. ResearchInstitute of Science for Safety)(e-mail) masashi-kamo@aist.go.jp
Collaborators:
Yuichi Iwasaki. National Institute of Advanced Industrial Science and Technology. ResearchInstitute of Science for Safety
Wataru Naito. National Institute of Advanced Industrial Science and Technology. ResearchInstitute of Science for Safety
Summary of Research:Species sensitivity distribution (SSD) has been internationally used to derive predicted no effectconcentration (PNEC) in ecological risk assessment. In this study, we aimed to (1) develop
statistical models to estimate log-normal SSD parameters (i.e., mean and standard deviation ofSSD) based on limited data and (2) quantify the uncertainties associated with risk assessments byusing SSD. For the former study (1), we first collected acute ecotoxicity data, and estimated SSDs
for 64 chemicals. We developed best models for estimating those SSD parameters based on
Akaike information criterion. In addition, we collected chronic ecotoxicity data, derived SSDs for 15
chemicals, and performed some preliminary analysis. For the latter study (2), we published a peer
reviewed paper based on the results obtained last year and presented the results at the SETAC
Europe 29th Annual Meeting. Based on standard deviations of acute SSDs estimated in the study(1), we also determined the magnitude of assessment factors required to determine more accurate
PNECs for the ecosystem protection.
Timeline:
March 1, 2019 – February 29, 2020
Topics:Poster presentation at JCIA LRI Annual Workshop 2019 “Exploring roles and simple estimation
methods of species sensitivity distribution for deriving PNECs” (Tokyo, August 30th, 2019)
Publications:
1. Kiyan Sorgog and Masashi Kamo. Quantifying the precision of ecological risk: Conventional
assessment factor method VS. species sensitivity distribution method. Ecotoxicology andEnvironmental Safety. Volume 183, 15, November 2019.
https://doi.org/10.1016/j.ecoenv.2019.109494
2. Poster presentation at the 29th SETAC Europe Annual Meeting at Helsinki, Finland from the26th till the 30th of May 2019, titled Assessment factor and SSD methods: which one provides
more protective threshold for the ecosystem against adverse effects of toxicants? by Kiyan
Sorgog, Masashi Kamo, Yuichi Iwasaki, Wataru Naito.
3. Poster presentation at 25th annual meeting of the Japanese society of environmental
toxicology, Tsukuba, Ibaraki, September 2019, titled Developing a new approach to predicting
the species sensitivity distribution by limited data by Kiyan Sorgog, Yuichi Iwasaki.
4. Poster presentation at 54th Annual Conference of Japan Society on Water Environment, titledComparison of performance ability of various assessment method by Kiyan Sorgog, Yuichi
Iwasaki (March 16-18th 2020 at campus of Iwate university).

9Other issues to be urgently addressed.
Title of Research:
17_S05-01

Development of Novel Analytical System of Biological Responses to
Chemicals by Deep Phenotyping Method

Principal Investigator:
Hiroyuki Kusuhara (Professor, University of Tokyo, Graduate School of Pharmaceutical Sciences,
Department of Molecular Pharmacokinetics)
7-3-1 Hongo, Bunkyo, Tokyo, 113-0033, Japan(tel) +81-3-5841-4770, (e-mail) kusuhara@mol.f.u-tokyo.ac.jp
Collaborators:
Tadahaya Mizuno (Assistant Professor, University of Tokyo, Graduate School of Pharmaceutical
Sciences, Department of Molecular Pharmacokinetics)
7-3-1 Hongo, Bunkyo, Tokyo, 113-0033, Japan(tel) +81-3-5841-4771, (e-mail) tadahaya@mol.f.u-tokyo.ac.jp
Setsuo Kinoshita (Contract Research Student, University of Tokyo, Graduate School of
Pharmaceutical Sciences, Department of Molecular Pharmacokinetics)
7-3-1 Hongo, Bunkyo, Tokyo, 113-0033, Japan
Summary of Research:
Today, organisms are always exposed to chemicals in every situation to some extent. Widevariety of chemicals exist around us and the chemicals affect organisms not one by one but
simultaneously like a complex. The fact prompts us to care “multiple exposure”. To achieve theappropriate and correct prediction of multiple exposure of chemicals, it is necessary tocomprehend the effects of chemicals even if those are unknown and difficult to be detected. In thisstudy, we tackle with the theme by omic approaches with innovative techniques in both dataacquisition and data analysis method.
This study consists of two pillars: (1) a novel acquisition method of proteome profile data by
2-dimensional electrophoresis (2DE) and (2) a new analysis method of profile data, orthogonallinear separation analysis (OLSA). In this 7th term, the last term, we tested the performance of 2DE
in detection of chemical features and showed capacity of OLSA to uncover novel aspects of anatural product.
We have carried out 2DE of HeLa cells treated with 6 different anti-cancer drugs under several
conditions obtained total 33 proteome profile data. Clustering analysis revealed good separationof each drug group composed of three biological replicates except for Cisplatin and doxorubicin.
Of note, combinatorial treatment of a MEK1/2 inhibitor (PD184352) and a microtubule inhibitor(vincristine) constructed a specific cluster and OLSA successfully extracted the combinatorial
effect as a specific vector that was not observed in each inhibitor alone.
As for data analysis, we have extended our profile data analysis method to achieve fully unbiased
analysis by implementation of parallel analysis. This extension enabled us to estimate theappropriate number of effects to be decomposed, without existing body of biological knowledge. In
fact, the basic effects generated by the renewed method were well consistent with gene groups
defined in gene ontology, compared with the original one, when Gaussian noise were added to thedata. To test the performance of our decomposition analysis approach, Rescinnamine, a natural
product was subjected to OLSA. We detected four main effects of the natural product and two ofthem were actually confirmed in vitro analysis such as luciferase assay and western blotting
analysis. All of these data suggest that our platform has the potential to boost deep understandingof combinatorial exposure of chemicals.
Timeline:
March 1, 2019 - February 29, 2020
Topics:
1. Poster presentation at JCIA LRI Annual Workshop 2019 “Development of Novel Analytical
10

Other issues to be urgently addressed.
System of Biological Responses to Chemicals by Deep Phenotyping Method” (Tokyo, August
30th, 2019)
Publications:
1. Akademi) ICCA-LRI workshop, June 19-20, 2019, Stresa, Italy
2. Journal) Kinoshita S, Biol Pharm Bull, 201911

Other issues to be urgently addressed.
Title of Research:
19_R05-01

Study on the contribution of microplastics to bioaccumulation andbiological magnification towards fish

Principal Investigator:
Tatarazako Norihisa（Professor, Ehime University, Graduate School of Agriculture）
3-5-7 Tarumi, Matsuyama, Ehime, 790-8566 Japan
Collaborators:
Tatsuhiro Niino (LSI Medience Corporation)
1000 Kamoshidacho, Aoba-ku, Yokohama-shi, Kanagawa 227-0033, Japan
Shin Takahashi（Ehime University, Graduate School of Agriculture）
3-5-7 Tarumi, Matsuyama, Ehime, 790-8566 Japan
Yoshifumi Horie (Akita Prefectural Univesity)
241-438 Kaidobata-Nishi Nakano Shimoshinjo Akita City 010-0195 Japan
Summary of Research:
The microplastics (MPs) are detected from sewage treated water or sea water, and harmful
chemical substances are known to adsorbed to those MPs. And there is a concern about thoseadsorbed chemicals may be taken more efficiently to organisms intermediated by MP and that
biological concentration or biological magnification is accelerated. Our study will try to clarify
whether the chemical substances adhered to MP are eluted, and absorbed / transferred /accumulated in the body of organisms.
This year, we reviewed the kind of solvent to be adsorbed on MP of PAHs and the quantitative
analysis procedure, and when the amount of adsorption was re-measured under new procedure,
the result was PE> PP> TF. A significant correlation was observed between the amount of PAHs
adsorbed and the octanol / water partition coefficient. Furthermore, the amount of PAHs adsorbed
on MP was higher in seawater than in pure water. It is necessary to confirm the acute toxicity of
MPB itself in medaka and daphnia in order to determine the set concentration for the accumulation
test. Medaka did not die with MPB of all sizes, whereas daphnia showed acute toxicity with MPB of
0.2 and 0.5 μm in diameter. Medaka was toxic to benzo [a] pyrene and pyrene in an extended
acute toxicity test. When larvae and Daphnia larvae were fed 5 μm-diameter fluorescent MPB andobserved under a microscope, both were confirmed to accumulate MPB in the digestive tract, but
no transfer to other internal organs and muscle tissues was observed. In addition, in order toexamine biomagnification, it was examined whether fluorescent MP was transferred to juvenile
medaka through daphnia. Although water flea was eaten by medaka, MPB was transferred, but noaccumulation of MPB was detected.
In the next fiscal year, we will determine the concentration of chemicals accumulated in fish byfeeding MPs with PAHs adsorbed. The transfer efficiency is determined from the total intake andthe amount of chemicals in the fish.
Timeline:
March 1, 2019 - February 29, 2020
Topics:Poster presentation at JCIA LRI Annual Workshop 2019 “Study on the contribution of microplastics
to bioaccumulation and biological magnification towards fish” (Tokyo, August 30th, 2019)
Publications:

1.

2.
3.

Norihisa Tatarazako, Kazufumi Misawa, Yukiyo Okazaki, SETAC Europe 29th Annual Meeting
26−30 May 2019 | Helsinki, Finland,poster
Yukiyo Okazaki, Takahiro Ishizaka, Norihisa Tatarazako, SETAC Europe 29th Annual Meeting
26−30 May 2019 | Helsinki, Finland,poster
Norihisa Tatarazako, Yukiyo Okazaki, SETAC North America 40th Annual Meeting 3–7 November
2019, Metro Toronto Convention Centre, Toronto, Ontario

12

4.

Other issues to be urgently addressed.

Yukiyo Okazaki, Norihisa Tatarazako, Shin Takahashi, Harumi Kasahara, Yaeko Sera, Tatsuhiro
Niino, SETAC North America 40th Annual Meeting 3–7 November 2019, Metro Toronto Convention
Centre, Toronto, Ontario

13

Other issues to be urgently addressed.
Title of Research:
19_R05-01

Establishment of medaka kinetic model for aged microplastic andadsorbed chemical
Principal Investigator:
Yuji Oshima (Kyushu University, Faculty of Agriculture, Laboratory of Marine Environmental
Science Professor)
744 Motooka, Nishi-ku, Fukuoka 8020345, Japan(tel&fax)+81-092-802-4607
Collaborators:
Yohei Shimasaki (Faculty of Agriculture, Kyushu University, Associated Professor)
Kang Ik Joon (School of Interdisciplinary Science and Innovation, Kyushu University, AssociatedProfessor)
Summary of Research:
Plastic has an extensive use in our daily life due to its low cost, lightweight and hard todecompose. Due to their high production and a quite slow degradation, plastic pollution has been
expanded worldwide in aquatic environments. Pollution of plastic from large items to small particle(i.e. microscopic plastic MP, < 5mm) were emerged. Thus, effect of MP and its vector effect with
toxicants are of concern. Many researchers have been performed in these topics. However, mostof study were performed using pristine MP. Thus, risk analysis of aged MP is required.
In this study, two kinds of aged PE plastics were prepared by exposure of ultraviolet for 180 and
460 hours. Weak changes on chemical characters were detected. No aging effect on sorption andadsorption of anthracene to MP was observed. Bioaccumulation factor, and elimination andabsorption constants were not changed in medaka fish co-exposed with anthracene and aged MP.
From results of one compartment analysis and simulation, vector effect of MP on accumulation ofanthracene on medaka fish may suggest. Further study on another type of aged MP is required.
Timeline:
March 1, 2019 - February 29, 2020
Topics:Poster presentation at JCIA LRI Annual Workshop 2019 “Establishment of medaka kinetic model
for aged microplastic and adsorbed chemical” (Tokyo, August 30th, 2019)14

Other issues to be urgently addressed
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)
Nakatani Hisayuki (Department of Chemistry and Materials Engineering, Nagasaki University)
Summary of Research:
The purpose of this study is to clarify by what mechanism and from what kind of plastic products
the secondary microplastics are generated from the perspective of polymer science and polymer
engineering. Various approaches of us lead the following results.
In the analysis using the pyrolysis method, individual component information was successfully
extracted from the plastic mixture samples by means of Py-APGC-MS measurements withselecting the ions characteristic of the object polymer and recording the ion chromatogram withhigh mass resolution.
In the morphological analysis of secondary microplastics, the microplastics recovered from rivers
and beaches showed cracks as in the outdoor-exposed polymers, where light-induced oxidation
takes place. On the other hand, no crack was observed in the microplastics recovered frommarine.
In a study considering microbial corrosion, the ”ABC degradation” mechanism was revealed: it
was found that a complex degradation takes place where the hydrophilization of polymer proceeds
owing to the photo-oxidative degradation (Abiotic) and microbial corrosion (Biotic), followed by the
micro-flake detachment (Cutting) due to the dissociation of the hydrophilic zone caused by water
ingress.
Chemiluminescence analysis shows that hydrophobic polyolefins such as HDPE and PP tends todecelerate the degradation owing to the suppression of temperature increase and oxygen diffusion
due to the presence of seawater.
Focusing on mechanical effects, it is was found that fatigue may cause the fragmentation ofplastic materials and that the jet mill could efficiently fine-grind plastics.
In the future, a database of literature and research results on the formation mechanism of
microplastics will be build. In addition, the microplastic formation behavior through long-term
outdoor exposure will be observed and the microplastic accelerated generation test methods will
be developed.
Timeline:
March 1, 2019 - February 29, 2020
Topics:Poster presentation at JCIA LRI Annual Workshop 2019 "Elucidation of the mechanism of
microplastic formation" (Tokyo, August 30th, 2019)
Publications:
1. Kuroda Shinichi, "Trends in Research on Microplastics", Materiaru Raifu Gakkaishi, 31 (1):10-14, 2019
15

2.
3.

Other issues to be urgently addressed
Kuroda Shinichi, "Microplastics - its problems and research trends", Jisedai Poriorefin
Sogokenkyu, 13: 17-22, 2019
Igarashi Toshiro, "Resource recycling of PE from the perspective of microplastics", Jisedai
Poriorefin Sogokenkyu, 13: 23-30, 201916

A n n u a l R e p o r t 2 019
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：http://www.nikkakyo.org/
URL for LRI : http://www.j-lri.org/