A n nu a lRepor t2 0 1 82018
Japan Chemical Industry Association

Annual Report 2018

Long-range Research InitiativeDevelopment and assessment of new risk assessment methods
Title of Research:
15_PT01-01

Development of new in vivo screening method to validate thelow-dose effects induced by estrogenic chemicals using estrogen
reporter micePrincipal Investigator:
Tsuyoshi Nakanishi (Laboratory of Hygienic Chemistry and Molecular Toxicology, Gifu
Pharmaceutical University)
Collaborators:
Hisamitsu Nagase (Laboratory of Hygienic Chemistry and Molecular Toxicology, Gifu
Pharmaceutical University)
Summary of Research:
So far we have been developing a new short-term in vivo screening test method for
xenoestrogens by using our transgenic mice carrying estrogen-responsible reporter gene (E-Rep
mice) and systemic in vivo imaging analysis as an end point. This method has been shown thepossibility of a high-superiority test method that can solve several problems of the uterotrophic
bioassay which is the only in vivo screening test method standardized as an OECD test guideline
No.440. In the current period, using our established test method, we evaluatedestrogenic/anti-estrogenic potential of Bisphenol A (BPA) which is a typical xenoestrogens.
As a result, in the state of estrogen depletion, BPA showed estrogenic effect at high doses, ofwhich action was not nonlinearity. On the other hand, in the state of maintaining the physiologicalestrogen level in the test animal, BPA showed anti-estrogenic effect.
These results indicate that it has been confirmed that BPA acts as a xenoestrogen in our new
method. In addition, our results suggest that our new test method using systemic in vivo imaging
analysis should perform the test in both the state of maintaining the physiological estrogen leveland of estrogen depletion.
Timeline:
April 1, 2018 – March 31, 2019
Topics:Poster presentation at JCIA LRI Annual Workshop 2018 “Development of new in vivo screening
method to validate the low-dose effects induced by estrogenic chemicals using estrogen reportermice” (Tokyo, August 31st, 2018)
Publications:
1. Törner K, Nakanishi T, Matsuura T, Kato Y, Watanabe H, Genomic integration andligand-dependent activation of the human estrogen receptor α in the crustacean Daphnia
magna, PLOS One, 13:e0198023 (2018)1

Development and assessment of new risk assessment methods
Title of Research:
16_PT01-02-3

Construction of prediction models for toxicities induced by drugs
and chemicals based on chemical structures and AOP
Principal Investigator:
Yoshihiro Uesawa (Department of Clinical Pharmaceutics, Meiji Pharmaceutical University)
Summary of Research:
Quantitative structure–activity relationship (QSAR) analysis applies structural and quantum
chemical, and physicochemical features, calculated from molecular geometry as explanatory
variables, to predict physiological activity. In current studies, the applications of QSAR prediction
models in a variety of adverse effects such as Delirium, pulmonary edema, nausea/vomiting,
respiratory depression, somnolence, rash, diarrhea, loss of appetite, nephritis, uterine cancer ...
etc., have been constructed by databases such as JADER and FAERS (JAPIC-AERS).
Furthermore, data set that includes FAERS and Tox21-AOP/MIE data was constructed andapplied to analyze the relationship between adverse effects and major MIEs such as nuclear
receptors and stress response pathways.
Timeline:
March 1, 2018 – February 28, 2019
Topics:
1. Poster presentation at JCIA LRI Annual Workshop 2018 “Construction of prediction models
for toxicities induced by drugs and chemicals based on chemical structures and AOP” (Tokyo,
August 31st, 2018)
2. Uesawa, Y., inventor; Meiji Pharmaceutical University, assignee. Prediction systems,
prediction methods, prediction programs, learning model input data generation systems, andlearning model input data generation programs. Pending Japanese patent application
2018-122565(PTC). 2018.
3. Matsuzaka, Y., Uesawa, Y.; Excellent Poster, CBI Annual Meeting 2018.
4. Asato, S, Kagaya, H., Uesawa, Y.; Excellent Presenter, The 4th Regulatory science forum foryoung people. 2018.
Publications:
1. Uesawa Y, "Quantitative structure-activity relationship analysis using deep learning based ona novel molecular image input technique", Bioorg Med Chem Lett, 28(20):3400-3403 (2018)
2. Matsuzaka Y, Uesawa Y, "Optimization of a Deep-Learning Method Based on theClassification of Images Generated by Parameterized Deep Snap, a Novel
Molecular-Image-Input Technique for Quantitative Structure–Activity Relationship (QSAR)
Analysis", Frontiers in Bioengineering and Biotechnology, in press (2019)3.
Ayano Shiota, Hajime Kagaya, Yoshihiro Uesawa, “Analysis of drug-induced somnolence
based on JADER with chemical structure information”, 138th annual meeting of thepharmaceutical society of Japan, Sendai Japan, 2018/3
4. Yuina Tatsuhiro, Hajime Kagaya, Yoshihiro Uesawa, “Analysis of structural andphysicochemical features of drugs that cause non-cardiogenic pulmonary edema using
Japanese Adverse Drug Event Report database (JADER)”, 138th annual meeting of thepharmaceutical society of Japan, Sendai Japan, 2018/3
5. Yoshiyasu Ozu, Hajime Kagaya, Yoshihiro Uesawa, “Classification of constipation-relateddiseases using Japanese Adverse Drug Event Report database (JADER)”, 138th annual
meeting of the pharmaceutical society of Japan, Sendai Japan, 2018/3.
6. Sumino Asato, Hajime Kagaya, Yoshihiro Uesawa, “Construction of a prediction model fornausea and vomiting based on JADER with chemical structures”, 138th annual meeting of thepharmaceutical society of Japan, Sendai Japan, 2018/3
7. Yuka Seta, Hajime Kagaya, Yoshihiro Uesawa, “The analysis on drug-induced delirium using
Japanese Adverse Drug Event Report database with chemical structure information”, 138th2

Development and assessment of new risk assessment methods
annual meeting of the pharmaceutical society of Japan, Sendai Japan, 2018/3

3

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:
Hiroshi Itagaki, PhD (Professor, Yokohama National University, Engineering)79-5 Tokiwadai, Hodogaya, Yokohama, Kanagawa, 240-8501, Japan
Masatoshi Watanabe, MD, PhD (Professor, Mie University, Medicine)1577 Kurimamachiya, Tsu, Mie, 514-8507, Japan
Summary of Research:
The aim of this study is to develop microdevices, so-called Body on a chip, for in vitro drug testing
to evaluate systemic toxicity of chemicals. This device may enable to replicate in vivo kinetics andinteractions between several tissues including the skin, the small intestine, and the lung for drug
absorption, liver and myocardial tissues for drug metabolism and exposure. The point of ourdevice is to engineer cell layer and spherical tissues on a chip as tissue models to acquire more
physiological responses.
In the second year of the project, we improved functionalities of spherical tissues. Data weobtained last year indicated that when cells form tissues and sufficient oxygen is supplied, not only
positive effects but also adverse effects were observed, probably due to generation of reactive
oxygen species. To alleviate the negative effects, we examined antioxidants. Indeed, the additionof antioxidants in culture medium further improved insulin secretion on pancreatic spheroids.
Permeability testing was performed with skin and intestine models that we fabricated last year. In
addition, interactions between two model tissues, liver and breast cancer, were evaluated on aseesaw-like stage, with which culture medium were perfused in the microdevice without any
external pump and air pressure supply. In this setup, a pro-drug was metabolized with the livermodel and inhibited growth of the cancer cells. Furthermore, we demonstrated that the modified
h-CLAT testing shows a positive reaction to a positive control substrate.
Timeline:
March 1, 2018 – February 28, 2019
Topics:Poster presentation at JCIA LRI Annual Workshop 2018 “Chip device for in vitro systemic
toxicology” (Tokyo, August 31st, 2018)
Publications:
1. D. Myasnikova, T. Osaki, K. Onishi, T. Kageyama, B. Zhang, J. Fukuda, Synergic effects ofoxygen supply and antioxidants on pancreatic β-cell spheroids, Scientific Reports, 9, 1802(2019)

4

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 (Associate Professor, Department of Integrative Physiology, Graduate
School of Medicine, Gunma University)
3-39-22 Showa-machi, Maebashi, 371-8511, Japan(tel) +81-27-220-7923, (e-mail) miya@gunma-u.ac.jp
Collaborators:
Noriyuki Koibuchi (Professor, Department of Integrative Physiology, Graduate School of Medicine,Gunma University)
Asahi Haijima (Assistant 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:
Detecting organ injury (e.g. by toxins or chemicals) at earlier stage is important to prevent theadverse outcomes. For such purpose, we have developed a novel screening system using serum.
This system is simple and minimally invasive, because we utilized serum to detect cell freecirculating DNA (cfDNA), whose origin may be identifiable if they harbor tissue-specific epigenetic
modification. Furthermore, our system may have a potential to detect fetal organ injury if cfDNA of
fetal origin in maternal serum is identifiable. Previously, we identified and selected several
candidate regions as the adult and fetal organ-specific markers (specific un-methylated regions).
In this term, first, to confirm whether these regions harbor tissue-specific epigenetic modification,
we performed bisulfite amplicon sequence (BSAS) assay with genome DNA, derived from several
fetal tissues. Second, we produced organ-specific injury mouse model using chemical exposure.
We could detect cfDNA harboring each organ-specific nature. The level of such organ-specific
cfDNAs increased with the increase in chemicals causing organ-specific injury. These results
indicate that our system may be useful to detect specific injured organs just by using serum.
Timeline:
March 1, 2018 – February 28, 2019
Topics:Poster presentation at JCIA LRI Annual Workshop 2018 “Development of novel screening system
to predict the effects of environmental factors in each organ by using cell-free circulating DNA inserum” (Tokyo, August 31st, 2018)
Publications:
1. Poster presentation at 21st Japan Society of Endocrine Disrupters Research Annual Meeting,title: Development of novel screening system to predict the effects of environmental factors ineach organ by using cell-free DNA in serum
2. Oral presentation at 89th The Japan Society for Hygiene Annual Meeting, title: Development of
a novel screening system to predict injured organs using cell-free DNA in serum5Development and assessment of new risk assessment methods
Title of Research:
18_S01-X01

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:
Noriyasu Imai (KOSÉ Corporation)
Megumi Sakuma, PhD (KOSÉ Corporation)
Momoko Shibata (KOSÉ Corporation)
Mika Watanabe (Hatano Research Institute, Food and Drug Safety Center)
Mayu Ikezumi (Hatano Research Institute, Food and Drug Safety Center)
Katsuhiro Uno, PhD (Hatano Research Institute, Food and Drug Safety Center)
Shinichi Watanabe (LION Corporation)
Junko Ueno (LION Corporation)
Summary of Research:
The assessment of skin sensitization potential is a major content of safety evaluation for novel
substances and was traditionally performed by animal tests. On the other hand, the developmentof non-animal tests for identifying the skin sensitization has been recently activated because ofanimal welfare and regulatory requirements. However, lipophilic chemicals are difficult to correctly
evaluate, since 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 gene expression of four markers related to induction of skinsensitization. Based on the comparison with the results of animal test, we confirmed that
EpiSensA had the great predictive performance for lipophilic test chemicals. Therefore, thevalidation study of EpiSensA was started from July 2018 at JaCVAM (Japanese Center for the
Validation of Alternative Methods) to adopt it for OECD test guideline.
At current study, the transferability to the three naive laboratories was evaluated based on three
sensitizers and a non-sensitizer distributed from Kao Corporation with non-coded. As the results,
the concurrent results of EpiSensA compared to animal test were observed with great
reproducibility at all laboratories. From these results, we concluded that the technical transfer tothree naive laboratories was successfully completed. From November 2018, the Phase I study forthe evaluation of with-in laboratory reproducibility have been performed based on 14 test
chemicals. After that, the Phase II study for between laboratory reproducibility will be performed
from March 2020, and the validation study of EpiSensA will finish at early 2021.
Timeline:
March 1, 2018 – February 28, 2019
Topics:Poster presentation at JCIA LRI Annual Workshop 2018 “The validation study of EpiSensA(Epidermal Sensitization Assay); the in vitro skin sensitization assay based on reconstructed
human epidermis” (Tokyo, August 31st, 2018)6

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
Collaborators:
Susumu Tomono, PhD(Assistant Professor, Aichi Medical University School of Medicine,
Department of Microbiology and Immunology)
1-1 Yazakokarimata, Nagakute, Aichi, 480-1195 Japan(tel) +81-561-62-3311 (e-mail)tomono@aichi-med-u.ac.jp
Summary of Research:
Purpose: To establish a method of evaluating the toxicity and carcinogenicity of respirable
nanomaterials, especially carbon nanotubes, that is less expensive and easier to perform thanconventional high-cost inhalation exposure. We developed transtracheal intrapulmonary spraying(TIPS) to administer test materials to the lungs of test animals. We tested several types ofmulti-walled carbon nanotubes (MWCNT): MWCNT-7 (Hodogaya, 40 layers), needle-likeaggregates of MWCNTs; MWCNT-N (Nikkiso, about 30 layers), another type of MWCNT that
forms needle-like aggregates; MWCNT-A (Company C, 150 layers), a third type of MWCNT that
forms needle-like aggregates; MWCNT-B (Company C, 15 layers), which because of its muchsmaller number of layers is flexible and produces cotton-like aggregates. We have shown thatthese four types of MWCNTs are carcinogenic to the lung. We are currently testing two-layered
carbon nanotubes (DWCNT) to determine if this type of carbon nanotube is also harmful to thelung and the pleural tissues as observed with the MWCNTs composed of several layers.
Methods: We conducted a preliminary dose setting trial using DWCNT from Sigma-Aldrich andfrom Company A. We determined that 0.5 mg per rat was an appropriate dose. We administered
DWCNT (1-3 nm diameter) (Company A) to 10-week old male rats. The test materials weresuspended in a solvent (saline + 0.5% dispersant PF68 PF copolymer) to give final concentrationsof 0.125 mg/ml and 0.0625 mg/ml, and 0.5 ml of the suspended material was administered to therats a total of 8 times, for final doses of 0.25 and 0.50 mg per rat. The control groups were (1)untreated and (2) administered vehicle alone. MWCNT-7, whose lung carcinogenicity wasdemonstrated using both a whole body exposure inhalation test and our TIPS administration
procedure was used as the carcinogenic positive control. Rats were killed at experimental week 3(1 week after the end of administration) and week 8 (6 week after the end of administration).
Histopathological analysis of the lung and pleura and biochemical analysis of the lung tissue,
tracheopulmonary lavage (BALF), and thoracic cavity lavage (PLF) was performed for each rat.
Inflammatory markers in supernatants and lavage cell pellets were also evaluated.
Results: Real time PCR and ELISA analyses of the lung tissue, pathological and histopathologicalobservation (distribution of sample, shape and degree of inflammation), and biochemical analysis(inflammatory cytokines, DNA damage, oxidative stress markers) indicated that damage of thelung tissue and visceral pleural mesothelium was absent or weak in the DWCNT treated rats. No

7

Research on the safety of new chemicals such as "Nano materials" etc.
grossly recognized neoplastic lesions were observed in rats killed at one year (52 weeks) after thestart of administration. Tissue samples from these rats are currently being prepared forhistopathological analysis.
New Studies: Using TIPS administration, we have started a study of the pleural toxicity of two new
types of materials. Both materials, like single walled carbon nanotubes, are constructed from asingle carbon sheet. The 8-week interim sacrifice is scheduled for this April 15-16.
Timeline:
March 1, 2018 – February 28, 2019
Topics:Poster presentation at JCIA LRI Annual Workshop 2018 “Establishment of mechanism based
assay protocol for hazard and carcinogenic risk of carbon based nanomaterials” (Tokyo, August
31st, 2018)
Publications:1.

2.

Elgazzar AM., Abdelgied M., Alexander D., Alexander W., Numano T., Iigo M., Naiki A.,
Takahashi S., Takase H., Hirose A., Kanno J., Elokle OM., Nasem AM., Tsuda H. Comparative
pulmonary toxicity of a DWCNT and MWCNT-7 in rats,Arch. Toxicol.,Oct. 10,
https ://doi.org/10.1007/s0020 4-018-2336-3, 2018
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., Tsuda H. Potassium octatitanate fibers induce
persistent lung and pleural injury and are possibly carcinogenic in male Fischer 344 rats, Cancer
Sci., May 2018, https://doi.org/10.1111/cas.13643

8

Title of Research:
15_S03-01

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

Establishment of in vitro evaluation system for the prediction of
respiratory sensitizing potential of chemicals
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
sensitizers and skin sensitizers, although the risk management systems for them are quite
different. Therefore, in the present study, we aim at developing a novel in vitro assay, which candiscriminate them by taking advantage of the fundamental differences between their modes offunction; development of helper T (Th) 2 immune responses, which is critically important for
respiratory sensitization.
So far, we established a new immunocompetent 3D co-culture system consisting of human
airway epithelial cells, immature dendritic cells (DCs) derived from human peripheral blood CD14+
monocytes, and human fibroblast cells. This co-culture system was demonstrated to predict the
respiratory sensitizing potential of chemicals by means of more enhanced expression of key
costimulatory molecule OX40Ligand (OX40L), which is important for Th2 differentiation, in DCs. In
this term, to further improve the versatility, we utilized iPS cell-derived DCs based on publishedprotocols, although their efficacy is not so good. Therefore, we initially tried to establish a novel
method to generate infinitely growing myeloid progenitors by stimulation with various cytokines
and/or lentiviral incorporation of genes related to cell cycle and survival. Finally, we havedeveloped several types of immortalized myeloid progenitors. By using these cells-derived DCs,
the co-culture system seems to be able to discriminate between skin and respiratory sensitizers as
in the case of primary DCs. Moreover, we also tried to develop a novel DC/T co-culture system,
which consists of sensitized DCs and allogenic naive CD4+ T cells. In this system, we have great
advantage that IL-4 up-regulation can be used as a marker for the prediction of respiratory
sensitizing potential. Indeed, our preliminary data suggest that selective up-regulation of IL-4 wasobserved by the stimulation with respiratory sensitizer as compared to that with skin sensitizer.
Timeline:
March 1, 2018 - February 28, 2019
Topics:Poster presentation at JCIA LRI Annual Workshop 2018 Establishment of a novel in vitro
evaluation system for the prediction of respiratory sensitizing potential of chemicals” (Tokyo,
August 31st, 2018)
Publications:
1. Yoshimoto T, Ohashi M, Hasegawa H, Orii N, Xu M, Owaki T, Mizoguchi I. Development of anovel alternative method by using 3D co-culture system for evaluation of respiratory sensitizing
potential. The 45th Annual Meeting of The Japanese Society of Toxicology, Workshop. (Osaka,Jul. 18-10, 2018)
2. Yoshimoto T, Hasegawa H, Orii N, Xu M, Mizoguchi I. Prediction of chemical respiratory
sensitizers by OX40L expression in dendritic cells using a new 3D co-culture system. The 4thInternational Conference on Toxicity Testing Alternatives & Translational Toxicology and the 2nd
9Research on the effects of chemical substances on children, elderly
people, and those with gene disorders
Asian Congress on Alternatives. (Guangzhou, Guangdong, China, Oct. 9-12, 2018)10

Research on the effects of chemical substances on children, elderly
people, and those with gene disorders
Title of Research:
12_PT03-01-6

Development of high sensitivity in vitro assessment system ofchemical-mediated hypersensitivity by using serine proteaseinhibitor-deficient cells
Subtitle: Analysis of in vitro assessment for respiratory allergy
Principal Investigator:
Akira Nakamura, Department of Immunology, Tohoku Medical and Pharmaceutical University
1-15-1 Fukumuro, Wakabayashi-ku, Sendai, Japan 981-8536(tel) +81-22-290-8725(e-mail) aki-n@tohoku-mpu.ac.jp
Summary of Research:
The allergic disorders triggered by various chemicals are separated into two types of diseases,
IgE-dependent immediate allergy and IgE-independent chronic allergy. Mast cells, basophils andeosinophils act as the effector cells in both allergic reactions. In particular, basophils andeosinophils rapidly infiltrate into respiratory tissue, and cause airway hyper-responsiveness andairway obstruction. These cells secrete abundant serine proteases as well as chemical mediatorsand cytokines. Serine proteases, such as elastases, disrupt the basement membrane, leading to
the infiltration of inflammatory cells. The serine proteases are repressed by the serine proteaseinhibitors. Because we found that basophils and eosinophils lacking a secretory leukoproteaseinhibitor SLPI produced more cytokines than wild type cells after stimulation with IgE or LPS.
Therefore, we have planned to generate the highly sensitive cell line by the deletion of serine
protease inhibitors using a human basophilic cell line, KU812-F. Because KU812-F expresses highlevels of Serpin b1 and b6, and we have generated stable Serpin b1 knockdown KU812-F celllines by using lentiviral-delivered sh (short hairpin) RNA. The knockdown KU812-F cells (KD cells)
produced IL-6 about 2 times higher than KU812-F after administration with toluene diisocyanate(TDI). KD cells also secreted IL-13 upon stimulation with TDI. In contrast, scramble and wild type
KU812-F cells did not produce IL-13. We investigated gene alteration between KD cells andscramble KU812-F cells in responses to TDI by mRNA sequencing analysis. We found significant
elevation of three genes as follows: CD69 (T cell activation marker), a chemokine receptor,
CXCR4, and sphingosine-1-phosphate receptor 4. We evaluated the relative fluorescenceintensities (RFI) in accordance with human Cell Line Activation Test (H-CLAT). KU812-F and KD
cells showed higher RFI of CD69 than scramble cells upon TDI and formaldehyde stimulation,
whereas all cell lines did not display any alteration of CD69 expression after DNCB and Methyl
Salicilate. We also found that the levels of CD69 mRNA are significantly augmented in KD cells
after TDI stimulation. In the present LRI research, we confirmed the high level of CD69 expression
in KD cells. Moreover, to establish a simpler method for the screening, we analyzed the CD69
expression using wild type KU812-F cells in response to 12 kinds of chemicals since wild typecells are also sensitive to chemical stimulation. The RFI values displayed more than 150% in WT
cells after administration with TDI and p-phenylenediamine. Collectively, our data suggested thatthe analysis of CD69 expression is a suitable application for the detection of respiratory sensitizes
by using KU812-F cells in addition to Serpin b1 knockdown KU812-F cells.
Timeline:
March 1, 2018 – February 28, 2019
Topics:Poster presentation at JCIA LRI Annual Workshop 2018 “Development of high sensitivity in vitro
assessment system of chemical-mediated hypersensitivity by using serine proteaseinhibitor-deficient cells.” (Tokyo, August 31st, 2018)11

Assessment on the effects on ecosystems and the environment
Title of Research:
16_PT04-01

Development of the platform to manage the monitoring data ofchemicals in environment, ChemTHEATRE

Principal Investigator:
Kei Nakayama, PhD (Assistant Professor, Center for Marine Environmental Studies, Ehime
University)
2-5 Bunkyo-cho, Matsuyama 790-8577, Japan(tel) +81-89-927-8132, (e-mail) kei_n@ehime-u.ac.jp
Collaborators:
Tatsuya Kunisue (Center for Marine Environmental Studies, Ehime University)
Itsuki C Handoh (College of Creative Studies and Institute of Science and Technology, Niigata
University)
Seiichi Uno (Faculty of Fisheries, Kagoshima University)
Tomohiko Isobe (Center for Health and Environmental Risk Research, National Institute forEnvironmental Studies)
Nobuaki Ohno (Graduate School of Simulation Studies, University of Hyogo)
Summary of Research:
We have launched a web platform to register and visualize the monitoring data of environmental
contaminants, named ‘ChemTHEATRE’ (Chemicals in the THEATRE: Tractable and Heuristic
E-Archive for Traceability and Responsible-care Engagement) at https://chem-theatre.com/. So far,the data of 83 projects have been registered to the database. The chemical concentration data inenvironmental and biological specimens can be found by sample type, chemical name, sampling
date and location, or other metadata. Users can export the query results for further analyses.
The registered data to ChemTHEATRE are also available at the websites of National Bioscience
Database Center, as follows,Integbio Database Catalog: https://integbio.jp/dbcatalog/
Life Science Database Cross Search: https://biosciencedbc.jp/dbsearch/
Life Science Database Archive: https://dbarchive.biosciencedbc.jp/en/chemtheatre/desc.html
Timeline:
March 1, 2018 – February 28, 2019
Topics:Poster presentation at JCIA LRI Annual Workshop 2018 “Development of the platform to manage
the monitoring data of chemicals in environment, ChemTHEATRE” (Tokyo, August 31st, 2018)
Publications:
1. Seiichi Uno, Machi Kawano, Tomohiko Isobe, Itsuki C Handoh, Nobuaki Ohno, Tatsuya
Kunisue, Kei Nakayama. “Monitoring of PAHs in shellfish after oil spill from the Sanchi tanker;
comparison of polluted conditions with other spill accident using ChemTHEATRE.” SETAC
North America 39th Annual Meeting, Sacramento, CA, USA, November 4–8, 2018.
2. Itsuki C Handoh, Toru Kawai, Tomohiko Isobe, Nobuaki Ohno, Seiichi Uno, Tatsuya Kunisue,
Kei Nakayama. “Bayesian uncertainties analysis of modelled chemical exposure of global fish
community: How can ChemTHEATRE improve the performance of FATE?” SETAC North
America 39th Annual Meeting, Sacramento, CA, USA, November 4–8, 2018.
3. Kei Nakayama, Binle Lin, Tomohiko Isobe, Seiichi Uno, Itsuki C Handoh, Nobuaki Ohno,
Tatsuya Kunisue. “ChemTHEATRE, a platform to browse the published or public data ofenvironmental monitoring and its use for risk assessment.” SETAC North America 39th
Annual Meeting, Sacramento, CA, USA, November 4–8, 2018.
4. Tomohiko Isobe, Kei Nakayama, Seiichi Uno, Itsuki C Handoh, Nobuaki Ohno, Tatsuya
Kunisue. “ChemTHEATRE: Opendata platform for environmental monitoring of POPs andother contaminants.” 11th SETAC Asia-Pacific Meeting, Daegu, South Korea, September
12

5.

6.

Assessment on the effects on ecosystems and the environment
16–19, 2018.
Tatsuya Kunisue, Kana Egashira, Tomohiko Isobe, Kei Nakayama, Takashi Matsuishi, Yuko
Tajima, Tadasu K Yamada, Shinsuke Tanabe. “Temporal trend analyses of POPs in three
toothed whale species stranded along the Japanese coastal waters: importance of samples
and data stored in es-BANK and ChemTHEATRE.” 38th International Symposium on
Halogenated Persistent Organic Pollutants & 10th International PCB Workshop: DIOXIN 2018,
Kraków, Poland, August 26–31, 2018.
Kei Nakayama, Tomohiko Isobe, Seiichi Uno, Itsuki C Handoh, Nobuaki Ohno, Tatsuya
Kunisue. “Introduction to ChemTHEATRE: a platform to utilize the published or public data ofenvironmental monitoring.” Water and Environment Technology Conference 2018 (WET2018),
Matsuyama, Japan, July 14–15, 2018.

13

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)
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:
The aim of the present study is to develop a new methodology to determine a suitablepredicted no effect concentrations (PNEC) by using a sensitivity distribution (SSD) in theecological risk assessment. The study is carried out in two perspectives. One is to develop astatistical method to estimate SSD with limited number of toxicity data [1], and the other is todevelop a method to quantify an uncertainty in the risk assessment through a comparison ofusing SSD and using conventional assessment factors (AF) [2] (termed SSD-method andAF-method, respectively). In [1], toxic data are collected to estimate SSD. Data selection are
carried out based on reliability of toxicity tests and number of species tested in SSD. As aresult, 28 chemicals were selected for SSD estimation. These SSD will be used as a learning
set for the SSD predictions. Descriptors of 28 chemicals such as logKow, boiling (melting)points, toxic values of model species in toxicity tests are collected. At the first step,correlations between these descriptors and mean (µ)/standard deviation (σ) of the SSDs areinvestigated, respectively. The correlations between the descriptors and µ are relatively high,compared to those between the descriptors and σ. These results suggest that the prediction
for σ is a harder task than the prediction for µ. In [2], we quantified the uncertainty which isinevitable for the SSD estimation and then developed the method to determine a magnitude of
AF under the uncertainty. Using the AF, the performance abilities of SSD-method andAF-method are compared. Measurement of the performance ability is as follows. For a given
toxicity data set, PNEC is determined, and if PNEC is greater than the protection goal,
determination of PNEC is counted as failure. These processes are repeated many times andthe number of failures is counted at various µ and σ of SSD. The performance ability of
AF-method is σ sensitive. In lower σ, conventional AF-method had a lower failure probability
compared to SSD-method. On the other hand, in higher σ, SSD-method has a lower failure
probability. Our result suggests that it is important to understand the factors affecting theperformance ability, and σ of SSD is surely one of them.
Timeline:
March 1, 2018 – February 28, 2019
Topics:Poster presentation at JCIA LRI Annual Workshop 2018 “Exploring roles and simple estimation
methods of species sensitivity distribution for deriving PNECs” (Tokyo, August 31st, 2018)
Publications:
1. Oral presentation at an annual meeting of the Japanese society of environmental toxicology,
titled Comparison of performance ability of various assessment method by Masashi kamo
(September 11th 2018 at satellite campus of Gifu university).

14

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 hidden 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. In the 5th term,
we carried out fine-tuning of these two.
As for 2DE, we have investigated intra-day deviation of profile data because it is indispensablefor the construction of database. HeLa cells treated with or without bortezomib were lysed and thespecimens were subjected to 2DE. The difference between days were smaller than that between
treatments (bortezomib and DMSO as a control). We have written the detailed protocol of our 2DE
method to minimize the dependency of the data on investigators.
As for data analysis, we have summarized the results so far and it was accepted by Scientific
Reports. By this method, it is expected that profile data of samples subjected to multiple
exposures can be separated in unsupervised manner, and it can be estimated what kind of action
is combined. In this term, we searched for data subjected to combined exposure from Gene
expression omnibus (GEO; https://www.ncbi.nlm.nih.gov/gds), and analyzed the data analysis
with OLSA. Six combined exposure data were found (ID: GSE10466, GSE16816, GSE17589,
GSE24065, GSE35230, GSE59882). Of these, combined exposure data of GSE16816,
GSE24065, and GSE59882 were expressed in a linear combination of vectors obtained by OLSA.
Timeline:
March 1, 2018 – February 28, 2019
Topics:Poster presentation at JCIA LRI Annual Workshop 2018 “Development of Novel Analytical System
of Biological Responses to Chemicals by Deep Phenotyping Method” (Tokyo, August 31st, 2018)
Publications:
1. The 2nd workshop for Japan-Korea Young Scientists on Pharmaceutics, Seul, 2018/7/9-11;
2. GPEN2018, Singapore, 2018/9/25-29
15

3.
4.
5.
6.
7.

Other issues to be urgently addressed.
33rd JSSX, Ishikawa, 2018/10/1-5
CBI 2018, Tokyo, 2018/10/9-10/11
第 40 回生体膜と薬物の相互作用シンポジウム, 2018/10/18-19, Miyagi
THE 13th SUGIYAMA LABORATORY (RIKEN) OPEN SYMPOSIUM, 2019/2/22, Yokohama
Journal) Mizuno T, Sci Rep, 201916

Other issues to be urgently addressed.
Title of Research:
18_R05-01

Study on the contribution of microplastics to bioaccumulation andbiological magnification towards fish
Principal Investigator:
Tatarazako Norihisa（Professor, Ehime University, Graduate School of Agriculture）
3-5-7 Tarumi, Matsuyama, Ehime, 790-8566 Japan
Collaborators:
Tatsuhiro Niino (LSI Medience Corporation)
1000 Kamoshidacho, Aoba-ku, Yokohama-shi, Kanagawa 227-0033, Japan
Shin Takahashi（Ehime University, Graduate School of Agriculture）
3-5-7 Tarumi, Matsuyama, Ehime, 790-8566 Japan
Yoshifumi Horie (Akita Prefectural Univesity)
241-438 Kaidobata-Nishi Nakano Shimoshinjo Akita City 010-0195 Japan
Summary of Research:
The microplastics (MPs) are 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. However, there are many
unclarified questions such as abundance in the water, transfer ratio to organisms or sediment,
lifetime and the mass balance of those chemical substances related with MPs in the environment.
Our study will try to clarify whether the chemical substances adhered to MP are eluted, andabsorbed / transferred / accumulated in the body of organisms.
In this fiscal year, we conducted quantitative analysis of the chemicals (13 kinds of polycyclicaromatic hydrocarbon) preparedly adhered to MP. The result showed that the concentration offorcibly adhered PAH to MP were correlated to molecular weight and hydrophobicity of PAH. The
adhered concentration were different between the materials of MP. Also, the materials of MP
differentiated the elution characteristics of water and seawater. Acute toxicity test of MP and theconsideration of feeding condition to fish as preliminary examinations for the biologicalaccumulation study were conducted. The medaka was not able to eat the MP which diameter was
more than 1.5mm, but they preferably ate the MP covered with feed which diameter was around
1.0mm. Further examination is needed for the acute toxicity testing.
From next fiscal year, we will conduct the quantitative analysis of organisms, which have taken
MPs and accumulated PAHs preparedly adhered to MP. The transition efficiency will be calculated
when the concentration of internal chemical substance and the amount of total chemical intake isprovided by this study.
Timeline:
June 1, 2018 – February 28, 2019
Topics:Poster presentation at JCIA LRI Annual Workshop 2018 “Study on the contribution of microplastics
to bioaccumulation and biological magnification towards fish” (Tokyo, August 31st, 2018)
Publications:
1. Norihisa Tatarazako, Yukiyo Okazaki, etc, 39th SETAC North America, Nov. 2018,
Sacramento17

A n n u a l R e p o r t 2 018
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/