A n nu a lRepor t2 0 1 32013
Japan Chemical Industry Association

Annual Report 2013

Long-range Research InitiativeDevelopment and assessment of new risk assessment methods

Research Field 1: Development and assessment of
new risk assessment methods
Development of human exposure assessment methods with the useof the probabilistic approach assisting voluntary risk management
by industrial sector
Akihiro Tokai, PhD (Professor, Osaka University, Graduate School of Engineering, Division of
Sustainable Energy and Environmental Engineering)

Human physiologically-based pharmacokinetic modeling ofindustrial chemicals with chimeric mice with humanized liver
Hiroshi Yamazaki, PhD (Professor, Showa Pharmaceutical University, Laboratory of Drug
Metabolism and Pharmacokinetics)

Establishment of animal testing for the prediction of respiratory
sensitizing potential of chemicals
Kohji Aoyama (Department of Hygiene and Health Promotion Medicine, Graduate School of
Medical and Dental Sciences, Kagoshima University)

Multi-functional testing method for attention ability by elaborating
the target detection method and application to developmental
neurotoxicology : testing for selective, sustained, shifted, andbisectional attention
Hiromi Wada (Hokkaido University, Graduate School of Letters, Division of Psychology)

Development of a user-friendly risk assessment tool for voluntary
environmental risk assessment and management
Bin-Le Lin (Research Institute of Science for Safety and Sustainability, National Institute ofAdvanced Industrial Science and Technology)

Research for establishing new method for risk assessment ofchemical toxicity using gene-modified ES cells
Takashi Morita（Professor of Department of Molecular Genetics, Osaka City University, Graduate
School of Medicine）

Development of mice visualizing "Metabolic reprograming" at earlyphase of tumorigenesis, and its application to caricinogenicity tests
Nobuhiro Tanuma (Div. Cancer Chemother., Miyagi Cancer Ctr. Res. Inst.)

Development of in vitro screening endocrine disruptor by steroid
profiling
Takeshi BAMBA (Department of Biotechnology, Graduate School of Engineering, Osaka
University)

Sophisticated hazard prediction by active QSAR modeling
Yoshimasa Takahashi (Professor, Dept. Comp. Sci. Eng., Toyohashi Univ. Tech.)
Annual Report 20131

Development and assessment of new risk assessment methods

Title of Research:
12_S01-01

Development of human exposure assessment methods
with the use of the probabilistic approach assisting
voluntary risk management by industrial sector
Principal Investigator:
Akihiro Tokai, PhD (Professor, Osaka University, Graduate School of Engineering, Division of
Sustainable Energy and Environmental Engineering)
2-1 Yamadaoka, Suita, Osaka 565-0871, Japan(tel)+81-06-6879-7676, (e-mail) tokai@see.eng.osaka-u.ac.jp
Collaborators:
Haruko Yamaguchi (ibid)(tel)+81-06-6879-7677,(e-mail) h.yamaguchi@em.see.eng.osaka-u.ac.jp
Summary of Research:
Risk management throughout life cycles of chemical substances becomes increasingly
implemented, as enactment of laws and regulations on chemicals management and voluntary
chemicals management in the industrial sector are advanced. For further development of thechemicals management, a probabilistic exposure assessment tool that estimates exposure levels
and their spatial/temporal variability or uncertainty on estimated error would facilitate an effective
risk management.
Thus, the purpose of this study is to develop an exposure assessment model that assists industrial
voluntary chemicals management for occupational exposure which is expected to have the highest
risk in the supply chain. With a reference to the Advanced REACH Tool (ART) constructed inEurope, we have developed a probabilistic assessment tool for the occupational exposure in
Japan, named SWEEs (integrated Score-based Workplace Exposure system) and validated thealgorithm by using some data in Japanese industries.
Timeline: November 1, 2012 –Topics:The 2nd Research Meeting of the New LRI of Japan Chemical Industry Association, Tokyo, August,2013. “Development of human exposure assessment methods with the use of the probabilistic
approach assisting voluntary risk management by industrial sector”, Oral and Poster presentation.
Publications:
Ishimaru, T., Yamaguchi, H., Tokai, A. and Nakakubo, T.（2013） Development of quantifying
method for the effect of metabolic inhibition during co-exposure by applying PBPK model: A caseof toluene and n-Hexane ， Proceeding of the SRA-JAPAN 26th annual meeting, B-6-3 (inJapanese).
Ichikawa, J., Yamaguchi, H., Tokai, A. and Nakakubo, T.（2013）Impact of lack of knowledge aboutmodel parameter on reduction of uncertainty factors by applying PBPK/PD model: A case ofchlorpyrifos，Proceeding of the SRA-JAPAN 26th annual meeting, B-3-2 (in Japanese).
Ishimaru,T., Yamaguchi, H., Tokai，A. and Nakakubo, T., (2013)，Development of practicalquantifying method applicable for risk assessment of metabolic inhibition during co-exposure in
workplaces by applying a PBPK model in humans, Society for Risk Analysis, Annual meeting,(2013.12.7-13,Baltimore) ※※student merit award for the Dose-Response Specialty Group (DRSG) of the SRA.
※travel award.
Annual Report 20132

Development and assessment of new risk assessment methods

Title of Research:
12_S01-02

Human physiologically-based pharmacokinetic modeling ofindustrial chemicals with chimeric mice with humanizedliver
Principal Investigator:
Hiroshi Yamazaki, PhD (Professor, 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.
Collaborators:
Norie Murayama, ibid, (e-mail) muraya_n@ac.shoyaku.ac.jp; Makiko Shimizu, ibid, (e-mail)shimizu@ac.shoyaku.ac.jp; Hiroshi Suemizu, CIEA, (e-mail) suemizu@ciea.or.jp; Masato
Nakamura, ibid, (e-mail) masatonakamuramdphdg@mail.com; Masato Kitajima, Fujitsu Kyushu
Systems, (e-mail) kitajima.masato@jp.fujitsu.com; Ryoji Takano, ibid, (e-mail)takano.r@jp.fujitsu.com
Summary of Research:
A simplified physiologically based pharmacokinetic (PBPK) model was defined in humans basedon metabolic parameters determined experimentally in vitro and/or in vivo and physiologicalparameters derived from the literature. In this study, the PBPK model basically consists of achemical absorption compartment, a metabolizing compartment, and a central compartment for awide of academic, regulatory, and industrial users. Test chemicals and primary metabolites,
melengestrol acetate (animal drug) and molinate (pesticide), were multi-dosed apparently
accumulated in human bodies by the present PBPK modeling. Using Humanized-liver mice, inwhich the liver has been repopulated with human hepatocytes is one of the challenge for
evaluation of species differences. In order to overcome limitation of available humanhepatocytes, the human hepatic cell line HepaRG were evaluated as promising donor cells forliver reconstitution in the TK-NOG mouse model. Taken together, the utility of this simplified PBPK
model with humanized mice could be also expanded to the industry researchers and regulatory
authorities.
Timeline: November 1, 2012 –Topics:An invited presentation in 2013 ICCA-LRI & NCATS Workshop, “What Is Normal? Implications for
Chemical Safety Assessment”, Santa Fe, New Mexico, USA
Publications:Y. Higuchi, K. Kawai, H. Yamazaki, M. Nakamura, F. Bree, C. Guillouzo, and H. Suemizu. The
human hepatic cell line HepaRG cells, possible cell source for steady generation of humanizedliver TK-NOG mice. Xenobiotica, in press (doi:10.3109/00498254.2013.836257)
A. Tsukada, H. Suemizu, N. Murayama, R. Takano, M. Shimizu, M. Nakamura, and H. Yamazaki.
Plasma concentrations of melengestrol acetate in humans extrapolated from the pharmacokinetics
established in in vivo experiments with rats and chimeric mice with humanized liver andphysiologically based pharmacokinetic modeling. Regul.Toxicol.Pharmacol. 65:316-324, 2013.
H. Yamazaki, H. Suemizu, N. Murayama, M. Utoh, N. Shibata, M. Nakamura, and F. P.
Guengerich. In vivo drug interactions of the teratogen thalidomide with midazolam: Heterotropic
cooperativity of human cytochrome P450 in humanized TK-NOG mice. Chem.Res.Toxicol.
26:486-489, 2013.

Annual Report 20133

Development and assessment of new risk assessment methods

Title of Research:
12_S01-03

Establishment of animal testing for the prediction of
respiratory sensitizing potential of chemicals
Principal Investigator:
Kohji Aoyama (Department of Hygiene and Health Promotion Medicine, Graduate School of
Medical and Dental Sciences, Kagoshima University)
8-35-1 Sakuragaoka, Kagoshima, 890-0031, Japan(tel)+81-99-275-5291 (e-mail) aoyama@m.kufm.kagoshima-u.ac.jp
Collaborators:
Hiroaki Kawaguchi (Laboratory of Veterinary Histopathology, Joint Faculty of Verterinary
Medicine, Kagoshima University)
1-21-1 korimoto, Kagoshima, 890-8580, Japan(tel)+81-99-285-8720 (e-mail) k3038952@kadai.jp
Kunihiko Yamashita (Corporate Research Center, R &D Management, Daicel Corporation)
1239 shinzaike, aboshi-ku, himeji, 671-1283, Japan(tel)+81-79-274-4061 (e-mail) ku_yamashita@daicel.co.jp
Summary of Research:
Respiratory allergy caused by occupational chemicals remains as a significant health concern
for industrial workers. However, to date there are no proper animal models for effectively
predicting their respiratory sensitizing potentials. The goals of this project are: (1) to develop theanimal models for evaluating the relative hazard of respiratory sensitizing chemicals; and (2) toprovide the useful information required for hazard assessment of these sensitizers in theworkplaces. we developed a small device that enables intratracheal administration of testing
substances in small animals such as mice. In this project, TDI, TMA and GA, three well-known
respiratory sensitizing chemicals and chicken ovalbumin (OVA), were evaluated. Known contact
sensitizing DNCB served as the negative control for respiratory allergens. Under light anesthesia
with diethyl ether, mice were sensitized by administering individual agents, each at 4 doses
including vehicle control, 5 times a week for a total of 3 weeks using a mouse intra-tracheal
installation device. Three days following the last installation, these mice were daily challenged
intratracheally with the corresponding agents for 3 days, and sacrificed 2 days thereafter. The
degree of airway inflammation was evaluated using semi-quantitative histological grading systems.
We found that histological scores, representing the degree of airway inflammation, increased withincreasing doses of OVA and TDI used for sensitization. Similar trends were also noted for TMAor GA-sensitized mice. They were also true for PAS staining in airway epithelial cells. In contrast,
such changes were not observed in mice treated with DNCB. Thus, our testing system confirmed
the relative sensitizing potency of these known respiratory sensitizers as previously reported incase-studies or epidemiologic studies. As the strategy for assessing the risk for respiratory
sensitizing agents in workplace, although the relationship between airway sensitization onset (asdetermined by various endpoints) and exposure dose/concentration should be first investigated,these evidence for setting exposure limit vary in substances and exposure conditions. Thus,
multi-factors, including experimental evidence, workplace exposure and clinical signs should beconsidered for properly and effectively controlling occupational respiratory allergy.
Timeline: November 1, 2012 –Topics:Presented at the 2th Annual Conferences of New LRI (31th August, 2013, Tokyo)

Annual Report 20134

Development and assessment of new risk assessment methods

Title of Research:
12_PT01-01

Multi-functional testing method for attention ability byelaborating the target detection method and application todevelopmental neurotoxicology: testing for selective,
sustained, shifted, and bisectional attention
Principal Investigator:
Hiromi Wada (Hokkaido University, Graduate School of Letters, Division of Psychology)
060-0810 Kita 10 Nishi 7, Kita-ku, Sapporo, Hokkaido(tel) +81-11-706-3321 (e-mail) wada@let.hokudai.ac.jp
Collaborators:
Hiroyuki Iso (Hyogo Health Science University, General Education Center)
650-0045 Minatojima 1-3-6, chuou-ku, Kobe, Hyogo(tel) +81-78-304-3123 (e-mail) iso@huhs.ac.jp
Summary of Research:Pregnant rats were treated with 0.01% or 0.015% Methimazole. The resulted pups were trainedwith the selective, sustained, shift, and divided attention tasks. The hypothyroid rats displayed
longer reaction times than the control rats in the selective attention task. In the sustained attentiontask, the hypothyroid rats exhibited increased variability of reaction times compared with thecontrol rats. In the shift attention task, the hypothyroid rats exhibited increased reaction times
when they were required to shift attention from one target to the other target. We conclude that the
hypothyroid rats were difficult to pay attention to a target quickly, to maintain attention to a target,
to shift attention from a target to another target. The hypothyroid rats reduced total T4 to 49-55%
and 35-38% of control levels at age of 21-22 in the treatments of 0.01% and 0.015% Methimazole,
respectively. Total T3 and TSH did not change at all. At age of 55-56, the hypothyroid rats
displayed increased TSH but normal levels of total T4. It is necessary to maintain total T4 level
more than 55% of the control level.
Timeline:

November 1, 2012 –Topics:2nd Meeting of JCIA-new LRI Poster presentation ” The testing methods for selective,
sustained, shifted, and divided attention by elaborating the target detection method”
Publications:
1, Wada Hiromi and Seto Yukina “Effects of perinatal hypothyroidism on shift attention in rats.”
Organohalogen Compounds, 74：1385-1388, 2012 (http://www.dioxin20xx.org/).
2, Wada Hiromi and Seto Yukina “Developmental effects of perinatal hypothyroidism on cognitive
functions: Focused on attention.” Psychiatria et Neurologia Japonica 114(8)：949－956，2012
3, Wada Hiromi and Seto Yukina “Effects of perinatal hypothyroidism on divided attention in rats.”
Scientific Committee on Neurotoxicology and Psychophysiology, Cape Town, South Africa,
March 2013.
4, Seto Yukina and Wada Hiromi “Effects of perinatal hypothyroidism on shift attention in rats.”
73nd Annual Meeting of the Japanese Society for Animal Psychology, Tsukuba, August 2013
5, Wada Hiromi, Yumoto Syoko, and Iso Hiroyuki. “Irreversible damages to auditory system
functions caused by perinatal hypothyroidism in rats.” Neurotoxicology and Teratology, 37:
18-22, 2013.
3. Innovation Kansai, Poster Announcement "Toxic judgment method of using pluripotent stem
cell" December 6, 2012, Osaka International Conference Hall
Annual Report 20135

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

12_PT01-02

Development of a user-friendly risk assessment tool forvoluntary environmental risk assessment and management
Principal Investigator:
Bin-Le Lin (Research Institute of Science for Safety and Sustainability, National Institute ofAdvanced Industrial Science and Technology). 16-1 Onogawa Tsukuba-shi, Ibaraki, 305-8569
Japan. (tel) +81-29861 8844, (e-mail) binle-lin@aist.go.jp
Collaborators:
Masashi Kamo (ditto). (tel) +81-29861 8029, (e-mail) masashi-kamo@aist.go.jp; Wataru Naito(ditto). (tel) +81-29861 8299, (e-mail) w-naito@aist.go.jp

Generalized Refined assessment
 All functions available

Assessment
Functions

Risk Assessment
 Probabilistic assessment
 Deterministic assessment
 Automatic determinationof UF Screening assessment
 Simplified GUI

SelectHazard Assessment
 Population –level effectassessment
 SSD
 No-effect concentration
for population –level
impact Screening under CSCL

Batch
Assessment
 CSCL-compliant
assessment
 Routine-task oriented
GUI

Exposure Assessment
 Tier 1 of risk
assessment 1 under
CSCL
 Exposure
concentration
distribution of

化学物質の濃度

Example of Hazard
Assessment

Example of Exposure
Assessment

Specialized

Capable of conducting assessment
according to the purpose, scenario
and level of detail for chemical
management

Example of Risk Assessment
ResultOutputPhysical property
data
Ecotoxicity study
data

Emission factors
Environmental
parameters

頻度

InitialAssessment

SimpleImage of Risk Assessment

Risk Management
 Relative comparison ofrisks
 Present checkpoints

環境濃度分布

Detailed
Assessment

Assessment Result

確率密度

RefinedBasic system

種の影響割合Assessment Mode

暴露濃度

個体群影響閾値濃度

For the realization of the WSSD 2020 goal, this tool supports the “acceleration/routinization of assessment” and“practical use/standardization of assessment models”.

Figure 1. Features and Functions of AIST-MeRAM
Summary of Research:
In the midst of the growing social needs for environmental risk assessment to address newnational/international regulations, this study has been promoting the development of theMulti-purpose Ecological Risk Assessment and Management Tool (AIST-MeRAM) that allows
business operators to select an assessment model on their own, and to perform a variety ofassessments easily and efficiently in order to meet their purpose.(Fig.1)
During the first term of New LRI, we made some improvements to the algorithm and interface,based on the hearings from the industry and government, and undertook the development of astandalone application. As a result, we released the standalone version of AIST-MeRAM(Japanese version: 0.9.12) that contains large amounts of toxicity data and is capable ofconducting many different types of ecological risk assessments as well as Kashinhou-compliant
screening assessment.
Annual Report 20136

Development and assessment of new risk assessment methods

Timeline: November 1, 2012 –Publications:1) Newspaper coverage: “’New LRI’: JCIA started taking actions. 130,000 ecotoxicity data”,
The Chemical Daily, 26 February 2013, p.10 (section of environment).
2) Newspaper coverage: “Development of a user-friendly tool for ecological risk assessment”,
Business Line, 26 April 2013, p.27.
3) Newspaper coverage: “AIST’s green innovation for future sustainable society: Ecological risk
assessment and management of chemicals – AIST-MeRAM”, Business Line, 25 July 2013,
p.30.
4) AIST-RISS Newsletter No.18. “AIST-MeRAM: Industry-academia-government needs
adaptive tool for ecological risk assessment and management”.
5) Lin, Naito and Kamo (October 2013) AIST OpenLab 2013: “AIST-MeRAM: Tool for
dissemination, optimization and standardization of ecological risk assessment of chemicals.
6) Lin, Hirata, Naito, Kamo (June 2013). “AIST-MeRAM: an easy-to-use tool for aquatic
environmental risk assessment and management of chemicals”, Water and Environment
Technology Conference, June 15-16 2013, Kaganei Campus TUAT.
nd7) Lin, Kamo and Naito (August 2013). Lecture and poster presentation at the 2 Annual
Meeting of New LRI. “AIST-MeRAM: Development of an industry needs adaptive tool for risk
assessment and management”.

Annual Report 20137

Development and assessment of new risk assessment methods

Title of Research:
12_PT01-03

Research for establishing new method for risk assessmentof chemical toxicity using gene-modified ES cells
Principal Investigator:
Takashi Morita(Professor of Department of Molecular Genetics, Osaka City University, Graduate
School of Medicine)
1-4-3 Asahimachi, Abeno-ku, Osaka, 545-8585, Japan (Tel)+81-6-6645-3925(Fax) +81-6-6645-3927, E-mail: tmorita@med.osaka-cu.ac.jp
Collaborators:
Kayo Yoshida, Associate Professor, Osaka City University,
E-mail:k-yoshida@med.osaka-cu.ac.jp
Akane Kizu, Research Associate, Osaka City University, E-mail:kizu@med.osaka-cu.ac.jp

Summary of Research:
We will establish a new method of assessment of many chemical risks by using embryonicstem cells and iPS cells with gene-modification. We will examine growth retardation, chromosome
aberration and developmental defects of stem cells. We cultured mouse ES cells in 96-well plates.
After adding carcinogenic chemicals (DEN, MNU, MMS), we examined the cell growth by MTT
assay. We detected cytotoxicity and difference of effect depending on the status of p53. Our
experiment revealed that (1) difference of cytotoxicity by chemical species, (2) that p53 mutation
resulted in resistance to the cells, (3) and that high frequencies of chromosome aberration in
p53-deficient ES cells. These result suggested that risk assessment using ES cells will become
high through-put system capable of characterizing mechanisms of toxicity and defense.

Timeline:

November 1, 2012 –Topics:1. First new LRI Annual research Report 2012 Aug,31
2. Chemical Industrial News Paper 2013Apr2
3. Second new LRI Annual research Report 2013 Aug,30

Publications:
1. Patent Application
Patent number: 2012-110115;
Inventors: Takashi Morita and Kayo Yoshida
Invention: The assessment method of the toxicological risk using a pluripotent stem cell
Applicant: Osaka City University,
Filing date: May 11, 2012
2. JST new technology, Oral presentation "Toxic judgment method of using pluripotent stem cell"
October 19, 2012, Tokyo JST headquarters
3. Innovation Kansai, Poster Announcement "Toxic judgment method of using pluripotent stem
cell" December 6, 2012, Osaka International Conference Hall

Annual Report 20138

Development and assessment of new risk assessment methods

Title of Research:
12_PT01-04

Development of mice visualizing "Metabolic reprograming"
at early phase of tumorigenesis, and its application to
caricinogenicity tests
Principal Investigator:
Nobuhiro Tanuma (Div. Cancer Chemother., Miyagi Cancer Ctr. Res. Inst.)
47-1 Noda-Yama, Medeshima-Shiode, Natori 981-1293, phone:+81-22-3811165,
e-mail: ntanuma@med.tohoku.ac.jp
Collaborators:
Toshio Watanabe (Grad. Sch. Humanities & Sci., Nara Women’s Univ.)
Kitauoya-Nishimachi, Nara 630-8506, phone:+81-742-203413,
e-mail: toshiwatana@cc.nara-wu.ac.jp
Gen Kondoh (Inst. Frontier Med. Sci., Kyoto Univ.)
53 Kawara-Machi, Shogo-In, Sakyo-Ku, Kyoto 606-8507, phone: +81-75-7514860,
e-mail: kondohg@frontier.kyoto-u.ac.jp
Hiroshi Shima (Div. Cancer Chemother., Miyagi Cancer Ctr. Res. Inst.)
e-mail: shima@med.tohoku.ac..jp

Summary of Research:
Increased flux of glycolysis is a common feature of tumors, and known as Warburg effect.
Together with alterations of other pathways, it mediates metabolic reprograming, now recognized
as a core hallmark of cancer. One of key molecules in such a reprograming is pyruvate kinase M(PKM) that exists as two isoforms, M1 and M2, generated by alternative splicing. Expression ofthese isoforms switches from M1- to M2-type during tumorigenesis so that normal
differentiated and proliferating/tumor cells express M1 and M2, respectively. This PKM-switch(from M1 to M2) is shown to be essential to organize Warburg effect. We have developed areporter-gene system, which enable us to visualize PKM-switch by cell-autonomous
fluorescence derived from the reporter gene. In this study, the reporter-gene was introduced
into mouse genome by BAC-transgenic method. We established three independent lines oftransgenic mice harboring the reporter gene. These transgenic mice are expected to visualize"Metabolic reprograming" at early phase of tumorigenesis, and to be applied to caricinogenicity
tests.
Timeline: November 1, 2012 –Topics:Reported in “1st Annual meeting of JICA New LRI: Development of mice visualizing "Metabolic
reprograming" at early phase of tumorigenesis, and its application to caricinogenicity tests

Annual Report 20139

Development and assessment of new risk assessment methods

Title of Research:
12_PT01-05

Development of in vitro screening endocrine disruptor bysteroid profiling
Principal Investigator:
Takeshi BAMBA (Department of Biotechnology, Graduate School of Engineering, Osaka
University)
2-1 Yamadaoka Suita, Osaka 565-0871 Japan
phone/fax: +81-6-6879-7418
e-mail: bamba@bio.eng.osaka-u.ac.jp

Summary of Research:
The aim of this study was to apply metabolic profiling to phenotype analysis of cells exposed tochemicals, and to develop a system to evaluate endocrine disruptors by multimarker profilingbased on chemical exposure-induced endogenous metabolite changes.
First, we constructed a platform to simultaneously analyze steroids in steroidogenesis pathways.
To construct a versatile evaluation system, gas chromatography/mass spectrometry (GC/MS),
which is known for its versatility, was used in this analysis. Seventeen steroids were
simultaneously analyzed under optimized preparation and GC/MS conditions.
Next, we constructed a screening system for endocrine disruptors. Following the protocol ofOECD TG 456, the human adrenocortical carcinoma cell line H295R exposed to forskolin andprochloraz was used as a positive control. Steroid profiles were obtained successfully.

Timeline: November 1, 2012 -Topics:2nd New LRI Annual Conferences (Yaesu First Financial Building, August 31, 2013) “Developmentof in vitro screening endocrine disruptor by steroid profiling”

Publications:
1. Bamba Takeshi, Masashi Okuno, Toshiyuki Yamashita, Eiichiro Fukusaki
Simultaneous analysis of steroids by GC/MS for evaluation of endocrine disruption
65th SBJ Annual Meeting, International Conference Center Hiroshima (Hiroshima),
September 20, 2013
2.

Masashi Okuno, Toshiyuki Yamashita, Eiichiro Fukusaki, Bamba Takeshi
Development of in vitro screening endocrine disruptor by steroid profiling
Metabolome Symposium 2013, Kyushu University School of Medicine Centennial Hall
(Fukuoka), October 3 – 4, 2013

Annual Report 201310

Development and assessment of new risk assessment methods

Title of Research:
12_PT01-06

Sophisticated hazard prediction by active QSAR modeling
Principal Investigator:
Yoshimasa Takahashi (Professor, Dept. Comp. Sci. Eng., Toyohashi Univ. Tech.)Collaborators:
Hiroaki kato (Asoc. Professor, Dept. Comp. Sci. Eng., Toyohashi Univ. Tech.)Tomoya Yamazaki (Master student, Toyohashi Univ. Tech.)
Keisuke Saito (Master student, Toyohashi Univ. Tech.)

Summary of Research:
In preceding work, we proposed a technique for active QSAR Modeling that is based on active
sampling of a temporary training data set, which gives us higher performance in the prediction.
However, the prediction result depends on the number of compounds for the temporary training
set used for making the model. And, it is difficult to know the optimal number of the neighbors to be
used, in advance. In the present work, we employed a threshold of the similarity at exploring theneighbors but not the number of neighbors to be searched. Computer experiment showed us thatthe method with the threshold of similarity gives a better performance. Besides, it was shown thata QSAR model obtained from the whole data could give us better prediction when the appropriate
neighbors are not available enough. Alternatively the performance of the method also depends on
the local data structure around a query of interest. But, it is impossible to see a data structure of
the local space around the query in advance. We employed kNN, RMSST (Rooted Minimum
Spanning Sub-Tree) and Centroid method to explore the neighbors. Once, we generated the
QSAR models with the different training sets that are obtained by the different neighbor searching
methods, and we evaluated the statistical performance of the models. We used the best
approximation model among them. Computational experiment with a data set of toxic chemicals
suggested that the current approach can provide us much better predictions for the case.

Timeline: November 1, 2012 -Topics:The Secondt New LRI Workshop, Tokyo, Aug., 2013

Publications:
1) Tomoya Yamazaki and Yoshimasa Takahashi, Toxicity prediction of chemical substances by
Active Sampling :Introduction of cutoff value by similarity, Proc. of the 22nd Symposium on
Environmental Chemistry, 2013, July, Tokyo.
2) Tomoya Yamazaki and Yoshimasa Takahashi, Toxicity prediction of chemical substances by
Active Sampling: Neighbor search method and model selection, Proc. of the 41th Symposiumon Structure-Activity Relationships, 2013, November, Osaka.

Annual Report 201311

Research on the safety of new chemical substances including nanomaterials

Research Field 2: Research on the safety of new
chemical substances including
nanomaterials
Evaluation of the relationship between physicochemical properties,
bio-distribution, and safety of nanomaterials
Yasuo Yoshioka (Laboratory of Toxicology and Safety Science, Graduate School of
Pharmaceutical Sciences, Osaka University)

Annual Report 201312

Research on the safety of new chemical substances including nanomaterials

Title of Research:
12_S02-01

Evaluation of the relationship between physicochemical
properties, bio-distribution, and safety of nanomaterials
Principal Investigator:
Yasuo Yoshioka (Laboratory of Toxicology and Safety Science, Graduate School of
Pharmaceutical Sciences, Osaka University)
1-6 Yamadaoka, Suita, Osaka 565-0871, Japan. (tel) +81-6-6879-8233(e-mail) yasuo@phs.osaka-u.ac.jp

Collaborators:
Kiyohito Yagi (Laboratory of Bio-Functional Molecular Chemistry, Graduate School of
Pharmaceutical Sciences, Osaka University)
1-6 Yamadaoka, Suita, Osaka 565-0871, Japan. (tel) +81-6-6879-8195(e-mail) yagi@phs.osaka-u.ac.jp
Makiko Kuwagata (Laboratory of Pathology, Toxicology Division, Hatano Research Institute, Foodand Drug Safety Center)729-5 Ochiai, Hadano, Kanagawa 257-8523, Japan. (tel) +81-463-82-4751(e-mail) kuwagata.m@fdsc.or.jp

Summary of Research:
Advances in nanotechnology have led to the recent development of many nanomaterials. However,
the increasing use of nanomaterials has prompted public concern regarding their potential safety.
Because nanomaterials have great potential to improve the quality of human life, it is essential toensure the safety of nanomaterials for the development of safety-assessed products. The safety ofnanomaterials is related to the dose, concentration, and duration of the exposure and theirabundance and persistence in tissue. Accordingly, a systematic and thorough analysis of theAbsorption, Distribution, Metabolism, and Excretion (ADME) of nanomaterials is essential as thebasis for determining the potential for risk to human health. In addition, understanding of the
ADME of nanomaterials is necessary in regard to their tissue toxicity. In this study, we examined
the absorption of nanomaterials via skin route. We used nickel particles with the diameter of 1000
nm (NP1000) or less than 100 nm (NP100). Nickel particles are generally used for industrial
products, such as a ceramic capacitor. In addition, nickel ion is known to induce metal allergy to
the skin exposure. Our results showed that the ion and the nickel particles used in this study could
not penetrate the skin in vivo and in vitro, although more precise investigation is needed. The data
could provide information to ensure the safety of nanomaterials.

Timeline: November 1, 2012 –Topics:Poster presentation at The 2nd LRI Annual Conferences “Evaluation of the relationship between
physicochemical properties, biodistribution, and safety of nanomaterials”

Annual Report 201313

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

Research Field 3: Research on the effects ofchemical substances on children,
elderly people, and those withgene disorders
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
Nakamura Akira (Department of Immunology, Kanazawa Medical University)

Annual Report 201314

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

Development of high sensitivity in vitro assessment systemof chemical-mediated hypersensitivity by using serine
protease inhibitor-deficient cells. Subtitle: Analysis of invitro assessment for respiratory allergy
Principal Investigator:
Nakamura Akira(Department of Immunology, Kanazawa Medical University)
1-1 Daigaku, Uchinada, Ishikawa, Japan 920-0293(tel) +81-76-218-8120(e-mail) aki-n@kanazawa-med.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. In 2011 LRI research, we found that a serine protease inhibitor, secretory leukoproteaseinhibitor (SLPI) are expressed in murine basophils and eosinophils. Both Slpi -/- (SLPI-deficient)basophils and eosinophils produced more cytokines than wild type (WT) cells after stimulation with
IgE or LPS. Therefore, we have planed to establish the highly sensitive cell lines of chemicalmediated hypersensitivity by the deletion of serine protease inhibitors including SLPI. In 2012LRI
study, we firstly examined the cytokine responses of murine Slpi -/- basophils and eosinophils. As
in the case of 2011LRI research, Slpi -/- basophils were susceptible for the cell toxicity of thechemicals, demonstrating that murine Slpi -/- basophils seemed to be inadequate for the screeningof the sensitizers. Conversely, Slpi -/- eosinophils showed high viabilities after administration withvarious chemicals. In addition, IL-6 production stimulated with beryllium sulfate was augmented in
Slpi -/- eosinophils, suggesting that eosinophils lacking serine protease inhibitors are suitable forthe screening of some chemicals. We next investigated the expression profile of the serine
protease inhibitors in mast cells, basophils, and eosinophils by DNA microarray analysis. The
array studies showed the results as follows. : Mast cells express Serpin b1a and b6a. Basophils
express Serpin b1a, b2, and SLPI. Therefore, these serine proteases would be the target genes
for the generation of cell lines. We also induced the toluene diisocyanate (TDI)-induced respiratory
inflammation. However, since the cellular infiltration was comparable between WT and Slpi -/- mice,the methods should be improved. Finally, we tested the cell viability and cytokine responses in thebasophilic cell line KU812. KU812 showed high viabilities against various chemicals. KU812 also
secreted cytokines in response to several chemicals, suggesting that KU812 lacking serine
protease inhibitors would be an ideal risk evaluation model against chemical substances.

Timeline: November 1, 2012 Topics:
Poster presentation entitled “Development of high sensitivity in vitro assessment system ofchemical-mediated hypersensitivity by using serine protease inhibitor-deficient cells.” at the 2th
annual conference of new LRI, Tokyo, Japan, August 2012

Annual Report 201315

Assessment on the effects on ecosystems and the environment

Research Field 4: Assessment on the effects on
ecosystems and the environment
Development of in silico Prediction Model on Environmental Fate ofChemical Substances
Tatsuya Takagi (Graduate School of Pharmaceutical Sciences, Osaka University; Genome
Information Research Center, Research Institute for Microbial Diseases, Osaka Univ.)

Comprehensive evaluation methods for chemicals registered in
PRTR.
Norihide Nakada (Research center for Environmental Quality Management, Graduate School ofEngineering, Kyoto University)

Title of Research: Development of Ecosystem Risk Impact
Assessment System Methods for Chemicals using Microcosm
Systems
Yuhei INAMORI (Fukushima University)

Annual Report 201316

Assessment on the effects on ecosystems and the environment

Title of Research:
12_S04-01

Development of in silico Prediction Model on Environmental
Fate of Chemical Substances
Principal Investigator:
Tatsuya Takagi, Graduate School of Pharmaceutical Sciences, Osaka University; Genome
Information Research Center, Research Institute for Microbial Diseases, Osaka Univ.
1-6, Yamadaoka, Suita, Osaka, Japan, 565-0871, (tel) +81-6-6879-8243,(e-mail) ttakagi@phs.osaka-u.ac.jp
Collaborators:
Norihito Kawashita, Graduate School of Pharmaceutical Sciences, Osaka University; Genome
Information Research Center, Research Institute for Microbial Diseases, Osaka Univ.
1-6, Yamadaoka, Suita, Osaka, Japan, 565-0871, (tel) +81-6-6879-8244, (e-mail)kawasita@gen-info.osaka-u.ac.jp
Kousuke Okamoto, Graduate School of Pharmaceutical Sciences, Osaka University;
1-6, Yamadaoka, Suita, Osaka, Japan, 565-0871, (tel) +81-6-6879-8242, (e-mail)okamotok@phs.osaka-u.ac.jp
Summary of Research:
In 2009, since the Act on the Evaluation of Chemical Substances and Regulation of Their
Manufacture, etc., was revised, chemical substances which are readily degradable have become
subject to control under the law. Chemical industries are required to examine various physical,
chemical, and biological properties of compounds such as toxicity, accumulation behavior, anddegradation property. If there is a procedure which enables us to predict any properties of new orexisting chemical substances without experiments, we are able to plan the efficient order of theexperiments based on the ranking of the predicted properties. Because most existing prediction
models on abiotic degradation, especially photolysis and hydrolysis are targeted to similar
chemical substances, the development of the prediction models on the photolysis and hydrolysis
of more various chemical substances would be contributed to obtain the effective ranking ofcandidates for experimentation.
The experimental results of the direct photolysis and hydrolysis under certain conditions
were collected and then the datasets of hydrolysis and direct photolysis consisted of the results of
143 and 106 compounds, respectively. Although the experimental results of indirect photolysis
were also checked, since there were few results whose experimental conditions were under the
EPA guideline, we decided that the prediction on photolysis of this project was focused on directone. We are intended to collect the information on photolysis and hydrolysis and to add it into thedataset continuously. The quantitative model on hydrolysis of “unstable” compounds whose
half-lives are less than a year was built by using publicly available software, and its prediction
performance was adequate. We are preparing to calculate an excited state that is regarded as a
key factor for the prediction of direct photolysis of a compound using publicly available software.
The trial system which provides the prediction models through the WWW was constructed. We areadding several functions and improving usability of this web site.
Timeline: November 1, 2012 –Topics:The 2th Annual Conference of New LRI, “Development of new tool for in silico prediction ofenvironmental fate of compounds”, Aug 30, 2013
Publications:
The 40th Symposium of Structure-Activity Relationships, Aichi, Japan, Nov 2012

Annual Report 201317

Assessment on the effects on ecosystems and the environment

Title of Research:
12_PT04-01

Comprehensive evaluation methods for chemicals
registered in PRTR.
Principal Investigator:
Norihide Nakada (Research center for Environmental Quality Management, Graduate School ofEngineering, Kyoto University)
520-0811, 1-2 Yumihama, Otsu, Shiga, +81-77-527-6220, nakada.norihide.8w@kyoto-u.ac.jp
Collaborators:
Shuhei Tanaka (Graduate School of Global Environmental Studies, Kyoto University)
606-8501, Yoshida-honmachi, Sakyo, Kyoto, +81-75-753-5151, t-shuhei@edn.env.kyoto-u.ac.jp
Yongkui Yang, (Research center for Environmental Quality Management, Graduate School ofEngineering, Kyoto University)
520-0811, 1-2 Yumihama, Otsu, Shiga, +81-77-527-6220, ykyang@biwa.eqc.kyoto-u.ac.jp

Summary of Research:
Chemicals are widely used in chemical industries. Huge amount of the chemical usage andconsequent discharge might result in their entering into the wastewater and even water treatment
units such as the biological and/or physicochemical processes. This research is aimed at
establishing the formation potential methods for toxic chemicals during those treatments above,and proposing new risk assessment methods.
In the first term, four analytical methods were established for each group including fourtrihalomethanes (THMs), four aldehydes, eight nitrosamines and six perfluorinated carboxylic
acids (PFCAs), respectively. Moreover, four fluorotelomer alcohols (FTOHs) as the precursors of
PFCAs were established. Besides, according to the reported mechanisms for forming the THMs,
aldehydes and nitrosames, the predicted precursors were selected among the chemicals
registered in PRTR. And their concentrations in wastewater were calculated based on the PPTR
data and annual wastewater in Japan. Three assessment methods were established for formation
potential tests, the ozonation treatment (two conditions: high and low dissolved ozone dose),chloramination treatment, and biological treatment (two conditions: aerobic and anaerobic),
respectively. Samples covered the chemicals in PPTR and actual wastewater samples. Fivechemicals were firstly selected as the potential precursors according to the high potential of
exposure in wastewater (calculated concentration in wastewater) and reported precursors inliteratures. The formation potential for the aldehydes and nitrosamines during the ozonation andchlorination treatments were investigated. The results showed during the ozonation treatment, thedimethylhydrazine formed significant formaldehyde and N-nitrosodimethylamine. Further, morechemicals will be tested and quantified for the formation potential. In the case of PFCAs formation
potential during the biological treatment, the formation and degradation rates were studied under
the anaerobic (10, 20 day, 1, 2, and 3 month) and the aerobic condition (10, 20 day, 1 month). The
results found the PFCAs formation was correlated with the degradation of FTOHs predictor.

Timeline: November 1, 2012 –Topics:Poster presentation at 2nd Annual Conference of LRI
Publications:
48th Annual Conference of Japan Society of Water Environment, Sendai, Japan, March 2014.

Annual Report 201318

Assessment on the effects on ecosystems and the environment

Title of Research:
12_PT04-02

Title of Research: Development of Ecosystem Risk Impact
Assessment System Methods for Chemicals using
Microcosm Systems
Principal Investigator:
Yuhei INAMORI (Fukushima University)
1 Kanayagawa, Fukushima 960-1296 Japan(tel)+81-24-548-5254 (e-mail) ina0120@sss.fukushima-u.ac.jp
Collaborators:
Kazuhito MURAKAMI (Chiba Institute of Technology)
2-17-1 Tsudanuma, Narashino, Chiba 275-0016 Japan(tel)+81-47-478-0455 (e-mail) kaz_murakami@sky.it-chiba.ac.jp
Takashi AMEMIYA (Yokohama National University)
79-1 Tokiwadai, Hodogaya-ku, Yokohama, Kanagawa 240-8501 Japan(tel)+81-45-339-4353 (e-mail) amemiyat@ynu.ac.jp
Ken-ichi SHIBATA (Yokohama National University)
79-1 Tokiwadai, Hodogaya-ku, Yokohama, Kanagawa 240-8501 Japan(tel) +81-45-339-4369 (e-mail) shibata-kenichi-ym@ynu.ac.jp
Ryuhei INAMORI (Fukushima University)
1 Kanayagawa, Fukushima 960-1296 Japan(tel) +81-24-548-5254 (e-mail) r_inamori@hotmail.com
Katsura SUGIURA (Sagami Women’s University), Kunihiko KAKAZU (Fukushima University),
Hideaki HAYASHI (Chiba Institute of Technology), Jun KUMADA (Fukushima University)
Summary of Research:
In this research, research and development in the environmental impact risk assessment of achemical substance was conducted paying attention to microcosm system which is an aquaticmodel ecosystem which consists of a producer (phytoplankton), a predator (zooplankton), and adecomposer (bacteria). The enactment as international guideline as OECD test of thegeneral-purpose microcosm test method from Japan is aimed at by making the P/R (quantity of
production/respiration) ratio which can indicate the change of the whole ecosystem into anassessment index. Although the environmental impact risk assessment examination of a chemical
substance has been conventionally carried out using a single species creature, as for the model
ecosystem examination which imitated the nature, the general-purpose standard test method is
not established by problems, such as stability, high cost nature and reproducibility. In addition, thefundamental manual concerning test operation is already built (Funds for the Overall Promotion ofEnvironmental Research -Ministry of Environment in FY2009-2011), and the ring test etc. which
carried out the test between several research institutions for wide use, are turned to carry out toestablish as the OECD standard test method.
Timeline: November 1, 2012 –Topics:2nd New LRI Research Report Meeting: “Development of Ecosystem Risk Impact Assessment
System Methods for Chemicals using Microcosm Systems”
Publications:
Kunihiko Kakazu, Hideto Usui, Jun Kumada, Katsura Sugiura, Ryuhei Inamori, Yuhei
Inamori：”Ecotoxicity Assessment of SDS with P,R (Production, Respiration) about Aquatic
Microcosm”, Journal of Water and Waste, Vol.54, No.9, pp.683-690 (2012).
Annual Report 201319

A n n u a l R e p o r t 2 013
Japan Chemical Industry Association
LRI oﬃce : 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/